oxidative phenol coupling in ascomycetes: regioselectivity

Oxidative Phenol Coupling in Ascomycetes:Regioselectivity of Coupling Enzymes

INAUGURALDISSERTATION

zur Erlangung des Doktorgradesder Fakultät für Chemie und Pharmazie

der Albert-Ludwigs-Universität Freiburg im Breisgau

vorgelegt von

Leon Maximilian Ernst Günter Fürtges

aus Essen2018

Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan WeberDekan: Prof. Dr. Manfred JungReferent: Prof. Dr. Michael MüllerKorreferent: Prof. Dr. Dirk Hoffmeister

Datum der mündlichen Prüfung: 13.04.2018

Declaration

Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und

ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus

anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter

Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche

Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Promotionsberater oder

anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder

mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem

Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch

im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.

Freiburg im Breisgau, den

Acknowledgements

I want to thank:

• First and for most Michael Müller for the interesting and dynamically evolving

topic, the good working environment and infrastructure, and for always being

open for discussions and new ideas.

• Prof. Dr. Dirk Hoffmeister and PD Dr. Wolfgang Hüttel

• Prof. Dr. Axel Brakhage, Dr. Thorsten Heinekamp, Dr. Daniel Scharf for their

support and know-how on fungal transformation and genetics.

• Dr. Daniel Drochner and Dr. Silke Foegen for their work laying the basis for this

thesis.

• Dr. Björn Grüning for his 24/7-Galaxy-hotline.

• Dr. Laura Mazzaferro for a lot of on- and off-topic discussions, and for all the

help while writing this thesis.

• Dr. Andreas Präg, Dr. Eduard Frick for their help with my first steps in the lab

and for grounding me when I got all too enthusiastic.

• Sascha Ferlaino for his help with NMR measurements and interpretation, and for

starting to ride to work with an e-bike.

• Julian for a lot of on- and off-topic discussions in the saddle.

• Stefanie2, Sebastian, and Johanna for the comfortable atmosphere in our office

and lab. And Stefanie for several nighttime baking attempts.

• The Fungi-Team AK Müller (Wiebke and Sebastian) for fruitful discussions and

help.

• Stefan, Jakob, and Philipp for numerous barbecues and Elpi nights.

• Daphné, Martin, Klaus and Andi for making me feel like home in 3Königstraße

and Rheinstraße.

• My family for being always there for me, for a lot of good memories and trips. My

parents for making education a top priority and always supporting my decisions.

My sisters for fun times in Freiburg, Berlin (Sage Club), and Essen (Turock).

• Jelena for making the process of writing this thesis so much easier!

Leon Fürtges

Abbreviations

17bHSDcl 17-β -hydroxysteroid dehydrogenase from C. lunata

AMM Aspergillus minimal medium

AMM-T Aspergillus minimal medium for transformation

BLAST basic local alignment search tool

bp basepair(s)

BSA bovine serum albumine

BVMO Baeyer–Villiger monooxygenase

BVO Baeyer–Villiger oxidation

CD circular dichroism

CDD Conserved Domain Database

cDNA complementary DNA

CRISPR/Cas9 clustered regularly interspaced short palindromic

repeats-Cas9

CYP cytochrome P450

de diastereomeric excess

DEBS deoxyerythronolide B synthase

DNA deoxyribonucleic acid

dNTP deoxynucleoside triphosphate

DTT dithiothreitol

dU deoxyuracil

DUF domain of unknown function

EDTA ethylenediaminetetraacetic acid

ee enantiomeric excess

ER endoplasmatic reticulum

ESIPT excited state intramolecular proton transfer

FAD flavin adenine dinucleotide

FAS protein fasciclin domain containing protein

FMN flavin mononucleotide

FMO flavin-dependent monooxygenase

gDNA genomic DNA

HMBC heteronuclear multiple-bond correlation

spectroscopy

HPLC high performance liquid chromatography

HPLC-MS high performance liquid chromatography coupled

with mass spectrometry

HR homology directed repair

HSQC heteronuclear single-quantum correlation

spectroscopy

INADEQUATE incredible natural-abundance double-quantum

transfer experiment

iPKS iterative type I polyketide synthase

IPTG isopropyl β -D-1-thiogalactopyranoside

JGI Joint Genome Institute

LB lysogeny broth

LMM Aspergillus minimal medium for laccase production

LTE lithium acetate-Tris-EDTA buffer

Mb mega basepairs

MβL metallo-β -lactamase

MCO multi-copper oxidase

mCPBA meta-chloroperoxybenzoic acid

MEA malt extract medium

MeOH methanol

mPKS modular type I polyketide synthase

NADH nicotinamide adenine dinucleotide

NADPH nicotinamide adenine dinucleotide phosphate

NCBI National Center for Biotechnology Information

NHEJ non-homologous end joining

NR-PKS non-reducing polyketide synthase

OD optical density

O-MT O-methyltransferase

ORF open reading frame

PBS phosphate buffered saline

PBST phosphate buffered saline with tween

PCR polymerase chain reaction

PEG polyethylenglycol [average molecular weight]

PHAR polyhydroxyanthracene reductase

PKS polyketide synthase

PR-PKS partially-reducing polyketide synthase

PT product template

qRT-PCR quantitative real time PCR

RNA ribonucleic acid

SD/∅His synthetic drop-out medium without histidin for

Saccharomyces cerevisiae

SDR short chain dehydrogenase/reductase

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel

electrophoresis

sgRNA singleguide ribonucleic acid

SMM Aspergillus minimal medium with sucrose

SSC saline sodium citrate

TAE Tris-acetic acid-EDTA buffer

TB terrific broth

TE thioesterase

USER uracil-specific excision reagent

YPAD yeast extract-peptone-adenine-dextrose medium

Contents

Summary 1

Zusammenfassung 5

1 Introduction 9

1.1 Polyketides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.2 Structural diversity of polyketide dimers . . . . . . . . . . . . . . . . . . 10

1.3 Biosynthesis of the natural product’s backbone: PKS . . . . . . . . . . . 21

1.4 Dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2 Results and Discussion 31

2.1 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.2 Vioxanthin biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2.3 Viriditoxin biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

2.4 In-silico analysis of polyketide dimers’ biosynthesis . . . . . . . . . . . . . 98

3 Concluding remarks and outlook 149

4 Experimental Section 155

4.1 Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

4.2 Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

4.3 Natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

4.4 Analytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

4.5 Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Bibliography 193

List of Publications 219

Appendix 221

Summary

Oxidative phenol coupling is a crucial step in the biosynthesis of natural products such

as morphine (1), gossypol or kotanin (2).[1–3] In the case of gossypol and kotanin

(2), the homodimers are formed regio- and stereoselectively.[3–5] Cytochrome P450

(CYP) enzymes, flavin-dependent monooxygenases (FMOs), and laccases have been

described to catalyze coupling steps, and CYP enzymes and FMOs have been reported to

control the regio- and stereochemistry of the dimerization as well.[3,6,7] To gain further

insights into the enzymatic control of the coupling’s selectivity, the biosyntheses of

O

O

O

O

OH OH O

OOHOH

pigmentosin A (24)

M *

O

O

OH OH O

(R)-semi-vioxanthin (4)

OO

OHOHO

OOH

O

OH O

vioxanthin (3)

P*

O

O

O

OO

OH OH

(S)-semi-viriditoxin (6)

O

OH

O

OH O

O

O

O

O

O

OOHOH

O

viriditoxin (5)

M *

O

O

O

O

OH OH O

OOHOH

P-pigmentosin A (7)

P *

P. citreonigrum

P. freii

A. ochraceus

T. rubrum

T. violaceum

H. immaculata

A. niger FGSC A1180

pSUC::vavH

A. viridinutans

C. cladosporioides

from A. viridinutans

Scheme 1. Graphical abstract.

2

dimeric naphtho-α-pyrones in ascomycetes was investigated. Vioxanthin [3, 8,8′-linked

(R)-semi-vioxanthin (4)] and viriditoxin [5, 6,6′-linked semi-viriditoxin (6)] are

biosynthesized regio- and stereoselectively by Trichophyton rubrum, Penicillium

citreonigrum, and Aspergillus ochraceus, and Aspergillus viridinutans, respectively. In

this work the biosynthetic gene clusters of the polyketide dimers were identified and

they contained a gene coding for a laccase as the dimerizing enzyme. Until now, no

member of this enzyme class has been reported to control the regio- or stereochemistry

of a catalyzed intermolecular phenol coupling reaction.

The laccase VavH from A. viridinutans was heterologously produced and was shown

to convert the non-native substrate (R)-semi-vioxanthin(4) selectively to the 6,6′-linked

dimer 7. While the regiochemistry is tightly controlled, the stereochemistry of 7 showed

the favored formation of one atropisomer, although with low diastereoselectivity

(30–50% de). The coupling pattern of the formed 6,6′-linked dimer 7 corresponds to

the regiochemistry of the laccase’s native product viriditoxin (5), suggesting enzymatic

control over the regiochemistry.

Subsequently, candidate gene clusters for the biosynthesis of tetrahydroxanthone

dimers such as secalonic acid A (8) or neosartorin (9, Scheme 4) were predicted using

the same approach. Homologous gene clusters were identified in the genome sequences

of Claviceps purpurea, Aspergillus aculeatus, Aspergillus fischeri, Aspergillus lentulus,

and Penicillium oxalicum, all containing a gene coding for a CYP enzyme. Members

of this enzyme class have been shown to catalyze the phenol coupling step in the

biosynthesis of dimeric polyketides in Streptomyces and Aspergillus controlling the regio-

and stereochemistry of the dimerization.[6,8,9]

Taking into account published results on the biosynthesis of xanthones and

observations made on conversions of (R)-3,6,8,9,10-pentahydroxy-3,4-dihydro-

3

O

O

OH OH OOH

OH

O

O

COOMe

HO

OH

MeOOC

OH O OH

H3C OH

O

OH

* P

neosartorin (9)chrysophanol (11) monodictyphenone (15)

OHOOH

Scheme 2. Proposed biosynthetic pathway to neosartorin (9) with theintermediates chrysophanol (11) and monodictyphenone (15).

anthracen-1(2H)-one (10), a new biosynthetic pathway was proposed. It relies

on chrysophanol (11) as substrate for an atypical Baeyer–Villiger monooxygenase

(BVMO) that stabilizes a more vulnerable tautomer of the anthraquinone. This theory

explains the incorporation of labeled chrysophanol (11) into the prenylated xanthone

tajixanthone (12) and the tetrahydroxanthone dimer secalonic acid D (13), the missing

BVMOs’ active site motifs, and the controlled reaction of the anthraquinone to either

janthinone (14) or monodictyphenone (15).[10,11]

Zusammenfassung

Oxidative Phenolkupplung ist ein wichtiger Schritt in der Biosynthese von Naturstoffen

wie Morphin (1), Gossypol, oder Kotanin (2).[1–3] Im Fall von Gossypol und Kotanin (2)

werden die Homodimere regio- und stereoselektive verknüpft.[3–5] Cytochrome P450

Enzyme (CYP Enzyme), Flavin-abhängige Monooxygenasen (FMO) und Laccasen sind

in der Lage diese Kupplungsschritte zu katalysieren. Im Fall von CYP Enzymen und FMO

wurde nachgewiesen, dass die Enzyme die Regio- und Stereochemie der Dimerisierung

kontrollieren können.[3,6,7] Um weitere Einblicke in die Enzym-kontrollierte Selektivität

O

O

O

O

OH OH O

OOHOH

Pigmentosin A (24)

M *

O

O

OH OH O

(R)-semi-Vioxanthin (4)

OO

OHOHO

OOH

O

OH O

Vioxanthin (3)

P*

O

O

O

OO

OH OH

(S)-semi-Viriditoxin (6)

O

OH

O

OH O

O

O

O

O

O

OOHOH

O

Viriditoxin (5)

M *

O

O

O

O

OH OH O

OOHOH

P-Pigmentosin A (7)

P *

P. citreonigrum

P. freii

A. ochraceus

T. rubrum

T. violaceum

H. immaculata

A. niger FGSC A1180

pSUC::vavH

A. viridinutans

C. cladosporioides

aus A. viridinutans

Scheme 3. Graphische Zusammenfassung.

6

der Phenolkupplung zu gewinnen, wurden die Biosynthesen von dimeren

Naphtho-γ-pyronen in Ascomyceten untersucht. Vioxanthin [3, 8,8′-verknüpftes

(R)-semi-Vioxanthin (4)] und Viriditoxin [5, 6,6′-verknüpftes semi-Viriditoxin (6)]

werden von Penicillium citreonigrum, Aspergillus ochraceus, und Aspergillus viridinutans

regio- und stereoslektiv produziert. In dieser Arbeit wurden die biosynthetischen

Gencluster dieser Polyketiddimere identifiziert. Die Gencluster enthalten jeweils ein

Gen, das für eine Laccase kodiert. Diese Enzymklasse war bisher nicht dafür bekannt

die Regio- und Stereochemie der von ihr katalysierten Phenolkupplung zu kontrollieren.

Die Laccase VavH aus A. viridinutans wurde heterolog in Aspergillus niger produziert.

In Umsetzungen mit dem Enzym wurde das nicht-physiologische Substrat

(R)-semi-Vioxanthin (4) selektiv zu dem 6,6′-verknüpften Dimer 7 gekuppelt. Im

Gegensatz zur streng regioselektiv katalysierten Dimerisierung, wird eines der

Atropisomer mit niedriger Diastereoselektivität (30–50% de) bevorzugt gebildet. Das

dem physiologischen Produkt Viriditoxin (5) entprechende Kupplungsmuster des

gebildet 6,6′-verknüpften Dimers deutet auf enzymatische Kontrolle der Regiochemie

hin.

Außerdem wurden mit dem bereits genutzten Ansatz auch putative

Biosynthesegencluster von Tetrahydroxyxanthondimeren wie Secalonsäure A (8) oder

Neosartorin (9) untersucht. In den veröffentlichten Genomsequenzen der

Tetrahydroxyxanthondimer-Produzenten Claviceps purpurea, Aspergillus aculeatus,

Aspergillus fischeri, Aspergillus lentulus und Penicillium oxalicum wurden homologe

Biosynthesegencluster identifiziert. Diese Gencluster enthalten die für die Biosynthese

der polyketidischen Grundstruktur nötigen Gene und ein vermutlich für die

Dimerisierung verantwortliches CYP Enzym. Die Enzymklasse der CYP Enzyme ist für

den Phenolkupplungsschritt in der Biosynthese von Polyketiddimeren in Streptomyces

7

und Aspergillus zuständig und kontrolliert die Regio- und Stereochemie dieser

Dimerisierung.[6,8,9]

Unter Zuhilfename publizierter Ergebnisse in Kombination mit Beobachtungen, die

bei der Umsetzung von (R)-3,6,8,9,10-Pentahydroxy-3,4-dihydroanthracen-1-(2H)-one

(10) gemacht wurden, wurde ein abgeänderter Biosyntheseweg vorgeschlagen.

Zentraler Bestandteil der neuen Biosyntheseroute ist das Anthrachinon Chrysophanol

(11), das als Substrat einer atypischen Baeyer–Villiger-Monooxygenase (BVMO) dient.

Von diesem Enzym soll ein Tautomer von Chrysophanol (11) stabilisiert werden,

so dass das sonst stabile Anthrachinon in einer Baeyer–Villiger-Oxidation gespalten

werden kann. Diese neue Biosyntheseroute berücksichtigt den Einbau von markiertem

Chrysophanol (11) in Tajixanthone (12) oder Secalonsäure D (13) und erklärt die Rolle

dieses Intermediates.[10,11] Dem an der Reaktion beteiligten Enzym (BVMO) wird keine

katalytische Funktion im klassischen Sinne zuteil. Es stabilisiert einen angreifbares

Tautomer und lenkt über diese Stabilisierung auch die Regiochemie der chemisch

ablaufenden BVO hin zu Janthinon (14) oder Monodictyphenon (15).

O

O

OH OH OOH

OH

O

O

COOMe

HO

OH

MeOOC

OH O OH

H3C OH

O

OH

* P

neosartorin (9)chrysophanol (11) monodictyphenone (15)

OHOOH

Scheme 4. Vorgeschlagener Biosyntheseweg von Neosartorin (9) inklusive derIntermediate Chrysophanol (11) und Monodictyphenone (15).

1 Introduction

1.1 Polyketides

A considerable number of currently used drugs are either polyketide natural products

or derived from them. Some well known examples are lovastatin (16), amphotericin

B, and tacrolimus.[12–15] Polyketides form an extensive group of structurally diverse

natural products, which consists of acetate building blocks linked in a head-to-tail

fumonisin B1 (17)

OOH

OH OH

NH2

O

OH

OOHO

O

OO OH

OH

O

erythromycin A (18)

O

O O

O

HOOH

OH

O

O

O

NHO

O

OH

(R)-mellein (19)

O

OH O

aflatoxin B1 (20)

OO

O

O

O

O OH

HO

OH

O

orsellinic acid (21)

Figure 1.1. Structural diversity of polyketides produced by fungi and bacteria.

10 1 Introduction

manner. The biosynthesis using simple building blocks in an assembly line-like manner

is catalyzed by a polyketide synthase (PKS) and yields a variety of natural products.

This carbon skeleton is further modified, leading to a diversification of the spectrum

of natural products. The modifications catalyzed by the so-called tailoring enzymes

include O- and C-methylation, hydroxylation, reduction, dehydration, glycosylation, or

dimerization, producing compounds such as fumonisin B1 (17), erythromycin A (18),

(R)-mellein (19), aflatoxin B1 (20), or orsellinic acid (21) (Figure 1.1).[6,8,16–28]

1.2 Structural diversity of polyketide dimers

Several polyketide dimers have been isolated from bacteria, fungi, and plants, however

for most of them the biosynthesis is only partially elucidated. The polyketide dimers

differ in the regio- and stereochemistry of the biaryl linkage. In some cases, the same

polyketide monomer is coupled at different positions by different producers [e.g.,

kotanin (2) and related bicoumarins (Figure 1.2)].[29–33]

O

O

O

O

O

O

O

O

O

O

O

OO

O

OO

O O

O

O

O

O O

O

M*

P*

P *

isokotanin A (23)desertorin C (22)kotanin (2)

Figure 1.2. Homodimeric bicoumarins kotanin (2), desertorin C (22), andisokotanin A (23) isolated from Aspergillus niger, Emericella desertorum, andAspergillus alliaceus, respectively.[31,32,34]

Studies of the naphtho-α-pyrone dimer vioxanthin’s (3) biosynthesis revealed its

polyketidic origin and synthesis through coupling of monomeric precursors.[22,35,36] The

1.2 Structural diversity of polyketide dimers 11

same monomeric precursor is coupled with a different regiochemistry in pigmentosin A

(24), showing possible similarities of the tricyclic polyketides’ biosynthesis with the well

studied biosynthesis of bicyclic bicoumarins.[37] For practical purposes, a numbering

derived from the bisnaphtho-α-pyrones, such as aschernaphtopyrone A (25) and

aschernaphtopyrone B (26) isolated from the sacle insect pathogenic fungus Aschersonia

paraphysata BCC 11964, was used in this work. It means, the numbering does

not necessarily correspond to the IUPAC numbering rules and for instance, biaryls

are referred to as 8,8′-like dimers (Figure 1.3).[38] Monomeric polyketides such as

(R)-pro-semi-vioxanthin (27) can be theoretically coupled to five different regioisomers,

although not all regioisomers have been isolated in nature (Figure 1.3).

12 1 Introduction

O

O

O

OOHOH

HO

OH

HO

OH

M *

aschernaphthopyrone A (25)

6

6'

OO

OHOHO

OHOH

HO

OH O

aschernaphthopyrone B (26)

M*

8

8'

O

O

O OH OH

OH

HO

OH OH O

*

O

O

HO OH

HO

OH OH O

O

OH

* *

OOH

OOHOH

HO

OH

HO

O

O

5

8'

5

5'

6

8'

O

HO

HO

OH

OH

OH O

*

6

8'

O

OH

O

OO

OHOHO

OOH

O

OH O

vioxanthin (3)

P*

8

8'

O

O

O

OOHOH

O

OH

O

OH

M *

pigmentosin A (24)

6

6'

not (yet) isolated

Figure 1.3. Possible regioisomers of bisnaphtho-α-pyrones.


1.2.1 8,8′-like dimers

The 8,8′-dimers are coupled in ortho-position to a phenolic hydroxy group and are

symmetrical dimers. Examples of this group have been isolated from bacteria, fungi,

and plants.[39–41] The julichromes [e.g., julichrome Q3-3 (28), Figure 1.4], isolated

among others from Streptomyces shiodaensis, are polyketide dimers with a variety of

different post-PKS decorations of their core structure. Even with different post-PKS

modifications the coupling pattern for the dimers remains unchanged, suggesting that

the regiochemistry of the phenol coupling step is not controlled by the substrate, but

by the involved coupling enzyme.[39,42–48] The biosynthetic gene cluster responsible for

julichrome production was identified, which contains all tailoring enzymes and a CYP

enzyme (JulI, EPJ36714).[6] With actinorhodin (29, Figure 1.4), another polyketide

dimer with the same 8,8′-like coupling pattern has been isolated from Streptomyces

coelicolor.[49] The biosynthesis of the compound has been thoroughly studied and

the biosynthetic gene cluster identified.[50–52] Although a candidate coupling enzyme

(ActVA-4, Q53906) has been identified, the phenol coupling step of actinorhodin’s (29)

biosynthesis remains poorly understood.[53]

OH

O

O

O

O

OH

OAc

OAc

H

H

H

H

H

H

H OH

HO

O

O OH

HO

julichrome Q3-3 (28)

OO

OH O

OH O

COOH

OHO

O OH

actinorhodin (29)

COOH

Figure 1.4. Polyketide dimers isolated from Streptomyces afghaniensis NC 5228(28) and S. coelicolor (29).

14 1 Introduction

Fungal homodimeric secondary metabolites, such as aurofusarin (30, Figure 1.5),

have been studied for more than a century, even though their structure was not known at

the time.[54] This dimeric naphtho-α-pyrone was isolated from Fusarium graminearum

(teleomorph Giberella zeae), Hypomyces rosellus, and Fusarium culmorum.[40,55–57]

After the elucidation of the structure, and with the advent of high through-put

genome sequencing, the biosynthesis of aurofusarin (30) has been investigated.[40,57]

Monomeric rubrofusarin (31) was isolated from F. culmorum together with the dimeric

aurofusarin (30) and used to synthesize the dimer by coupling two rubrofusarin (31)

units.[57,58] Feeding studies with labeled acetate showed incorporation of the acetate

label into rubrofusarin (31) and helped to elucidate the folding mode of the poly-β -keto

chain.[59] These finding confirmed the polyketidic origin of rubrofusarin (31) and

its dimer aurofusarin (30). Knock-out studies reaffirmed the previous biochemical

work, however a single coupling enzyme could not be identified.[60–63] Instead, the

dimerization was found to be dependent on four enzymes.[60–63]

A large group of fungal dimers are the nor-toralactone (32)-derived naphtho-α-pyrone

dimers. Several of these dimers show a 8,8′-coupling pattern, such as vioxanthin (3) and

xanthomegnin (33), which have been isolated from the filamentous fungi Penicillium

citreonigrum (formerly Penicillium citreo-viride), Penicillium freii, Aspergillus ochraceus,

O

OO OH O

O

OO

O

O

OH O

aurofusarin (30)

OO

OH OH O

2

rubrofusarin (31)

Figure 1.5. Aurofusarin (30), a homodimeric polyketide produced by variousFusarium species. Rubrofusarin (31) is the last isolated intermediate in thebiosynthesis of aurofusarin (30).[63]


O

OOHOH

HO

nor-toralactone (32)

OO

OOHO

OOH

O

OH O

viomellein (34)

R *

OO

OHOHO

OOH

O

OH O

xanthomegnin (33)

R *

O

O

O

Figure 1.6. Nor-toralactone (32)-derived 8,8′-coupled dimers.

and the dermatophytes Trichophyton violaceum and Trichophyton rubrum (formerly

Trichophyton megnini) (Figure 1.6).[64–68] The polyketidic origin of the natural products

was confirmed by the incorporation of labeled acetate into the dimer viomellein (34),

an oxidized derivative of vioxanthin (3).[35]

The aschernaphthopyrones A (25) and B (26) are nor-toralactone (32) derived

dimers with different regiochemistry, which have been isolated from the scale insect

pathogen Aschersonia paraphysata BCC11964 (Figure 1.3).[38] A similar observation

has been made for tetrahydroxanthone dimers, such as ascherxanthone B (35) and

secalonic acid A (8)

OH

OH

O

O

OH

OHO

O

OH

OH

O

O

ascherxanthone B (35)

MeOOC OH

OHCOOMe

OH O OH

OH OHO

O

O

Figure 1.7. 8,8′-like coupled tetrahydroxanthone dimers.

16 1 Introduction

secalonic acid A (8, Figure 1.7). The latter has been isolated from Claviceps purpurea

together with a group of similar tetrahydroxanthone dimers, called ergochromes.[69]

The coupling pattern identified in these dimers corresponds to a 8,8′-like biaryl

linkage.[70] However, a 6,6′-like linkage had previously been observed by Franck

et al. for secalonic acid A–C (8).[71] This contradiction was later explained by the

regioisomerization of secalonic acid A (8) in DMSO.[72] The regioisomerization of the

8,8′-like linked secalonic acid A (8) to the unsymmetrically linked penicillixanthone A

(36) and 6,6′-like linked secalonic acid A (4,4′-8) has been observed (Scheme 1.1).[72]

Tautomerization of the enol function to a β -γ-unsaturated ketone opens the cyclic ether

of the xanthone structure, allowing rotation of the the aromatic ring.

O

O

O

OO

O

O OH

OH O OH

OH

HO

O

HO

OH O OH

OOH OH

OH O OH

OH

COOMe

COOMeOH

MeOOC OH

COOMeOH

MeOOCOH

MeOOCOH

4,4'-secalonic acid A (4,4'-8) secalonic acid A (8)

penicillixanthone (36)

Scheme 1.1. Regioisomerization of the tetrahydroxanthone dimer secalonic acid A(8) observed at room temperature in DMSO.[72]


1.2.2 6,6′-like dimers

O

OH

OOH

OH

OHO

O

OH

rubellin A (39)

O

O

OH O

O OH

OO O

OH H

gonytolide A (38)

O

O

OHOOH

OHOOH

O

O

O

OO

O

OO

P * M *

phomoxanthone A (37)

Figure 1.8. 6,6′-like coupled tetrahydroxanthone dimers.

While tetrahydroxanthone dimers such as secalonic acid A (8) have been assigned a

8,8′-like coupling pattern despite of the observed regioisomerization, other

tetrahydroxanthone dimers such as phomoxanthone A (37), gonytolides A (38),

D and F, or rubellins A (39), B–D have been determined to be 6,6′-like coupled

homodimers.[73–75] In the case of gonytolides A (38), D, F and rubellins A (39), B–D,

no regioisomerization is possible, trapping the 6,6′-like coupled isomer.

O

O

O

O

O

O

OOHOH

O

O

OH OH O

M *

viriditoxin (5)

Figure 1.9. The 6,6′-couplednaphtho-α-pyrone viriditoxin(5).[76,77]

For bisnaphtho-α-pyrones, no regioisomerization

has been postulated or observed. Nevertheless,

6,6′-coupled nor-toralactone (32) dimers have

been described. Apart from 6,6′-coupled

aschernaphthopyrone A (25), other naphtho-α-pyrone

dimers have been isolated from fungi. Pigmentosin A

(24), a 6,6′-coupled (R)-semi-vioxanthin (4) dimer,

has been isolated from an extract of the lichen

Hypotrachyna immaculata.[37] Viriditoxin (5) was

isolated from A. viridinutans and Spicaria divaricata.[76,77] A 8,8′-linkage was proposed

18 1 Introduction

for the compound, but later revised to a 6,6′-coupling.[76–78] This shows the difficulty

of correctly determining the regiochemistry of these homodimers.

The group of perylenequinones are 6,6′-like coupled dimers, which have been

connected with one or two additional linkages. Perlyene structures have been isolated

from sediments, petroleum, fossil crinoids, and tropical termite mounds.[79,80] It has

been hypothesized that the isolated compounds are derived from perylenequinone

pigments from ligninolytic fungi.[81] Perylenequinones are produced by a variety

of different phytopathogenic fungi and include the compounds cercosporin (40),

elsinochrome A (41), hypocrellin A (42), hypomycins, phleichromes, calphostins,

and phaeosphaerins (Figure 1.10).[82–88]

OH

OH

O

OOH

OH

O

O

O

O

cercosporin (40)

O

O

OH

OH

O

O

O

O

elsinochrome A (41)

O

O

OHO

O

O

OH

O

O

OH O

O

hypocrellin A (42)

Figure 1.10. Perylenequinones isolated from phytopathogenic fungi.


1.2.3 Other (rare) linkages

The bisnaphtho-γ-pyrones show a broad variety of different biaryl linkages. The

compounds isoustilaginoidin A (43) and nigerone (44) are symmetrically coupled

homodimers (Figure 1.11).[89,90] Isoustilaginoidin A (43) shows a 6,6′-like coupling

pattern, while nigerone (44) is a 5,5′-like linked dimer. Apart from the symmetrically

linked homodimers, unsymmetrical heterodimers, such as fonsecinone B (45), have

been isolated (Figure 1.11).

The regioisomerization of tetrahydroxanthones leads to the formation of two possible

symmetrical isomers and one unsymmetrical isomer, which is coupled in a 6,8′-like

pattern. Examples for natural products that have been isolated as this isomer are

phomoxanthone B or lentulin A (46) from Aspergillus lentulus.[73,91] The biosynthesis

of tetrahydroxanthones has been linked to anthraquinone precursors. Cladofulvin (47)

is a dimeric anthraquinone that is coupled unsymmetrically in a 6,8′-like pattern and

has been isolated from Cladosporium cladosporioides Cooke.[92]

O

O

O

O OH O

OOH

O

O

nigerone (44)

O

OHO

OH

HO

OH OH

OH O

O

isoustilaginoidin A (43)

P * M *

O

O

O

O

O O

OH

O

OH

OOH

M *

fonsecinone B (45)

Figure 1.11. Differently-linked naphtho-γ-pyrones isolated from filamentous fungi.

20 1 Introduction

O

O

O

OH

OHOOH

OH

MeOOCHO

COOMeOH

lentulin A (46)

HO

OH O

O

O

HO

O

OH

HO

HO

cladofulvin (47)

Figure 1.12. The anthraquinone-derived dimers lentulin A (46) from Aspergilluslentulus and cladofulvin (47) from Cladosporium cladosporioides.[91,92]

The different linkages observed in polyketide dimers probably originate from the

diversity of the involved coupling enzymes, while the core structure is synthesized via

a conserved or related polyketide biosynthetic pathway.

1.3 Biosynthesis of the natural product’s backbone: PKS 21

1.3 Biosynthesis of the natural product’s backbone: PKS

The hypothesis of Collie that polyphenol natural products are synthesized by iteratively

fusing acetate units did not immediately initiate research in the field of polyketide

biosynthesis.[93] Collie’s hypothesis was confirmed by demonstrating the incorporation

of [1-14C]-acetate into orsellinic acid (21).[94] The first PKS genes were identified in

Streptomyces strains.[23,95,96]

PKSs consist of different domains, each domain catalyzing a separate reaction on

the substrate that is tethered to the enzyme. These enzymes have been organized

in three main classes according to their domain architecture. Type I PKS consist of

separate domains that sequentially modify the growing poly-β -keto chain. This class

is further divided into modular and iterative type I PKSs. The majority of modular

type I polyketide synthases (mPKSs) have been identified in bacteria and include

functionally diverse domains in a large single polypeptide. The deoxyerythronolide

B synthase (DEBS) consists of eight modules encoded by three open reading frames

(ORFs), including a loading and a releasing module. The modules contain a set

of domains, which extend the poly-β -keto chain and modify it. Each module acts

on the substrate sequentially, catalyzing only one reaction step in the biosynthesis

of 6-deoxyerythronolide. According to the so-called DEBS-paradigm, the domain

architecture of a mPKS allows to predict the structure of the product by counting the

domains.[97]

In contrast to mPKS, iterative type I polyketide synthase (iPKS)s are encoded by a

single ORF and contain a set of functionally diverse domains, which act on the substrate

repeatedly (Scheme 1.2). Unlike mPKS, the programming of iPKS is not directly

reflected in its sequence, making it difficult to predict the structure of iPKS product

by analyzing the enzymes domain architecture. Nevertheless, Crawford et al. recently

22 1 Introduction

AT ACP KS AT

KR

ACP

S SO O

HO

KS AT

KR

ACP

SO

HO

HO

KS KS

ACP

PKS

S

SH

O

O O O

O

O O O O

O R

O

OO OH O

OH

O

OH

O

NH2

OH

O

SCoA

HO

O

O

OH

OH

3HO

O

SCoA

O

HO

O

SCoA

O

HO

O

SCoA

O

HO

O

SCoA

O

O

SCoA

9

loadingmodule 1

module 2

as modular type I

cs iterative type II

ds iterative type III

SS O O

HO

OH

HO

O O

SCoA8

SAM Bbs

b

ER

KS AT DH MT KR ACP LovB

LovC

O

O

H

O

O

H

HO

lovastatin B16s

bs iterative type I

doxorubicin

naringenin

Scheme 1.2. Domain architecture of different polyketide synthase classes.[98]

1.3 Biosynthesis of the natural product’s backbone: PKS 23

identified a domain in the non-reducing polyketide synthase (NR-PKS) subgroup of

iPKS.[99,100] The so-called product template (PT) domain controls the regiochemistry

of the first ring cyclization. The PT domains from NR-PKSs that have been linked

to characterized natural products, were used to further divide them in subgroups

depending on the PT domain’s sequence similarity.[101] In contrast to other iPKSs,

NR-PKSs do not necessarily contain a thioesterase (TE) domain. Some of the NR-PKSs

rely on an external TE for product release, which resembles metallo-β -lactamase

(MβL)s on the amino acid sequence level.[102,103] These insights provide the opportunity

to predict the structure of an iPKS product by analyzing the enzyme’s function.

Two additional archetypical classes of PKSs have been described. They differ in their

domain structure and mode of action (Scheme 1.2). PKS summarized as type II PKS

are enzymes complexes that consist of discrete separately encoded domains, which can

dissociate. They work iteratively and have only been identified in procaryotes. The

third group of PKS are mainly found in plants, but some occurrences in procaryotes

and fungi have been described.[104–106] These enzymes use variable complex starter

units, but do not modify the carbon backbone of the poly-β -keto chain. They iteratively

fuse malonate extender units to the starter molecule. The length of the poly-β -keto

chain is determined by the size of the active site.

This classification is challenged by recent research discovering PKS enzymes that do

not fit into the established classification.[107,108] These enzymes combine archetypical

features of the three major PKS classes. Nevertheless, the classification remains useful

to summarize PKSs into groups of similar mode of action and product types.

24 1 Introduction

1.4 Dimerization

The core of polyketidic natural products is often modified by so-called tailoring enzymes.

One of the possible post-PKS modifications is intra- and intermolecular oxidative phenol

coupling. In the simplest scenario two mono-substituted phenols are coupled. To

achieve this biaryl formation a proton is abstracted from the phenol and a radical is

generated via one-electron oxidation. The phenoxyradicals can be coupled at different

positions by the formation of new C–C or C–O bonds (Scheme 1.3). The regiochemistry

of the produced biaryls depends on the spin density of the different atoms of the phenols

involved in the coupling reaction. The chemical biaryl formation favors C–C over C–O

linked biaryls, because the spin density of the carbon atoms in ortho and para position

to the phenolic hydroxy group is higher.[109] The phenoxyradicals are equally likely

to be coupled at the ortho or para position, yielding a mixture of C–C linked biaryls

with different regiochemistry (Scheme 1.3). If substituted phenols are used to generate

biaryls, the substituents’ influence on the phenol’s spin density may favor the formation

OH

R

O

R

O

R

O

RR

R

R

R

HOOH

OH

OH

HO

OHR

R

ortho-para

ortho-ortho

para-para

2x

e-

H+

Scheme 1.3. Biaryls arising from coupling of phenoxyradicals.

1.4 Dimerization 25

of one regioisomer or prevent the formation of another. When substituted phenols are

coupled, ortho-disubstituted biaryls may be generated. The coupling of rotationally

hindered biaryls will generate a mixture of atropisomers. Without stereochemical

information from the coupled molecules or a chemical or enzymatic catalyst a racemic

mixture will be formed.

Intramolecular phenol coupling has been observed, e.g., in the biosynthesis of

vancomycin (48) and morphine (1) (Figure 1.13).[110–115] In these cases, the positions

at which the phenol coupling can take place are restricted by the substitution pattern

of the involved phenols and the general structure of the molecule that is cyclized. In

an intramolecular phenol coupling the regiochemistry is influenced by the rest of the

molecule, and the stereochemistry as well. Steric constraints may force the molecule’s

backbone in a specific conformation favoring the formation of a specific stereo- and

O

HO

HO

H

H

N

HN

O

NH

HO

O

NH

O

HN

NH2

O

Cl

O

O

O

O

NH

Cl

HN

OOH

HN

O

OH

O

OHHOOH

O

O

OOH

OHH2N

HO

HO

morphine (1) vancomycin (48)

Figure 1.13. The alkaloid morphine (1) from Papaver somniferum and theglycopeptide antibiotic vancomycin (48) from Amycolatopsis orientalis. C–C bondsbuilt up via phenol coupling are highlighted in red.

26 1 Introduction

regioisomer. These conformational influences may also play a role in the coupling of

two separate molecules.

The enzymes responsible for this coupling step catalyze the initial one-electron

oxidation to generate a phenoxyradical. CYP enzymes are able to generate these radicals

and their direct involvement in phenol coupling reactions has been

demonstrated.[6,8,112,113,116] Flavin-dependent monooxygenase catalyze one-electron

oxidations of their substrates as well and have been linked to dimer formation.[7,117]

Laccases and peroxidases, copper dependent oxidases, have been isolated from fungi

and plants, and were shown to produce phenoxyradicals.[118,119]

CYP enzymes (EC1.14.13, EC 1.14.14 and EC 1.14.15) constitute a large and

heterogeneous enzyme class. The superfamily of CYP has been identified in mammals,

plants, fungi, bacteria, and even viruses.[121,122] There are different CYP systems in

different organisms and they all share the tetrapyrrole heme-thiolate co-factor that

binds an iron ion in the active site. Bacterial CYP enzymes (class I) are soluble proteins,

which are dependent on ferredoxin and ferredoxin reductase to catalyze one-electron

oxidations. An illustrative example of this class is the CYP responsible for the phenol

coupling step in the biosynthesis of julichromes.[6] Microsomal CYP systems (class II)

are related to bacterial CYP systems but depend on different reducing enzymes.[123,124]

In Rhodococcus sp. a CYP class was identified that combines the reductase and the

monooxygenase in a single polypeptide.[125] These fusion enzymes are additionally

dependent on the co-factor flavin mononucleotide (FMN) to reduce the CYP enzyme

after oxidation of the substrate. Lastly, the class III are membrane bound CYP enzymes

found in eucaryotes and require reduction via a cytochrome reductase system. This

class includes fungal CYP enzymes, which are involved in the biosynthesis of natural

1.4 Dimerization 27

O

OH

OHO O

O

OHO

O

OO

O

O

O

O

O

kotanin (2)demethylsiderin

P *O

OHO

HO

O

O

O

O

orlandin

P *

2KtnB KtnC

Scheme 1.4. Biosynthesis of the homodimeric bicoumarin kotanin (2).[8,9,120]

products such as kotanin (2, Scheme 1.4).[8,9,124] The enzymes can control the regio-

and stereochemistry of the biaryl formation.[8,9]

FMOs (EC 1.13.12 and EC 1.14.13) use mostly a non-covalently bound flavin

co-factor to catalyze a variety of reactions and transfer one atom from molecular

oxygen onto their substrate.[126] The remaining oxygen atom is subsequently reduced

to water. During this process, a highly reactive hydroperoxyflavin species is formed.[127]

This species rapidly decomposes to oxidized flavin and hydrogenperoxide, but in the

enzyme the species is stabilized and allows the transfer of a single oxygen atom

onto the substrate.[128] The oxidized flavin has to be reduced to start a new reaction

cycle, which is accomplished by NAD(P)H. However, there are a few examples of

substrates that act as a reducing agent (e.g., lactate monooxygenase).[129] These

enzymes participate in catabolism of xenobiotics, hydroxylations, Baeyer–Villiger

oxidations (BVOs), epoxidations, sulfoxidations, and halogenations.[128,130–132]

Up to now, only one example of a homodimer synthesized by an FMO has been

reported.[7] The C–N coupled marinopyrroles A–C (49, 50 and 51) and E (52) have

been isolated from Streptomyces sp. CNQ418 (Figure 1.14). Gene deletions showed that

two FMOs are involved in the coupling step. The FMOs involved in their biosynthesis

control the regiochemistry and the dimers are coupled stereoselectively.[7] The authors

28 1 Introduction

hypothesize that a halogenation catalyzed by the FMOs facilitates N,C-bipyrrole

homocoupling.[7]

O

HN

NO

Cl

Cl

Cl

ClOH

OH O

HN

NO

R1

OH

Cl

Cl

Cl

Cl

OHM * M *

marinopyrrole A (49), R=H marinopyrrole C (51), R1=Cl, R2=H

R

R2

marinopyrrole B (50), R=Br marinopyrrole E (52), R1=H, R2=Br

Figure 1.14. Pyrrole dimers isolated from marine dwelling Streptomyces sp.CNQ418.[7]

Laccases belong to the multi-copper oxidase (MCO) family (EC 1.10.3.2). With the

help of three copper centers (T1–3) they catalyze a four-electron reduction of dioxygen

to water via four one-electron oxidations of substrate molecules, which are oxidized

to a radical. The electrons are channeled from the T1 copper center to the trinuclear

T2/T3 copper center, where the reduction of O2 takes place.[133]

The first laccase was reported in 1886 by Yoshida in the sap of Rhus venicifera.[134]

Ten years later, the first laccase of fungal origin was described.[135] Since then, laccases

have been identified in various higher plants, almost all wood-rotting fungi, bacteria,

and insects.[136–139] Laccases appear to play a role in the biosynthesis of pinoresinol and

other lignans.[2,141] Umezawa et al. used cell free extracts of Forsythia intermedia to

convert achiral coniferyl alcohol (53) to (−)-secoisolariciresinol (54, Scheme 1.5).[140]

Interestingly, the conversion exclusively yielded one of the two possible

stereoisomers.[140] A stereoselective oxidase was assumed to be responsible for the

selective formation of only one stereoisomer in Forsynthia sp.’s lignan biosynthesis.[142]

Later on, Davin et al. demonstrated that a protein lacking an active site enables

1.4 Dimerization 29

OH

OH

O

2x

coniferyl alcohol (53)

OH

OH

O

OH

O

HO

()-secoisolariciresinol (54)

Scheme 1.5. Biosynthesis of (−)-secoisolariciresinol (54) from coniferyl alcohol(53).[140]

(“directs”) the stereoselective coupling of phenoxyradicals to the corresponding lignan,

while the reaction itself is catalyzed by a non-selective laccase.[143] Phenol coupling

reactions that have been carried out without such a dirigent protein lead to a mixture

of C–C and C–O coupled regioisomers.[144] Additionally, a peroxidase (also a MCO

enzyme) has been shown to catalyze the phenol coupling step in the biosynthesis of

the homodimer gossypol.[141] The regio- and stereochemistry of the phenol coupling

probably is controlled by a dirigent protein.[5]

2 Results and Discussion

2.1 Objectives

The main goal of this work was the identification of the enzymes catalyzing the

coupling step in the biosynthesis of biarylic polyketide natural products in fungi. A

further aim was the elucidation of the regio- and stereocontrol of the reaction. To

identify the enzymes involved in the biaryl coupling, we assumed that their coding

genes are organized in biosynthetic clusters, together with the gene encoding the iPKS

responsible for the synthesis of the core structure (Figure 2.1).

First, publications on natural products and their biosynthesis were gathered. The

therein described producers of homodimeric polyketide natural products were targeted

and the databases of National Center for Biotechnology Information (NCBI) and the

Joint Genome Institute (JGI) were screened for their genomes. The process of cluster

identification started with the identification of all candidate core genes (iPKS) in the

genome. These iPKS can be divided into different groups, producing polyketides

with different characteristics. Depending on the chemical structure of the natural

product’s backbone, the iPKS with appropriate predicted function were selected. The

putative function of genes surrounding the candidate iPKS was deduced by comparison

with already characterized homologues, or homologues present in other putative

biosynthetic gene clusters connected to structurally similar compounds. Phenol coupling

32 2 Results and Discussion

literature

• natural products

• biosynthesiselucidation

bioinformatics

• functional genecluster annotation

• homologous geneclusters

activity assays

• substrate range

• productcharacterization

biosynthetic gene cluster

iPKS• PR-PKS• NR-PKS + SDR

PCE• CYP• FMO• laccase

Tailoring• O-MT• BVMO• oxygenase

Figure 2.1. Scheme of the steps leading to the identification of putative biosytheticgene clusters.

enzymes (CYP enzymes, FMOs, and laccases) and other tailoring enzymes, such as

O-methyltransferases (O-MTs), BVMOs, reductases or oxygenases, were on focus. The

clusters that were taken into consideration were those that contain genes coding for all

or most of the tailoring enzymes needed in the proposed biosynthetic pathway. For these

clusters, the coupling enzyme was heterologously produced in an adequate expression

system and tested for coupling activity with its proposed substrate or analogues thereof

to confirm the in silico analysis.

2.2 Vioxanthin biosynthesis 33

2.2 Vioxanthin biosynthesis

O

OR

OH OH O

OO

OH

OOH

O

OH O

OHO

?

(R)-semi-vioxanthin (4), R=CH3 vioxanthin (3)

*P

(R)-pro-semi-vioxanthin (27), R=H

Scheme 2.1. Candidate coupling substrates for the dimerization step in thebiosynthesis of vioxanthin (3).

The ascomycetes Penicillium citreonigrum ATCC 42743™and Aspergillus ochraceus DSM

2499 have been reported to produce vioxanthin (3).[64,65] The biosynthesis of this

biaryl has been shown to proceed via the coupling of the monomeric precursor

(R)-semi-vioxanthin (4).[22] However, the enzyme(s) responsible for this coupling

step, and the biosynthesis in general, remain unknown. To identify these enzymes and

taking into account the chemical structure of vioxanthin (3), we proposed a minimal

biosynthetic gene cluster composition (Figure 2.2). The biosynthesis of the monomer

backbone is likely catalyzed by a partially-reducing polyketide synthase (PR-PKS), or

a NR-PKS in combination with a reductase. Considering the known biosynthesis of

other biaryls, CYP, FMO or laccases could be responsible for the phenol coupling step.

Moreover, at least an O-MT is needed for the tailoring.


2.2.1 Producers of vioxanthin and related compounds: genome analysis

At the beginning of this work, the genome sequences of the reported vioxanthin (3)

producers were not available. For this reason, we searched for producers of related

compounds. The dermatophyte Trichophyton rubrum was reported to produce

xanthomegnin (33), a quinone derivative of vioxanthin (3), and its genome was

already available at the NCBI (BioProject PRJNA65025). The genome of T. rubrum

has a size of circa 22.5 mega basepairs (Mb) and has been assembled to 35 scaffolds.

Together with the genome sequence, a gene annotation and predicted protein sequences

have been published.[145]

In order to identify putative vioxanthin (3) biosynthetic gene clusters containing

an iPKS, the T. rubrum’s genome sequence was analyzed with the online tool

antiSMASH.[146] A total of 58 biosynthetic gene clusters were predicted, six of which

contain an iPKS. (R)-semi-Vioxanthin (4) is reduced once in the lactone ring compared

to toralactone (55). The reduction can either be catalyzed by an external short chain




Tailoring• O-MT

OO

OH

OOH

O

OH O

OHO

vioxanthin (3)

*P

polyketide synthasereductaseO-methyltransferasecoupling enzyme

Figure 2.2. Possible composition of a putative biosynthetic gene cluster ofvioxanthin (3).


dehydrogenase/reductase (SDR) or by a reducing module of the core unit producing

iPKS. The reducing domain of the iPKS would only act on the substrate once, which

suggests that the iPKS is part of the PR-PKS family. CYP are known to be involved in

oxidative intermolecular phenol coupling and in several cases they have been shown

to determine the regio- and stereochemistry of the coupling.[6,8] Therefore, the first

attempts to identify a biosynthetic gene cluster for vioxanthin (3) focused on clusters

containing genes coding for an iPKS and a CYP enzyme. Using these search parameters

three PR-PKS gene clusters were identified that contained a gene coding for a CYP

enzyme. With the assumption that structurally similar natural products are produced by

gene clusters containing homologous genes, the genome of the vioxanthin (3) producer

(P. citreonigrum) was sequenced by BaseClear B.V. (Leiden, Netherlands) using Illumina

sequencing technology.

The genome sequencing and assembly of P. citreonigrum yielded 1855 scaffolds and

a total genome size of 35 Mb. It was annotated using the AUGUSTUS web server and

subsequently analyzed using antiSMASH. The online tool identified 126 biosynthetic

TERG_05583TERG_05582 TERG_05584 TERG_05585 TERG_05586 TERG_05587

Trichophyton rubrum

PCV_6005 PCV_6006 PCV_6007 PCV_6008 PCV_6009 PCV_6010

Penicillium citreonigrum

Cluster I

Cluster II


vpcF vpcH vpcG vpcB vpcA vpcJ vpcC vpcD vpcE

Trichophyton rubrum

xtrH xtrG xtrF xtrE xtrD xtrC xtrB xtrAxtrI

Figure 2.3. The gene clusters containing homologous iPKSs from P. citreonigrumand T. rubrum.


gene clusters, 27 of which are clusters containing a gene coding for an iPKS. The protein

sequences of the iPKS encoding genes of P. citreonigrum were compared with the iPKS

protein sequences of T. rubrum. The comparison yielded two pairs of homologous

iPKS enzymes [PCV_6009/TERG_05583 (Cluster I) and VpcA/XtrA (Cluster II)], one of

which contains reducing domains (PCV_6009/TERG_05583, Table 2.1), predicted by

the Conserved Domain Database (CDD) web tool.[147,148]

Characterization of Cluster I’s CYP enzymes PCV_6005 and PCV_6007

Cluster I from T. rubrum and P. citreonigrum contain CYP enzymes, which could catalyze

the coupling step in xanthomegnin (33) and vioxanthin (3) biosynthesis (Table 2.1).

The PR-PKS TERG_05583 from T. rubrum and PCV_6009 from P. citreonigrum could be

capable to synthesize the core unit and carry out the necessary reduction. However, an

O-MT is missing in both clusters. Nevertheless, mRNA of P. citreonigrum was isolated

and transcribed to cDNA. The intronless cDNA-derived genes PCV_6005 and PCV_6007

both encoding CYP enzymes were cloned into pESC-His and expressed in Saccharomyces

cerevisiae. S. cerevisiae cultures were used to convert (R)-semi-vioxanthin (4) in whole

cell conversions. No dimer was isolated.

Table 2.1. iPKS clusters (I) of PCV_6009 from P. citreonigrum and TERG_05583from T. rubrum.

P. citreonigrum Putative function Sequence T. rubrumidentity [%]

PCV_6005 CYP (PatH) 44.1 TERG_05587PCV_6006 6-MSA decarboxylasePCV_6007 CYP (PatI) 42.2 TERG_05587PCV_6008 hypothetical proteinPCV_6009 PR-PKS 59.6 TERG_05583PCV_6010 transcription factor


CoA

O

HO CoA

O O

OH

OH

O OH

OH

OH

OH

OH

OH

OH

OH

3

O

OHO

O

CYP619C2

CYP619C3

6-MSAS 6-MSAD

CYP619C2

6-MSA m-cresol (58) toluhydroquinone (57)

m-hydroxybenzyl alcohol (59)

gentisyl alcohol (60) patulin (56)

Figure 2.4. Excerpt from the biosynthesis of patulin (56).

Sequence analysis of the CYP enzymes PCV_6005 and PCV_6007 from P. citreonigrum

revealed sequence homology with CYP enzymes involved in the biosynthesis of patulin

(56).[149,150] Therefore, conversions were also carried out with substrates from patulin’s

(56) biosynthesis. PCV_6005 is homologous to the characterized CYP619C3 enzyme

from Aspergillus clavatus NRRL1 (sequence identity 77.6%). PCV_6007 has a sequence

identity of 75.8% compared to CYP619C2 from A. clavatus NRRL1. CYP619C2 forms

toluhydroquinone (57) via hydroxylation of m-cresol (58). Toluhydroquinone (57) is

a shunt product and not incorporated into patulin (56, Figure 2.4). CYP619C2 also

catalyzes the hydroxylation of m-hydroxybenzyl alcohol (59) to gentisyl alcohol (60),

which is converted to patulin (56) via several modifying steps. m-Cresol (58) is a


substrate of CYP619C3 as well. The enzyme converts m-cresol (58) to m-hydroxybenzyl

alcohol (59, Figure 2.4).

Conversions of m-cresol (58) with PCV_6005 did not yield any product

(subsection 3.1 Figure 1). PCV_6007, on the other hand, converted m-cresol (58) to

toluhydroquinone (57) and m-hydroxybenzyl alcohol (59) to an unknown compound,

most likely gentisyl alcohol (60) (appendix: subsection 3.1 Figure 2). The two

candidate genes for the biosythesis of vioxanthin (3) could therefore be excluded.

Characterization of Cluster II

As the cluster I candidate was ruled out as putative biosynthetic gene cluster of

vioxanthin (3), the gene cluster II with homologous iPKS (VpcA/XtrA) in both organisms

was analyzed in detail. Apart from the central NR-PKS the cluster in T. rubrum is

comprised of a FMO, an O-MT, a fasciclin domain containing protein (FAS protein), a

laccase, a SDR, a transporter, and a transcription factor (Table 2.2). A similar cluster

was identified in P. citreonigrum. However, the cluster from P. citreonigrum did not

contain homologues of the laccase, FAS protein, and SDR. The missing genes were

identified on a different small contig (9 kb). As the two contigs were suspected to be

contiguous, primers were designed to detect an assembly gap. A DNA fragment of

576 bp length was amplified using the primer pair gapclose-304.2 and gapclose-684.4,

confirming the continuity of the two scaffolds. Thus, both clusters contain two putative

coupling enzymes, namely a FMO and a laccase.

To gather more genetic information, the genome of the vioxanthin (3)-producing

A. ochraceus DSM 2499 was sequenced by BaseClear B.V. (Leiden, Netherlands)

using Illumina sequencing technology. The sequencing yielded 1504 scaffolds and

a total genome size of 35.5 Mb. The genome was annotated using AUGUSTUS and


secondary metabolite gene clusters predicted with antiSMASH. It contains 125 predicted

biosynthetic gene clusters, 24 of with a gene encoding for an iPKS.

Table 2.2. Homologous genes of the putative biosynthetic gene cluster (II) ofvioxanthin (3) in T. rubrum, P. citreonigrum, and A. ochraceus.

T. rubrum P. citreonigrum A. ochraceus Putative function

XtrA VpcA VaoA NR-PKSXtrB VpcB VaoB FMOXtrC VpcC VaoC SDRXtrD VpcD VaoD FAS proteinXtrE VpcE VaoE laccaseXtrF VpcF VaoF O-MTXtrG VpcG VaoG MFS transporterXtrH VpcH VaoH transcription factorXtrI FAD/FMN-binding protein

VpcJ VaoJ hemerythrin-like protein

The protein identifiers of the gene cluster of T. rubrum were added to theappendix (Table 4, p.230). The genomes of P. citreonigrum and A. ochraceuswere sequenced for this work and have not been published yet. Thesequences were added to the appendix (appendix, section 2.2, p.238).

The comparison of the iPKSs of A. ochraceus with the iPKSs of T. rubrum and

P. citreonigrum showed two genes coding for iPKS with more than 70% sequence

Cluster II

Aspergillus ochraceus

vaoF vaoE vaoD vaoC vaoG vaoB vaoA vaoHvaoJ


vpcF vpcH vpcG vpcB vpcA vpcJ vpcC vpcD vpcE

Trichophyton rubrum

xtrI xtrG xtrF xtrE xtrD xtrC xtrB xtrAxtrH

Figure 2.5. Graphical overview of the putative vioxanthin (3) biosynthetic geneclusters (II) in T. rubrum, P. citreonigrum, and A. ochraceus.


identity that are present in all three genomes. One iPKS gene is part of a gene cluster

with genes coding for homologous tailoring enzymes in all three organisms (Table 2.2,

Figure 2.5). A detailed table with sequence identities is added to the appendix (Table 7).

The cluster identified in A. ochraceus does also contain two putative coupling enzymes.

S

O O O

OOO

O

enzyme4

9

O

O

OH OH O4

9


Scheme 2.2. Proposed folding mode of the poly-β -keto chain in the biosynthesisof (R)-semi-vioxanthin (4).[35]

Additional evidence for the putative biosynthetic gene cluster of vioxanthin (3)

was gathered by the analysis of the so-called PT domains of the candidate

NR-PKSs, which has been shown to control the regiochemistry of the first ring

cyclization in the biosynthesis of aromatic polyketides.[99,101,151] The folding mode for

(R)-semi-vioxanthin (4) proposed by Simpson et al. suggests a C4–C9 cyclization pattern

(Scheme 2.2).[35] This pattern is found in natural products produced by NR-PKSs of

the PT domain family IV. To confirm if the selected gene clusters contain a NR-PKS

with a PT domain that belongs to family IV, a phylogenetic tree consisting of the

55 PT domains was constructed according to the published parameters (Figure 2.6,

appendix: subsection 1.4 Table 8).[151] The sequences to construct the phylogenetic

tree were chosen by the authors based on published data connecting NR-PKSs with

secondary metabolites.[151] The PT domains were identified using the CDD website of

NCBI. The PT domain amino acid sequences were aligned using the ClustalW algorithm

and the phylogenetic tree was constructed using the minimal evolution procedure.

The PT domains of XtrA from T. rubrum, VpcA from P. citreonigrum, and VaoA from


1 23 4

56

7Fsr1

PKS1

XtrA

VaoA

VpcA

CTB1

11

12

13

14

15

16

1718

19202122

2324252627

28293031

32333435

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

5152

53

54 55

III

III

IV

V

VI

VII

VIII

C2-C7C4-C9C6-C11C1-C6C3-C8

0.2

VpfA

Figure 2.6. Phylogenetic tree constructed using 55 NR-PKSs with identifiedproducts and the PT domains of XtrA, VpcA and VaoA. The accession numbers ofthe selected NR-PKSs and the products they have been linked to, are listed in theappendix.

A. ochraceus grouped with PT domains of NR-PKSs linked to natural products with

a C4–C9 ring cyclization pattern, supporting the proposed folding mode (Figure 2.6,

Scheme 2.2).

The closest relatives (Figure 2.6, Fsr1, PKS1) of the PT domains of the analyzed

NR-PKS XtrA, VpcA, and VaoA are linked to the biosynthesis of the naphthoquinone

fusarubin (61).[152,153] In the biosynthesis of fusarubin (61), the intermediate


6-O-demethylfusarubinaldehyde (62), structurally resembling (R)-semi-vioxanthin (4),

is released from the NR-PKS via a thiol reductase domain (Figure 2.7).[152,153]

O

O

O

O OH

OH

OH

fusarubin (61)

O

OOHOH

HO


OHOH

HO

O

OH

6-O-demethylfusarubinaldehyde (62)

Figure 2.7. The naphthoquinone fusarubin (61) and the naphtho-α-pyronenor-toralactone (32).

The NR-PKS CnCTB1 (Figure 2.6) from Cercospora nicotianae is responsible for the

biosynthesis of the monomer of the perylenequinone cercosporin (40). Newman et

al. showed that CnCTB1 produces the naphtho-α-pyrone nor-toralactone (32), which

could be easily converted to (R)-semi-vioxanthin (4) by regioselective O-methylation

and a single reduction step.[154] Although the PT domain of CnCTB1 has a low sequence

identity (26%) compared to the PT domains of XtrA, VpcA, and VaoA, its product is the

closest structural relative to (R)-semi-vioxanthin (4), indicating a similar function and

a similar product produced by the NR-PKSs.

The biosynthetic gene cluster for the naphtho-γ-pyrone aurofusarin (30) isolated

from Fusarium graminearum has been studied in detail.[18,60–63,155,156] The central

NR-PKS produces nor-rubrofusarin, which is not related to the vioxanthin (3) NR-PKSs,

and its PT domain belongs to family III (Figure 2.6, branch 12).[18,151] Even though

the iPKS and the synthesized core structure are different, the tailoring steps in the

biosynthesis of aurofusarin (30) and vioxanthin (3) are similar. Both core structures

carry a methoxy group at C7 and are coupled at C8 (Figure 2.8). Additionally, for both

aurofusarin (30) and xanthomegnin (33) the coupled aryl ring is oxidized to yield a

quinone structure. These structural similarities could arise from homologous enzymes


OO O

O

O

O

O

O

O

O

O

O

O OH

OOH

OHOHO

O

O

OH OH O

O

O

OO

O

OH O

O OH

O

P*

vioxanthin (3) xanthomegnin (33)

aurofusarin (30)

8

8'

8

8'

8

8'

O OH

OOHOHO

OHO

sporandol (63)

8

8'

Figure 2.8. Shared structural features of the naphtho-α-pyrone vioxanthin (3),xanthomegnin (33), the naphtho-γ-pyrone aurofusarin (30), and sporandol (63).

carrying out similar tailoring steps in the two biosynthetic gene clusters. The O-MT

AurJ methylates nor-rubrofusarin to yield rubrofusarin (31).[18,61] Accordingly, the

homologous O-MTs XtrF, VpcF, and VaoF are proposed to catalyze a similar methylation

in the biosynthesis of vioxanthin (3).

To test this hypothesis a sequence comparison of identified O-MTs from biosynthetic

gene clusters responsible for the production of compounds structurally related to

(R)-semi-vioxanthin (4) was carried out (Figure 2.9, Table 2.3). The O-MTs identified in

the putative biosynthetic gene cluster of vioxanthin (3) show a sequence identity

of 37.5–57.5% compared to the O-MTs AnAunD, AcAunD, BfoD, SpoM and the

bifunctional O-MT/FMO CnCTB3. The O-MTs AnAunD from A. niger NRRL3, AcAunD

from Aspergillus carbonarius ITEM 5010, and BfoD from Aspergillus brasiliensis are


O

O

O O

O

O OH O

O

OH

O

O

OH

HO

O

OOHO

O

O

OOH

M *

bifonsecin B (65)aurasperone A (64)

P *

OO

OH

O

OH

cercosporin pathway

intermediate

OO

OH OH O

rubrofusarin (31)

O

OH OH O

O


AnAunD, AcAunD, BfoD,

CTB3, AurJ

AnAunE, AcAunE, BfoE

CTB2

Figure 2.9. Regiochemistry of the catalyzed methylations of O-MTs selected forthe sequence comparison with the O-MTs XtrF, VpcF, and VaoF from putativebiosynthetic gene clusters of vioxanthin (3).[18,154,157]

involved in the biosynthesis of the naphtho-γ-pyrone dimers aurasperone A (64)

and bifonsecin B (65).[157] They have been shown to selectively methylate the

phenolic hydroxy group at C8 [corresponding to C7 in (R)-semi-vioxanthin (4)],

while the O-MTs AnAunE, AcAunE, and BfoE methylate the phenolic hydroxy group

at C6 [corresponding to C9 in (R)-semi-vioxanthin (4), Figure 2.9].[157] Similar

regioselectivity was demonstrated for CnCTB3 in the biosynthesis of cercosporin

(40).[154] The O-MT SpoM is part of the biosynthetic gene cluster of sporandol (63)

in Chrysosporium merdarium.[158] The enzyme selectively converts 6-hydroxymusizin

(66) to torachrysone (67), corresponding to a methylation at the C7-hydroxy group

of (R)-semi-vioxanthin (4, Scheme 2.3).[159] The observed sequence homology of the

O-MTs XtrF, VpcF, and VaoF identified in the putative biosynthetic gene clusters of

xanthomegnin (33) and vioxanthin (3) compared to the O-MTs AnAunD, AcAunD,


BfoD, and the bifunctional O-MT/FMO CnCTB3 indicates a similar regioselectivity (C7)

for the analyzed enzymes. Compared to O-MTs with other regioselectivity (C9), the

enzymes XtrF, VpcF, and VaoF showed low sequence identity (10.9–26.2%, Figure 2.9,

Table 2.3).

The similar regiochemistry of the phenol coupling step in the biosynthesis of

aurofusarin (30), sporandol (63), and vioxanthin (3) indicates that the enzymes

involved in this biosynthetic step could be similar as well. The laccases Gip1 and SpoL

share 58.7–65.8% sequence identity with the laccases from the putative vioxanthin (3)

gene clusters (Table 2.4). Kim et al. constructed a knock-out of gip1 in F. graminearum,

which was deficient in production of aurofusarin (30), while the monomer rubrofusarin

(31) was still produced.[60] The laccase MDE from Talaromyces pinophilus has been

shown to catalyze the dimerization of monapinone (68) to the dimeric dinapinones A1

and A2 (69 and 70).[162] These dimers show the same coupling pattern as observed for

HO

OH OH O

O

OH OH OPKS O-MT

*

O OH

OOHOHO

OHO

SCoA

HO SCoA

O O

O

6

SpoP SpoM2

6-hydroxymusizin (66) torachrysone (67)

sporandol (63)

Fas protein/laccase

SpoF/SpoL

Scheme 2.3. Proposed biosynthetic pathway for sporandol (63) inC. merdarium.[159]


Table 2.3. Sequence comparison of the O-MTs XtrF, VpcF, and VaoF fromthe putative biosynthetic gene clusters of vioxanthin (3), xanthomegnin (33),and characterized O-MTs from the biosynthetic gene clusters of sporandol (63),aurasperone A (64), bifonsecin B (65) and cercosporin (40). The sequence identitiyof the amino acid sequences is shown [%].

O-MTs VpcF VaoF XtrF SpoM AurJ AnAunD BfoD AcAunD CnCTB3 AnAunE BfoE AcAunE

VaoF 73.2XtrF 68.0 64.3SpoM 57.4 53.5 52.9AurJ 47.2 45.3 44.6 42.5AnAunD 38.6 36.7 38.5 35.2 38.1BfoD 38.6 36.9 39.8 36.2 37.7 83.5AcAunD 43.3 42.0 43.2 40.2 40.4 63.0 62.0CTB3 39.6 37.6 41.2 35.2 38.5 34.6 34.3 35.9AnAunE 11.6 11.6 12.4 14.2 13.3 12.2 11.9 12.0 11.1BfoE 11.6 10.9 11.9 14.0 14.0 11.7 11.7 12.2 10.8 92.5AnAunE 12.5 12.0 11.6 13.2 13.7 11.4 11.4 11.9 11.5 81.4 80.1CTB2 26.4 26.9 26.2 27.1 27.1 23.7 23.9 27.3 23.5 11.7 11.7 11.6

AurJ from F. graminearum (I1RF60), AnAunD (Aspni_NRRL3_1|2868) and AnAunE (Aspni_NRRL3_1|2869) from A. niger NRRL3, AcAunD(Aspca3|50066) and AcAunE (Aspca3|131073) from A. carbonarius ITEM 5010, BfoD (Aspbr1|39215) and BfoE (Aspbr1|438143) fromA. brasiliensis CBS 101740, CnCTB2 (ABK64180) and CnCTB3 (ABC79591) from C. nicotianae.[155,157,160,161]

Table 2.4. Sequence comparison of the laccases XtrE, VpcE, and VaoE from theputative biosynthetic gene clusters of xantomegnin (33) and vioxanthin (3), Gip1involved in the dimerization in aurofusarin (30) biosynthesis, SpoL from thebiosynthetic gene cluster of sporandol (63), and MDE responsible for the phenolcoupling step in the biosynthesis of the 8,8′-coupled dinapinones A1 and A2 (69and 70). The ligninolytic laccase LCC2 from Trametes versicolor was added to showthe general sequence similarity of the enzyme class. The sequence identitiy of theamino acid sequences is shown [%].

laccases VaoE VpcE XtrE SpoL Gip1 MDE LCC2

VpcE 82.7XtrE 77.5 78.5SpoL 63.6 60.6 62.5Gip1 65.8 58.7 63.0 55.4MDE 53.5 50.4 49.1 47.5 49.8LCC2 21.4 20.6 20.5 18.1 18.3 19.8LacTL2 15.7 15.3 15.9 16.3 16.3 16.5 20.4

Gip1 from F. graminearum (I1RF62), MDE from Talaromycespinophilus (BAW99827), LCC2 from T. versicolor (Q12718).


R

R

*PO

O

O OH OH

O

O

OH OH O

dinapinone A2 (70)

*MO

O

R

O OH OH

O

O

OH OH O

R

dinapinone A1 (69)

R=OH OH

O

O R

OOHOH

monapinone (68)

2MDE

Scheme 2.4. Conversion of monapinone (68) to dinapinones A1 and A2 (69 and70) by MDE.[162]

aurofusarin (30), xanthomegnin (33), and vioxanthin (3). Similar to Gip1, MDE shows

high sequence identity (49.4–54.1%) compared to XtrE, VpcE, and VaoE, suggesting a

similar function of the laccases in the respective biosynthetic pathways.

Adjacent to the laccase encoding genes, a gene coding for a FAS protein (AurS, SpoF)

was identified in the aurofusarin (30) and sporandol (63) biosynthetic gene clusters. It

has been demonstrated that AurS is essential for dimerization.[63] The authors have

proposed that Gip1 and AurS together with the FMO AurF and the FAD/FMN-binding

protein AurO form an enzyme complex that catalyzes the formation of aurofusarin

(30) from the monomer rubrofusarin (31).[63] Genes coding for homologues of Gip1,

AurS and AurF from F. graminearum were identified in the putative biosynthetic gene

cluster of vioxanthin (3) and xanthomegnin (33) in the three producers (Table 2.5).

A homologue of AurO has only been identified in the putative biosynthetic gene

cluster of xanthomegnin (33) in T. rubrum. It is possible that AurO and XtrI are

involved in the formation of the quinone moiety of xanthomegnin (33). Even though


xanthomegnin (33) has been isolated from P. citreonigrum and A. ochraceus as well,

which do not possess an AurO-homologue, this enzyme might be responsible for

the oxidation of the hydroquinone synthesized via para-hydroxylation. In the case

of P. citreonigrum and A. ochraceus, the formed hydroquinone could be oxidized to

xanthomegnin (33) non-enzymatically by oxygen. If AurO and XtrI catalyze the

oxidation of the hydroquinone moiety, a different ratio of the dimeric products can be

expected, favoring xanthomegnin (33) over vioxanthin (3) production.

Knock-out experiments have shown that both laccases and FMOs are involved in the

phenol coupling step in the biosynthesis of homodimeric natural products.[7,61,63,141] It

cannot clearly be deduced by sequence comparison alone, which enzyme catalyzes the

phenol coupling step.

Table 2.5. Homologous enzymes from the putative aurofusarin (30), vioxanthin (3)and xanthomegnin (33) dimerizing complex.

F. graminearum T. rubrum P. citreonigrum A. ochraceus Putative(30)[60–63] (33) (3) (3) function

Gip1 XtrE VpcE VaoE laccaseAurS XtrD VpcD VaoD FAS proteinAurF XtrB VpcB VaoB FMOAurO XtrI FAD/FMN

binding protein

Gip1 (I1RF62), AurS (I1RF63), AurF (I1RF61), AurO (I1RF55) fromF. graminearum.[60–63]


2.2.2 Deletion mutants

To verify the involvement of these biosynthetic gene clusters in the biosynthesis of

vioxanthin (3) knock-out mutants of A. ochraceus were pursued. The NR-PKS (vaoA)

and the laccase (vaoE) gene as a putative coupling enzyme, were targeted. In first

place, knock-out mutants were attempted in cooperation with the group of Prof. Dr.

Axel Brakhage at the Heinz-Knöll-Institut in Jena.

The deletion experiments, targeting the NR-PKS-encoding vaoA and the

laccase-encoding vaoE in A. ochraceus, were carried out using a genetically unmodified

wild type strain, leading to some difficulties to obtain the desired knock-out mutants.

The transformation of both knock-out constructs resulted in several transformants with

a resistance against pyrathiamine. The mutants were screened via Southern Blot or

PCR. No mutant carrying a knock-out of the targeted NR-PKS gene, nor the laccase

gene was isolated. Most of the pyrathiamine resistant mutants carried an ectopic,

undirected integration of the resistance cassette, likely as a result of the high DNA

repair activity of the non-homologous end joining (NHEJ) pathway.[163,164] In some

cases the resistance cassette was integrated with one of the homologous flanking

regions, either up- or downstream of the gene, without disrupting it. This could be

due to the DNA repair activity via the homology directed repair (HR) pathway.[165]

Consequently, no genotype deficient in the production of vioxanthin (3) or the phenol

coupling of (R)-semi-vioxanthin (4) was identified.

The second approach suited for genetically unmodified wild type ascomycete strains

was the construction of gene knock-outs with an adapted clustered regularly interspaced

short palindromic repeats-Cas9 (CRISPR/Cas9) system. The CRISPR/Cas9 system is a

bacterial and archeal adaptive immune system and has been developed into a powerful

genome editing tool in recent years.[166–169] The system uses a small 20 bp singleguide


ribonucleic acid (sgRNA) sequence that guides the endonuclease Cas9 to a part of

the genome containing a homologous sequence to the sgRNA. Nødvig et al. modified

the established CRISPR/Cas9 system for the use in ascomycetes.[170] Prof. Dr. U. H.

Mortensen from the Technical University of Denmark kindly provided the developed

plasmids. The system allows targeted genome editing in wild type strains by causing

directed double strand breaks that are repaired by the error prone NHEJ pathway,

leading to insertion and deletion events in the process of DNA repair. These insertions

and deletions can cause a frame shift in the gene of interest, rendering the gene product

inactive.

The cloning approach was adapted from Nødvig et al. for InFusion cloning, an

already frequently used cloning technique at our lab (Figure 2.10), in order to avoid

the use of the expensive USER™ system.[170] The plasmid pFC333::vaoE was used to

transform A. ochraceus following the procedure described in subsection 4.2.12. No

TtrpCPgdpA HDVsgRNA backboneHH sgRNA

pFC333P1

P2

P3

P4

Cas9Ptef1 Ttef1AMA

oriampR ble

PacI

PacI

PgdpA HH

TtrpCHDVsgRNA backbonesgRNAHH

pFC333

Cas9Ptef1 Ttef1AMA

oriampR ble

TtrpCPgdpA HDVsgRNA backboneHH sgRNA

Figure 2.10. InFusion cloning approach to construct CRISPR/Cas9 plasmids forknock-outs in A. ochraceus.


transformants grew on the selection plate. Hence, no successful gene disruption mutant

of the laccase gene vaoE was identified.


2.2.3 Heterologous expression

To confirm the involvement of the putative gene clusters in the biosynthesis of

vioxanthin (3) in a direct way, the heterologous expression of the gene vaoE coding for a

laccase was attempted. complementary DNA (cDNA) was isolated from A. ochraceus to

amplify the laccase-encoding gene without introns. The intronless gene vaoE was cloned

into the plasmids pET19b and pET28b and produced as N- and C-terminally tagged

protein in the heterologous expression host Escherichia coli BL21 GOLD. No enzyme

production was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) analysis. To detect very small amounts of protein a Western Blot was

performed. No protein was detected in the blot, neither as soluble protein nor as

insoluble protein. Afterwards, the intronless cDNA-derived gene vaoE was expressed

in S. cerevisiae without an affinity tag. The enzymatic activity was tested with whole

cells after induction with galactose, by feeding the monomer (R)-semi-vioxanthin (4).

The samples were analyzed via high performance liquid chromatography (HPLC), but

no dimer was detected by this means. The heterologous expression in E. coli and

S. cerevisiae failed to produce active protein. As laccases are glycoproteins, missing or

incorrect glycosylation may have prevented the production of active laccase.[171] Even

though S. cerevisiae is able to glycosylate proteins, in some cases hyperglycosilation

has been reported.[172] To overcome this problem A. niger was chosen as heterologous

expression host. As this microorganism is closely related to the producer of the enzyme,

a native glycosylation pattern is expected.

For the functional expression of vaoE in A. niger, a non integrative plasmid

was needed. In first place, the plasmid pKGH2 available at our laboratory was

used.[173] pKGH2 is a modification of pMA171, by the introduction of Tet-on promoter

system.[8,174] The heterologous production of the laccase was carried out using the


cDNA-derived vaoE gene and the newly constructed expression plasmid. Conversions of

(R)-semi-vioxanthin (4) with the lysate of A. niger expression cultures were analyzed via

high performance liquid chromatography coupled with mass spectrometry (HPLC-MS).

No dimer was detected in the analysis of the conversions. Possible reasons for the

absent activity of heterologously expressed VaoE could be the genetic instability of

the Tet-on expression system used in pKGH2, or unsuccessful cell disruption. The

plasmid pKGH2 contains the same promoter sequence twice. In A. niger the plasmid

can be destroyed by the HR pathway, which is the main DNA repair mechanism in

the genetically modified A. niger strain (FGSC A1180, ∆kusA) used in this study.[175]

Additionally, cell disruption via sonication or french press did not yield protein amounts

that were detectable with SDS-PAGE analysis.

A new expression plasmid was designed that does not contain homologous sequences

and uses a different inducible promoter. The promoter of the β -fructofuranosidase

gene (sucA) of Aspergillus carbonarius and the terminator of the nucleolar protein cgrA

were chosen for the plasmid. The AMA1 sequence, f1-ori, the hygromycin resistance

cassette, and the ampicillin resistance gene of the CRISPR/Cas9 plasmid pFC332 were

fused to the chosen promoter and terminator. The gene of interest can be cloned into

the plasmid after linearization of the plasmid with NheI, SnaBI, or BamHI. The plasmid

was named pSUC.

The laccase encoding vaoE gene was cloned into pSUC using NheI and SnaBI

to linearize the plasmid. A. niger was transformed with pSUC::vaoE. The

expression of pSUC::vaoE was carried out similar to the expression of pKGH2::vaoE.

Sucrose was used to induce the expression of vaoE. The cells were sonicated and

(R)-semi-vioxanthin (4) was added for conversions. No dimer was detected after

addition of (R)-semi-vioxanthin (4) to the lysate. Even when an improper expression


leading to a non-functional protein could not be excluded, other reasons might be

responsible for the lack of coupling activity.

Adjacent to vaoE a gene encoding for a FAS protein was identified. The function of

this protein is unknown and no characterized homologue has been described. The FAS1

domain has been identified in several secreted or membrane-anchored proteins, such

as the human transforming growth factor-β -induced gene β ig-h3, Algal-cell adhesion

molecule (CAM) and fasciclin-like arabinogalactan-proteins (FLAs) from wheat and

rice.[176–178] As laccases are secreted proteins as well, the FAS protein could be needed

to attach the laccase to the cell wall or act as a dirigent protein. The combination of an

unselective laccase and a “dirigent protein” to generate an enantioenriched product has

been discovered in the biosynthesis of pinoresinol.[179] Similarly, in the biosynthetic

gene cluster of aurofusarin (30), a laccase encoding gene (gip1) and an adjacent FAS

protein encoding gene (aurS) were identified.[63] Knock-out mutants of the genes

were deficient in dimer production, confirming the involvement of the laccase and

FAS protein in the coupling step.[61,63] Therefore, it is possible that the FAS protein is

interacting with other enzymes to form an enzyme complex, which might be unstable

if one of the enzymes is missing.

Hence, the gene coding for the FAS protein in the biosynthetic gene cluster (vaoD)

was heterologously produced in A. niger. The lysate of the expression culture of vaoD

was combined with a lysate from an expression culture of vaoE. (R)-semi-Vioxanthin

(4) was added to the combined lysates for conversions, but no dimer was detected.

This could be due to other partners of the putative enzyme complex that are missing,

or that the partners of the complex only interact correctly in their native environment.

As the genes vaoD and vaoE are likely co-expressed in A. ochraceus, a similar expression

approach might be needed to produce an active coupling enzyme/enzyme complex.


2.2.4 Conversions with lysate of Penicillium citreonigrum

Without detectable coupling activity of the studied enzymes, it was unclear if the

expression did not yield any or only inactive enzyme, or if the enzyme is correctly

produced but damaged upon cell disruption. VaoE contains a signal peptide, predicted

by the online bioinformatics tool SignalP 4.1.[180] If VaoE is secreted by A. niger, the

conditions determined by the medium and secreted metabolites of A. niger could lead

to inactivation of VaoE. On the other hand, if VaoE is a secreted enzyme, the mycelium

would not have to be sonicated simplifying the heterologous production.

To test the hypothesis of VaoE being secreted and the influence of the cell disruption

method on the enzyme activity, a culture of P. citreonigrum was grown for four days.

In this time frame only traces of the dimer are produced by the fungus. The culture

supernatant was separated from the mycelium and both were tested separately for

coupling activity by the addition of (R)-semi-vioxanthin (4). No dimer production

was observed in HPLC-MS analysis. Part of the mycelium was sonicated. The lysate

was separated from the cell debris and both lysate and debris tested for coupling

activity. The debris showed minor activity, whereas the lysate completely converted

(R)-semi-vioxanthin (4) to vioxanthin (3). One product with the same retention time,

m/z, and UV spectrum as vioxanthin (3) isolated from P. citreonigrum was detected

(Figure 2.11). The minor activity of the cell debris was attributed to the remaining

lysate in the debris. Conversions using (R)-pro-semi-vioxanthin (27) as substrate did

not yield any product (Figure 2.11).

These results show that the chosen cell disruption method and the buffer system

are suited to produce active coupling enzyme. Therefore, missing activity of the

heterologously produced enzyme is likely due to the necessary combination of all

members of the putative enzyme complex formed to dimerize (R)-semi-vioxanthin (4).


0

100

200

300

400

4 3

Abs

orpt

ion

[mA

U]

(a) Standard of (R)-semi-vioxanthin (4) and vioxanthin (3)

0

0.5

1

1.5

2·108

4 3

Inte

nsit

y[c

ps] m/z 273

m/z 545

0

100

200

300

400

(b) Conversion of (R)-semi-vioxanthin (4) with lysate of P. citreonigrum

0

2

4

6·106

m/z 273m/z 545

0 2 4 6 8 10 12

0

200

400

27

Time [min]

(c) Conversion of (R)-pro-semi-vioxanthin (27) with lysate of P. citreonigrum

0 5 10 15

0

1

2

3

·107

Time [min]

m/z 259m/z 517

Figure 2.11. HPLC-DAD-MS analysis of the conversion of (R)-semi-vioxanthin (4)and (R)-pro-semi-vioxanthin (27) with P. citreonigrum lysate: (a) standard of themonomer (R)-semi-vioxanthin (4, m/z 273) and the dimer vioxanthin (3, m/z545), (b) conversion of (R)-semi-vioxanthin (4) with lysate of P. citreonigrum, (c)conversion of (R)-pro-semi-vioxanthin (27) with lysate of P. citreonigrum. Detectionwas carried out with DAD (left column, λ 360–400 nm) and mass spectrometry(right column, −Q1, Method 3, ISAspher Phenyl 100-5).


As previously mentioned, a similar enzyme complex has been proposed for the

dimerization of aurofusarin (30).[63]


2.2.5 Folding mode of (R)-semi-vioxanthin

First labeling studies with [1-13C]-acetate and labeled (R)-semi-vioxanthin (4) showed

that vioxanthin (3) and the related quinone xanthomegnin (33) are synthesized via the

polyketide pathway and consist of two coupled monomeric precursor molecules.[22,35]

The folding mode of (R)-semi-vioxanthin (4) proposed by Simpson is shown in

Scheme 2.2. Yet, this proposal was until now not experimentally confirmed.

To verify this, 80 mg of [1,2-13C2]-acetate were fed to a culture of

P. citreonigrum resulting in the isolation of 6.8 mg of (R)-semi-vioxanthin (4) that were

purified via flash chromatography on acid washed silica with CH2Cl2 and methanol

(MeOH) as eluents. 1H and 13C NMR, HMBC, HSQC, and INADEQUATE spectra of

the labeled compound were measured. The 1H NMR spectrum confirmed the identity

of the compound by comparison with published NMR data (appendix Figure 3).[22]

HMBC and HSQC allowed to unambiguously assign the signals of C6/C8, C7/C9, and

H6/H8 that were not assigned previously (Table 2.6).[181] The INADEQUATE spectrum

showed the coupling of adjacent [13C]-nuclei, if they were derived from a single

[1,2-13C2]-acetate unit (Figure 2.12). The spectrum confirmed the proposed C4–C9

folding mode. These findings further strengthen the results of the PT domain analysis

to identify the biosynthetic gene cluster of vioxanthin (3), which grouped the NR-PKS

putatively involved in the biosynthesis of (R)-semi-vioxanthin (4) (Figure 2.6).

A quantitative 13C NMR-spectrum was measured to determine the incorporation rate

of labeled acetate and whether the label is enriched in all parts of the molecule. For

each [13C]-nucleus either a singlet or a doublet is visible in the spectrum, depending on

whether the [13C]-nucleus is a naturally occurring isotope or part of an incorporated

labeled [1,2-13C2]-acetate molecule. For each signal the integral of the doublet was

compared to the integral of the singlet. No doublet was observed for the methoxy


F2 [ppm]150 100 50

F1[ppm]

150

100

500

C1 C7C10 C9 C5a C4a

C5

C9a

C8

C6C10a O-CH3

C4CH3

C3

O

OOHOH

O


Figure 2.12. INADEQUATE NMR spectrum of (R)-semi-vioxanthin (4, CDCl3, 21 °C,100 MHz). [1,2-13C2]-acetate units incorporated into (R)-semi-vioxanthin (4)isolated from P. citreonigrum.

group. The frequency of naturally occurring [13C]-nuclei was assumed to be 1.1%. The

incorporation rate was calculated as the sum of the doublet integrals divided by the

singlet’s integral times 1.1% (Equation 2.1).

incorporation rate =∑∫

doublet∫singlet

×1.1% (2.1)

The incorporation rate ranges from 2.5–11% (Table 2.6). The signal of C3 in the

quantitative 13C NMR spectrum is overlaid by the solvent signal (CDCl3) and thus can


not be properly integrated. The methyl group adjacent to C3 shows an incorporation

rate of 11%. Fungal iPKSs are known to show a preference for acetate, explaining the

enrichment of the 13C-label at these positions.[182]

Table 2.6. Integrals and incorporation rates of [13C]-labels in(R)-semi-vioxanthin (4). CDCl3, 21 °C, 1H NMR 400 MHz, 13C NMR 100 MHz.Position (R)-semi-Vioxanthin (4) Doublet integral Singlet integral Incorporation rate

1H [ppm] 13C [ppm] [%]

1 – 171.6 2.08 0.310 7.43 4.74 76.5 n.d. n.d. n.d.4 2.96 34.7 1.75 0.530 3.64a – 132.8 1.30 0.225 6.45 6.87 116.1 1.88 0.219 9.45a – 140.1 1.30 0.607 2.46 6.55 99.4 1.70 0.330 5.77 – 162.5 1.80 0.359 5.58 6.51 101.5 1.84 0.416 4.99 9.46 158.5 1.58 0.347 5.09a – 108.5 0.957 0.367 2.910 13.76 162.9 1.48 0.642 2.510a – 99.3 1.04 0.353 3.2CH3 1.54 20.7 2.38 0.239 11OCH3 3.87 55.4 – – –


2.2.6 Biosynthetic hypothesis

The genome analysis of T. rubrum, P. citreonigrum, and A. ochraceus combined with

previous and current results allows the proposal of an improved biosynthetic pathway

for vioxanthin (3, Scheme 2.6, Scheme 2.7). Labeling studies with [1,2-13C2]-acetate

showed homogeneous incorporation of the labeled C2-unit. This result suggests that

(R)-semi-vioxanthin (4) is synthesized using an acetate starter unit and six malonate

extender units, rather than using enantiomerically pure hydroxybutyric acid as a

starter unit. The NR-PKS VpcA is proposed to produce the carbon backbone, while the

reduction is carried out by the SDR VpcC. The PT domain of the NR-PKS VpcA has been

grouped with PT domains of family IV (Figure 2.6). The phylogenetic classification

has been confirmed by labeling studies with [1,2-13C2]-acetate, which elucidated the

folding mode of (R)-semi-vioxanthin (4, Scheme 2.2). Other NR-PKS with PT domains

of family IV have been linked to the biosynthesis of structurally similar compounds,

such as cercosporin (40) or fusarubin (61).[152,154]

While the PT domain of NR-PKSs controls the pattern of the first cyclization of the

poly-β -ketide chain, the TE domain determines the pattern of the last cyclization.

Different release mechanisms have been reported for iPKSs, differing in the nucleophile

used to cleave off the polyketide from the iPKS protein.[183] The product released

from the NR-PKSs XtrA, VpcA, and VaoA has not been identified yet. The possible

products released from the NR-PKSs are shown in Scheme 2.5. If a water molecule

attacks the ester bond attaching the polyketide to the enzyme, a bicyclic intermediate

with a keto-sidechain is released. A second possibility is a product release following

the mechanism described for nor-toralactone (32) from the NR-PKS CnCTB1.[184]

In this case an enol is the nucleophile cleaving the thioester linkage of the NR-PKS

product to the enzyme. A third possible product that might be released from the


S

SAT KS AT PT PP PP TE

XtrA, VpcA, VaoA

O

HO

OH

HO

OH

(a) (b)

S


XtrA, VpcA, VaoA

O

HO

HO

OH

OH

H2O

O

OOOHOH

HO

OH OH

HOOOH

S


XtrA, VpcA, VaoA

O

O

O

OH

OH

(c)

S


XtrA, VpcA, VaoA

O

HO

OH

HO

OH

O

OOHOH

HO

OH

SDR

nor-toralactone (32) 72

(R)-pro-semi-vioxanthin (27)

[H]

Scheme 2.5. Possible scenarios and products released from the NR-PKSs XtrA,VpcA, and VaoA from the putative biosynthetic gene clusters of xanthomegnin (33)and vioxanthin (3).


NR-PKS is (R)-pro-semi-vioxanthin (27). This scenario would include a reduction of

the poly-β -keto chain by a separate SDR (XtrC, VpcC, VaoC), while it is still tethered to

the NR-PKS enzyme (Scheme 2.5).

To gain further insight into the release mechanism of the NR-PKSs XtrA, VpcA, and

VaoA, the TE domains of the enzymes were compared to TE domains of NR-PKS linked

to specific polyketide products. The NR-PKS Fsr1 from Fusarium fujikuroi contains a

PT domain of family IV closely related to the PT domains of XtrA, VpcA, and VaoA

and releases 6-O-demethylfusarubinaldehyde [(62), Figure 2.7] as a product.[153]

The NR-PKS CnCTB1 from C. nicotianae has been shown to produce nor-toralactone

(32).[154] The NR-PKSs AflC and StcA are involved in the biosynthesis of aflatoxins and

sterigmatocystin and contain a PT domain of family V.[185,186] The enzymes have been

proposed to produce norsolorinic acid anthrone.[187,188]

Table 2.7. Sequence comparison of TE domains of characterized iPKSs XtrA, VpcA,and VaoA from the putative biosynthetic gene clusters of vioxanthin (3) fromT. rubrum, P. citreonigrum, and A. ochraceus. The sequence identitiy of the aminoacid sequences is shown [%].

TE XtrA VpcA VaoA CnCTB1 PKS12 PKS27 AflC StcA Fsr1

VpcA 74.7VaoA 73.5 81.8CTB1 56.7 54.8 61.5PKS12 39.1 38.2 37.4 48.6PKS27 48.6 49.8 50.8 52.4 35.6AflC 42.9 42.1 43.2 50.5 34.3 42.5StcA 46.4 45.6 47.4 48.1 31.5 41.3 74.6Fsr1 10.1 10.2 9.1 11.8 9.7 10.6 8.2 9.1PKS1 10.4 10.9 9.8 8.4 8.1 9.9 7.4 9.8 82.4

CTB1 from C. nicotianae (AAT69682), PKS12 from F. graminearum PH-1(I1RF58), PKS27 from Aspergillus flavus NRRL 3357 (B8MYS6), AflC fromAspergillus parasiticus SU-1 (Q12053), StcA from Aspergillus nidulans FGSC4(Q12397), Fsr1 from F. fujikuroi IMI 58289 (S0DTP6), and PKS1from Nectria haematococca mpVI 77-13-4(XP_003039929).[151,153]


OO

O OH O

OO

O

OH O

O

O

3,4-dehydroxanthomegnin (71)

Figure 2.13.3,4-Dehydroxanthomegnin (71)isolated from P. citreonigrum.[64]

The TE domains of XtrA, VpcA, and VaoA

show sequence homology to CnCTB1 (54.8–61.5%,

Table 2.7), suggesting a similar release mechanism.

Even though the sequence identity of the

NR-PKSs from the putative xanthomegnin (33)

and vioxanthin (3) biosynthetic gene clusters show

a similar sequence homology to the NR-PKSs

PKS27, AflC, and StcA (43.2–50.8%, Table 2.7),

the release mechanism of these anthraquinone producing NR-PKSs is unlikely for the

biosynthesis of naphtho-α-pyrones. The release mechanism of PKS27, AflC, and StcA

uses a C-nucleophile to cleave the polyketide off from the iPKS enzyme, while in the

case of naphtho-α-pyrone biosynthesis, the polyketide is either cleaved from the iPKS by

a water molecule or an intramolecular O-nucleophile. The formed unsaturated lactone

has been observed in 3,4-dehydroxanthomegnin (71) isolated from P. citreonigrum

(Figure 2.13).[64] Therefore, the product released from XtrA, VpcA, and VaoA is likely

nor-toralactone (32).

The released nor-toralactone (32) or the naphthoic acid derivative 72 is reduced

by a SDR (e.g., VpcC). The naphthoic acid derivative 72 and nor-toralactone

are interconvertible by the addition/elimination of water, making it difficult to

determine the substrate of the reduction (Scheme 2.6). If the substrate of the

SDR is the naphthoic acid derivative 72, the reduction product has to cyclize to

the lactone (R)-pro-semi-vioxanthin (27) chemically. In turn, if the NR-PKS VpcA ejects

nor-toralactone (32), the cyclization is carried out by the TE domain of the NR-PKS. In

this case, the SDR VpcC would reduce nor-toralactone (32) to (R)-pro-semi-vioxanthin

(27). A comparison of characterized SDRs with the SDRs from the putative biosynthetic


CTB6

O

OOO

tert-butyl 3,5-dioxohexanoate

O

OH OH

OO

HO

OH OH

OH

OH

HO

OH OH O

OHO O

OOHOH

HO


cercosporin pathway

intermediate

emodin hydroquinone (88)

CMCR-S1 MdpC

possible substrates of XtrC, VpcC, and VaoC

H2O

H2O

72

Figure 2.14. Substrates of the characterized SDRs CMCR-S1 and MdpC in the firstrow.[20,189–191] ∆CTB6 mutants accumulate the shown intermediate.[154] Therefore,CnCTB6 is proposed to reduce the keto-side chain in the biosynthesis of cercosporin(40).[154] The possible substrates for the SDRs XtrC, VpcC, and VaoC are shown inthe second row.

gene clusters of xanthomegnin (33) and vioxanthin (3) did not yield sufficient results

to determine the putative substrate of XtrC, VpcC, and VaoC (Table 2.8). However, the

isolation of 3,4-dehydroxanthomegnin (71) suggests nor-toralactone as product of the

NR-PKSs XtrA, VpcA, and VaoA and consequently as substrate for the SDRs XtrC, VpcC,

and VaoC.[64] A third scenario is a reduction catalyzed by an SDR while the poly-β -keto

chain is still tethered to the NR-PKS enzyme that would release (R)-pro-semi-vioxanthin

(27).

Feeding studies with [13C]-pro-semi-vioxanthin (27) carried out by Bode showed

the incorporation of the label into (R)-semi-vioxanthin (4) and vioxanthin (3).[22] This

suggests the O-MT synthesizing (R)-semi-vioxanthin (4) accepts (R)-pro-semi-vioxanthin

(27) as the substrate.


O

O

OH OH O

O

HO

OH OH O

O

OOHOH

HO

SCoA

HO SCoA

OO

O

6

VpcA

VpcA / VpcC




VpcFVpcA

HO

OH OH O

OHO

VpcC

HO

OH OH O

OH

VpcC

OH

O

HO

OH OH O


H2O

72H2O

H2O

VpcF

O

HO

OH OH O


O

O

OH OH O


VpcF

Scheme 2.6. Proposed biosynthetic pathway of (R)-semi-vioxanthin (4) inT. rubrum, P. citreonigrum, and A. ochraceus. The pathway hypothesis involvingthe intermediate nor-toralactone (32) is depicted in blue. The pathway hypothesisinvolving a keto-reduction and subsequent lactonization is depicted in red.


Table 2.8. Sequence comparison of SDRs XtrC, VpcC, and VaoC from theputative biosynthetic gene clusters of xanthomegnin (33) and vioxanthin (3)with characterized SDRs. The sequence identitiy of the amino acid sequences isshown [%].

SDRs XtrC VpcC VaoC CMCR-S1 MdpC

VpcC 77.4VaoC 75.7 85.4CMCR-S1 22.0 22.1 22.7MdpC 19.2 20.3 19.2 23.4CTB6 8.7 9.8 9.8 10.5 8.7

XtrC from T. rubrum (TERG_02848), CMCR-S1 from Candidamagnoliae (BAB21578), MdpC from Aspergillus nidulans(Q5BH34), CnCTB6 from C. nicotianae (ABK64183).

The coupling enzyme does not convert (R)-pro-semi-vioxanthin (27) to the

corresponding dimer, only (R)-semi-vioxanthin (4) is coupled to vioxanthin (3).

This observation is in accordance with feeding experiments of [13C]-labeled

(R)-pro-semi-vioxanthin (27).[181] The FAS protein encoded in the cluster might be

involved in the coupling step of vioxanthin’s (3) biosynthesis. Similar to previous

examples (pinoresinol, gossypol) the coupling enzyme alone might not control the

regio- and stereoselectivity of the biaryl formation. In the case of vioxanthin (3), the

FAS protein could determine these selectivities. Additionally, conversion experiments

with heterologously produced laccase show that the laccase alone might not be able

to dimerize (R)-semi-vioxanthin (4). The coupling has been observed in the lysate of

P. citreonigrum, suggesting that at least one other enzyme is involved in this reaction.

The formation of the quinone moiety is possible on two stages of the biosynthesis;

prior to biaryl formation or after the coupling step.[192] A para-hydroxylation, likely

by a FMO, of the monomeric (R)-semi-vioxanthin (4) would imply a lower substrate

specificity of the coupling enzyme to generate heterodimers, such as rubrosulphin


O O

O

OH OH O OOHO

O

O(R)-semi-vioxanthin (4) (semi)-xanthomegnin

VpcB

O

O

O

O

O

O

OO

OO

OO

O

O

O

O

OO

O

OHO

OHO

O OH

OH O

OH O

O OH

OH O

OO

OHOHO

OOH

O

OH O

xanthomegnin (33)

vioxanthin (3) viomellein (34)

rubrosulphin (73)

VpcE

VpcD2x

VcpB

VpcB

P*

VpcE

VpcD2x

Scheme 2.7. Proposed biosynthetic pathway of vioxanthin (3) and related dimersin P. citreonigrum.[192] The pathway for xanthomegnin (33) based on a dimerizationpreceding para-hydroxylation is colored in blue. If para-hydroxylation takes placeon the stage of the monomer, the biosynthesis of xanthomegnin (33) follows theroute colored in red.


(73, Scheme 2.7). This theory is challenged by the conversion experiments with

(R)-pro-semi-vioxanthin (27), which is not converted by the lysate of P. citreonigrum.

The second possibility is the para-hydroxylation of the homodimer vioxanthin (3).

Xanthoepocin hypothesis

The dimeric xanthoepocin (74) is structurally closely related to vioxanthin (3) and

xanthomegnin (33, Figure 2.15). The structural similarities of xanthoepocin (74)

and xanthomegnin (33) might translate to similar biosynthetic pathways and similar

enzymes involved in the compounds’ biosyntheses. Following this hypothesis a gene

cluster homologous to the putative biosynthetic gene clusters of vioxanthin (3) and

xanthomegnin (33) from T. rubrum, P. citreonigrum, and A. ochraceus was proposed to

be responsible for the biosynthesis of xanthoepocin (74).

Xanthoepocin (74) has been isolated from Penicillium simplicissimus, Penicillium

excelsum, and Penicillium arizonense, whose genome sequence has been published

OO

O

OO OH OH

O

OH

O

OH O

xanthoepocin (74)

OO

OHOHO

OOH

O

OH O

vioxanthin (3)

P

*

R *

polyketide synthasereductaseO-methyltransferasecoupling enzyme

O

O FMO

OO

O OH O

O

O

O

OH O

xanthomegnin (33)

O

O

Figure 2.15. The structure of xanthoepocin (74) in comparison with vioxanthin (3)and xanthomegnin (33).


recently (BioProject PRJNA395479).[193–195] To identify a putative xanthoepocin

(74) biosynthetic gene cluster, the published genome sequence was annotated with

AUGUSTUS and secondary metabolite gene clusters predicted with antiSMASH.

Comparison of the predicted iPKS gene clusters with the identified putative biosynthetic

gene clusters of vioxanthin (3) and xanthomegnin (33) showed one homologous gene

cluster in P. arizonense (Table 2.9).

Table 2.9. Comparison of the putative biosynthetic gene clusters of xanthoepocin(74), vioxanthin (3), xanthomegnin (33). Depiction of the putative biosyntheticgene cluster of xanthoepocin (74) from P. arizonense.

P. arizonense P. citreonigrum T. rubrum putative function

XpaA XtrA VpcA NR-PKSXpaF XtrF VpcF O-MTXpaK NADH-binding proteinXpaB XtrB VpcB FMOXpaE XtrE VpcE laccaseXpaD XtrD VpcD FAS proteinXpaL hypothetical proteinXpaM transcription factorXpaI XtrI FAD/FMN-binding proteinXpaH XtrH VpcH transcription factorXpaG XtrG VpcG MFS transporter

The protein identifiers of the putative biosynthetic gene cluster ofxanthoepocin (74) from P. arizonense were added to the appendix (subsection 1.2,Table 5, p.230).

xpaG xpaH xpaI xpaM xpaL xpaD xpaE xpaB xpaK xpaF xpaA

Penicillium arizonense

The biosynthetic pathway proposed for xanthoepocin (74) is based on the pathway

hypothesis for vioxanthin (3) and xanthomegnin (33) biosynthesis in T. rubrum,

P. citreonigrum, and A. ochraceus. Consequently, the NR-PKS XpaA likely synthesizes the

carbon backbone of xanthoepocin (74). Because of its sequence similarity (37.0–47.7%)

compared to characterized regioselective O-MTs (BfoD, AnAunD, AcAunD, AurJ), the


O-MT XpaF is proposed to transfer a methyl group to the phenolic hydroxy group at

C7. The FMO XpaB probably catalyzes a para-hydroxylation, similar to its counterparts

XtrB, VpcB, and VaoB in the biosynthesis of xanthomegnin (33) and vioxanthin (3).

Xanthomegnin (33) and vioxanthin (3) contain an α,β -unsaturated lactone moiety.

It is proposed that the SDRs XtrC, VpcC, and VaoC are responsible for the necessary

reduction of an unsaturated precursor. In contrast, xanthoepocin (74) contains a fully

unsaturated lactone and, accordingly, an enzyme capable of catalyzing such a reduction

is missing in the putative biosynthetic gene cluster of xanthoepocin (74, Table 2.9).

XpaK, XpaL, and XpaM do not show sequence similarities to enzymes of the identified

biosynthetic gene clusters of xanthomegnin (33) and vioxanthin (3). XpaM contains

conserved domains typically observed for transcription factors. For XpaL no conserved

domains were identified and, therefore, no function was assigned to the protein. XpaK

is predicted to bind NADH and could be involved in the formation of the epoxide

structures of xanthoepocin (74).

Table 2.10. Sequence comparison of the laccase XpaE from P. arizonense with thelaccases Gip1 from the aurofusarin (30) biosynthetic gene cluster, MDE from thedinapinone (69 and 70) biosynthesis, and XtrE, VpcE, and VaoE from the putativebiosynthetic gene clusters of xanthomegnin (74) and vioxanthin (3). The laccaseLCC2 from T. versicolor was added as a non-dimerizing laccase. The sequenceidentitiy of the amino acid sequences is shown [%].

Laccases VpcE VaoE XtrE XpaE Gip1 MDEVaoE 82.7XtrE 78.5 77.5XpaE 61.0 68.5 65.4Gip1 59.0 65.7 62.9 61.0MDE 50.4 53.5 49.0 51.1 49.8LCC2 19.8 19.8 19.1 18.2 17.5 19.8

XpaE from P. arizonense (OGE58328), Gip1 fromF. graminearum (I1RF62), MDE from T. pinophilus(BAW99827), LCC2 from T. versicolor (Q12718).


Based on the similar coupling pattern of xanthomegnin (33), vioxanthin (3), and

xanthoepocin (74), the coupling enzymes responsible for the dimerization in the

biosyntheses of these secondary metabolites are proposed to show high sequence

similarities (>50%). Sequence comparison of the laccase XpaE, identified in the

putative biosynthetic gene cluster of xanthoepocin (74), with the laccases XtrE, VpcE,

VaoE, and Gip1 confirmed the hypothesis (Table 2.10).

Adjacent to the laccase encoding gene xpaE a gene coding for a FAS protein (XpaD)

was identified. Similar combinations were identified in the biosynthetic gene clusters of

aurofusarin (30) in F. graminearum (aurS) and in the putative biosynthetic gene clusters

of xanthomegnin (33) and vioxanthin (3) in T. rubrum (xtrD), P. citreonigrum (vpcD),

andA. ochraceus (vaoD). For the biosynthesis of aurofusarin (30) it was demonstrated

that AurS is essential for the dimerization step in the biosynthesis.[63] A similar role is

proposed for its homologue XpaE.

2.3 Viriditoxin biosynthesis 73

2.3 Viriditoxin biosynthesis

O

OR1

OH OH O

O

OH

O

OH O

?

semi-viriditoxin (6)

R2

O

R2

O

O

R2

O

OOHOH

O

viriditoxin (5)

M *

Viriditoxin (5) has been isolated from Aspergillus viridinutans, Aspergillus frankstonensis,

Cladosporium cladosporioides CBS 112388, Paecilomyces variotii, Neosartorya denticulate,

and Penicillium mononematosum.[196–202] The first published structure of viriditoxin

(5) showed a biaryl bond with a similar regiochemistry as vioxanthin (3).[203] The

structure was later revised from a 8,8′- to 6,6′-coupling pattern.[78] No further findings

on the biosynthesis of viriditoxin (5) have been published yet. The isolation of

cladosporinone (75) together with the keto-derivatives 76 and 77 of viriditoxin (5)

suggested a biosynthesis involving a BVO like step creating the terminal methyl ester

moiety (Figure 2.16).[199]

M * M * M *O

O

OH OH

OH O

O O

O

O

OHO

O

O

O

O

O

OO

O

O

O

O

O

OOHOH

O

O

O

O

OH OH O

OOHOH

OH OH

cladosporinone (75) 76 77

Figure 2.16. Cladosporinone (75) and the keto-derivatives 76 and 77 isolatedfrom C. cladosporioides.[199]


Viriditoxin (5) was chosen for the study of the biosynthesis of biaryls, because of

the structural similarities to (R)-semi-vioxanthin (4) of the monomeric units coupled

to the biaryl viriditoxin (5), and the different regiochemistry of the biaryl axis

compared to vioxanthin (3). As the monomeric precursors of viriditoxin (5) and

vioxanthin (3) are structurally related, we assumed that the coupling enzyme from

the viriditoxin (5) biosynthesis will accept (R)-semi-vioxanthin (4) as a substrate. The

dimerization of (R)-semi-vioxanthin (4) to a 6,6′-coupling product would prove that the

regiochemistry of the coupling step is enzymatically controlled and not pre-determined

by the substrate. Taking into account viriditoxin’s (5) structure and its structural

similarities to vioxanthin (3), we proposed a composition for the biosynthetic gene

cluster of the naphtho-α-pyrone dimer (Figure 2.17). For the synthesis of the carbon

backbone of the monomeric precursor semi-viriditoxin (6) an iPKS is expected. Similar

to vioxanthin (3) viriditoxin (5) contains a secondary alcohol that is likely formed by

reduction by a SDR from an achiral precursor. For the formation of the methoxy group




Tailoring• O-MT• BVMO

polyketide synthasereductase (in case of NR-PKS)O-methyltransferaseBaeyer-Villiger monooxygenase

O

OH

O

OH O

O

O

O

O

O

OOHOH

O

viriditoxin (5)

M

coupling enzyme

*

Figure 2.17. Possible composition of a putative biosynthetic gene cluster ofviriditoxin (5).


an O-MT is necessary and the dimerization is proposed to be catalyzed by a coupling

enzyme (CYP, FMO, laccase).


2.3.1 Cluster analysis

To investigate the biosynthesis of viriditoxin (5), the ascomycete A. viridinutans was

chosen. The fungus has been reported to produce the dimer and optimized parameters

for the production of viriditoxin (5) by A. viridinutans have been published.[76,196] The

genome of A. viridinutans was sequenced by BaseClear B.V. (Leiden, Netherlands) and

yielded a genome of 30 Mb assembled to 1579 scaffolds. Moreover, the genome of the

viriditoxin (5) and cladosporinone producer Cladosporium cladosporioides CBS 112388

has recently been published to the database of NCBI (BioProject PRJNA355122).[204]

AUGUSTUS was used for gene prediction and the annotated genomes analyzed with

antiSMASH. The genome of A. viridinutans contains 89 predicted secondary metabolite

gene clusters, including 12 iPKS gene clusters. The genome of C. cladosporioides

contains 54 putative secondary metabolite gene clusters, including seven iPKS

gene clusters. The comparison of the predicted iPKS gene clusters yielded a pair

of homologous gene clusters containing the genes coding for the NR-PKSs VavA

(A. viridinutans) and VccA (C. cladosporioides).

Analogously to vioxanthin (3), viriditoxin (5) was assumed to share the C4–C9

folding pattern (Scheme 2.8). The same folding mode was also recently suggested

after the isolation of viriditoxin (5) and derivatives from C. cladosporioides.[199] Thus,

the iPKS of viriditoxin’s (5) biosynthetic gene cluster was expected to contain a PT

S

O O O

OOO

O

enzyme4

9

O

semi-viriditoxin (6)

O

O

OH OH O4

9 O

O

Scheme 2.8. Folding mode proposed for the biosynthesis of semi-viriditoxin (6).


1 23 4

56

7Fsr1PKS1

VccA

XpaA

VavA

CTB1

11

1213

14

15

16

1718

192021

2223242526272829

3031

323334

3536

37

38

39

4041

42

43

44

45

46

47

48

49

50

51 52

53

54 55

I IIIII

IV

V

VI

VII

VIII

C2-C7C4-C9C6-C11C1-C6C3-C8

XtrA

0.2

VpcA

VaoA

VpfA

Figure 2.18. Phylogenetic tree constructed using 55 NR-PKSs with identifiedproducts and the PT domains of VavA and VccA. The accession numbers of theselected NR-PKSs and the products they have been linked to, are listed in theappendix.

domain of the family IV, catalyzing a C4–C9 first ring cyclization. The PT domains

of the predicted iPKSs were identified using the CDD of NCBI. The PT domains were

aligned and a phylogenetic tree was constructed using the published parameters and

reference sequences.[151] The PT domains of the NR-PKSs VavA and VccA grouped with

PT domains of family IV, indicating a C4-C9 cyclization pattern for the product of VavA

and VccA (Figure 2.18).


Similar to the PT domains of the putative vioxanthin (3) NR-PKS, on the sequence

level the PT domains of VavA and VccA are the closest relatives of PT domains of

NR-PKS responsible for the biosynthesis of fusarubin (61, Figure 2.18, Fsr1, PKS1).[152]

The intermediate released from the fusarubin (61) NR-PKS structurally resembles

semi-viriditoxin (6). However, nor-toralactone (32), the product of CnCTB1 from

C. nicotianae, is structurally more closely related to semi-viriditoxin (6).

The gene cluster containing vavA (A. viridinutans) consists of eight genes encoding

the tailoring enzymes necessary for viriditoxin (5) production. Homologous genes

were identified in the genome sequence of C. cladosporioides as well (Table 2.11,

Figure 2.19). Even though their core structures and the NR-PKSs differ significantly,

viriditoxin (5) and aurofusarin (30) share the methoxy group at C7. In the biosynthesis

of aurofusarin (30) the O-MT AurJ catalyzes the methylation of the phenolic hydroxy

group at C7.[18,61] A homologue of this enzyme is encoded by vavG and vccG, likely

responsible for a similar methylation in viriditoxin (5) biosynthesis.

Viriditoxin (5) and vioxanthin (3) both carry a secondary alcohol at C3. Together

with the proposed folding mode of viriditoxin (5) and grouping of VavA and VccA’s PT

domain, a similar biosynthetic origin of the alcohols is likely. This is reflected in the high

sequence similarities of the SDRs encoded in the gene clusters (55–71%, Table 2.12).

The isolation of ketone derivatives of viriditoxin (5) might help elucidate the

formation of the side chain methyl ester. The putative biosynthetic gene cluster of

vavAvavBvavCvavDvavEvavFvavGvavH

Aspergillus viridinutans

vccH vccAPKSvccBvccCvccDvccEvccFvccG

Cladosporium cladosporioides

Figure 2.19. Graphical overview of the putative biosynthetic gene cluster ofviriditoxin (5) from A. viridinutans and C. cladosporioides.


Table 2.11. Genes of the putative biosynthetic gene cluster of viriditoxin (5) fromA. viridinutans and C. cladosporioides.

A. viridinutans, Sequence identity Putative function Homologues Sequence identityC. cladosporioides [%] [%]

VavA, VccA 60.6 NR-PKS XtrA, VpcA, VaoA 45.7–47.1VavB, VccB 34.2 transcription factorVavC, VccC 70.6 MFS transporter XtrG, VpcG, VaoG 41.7–43.7VavD, VccD 68.2 SDR XtrC, VpcC, VaoC 54.8–71.5VavE, VccE 68.3 BVMOVavF, VccF 41.3 esteraseVavG, VccG 62.8 O-MT XtrF, VpcF, VaoF 46.0–49.7VavH, VccH 66.8 laccase XtrE, VpcE, VaoE 39.8–53.2

viriditoxin (5) is thus expected to contain a gene coding for an enzyme capable of

catalyzing a BVO. The genes vavD and vccD were analyzed using the CDD of NCBI and

the online tool Cofactory.[205] A pyridine nucleotide-disulphide oxidoreductase-like

sequence motif was identified. Congruently, the cofactor binding domains for FAD and

NADH were identified by Cofactory. Type I BVMOs use FAD and NADPH to catalyze

the oxidation of their substrate. Moreover, the signature motif F-x-G-x-x-x-H-x-x-x-W-P,

which was also identified in the amino acid sequence of VavD and VccD, suggests their

role as BVMOs.[206]

Analogously to the gene clusters for vioxanthin (3), the putative biosynthetic gene

clusters of viriditoxin (5) contain the genes vavH and vccH coding for a laccase. In

Table 2.12. Sequence comparison of SDRs from the putative biosynthetic geneclusters of xanthomegnin (33), vioxanthin (3), and viriditoxin (5). The sequenceidentitiy of the amino acid sequences is shown [%].

SDRs VpcC XtrC VaoC VccE

XtrC 77.7VaoC 85.4 75.4VccE 68.3 62.4 71.5VavE 60.7 54.8 58.3 66.4


Table 2.13. Sequence comparison of laccases. Included are laccases VavH andVccH from the putative viriditoxin (5), xanthomegnin (33) and vioxanthin (3)gene clusters, MDE from the dinapinone A1/2 (69 and 70) biosynthesis, Gip1 fromthe aurofusarin (30) biosynthesis, LacTL2 from the dalesconol biosynthesis, andLCC2 from T. versicolor. The sequence identitiy of the amino acid sequences isshown [%].

Laccases VpcE VaoE XtrE XpaE Gip1 MDE VavH VccH LCC2

VaoE 82.7XtrE 78.5 77.5XpaE 61.0 68.5 65.4Gip1 59.0 65.7 62.9 61.0MDE 50.4 53.5 49.0 51.1 49.8VavH 46.9 53.1 50.2 45.7 47.0 48.0VccH 39.8 42.3 40.6 37.1 39.4 36.0 66.8LCC2 18.8 18.9 18.3 18.0 17.1 19.5 17.4 12.1LacTL2 14.3 15.3 15.1 15.2 16.0 16.7 17.1 10.4 21.1

Gip1 from F. graminearum (I1RF62), MDE from T. pinophilus (BAW99827),LCC2 from T. versicolor (Q12718), and LacTL2 from Daldinia eschscholzii(AFO12488).[60,61,162,207–209]

the biosynthesis of aurofusarin (30) the laccase Gip1 is involved in the penol coupling

step together with three additional enzymes.[60–63] However, the biaryls aurofusarin

(30) and viriditoxin (5) are coupled with different regiochemistries. While the laccase

Gip1 and the laccase VavH/VccH show a sequence identity of 47/40%, the putative

vioxanthin (3) laccases show sequence identities of circa 60% compared to Gip1

(Table 2.13). This could indicate a similar function for the synthesis of biarylic natural

products. Additionally, the putative biosynthetic gene clusters of viriditoxin (5) do

not contain a FAS protein, in contrast to the aurofusarin (30) and vioxanthin (3) gene

clusters. FAS protein coding genes have been identified adjacent to genes encoding for

laccases in the genomes of organisms that produce 8,8′-coupled natural products. It

is possible that the FAS proteins are involved in the coupling step and either have a


dirigent role guiding the activated monomers to form a 8,8′-dimer, or they modulate

the activity of the coupling enzyme to generate the disfavored coupling pattern.



To test the activity of the laccase VavH, the gene coding for the laccase was expressed

in A. niger using the newly designed expression plasmid pSUC. The fungal lysate was

tested for coupling activity with the non-native substrates (R)-pro-semi-vioxanthin (27)

and (R)-semi-vioxanthin (4).

While the activity assay with unlabeled (R)-pro-semi-vioxanthin (27) did not yield

a new product, for the conversion with unlabeled (R)-semi-vioxanthin (4), a product

with a mass corresponding to a dimer (−Q1, m/z 545) was detected (Figure 2.20). The

retention time of the compound is different from the retention time of the 8,8′-dimer

vioxanthin (3). Heterologously produced VavH completely converted the added

(R)-semi-vioxanthin (4) to the dimer 7 in 90 minutes (Figure 2.20). In conversions

with VavH using [13C]-labeled (R)-semi-vioxanthin([13C]-4) the detected dimer mass

was shifted (+2 m/z, Figure 2.21). The mass shift corresponds to the number of

incorporated of 1,2-[13C2]-acetate into (R)-semi-vioxanthin (4).

The initially low substrate concentrations (0.02–0.04 mM ≡ 1.4 µg) were increased

to 0.6 mM ≡ 162.4 µg of previously isolated [13C]-labeled (R)-semi-vioxanthin ([13C]-4,

subsection 2.2.5) for further characterization of 7 via NMR. A total of 4 mg of

monomer was converted. 1H NMR, 13C NMR, HMBC and HSQC spectra of 7 product

were measured. The 1H NMR of the 7 showed a significant low field shift for the

proton at C5 compared to (R)-semi-vioxanthin (4) and vioxanthin (3). In general

a low field shift of all signals was observed, when compared to vioxanthin (3)

(Table 2.14). The chemical shifts of the identified dimer proton signals agree with the

published NMR data of pigmentosin A (24) from Hypotrachyna immaculata.[37] The

same is observed for 7’s signals in the 13C NMR spectrum. A set of 15 signals was

identified in the 13C-spectrum, indicating a symmetrical dimer structure. This rules out


0

100

200

300

400

4 3

Abs

orpt

ion

[mA

U]

(a) Standard of (R)-semi-vioxanthin (4) and vioxanthin (3)

0

0.5

1

·108

43

Inte

nsit

y[c

ps] m/z 273

m/z 545

40

60

80

100

(b) Conversion of (R)-semi-vioxanthin (4) with A. niger pSUC::vavH

30 min

0

1

2

3·107

30 min

40

60

80

100 60 min

0

1

2

3·107

60 min

0 2 4 6 8 10 12

40

60

80

100

Time [min]

90 min

0 5 10 15

0

1

2

3·107

Time [min]

90 min

Figure 2.20. HPLC-MS analysis of conversions with the laccase VavHusing (R)-semi-vioxanthin (4) as substrate: (a) standard of the monomer(R)-semi-vioxanthin (4, m/z 273) and the dimer vioxanthin (3, m/z 545).(b) Conversion of unlabeled (R)-semi-vioxanthin (4) with lysate from A. nigerpSUC::vavH after different reaction times. Detection was carried out with DAD(left column, λ 360–400 nm) and mass spectrometry (right column, −Q1, Method3, ISAspher Phenyl 100-5).


540 550 560

0

20

40

60 545

m/z [Da]

Inte

nsit

y[%

]

540 550 560

545

m/z [Da]

Figure 2.21. MS-spectra of the dimer 7 formed in conversions withthe laccase VavH using (R)-semi-vioxanthin (4, left) and [13C]-labeled(R)-semi-vioxanthin ([13C]-4, right) as substrate. −Q1, Method 3, ISAspher Phenyl100-5.

Table 2.14. 1H- and 13C NMR data for the dimer 7 of VavH conversionsof [13C]-labeled (R)-semi-vioxanthin ([13C]-4), pigmentosin A (24) andvioxanthin (3).CDCl3, 21 °C, 1H NMR (400 MHz), 13C NMR (100 MHz) for6,6′-dimer 7 and vioxanthin (3).[22,181] Spectra for pigmentosin A (24) werepublished using the same parameters without details on temperature.[210]

Position Dimer 7 (6,6′) Pigmentosin A (6,6′) Vioxanthin (8,8′)1H [ppm] 13C [ppm] 1H [ppm] 13C [ppm] 1H [ppm] 13C [ppm]

CH3 1.45 20.7 1.45 20.7 1.55 20.7OCH3 3.77 56.1 3.76 56.2 3.84 56.01 – 171.6 – 171.7 – 171.63 4.66 76.5 4.66 76.3 4.76 76.54 2.75 34.7 2.74 34.9 3.01 34.74a – 133.0 – 133.0 – 132.85 6.24 113.7 6.23 113.8 6.95 116.15a – 139.1 – 139.1 – 140.06 – 109.9 – 109.9 6.70 98.17 – 160.0 – 160.8 – 161.48 6.79 98.0 6.78 98.0 – 108.19/9-OH 9.82 159.2 9.82 159.1 9.69 155.49a – 108.0 – 108.0 – 108.510/10-OH 13.96 163.3 13.95 163.3 13.79 162.810a – 98.9 – 98.9 – 99.3


unsymmetrical coupling products, e.g. a 6,8′-dimer. To determine the regiochemistry

of 7, 2D NMR spectra were measured. The combination of HSQC and HMBC data

O

O

O

O

H3C

H3C

OH

OH OH O

CH3

CH3

OOH

H

H

H H

H HHH

H

H

1

4'6'8'

9

5 37

2'10'

*

Figure 2.22. HMBC correlations observedfor the newly formed dimer 7. CDCl3, 21 °C,1H NMR 400 MHz, 13C NMR 100 MHz(appendix, subsection 3.2, Figure 10 andFigure 11).

allowed the assignment of all protons

and their carbon atom partner. By

HMBC analysis, the aromatic protons

were determined to be at C8 and

C5 (Figure 2.22). The high field

shifted aromatic proton couples with the

[13C]-nuclei at C9, C9a, C7, C6. With

this coupling pattern a proton is placed

at C8. The second aromatic proton

(6.95 ppm) couples with C6, C5a, C9a,

C4 locating it at C5. A coupling of the

phenolic proton at C9 with the carbon

atom partner (98.0 ppm) of the high field

shifted aromatic proton (6.79 ppm) further confirms its placement at C8. The missing

signal of a third aromatic proton and the chemical shift of C6 (109.9 ppm) in the

13C NMR spectrum show that the dimer is coupled at C6. Hence, VavH accepts

(R)-semi-vioxanthin (4) as a substrate and converts it exclusively to a 6,6′-coupled

dimer. The natural product viriditoxin (5), in whose biosynthesis VavH is proposed to

be involved, is a 6,6′-coupled dimer as well. (R)-semi-Vioxanthin (4) is dimerized to

yield a 8,8′-dimer in the biosynthesis of vioxanthin (3), indicating that not only the

substrate might have an influence on the regiochemistry of the coupling reaction, but

also the enzyme. Therefore, we propose that VavH catalyzes the phenol coupling step

in viriditoxin’s (5) biosynthesis and also controls the regiochemistry of the reaction.


The high sequence identity of VavH compared to the laccases identified in the putative

vioxanthin (3) gene clusters (47.3–52.9%, Table 2.13) indicates a similar role for these

still uncharacterized enzymes.

Regarding the stereoselectivity, the biosyntheses of viriditoxin (5) and vioxanthin (3)

yield in the corresponding producing fungi preferentially one regio- and stereoisomer

with a diastereomeric excess (de) of 90% for vioxanthin (3).[181] The de for

viriditoxin has not yet been determined. The peak in the UV/Vis chromatogram

corresponding to the formed dimer shows a slight shoulder, while a single peak

was detected for the m/z of the dimer (m/z 545) formed in the VavH-catalyzed

coupling of unlabeled (R)-semi-vioxanthin (4). This could indicate the presence of

two compounds with similar physico-chemical properties, for instance, the conversion

of (R)-semi-vioxanthin (4) to two atropisomeric biaryls. The 1H NMR spectrum of

13.95

74.0098

25.9902

(a)Phenolicproton atC10

6.80 6.78

65.4014

34.5986

(b)Aromaticproton atC8

6.26 6.24 6.22

68.6202

31.3799

(c) Aromaticproton at C5

Figure 2.23. Selected 1H NMR signals of the atropisomeric 6,6′-dimer 7 of theconversion of (R)-semi-vioxanthin (4) with heterologously produced VavH (CDCl3,21 °C, 400 MHz).


the dimer 7 suggests the presence of two diastereomers. The signals of the phenolic

proton at C10 and the aromatic protons at C5 and C8 show a second minor signal

(Figure 2.23). The signals’ integrals (main:minor) have a ratio of 2:1 to 3:1. The signals

are not fully separated making the correct integration difficult. The main product of

the phenol coupling thus has a de of 30–50%. The configuration of the biaryl axis could

not be deduced from the NMR data.

−100

0

100

θ[m

°]

dimer 7pigmentosin A

250 300 350 400 450

0

50

100

λ [nm]

Rel

ativ

eab

sorb

ance

[%]

O

O

O

O

OH OH O

OOHOH

pigmentosin A (24)

M *

O

O

O

O

OH OH O

OOHOH

P-pigmentosin A (7)

P *

Figure 2.24. circular dichroism (CD) and UV spectra of the P-configured dimer7 and M-pigmentosin A (24) in MeOH. The spectra of the M-atropisomer ofpigmentosin A (24) were published by Grove et al.[210] The absorption spectra ofthe pigmentosin diastereomers were normalized setting the absorption maximumas 100% relative absorbance.

Pigmentosin A (24), synthesized by Grove et al. and compared to natural

pigmentosin A (24) isolated from H. immaculata, shows a Cotton effect with a


minimum at λ 275 nm and a maximum at λ 250 nm in its CD spectrum.[37,210]

The coupling product of VavH shows a CD spectrum that has a Cotton effect at the same

wavelength, but with inverse values (Figure 2.24). According to these results the main

VavH-coupling product (7) has P-configuration of the biaryl axis, whereas pigmentosin

A (24) and the minor coupling product show the opposite M-configuration. The UV

spectrum of the coupling product matches the spectra of (R)-semi-vioxanthin (4),

vioxanthin (3), and pigmentosin A (24, Figure 2.24).

The structural elucidation of the coupling products showed that the laccase VavH

is able to selectively catalyze the phenol coupling of (R)-semi-vioxanthin (4) to

6,6′-dimers, hence the laccase is able to control the regiochemistry of the coupling.

The stereochemistry, in turn, is influenced to a lesser extent by the heterologously

produced enzyme. Interestingly, the main coupling product has the opposite

configuration of the biarylic bond compared to the putative native product viriditoxin

(5) from A. viridinutans.[78] This deviation in the stereochemistry might be due to a

non-native substrate used for the conversions with VavH. The methyl ester group of

(semi)-viriditoxin (6) could lead to a different positioning of the monomers in the

laccase’s active side, compared to (R)-semi-vioxanthin (4). While the configuration of

the biaryl axis of viriditoxin (5) has been determined via CD, no de has been published

for the natural product.[78] Therefore, viriditoxin (5) might be produced with a (low)

diastereoselectivity that is dependent on the monomer.


2.3.3 Biosynthetic hypothesis

The genome analysis of A. viridinutans and C. cladosporioides, and the heterologous

expression of the laccase-encoding gene vavH, provided new insights into the

biosynthesis of the dimeric naphtho-α-pyrone viriditoxin (5). Analogous to the

proposed biosynthesis of vioxanthin (3) the carbon backbone of the monomeric

precursor semi-viriditoxin (6) is likely produced by the NR-PKS VavA from

A. viridinutans and VccA from C. cladosporioides. VavA and VccA contain a family IV PT

domain and therefore show a different folding mode for their product compared to other

oktaketide producing NR-PKS, such as the atrochrysonecarboxylic acid (78)-producing

MdpG (Figure 2.18). The reduction taking place in the biosynthesis of viriditoxin

(5) is likely catalyzed by the SDRs VavE and VccE. However, similar to vioxanthin (3)

biosynthesis the substrate of the SDRs is unknown. The released product is subsequently

methylated at the phenolic hydroxy group at C7 by the O-MTs VavG or VccG.

The isolation of viriditoxin and related compounds from C. cladosporioides gave

new insights into the formation of the methyl ester of viriditoxin (5).[199] Together

with viriditoxin (5), the homodimeric ketone-derivative 77, the heterodimeric

ketone-derivative 76 and cladosporinone (75) were isolated (Figure 2.16).[199] The

detection of these compounds suggests a BVO of the side chain ketone to yield a methyl

ester. The putative BVMOs VavD and VccD are likely responsible for this reaction step.

Similar to the biosynthesis of the structurally related polyketide dimer vioxanthin

(3), the hypothesis for the biosynthesis of viriditoxin (5) includes the oxidative phenol

coupling of two molecules of semi-viriditoxin (6) as the final step (Scheme 2.10). The

detection of the monomeric precursor semi-viriditoxin (6) in the chloroform extract

of A. viridinutans reaffirms this hypothesis (Figure 2.25). Additionally, the observed

conversion of the non-native substrate (R)-semi-vioxanthin (4) by the laccase VavH


O

O

OH OH O

O

HO

OH OH O

O

OOHOH

HO

SCoA

HO SCoA

OO

O

7

VavA / VccA

VavA / VccA

79

VavG / VccGVavA / VccA

HO

OH OH O

OHO

VavE / VccE

HO

OH OH O

OH

VavE / VccE

OH

O

HO

OH OH O

H2O

H2O

H2O

VavG / VccG

O

HO

OH OH O

O

O

OH OH O

79

VavG / VccG

O

OOO

O O

O O

VavE / VccE

Scheme 2.9. Hypothesis for the biosynthesis of viriditoxin (5) showing the pathwayto the the monomeric intermediate 79.


0

2

4

·106In

tens

ity

[cps

]

m/z 333m/z 663

0 5 10 15 20 25 30 35

0

500

1,000

Time [min]

Abs

orpt

ion

[mA

U]

λ360−400 nm

Figure 2.25. HPLC chromatogram of an A. viridinutans extract. HPLC-MS analysisof an A. viridinutans extract. Detection was carried out with DAD (bottom) andmass spectrometry (top, +Q1, Method 2, Zorbax Eclipse XDB-C8).

to the 6,6′-coupled dimer 7 supports the proposed coupling of semi-viriditoxin (6,

Figure 2.20). The demethyl derivative (R)-pro-semi-vioxanthin (27) was not accepted

as a substrate by VavH. Consequently, the demethyl derivative of semi-viriditoxin (6) is

not proposed as the physiological substrate of VavH.

VavH is able to control the regio- and to a lesser extent the stereochemistry

of the coupling reaction. The similar structures of the monomeric precursors

(R)-semi-vioxanthin (4) and semi-viriditoxin (6) suggest a similar reactivity in a phenol

coupling reaction. A chemical dimerization of the monomers would likely yield a

similar mixture of regioisomers. However, the laccase VavH is able to generate a dimer


with a regiochemistry different from the regiochemistry observed in vioxanthin (3).

The similar reactivity of the monomeric precursors and the differing regiochemistry

of the dimers suggest that the regiochemistry is controlled by the involved coupling

enzymes.

O

O

O

OO

OH OH

(S)-semi-viriditoxin (6)

VavD / VccDO

O

OH OH O

O

79

7677

O

O

O

O

OH OH

OHOH

O

O

O

O

M *

O

O

O

OO

OH OH

OHOH

O

O

O

O

M *

viriditoxin (5)

O

O

O

OO

OH OH

OHOH

O O

O

O

O

M *

VavH / VccH VavH / VccHVavH / VccH

Scheme 2.10. Proposed biosynthetic pathway for viriditoxin (5) inA. viridinutans.[199]

However, the electrochemically catalyzed phenol coupling of 1-naphthol (80) yields

a mixture of regioisomers with the para-para-coupled product being preferred over

the ortho-para- and the ortho-ortho-coupled products.[211] 2-Methoxynaphthalene (81)

is exclusively coupled to the para-para-coupled biaryl. These observations indicate a

chemically preferred regiochemistry of the phenol coupling, which is not altered by

the enzyme in the case of 6,6′-coupled natural products. The enzyme might partially


control the regiochemistry of the reaction, while its main role is the generation of

monomer radicals that dimerize to the thermodynamically favored regioisomer.


2.3.4 Pigmentosin A hypothesis

The dimeric naphthopyrone pigmentosin A (24) has been isolated from the lichen

H. immaculata. The dimer contains two (R)-semi-vioxanthin (4) subunits suggesting

that pigmentosin A (24) and vioxanthin (3) share the same monomeric precursor

and do have a similar biosynthetic pathway. Therefore, a gene cluster responsible for

pigmentosin A (24) biosynthesis is predicted to contain genes coding for homologues

of the putative xanthomegnin (33) and vioxanthin (3) biosynthesis enzymes. However,

the phenol coupling enzyme likely is an exception. While the putative biosynthetic

gene clusters of 8,8′-linked dimers contain the combination of a laccase encoding gene

and an adjacent FAS protein encoding gene, the latter is missing in the characterized

biosynthetic gene cluster of the 6,6′-linked viriditoxin (5). The biosynthetic gene

cluster of pigmentosin A (24) is predicted to contain a laccase homologous to VavH

(A. viridinutans) without an adjacent FAS protein (Scheme 2.11).

OO

OH

OOH

O

OH O

OHO

vioxanthin (3)

*P

O

O

O

O

OH OH O

OOHOH

pigmentosin A (24)

M *

O

O

OH OH O

(R)-semi-vioxanthin (4)laccase

FAS proteinlaccase

6

6'

8

8'

Scheme 2.11. Hypothesis for the phenol coupling enzyme(s) involved inthe dimerization of (R)-semi-vioxanthin (4) yielding pigmentosin A (24) orvioxanthin (3).


2.3.5 Bicoumarin biosynthesis

During the investigation of the biosynthesis of viriditoxin (5), a bicoumarin secondary

metabolite was isolated from an extract of an A. viridinutans culture. The HPLC-MS

chromatogram of the extract showed the expected peaks for viriditoxin (5) and the

monomer semi-viriditoxin (6). Additionally, a bicoumarin [kotanin (2 or a regioisomer]

was detected (Figure 2.28). The compound has a m/z 439 (+Q1), matching the m/z

of kotanin-like bicoumarins. The UV-spectrum resembles the spectrum published for

kotanin-like bicoumarins.[120]

The genome of A. viridinutans was screened for homologous clusters of the

characterized kotanin biosynthetic gene cluster from A. niger CBS 513.88. The identified

putative bicoumarin biosynthetic gene cluster contains a NR-PKS, two O-MT, and a

CYP enzyme (Table 2.15).

O

O

O

O

OH

OO

O

OH

O

O

OH

O

O

OH

O

P *

M *

kotanin (2) desertorin C (22)

Figure 2.26. The bicoumarins kotanin (2) and desertorin C (22) isolated fromA. niger and E. desertorum, respectively.


Table 2.15. Comparison of the putative kotanin biosynthetic gene cluster fromA. viridinutans with the characterized kotanin biosynthetic gene cluster of A. nigerCBS 513.88. Graphical depiction of the putative bicoumarin biosynthetic genecluster from A. viridinutans.

Enzyme Putative function Homologue Sequence identity [%]

KavA O-MT KtnA (A2QK64) 84.6KavB O-MT KtnB (A2QK65) 91.5KavC CYP enzyme KtnC (A2QK67) 83.4KavS NR-PKS KtnS (A2QK66) 88.0

kavA kavB kavS kavC

The construction of a phylogentic tree using characterized CYP enzymes from

bicourmain clusters shows that the newly identified CYP enzyme KavC likely dimerizes

monomeric precursors to the bicoumarin kotanin (2, Figure 2.27). This hypothesis

was confirmed by comparison of the detected bicoumarin from A. viridinutans with

bicoumarin reference standards. The detected bicoumarin shows a similar retention

time to kotanin (2, Figure 2.28).

KavC - A. viridinutans

KtnC - A. niger

DesC - E. desertorum

BicC - A. alliaceus

BAJ04470 - A. oryzae

XP_002376728 - A. flavus

XP_001275037 - A. clavatus

0.1

Figure 2.27. Phylogentic tree of characterized CYP enzymes involved inthe biosynthesis of regiochemically different bicoumarins and KavC fromA. viridinutans.[9] KtnC from A. niger (XP_001402310), DesC from Emericelladesertorum (A0A0N9HKQ7), BicC from Aspergillus alliaceus (ALG03239).


0

0.5

1

1.5·108

Inte

nsit

y[c

ps]

(a) Standard of kotanin (2)

m/z 439

0

0.5

1

1.5·108

13.17

0

2

4

6·108

Inte

nsit

y[c

ps]

(b) Standard of desertorin C (22)

m/z 439

0

2

4

6·108

13.47

0 10 20 30

0

0.5

1

1.5·108

Time [min]

Inte

nsit

y[c

ps]

(c) Extract of A. viridinutans

m/z 439

11 12 13 14 15

0

0.5

1

1.5·108

13.21

Time [min]

Figure 2.28. HPLC-DAD-MS analysis of an A. viridinutans extract using publishedparameters.[9,120] The extract (c) is compared to (a) kotanin (2) and (b) desertorinC (22). Detection was carried out with mass spectrometry (+Q1, Method 4, ZorbaxEclipse XDB-C8).


2.4 In-silico analysis of polyketide dimers’ biosynthesis

Apart from the biosynthesis of vioxanthin (3) and viriditoxin (5), the biosynthesis

of other dimeric polyketide natural products was studied to identify new coupling

enzymes. Literature reporting the isolation of polyketide dimers was used to identify

producers. If a genome sequence of the producer (or a closely related organism) was

available, it was screened for iPKS clusters containing genes coding for the enzymes

proposed in the biosynthetic hypothesis for the dimeric compound.

2.4.1 Perylenequinone biosynthesis

O

OOHOH

HO

OH O

O

O

OOH

OH

OH

?

nor-toralactone (32) cercosporin (40)

O

O

During the analysis of the putative biosynthetic gene clusters of vioxanthin (3)

and viriditoxin (5), the central iPKS’s PT domain was compared to CnCTB1’s PT

domain from Cercospora nicotianae, because of structural similarities of its product

nor-toralactone (32). The biosynthetic gene cluster of cercosporin (40) has been

identified in C. nicotianae, while the coupling enzyme responsible for the dimerization

step has not been determined.[154,212]

Several perylenequinone producers have been reported and their genome sequences

have been published. The iPKS EfPKS1 from Elsinoë fawcettii linked to perylenequinone

biosynthesis in knock-out experiments, is likely not responsible for the synthesis

of the natural product’s backbone and could be involved in the regulation of the

2.4 In-silico analysis of polyketide dimers’ biosynthesis 99

OH

OH

O

OOH

OH

O

O

O

O

cercosporin (40)

O

O

OH

OH

O

O

O

O

elsinochrome A (41)

O

O

OHO

O

O

OH

O

O

OH O

O

hypocrellin A (42)

Figure 2.29. Perylenequinones from Cercospora nicotianae, Cercospora beticola,Cercospora zeae-maydis (40, single genes published for C. nicotianae, full genomefor C. beticola and C. zeae-maydis), Parastagonospora nodorum (41, publishedgenome), Elsinoë fawcettii (41, single genes published), and Shiraia sp. Slf14 (42,published genome).

biosynthesis.[213,214] Knock-outs of the genes coding for the iPKS EfPKS1 and the

transcription factor EfTSF1 stopped the production of perylenequinone secondary

metabolites by E. fawcettii.[213,214] The transcription of EfPKS1 coincides with the

production of perylenequinones.[214] The characterized NR-PKS CnCTB1 is not

homologous to EfPKS1 and the the PT domains of the NR-PKSs were not grouped in the

same PT domain family in a phylogenetic tree constructed using published parameters

(Figure 2.30).[151] With this information available the genomes of the perylenequinone

producers Cercospora beticola (PRJNA430274), Cercospora canescens (PRJNA183604),

Cercospora zeina (PRJNA355276), Cercospora zeae-maydis (JGI Project ID 401984),

Parastagonospora nodorum SN15 (BioProject PRJNA43259), and Shiraia sp. Slf14

(BioProject PRJNA222884) were analyzed.[88,215–219]

To identify putative biosynthetic gene clusters of polyketide dimers in the genomes

of P. nodorum SN15 and Shiraia sp. Slf14, the predicted genes were submitted to

antiSMASH for cluster prediction. Fifteen iPKS gene clusters were predicted for

P. nodorum SN15 and the genome of Shiraia sp. Slf14 contains 9 iPKS gene clusters.

The protein sequences of the predicted iPKSs from P. nodorum SN15 and Shiraia sp.


1 2 3 4

56

7Fsr1

PKS1VccA

XpaA

VavA

CnCTB1

1112

131415

16

171819/EfPKS120

2122232425

2627282930

31

3233

3435

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51 52

5354 55

I

II

III

IV

V

VI

VII

VIII

C2-C7C4-C9C6-C11C1-C6C3-C8

XtrA

VpcA

VaoA

VpfA

0.2

CbCTB1

CzmCTB1

CzCTB1

HypA

ElcA

EaEfPKS1ShEfPKS1PnEfPKS1

Figure 2.30. Phylogenetic tree of PT domains of characterized NR-PKS,the NR-PKSs putatively involved in the biosynthesis of xanthomegnin (33),vioxanthin (3), and viriditoxin (5), and the PT domains of EfPKS1 and CnCTB1homologues from P. nodorum and Shiraia sp. Slf14.


Slf14 were compared with the identified iPKS from C. nicotianae (CnCTB1) and EfPKS1

from E. fawcettii using NCBI’s stand-alone BLAST tool.[220] The genomes of C. beticola,

C. zeina, and C. zeae-maydis have been published with a gene annotation. For these

organisms the complete proteome was compared with the published cercosporin (40)

biosynthetic gene cluster from C. nicotianae and the putative elsinochrome A (41)

biosynthetic gene cluster from E. fawcettii.

The genomes of P. nodorum SN15, Shiraia sp. Slf14, C. beticola, C. zeina,

and C. zeae-maydis contain genes coding for homologues of EfPKS1 and CnCTB1

(Table 2.16). The existence of a CnCTB1 homologue and a putative biosynthetic

gene cluster of perylenequinones in Shiraia sp. Slf14 was also published by Yang et

al. and recently confirmed to be responsible for hypocrellin A (42) production by

knock-out with CRISPR/Cas9.[219,221] The CnCTB1 homologue ElcA from P. nodorum

has been shown to produce toralactone (55), the precursor of elsinochromes.[222]

An inactive CnCTB7 homologue (CzCTB7) has recently been identified in C. zeina.

Complementation of the gene with Cnctb7 initiated cercosporin (40) production by

C. zeina. The existence of an EfPKS1-homologue containing cluster suggests a similar

regulatory circuit for perylenequinone production in P. nodorum Sn15, Shiraia sp. Slf14,

C. beticola, C. zeina, and C. zeae-maydis compared to E. fawcettii.[223]

The NR-PKS CnCTB1 from C. nicotianae and ElcA from P. nodorum synthesize

nor-toralactone (32).[154,222] Similar enzymes, in terms of PT domain, such as XtrA from

T. rubrum, VpcA from P. citreonigrum, VaoA from A. ochraceus, and HypA from Shiraia

sp. Slf14 likely produce the same or structurally related compounds (Figure 2.18).

Shiraia sp. Slf14 produces hypocrellin A (42), a perylenenquinone structurally closely

related to cercosporin (40), and the elsinochromes.


Czmctb6Czmctb4Czmctb2Czmctb1Czmctb3Czmctb5Czmctb7Czmctb8Czmatr1czmFczmG

Cercospora zeae-maydis

Czctb6Czctb4Czctb2Czctb1Czctb3Czctb5Czctb7Czctb8Czatr1cczFcczG

Cercospora zeina

Cbctb6Cbctb4Cbctb2Cbctb1Cbctb3Cbctb5Cbctb7Cbctb8Cbatr1ccbFccbG

Cercospora beticola

elcE elcR elcD elcC elcB elcA elcF elcG

Parastagonospora nodorum SN15

hypAhypBhypChypDhypRhypE hypH hypF hypG

Shiraia sp. Slf14

Figure 2.31. Graphical overview of the perylenequinone clusters from C. beticola,C. zeina, C. zeae-maydis, P. nodorum, and Shiraia Slf14.[219,222,224,225]

The NR-PKS PKS12 from F. pseudograminearum PH-1 produces YWA1 and its PT

domain is assigned to a different family than the PT domains of the vioxanthin (3)

and perylenequinone NR-PKSs. However, the cluster is well characterized and shares

tailoring enzymes with the other clusters and was therefore included in the comparison.

The putative perylenequinone gene clusters in P. nodorum SN15 and Shiraia sp.

Slf14 share a total of 4 genes with the characterized cercosporin (40) gene cluster in

C. nicotianae. Apart from the CnCTB1 homologue encoding gene, the genomes also

contain genes coding for homologues of CnCTB2, CnCTB3, and CnCTB5. Homologues

of the genes coding for the transporter CnCTB4, the SDR CnCTB6, the oxidoreductase

CnCTB7, and the transcription factor CnCTB8 are missing. Instead, the gene clusters of

P. nodorum SN15 and Shiraia sp. Slf14 contain genes coding for a non-homologous

transporter ElcC/HypC, a different transcription factor ElcR/HypR, a FAS protein

ElcF/HypF and a laccase ElcG/HypG (Figure 2.31, Table 2.16). The gene cluster of

2.4 In-silico analysis of polyketide dimers’ biosynthesis 103Ta

ble

2.16

.H

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tran

scri

ptio

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DT1

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etic

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etic

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Shiraia sp. Slf14 additionally contains a gene encoding a putative NAD-binding protein

HypH. Apart from the central NR-PKS the clusters share three of the tailoring enzymes.

Interestingly, the putative biosynthetic gene clusters of cercosporin (40) in

C. beticola, C. zeina, and C. zeae-maydis also contain homologues of the FAS proteins

ElcF/HypF and the laccases ElcG/HypG (Figure 2.31, Table 2.16). As the newly

identified homologous cercosporin (40) biosynthetic gene clusters share a high

sequence similiarity (52.8–99.5%) with the proteins CnCTB1–CnCTB8 encoded by

the characterized gene cluster from C. nicotianae, the cluster comparison is carried out

with the proteins CnCTB1–CnCTB8.

The bifunctional CnCTB3 enzyme from C. nicotianae contains a FMO and an O-MT

domain.[226] The O-MT domain is responsible for the methylation of the oxygen at

C7.[154] The enzyme CnCTB3 shares 38.4–46.3% sequence identity with the O-MTs VpcF

from P. citreonigrum, AurJ from F. graminearum, ElcB from P. nodorum SN15, and HypB

from Shiraia sp. Slf14 (Table 2.17). In the case of the two latter fungi, the homologous

enzyme is bifunctional as well. The second methyltransferase CnCTB2 from the

cercosporin (40) biosynthetic gene cluster has a lower sequence identity (23.0–26.8%)

compared to VpcF, AurJ, ElcB, and HypB (Table 2.17). CnCTB2 was shown to methylate

the phenolic hydroxy group formed by the oxidative decarboxylation catalyzed by

CnCTB3.[154] The higher sequence similarities of HypB and ElcB to O-MTs with a

regioselectivity for methylation at the phenolic hydroxy group at C7 suggests a similar

selectivity for the uncharacterized O-MTs HypB and ElcB. Because of their high sequence

similarity (52.3–54.0%) to CnCTB2, the O-MTs HypD and ElcD likely catalyze an

O-methylation with a similar regiochemistry as the modification catalyzed by CnCTB2

(Table 2.17).


Table 2.17. Sequence comparison of characterized O-MTs BfoD, BfoE, AnAunD,AnAunE, AurJ, CnCTB3, CnCTB2, the O-MT VpcF from the putative biosyntheticgene cluster of vioxanthin (3), and the O-MTs ElcB, ElcD, HypD, and HypE. Thesequence identitiy of the amino acid sequences is shown [%].O-MT BfoD AnAunD ElcB HypB CnCTB3 AurJ VpcF ElcD HypD CnCTB2 BfoE

AnAunD 83.5ElcB 31.7 30.8HypB 32.9 33.3 64.0CnCTB3 33.9 34.4 44.7 46.3AurJ 37.3 37.8 37.2 37.6 38.4VpcF 39.5 40.0 37.7 40.9 40.4 48.1ElcD 24.4 24.6 20.9 22.1 23.0 25.0 23.9HypD 23.9 24.8 21.6 23.4 24.0 25.0 25.3 75.8CnCTB2 24.7 24.4 23.0 23.5 23.4 26.8 25.8 52.3 54.0BfoE 13.9 12.1 11.8 13.0 11.7 11.8 11.8 13.8 14.3 12.2AnAunE 14.7 12.8 12.0 13.5 12.2 11.5 11.8 14.8 15.4 13.4 92.2

AurJ from F. graminearum (I1RF60), AnAunD (Aspni_NRRL3_1|2868) and AnAunE (Aspni_NRRL3_1|2869)from A. niger NRRL3, BfoD (Aspbr1|39215) and BfoE (Aspbr1|438143) from A. brasiliensis CBS 101740,CTB2 (ABK64180) and CnCTB3 (ABC79591) from C. nicotianae, ElcD (EAT83778) and ElcB (EAT83780) fromP. nodorum, HypB and HypD from Shiraia sp. Slf14, and VpcF from P. citreonigrum.[155,157,160,161,222]

Recently, a biosynthetic route was proposed for cercosporin (40).[154] CnCTB5 and

CnCTB7 were predicted as putative coupling enzymes. In the biosynthetic gene clusters

of P. nodorum SN15 and Shiraia sp. Slf14, no homologues of these putative coupling

enzymes were identified. Instead, a gene encoding a laccase and a gene encoding

a FAS protein are present in the cluster. This combination has also been identified

in the biosynthetic gene cluster of aurofusarin (30) and in the putative biosynthetic

gene clusters of vioxanthin (3) in T. rubrum, P. citreonigrum, and A. ochraceus (see

subsection 2.2.1).[61] While no homologues of the laccases ElcG/HypG and the FAS

protein ElcF/HypF have been identified in C. nicotianae, a gene encoding a similar

laccase (cccG, ccbG, cczG, cczmG) and an adjacent gene coding for a FAS protein (cccF,

ccbG, cczG, cczmG) were identified in the genomes of C. canescens, C. beticola, C. zeina,

and C. zeae-maydis, cercosporin (40) producers related to C. nicotianae.[217,224,227–230]

Homologues of the enzymes encoded by Cnctb1–Cnctb8 were also identified in genome

of C. canescens (Cnctb3 encoding homologue is missing). However, the contigs with


the homologue encoding genes are not contiguous. The identified gene coding for

a homologue of the laccases ElcG/HypG was identified on a contig without any

genes coding for homologues of CnCTB1–CTB8. Nevertheless, the examples of split

biosynthetic gene clusters like the biosynthetic gene cluster of the naphtho-γ-pyrone

bifonsecin B, aurasperone B and rubrasperone B in different Aspergillus species show

that biosynthetic gene clusters do not need to be physically clustered.[231] In the case of

the split naphtho-γ-pyrone biosynthetic gene clusters, the genes coding for the enzymes

producing the core structure of the dimers are clustered with the tailoring O-MTs AunD

and AunE, and the dimerizing CYP enzyme AunB.[231] The identification of homologous

clusters in the related fungi C. beticola, C. zeina, and C. zeae-maydis that the genes

coding for the laccases CcbG, CczG, and CczmG and the FAS proteins CcbF, CczF, and

CczmF are situated in direct proximity of the CTB1-containing cluster (Figure 2.31).

Therefore, the identified homologous laccases CccG, CcbG, CczG, and CczmG and

FAS proteins CccF, CcbF, CczF, and CczmF are likely involved in the biosynthesis

of cercosporin (40) in C. canescens. Genes coding for homologues of the laccases

ElcG/HypG/PeaG/CccG (and homologues) and the FAS proteins ElcF/HypF/PeaF/CccF

(and homologues) are expected to be present in the genome of C. nicotianae.

In the case of aurofusarin (30) biosynthesis the enzyme responsible for the phenol

coupling step has not yet been identified. The separate knock-out of four genes lead

to mutants of F. graminearum deficient in dimer production, while the monomeric

precursors were still synthesized.[60–63] Homologues of Gip1, AurF and AurS have been

identified in T. rubrum, P. citreonigrum, and A. ochraceus (Table 2.5).

As stated earlier, laccases and FMOs are both capable of catalyzing an intermolecular

phenol coupling reaction. The putative biosynthetic gene clusters of elsinochrome

A–C (41) and hypocrellin A (42) do not contain a homologous enzyme to AurF. This


enzyme is proposed to catalyze para-hydroxylation generating the quinone moiety of

aurofusarin (30). Additionally, the FMO AurF is involved in the phenol coupling step

in aurofusarin’s (30) biosynthesis.[61] The FAD-binding proteins CnCTB5, ElcE, and

HypE share 7.5–10.1% sequence identity with AurF. Hence, it is unlikely that they

have a similar function as AurF. In contrast, Gip1 homologues were identified in the

genomes of T. rubrum, P. citreonigrum, A. ochraceus, C. canescens, C. beticola, C. zeina,

C. zeae-maydis, P. nodorum, and Shiraia sp. Slf14 (Table 2.18, Table 2.16).

Table 2.18. Comparison of laccases from different biosynthetic gene clusters. Thesequence identitiy of the amino acid sequences is shown [%].

Laccases ElcG HypG PeaG CccG VavH VccH VpcE VaoE XtrE XpaE Gip1 MDE LCC2

HypG 76.3PeaG 59.8 60.9CccG 48.3 51.0 51.2VavH 31.2 32.1 29.5 32.2VccH 23.8 24.2 22.4 24.4 66.8VpcE 32.1 32.2 29.4 32.4 46.9 39.8VaoE 31.9 32.5 30.6 32.7 53.1 42.3 82.7XtrE 30.8 31.7 29.3 33.3 50.2 40.6 78.5 77.5XpaE 33.1 32.9 29.6 33.1 45.7 37.1 61.0 68.5 65.4Gip1 33.8 32.9 31.4 32.0 47.1 38.6 58.7 65.7 62.9 61.2MDE 31.6 33.3 31.0 32.1 48.0 36.0 50.4 53.5 49.0 51.1 49.8LCC2 17.5 18.2 18.2 17.0 17.2 11.6 18.9 18.9 18.3 17.9 16.7 19.3LacTL2 17.1 18.1 15.9 16.3 16.2 10.5 14.7 16.3 16.1 15.7 16.5 17.5 19.9

ElcG from P. nodorum (EAT83784), PeaG from E. ampelina (Elsamp1|43111), XtrE from T. rubrum (TERG_02846), XpaE from P. arizonense(OGE58328), Gip1 from F. graminearum (I1RF62),MDE from T. pinophilus (BAW99827), LCC2 from T. versicolor (Q12718).[60,61,162,208,209]

The genomes of Shiraia sp. Slf14 (HypG) and C. canescens (CccG) were annotated in this work.

The laccases identified in the seven analyzed clusters share a sequence identity

of 30.3–82.7% (Table 2.18). Included in this comparison is VavH, which is able

to catalyze an intermolecular phenol coupling of (R)-semi-vioxanthin (4) with a

regiochemistry similar to cercosporin (40). The laccase PeaG was identified in the

genome of E. ampelina, a fungus related to the elsinochrome producer E. fawcettii. To

test the putative dimerizing laccases against a characterized non-dimerizing laccase,

LCC2 from Trametes versicolor was included in the sequence comparison as well.[208,209]

While the putative dimerizing enzymes share a sequence identity of at least 30%, LCC2

represents the basic sequence similarity of the enzyme class. The knock-out of gip1,


the shown activity of the heterologously expressed vavH (subsection 2.3.2), and the

observed sequence similarity of the identified laccase suggest that these enzymes are

responsible for the phenol coupling step in different biosyntheses.

In the case of aurofusarin (30), elsinochromes (41), hypocrellin A (42), and

vioxanthin (3), the laccase gene is adjacent to a FAS protein encoding gene. This gene

is absent in the putative biosynthetic gene cluster of viriditoxin (5) in A. viridinutans.

The phenol coupling step in the biosynthesis of aurofusarin (30) is one of the final steps

of the biosynthesis. A quinone or hydroquinone derivative of rubrofusarin (31) is likely

coupled by Gip1 to yield aurofusarin (30) or a hydroquinone derivative of the dimer.

The para-hydroxylation might be catalyzed by a FMO (AurF) and the oxidation of the

hydroquinone by a FAD-binding protein (AurO). Of the four gene knock-outs that lead

to mutants deficient in dimer production, AurS has not yet been assigned a function.

The same holds true for the FAS proteins XtrD (T. rubrum), VpcD (P. citreonigrum), VaoD

(A. ochraceus), ElcF (P. nodorum), HypF (Shiraia sp. Slf14), and PeaF (E. ampelina).

A FAS protein encoding gene has been identified in the putative biosynthetic gene

clusters of 8,8′-coupled dimers such as vioxanthin (3), while a similar gene is missing

in the putative biosynthetic gene cluster of the 6,6′-coupled viriditoxin (5). The gene

clusters responsible for the biosynthesis of hypocrellin A (42) and the elsinochromes

(41) contain a FAS protein, even though the natural product is a 6,6′-coupled dimer. A

sequence comparison of FAS proteins shows that the proteins form two distinct groups

that are only distantly related to each other, while members of the groups are closely

related to each other (Table 2.19).


Table 2.19. Comparison of FAS proteins from different biosynthetic gene clusters.The sequence identitiy of the amino acid sequences is shown [%].

FAS proteins AurS XtrD VpcD VaoD ElcF HypF

XtrD 37.7VpcD 38.4 55.9VaoD 36.0 53.9 63.9ElcF 15.2 13.4 14.9 14.0HypF 16.8 15.3 16.5 16.1 58.2PeaF 14.1 16.0 16.2 14.8 47.8 51.9CccF 11.4 12.7 15.5 13.1 35.6 38.3 40.2

AurS from F. graminearum (I1RF63), XtrD from T. rubrum(TERG_02847), ElcF from P. nodorum (EAT83783).[63,222]

Biosynthetic hypothesis

The biosynthesis of the perylenequinone cercosporin (40) has been studied in detail and

a biosynthetic pathway has been proposed by Newman et al. describing the biosynthesis

with the exception of the final dimerization step (Scheme 2.12).[154] The coupling step

is proposed to be carried out by the oxidoreductases CnCTB5 and CnCTB7.[154]

The biosynthetic gene clusters of hypocrellin A (42) in Shiraia sp. Slf14 and the

elsinochromes (41) in P. nodorum do not contain homologues of the oxidoreductase

CnCTB7. However, homologues of the flavin-binding CnCTB5 have been identified

(Table 2.16). The enzymes share 57.9–79.5% sequence identity, indicating a similar

function in the biosynthesis of perylenequinones. The gene clusters additionally contain

a laccase and a FAS protein. Homologues of this laccase have not been described

for C. nicotianae, but have been identified in the genomes of C. canescens, C. beticola,

C. zeina, and C. zeae-maydis. The genes are not part of the cluster but are situated 15

kilobasepairs downstream of the gene encoding the CnCTB7 homologue (Figure 2.31).

However, adjacent to the genes encoding the laccase and FAS protein a homologue of


SCoA

O

HO SCoA

OO6

O

OOHOH

HO

O

OOHOH

O

OHOH

O

OOH

OHOH

O

OO

OHOH

O

OHO

O

O

OH O

OOH

O

O

O

O

O

O

O

OH

OH

OH

OH

O

elsinochrome A (41)

O

O

OH

OH

OH

COMe

H

O

O

O

nor-toralactone (32) toralactone (55)

cercosporin (40)

2x

CTB1 CTB3

CTB3

CTB2CTB6?

hypocrellin A (42)

Scheme 2.12. Biosynthesis of the perylenequinone cercosporin (40) and thestructurally related hypocrellin A (42) and elsinochrome A (41) proposed byNewman et al.[154]

the transporter ATR1 was identified. This transporter has been reported to be involved

in the hyphal export of cercosporin (40, Figure 2.31, Table 2.16).[232] Additionally, in

the biosynthesis of naphtho-γ-pyrones in different Aspergillus species the biosynthetic

gene cluster is split. The genes encoding enzymes responsible for the biosynthesis of the

core structure are not physically clustered with the genes encoding the enzymes carrying

out the tailoring (including dimerization) of the core structure.[231] Therefore, the

laccase and the FAS protein are likely involved in the biosynthesis of perylenequinones.


2.4.2 Xanthone biosynthesis

HO

OH O

O

OH

OHMeOOC

COOMeOH

OHO

OH O OH

OHO

O

?

emodin (83) secalonic acid A (8)

OO

O OHO

COOMe Cl

Cl

(+)-geodin (84)

Scheme 2.13. Emodin (83)-derived natural products.

Xanthone natural products have been isolated from bacteria, fungi, and plants.[233–235]

The biosyntheses of some xanthones, such as bikaverin (82) from Fusarium oxysporum,

prenylated xanthones in Aspergillus nidulans, and secalonic acids in Penicillium oxalicum,

have been studied via labeling and knock-out studies (Figure 2.32).[10,11,236–243]

While the xanthone structure in bikaverin (82) is formed by a unique folding of

the poly-β -keto chain, the prenylated xanthone tajixanthone (12, Figure 2.32) and

the secalonic acids are derived from the anthraquinone emodin (83).[10,11] To obtain

a xanthone core structure from emodin (83), the anthraquinone skeleton has to be

cleaved oxidatively. This hypothesis has been confirmed by labeling studies in the

biosynthesis of tajixanthone (12).[239]


O

OOH

OHOOH

OHO

OH

OH

COOMe

MeOOC


O

O

COOMe

HO

O

MeOOC

OH O OH

OH

O

OH

* P

O

O

O

OH

OHOOH

OH

MeOOCHO

COOMeOH

lentulin A (46) neosartorin (9)

O

O

O

OH

O

OH

tajixanthone (12)

OO

O

O OH

OH

O

O

bikaverin (82)

O

Figure 2.32. Natural products with a xanthone core isolated from aspergillusaculeatus (8), Aspergillus fischeri (9), Aspergillus lentulus (46), Aspergillusvariecolor (12), Claviceps purpurea (8), P. oxalcium (8), and F. oxysporum(82).[71,91,234,244–247]

Cluster identification

In order to identify putative biosynthetic gene clusters of tetrahydroxanthone dimers,

ascomycetes that have been reported to produce the dimers of interest have been

on focus. Wezeman et al. reported on various fungi producing tetrahydroxanthone

dimers similar to the secalonic acids.[248] The filamentous fungus C. purpurea produces

the ergochrome group of tetrahydroxanthone dimers, also referred to as secalonic

acids. Aspergillus aculeatus, Aspergillus fischeri, Aspergillus lentulus, and P. oxalcium

produce different dimeric tetrahydroxanthones as well (Figure 2.32).[72,244–246] The

genomes of these fungi have been sequenced, assembled and published to NCBI and

the JGI.[160,249–253]

The characterization of the biosynthetic gene cluster of the benzophenone

monodictyphenone (15) and tajixanthone (12) confirmed emodin (83) as an


O

O

OH

OH O

OOH

OH

MeOOC OH

COOMeOH


polyketide synthasereductasemethyltransferase/esteraseBaeyer–Villiger monooxygenasehydroxylasedecarboxylaseanthrone oxygenase


iPKS• NR-PKS


Tailoring• MT/esterase• BVMO• SDR• anthrone

oxygenase• hydroxylase• decarboxylase

coupling enzyme

Figure 2.33. Possible composition of a dimeric xanthone biosynthetic gene cluster.

biosynthetic intermediate.[10,237,238] Labeled emodin (83) was also incorporated

into tetrahydroxanthone biosynthesis in C. purpurea.[254] As emodin (83) is a

shared pathway intermediate, the enzymes responsible for its biosynthesis are likely

homologous. The NR-PKS MdpG from the tajixanthone-producing (12) A. nidulans

has been reported to produce atrochrysonecarboxylic acid (78).[240,241,255] Analysis

of MdpG’s PT domain grouped it with other enzymes known to be part of emodin

(83)-derived secondary metabolites.[151] To identify similar biosynthetic gene clusters of

tetrahydroxanthone dimers, the genomes of the fungi mentioned above were analyzed

with antiSMASH to identify putative iPKS-clusters.

The predicted iPKSs were screened for enzymes containing a PT domain of

group V. The genes surrounding the NR-PKS with such a PT domain were analyzed

by BLASTp-comparison to the non-redundant protein sequence database of NCBI

(nr-database) to find homologous enzymes with an assigned function. The CDD was


used to predict the function of enzymes without characterized homologues. The

bioinformatic analysis yielded five putative biosynthetic gene clusters of dimeric

tetrahydroxanthones and one putative biosynthetic gene cluster of a monomeric

xanthone produced by Monilina fructicola (Figure 2.34, Table 2.20).

The clusters contain a NR-PKS with a PT domain of family V, which does not possess

a TE. The PKS product is cleaved off by an external TE, that resembles MβLs on

the amino acid sequence level.[103] A MβL encoding gene was identified adjacent

to the NR-PKS. Additionally, the clusters contain a gene coding for a homologue of

HypC, GedH, and MdpH. HypC has been shown to be responsible for the oxidation of

norsolorinic acid anthrone to norsolorinic acid.[188] A domain of unknown function

(DUF)1772 was identified in the sequence of HypC by CDD analysis. The emodin

anthrone oxidase GedH from the (+)-geodin (84) biosynthesis cluster of Aspergillus

eaaA eaaJ eaaY eaaHeaaBeaaIeaaVeaaUeaaNeaaWeaaF eaaK eaaD eaaX eaaS eaaT eaaO eaaQ

Aspergillus aculeatus

epoA epoJ epoY epoH epoKepoD epoX epoSepoBepoIepoVepoUepoNepoWepoF epoO epoP epoQ

Penicillium oxalicum

ecpAecpBecpCecpDecpEecpFecpGecpHecpIecpJecpKecpLecpMecpOecpPecpQecpRecpSecpTecpU ecpN

Claviceps purpurea

nafAnafBnafCnafDnafKnafNnafFnafJnafInafOnafZnafQnafPnafTnafSnafUnafHnafV

Aspergillus fischeri

nalAnalBnalCnalDnalKnalNnalFnalJnalInalOnalZnalQnalPnalTnalSnalUnalH

Aspergillus lentulus

mfrAmfrBmfrCmfrDmfrKmfrEmfrFmfrNmfrOmfrJmfrImfrUmfrHmfr1

Monilinia fructicola

Figure 2.34. Graphical overview of the putative xanthone clusters fromA. aculeatus, P. oxalcium, C. purpurea, A. fischeri, A. lentulus, and M. fructicola.


terreus catalyzes the oxidation of emodin anthrone (85) to emodin (83).[17] MdpH

is proposed to catalyze the decarboxylation of atrochrysonecarboxylic acid (78) to

atrochrysone (86) and prevent the dehydration to endocrocin (87, Scheme 2.14).[240]

However, the annotation of mdpH is likely incorrect, as the gene product contains two

domains [DUF 1772 and EthD-like domain], which are encoded by two separate genes

in similar clusters responsible for the production of xanthones. The gene mdpH can

be split into two separate ORFs that encode a EthD-like domain (mdpH-ORF1) and

the DUF1772 (mdpH-ORF2). The new DUF1772-containing ORF of MdpH is likely

responsible for the oxidation of emodin anthrone (85), while the EthD-containing ORF

codes for a decarboxylase.

The combination of NR-PKS, MβL, an atrochrysonecarboxylic acid (78)

decarboxylase, and an emodin anthrone (85) oxidase suggests that the identified

gene clusters are responsible for the production of emodin (83) as an intermediate in

xanthone biosynthesis.

HO

OH OH O

OH

OH

O

atrochrysonecarboxylic acid (78)CO2

OOHOH

HO OH

MdpH

atrochrysone (86)

H2O

[O]

HO

OH O OH

OH

O

endocrocin anthrone

HO

OH O OH

OH

O

O

endocrocin (87)

MdpH

Scheme 2.14. Conversion of atrochrysonecarboxylic acid (78) to atrochrysone(86) by MdpH. The conversion of atrochrysonecarboxylic acid (78) to endocrocin(87) is likely inhibited by MdpH.


Table2.20.

Hom

ologousgenes

ofthe

putativebiosynthetic

geneclusters

ofdifferent

dimeric

xanthonenatural

productsfrom

A.

aculeatus,P.

oxalcium,

C.

purpurea,A

.fischeri,

A.

lentulus,and

M.

fructicolaand

theirB

ioProject/assembly

identifier.A

.aculeatusP.oxalcium

C.purpurea

A.fi

scheriA

.lentulusM

.fructicolaPu

tativefu

nction

EaaA(X

P_020053312)EpoA

(EPS34273)EcpA

(CC

E31584)N

afA(X

P_001266579)N

alA(G

AQ

08560)M

frAN

R-PK

SEaaB

(XP_020053311)

EpoB(EPS34274)

EcpB(C

CE31583)

NafB

(XP_001266578)

NalB

(GA

Q08561)

MfrB

Mβ

LEcpC

(CC

E31582)N

afC(X

P_001266577)N

alC(G

AQ

08562)M

frCem

odinanthrone

oxidaseEaaD

(XP_020053317)

EpoD(EPS34268)

EcpD(C

CE31581)

NafD

(XP_001266576)

NalD

(GA

Q08563)

MfrD

dehydratase/decarboxylase

EcpE(C

CE31580)

MfrE

transcriptionfactor

EaaF(X

P_020053305)EpoF

(EPS34280)EcpF

(CC

E31579)N

afF(X

P_001266573)N

alF(G

AQ

08566)M

frFM

dpAhom

ologueEcpG

(CC

E31578)FM

OEaaH

(XP_020053315)

EpoH(EPS34270)

EcpH(C

CE31577)

NafH

(XP_001266563)

NalH

(GA

Q08576)

MfrH

emodin

reductaseEaaI

(XP_020053310)

EpoI(EPS34275)

EcpI(C

CE31576)

NafI

(XP_001266571)

NalI

(GA

Q08568)

MfrI

emodin

hydroquinonereductase

EaaJ(X

P_020053313)EpoJ

(EPS34272)EcpJ

(CC

E31575)N

afJ(X

P_001266572)N

alJ(G

AQ

08567)M

frJM

dpCproduct

dehydrataseEaaK

(XP_020053316)

EpoK(EPS34269)

EcpK(C

CE31574)

NafK

(XP_001266575)

NalK

(GA

Q08564)

MfrK

put.BV

MO

EcpL(C

CE31573)

hypotheticalproteinEcpM

(CC

E31572)hypotheticalprotein

EaaN(X

P_020053307)EpoN

(EPS34278)EcpN

(CC

E31571)N

afN(X

P_001266574)N

alN(G

AQ

08565)M

frNO

-MT

EaaO(X

P_020053321)EpoO

(EPS34265)EcpO

(CC

E31570)N

afO(X

P_001266570)N

alO(G

AQ

08569)M

frOFA

D-binding

proteinEpoP

(EPS34264)EcpP

(CC

E31569)N

afP(X

P_001266567)N

alP(G

AQ

08572)M

FStransporter

EaaQ(X

P_020053322)EpoQ

(EPS34263)EcpQ

(CC

E31568)N

afQ(X

P_001266568)N

alQ(G

AQ

08571)transcription

factorEcpR

(CC


EaaS(X

P_020053319)EpoS

(EPS34266)EcpS

(CC

E31566)N

afS(X

P_001266565)N

alS(G

AQ

08574)cytochrom

eP450

EaaT(X

P_020053320)EcpT

(CC

E31565)N

afT(X

P_001266566)N

alT(G

AQ

08573)SD

REaaU

(XP_020053308)

EpoU(EPS34277)

EcpU(C

CE31564)

NafU

(XP_001266564)

NalU

(GA

Q08575)

MfrU

hypotheticalproteinEaaV

(XP_020053309)

EpoV(EPS34276)

NafV

(XP_001266562)

NTF2-like

proteinEaaW

(XP_020053306)

EpoW(EPS34279)

transcriptionfactor

EaaX(X

P_020053318)EpoX

(EPS34267)SD

REaaY

(XP_020053314)

HP20-like

chaperoneN

afZ(X

P_001266569)N

alZ(G

AQ

08570)hypotheticalprotein

Mfr1

halogenase

Thegenom

esequences

analyzedin

thisw

orkare

availableat

NC

BI:A.aculeatus

(PRJN

A374040), [160]

P.oxalcium(PR

JNA

60877), [249]C.purpurea

(PRJEA

76493), [250]A.fischeri(PR

JNA

18475), [251]A.lentulus

(PRJD

B4311), [252]

andM

.fructicola(SA

MN

03448619). [253]

Thegenom

eofM

.fructicolaw

asannotated

forthis

work.

Thegene

andprotein

sequencesare

includedin

theappendix.


The in silico identification of the biosynthetic gene cluster of the ergochromes in

C. purpurea was confirmed by a publication of Neubauer et al.[256] The central NR-PKS

encoding gene ecpA has been over-expressed via the insertion of an inducible promoter

and knocked-out to verify the predicted function of the gene product.[256] The cluster

shares the enzymes needed for the biosynthesis of emodin hydroquinone (88) with

the characterized monodictyphenone (15) gene cluster from A. nidulans (Table 2.21)

and additionally it contains several genes coding for tailoring enzymes. These include

a reductase that is likely responsible for the dearomatization of ring A and a FMO

that probably catalyzes the para-hydroxylation of ring A. In agreement with other

biosynthetic pathways, the final step of tetrahydroxanthone dimer biosynthesis is the

dimerization (subsection 2.2.4).


Pathway hypothesis

O

OOH

OHOOH

OHO

OH

OH

COOMe

MeOOC

O

OOO

O

O

O

O

O OH

Cl

OH

O OHO O

HO

OH O

O

O

HO

O

OH

HO

HO

cladofulvin (47)

HOOH O

OH

OHO

secalonic acid A (8)trypacidin (90)

pestheic acid (89) monodictyphenone (15)

Figure 2.35. Emodin (83)-derived natural products of characterizedbiosynthetic gene clusters; trypacidin (90) from Aspergillus fumigatus, secalonicacid A (8) from C. purpurea, pestheic acid (89) from Pestalotiopsis fici,monodictyphenone (15) from Aspergillus nidulans, and cladofulvin (47) fromC. cladosporioides.[240,250,257–259]

The reaction sequence leading to emodin (83) is generally agreed upon and supported

by heterologous expression of the involved genes, labeling and knock-out studies. The

reductase MdpC from A. nidulans was demonstrated to convert emodin hydroquinone

(88) to (R)-3,8-dihydroxy-6-methyl-3,4-dihydroanthracene-1,9,10(2H)-trione

(10).[20,190] Emodin (83) is not accepted as a substrate. The same was demonstrated

for the conversion of versicolorin A hydroquinone to an analogue of 10 in aflatoxin B1

(20) biosynthesis.[20] Therefore, an unidentified enzyme might be responsible for the

reduction of emodin (83) to emodin hydroquinone (88).

Knock-outs of mdpC, mdpJ, mdpK, and mdpL, from the biosynthetic gene cluster of

monodictyphenone (15) and tajixanthone (12), yielded mutants deficient in production


S

O

CoA

HO S

OO

CoA7

OH OH O

OH

S

O

HO

Enzyme

HO

OH OH O

OH

OH

O

CO2

OOHOH

HO OH

OOH OH

HOHO

OH O

O

OH

NR-PKS MßL

atrochrysonecarboxylic acid [78]

DUF1772

DUF1772

emodin anthrone

oxidase

H2O

[O]

H2O

emodin [83] atrochrysone [86]emodin anthrone [85]

MdpG MdpF

MdpH - ORF2

MdpH - ORF2MdpH - ORF1

Scheme 2.15. Biosynthesis of xanthones: Proposed biosynthetic pathway to theintermediate emodin (83).

of monodictyphenone (15) and tajixanthone (12).[240,241] Chrysophanol (11) was

detected as a main shunt product for the knock-out mutant of mdpL.[241] This gene

is proposed to encode a Baeyer–Villiger monooxygenase (BVMO). Knock-outs of the

three other genes accumulate emodin (83) as their main shunt product.[241] The

accumulation of this anthraquinone shunt product can be explained by the inability of

the ∆mdpC mutants to convert emodin hydroquinone (88) to the secondary alcohol

10. The hydroquinone easily re-oxidizes to emodin (83) in the presence of O2. An

SDR sequence motif was identified in the sequence of MdpK by CDD analysis. MdpJ

has been annotated as a glutathione-S-transferase. As ∆mdpJ and ∆mdpK mutants

accumulate emodin (83), the encoded enzymes will catalyze reactions prior to the BVO

of the quinone ring.

The biosynthetic gene cluster of cladofulvin (47) from Cladosporium fulvum has

been characterized via knock-outs.[258] On replacement of the CYP encoding gene


claM, the production of cladofulvin (47) was stopped and a monomeric anthraquinone

precursor was detected.[258] These anthraquinones do not carry a hydroxy function

on C6, similar to chrysophanol (11). The gene cluster responsible for the biosynthesis

of cladofulvin (47) contains homologues of MdpG, MdpF, MdpH, MdpK, MdpC, and

MdpB, as well as a homologue of the co-activator MdpA (Table 2.21, Scheme 2.15).

The identified enzyme combination (ClaG, ClaF, ClaH) allows the production of emodin

(83, Scheme 2.16). ClaK acts as a putative quinone reductase generating the substrate

for the reduction catalyzed by ClaC (Scheme 2.16). The example of cladofulvin

(47) biosynthesis demonstrates that ClaK or its homologue MdpK do not act on a

post-Baeyer–Villiger-substrate.

Homologues of the hitherto discussed enzymes have been identified in all

putative dimeric xanthone clusters. However, no homologues of the putative

glutathione-S-transferase MdpJ are present in the analyzed genomes. Comparing

the characterized biosynthetic gene cluster of prenylated xanthones in A. nidulans with

the characterized biosynthetic gene clusters of pestheic acid (89), cladofulvin (47),

and trypacidin that are responsible for the biosynthesis of natural products based on

emodin (83), shows that homologues of MdpG, MdpF, MdpH, MdpK, and MdpL are

present in all analyzed biosynthetic gene clusters of xanthones (Table 2.21, Figure 2.35),

while MdpJ is an enzyme unique for the biosynthesis of monodictyphenone (15). As

emodin hydroquinone (88) is a common intermediate of the analyzed biosyntheses, the

production of this intermediate is based on enzymes encoded in all genomes of emodin

hydroquinone (88)-based natural product producers. Thus, MdpJ is likely acting on a

post-emodin hydroquinone (88) intermediate in the biosynthesis of monodictyphenone

(15), making MdpK a good candidate to be responsible for the reduction of emodin

(83) to emodin hydroquinone (88).


S

O

CoA

HO S

OO

CoA7

OH OH O

OH

S

O

HO

Enzyme

HO

OH OH O

OH

OH

O

CO2

OOHOH

HO OH

OOH OH

HOHO

OH O

O

OH

atrochrysonecarboxylic acid (78)

H2O

[O]

H2O

emodin (83) atrochrysone (86)emodin anthrone (85)

ClaG ClaF

ClaH

ClaHClaH

ClaK

HO

OH OH

OH

OH

emodin hydroquinone (88)

ClaC

HO

O OH

OH

OH

10

HO

OH O

O

O

HO

O

OH

HO

HO

cladofulvin (47)

[O]

HO

O O

O

OH

ClaM

H2O

Scheme 2.16. Adapted hypothesis for the biosynthesis of cladofulvin (47) basedon the comparison of different biosynthetic gene clusters of xanthone naturalproducts.[258]

122 2 Results and DiscussionTable

2.21.H

omologous

proteinsofthe

putativebiosynthetic

geneclusters

ofergochromes

fromC.purpurea

andcharacterized

biosyntheticgene

clustersfrom

otherem

odin(83)-derived

naturalproducts.A

.nidulans [240]P.fi

ci [257]C

.fulvum[258]

A.fum

igatus [259]C

.purpurea[250]

Putative

fun

ctionm

onodictyphenone(15)

pestheicacid

(89)cladofulvin

(47)trypacidin

secalonicacid

A(8)

MdpA

(XP_657749)

ClaA

(Clafu1|184393)

TpcD(X

P_751378)EcpF

(CC

E31579)transcriptionalco-activator

MdpB

(C8V

Q71)

ClaB

(Clafu1|184392)

EcpJ(C

CE31575)

MdpC

productdehydratase

MdpC

(XP_657750)

ClaC

(Clafu1|184391)

EcpI(C

CE31576)

emodin

hydroquinonereductase

MdpD

(XP_657751)

EcpO(C

CE31570)

FAD

-bindingprotein

MdpE

(XP_657752)

transcriptionfactor

MdpF

(XP_657753)

PtaB(A

GO

59041)C

laF(C

lafu1|184396)TpcB

(XP_751376)

EcpB(C

CE31583)

Mβ

LM

dpG(X

P_657754)PtaA

(AG

O59040)

ClaG

(Clafu1|184395)

TpcC(X

P_751377)EcpA

(CC

E31584)N

R-PK

SM

dpH(C

8VQ

72)PtaD

(AG

O59044)

ClaH

(Clafu1|190728)

TpcK(X

P_751385)EcpD

(CC

E31581)em

odinanthrone

oxidase,dehydratase/decarboxylase

MdpI

(CB

F90094)A

MP-binding

proteinM

dpJ(C

8VQ

63)glutathione-S-transferase

MdpK

(C8V

Q62)

PtaF(A

GO

59035)C

laK(C

lafu1|184397)TpcG

(XP_751381)

EcpH(C

CE31577)

emodin

reductaseM

dpL(X

P_657756)PtaJ

(AG

O59039)

TpcI(X

P_751383)EcpK

(CC

E31574)put.

BVM

OPtaC

(AG

O59043)

TpcL(X

P_751386)EcpC

(CC

E31582)em

odinanthrone

oxidasePtaG

(AG

O59045)

EcpL(C

CE31573)

hypotheticalproteinEcpM

(CC


EcpU(C

CE31564)

hypotheticalproteinPtaH

(AG

O59036)

TpcM(X

P_751387)EcpN

(CC

E31571)m

ethyltransferasePtaI

(AG

O59038)

methyltransferase

PtaL(A

GO

59047)oxidoreductase

PtaK(A

GO

59052)TpcJ

(XP_751384)

MC

OPtaE

(AG

O59042)

MC

OPtaM

(AG

O59046)

FAD

-bindingprotein

ClaM

(Clafu1|184398)

EcpS(C

CE31566)

CYP

enzyme

ClaE

(Clafu1|184394)

transcriptionfactor

ClaN

(Clafu1|184399)

SDR

TpcE(X

P_751379)transcription

factorTpcA

(XP_751375)

O-M

TTpcF

(XP_751380)

glutathione-S-transferaseTpcH

(XP_751382)

O-M

TEcpE

(CC

E31580)transcription

factorEcpG

(CC

E31578)FM

OEcpP

(CC

E31569)M

FStransporter

EcpQ(C

CE31568)

transcriptionfactor

EcpR(C

CE31567)

hypotheticalprotein


Aside from the characterized biosynthetic gene cluster of monodictyphenone (15) in

A. nidulans, several other characterized gene clusters of emodin (83)-derived natural

products have been published.[256–259] The diphenyl ether pestheic acid (89) was

isolated from the endophytic fungus Pestalotiopsis fici CGMCC3.15140.[257] The gene

cluster responsible for the biosynthesis of pestheic acid (89) has been identified via

knock-outs of the NR-PKS encoding gene ptaA and the halogenase encoding gene

ptaM. The proposed biosynthetic pathway of pestheic acid (89) is similar to the

biosynthetic pathway of monodictyphenone (15) up to the intermediate emodin

hydroquinone (88). The cluster contains a NR-PKS PtaA, an external TE PtaB

(MβL), a dehydratase/decarboxylase PtaD, an anthrone oxidase PtaD, and an emodin

(83) reductase PtaF (Table 2.21). These enzymes produce the intermediate emodin

hydroquinone (88). This is the last intermediate shared by the biosynthetic pathways

of monodictyphenone (15), tetrahydroxanthone dimers, and pestheic acid (89).

The biosynthetic gene clusters of the grisans trypacidin (90) from A. fumigatus and

(+)-geodin (84) from A. terreus have been identified.[259,260] Both clusters contain

genes responsible for the biosynthesis of emodin hydroquinone (88). Similar to the

biosynthesis of pestheic acid (89) no MdpC-homologue is involved in the formation of

the BVO product, because the final product of the biosynthesis still contains the hydroxy

group at C3. Oxidative quinone ring cleavage in anthraquinones was demonstrated

using cell-free extracts of the (+)-geodin (84) producer A. terreus.[261] They observed

conversion of questin (91) to demethylsulochrin (92, Scheme 2.17). The activity was

NADPH-dependent and lost if the cell-free extract was fractionated. This suggests

the involvement of at least two enzymes in the oxidative ring cleavage. As the BVO

requires NADPH, it is likely that a reductase is the unknown enzyme. MdpC activity

has been reported to be dependent on emodin hydroquinone (88) formation.[20] The


hydroquinone has been generated chemically by the addition of Na2S2O4. As the

biosynthetic gene cluster of (+)-geodin (84) contains a MdpK homologue, but not a

MdpC homologue, the addition of NADPH likely drives the conversion of emodin (83)

to emodin hydroquinone (88).

HO

O O

O

OH OHO

HO

OHOHO

O

questin (91) demethylsulochrin (92)

[NADPH]

Scheme 2.17. NADPH-dependent conversion of questin (91) to demethylsulochrin(92).[261]

The hydroquinone generated by the putative emodin (83) reductase MdpK and

homologues serves as a substrate for MdpC and homologues. When mdpC is deleted

the mutants are not able to synthesize the highly reactive secondary alcohol 10. The

MdpC substrate emodin hydroquinone (88) is oxidized under aerobic conditions and

leads to accumulation of emodin (83).[241] The SDR MdpC stereoselectively converts

emodin hydroquinone (88) to the secondary alcohol 10.[20] This product is highly

reactive and easily decomposes to chrysophanol (11). Knock-outs of mdpC accumulated

chrysophanol (11), therefore MdpC is believed to create the possibly unstable substrate

of the putative BVMO MdpL.[241]

Homologues of the well characterized enzyme MdpC have been identified as part of

confirmed and putative biosynthetic gene clusters of aflatoxin, sterigmatocystin, and

anthraquinone-derived natural products.[20,262] These clusters include the biosynthetic

gene cluster of shamixanthone and tajixanthone (12), as well as the biosynthetic gene

clusters of cladofulvin (47) and neosartorin (9).[240,241,258] The biosynthetic routes

to these natural products share emodin (83), emodin hydroquinone (88), and 10 as


R1O

OR2 O OH

O

R1O

OH

OHOR2 OH

O

HO

OH

OH OH

O

OOR2

R1O

O

OH

OHO

HO O OO

O

Cl

HO

pestheic acid g89=

O

OO

O

OO

OH

Cl

Cl

O

O

O

O O

O

O

trypacidin g90=

O

OOH

OHOOH

OHO

OH

OH

COOMe

MeOOC

HO

OH O

O

O

HO

O

OH

HO

HO

cladofulvin g47=

secalonic acid g8=

O

OOHO O

8-hydroxy-1-methoxycarbonyl-6-methylxanthone

MdpK

MdpC

DUF4243

DUF4243

g+=-geodin g84=10

g107=

88 gR1=R2=H=83 gR1=R2=H=

Scheme 2.18. Emodin hydroquinone (88) as branching point in emodin(83)-derived natural products.


an intermediate. 10 is a versatile shared intermediate and represents an important

branching point (Scheme 2.19).

The product of the MdpC-catalyzed reduction of emodin hydroquinone (88)

is unstable and easily decomposes to chrysophanol (11).[263] Apart from the

re-aromatization via dehydration, dimerization of two molecules of 10 has been

observed.[263,264] The dimer (−)-flavoskyrin [(−)-93] has been identified via 1H NMR.

10 has been readily converted to 4,5-dihydroxy-2-methyl-9H-xanthen-9-one (94) as

well.[263,265]

2

3412

111

1013

149

56

78

OH

HO

O OH

O

15

A B C14

135

6

78911

12

12

3

4

OHO

OH

10O

O

OH

15

A

C

emodin (83) monodictyphenone (15)

Figure 2.36. Numbering of emodin (83) and the emodin (83) numberingtransferred to monodictyphenone (15).

The conversion of the secondary alcohol 10 to the xanthone 94 can be explained

with the mechanism presented in Scheme 2.20. 10 is oxidized to an epoxide by

atmospheric oxygen. We propose this epoxidation is formed via a mechanism similar to

vitamin K epoxidation. The rings B and C represent a vitamin K-like substructure.[263]

Subsequently, a BVO at the quinone ring B takes place. The nucleophilic attack of the

carbonyl takes place at C9, because the carbonyl is activated through a hydrogen bond

to the phenolic alcohol at C8. After hydrolysis of the formed ester, the alcohol at C14

attacks at C11 opening the epoxide. This leads to decarboxylation and triggers the


HO

OH O

O

OH

emodin 5839

HO

O OH

OH

OH

HO

O OH

OH

OH

CH3

H2O

OH

O

O

CH3

OH

emodin hydroquinone 5889

MdpC product 5109

chrysophanol 5119

OOHO

HO

OH

O OH

O OH

CH3

CH3

OH

O

O

COOMe

HO

O

MeOOC

OH O OH

H3C OH

O

OH

* P

neosartorin 589

O

OOHO O

CH3

8-hydroxy-1-methoxycarbonyl-6-methylxanthone

HO

OH O

O

O

HO CH3

O

OH

CH3

HO

HO

cladofulvin 5479

O

O

OHO

H3C

OH

tajixanthone 5129

flavoskyrin 5939

MdpC

MdpK

O

OH

O

OH

CH3OH

CO2

1,4,5-trihydroxy-2-methyl-9H-xanthen-9-one

51079 O 5959

O

Scheme 2.19. MdpC product as an intersection of emodin (83)-derived naturalproducts.


elimination of water. Additional water elimination and enolization of the keto group at

C1 re-aromatizes the ring C yielding the xanthone 94.

The conversion of 10 to 94 is not catalyzed by an enzyme and can be directed

by pH changes. High pH values favor the formation of 94, while lower pH values

give rise to other decomposition products like chrysophanol (11) and (−)-flavoskyrin

[(−)-93].[264,265] A similar epoxide-based mechanism for xanthone formation including

decarboxylation was proposed and investigated in xantholipin biosynthesis.[266]

Chrysophanol (11) is formed via dehydration, and (−)-flavoskyrin [(−)-93] via a

hetero-Diels–Alder reaction.[267,268] The bisanthraquinone (−)-flavoskyrin [(−)-93]

has been isolated from Penicillium islandicum.[269] However, the observation that 10

easily dimerizes forming (−)-flavoskyrin [(−)-93] casts doubt on the isolation of an

enzymatically synthesized compound. The dimer may have formed in the process of

natural product isolation.

Interestingly, putative emodin (83)-derived xanthones have been isolated from the

endophytic fungus Talaromyces islandicus EN-501 and the lichen Cladonia incrassata,

which show the same xanthone core structure as 94 (Figure 2.37).[270,271] Similar

to 94, the isolated natural products lost the carboxy group formed during BVO. This

O

OH OH

O

O

OH

O

OH

OH

4,5-dihydroxy- 1,4,5-trihydroxy-

O

O

Cl

Cl

Cl

OH

OH

2-methyl-9H-xanthen-9-one

2-methyl-9H-xanthen-9-one

cladoxanthone A (96)

(94) (95)

Figure 2.37. Unusual xanthones isolated from Talaromyces islandicus EN-501 andthe lichen Cladonia incrassata.[270,271]


O OH

HO

O

OH

H

O2 OHOO

O O OH

HO

H+

O

O

O

O O

HO

H

OO

O

OH

O

OHOO

O

OO

OHHO

OH

HO

O

O

HOO

O

HO

OH

HO

O

O

OHO

HO

CO2H2O

O

O

OHOH

H H

HH

[O]

+H2O

H2O

11

94

Scheme 2.20. Postulated mechanism for the formation of the xanthone 94 fromthe MdpC reduction product 10.[263,265]

group is retained in most emodin (83)-derived natural products.[248,272] The different

regiochemistry of the BVO of the observed xanthone (94) and the natural products

1,4,5-trihydroxy-2-methyl-9H-xanthen-9-one (95) and cladoxanthone A (96) compared

to xanthone natural products such as 4-chloropinselin (97), can be explained by

a hydrogen bond of the phenol in ring C with the C9-keto group (Scheme 2.21).

This activation is likely directed towards the C10-keto group by the enzyme in

tetrahydroxanthone and 4-chloropinselin (97) biosynthesis.

The xanthone sterigmatocystin has been isolated from Aspergillus versicolor and linked

to aflatoxin biosynthesis.[273–275] In contrast to xanthone substructures observed in

ergochrome dimers, sterigmatocystin does not contain the carboxylic acid group formed


O

OH

O

HO

OH

OH

OH

O

HO

OH

H

BVO with

enzymatic control

BVO without

enzymatic controlO

OH OH

OH2O

H2O

10

10

94

9C

HO

OHO O

Cl

O

O

4-chloropinselin (97)

Scheme 2.21. Different regiochemistry of a BVO with the secondary alcohol 10 assubstrate.

in the BVO. Therefore, this xanthone substructure could be synthesized following a

similar mechanism proposed for the formation of the chemically formed xanthone 94.

MdpL, the enzyme proposed to catalyze this oxidative opening of the quinone

ring, does not contain a characterized sequence motif. The conserved DUF4243

was identified in the sequence of MdpL by CDD analysis. This domain is found in

many enzymes. from fungi and bacteria in NCBI’s non-redundant protein database.

Because a gene deletion of mdpL eliminated xanthone production completely and led

to an accumulation of chrysophanol (11), MdpL likely is responsible for the oxidative

quinone ring cleavage.[240] In knock-outs of mdpL the substrate of MdpC is generated

and converted to 10. This compound easily decomposes to chrysophanol (11).[20] The

accumulation of the stable chrysophanol (11) in mdpL knock-out mutants suggests 10

as MdpL’s substrate.

The proposed biosynthetic pathway describing chrysophanol (11) as a shunt product

is challenged by experiments showing the incorporation of [2H3]-chrysophanol (11) in


tajixanthone (12).[10] The putative shunt product chrysophanol (11) has also been also

incorporated in secalonic acid D (13).[11] An enzymatically catalyzed hydroxylation

in meta-position to the remaining hydroxy-group is unlikely. So incorporation of

chrysophanol (11) cannot be explained by a hydroxylation generating emodin (83). In

a labeling study the competitive incorporation of chrysophanol (11) and emodin (83)

has been determined. The label derived from the incorporation of chrysophanol (11)

was three times stronger than the emodin (83)-derived label.[11]

The conversion of dantron (98) added to cultures of Streptomyces aureofaciens

to 1,10-dihydroxydibenzo[b,e]oxepine-6,11-dione (99, Scheme 2.22) has been

reported.[276,277] Conversions with the emodin anthrone (85) oxygenase GedH from

A. terreus showed more product formation with chrysophanol anthrone as substrate,

compared to emodin anthrone (85).[17] Thus, chrysophanol (11) has to be considered

as a putative intermediate of anthraquinone-based biosynthetic pathways, rather than

rejecting it as a dead end shunt product.

Studies on the tautomerization of different anthraquinones show that a 1,10-quinone

tautomer of chrysophanol (11) was detected in published UV/Vis spectra of the

compound.[278] Excited state intramolecular proton transfer (ESIPT) has been

confirmed to take place in 1,8-dihydroxyanthraquinone upon excitation with a

O

O

OHOH

O

O

O

OHOH

S. aureofaciens

dantron (98) 1,10-dihydroxydibenzo

[b,e]oxepine-6,11-dione(99)

Scheme 2.22. Conversion of dantron (98) by S. aureofaciens.[276,277]


laser (λ 406.7 nm, 413.1 nm, 457.9 nm) and measuring fluorescence and Raman

scattering.[279] If molecules are photoexcited, some molecules can relax to the

ground state by tautomerization described as ESIPT. The existence of such a

tautomer would explain the different migration tendencies of ring A and C observed

in the biosynthesis of different natural products, e.g., neosartorin (9). The

tautomer 8,9-dihydroxy-3-methylanthracene-1,10-dione (100) favors a BVO at C10

(Scheme 2.23). Additionally, the aromaticity of ring A explains its migration during the

oxidation. The product of a BVO on this tautomer yields monodictyphenone-ε-lactone

(101). If the tautomer 8,9-dihydroxy-6-methylanthracene-1,10-dione (102) is

converted in a BVO at C10, ring C migrates due to its aromaticity. The product

is janthinone-ε-lactone (103). Janthinone (14) has been isolated from Penicillium

janthinellum together with emodin (83).[280]

The pathway hypothesis accounts for the observed incorporation of labeled

chrysophanol (11) in chloromonilicin (104), tajixanthone (12), and secalonic acid

D (13).[10,11,281] It also explains the missing active site of the putative BVMOs that

do not catalyze a reaction, but stabilize a vulnerable tautomer of an otherwise stable

intermediate. The two different tautomers formed by chrysophanol (11) yield BVO

products with different regiochemistries, which is reflected in the natural product pair

monodictyphenone (15) and janthinone (14).

The tautomerization of hydroquinoid substructures are likely also responsible for the

formation of the emodin (83)-derived xanthone chloromonilicin (104) isolated from

M. fructicola.[282] A variety of precursors of chloromonilicin (104) was isolated from

the same fungus.[283] A putative reaction sequence would nominate 4-chloropinselin

(97) as precursor of chloromonilicin (104, Scheme 2.24).


O

O

HO

OHOH OH

OH

OH

HO

OH

HO

O OH

OH

OH

OHO

OH

OH

HO

OO

O

OH

HO

OOH OH

O

O O

OHOHOHOH OO

OO

OOH

OO

OOHOH

OH

emodin D83U emodin hydroquinone D88U

10chrysophanol D11U

monodictyphenone ε-lactone D101Ujanthinone ε-lactone D103U

MdpK

MdpC

[O]

H2O

[O] [O]

DUF4243

DUF4243

100102

Schätzle et al. 2012Conradt et al. 2015Saha et al. 2018

hypothesis in this work

DMdpLU

DMdpLU

Scheme 2.23. Proposed biosynthetic pathway leading from emodin (83) to lactonederivatives of monodictyphenone (101) and janthinone (103).


HO

OHO O

Cl

O

O

O

OHO O

Cl

O

O

chloromonilicin (104)

OH

Cl

O

OOO

O

O

[O]

4-chloropinselin (97)

Scheme 2.24. Putative scheme for the biosynthetic conversion of 4-chloropinselin(97) to chloromonilicin (104) based on tautomerization of 4-chloropinselin(97).[283,284]

Labeling studies with deuterated chrysophanol (11) showed incorporation of the

labeled anthraquinone in janthinone (14).[284] Feeding of deuterated janthinone

methyl ester (D-105) to cultures of M. fructicola led to the isolation of deuterated

chloromonilicin (104), confirming janthinone methyl ester (105) and chrysophanol

(11) as pathway intermediates.[281]

However, the pathway hypothesis does not account for the diol structures on ring C

of ergochromes DD (106, Figure 2.38, d), CC, AC, AD, BC, BD, CD, and ergoxanthin.

Other ergochromes such as secalonic acid D (13) carry an enol function in ring C,

instead of the anti-diol (Figure 2.38, d). This could be explained by an anti-elimination

of water in the enol-ergochromes, which is only possible if ring C contains a syn-diol

(Figure 2.38, a). The diol may be formed by the opening of an epoxide that arose from

a vitamin K like epoxidation described above for the formation of the xanthone 94

(Figure 2.38, c). Alternatively, the diol could be a product of a selective hydroxylation

or an addition of water to the enol double bond (Figure 2.38, b). The diol is only

described for ergochromes isolated from C. purpurea and diversonol from Penicillium

diversum.[248,272] Other tetrahydroxanthones contain an enol function in ring C.


O

OHOOH

O

OHOOH

OHCOOMe

COOMeOH

OH

OH

H2O

H2OO

OOH

OHCOOMe

OH

O

OOH

OHCOOMe

OH

a)

O

OOH

OHCOOMe

OH

O

OHO

OHCOOMe

OH

O

OHO

OHCOOMe

OHOH

OH

b)

H2O

H2O

OH

O

OH

COOMeHO

O OH

OH

O

OH

COOMeHO

O OH

O

OHOOH

OHCOOMe

OH

O

OHOOH

OHCOOMe

OH

H2O

H2OO

OOH

OHCOOMe

OH

O

OOH

OHCOOMe

OH

c)

O

O

O

O

OH

OH OHO

OH O

OH O OH

OH O OH

OH

OH

COOMeOH

OHMeOOC

COOMe

MeOOC

OH

OH

secalonic acid D (13) ergochrome DD (106)

d)

Figure 2.38. Possible origins of the anti-diol function of ergochromes. A)anti-Elimination only takes place in syn-diols. B) Addition of water to theenol-double bond leads to the formation of an anti-diol. C) Previously formedepoxide is opened upon ring closure leading to the formation of and anti-diol. Theanti-diols colored in red, the enol possibly formed from a syn-diol is colored in blue.The methyl group in ring A and the carboxy methyl ester group are configured antiin all ergochromes.


O OOH

OH

O

HO

OHOH

OHO

OHOH

O

O

HO

OHOH

OHO

OOH

OH

OHO

O OH

O

O

HO

OHO

OO

OOH

O

OHO

O O

O O

O COOMe

OH O OH OH O

MeOOC O

OH

OOH

OH

OO

OH

O

O

HO

OHOH

O

janthinone (14) monodictyphenone (15)

FMO FMO

[O] [O]

MT MT

janthinone methyl ester (105)

Scheme 2.25. Proposed biosyntheticpathway from janthinone (14) andmonodictyphenone (15) to xanthones.

After BVO the product’s

intramolecular ester is hydrolyzed

and the carboxy function is methylated.

The compound is then hydroxylated

at C5 or C4 of the ring carrying

the carboxy methyl ester function

(numbering from Figure 2.36). The

formed hydroquinone is oxidized to

the quinone enabling an attack of the

phenolic hydroxy group at C12 or C13

of the ring without carboxy methyl ester

function (Scheme 2.25). After xanthone

formation the ring carrying the carboxy

methyl ester function is reduced twice

to yield a tetrahydroxanthone core

structure. Depending on the starting

substrate ring A from emodin is aromatic

[monodictyphenone (15)], or ring C

retains its aromaticity [janthinone

(14)].

The dimerization of the

tetrahydroxanthones is the proposed

final step of the biosynthesis. Two

molecules of tetrahydroxanthone

monomers are coupled via oxidative phenol coupling of their aromatic rings.


O

O

OOH OH

OH O OH

COOMe MeOOC OH

OH

O

HO

O

OH O OH

O

COOMe

OH

HO

MeOOC OH

O

O

OH

OH O

OOH

OH

MeOOC OH

COOMeOH

4,4'-secalonic acid A (4,4'-8)

penicillixanthone A (36)


Scheme 2.26. Regioisomerization of the tetrahydroxanthone dimer secalonic acidA (8).[72]

Homodimers coupled on their ring A have been described, as well as compounds

coupled on their ring C. The former do not show atropisomerism, because the rotation

of the biaryl bond is not sufficiently hindered. Additionally, it was demonstrated

for secalonic acid A (8) that the dimer regioisomerizes within hours in DMSO at

room temperature (Scheme 2.26).[72] A keto-enol-tautomerization opens the central

xanthone ring and allows rotation of the non-aromatic ring (Scheme 2.26). The ring is

closed by an attack of the formerly free phenolic oxygen of the aromatic ring. The

instability of the central ring leads to the isomerization of 4,4′-linked secalonic acid A

(4,4′-8) to 2,2′-linked secalonic acid A (8). Therefore, natural products that have been

mainly isolated as 2,2′-coupled dimers, could have been synthesized with a different

regiochemistry and isomerization during isolation has lead to the formation of the

more stable 2,2′-regioisomer. However, compounds such as ergochrome DD (106) do


not contain the enol function preventing the regioisomerization of the dimer. These

compounds likely have been synthesized with a 2,2′-linkage.

In contrast to homodimers coupled with their ring A, homodimers coupled on

their ring C exhibit atropisomerism.[285] The same holds true for heterodimers,

such as neosartorin (9).[286] These compounds are sterically hindered sufficiently

to inhibit isomerization of the atropisomers. Even though, the compounds are able

to regioisomerize as well, the stereochemistry of the biaryl linkage will not change in

this process. Therefore, these dimers are coupled in a stereoselective manner by the

producing organism.

The identified putative biosynthetic gene clusters of dimeric xanthones in A. aculeatus,

A. fischeri, A. lentulus, C. purpurea, and P. oxalcium contain two genes coding for

enzymes that have been shown to catalyze oxidative phenol coupling. The first

candidate gene is coding for a FMO. In the case of marinopyrroles’ (49, 50, 51 and

52) biosynthesis, a FMO catalyzes the N,C-bipyrrole homocoupling.[7] The genes eaaO,

nafO, nalO, ecpO, and epoO encode FAD-binding enzymes that are homologues of

MdpD from the biosynthetic gene cluster of monodictyphenone (15) from A. nidulans.

Knock-out mutants of mdpD have been shown to produce monodictyphenone (15),

but not prenylated xanthones or hydroxylated monodictyphenone derivatives.[241]

The hydroxylation that is probably catalyzed by MdpD takes place on ring C in

ortho-position to the methyl and carboxy group. Homologues of MdpD likely catalyze

a similar reaction, in this case para-hydroxylation of the carboxy group bearing ring

in ortho-position to the carboxy group (Scheme 2.25). This hydroxylation creates a

hydroquinone that is easily oxidized to the corresponding quinone enabling xanthone

ring formation via an oxa-Michael addition. Therefore, the FAD-binding enzymes are


probably not the responsible enzymes for biaryl coupling in the biosynthesis of dimeric

tetrahydroxanthones.

The second candidate gene is coding for a CYP. These enzymes are known to catalyze

oxidative phenol coupling in the biosynthesis of kotanin (2) in A. niger.[8] The enzyme

KtnC controls the regio- and stereoselectivity of the phenol coupling step to kotanin

(2).[9] However, the enzymes encoded by the genes eaaS, nafS, nalS, ecpS, and epoS do

not show homology to KtnC. Instead, they are related to the CYP enzyme ClaM, which

is responsible for the phenol coupling step in the biosynthesis of cladofulvin (47).[258]

The CYP enzyme has been proposed to couple two anthraquinone moieties, which are

structurally more similar to the anthraquinone-derived xanthone monomers.[258]

The plant pathogen M. fructicola produces the monomeric xanthone

8-hydroxy-1-methoxycarbonyl-6-methylxanthone (107, Scheme 2.18, Scheme 2.19),

4-chloropinselin (97, Scheme 2.24), and chloromonicilin (104, Scheme 2.24).[282,283]

The published genome of M. fructicola (assembly ID: ASM216254v1) was annotated

using the AUGUSTUS tool in Galaxy to identify the putative biosynthetic gene cluster of

chloromonicilin (104).[253] An iPKS containing gene cluster homologous to previously

predicted xanthone gene clusters was identified in the genome of M. fructicola. This

gene cluster contains a gene coding for a MdpD homologue, but does not contain a CYP

enzyme homologue. The xanthone 107 is hydroxylated in ortho-position to the carboxy

methyl group in the biosynthesis of chloromonicilin (104), likely by the FMO encoded

by mfrO (Table 2.20). Therefore, the CYP enzymes EaaS, NafS, NalS, EcpS, and EpoS

in the identified gene clusters from A. aculeatus, A. fischeri, A. lentulus, C. purpurea,

and P. oxalcium are good candidates to take up the role of coupling enzymes in dimeric

tetrahydroxanthone biosynthesis.


Table 2.22. Sequence comparison of characterized phenol coupling enzymes fromthe biosynthesis of bicoumarins, cladofulvin (47), and the CYP enzymes from theputative biosynthetic gene clusters of dimeric tetrahydroxanthones. The sequenceidentity of the amino acid sequences is shown [%].

CYP enzymes NafS NalS EaaS EpoS EcpS ClaM KavC KtnC

NalS 95.1EaaS 48.4 47.9EpoS 46.8 46.2 86.9EcpS 39.3 39.4 47.7 46.4ClaM 39.2 39.6 36.5 35.6 32.2KavC 10.6 10.2 13.3 13.0 10.8 11.7KtnC 10.6 10.2 12.7 12.5 10.8 11.9 83.6DesC 10.0 9.8 10.6 10.1 9.7 9.3 45.5 44.3

NalS from A. lentulus (GAQ08574), EaaS from A. aculeatus (XP_020053319),EpoS from P. oxalcium (EPS64277), EcpS from C. purpurea (CCE31566),ClaM from C. fulvum (Clafu1|184398), KtnC from A. niger (XP_001402310),DesC from Emericella desertorum (A0A0N9HKQ7).

Mutants of the tetrahydroxanthone dimer producer C. purpurea that overexpress

a transcription factor of the putative biosynthetic gene cluster of ergochromes have

been created.[256] The mutants have been analyzed with quantitative real time PCR

(qRT-PCR) to identify genes, whose expression is controlled by the transcription

factor. Most of the cluster genes are co-expressed with the exception of the CYP

enzyme encoding gene ecpS (locus tag CPUR_05419), which is proposed as coupling

enzyme in this work. However, no expression of ecpS has been detected, while the

fungus produced dimeric ergochromes.[256] The missing transcription while dimer

production was detected casts doubt on their putative role as phenol coupling enzymes

in the biosynthesis of dimeric tetrahydroxanthones. Nevertheless, the identification

of the CYP enzyme ClaM (C. cladosporioides) as the coupling enzyme in cladofulvin

(47) biosynthesis and SKY_4059 (VA03HOR0624_4059), a ClaM-homologue from


Cyanodermella asteris supposedly involved in the biosynthesis of skyrin, show that

this enzyme class is responsible for the dimerization step in dimeric anthraquinone

biosynthesis.[287]

Integrating the new hypothesis for the BVO and the oxidative phenol coupling

into the biosynthetic pathway of tetrahydroxanthone dimers, leads to the following

proposed reaction sequence (Scheme 2.27). The central NR-PKS produces a heptaketide

from an acetate starter molecule and six malonate extender units. The polyketide is

cyclized by the PT domain of the NR-PKS enzyme and cleaved off the enzyme by

an external thioesterase (MβL). The NR-PKS product atrochrysonecarboxylic acid

(78) is decarboxylated by an EthD-like enzyme to yield atrochrysone (86), which is

subsequently dehydrated to emodin anthrone (85). The DUF1772 containing emodin

anthrone (85) oxidase converts emodin anthrone (85) to emodin (83). The quinone

is reduced by an emodin quinone reductase (e.g., MdpK) to provide MdpC with its

substrate emdoin hydroquinone (88). The SDR MdpC reduces a tautomer of emodin

hydroquinone (88) to the secondary alcohol 10, a reactive intermediate that easily

decomposes to the quinone chrysophanol (11), the anthraquinone dimer flavoskyrin

(93), or the xanthone 94.

The new pathway hypothesis is based on the assumed tautomerization of

chrysophanol (11) to provide the substrate for the subsequent BVO. A similar

tautomerization is the basis for MdpC activity. According to the proposed

chrysophanol (11)-based pathway two different 1,10-quinone tautomers of the

1,8-dihydroxyanthraquinone yield two different products (Scheme 2.23). The respective

tautomer directs the BVO to occur at C10 and the aromaticity of the ring substituents

directs the migration of the involved substituents. The 3-methyl-1,10-quinone


S

O

CoA

HO S

OO

CoA7

OH OH O

OH

S

O

HO

Enzyme

HO

OH OH O

OH

OH

O

CO4

OOHOH

HO OH

OOH OH

HOHO

OH O

O

OH

atrochrysonecarboxylic acid F781

H4O

[O]

H4O

emodin F831 atrochrysone F861emodin anthrone F851

NR3PKS ML

EthD3like

EthD3likeDUF,774

NADFP1H3binding

HO

OH OH

OH

OH

emodin hydroquinone F881

HO

O OH

OH

OH

10

SDR

OH

O

O

CH5

OH

chrysophanol F111

OOHO

HO

OH

O OH

O OH

CH5

CH5

OH

flavoskyrin F931

O

OH

O

OH

CH5OH

,u9u53trihydroxy343methyl39H3xanthen393one

F951

[O]

H4O

enzyme

protein

enzyme

FMdpC3like1

Scheme 2.27. Proposed biosynthetic pathway for tetrahydroxanthones up to theformation of the secondary alcohol 10 and the decomposition products of 10.


tautomer of chrysophanol (11) is converted to monodictyphenone (15). The

6-methyl-1,10-quinone tautomer yields the regioisomer janthinone (14).

Following the BVO and hydrolysis of the formed ester, the carboxy group is

converted to a methyl ester and the ring bearing the carboxy group is hydroxylated

in ortho-position to the carboxy methyl ester moiety. The formed hydroquinone is

oxidized to the corresponding quinone, enabling xanthone ring formation via an

oxa-Michael addition (Scheme 2.25). The xanthone is then reduced twice to yield a

tetrahydroxanthone that is likely coupled by a CYP enzyme to a xanthone dimer such

as neosartorin (9, Scheme 2.28).


H2O

[O]

OOH OH

Ochrysophanol (11)

OO

O

OH

HO

OHO

OH

O O

OOH

OO

OOHOH

OH

OH

monodictyphenone -lactone (100) janthinone -lactone (102)

HO

DUF4243

10

H2OOOH

OH

OHO

OH

monodictyphenone (15)

OOH

OH

OO

OH

MT

monodictyphenone

FMO

OOH

OH

OHO

O OH

[O]

OOH

O

OHO

O O

O

OH O

MeOOC O

OH

O

OH O

MeOOC OH

OH

HO

O

HO

OH O OH

COOMe

OH

OH

MeOOC

O

O

penicillixanthone A (36)

CYP [H]2

methyl ester

Scheme 2.28. Proposed biosynthetic pathway for tetrahydroxanthones startingfrom the secondary alcohol 10.


2.4.3 PHAR

The filamentous fungus Curvularia lunata (teleomorph Cochliobolus

lunatus) has been reported to produce the anthraquinone

1,3,8-trihydroxy-6-methoxyanthracene-9,10-dione (108) and the anthraquinone dimer

cytoskyrin A (109).[288] Because of its 17β -hydroxysteroid dehydrogenase activity,

the fungus has been thoroughly studied. After this enzymatic activity has first been

observed, the enzyme responsible for the conversion of steroids (e.g., estrone) has

been isolated, crystallized, and characterized.[289–292] The supposed physiological role

of the isolated 17-β -hydroxysteroid dehydrogenase from C. lunata (17bHSDcl) has

been investigated.[293] Phylogenetic analysis showed that 17bHSDcl is related to the

oxidoreductase Ver-1 from aflatoxin biosynthesis.[294] It has been also proposed that

17bHSDcl is involved in melanin or mycotoxin biosynthesis.[292]

The SDR MdpC from A. nidulans has been shown to catalyze the reduction of

emodin hydroquinone (88) to the secondary alcohol 10.[20] A similar activity has

been described for the reductase AflM (Ver-1) with the hydroquinone of versicolorin

A.[20] As previously reported, 17bHSDcl is related to AflM and therefore related

to MdpC as well.[293] The genomes of two C. lunata strains have been sequenced,

O

O

OH OH

HO O

O

HO

OH

O

OHO

OHO

O

OH

O OH

cytoskyrin A (109)1,3,8-trihydroxy-6-methoxy-

anthracene-9,10-dione (108)

Figure 2.39. Anthraquinone natural products isolated from C. lunata.[288]


annotated and published to NCBI [C. lunata CX-3 (BioProject PRJNA182303), C. lunata

m118 (BioProject PRJNA207848)].[295,296] As previous work on 17bHSDcl has been

performed on C. lunata m118, this strain was chosen for genome analysis and genes

adjacent to 17bHSDcl were analyzed to establish a biosynthetic hypothesis.

Table 2.23. Genes of the 17bHSDcl containing gene cluster from C. lunatam118.[262]

Enzyme Putative function Homologous enzymes

CluA NR-PKS MdpGCluB MβL MdpFCluC dehydratase/decarboxylaseCluD dehydratase/decarboxylaseCluE PHAR-product dehydratase MdpBCluF emodin reductase MdpKPHAR (17bHSDcl) emodin hydroquinone reductase MdpCCluH transporterCluI transcription factorCluJ emodin anthrone oxidase MdpH

The gene encoding 17bHSDcl is surrounded by genes involved in the biosynthesis

of emodin (83) and chrysophanol (11).[262] The NR-PKS CluA of the identified gene

cluster does not contain a TE domain and relies on an external TE (CluB) for product

release. This is typical for NR-PKS enzymes with a PT domain of family V, such

as CluA.[297] The enzymes CluC and CluD likely catalyze the decarboxylation and

dehydration of CluA’s product. As homologues of the clusters enzymes are involved in

the biosynthesis of emodin (83) and chrysophanol (11), the product of CluA probably

is atrochrysonecarboxylic acid (78). The decarboxylation and dehydration would

then yield emodin anthrone (85). The emodin anthrone oxidase CluJ converts the

anthrone to emodin (83), which is reduced to emodin hydroquinone (88) by CluF. The

MdpC-homologue 17bHSDcl is proposed to reduce emodin hydroquinone (88) to the


secondary alcohol 10. The reactive intermediate is finally dehydrated by CluE to yield

chrysophanol (11).

This pathway hypothesis was confirmed by conversions of emodin hydroquinone (88)

carried out with heterologously in E. coli produced 17bHSDcl. The hydroquinone was

converted to the secondary alcohol 10 also described as a product of MdpC-catalyzed

reduction of emodin hydroquinone (88).[262]Because of the genetic environment of

17bHSDcl and the observed reductive activity towards a substrate, whose biosynthesis

could be carried out by the surrounding genes, it is proposed to more appropriately

refer to 17bHSDcl as polyhydroxyanthracene reductase (PHAR).[262] The isolation

of emodin (83) or chrysophanol (88) has not yet been reported, instead the

anthraquinone derivatives 108 and cytoskyrin A (109) have been described as products

of C. lunata.[288] The anthraquinones do not have an emodin (83)-derived carbon

skeleton, as the methyl group at C6 is replaced by a hydroxy or methoxy group.

O

O

O

O

OH

OH

O

OOH

HO R

OH

M *

(110), R=OH

(111), R=OCH3

Figure 2.40. Anthraquinonedimers isolated from Bulgariainquinans.[298]

Without a biosynthetic hypothesis for the

monomer 108 it is difficult to identify a putative

biosynthetic gene cluster in the genome of

C. lunata m118. Additionally, the C. lunata strain

used for the isolation of the anthraquinone natural

products is not specified in the corresponding

publication. Thus, it is possible that the strains,

whose genome is sequenced and published, are

not able to produce these compounds. The

identification of a biosynthetic gene cluster of

cytoskyrin A (109) might be aided by the

analysis of the genome of the ascomycete Bulgaria


inquinans. The fungus has been reported to produce the anthraquinone dimers 110

and 111 (Figure 2.40) likely sharing the monomeric precursor 108 with cytoskyrin A

(109).[298]

Even though no candidate gene cluster for the biosynthesis of cytoskyrin A (109)

was identified, it is proposed that an enzyme similar to PHAR and MdpC is involved

in its biosynthesis. The observation of dimerization of 10 to (−)-flavoskyrin [(−)-93]

shows that a reactive intermediate could be used to generate a dimer similar to

cytoskyrin A (109).[264] A semi-synthetic approach using PHAR to generate the reactive

intermediate 10 has led to the synthesis of (−)-rugulosin and (−)-2,2’-epi-cytoskyrin A

[(−)-2,2’-epi-109].[264]

3 Concluding remarks and outlook

Contents

1.1 Polyketides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2 Structural diversity of polyketide dimers . . . . . . . . . . . . . . . . . . . . 101.3 Biosynthesis of the natural product’s backbone: PKS . . . . . . . . . . . . . . 211.4 Dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

In this work, the genomes of the ascomycetes T. rubrum, P. citreonigrum, A. ochraceus,

A. viridinutans, C. purpurea, A. aculeatus, A. fischeri, A. lentulus, P. oxalcium, and

M. fructicola were analyzed focusing on the production of secondary metabolites of

interest. PKS-containing clusters for the biosynthesis of vioxanthin (3), viriditoxin

(5), ergochromes, lentulins, chloromonilicin (104) were identified and, together with

previous and current experimental results, the encoded enzymes were linked to the

corresponding steps of the pathway.

Of particular interest was the characterization of the laccase VavH from

A. viridinutans. This enzyme was identified in the putative biosynthetic gene cluster

of viriditoxin (5). It was shown to catalyze the oxidative phenol coupling of

the non-native monomer (R)-semi-vioxanthin (4) exclusively to a 6,6′-linked biaryl.

Laccases generate radicals that can undergo a variety of subsequent reactions.[299–301]

150 3 Concluding remarks and outlook

OH

OH

OHOH

OH

OH

OH

2ortho-ortho (11.3 d)

ortho-para (36.1 d)para-para (43.6 d)

1-naphthol (80) -2 e-

-2 H+

2

O

-2 e-

-2 H+

CH3

2-methoxynaphthalene (81)

OCH3

OCH3

ortho-ortho (96.6 d)

O

OH

O

OH O

RH3C

O

R

OOHOH

OH3C

pigmentosin A (24), R=CH3

M *

O

Oviriditoxin (5), R=

Scheme 3.1. Electrochemically catalyzed phenol coupling of 1-naphthol (80) and2-methoxynaphthalene (81).[211]

Often, the laccase does not control the fate of the radical intermediates, and a mixture

of different oxidation products (e.g., quinones, hydroxylderivatives, oligomers) is

thus formed.[118,302,303] This is the case for the conversion of 1-naphthol (80) with

a fungal laccase from Rhizoctonia praticola, which yields a mixture of different C–O-

and C–C-coupled dimers, oligomers and oxidation products.[303] In other cases, the

high reactivity of the generated radical intermediates is guided towards the formation

of a single product by the laccase. Remarkably, the heterologously produced laccase

VavH from A. viridinutans coupled the monomeric substrate (R)-semi-vioxanthin(4) to

a single dimeric compound without generating quinone derivatives. This observation

151

and the fact that this enzyme is not secreted to the extracellular medium is congruent

with the proposed physiological role in the biosynthesis of viriditoxin (5). In contrast,

laccases involved in catabolism and detoxification of xenobiotics are secreted and show

higher functional promiscuity.

Regarding the regiochemistry of the reaction, VavH renders exclusively the 6,6′-linked

biaryl pigmentosin A (24), while the same monomer is coupled to the 8,8′-linked

vioxanthin (3) in T. rubrum, P. citreonigrum, and A. ochraceus. VavH coupled the

non-native substrate (R)-semi-vioxanthin (4) with a similar regiochemistry as the native

substrate semi-viriditoxin (6), suggesting that the regiochemistry of the dimerization

is (partially) controlled by the coupling enzyme. The non-enzymatic dimerization

of 1-naphthol (80) generates a mixture of regiochemically different biaryls (43.6%

para-para product or “6,6′-like”), while the dimerization of 2-methoxynaphthalene

(81) yields purely the ortho-ortho-linked product (or “8,8′-like”, Scheme 3.1).[211] The

substructures of 1-naphthol (80) and 2-methoxynaphthalene (81) are also present in

semi-viriditoxin (6) and (R)-semi-vioxanthin (4, Scheme 3.1). Therefore, a chemically

catalyzed oxidative phenol coupling of these naphtho-α-pyrones would preferably lead

to the coupling pattern observed in viriditoxin (5) and pigmentosin A (24). On the

other hand, the dimerization of 1-naphthol (80) and 2-naphtol enzymatically catalyzed

by radical-generating enzymes, yield the 6,6′-like, 8,8′-like and 6,8′-like dimers.[118]

These observations indicate that the regiochemistry of the phenol coupling catalyzed

by VavH is controlled by a combination of substrate and coupling enzyme. In the

case of 8,8′-coupled dimers, the regiochemistry of the formed linkage is not favored

chemically. Therefore, the enzyme(s) responsible for the dimerization likely play(s) a

more important role in the determination of the regiochemistry of the biaryl axis.

152 3 Concluding remarks and outlook

In P. citreonigrum, vioxanthin (3) is produced with a de of 90%, while for viriditoxin

(5) the favored formation of the M-atropisomer has been reported as well.[78,181] The

OH O

O

OH

HO

O

dalesconol A (112), R=H

R

dalesconol B (113), R=OH

Figure 3.1. Dalesconols A andB (112 and 113) isolated fromDaldinia eschscholzii IFB-TL01.[207]

heterologously-produced VavH coupled

the non-physiological substrate

(R)-semi-vioxanthin (4) selectively (de 30-50%) to

the P-atropisomer 7 of pigmentosin A (24). This

selectivity could be caused by the stereochemical

information of the chiral center at C3, while the

coupling enzyme’s role is the generation of a

radical via one-electron oxidation. Analogously,

the ascomycete Daldinia eschscholzii IFB-TL01

produces the polyhydroxynaphtalene-derived

(±)-dalesconols A and B (112 and 113) with a ca. 67% enantiomeric excess (ee) of the

(−)-enantiomers.[207] The reported enantiomeric excess was found to arise from an

interplay of the dominance of particular conformers of naphthol dimer intermediates

and the active site of the radical-generating laccase.[207]

The BVO of the anthraquinone core in the biosynthesis of aflatoxins and

tetrahydroxanthones remains poorly understood. Revisiting the biosynthesis of

tetrahydroxanthones, such as secalonic acid A (8) and neosartorin (9), a new

mechanistic hypothesis for this cryptic biosynthetic step was proposed. The observation

of tautomers of polyhydroxynaphthalenes and anthrahydroquinones, such as emodin

hydroquinone (88), enabling their reduction by a SDR (MdpC, PHAR and homologues)

transferred to the anthraquinone chrysophanol (11).[20,190,262,304] This compound has

been proposed as intermediate due to incorporation of labeled chrysophanol (11)

into secalonic acid D.[11] Tautomerization renders the otherwise stable compound

153

vulnerable to oxidative cleavage, yielding either janthinone-ε-lactone (103) or the

regioisomer monodictyphenone-ε-lactone (101). The tautomerization of naphtho-

and anthraquinones seems to be a wide spread phenomenon that has been efficiently

exploited by nature.[305]

These findings renewed the knowledge on the biosyntheses of emodin (83)-derived

xanthones and naphtho-α-pyrones in ascomycetes in terms of the pathways and the

enzymes involved. In the bigger picture, it sheds new light on the chemical and

biochemical forces governing the selectivity of C–C bond formation in nature.

4 Experimental Section

4.1 Microbiology

Strains

Table 4.1. Microorganisms.

Strain Application

Escherichia coli DH5α cloningEscherichia coli BL21 GOLD heterologous expressionEscherichia coli BL21 pL1SL2 heterologous expressionSaccharomyces cerevisiae heterologous expressionAspergillus niger FGSC A1180 heterologous expressionAspergillus ochraceus DSM 2499 natural product production, knock-outsAspergillus viridinutans ATCC® 16901™, natural product productionCBS 127.56, NRRL 4365Penicillium citreonigrum ATCC® 42743™, natural product production,formerly Penicillium citreo-viride feeding studies

4.1.1 Media

Media and chemicals used in this work were obtained from Carl Roth GmbH Co. KG,

Karlsruhe or Sigma Aldrich Chemie GmbH, München. For submerged cultures, the

micoroganisms were grown in Erlenmeyer flasks with baffled bottom or chicanes. The

flasks were filled with media to a maximum of one fifth of the flask’s total capacity.

156 4 Experimental Section

For solid cultures, liquid medium was mixed with agar agar (15 g/L) and autoclaved

(121 °C, 2.1 bar, 15 min). The mixture was cooled to 60–70 °C. Then the antibiotic was

added in the appropriate amount, if needed. 10 mL of the liquid agar medium was

poured into polystyrole plates (Ø 94 mm, Carl Roth GmbH Co. KG, Karlsruhe). After

the agar had cooled down, the plates were stored at 4 °C until usage.

Hutner’s trace elements solution

All components were prepared as separate solution (Table 4.3). All solutions were

mixed except the disodium ethylenediaminetetraacetic acid (EDTA) solution. The

mixture was heated to boiling. Then the disodium EDTA solution was added. The color

of the hot solution should be green. After all the ingredients were fully dissolved, the

solution was cooled to roughly 70 °C. The pH was adjusted to 6.7 by adding 85 mL hot

KOH (200 g/L). If the the pH exceeds 7, the solution has to be discarded. When the

solution reached room temperature, the volume was adjusted to 1000 mL by adding

water. The final solution should still be green, but turn purple over the next few days.

A brown precipitate may form.

4.1.2 Spore and cell suspensions

Inoculated plates were incubated at 25 °C until a sufficient amount of spores or

mycelium was visible on the plate. Then 5 mL of sterile H2O were added per plate.

Spores were harvested using a Drigalski spatula. Before storage at 4 °C the spore

suspension was filtered through a cell strainer (mesh size 40–70 µm). Cells were

harvested by scraping mycelium off the plates surface with a spatula and stored at 4 °C.

4.1 Microbiology 157

Table 4.2. Media used for microbial cultures.

Media Contents [g/L]

TB 12 tryptone24 yeast extract4 mL glycerolin 900 mLautoclave (121 °C, 2.1 bar, 15 min)add 100 mL sterile phosphate buffer (2.31 KH2PO4, 12.54 K2HPO4)

HA 10 malt extract4 yeast extract4 glucose

MEA 20 malt extract20 glucose6 peptone

AMM 6 NaNO30.52 KCl0.815 KH2PO41.045 K2HPO4ddH2O to 950 mLautoclave (121 °C, 2.1 bar, 15 min)0.5 MgSO4 x 7 H2O10 glucose1 mL/L Hutner’s trace elements

AMM agar 15 agar agar (w/v) in Aspergillus minimal medium (AMM)AMM-T agar 15 agar agar (w/v) in AMM

1 sorbitolLMM AMM 0.3 mM CuSO4SMM AMM

glucose replaced by sucroseSD/∅His 6.7 yeast nitrogen base without amino acids

20 dextrose1.92 yeast synthetic drop-out medium supplements withouthistidine20 agar agar

YPAD broth 0.075 L-adenine hemisulfate10 yeast extract20 peptone20 dextrose


Table 4.3. Hutner’s trace elements.

Components Content [g] Volume of H2O [mL]

Na2EDTA 50 250ZnSO4 x 7 H2O 22 100H3BO3 11.4 200MnCl2 x 4 H2O 5.06 50CoCl2 x 6 H2O 1.61 50CuSO4 x 5 H2O 1.57 50(NH4)6Mo7O24 x 4 H2O 1.10 50FeSO4 x 7 H2O 4.99 50

4.2 Molecular Biology

4.2.1 Kits

Table 4.4. Kits.

Kit Manufacturer

RNeasy® Plant Mini Kit Quiagen GmbH, HildenInvisorb® Fragment CleanUp Kit STRATEC Biomedical AG, BirkenfeldGenElute™Plant Genomic DNA Miniprep Kit Sigma Aldrich Chemie GmbH,

MünchenGeneJET Plasmid Miniprep Kit Fisher Scientific GmbH, SchwerteZyppy™Plasmid Miniprep Kit Zymo Research Europe, Freiburg

4.2.2 Restriction Digest

All enzymes and buffers were supplied by New England BioLabs GmbH (Frankfurt am

Main). CutSmart buffer was used as standard restriction digest buffer. One µg of DNA

was digested with 1 µL of restriction enzyme in a total volume of 50 µL containing

5 µL of CutSmart buffer.

4.2 Molecular Biology 159

4.2.3 Cloning

Cloning with T4-Ligase

Vector and insert were digested with similar restriction enzymes. The amount of total

DNA used for ligation is 100–200 µg. The ratio of m(insert) to m(vector) was chosen

to be 3–5. Two µL of T4-ligase buffer and 0.5 µL of T4-ligase were added to the DNA

and the mixture was filled up with ddH2O to a total volume of 20 µL. The mixture was

either incubated for 10 min at room temperature or overnight at 16 °C.

Cloning with InFusion

The vector was linearized via PCR or restriction digest. The insert was fitted with

the needed 15 bp overlaps for InFusion cloning via PCR. The manufacturer’s protocol

was slightly changed, as the total reaction volume was cut to 5 µL. Therefore, 1 µL

of InFusion master mix was used for each reaction. The amount of added insert

and linearized vector was calculated using the online ressources offered by Takara

Clontech. The mixture was incubated at 50 °C for 60 min and then used directly for

transformation.

Gibson Cloning

The Gibson cloning master mix was prepared from the components listed in Table 4.6.

5 µL master mix was used for each cloning reaction. The vector was linearized via PCR

or restriction digest. The insert was fitted with the needed 15 bp overlaps for Gibson

cloning via PCR. The DNA was added in a total volume of 1.7 µL to the master mix and

incubated at 45 °C for 30 min. Then the temperature was increased to 50 °C for another


30 min. The whole volume of the Gibson cloning reaction was used for transformation

of E. coli directly.

Table 4.5. Isothermal Buffercomponents.

Component Volume [µL]

50% PEG-8000 5002 M Tris 250500 mM MgCl2 1001 M DTT & 100 mM NAD 5010 mM dNTPs 100sterile ddH2O 10

Table 4.6. Gibson master mixcomponents.

Component Volume [µL]

5x Isothermal Buffer 100Taq ligase 50T5-exonuclease 2Phusion polymerase 6.3sterile ddH2O 217

4.2.4 PCR

PCR reactions were carried out using a general protocol, which was adjusted to the

used primers and templates (Table 4.7). QuickLoad Taq (New England BioLabs GmbH,

Frankfurt am Main) was used, if the PCR product was not used for cloning or sequencing.

In these cases Phusion Master Mix (Fisher Scientific GmbH, Schwerte) was used.

Table 4.7. Temperatures, duration and repetition of steps during PCR.

Temperature [°C] Duration [s] Repetition

95 hold95 3095 15

30Tm of Primer –5 1572 for Phusion Master Mix 20/kb for Phusion Master Mix68 for QuickLoad Taq 60/kb for QuickLoad TaqTPhusion/QuickLoadTaq 2 x previous step8 hold


Primer design

Primers were designed using Geneious version 7.1.8[306] and the organism’s or the

plasmid’s DNA sequence. For gene specific primers, 21 bp at the start and end of the

gene were selected. The primers were checked for hairpin structures and primer homo-

and heterodimers using Thermo Fisher Scientific’s Multiple Primer Analyzer online tool.

The tool was also used to determine the melting temperature of the primers.

Primers used for InFusion cloning were also designed and tested with the same

software and online tools. InFusion cloning depends on a 15 bp homologous overlap

of the DNA fragments that should be ligated. If the plasmid used for cloning was

linearized via restriction digest, the created overhangs were taken into account when

designing the primers by adding the needed overlap to the gene of interest. Only the

sequence of a 5′-overhang was included into the primer. 3′-overhangs were removed

by a 3′-exonuclease to create the needed single strand 15 bp overlap. As this 15 bp

sequence was fixed the gene specific part of the primer was used to adjust the melting

temperature, GC content and secondary structure of the primer pair. The melting

temperature should not differ more than 1 °C between the primers used for a single

gene. GC content should be in the range of 45–55% and the secondary structure should

not contain hairpins.

Primers were ordered at Eurofins Genomics GmbH, Ebersberg. The primers were

dissolved in ddH2O to a concentration of 100 pmol/µL and stored at −20 °C. For PCR

the primers were diluted 1:10 with ddH2O.

4.2.5 Electrophoresis

Agarose gel electrophoresis was conducted using a Consort E802 power supply. The

voltage (100–130 V) and run time was adjusted to the sample analyzed. Agarose was


supplied by Carl Roth GmbH Co. KG, Karlsruhe. Tris-acetic acid-EDTA buffer (TAE)

buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) was used as electrophoresis buffer.

SDS-PAGE was conducted using a Consort E835 power supply and a Bio-Rad

Mini-PROTEAN® Tetra System. Samples were prepared adding an appropriate amount

of 6 × loading buffer stock solution. The final concentrations of the loading buffer’s

components are listed in Table 4.8. After heating to 95 °C for 5 min, the samples were

transferred to the gel.

Table 4.8. SDS-PAGE loading buffer.

Components Content [mM]

Tris-HCl 62.5sodium dodecyl sulfate (SDS) 2.5%Bromophenol Blue 0.002%β -mercaptoethanol 713.5 mMglycerol 10%

4.2.6 Plasmids

Plasmids used in this study were either commercially available (pET19b, pET28b,

pESC-His) or newly constructed for this work (pKGH2, pSUC).

4.2.7 pKGH2

The plasmid pKGH2 was constructed by Dr. K. G. Hugentobler and used for heterologous

expression in A. niger.[173] It is based on the plasmid pMA171 that does not contain

a promotor and terminator sequence. pKGH2 was generated by adding the Tet-on

inducible promotor system to the plasmid pMA171 that was linearized by restriction

digest with NotI.[173,174] The plasmid contains a ampicillin and a hygromycin resistance


gene. After transformation of A. niger, the protein production was induced by the

addition of 100 µL of tetracyclin [20 mg/mL].

4.2.8 Construction of pSUC

The plasmid pFC332 was digested with PacI and BamHI. After agarose

gel-electrophoresis the fragment with a size of 10130 bp was isolated. Using the

primers pSUC-PsucA-FWD and pSUC-PsucA-REV and genomic DNA (gDNA) from

Aspergillus carbonarius a fragment of 1289 bp was amplified. A PCR with the primers

pSUC-TcgrA-FWD and pSUC-TcgrA-REV using pKGH2 as template yielded a fragment

of 325 bp. The three fragments were cloned using Gibson cloning. The sequence of

the obtained 11695 bp plasmid was confirmed via sequencing at GATC Biotech AG

(Konstanz).

500

1,000

1,500

2,0002,5003,000

3,500

4,000

4,500

5,000

5,500

6,000

6,500

7,000

7,500

8,000

8,500

9,0009,500 10,000

10,500

11,000

11,500

12,000

12,50013,000

13,50013,892

pKGH213,892 bp

AMA1

PgpdA

Tet-ontrans-

activator

TcgrA

tetO7

Pmin

TtrpC

pBR322ori

ampRori

PgpdA

hygR

TtrpC

Figure 4.1. Expression vector pKGH2.

250

500

750

1,000

1,250

1,500

1,7502,0002,2502,500

2,7503,000

3,250

3,500

3,750

4,000

4,250

4,500

4,750

5,000

5,250

5,500

5,750

6,000

6,250

6,500

6,75

0

7,000

7,250

7,500

7,7508,000 8,250 8,500

8,7509,000

9,250

9,500

9,750

10,000

10,250

10,50010,750

11,00011,250

11,50011,695

pSUC11,695 bp

AMA1

PsucA

TcgrA

PtrpC

hygR

ori

ampR

Figure 4.2. Expression vector pSUC.


Table 4.9. Primers used for pSUC construction.

Primer Sequence

pSUC-TcgrA-FWD GCTAGCTACGTACAAGGATCCACTAGTACAGCAGApSUC-TcgrA-REV TTGAATCGCGCATTGTTCGAATGATTCATGACGTATATTCACCpSUC-PsucA-FWD CGCTGAGGGTTTAATTAAGATCACGTGGATGGCCTpSUC-PsucA-REV TTGTACGTAGCTAGCGTGGGAGGGCCTTGGAG

4.2.9 Isolation of RNA

For the isolation of RNA from Ascomycetes, an overnight culture of the fungus was

filtered with Miracloth (Merck, Darmstadt) and rinsed with ddH2O. The washed

mycelium was ground with liquid nitrogen. Approximately 100 mg of the mycelium

powder was suspended in 450 µl RLT buffer and RNA extraction was done according

to the manual of the used RNeasy Plant Mini Kit (Qiagen, Hilden). Additionally, an

on-column gDNA was carried out. The final elution step of RNA was carried out with

30 µl of ddH2O. The isolated RNA was stored at −20 °C.

4.2.10 cDNA synthesis

Approximately 400 µg of RNA were used for a single reaction with a volume of

50 µL. The RNA was mixed with 2.5 µL PolyT Primer [100 pmol/µl] and 2 µL

deoxynucleoside triphosphate (dNTP)s (10 mM of each nucleotide, New England

BioLabs GmbH, Frankfurt am Main) and incubated at 65 °C for 5 min. The mixture was

chilled on ice for 2 min. Then 4 µL SuperScript™ III buffer, 4 µL MgCl2 (25 mM), 2 µL

dithiothreitol (DTT) (0.1 M), 1 µL RNaseOut and 1 µL SuperScript™ III (Fisher Scientific

GmbH, Schwerte) were added. After incubation at 50 °C for 50 min SuperScript™ III


was inactivated by incubation at 85 °C for 5 min. After inactivation of the reverse

transcriptase, the cDNA was stored at −20 °C.

4.2.11 Isolation of gDNA

For the isolation of gDNA from Ascomycetes an overnight culture of

the fungus was filtered with Miracloth (Merck, Darmstadt) and rinsed

Table 4.10. Buffers used for gDNAisolation.

Buffers Contents [M]

Lysis buffer 3% SDS

0.05 EDTA

0.05 Tris

adjust pH to 7.0 with HCl

with ddH2O. The washed mycelium

was ground with liquid nitrogen.

Approximately 100 mg of the mycelium

powder was suspended in 450 µl of lysis

buffer (0.03% (w/w) SDS, 0.05 M EDTA,

0.1 M Tris, pH 7). After addition of 5 µL

RNase A [10 µg/ml] the suspension was

incubated at 65 °C for 60 min. Then the

samples were chilled on ice for 5 min. To

precipitate proteins, 225 µL MCP Protein Precipitation Solution (Epicentre®, USA)

were added and the samples centrifuged at 11000 × g for 10 min. The supernatant

was transferred to a new micro-centrifuge tube. To precipitate the gDNA, 750 µL of

2-propanol were mixed into the solution. The precipitated gDNA is centrifuged at

12000 × g and the resulting gDNA pellet is washed with 500 µL 70% ethanol. After

the washing step the samples were centrifuged at 12000 × g for 10 min. The gDNA

pellet was then dried at room temperature. When the residual ethanol had evaporated,

the gDNA was dissolved in 100–200 µL ddH2O and stored at −20 °C.


4.2.12 Transformations of microorganisms

Antibiotics

Antibiotics and their work concentrations used for the selection of transformed

microorganism are listed in Table 4.11.

Table 4.11. Antibiotics used for selection.

Antibiotic Final concentration Supplier[µg/ml]

ampicillin 100 Carl Roth GmbH Co. KG, Karlsruhekanamycin A 50 Carl Roth GmbH Co. KG, Karlsruhechloramphenicol 34 Carl Roth GmbH Co. KG, Karlsruhedoxycyclin 100 Thermo Fisher (Kandel) GmbH,

KarlsruheHygromycin B Gold™ 100 InvivoGen Europe, Francepyrithiamine 10 Sigma Aldrich Chemie GmbH,

Münchenphleomycin 30 InvivoGen Europe, France

Escherichia coli

After completed ligation 5 µL of the ligation reaction were mixed with 100 µL of

competent cells and incubated for 15 min on ice. The cells were heat shocked at

42 °C for 30 seconds and incubated on ice for 5 min. 400 µL SOC medium were added

to the cells and the mixture was incubated at 37 °C for 60 min. After incubation the

cells were spread on an LB-agar plate with appropriate antibiotic added to the medium.


Five mL of yeast extract-peptone-adenine-dextrose medium (YPAD) broth were

inoculated with S. cerevisiae and incubated at 30 °C and 140 rpm overnight. The


culture was then diluted 1:20 in 50 mL YPAD broth to an OD600 of 0.25. After

incubation at 30 °C and 140 rpm for 4–5 h until an OD600 of 1.0 was reached. The

cells were centrifuged at room temperature and 1000 × g for 5 min. The pellet was

resuspended in 10 mL of lithium acetate-Tris-EDTA buffer (LTE) buffer and centrifuged

at room temperature and 1000 × g for 5 min. After resuspending the cells in 500 µL of

LTE buffer 50 µL of cell suspension were transferred into microcentrifuge tubes.

The transformation of S. cerevisiae was carried out using one 50 µL aliquot of cell

suspension. 1–3 µg of DNA and 300 µL of transformation mix was added to the cells.

This mixture was incubated at 30 °C for 30 min. Following an additional incubation at

42 °C for 15 min the cells were transferred to an amino acid drop-out plate (SD/∅His

agar). The plates were incubated at 30 °C for 2–3 days.

Table 4.12. Buffers used for S. cerevisiae transformation.


LTE buffer 0.1 lithium acetate0.01 Trisadjust pH to 7.5 with HCl0.001 EDTA

Transformation mix 40% polyethylene glycol 40000.1 lithium acetate0.01 Trisadjust pH to 7.5 with HCl0.001 EDTA

Alternatively, S. cerevisiae transformations were carried out using the Fast™ Yeast

Transformation kit from G Biosciences® (USA). Competent S. cerevisiae cells were

prepared and transformed following the kit’s protocol.


Aspergillus niger FGSC A1180

100 mL malt extract medium (MEA) were inoculated with circa 107 fresh spores and

incubated at 26 °C and 180 rpm for 16–20 h. The mycelium was filtered through

autoclaved Miracloth (Merck KGaA, Darmstadt) and washed with approximately 50 mL

of Solution I (Sol. I, Table 4.13). Approximately 600 mg of filtered and washed

mycelium were transferred to 40 mL of a sterile solution of 2 g/40 mL VinoTaste®

Pro (Novozymes A/S, Denmark) and incubated at 30 °C and 80 rpm for 2 h. Every

30 min the suspension was mixed with a pipette. After incubation the mycelium was

filtered through Miracloth (Merck KGaA, Darmstadt) and the filtrate transferred to a

50 mL centrifugation tube. The solution containing the protoplasts was centrifuged

at 4 °C with 2880 × g for 5 min. The pellet was suspended in 20 mL of Solution II

(Sol. II, Table 4.13) and centrifuged at 4 °C with 2880 × g for five min. The pellet

was suspended in 10 mL Sol. II. After suspending the protoplasts, the cell count was

determined using a Neubauer counting chamber. The cell suspension was centrifuged

at 4 °C with 2880 × g for 5 min and suspended in Sol. II to a concentration of 108

protoplasts/ml.

75 µL of the protoplast suspension were mixed with of DNA solution (1–10 µg/15 µL)

and 20 µL Solution (Sol. III, Table 4.13). For the positive and negative control, 15µl

ddH2O were added to the cells, instead of DNA. The mixtures were incubated on ice

for 20 min. Then 750 µL Sol. III were added and the solutions incubated on ice

for 5 min. Finally, 1000 µL Sol. II were added and the mixture poured into 40 mL

Aspergillus minimal medium for transformation (AMM-T) agar. 40 µL of Hygromycin

Gold™(Invivogen, USA) were added to the transformants and negative control agar

mixtures. The agar was poured onto AMM-T agar plates. The plates were incubated


at 26 °C for 3–5 days. The colonies were spread separately on AMM agar plates with

Hygromycin B Gold™.

Table 4.13. Buffers used for Aspergillus transformation.


Solution I 0.6 KClSolution II 0.6 KCl

0.05 CaCl20.01 TrispH 7.5

Solution III 25% PEG 8000 (w/v) in Solution II

Aspergillus ochraceus

The protocol for the transformation of A. niger was slightly modified for the use with

A. ochraceus. Instead of 2 g/40 mL Sol. I, 3–4 g of VinoTaste® Pro (Novozymes A/S,

Denmark) were used for protoplastation.


4.2.13 Knock-outs

Knock-outs in this work were produced using two different approaches. The first

approach was by replacement of the gene of interest via homologous recombination.

The second approach was by using a CRISPR/Cas9 system adopted to Ascomycetes

developed by Nødvig et al.[170]

Homologous recombination

Firstly, primers were designed for the amplification of a 1000 bp fragment of the

gene’s of interest 5′- and 3′-region. The primers were named P1–P4. P1 was the

forward primer for the 5′-region and P4 the reverse primer for the 3′-region. Primers

P2 and P3 introduced an overlap to the resistance cassette used for the selection of

the transformants. In this work a resistance cassette for hygromycin was used. The

resistance cassette was amplified using the primers P5 and P6. The three DNA fragments

were connected using a fusion PCR protocol shown in Table 4.14. The Ascomycete of

interest was transformed with the approximately 4 kb fragment as described above.

Transformants were tested via Southern blot or PCR.

CRISPR/Cas9

The CRISPR/Cas9 plasmids used in this work were kindly provided by Prof. U. H.

Mortensen from the Technical University of Denmark, Kongens Lyngby, Denmark.

The plasmid pCRISPR1-1a was used to amplify two DNA fragments. They contain

the promoter PgdpA, the sequence for two ribozymes, the sgRNA needed for the

CRISPR/Cas9 system and the terminator TtrpC.

The CRISPR/Cas9 uses a 20 bp RNA sequence as a guide to identify a cutting site.

This RNA sequence (sgRNA) has to be transcribed without cap structure and poly A-tail.


Table 4.14. Temperatures, duration and repetition of steps during fusion PCR.

Temperature [°C] Duration [s] Repetition

98 hold98 3098 15

1062 12072 12098 15

3262 1572 9072 908 hold

This can be achieved by putting the sgRNA under the control of a RNA-polymerase

III promoter. Because these promoters are ill defined in Ascomycetes, Nødvig et al.

used a well defined RNA-polymerase II promoter (PgpdA). This made it necessary

to use to ribozymes to get rid of the cap structure and the poly A-tail added by the

RNA-polymerase II.

CRISPR-P1.333 introduced a 15 bp overlap homologous to the plasmid pFC333,

which has been cut with PacI. CRISPR-P2.vaoE introduced a 6 bp gene specific sequence

for the correct folding and function of the ribozymes. CRISPR-P3.vaoE introduced the

gene specific sgRNA. CRISPR-P4.333 added a 15 bp overlap homologous to pFC333,

which was cut with PacI. Using CRISPR1-1a as template, amplification using the

primers CRISPR-P1.333/CRISPR-P2.vaoE yielded a product of 546 bp. The primers

CRISPR-P3.vaoE/CRISPR-P4.333 yielded a product of 442 bp.

The plasmid pFC333 was cut with PacI as described above. Together with the 546 bp

and 442 bp fragments the linearized plasmid was ligated with InFusion Cloning to yield

pFC333::vaoE. E. coli was transformed with pFC333::vaoE as described above. After


plasmid isolation from E. coli, A. ochraceus was transformed with pFC333::vaoE. The

transformants were plated onto AMM-T agar containing 100 µg Hygromycin Gold™and

30 µg/ml phleomycin.

Table 4.15. Primers used for pFC333::vaoE construction.

Primer Sequence

CRISPR-P1.333 CGCTGAGGGTTTAATGCGTAAGCTCCCTAATTGGCCCRISPR-P2.vaoE ACTCGTTTCGTCCTCACGGACTCATCAGTTGACTCGGTGATGTCRISPR-P3.vaoE GAGGACGAAACGAGTAAGCTCGTCTTGACTCTGACATGGGAGCTGTTTTAGAGCRISPR-P4.333 GGCTGAGGTCTTAATGAGCCAAGAGCGGATTCCTCA

4.2.14 Southern Blot

After isolation of the studied organism’s gDNA a restriction digest was set up overnight.

The chosen restriction enzyme or mixture of restriction enzymes generated DNA

fragments of different sizes depending whether the tested organism was the wild

type genotype or had undergone genetic modifications, such as deletion or insertion.

The digest was performed overnight at a temperature and with a buffer suitable for the

used enzymes.

To analyze the products, an agarose gel electrophoresis was set up. The

electrophoresis was run, until the digested gDNA was spread over the whole length of

the gel. The gel was then put into a flat dish with depurination solution and rocked for

eight min at room temperature. This step was repeated once. The gel was rinsed with

ddH2O between the steps.

The procedure was repeated twice with denaturation solution and a rocking time of

15 min at room temperature. Between the steps the gel was rinsed with ddH2O. Then


the flat dish was filled with neutralization solution and the gel was rocked for 15 min

at room temperature, twice. Between the steps the gel was rinsed with ddH2O.

The gel was set up for transfer as shown in Figure 4.3. As membrane Roti®-Nylon

plus (pore size 0.45 µm, Roth, Karlsruhe) and as blotting paper Roti®-blotting paper

(1.0 mm thickness, 580 x 600 mm, Roth, Karlsruhe) were used. To prevent transfer

from bypassing the membrane and gel strips of x-ray film were placed around the gel.

The transfer was conducted overnight at room temperature.

The transferred DNA was cross-linked to the membrane with UV-light (2 min,

1200 µJ/cm2, Hoefer UVC 500 UV-Crosslinker, Hoefer Pharmacia Biotech Inc., San

Francisco, USA). The dry membrane can then be stored at room temperature until

hybridization.

The pre-hybridization was performed in a hybridization tube with 20 mL

hybridization buffer and 1 mL 10x Western Blocking Reagent (Roche Diagnostics

GmbH, Mannheim) for one hour at 65 °C with agitation (5 rpm) in an hybridization

oven (HIR 10 M, Grant Boekel, USA). After pre-hybridization the denatured probe was

added to the tube and the mixture incubated at 65 °C with agitation (5 rpm) in an

hybridization oven overnight.

The membrane was washed twice with wash buffer I using the same hybridization

tube. For each washing step the tube was incubated at 65 °C with agitation (5 rpm) for

5 min in an hybridization oven. Then the membrane was washed twice with washing

buffer II. For each washing step the tube was incubated at 65 °C with agitation (5 rpm)

for 20 min in an hybridization oven. Lastly, the membrane was washed with 50 mL

maleic acid buffer and incubated at room temperature for 5 min with agitation (5 rpm).

The membrane was incubated with 20 mL blocking solution (with Tween 20) and

incubated at room temperature for 30 min with agitation (5 rpm). The membrane was


glass

paper towels

blotting paper

membraneagarose gel

blotting paper

10 x SSC

weight

Figure 4.3. Southern Blot transfer set up.

incubated with blocking solution (without Tween 20) and 0.5 µL anti-Digoxigenin-AP,

Fab fragments (Roche Diagnostics, Mannheim) was added. The mixture was incubated

at room temperature with agitation (5 rpm) for 30 min.

After hybridization, the membrane was washed twice with 50 mL maleic acid

buffer (without Tween 20). For each washing step, the tube was incubated at room

temperature with agitation (5 rpm) for 15 min. To prepare the membrane for detection,

it was washed with detection buffer and shaken carefully at room temperature until no

more bubbles were visible in the solution.

For detection, the membrane was placed between two plastic foils and dried by

pushing the remaining detection buffer out of the plastic foils. Several drops of


CPD-Star (Roche Diagnostics, Mannheim) were added to the membrane between the

plastic foils. The membrane was then incubated in darkness for 5 min.

Table 4.16. Transfer buffers used for Southern Blot.


depurination solution 0.25 HCldenaturation solution 1.5 NaCl

0.5 NaOHneutralization solution 1.5 NaCl

0.5 Trisadjust pH to 7.5 with HCl

20x saline sodium citrate (SSC) 3 NaCl0.5 Na citrateadjust pH to 7.0 with NaOH

transfer buffer 10x SSC


Escherichia coli

For heterologous expression of the genes of interest different E. coli strains were used

(Table 4.1). E. coli BL21 pL1SL2 contains plasmids encoding two chaperone proteins

from Streptomyces. 5 mL of LB were inoculated with a clone picked from a E. coli

transformation plate with the appropriate amount of kanamycin and ampicillin. The

culture was incubated overnight at 37 °C and 180 rpm.

200 mL were inoculated with 2 mL of the overnight culture. The main culture was

incubated at 37 °C and 180 rpm until the OD600 reached 0.6. Then 100 µL of 0.4 M

isopropyl β -D-1-thiogalactopyranoside (IPTG) were added to induce gene expression.

After induction, the culture was incubated at 37 °C and 180 rpm for 24 h.


Table 4.17. Hybridization and detection buffers used for Southern Blot.


Hybridization buffer 5x SSC0.1% SDSdextrane sulfate 50 g/L

maleic acid buffer 0.1 maleic acid0.15 NaCladjust pH to 7.5 with NaOH0.3% Tween 20

wash buffer I 1x SSC0.1% SDS

wash buffer II 0.5x SSC0.1% SDS

blocking solution for 40 mL36 mL maleic acid buffer4 mL 10x Western Blocking Reagent(Roche Diagnostics GmbH, Mannheim)

detection buffer 0.1 Tris0.1 NaCladjust pH to 9.5 with NaOH

antibody solution 0.5 µL anti-Digoxigenin antibody Fab-fragment(Roche, Mannheim)in 20 mL blocking solution without Tween 20


5 mL SD/∅His were inoculated with a clone picked from a S. cerevisiae transformation

plate. The culture was incubated overnight at 30 °C and 200 rpm. After incubation the

culture was centrifuged at 3214 × g and 4 °C for 5 min. The supernatant was decanted

and the cells were resuspended in K2HPO4 buffer with 20% glycerol.

The main culture was set up by inoculating 5 mL SD/∅His with 50 µL of the overnight

culture’s glycerol stock. The substrate to be tested with the expressed gene was added

and the culture incubated for 4 days at 30 °C and 200 rpm.


Aspergillus niger

100 mL of Aspergillus minimal medium for laccase production (LMM) were inoculated

with 107 spores of A. niger containing a vector with the gene of interest. The appropriate

antibiotic was added to the medium. The culture was incubated at 30 °C and 120 rpm

for 48 h. 100 µL tetracyclin [20 mg/mL] were added and the expression of the gene of

interest was started. The culture was incubated at 30 °C and 120 rpm for 24 h.

The mycelium was harvested by filtration through Miracloth (Merck KGaA,

Darmstadt) and rinsed with ddH2O. To get rid of most of the remaining liquid, the

mycelium was squeezed and then transferred to a plastic centrifuge tube. The mycelium

was stored at −20 °C.

4.2.16 Cell disruption of Aspergillus niger

1–1.5 g of mycelium were transferred to a plastic centrifuge tube. Sodium acetate buffer

(100 mM, pH 5.0) was added to a total volume of 15 mL. The mixture was poured into

a stainless steel beaker and placed on ice. The cell disruption was carried out using

ultra-sonication with a Branson Sonifier 250 (Branson Ultrasonics Corp., USA) and a

flat tip. The ultra-sonication was performed for 5 min (duty cycle 30%, output control

7). The homogeneous lysate was then transferred to a new plastic centrifuge tube and

placed on ice.

The samples were centrifuged at 4 °C and 15557 × g for 30 min and the supernatant

was transferred to a new plastic centrifuge tube. For storage at −20 °C glycerol was

added to a final concentration of 10% (v/v).


+

−

Filter paper

Filter paper

Membrane

Gel

Figure 4.4. Western Blot transfer set up.

4.2.17 Western Blot

Previous to Western Blot a SDS-PAGE was performed with the lysate containing the

protein of interest. The SDS-PAGE gel was not dyed but transferred to a bracket

together with a Protran BA 83 nitrocellulose membrane (GE Healthcare Europe

GmbH, Freiburg) as shown in Figure 4.4. The bracket was placed into a SDS-PAGE

chamber (Mini-PROTEAN® Tetra System, Bio-Rad Laboratories GmbH, München). The

SDS-PAGE chamber was filled with Western Blot transfer buffer and a magnetic stirrer

was added to enhance cooling. The chamber was placed into a sytrofoam box filled

with ice for cooling. The transfer was carried out at 100 V for 240 min.

After the transfer, the membrane was incubated in blocking solution for 60 min while

shaking. Then, the membrane was washed four times with phosphate buffered saline

with tween (PBST) for 10 min. The washed membrane was incubated at 4 °C for

60 min with hybridization solution in a plastic centrifuge tube. The membrane was

washed four times in PBST.


For detection, PBST was decanted. The membrane was put between two transparent

plastic foils. 1 mL of Reagent A and 1 mL of Reagent B (Western Blotting Luminol

Reagent: SC-2048, Santa Cruz Biotechnology, Inc., Heidelberg) were added. A

picture was taken 5–10 min after adding the luminol reagent with a LAS-3000 Imager

(FUJIFILM Europe GmbH, Düsseldorf).

Table 4.18. Buffers used for Western Blot.


10x Transfer buffer w/o MeOH 0.25 Tris1.92 glycine

1x Transfer buffer 0.025 Tris0.192 glycine20% (v/v) MeOH

10x Phosphate buffered saline (PBS) 1.37 NaCl0.027 KCl0.081 Na2HPO40.0147 KH2PO4800 mL ddH2Oadjust pH to 7.4 with HClad 1000 mL with ddH2Oautoclave (121 °C, 2.1 bar, 15 min)

PBST 0.137 NaCl0.0027 KCl0.0081 Na2HPO40.00147 KH2PO41 mL/L Tween 20

Blocking solution 2.5 g milk powder1.25 g BSAad 50 mL with PBST

Hybridisation solution 150 mg BSA1 µL anti-His antibody6 mL PBST


4.3 Natural products

(R)-semi-Vioxanthin and vioxanthin

100 mL of HA medium were inoculated with 0.5 mL of cell suspension of

P. citreonigrum or 0.5 mL of spore suspension of A. ochraceus. 150 mg XAD-7 were

added to increase natural product production. The production conditions are described

in Table 4.19.

After incubation, 2 mL of 2 M HCl were added to each 100 mL culture to adjust the

pH to 2–3. The mycelium and XAD-7 were separated from the culture supernatant

using Miracloth® (Merck KGaA, Darmstadt). The supernatant was extracted separately

from mycelium and XAD-7.

The mycelium was extracted with 100 mL of CHCl3 and MeOH (v/v1:1) in a round

bottom flask. The mixture was stirred for 1 h at room temperature. Then, the mycelium

was separated from the organic phase and extracted with 100 mL of CHCl3 and MeOH

(v/v 1:1) for 1 h. The pH was adjusted to 2–3 using 2 mL of 2 M HCl. After the

second extraction, the organic phase was filtered and the mycelium discarded. The

organic phases of both extractions were pooled and reduced under vacuum until

only an aqueous residue remains. This residue was extracted twice using 20 mL of

CH2Cl2/100 mL culture. The organic phases were pooled and dried using Na2SO4.

After filtration, the organic phase was removed under vacuum to yield a raw extract.

The supernatant was saturated with NaCl and filtered to remove precipitated proteins.

The filtrate was extracted twice with 100 mL ethyl acetate. The organic phase was

dried using Na2SO4. After filtration the organic phase was removed under vacuum to

yield a raw extract.

4.3 Natural products 181

The natural products extracted from the mycelium and culture supernatant were

separated using flash chromatography with acid washed silica gel. A mixture of CH2Cl2

and MeOH (v/v 97:3) was used as mobile phase. The separation was monitored using

an UV lamp (λ 366 nm). At this excitation wavelength (R)-semi-vioxanthin (4) shows a

white to turquoise fluorescence. Vioxanthin (3) was spotted by its green fluorescence.

Table 4.19. Incubation times and conditions for the production of biaryls byP. citreonigrum and A. ochraceus in HA medium.

Fungus T Natural product t Rf[°C] [h] CH2Cl2/MeOH

(97:3, v/v)

P. citreonigrum 21 (R)-semi-vioxanthin (4) 96 0.8vioxanthin (3) 168 0.25

A. ochraceus 22 (R)-semi-vioxanthin (4) 120 0.8vioxanthin (3) 240 0.25

4.3.1 Viriditoxin

For cultivation on solid medium, 25 mL tap water were added to 50 g sorghum and

autoclaved in a 500 mL glass flask. The solid medium was inoculated with 1 mL spore

suspension of A. viridinutans and incubated at room temperature for 28 days.

For cultivation in liquid medium, 200 mL medium containing 40 g sucrose and 20 g

yeast extract per liter were inoculated with 1 mL spore suspension of A. viridinutans.

The culture was incubated at 30 °C and 120 rpm for 130 h.

For the isolation of natural products, the pH of the culture was adjusted to 2–3 using

2 M HCl. The extraction was carried out using 100 mL of CHCl3 and MeOH (v/v 9:1)

and mixing the culture with an Ultra TURRAX® for 5 min. The mixture was filtered


and the organic phase separated from the aqueous medium. The organic phase was

dried using Na2SO4. After filtration, the organic phase was removed under vacuum.

4.4 Analytics 183

4.4 Analytics

4.4.1 HPLC

For HPLC analysis, the samples were extracted with 100 µL CHCl3 or ethyl acetate.

The solvent was evaporated under vacuum and the residue dissolved with 80 µL

acetonitrile (ACN)/ 0.1% formic acid (FA) (v/v 65:35). Before transferring the samples

into HPLC glass vials, the samples were filtered with a MULTOCLEAR 04 filter (PVDF,

pore size 0.2 µm, diameter 4 mm, CS - Chromatographie Service GmbH, Langerwehe)

via centrifugation at 4000 × g for 2 min. The HPLC analyses were conducted using an

Agilent 1100 or 1200 HPLC system with DAD detector. The columns LiChrospher 100

RP18 EC (Col. 1, 5 µm particle size, 250 × 2 mm, CS - Chromatographie Service GmbH,

Langerwehe), Zorbax Eclipse XDB-C8 (Col. 2, 150 × 4.6 mm, Agilent Technologies,

USA), and ISAspher Phenyl 100-5 (Col. 3, 5 µm particle size, 150 × 3 mm, Isera,

Düren) were used. The methods used for HPLC analysis are listed in Table 4.20.

Table 4.20. HPLC methods used for the analysis of fungal extracts and enzymeconversions.

Method 1 (Col. 1) Method 2 (Col. 2) Method 3 (Col. 3)[307] Method 4 (Col. 2)[9,120]

Time [min] ACN [%] Time [min] ACN [%] Time [min] ACN [%] Time [min] ACN [%]

0 65 0 65 0 25 0 303 65 0.5 65 3 25 0.3 3013 75 7 75 26 77 1.8 3018 75 10 75 29 100 12 5523 65 11 90 31 100 14 100

12 90 32 25 24 10013 65 37 25 30 3017 65

Flow [mL · min−1] 0.5 1.5 0.5 1


4.4.2 HPLC-MS

Samples were prepared as described for HPLC analysis.

Analyses of the samples were conducted with HPLC-MS system consisting of an

Agilent 1100 HPLC system and an AB Sciex QTRAP® 4500 mass spectrometer. The

columns LiChrospher 100 RP18 EC (Col. 1, 5 µm particle size, 250 × 2 mm, CS -

Chromatographie Service GmbH, Langerwehe), Zorbax Eclipse XDB-C8 (Col. 2, 150 ×

4.6 mm, Agilent Technologies, USA), and ISAspher Phenyl 100-5 (Col. 3, 5 µm particle

size, 150 × 3 mm, Isera, Düren) were used. All methods use ACN and 0.1% formic

acid as solvents.

Table 4.21. MS parameters used for the detection of natural products.

Parameter Value

Polarity negativCurtain gas 25Temperature 400 °CGas 1 30Gas 2 55Declustering Potential −90 VEntrance Potential −8 VVoltage −4500 V

4.4.3 Circular dichroism

CD spectra were measured with a Jasco J-810 spectrometer. The samples were purified

via HPLC and dissolved in MeOH after evaporation of the eluent. The samples were

measured using a 10 mm quartz cuvette. Each sample was measured five times with

a scan speed of 1000 nm · min−1 and a background correction was carried out using

MeOH as blank. The measurements were conducted in the range of λ 225–450 nm.

4.4 Analytics 185

4.4.4 NMR

NMR measurements were conducted on a Avance DRX 400 from Bruker (Rheinstetten,

Germany). 1H NMR spectra were measured at 400 MHz, 13C NMR-spectra at 100 MHz.

All spectra were measured at room temperature. CDCl3 was used as solvent and all

spectra calibrated to: 1H: 7.26 ppm, 13C: 77.36 ppm. Coupling constants are given in

Hz and chemical shifts (δ) in ppm compared to tetramethylsilan.


1H NMR: (400 MHz, CDCl3), δ = 1.54 (d, 3JHH = 6.3 Hz, 3 H, CH3),2.96 (m, 2 H, 4-CH2), 3.87 (s, 3 H, OCH3), 4.74 (m, 1 H, CH),6.51 (d, 4JHH = 2.2 Hz, 1 H, 8-CHar), 6.55 (d, 4JHH = 2.2 Hz,1 H, 6-CHar), 6.87 (s, 1 H, 5-CHar), 9.46 (s, 1 H, 9-OH), 13.76(s, 1 H, 10-OH).

13C NMR (100 MHz, CDCl3), δ = 20.7 (CH3), 34.7 (C-4), 55.4 (OCH3),76.5 (C-3), 99.3 (C-10a), 99.4 (C-6), 101.5 (C-8), 108.5(C-9a), 116.1 (C-5), 132.8 (C-4a), 140.1 (C-5a), 158.5 (C-9),162.5 (C-7), 162.9 (C-10), 171.6 (C-1).

Coupling product 7

1H NMR: (400 MHz, CDCl3), δ = 1.45 (d, 3JHH = 6.34 Hz, 6 H, CH3),2.76 (d, 3JHH = 5.96 Hz, 4 H, 4-CH2), 3.77 (s, 6 H, OCH3), 4.66(m, 2 H, 3-CH), 6.24 (s, 2 H, 5-CHar), 6.79 (s, 2 H, 8-CHar),9.82 (s, 2 H, 9-OH), 13.96 (d, JHH = 1 Hz, 2 H, 10-OH).

13C NMR (100 MHz, CDCl3), δ = 20.7 (CH3), 34.8 (C-4), 56.2 (OCH3),76.5 (C-3), 98.0 (C-8), 98.9 (C-10a), 108.0 (C-9a), 109.6 (C-6),113.8 (C-5), 132.9 (C-4a), 139.2 (C-5a), 159.1 (C-9), 161.0(C-7), 163.3 (C-10), 171.7 (C-1).


4.5 Bioinformatics

4.5.1 Software

Software used in this work is listed in Table 4.22.

Table 4.22. Bioinformatics software.

Software Application

Geneious (version 6–7) primer design, alignments, cluster graphicsMEGA (version 6–7) alignments, phylogenetic analysisNCBI standalone BLAST tool BLAST(version 2.2.27+–2.2.30+)Galaxy (web) alignments, BLAST, gene prediction, cluster

analysis, transcriptome analysisAUGUSTUS (web) gene predictionantiSMASH (web) (version 2–4) cluster analysisCRISPy-web (web) PAM-site prediction

4.5.2 Alignments

Multiple sequence alignments (DNA and protein sequences) were built using the

ClustalW algorithm. The algorithm was used in connection with Geneious 7.1.5 or

MEGA version 6 or MEGA version 7.[308,309] Sequence similarities [%] were calculated

by Geneious 7.1.5.

4.5.3 BLAST

To identify similar sequences the NCBI web service BLAST was used. The search

included the complete nr-database of NCBI. Hits were selected from the results

depending on the amount of similarity compared to the query sequence. Firstly,

the sequence similarity [%] was taken into consideration. Secondly, the e-value was

chosen to be as low as possible. Only hits were used that had a high sequence similarity

4.5 Bioinformatics 187

(>50%) with the query sequence and additionally presented low e-values (ideally 0.0).

For unpublished sequence databases, the BLAST standalone tool was used (BLAST

2.2.27+–2.2.30+).

4.5.4 Tree building

Phylogenetic trees were built using MEGA version 6 or 7. The trees were constructed

with the Minimal Evolution method (Poisson model, uniform rates, complete deletion)

and their robustness estimated with the bootstrap method (1000 repetitions).

4.5.5 Galaxy

The online bioinformatics platform Galaxy (https://galaxy.uni-freiburg.de) was

used for bioinformatic analysis and data set formatting and preparation. The available

bioinformatic tools were used to design automated workflows to identify candidate

coupling enzymes for further investigation.

CYP Finder

The workflow was designed to find all enzymes with cytochrome P450 characteristics.

It uses protein sequences and identifies proteins with a transmembrane domain on the

N-terminus, a PROSITE heme binding motif (PS00086) and an E-x-x-R motif. Enzymes

missing the N-terminal transmembrane domain or the E-x-x-R motif are still identified,

as long as they possess a heme binding motif.

Firstly, enzymes longer than 600 amino acids were excluded from the analysis, as

most fungal CYP enzymes are class II CYP enzymes.[124] They depend on a separate CYP

reductase for enzyme regeneration. CYP enzymes of class VIII, such as Fum6 from the

fumosinin biosynthetic gene cluster in Fusarium verticillioides, are not detected.[310–312]


They are fusion enzymes of the CYP monooxygenase and the CYP reductase units, with

the flavin co-factor binding unit at the N-terminus. The workflow can easily be modified

to identify these enzymes as well, by including enzymes with up to 1000 amino acids

in the search and adjusting the defined position of the heme binding motif.

The selected sequences (sequence length < 600 amino acids) were simultaneously

screened for a N-terminal transmembrane domain with the tool TMHMM 2.0, for a

PROSITE defined heme binding motif (PS00086) and an E-x-x-R motif with fuzzpro.

The three separate outputs were re-formatted and sequences lacking the domains or

possessing them outside of the defined region of the enzyme sequence were excluded.

The transmembrane domain had to be identified in the first 70 amino acids. The

E-x-x-R motif had to be situated between the amino acids 330–480 and the heme

binding between amino acids 340–510.

The results of the fuzzpro runs [E-x-x-R and heme binding (PS00086)] were

compared. Sequences not containing both motifs in the defined ranges were excluded.

If two E-x-x-R motifs and a heme binding motif were identified in the defined range in

a single sequence, the tool comparing the fuzzpro results created two data set entries.

These duplicates were removed by counting the instances of every sequence identifier

in the data set. The tool output contained the number of occurrences of a data set entry

and the data set entry itself.

Different combinations of motifs were collected by the workflow. It identified

sequences containing a heme binding domain in the defined range, sequences

containing a heme binding domain and an E-x-x-R motif in the defined range,

sequences containing a heme binding domain in the defined range and a N-terminal

transmembrane domain, and it identifies sequences containing all three motifs in the

defined range.


Another comparison adds the enzyme sequences to the putative CYP enzymes from

the tool result lists. The enzyme sequences identified as CYP enzymes by the workflow

were then compared to NCBI’s non-redundant protein database (nr) with the blastp

tool. Additionally, the sequences were analyzed with NCBI’s rpsblast, which identified

conserved domains in amino acid sequences.


sequences

<600

AA

TMHMM2.0

fuzzp

roElxlxlR

fuzzp

rohem

ebindin

g

Find&Replace

F:iR:o

Find&Replace

F:^oR:

Find&Replace

F:or8R:

Find&Replace

F:^,[^g]8R:

Convert

Dashes

toTab

Cut

c12c62c7Filterc3<

70

SelectNotM

atching^SeqName

SelectNotM

atching^SeqName

Filter

330<c3<

480

Filter

340<c3<

510

Compare

1st:c12nd:c1

Count

c1

FASTA

totabular

Compare

1st:c12nd:c1

Compare

1st:c12nd:c1

Compare

1st:c12nd:c1

Compare

1st:c12nd:c1

Cut

c12c22c3

Cut

c12c2

Cut

c12c22c3

Cut

c12c2

Compare

1st:c12nd:c1

Compare

1st:c12nd:c1

tabularto

FASTA

tabularto

FASTA

tabularto

FASTA

tabularto

FASTA

Cut

c12c2

Cut

c12c2

NCBIblastp

NCBIblastp

NCBIblastp

NCBIblastp

NCBIrp

sblast

NCBIrp

sblast

NCBIrp

sblast

NCBIrp

sblast

Cut

c12c22c32c112c122c25

Cut

c12c22c32c112c122c25

Cut

c12c22c32c112c122c25

Cut

c12c22c32c112c122c25

Figure

4.5.G

alaxyw

orkflowto

identifyfungalcytochrom

eP450

enzymes.


Laccase Finder

The Laccase Finder workflow was designed to identify enzymes with laccase-like

sequence characteristics. The sequences were screened for signal peptide sequences

and the PROSITE motifs PS00080 and PS00079. The motifs have been identified

in multi-copper oxidases and PS00080 has been linked to copper binding, while

PS00079 is a signature sequence not specific for copper binding enzymes.[313] Enzymes

containing PS00079 do not necessarily bind copper and may have lost the ability to do

so.

The results of the motif identification with fuzzpro were compared to the reformatted

results from SignalP 3.0. Sequences that contained both features were used to search

for homologous enzymes via NCBI’s blastp and were analyzed with NCBI’s rpsblast tool.


sequences

<600

AA

SignalP

3.0

fuzzp

roPS00080

fuzzp

roPS00079

SelectNotM

atching^SeqName

SelectNotM

atching^SeqName

SelectNotM

atching^rID

Cut

c1,c6

FASTA

totabular

tabularto

FASTA

NCBIblastp

NCBIrp

sblast

Cut

c1,c2,c3,c11,c12,c25

NCBIblastp

NCBIrp

sblast

Cut

c1,c2,c3,c11,c12,c25

Cut

c1,c2,c3

Cut

c1,c2,c3

Compare

1st:c12nd:c1

Compare

1st:c12nd:c1

Cut

c1,c2

Cut

c1,c2

tabularto

FASTA

Compare

1st:c12nd:c1

Compare

1st:c12nd:c1

SelectNotM

atching^rID

Figure

4.6.G

alaxyw

orkflowto

identifyfungallaccase

enzymes.

Bibliography

[1] D. H. R. Barton, G. W. Kirby, W. Steglich, G. M. Thomas, A. R. Battersby,

T. A. Dobson, H. Ramuz, J. Chem. Soc. 1965, 2423.

[2] J. A. Veech, R. D. Stipanovic, A. A. Bell, J. Chem. Soc. Chem. Commun. 1976,

144–145.

[3] W. Hüttel, M. Müller, ChemBioChem 2007, 8, 521–529.

[4] R. D. Stipanovic, L. S. Puckhaber, A. A. Bell, A. E. Percival, J. Jacobs, J. Agric.

Food Chem. 2005, 53, 6266–6271.

[5] J. Liu, R. D. Stipanovic, A. A. Bell, L. S. Puckhaber, C. W. Magill, Phytochemistry

2008, 69, 3038–3042.

[6] A. Präg, B. A. Grüning, M. Häckh, S. Lüdeke, M. Wilde, A. Luzhetskyy, M.

Richter, M. Luzhetska, S. Günther, M. Müller, J. Am. Chem. Soc. 2014, 136,

6195–6198.

[7] K. Yamanaka, K. S. Ryan, T. A. M. Gulder, C. C. Hughes, B. S. Moore, J. Am.

Chem. Soc. 2012, 134, 12434–12437.

[8] C. Gil Girol, K. M. Fisch, T. Heinekamp, S. Günther, W. Hüttel, J. Piel, A. A.

Brakhage, M. Müller, Angew. Chem. Int. Ed. 2012, 51, 9788–9791.

194 Bibliography

[9] L. S. Mazzaferro, W. Hüttel, A. Fries, M. Müller, J. Am. Chem. Soc. 2015, 137,

12289–12295.

[10] S. A. Ahmed, E. Bardshiri, T. J. Simpson, E. Bardshiri, J. Chem. Soc. Chem.

Commun. 1987, 883–884.

[11] B. Franck, G. Bringmann, G. Flohr, Angew. Chem. Int. Ed. Engl. 1980, 19,

460–461.

[12] L. Hendrickson, C. Ray Davis, C. Roach, Di Kim Nguyen, T. Aldrich, P. C. McAda,

C. D. Reeves, Chem. Biol. 1999, 6, 429–439.

[13] C. R. Hutchinson, J. Kennedy, C. Park, S. Kendrew, K. Auclair, J. Vederas,

Antonie van Leeuwenhoek 2000, 78, 287–295.

[14] P. Caffrey, S. Lynch, E. Flood, S. Finnan, M. Oliynyk, Chem. Biol. 2001, 8,

713–723.

[15] H. Motamedi, A. Shafiee, Eur. J. Biochem. 1998, 256, 528–534.

[16] Z.-G. Chen, I. Fujii, Y. Ebizuka, U. Sankawa, Arch. Microbiol. 1992, 158, 29–34.

[17] Z.-G. Chen, I. Fujii, Y. Ebizuka, U. Sankawa, Phytochemistry 1995, 38, 299–305.

[18] P. Rugbjerg, M. Naesby, U. H. Mortensen, R. J. N. Frandsen, Microb. Cell Fact.

2013, 12, 31.

[19] B. Shen, C. R. Hutchinson, J. Biol. Chem. 1994, 269, 30726–30733.

[20] D. Conradt, M. A. Schätzle, J. Haas, C. A. Townsend, M. Müller, J. Am. Chem.

Soc. 2015, 137, 10867–10869.

[21] S. Torkkell, K. Ylihonko, J. Hakala, M. Skurnik, P. Mäntsälä, Mol. Gen. Genet.

1997, 256, 203–209.

[22] S. E. Bode, D. Drochner, M. Müller, Angew. Chem. Int. Ed. 2007, 46, 5916–5920.

195

[23] J. Cortes, S. F. Haydock, G. A. Roberts, D. J. Bevitt, P. F. Leadlay, Nature 1990,

348, 176–178.

[24] H. Sun, C. L. Ho, F. Ding, I. Soehano, X.-W. Liu, Z.-X. Liang, J. Am. Chem. Soc.

2012, 134, 11924–11927.

[25] K. Yabe, H. Nakajima, Appl. Microbiol. Biotechnol. 2004, 64, 745–755.

[26] G. M. Gaucher, M. G. Shepherd, Biochem. Biophys. Res. Commun. 1968, 32,

664–671.

[27] M. K. Kharel, P. Pahari, K. A. Shaaban, G. Wang, C. Morris, J. Rohr, Org. Biomol.

Chem. 2012, 10, 4256–4265.

[28] A. Gutmann, M. Schiller, M. Gruber-Khadjawi, B. Nidetzky, Org. Biomol. Chem.

2017, 15, 7917–7924.

[29] H. G. Cutler, F. G. Crumley, R. H. Cox, O. Hernandez, R. J. Cole, J. W. Dorner,

J. Agric. Food Chem. 1979, 27, 592–595.

[30] K. Nozawa, H. Seyea, S. Nakajima, S.-I. Udagawa, K.-I. Kawai, J. Chem. Soc.

Perkin Trans. 1 1987, 1735–1738.

[31] J. A. Laakso, E. D. Narske, J. B. Gloer, D. T. Wicklow, P. F. Dowd, J. Nat. Prod.

1994, 57, 128–133.

[32] K. Nozawa, S. Nakajima, K.-I. Kawai, S.-I. Udagawa, M. Miyaji, Phytochemistry

1994, 35, 1049–1051.

[33] M. R. TePaske, J. B. Gloer, D. T. Wicklow, P. F. Dowd, J. Nat. Prod. 1992, 55,

1080–1086.

[34] J. B. Stothers, A. Stoessl, Can. J. Chem. 1988, 66, 2816–2818.

[35] T. J. Simpson, J. Chem. Soc. Perkin Trans. 1 1977, 592–595.

196 Bibliography

[36] D. Drochner, M. Müller, Eur. J. Org. Chem. 2001, 2001, 211–215.

[37] J. A. Elix, J. H. Wardlaw, Aust. J. Chem. 2004, 57, 681–683.

[38] M. Isaka, A. Yangchum, P. Rachtawee, S. Komwijit, A. Lutthisungneon, J. Nat.

Prod. 2010, 73, 688–692.

[39] N. Tsuji, Tetrahedron 1968, 24, 1765–1776.

[40] P. M. Baker, J. C. Roberts, J. Chem. Soc. C 1966, 2234–2237.

[41] R. G. Coelho, W. Vilegas, K. F. Devienne, M. S. G. Raddi, Fitoterapia 2000, 71,

497–500.

[42] N. Tsuji, K. Nagashima, Tetrahedron 1968, 24, 4233–4247.

[43] N. Tsuji, K. Nagashima, T. Kimura, H. Kyotani, Tetrahedron 1969, 25,

2999–3005.






[49] H. Brockmann, H. Pini, Naturwissenschaften 1947, 34, 190.

[50] S. Okamoto, T. Taguchi, K. Ochi, K. Ichinose, Chem. Biol. 2009, 16, 226–236.

[51] T. Taguchi, M. Yabe, H. Odaki, M. Shinozaki, M. Metsä-Ketelä, T. Arai, S.

Okamoto, K. Ichinose, Chem. Biol. 2013, 20, 510–520.

[52] T. Taguchi, T. Awakawa, Y. Nishihara, M. Kawamura, Y. Ohnishi, K. Ichinose,

ChemBioChem 2017, 18, 316–323.

197

[53] T. Taguchi, T. Ebihara, A. Furukawa, Y. Hidaka, R. Ariga, S. Okamoto, K.

Ichinose, Bioorg. Med. Chem. Lett. 2012, 22, 5041–5045.

[54] E. A. Bessey, Flora 1904, 93, 301–334.

[55] J. N. Ashley, B. C. Hobbs, H. Raistrick, Biochem. J. 1937, 31, 385–397.

[56] G. R. Birchall, K. Bowden, U. Weiss, W. B. Whalley, J. Chem. Soc. C 1966,

2237–2239.

[57] S. Shibata, E. Morishita, T. Takeda, K. Sakata, Tetrahedron Lett. 1966, 7,

4855–4860.

[58] E. Morishita, T. Takeda, S. Shibata, Chem. Pharm. Bull. 1968, 16, 411–413.

[59] F. J. Leeper, J. Staunton, J. Chem. Soc. Perkin Trans. 1 1984, 2919–2925.

[60] J.-E. Kim, K.-H. Han, J. Jin, H. Kim, J.-C. Kim, S.-H. Yun, Y.-W. Lee, Appl.

Environ. Microbiol. 2005, 71, 1701–1708.

[61] R. J. N. Frandsen, N. J. Nielsen, N. Maolanon, J. C. Sorensen, S. Olsson,

J. Nielsen, H. Giese, Mol. Microbiol. 2006, 61, 1069–1080.

[62] J.-E. Kim, J.-C. Kim, J.-M. Jin, S.-H. Yun, Y.-W. Lee, Plant Pathol. J. 2008, 24,

8–16.

[63] R. J. N. Frandsen, C. Schütt, B. W. Lund, D. Staerk, J. Nielsen, S. Olsson,

H. Giese, J. Biol. Chem. 2011, 286, 10419–10428.

[64] A. Zeeck, P. Ruß, H. Laatsch, W. Loeffler, H. Wehrle, H. Zähner, H. Holst, Chem.

Ber. 1979, 112, 957–978.

[65] M. E. Stack, P. B. Mislivec, T. Denizel, R. Gibson, A. E. Pohland, J. Food Prot.

1983, 46, 965–968.

[66] F. Lund, J. C. Frisvad, Mycol. Res. 1994, 98, 481–492.

198 Bibliography

[67] F. Blank, A. S. Ng, G. Just, Can. J. Chem. 1966, 44, 2873–2879.

[68] F. Blank, W. C. Day, G. Just, J. Invest. Dermatol. 1963, 40, 133–137.

[69] B. Franck, E. M. Gottschalk, Angew. Chem. Int. Ed. Engl. 1964, 3, 441.

[70] J. W. Hooper, W. Marlow, W. B. Whalley, A. D. Borthwick, R. Bowden, J. Chem.

Soc. C 1971, 3580–3590.

[71] B. Franck, G. Baumann, U. Ohnsorge, Tetrahedron Lett. 1965, 6, 2031–2037.

[72] T. Qin, T. Iwata, T. T. Ransom, J. A. Beutler, J. A. Porco, J. Am. Chem. Soc.

2015, 137, 15225–15233.

[73] M. Isaka, A. Jaturapat, K. Rukseree, K. Danwisetkanjana, M. Tanticharoen,

Y. Thebtaranonth, J. Nat. Prod. 2001, 64, 1015–1018.

[74] H. Kikuchi, M. Isobe, S. Kurata, Y. Katou, Y. Oshima, Tetrahedron 2012, 68,

6218–6223.

[75] S. Miethbauer, W. Günther, K.-U. Schmidtke, I. Heiser, S. Gräfe, B. Gitter,

B. Liebermann, J. Nat. Prod. 2008, 71, 1371–1375.

[76] E. B. Lillehoj, M. S. Milburn, Appl. Microbiol. 1973, 26, 202–206.

[77] S. Mizuba, C. Hsu, J. Jiu, J. Antibiot. 1977, 30, 670–672.

[78] K. Suzuki, K. Nozawa, S. Nakajima, K.-I. Kawai, Chem. Pharm. Bull. 1990, 38,

3180–3181.

[79] W. L. Orr, J. R. Grady, Geochim. Cosmochim. Acta 1967, 31, 1201–1209.

[80] W. Wilcke, W. Amelung, C. Martius, M. V. B. Garcia, W. Zech, J. Plant Nutr. Soil

Sci. 2000, 163, 27–30.

199

[81] K. Grice, H. Lu, P. Atahan, M. Asif, C. Hallmann, P. Greenwood, E. Maslen,

S. Tulipani, K. Williford, J. Dodson, Geochim. Cosmochim. Acta 2009, 73,

6531–6543.

[82] S. Kuyama, T. Tamura, J. Am. Chem. Soc. 1957, 79, 5725–5726.

[83] U. Weiss, H. Flon, W. C. Burger, Arch. Biochem. Biophys. 1957, 69, 311–319.

[84] T. Yoshihara, T. Shimanuki, T. Araki, S. Sakamura, Agric. Biol. Chem. 1975, 39,

1683–1684.

[85] H.-Y. Zhang, W. Liu, W.-Z. Liu, J.-L. Xie, Photochem. Photobiol. 2001, 74,

191–195.

[86] H. Wu, X.-F. Lao, Q.-W. Wang, R.-R. Lu, C. Shen, F. Zhang, M. Liu, L. Jia, J. Nat.

Prod. 1989, 52, 948–951.

[87] E. Kobayashi, K. Ando, H. Nakano, T. Iida, H. Ohno, M. Morimoto, T. Tamaoki,

J. Antibiot. 1989, 42, 1470–1474.

[88] G. Li, H. Wang, R. Zhu, L. Sun, L. Wang, M. Li, Y. Li, Y. Liu, Z. Zhao, H. Lou, J.

Nat. Prod. 2012, 75, 142–147.

[89] M. Matsumoto, H. Minato, E. Kondo, T. Mitsugi, K. Katagiri, J. Antibiot. 1975,

28, 602–604.

[90] D. P. Gorst-Allman, P. S. Stern, C. J. Rabie, J. Chem. Soc. Perkin Trans. 1 1980,

2474–2479.

[91] T.-X. Li, M.-H. Yang, Y. Wang, X.-B. Wang, J. Luo, J.-G. Luo, L.-Y. Kong, Sci. Rep.

2016, 6, 38958.

[92] D. G. Davies, P. Hodge, J. Chem. Soc. Perkin Trans. 1 1974, 2403–2405.

[93] J. N. Collie, J. Chem. Soc. Trans. 1907, 91, 1806–1813.

200 Bibliography

[94] A. J. Birch, R. A. Massy-Westropp, C. J. Moye, Aust. J. Chem. 1955, 8, 539.

[95] M. J. Bibb, S. Biró, H. Motamedi, J. F. Collins, C. R. Hutchinson, EMBO J. 1989,

8, 2727–2736.

[96] D. H. Sherman, F. Malpartida, M. J. Bibb, H. M. Kieser, D. A. Hopwood, EMBO

J. 1989, 8, 2717–2725.

[97] L. Katz in Polyketides, aminocoumarins and carbohydrates, (Ed.: D. A. Hopwood),

Methods in Enzymology, Elsevier, Acad. Press, Amsterdam, 2009, pp. 113–142.

[98] C. Hertweck, Angew. Chem. Int. Ed. 2009, 48, 4688–4716.

[99] J. M. Crawford, P. M. Thomas, J. R. Scheerer, A. L. Vagstad, N. L. Kelleher,

C. A. Townsend, Science 2008, 320, 243–246.

[100] J. M. Crawford, T. P. Korman, J. W. Labonte, A. L. Vagstad, E. A. Hill, O.

Kamari-Bidkorpeh, S.-C. Tsai, C. A. Townsend, Nature 2009, 461, 1139–1143.

[101] Y. Li, W. Xu, Y. Tang, J. Biol. Chem. 2010, 285, 22764–22773.

[102] E. Szewczyk, Y.-M. Chiang, C. E. Oakley, A. D. Davidson, C. C. C. Wang,

B. R. Oakley, Appl. Environ. Microbiol. 2008, 74, 7607–7612.

[103] T. Awakawa, K. Yokota, N. Funa, F. Doi, N. Mori, H. Watanabe, S. Horinouchi,

Chem. Biol. 2009, 16, 613–623.

[104] B. S. Moore, J. N. Hopke, ChemBioChem 2001, 2, 35–38.

[105] V. Pfeifer, G. J. Nicholson, J. Ries, J. Recktenwald, A. B. Schefer, R. M. Shawky,

J. Schröder, W. Wohlleben, S. Pelzer, J. Biol. Chem. 2001, 276, 38370–38377.

[106] Y. Seshime, P. R. Juvvadi, I. Fujii, K. Kitamoto, Biochem. Biophys. Res. Commun.

2005, 331, 253–260.

[107] B. Shen, Curr. Opin. Chem. Biol. 2003, 7, 285–295.

201

[108] R. Müller, Chem. Biol. 2004, 11, 4–6.

[109] A. I. Scott, Q. Rev. Chem. Soc. 1965, 19, 1–35.

[110] M. H. Zenk, R. Gerardy, R. Stadler, J. Chem. Soc. Chem. Commun. 1989,

1725–1727.

[111] R. Gerardy, M. H. Zenk, Phytochemistry 1992, 32, 79–86.

[112] D. Bischoff, S. Pelzer, B. Bister, G. J. Nicholson, S. Stockert, M. Schirle,

W. Wohlleben, G. Jung, R. D. Süssmuth, Angew. Chem. Int. Ed. 2001, 40,

4688–4691.

[113] D. Bischoff, S. Pelzer, A. Höltzel, G. J. Nicholson, S. Stockert, W. Wohlleben,

G. Jung, R. D. Süssmuth, Angew. Chem. Int. Ed. 2001, 40, 1693–1696.

[114] O. Pylypenko, F. Vitali, K. Zerbe, J. A. Robinson, I. Schlichting, J. Biol. Chem.

2003, 278, 46727–46733.

[115] D. Bischoff, B. Bister, M. Bertazzo, V. Pfeifer, E. Stegmann, G. J. Nicholson,

S. Keller, S. Pelzer, W. Wohlleben, R. D. Süssmuth, ChemBioChem 2005, 6,

267–272.

[116] R. D. Süssmuth, S. Pelzer, G. Nicholson, T. Walk, W. Wohlleben, G. Jung, Angew.

Chem. Int. Ed. 1999, 38, 1976–1979.

[117] K. Crozier-Reabe, G. R. Moran, Int. J. Mol. Sci. 2012, 13, 15601–15639.

[118] F. Xu, D. E. Koch, I. C. Kong, R. P. Hunter, A. Bhandari, Water Res. 2005, 39,

2358–2368.

[119] G. Benfield, S. M. Bocks, K. Bromley, B. R. Brown, Phytochemistry 1964, 3,

79–88.

202 Bibliography

[120] W. Hüttel, Dissertation, Rheinischen Friedrich-Wilhelms-Universität Bonn,

Bonn, 2005.

[121] M. Parvez, L. B. Qhanya, N. T. Mthakathi, I. K. R. Kgosiemang, H. D. Bamal,

N. S. Pagadala, T. Xie, H. Yang, H. Chen, C. W. Theron, et al., Sci. Rep. 2016, 6,

33099.

[122] D. C. Lamb, L. Lei, A. G. S. Warrilow, G. I. Lepesheva, J. G. L. Mullins, M. R.

Waterman, S. L. Kelly, J. Virol. 2009, 83, 8266–8269.

[123] I. Hanukoglu, Adv. Mol. Cell Biol. 1996, 14, 29–56.

[124] B. Crešnar, S. Petric, Biochim. Biophys. Acta 2011, 1814, 29–35.

[125] G. A. Roberts, G. Grogan, A. Greter, S. L. Flitsch, N. J. Turner, J. Bacteriol. 2002,

184, 3898–3908.

[126] M. Mewies, W. S. McIntire, N. S. Scrutton, Protein Sci. 1998, 7, 7–20.

[127] V. Massey, J. Biol. Chem. 1994, 269, 22459–22462.

[128] B. Entsch, W. J. van Berkel, FASEB J. 1995, 9, 476–483.

[129] W. B. Sutton, J. Biol. Chem. 1957, 226, 395–405.

[130] N. Hajjar, E. Hodgson, Science 1980, 209, 1134–1136.

[131] C. T. Walsh, Y.-C. J. Chen, Angew. Chem. Int. Ed. Engl. 1988, 27, 333–343.

[132] C. Dong, A. Kotzsch, M. Dorward, K. H. van Pée, J. H. Naismith, Acta Crystallogr.

Sect. D: Biol. Crystallogr. 2004, 60, 1438–1440.

[133] S. M. Jones, E. I. Solomon, Cell. Mol. Life Sci. 2015, 72, 869–883.

[134] H. Yoshida, J. Chem. Soc. Trans. 1883, 43, 472–486.

[135] G. Bertrand, C. R. Hebd. Seances Acad. Sci. 1896, 123, 463–465.

203

[136] W. Bao, D. M. O’malley, R. Whetten, R. R. Sederoff, Science 1993, 260,

672–674.

[137] S. A. Roberts, A. Weichsel, G. Grass, K. Thakali, J. T. Hazzard, G. Tollin, C.

Rensing, W. R. Montfort, Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 2766–2771.

[138] N. M. Parkinson, C. M. Conyers, J. N. Keen, A. D. MacNicoll, I. Smith, R. J.

Weaver, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2003, 134, 513–520.

[139] N. T. Dittmer, R. J. Suderman, H. Jiang, Y.-C. Zhu, M. J. Gorman, K. J. Kramer,

M. R. Kanost, Insect Biochem. Mol. Biol. 2004, 34, 29–41.

[140] T. Umezawa, L. B. Davin, N. G. Lewis, Biochem. Biophys. Res. Commun. 1990,

171, 1008–1014.

[141] C. R. Benedict, J. Liu, R. D. Stipanovic, Phytochemistry 2006, 67, 356–361.

[142] P. W. Paré, H.-B. Wang, L. B. Davin, N. G. Lewis, Tetrahedron Lett. 1994, 35,

4731–4734.

[143] L. B. Davin, N. G. Lewis, An. Acad. Bras. Cienc. Suppl. 3 1995, 67, 363–378.

[144] P. Gavezzotti, C. Navarra, S. Caufin, B. Danieli, P. Magrone, D. Monti, S. Riva,

Adv. Synth. Catal. 2011, 353, 2421–2430.

[145] D. A. Martinez, B. G. Oliver, Y. Gräser, J. M. Goldberg, W. Li,

N. M. Martinez-Rossi, M. Monod, E. Shelest, R. C. Barton, E. Birch, et al.,

MBio 2012, 3, e00259–12.

[146] T. Weber, K. Blin, S. Duddela, D. Krug, H. U. Kim, R. Bruccoleri, S. Y. Lee,

M. A. Fischbach, R. Müller, W. Wohlleben, et al., Nucleic Acids Res. 2015, 43,

W237–W243.

[147] A. Marchler-Bauer, S. H. Bryant, Nucleic Acids Res. 2004, 32, W327–W331.

204 Bibliography

[148] A. Marchler-Bauer, M. K. Derbyshire, N. R. Gonzales, S. Lu, F. Chitsaz, L. Y. Geer,

R. C. Geer, J. He, M. Gwadz, D. I. Hurwitz, et al., Nucleic Acids Res. 2015, 43,

D222–D226.

[149] S. White, J. O’Callaghan, A. D. W. Dobson, FEMS Microbiol. Lett. 2006, 255,

17–26.

[150] M. P. Artigot, N. Loiseau, J. Laffitte, L. Mas-Reguieg, S. Tadrist, I. P. Oswald,

O. Puel, Microbiology (Reading Engl.) 2009, 155, 1738–1747.

[151] L. Liu, Z. Zhang, C.-L. Shao, J.-L. Wang, H. Bai, C.-Y. Wang, Sci. Rep. 2015, 5,

10463.

[152] T. Awakawa, T. Kaji, T. Wakimoto, I. Abe, Bioorg. Med. Chem. Lett. 2012, 22,

4338–4340.

[153] L. Studt, P. Wiemann, K. Kleigrewe, H.-U. Humpf, B. Tudzynski, Appl. Environ.

Microbiol. 2012, 78, 4468–4480.

[154] A. G. Newman, C. A. Townsend, J. Am. Chem. Soc. 2016, 138, 4219–4228.

[155] S. Malz, M. N. Grell, C. Thrane, F. J. Maier, P. Rosager, A. Felk, K. S. Albertsen,

S. Salomon, L. Bohn, W. Schäfer, et al., Fungal Genet. Biol. 2005, 42, 420–433.

[156] J.-E. Kim, J. Jin, H. Kim, J.-C. Kim, S.-H. Yun, Y.-W. Lee, Appl. Environ. Microbiol.

2006, 72, 1645–1652.

[157] S. Obermaier, Activity and regioselectivity of AunD, AunE and their homologues

AucD, AucE, BfoD, and BfoE determined by the construction of knock-out

mutants. Manuscript in preparation. (Collab.: L. Fürtges), 2018.

[158] W. Thiele, Identification of the putative biosynthetic gene cluster of sporandol

in Chrysosporium merdarium. (Collab.: L. Fürtges), 2017.

205

[159] J. Markovic, Master Thesis, Albert-Ludwigs-Universität Freiburg, Freiburg,

2018.

[160] R. P. de Vries, R. Riley, A. Wiebenga, G. Aguilar-Osorio, S. Amillis, C. A. Uchima,

G. Anderluh, M. Asadollahi, M. Askin, K. Barry, et al., Genome Biol. 2017, 18,

28.

[161] H. Chen, M.-H. Lee, M. E. Daub, K.-R. Chung, Mol. Microbiol. 2007, 64,

755–770.

[162] M. Kawaguchi, T. Ohshiro, M. Toyoda, S. Ohte, J. Inokoshi, I. Fujii, H. Tomoda,

Angew. Chem. 2018, DOI \url10.1002/ange.201800415.

[163] E. van Dyck, A. Z. Stasiak, A. Stasiak, S. C. West, Nature 1999, 398, 728–731.

[164] V. Meyer, M. Arentshorst, A. El-Ghezal, A.-C. Drews, R. Kooistra, C. A. M. J. J.

van den Hondel, A. F. J. Ram, J. Biotechnol. 2007, 128, 770–775.

[165] D. Jiang, W. Zhu, Y. Wang, C. Sun, K.-Q. Zhang, J. Yang, Biotechnol. Adv. 2013,

31, 1562–1574.

[166] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, E. Charpentier,

Science 2012, 337, 816–821.

[167] P. Mali, L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville,

G. M. Church, Science 2013, 339, 823–826.

[168] W. Y. Hwang, Y. Fu, D. Reyon, M. L. Maeder, S. Q. Tsai, J. D. Sander, R. T.

Peterson, J.-R. J. Yeh, J. K. Joung, Nat. Biotechnol. 2013, 31, 227–229.

[169] S. W. Cho, S. Kim, J. M. Kim, J.-S. Kim, Nat. Biotechnol. 2013, 31, 230–232.

[170] C. S. Nødvig, J. B. Nielsen, M. E. Kogle, U. H. Mortensen, PloS ONE 2015, 10,

e0133085.

http://dx.doi.org/\url10.1002/ange.201800415

206 Bibliography

[171] O. V. Morozova, G. P. Shumakovich, M. A. Gorbacheva, S. V. Shleev, A. I.

Yaropolov, Biochemistry (Moscow) 2007, 72, 1136–1150.

[172] M. A. Romanos, C. A. Scorer, J. J. Clare, Yeast 1992, 8, 423–488.

[173] K. G. Hugentobler, Cloning of the expression plasmid pKGH2 with the Tet-on

expression system based on pMA171. The plasmid pMA171 was linearized

with the restriction enzyme NotI. Subsequently, the Tet-on inducible promotor

system provided by V. Meyer was cloned into the linearized plasmid pMA171

with InFusion cloning. (Collab.: L. Fürtges), 2015.

[174] V. Meyer, F. Wanka, J. van Gent, M. Arentshorst, C. A. M. J. J. van den Hondel,

A. F. J. Ram, Appl. Environ. Microbiol. 2011, 77, 2975–2983.

[175] F. Wanka, T. Cairns, S. Boecker, C. Berens, A. Happel, X. Zheng, J. Sun, S.

Krappmann, V. Meyer, Fungal Genet. Biol. 2016, 89, 72–83.

[176] J. E. Kim, S. J. Kim, B. H. Lee, R. W. Park, K. S. Kim, I. S. Kim, J. Biol. Chem.

2000, 275, 30907–30915.

[177] O. Huber, M. Sumper, EMBO J. 1994, 13, 4212–4222.

[178] A. Faik, J. Abouzouhair, F. Sarhan, Mol. Genet. Genomics 2006, 276, 478–494.

[179] L. B. Davin, H.-B. Wang, A. L. Crowell, D. L. Bedgar, D. M. Martin, S. Sarkanen,

N. G. Lewis, Science 1997, 275, 362–367.

[180] T. N. Petersen, S. Brunak, G. von Heijne, H. Nielsen, Nat. Methods 2011, 8,

785–786.

[181] S. E. Bode, Dissertation, Albert-Ludwigs-Universität Freiburg, Freiburg, 2007.

[182] J. M. Crawford, B. C. R. Dancy, E. A. Hill, D. W. Udwary, C. A. Townsend, Proc.

Natl. Acad. Sci. U. S. A. 2006, 103, 16728–16733.

207

[183] L. Du, L. Lou, Nat. Prod. Rep. 2010, 27, 255–278.

[184] A. G. Newman, A. L. Vagstad, K. Belecki, J. R. Scheerer, C. A. Townsend, Chem.

Commun. 2012, 48, 11772–11774.

[185] J. H. Yu, T. J. Leonard, J. Bacteriol. 1995, 177, 4792–4800.

[186] J. W. Cary, K. C. Ehrlich, S. B. Beltz, P. Harris-Coward, M. A. Klich, Mycologia

2009, 101, 352–362.

[187] K. E. Papa, Microbiology (Reading Engl.) 1982, 128, 1345–1348.

[188] K. C. Ehrlich, P. Li, L. Scharfenstein, P.-K. Chang, Appl. Environ. Microbiol. 2010,

76, 3374–3377.

[189] J. Haas, M. Häckh, V. Justus, M. Müller, S. Lüdeke, Org. Biomol. Chem. 2017,

15, 10256–10264.

[190] M. A. Schätzle, S. M. Husain, S. Ferlaino, M. Müller, J. Am. Chem. Soc. 2012,

134, 14742–14745.

[191] H.-Q. Chen, M.-H. Lee, K.-R. Chung, Microbiology (Reading Engl.) 2007, 153,

2781–2790.

[192] D. Drochner, Dissertation, Rheinischen Friedrich-Wilhelms-Universität Bonn,

Bonn, 2003.

[193] Y. Igarashi, Y. Kuwamori, K. Takagi, T. Ando, R. Fudou, T. Furumai, T. Oki, J.

Antibiot. 2000, 53, 928–933.

[194] M. H. Taniwaki, J. I. Pitt, B. T. Iamanaka, F. P. Massi, M. H. P. Fungaro,

J. C. Frisvad, PloS ONE 2015, 10, e0143189.

[195] S. Grijseels, J. C. Nielsen, M. Randelovic, J. Nielsen, K. F. Nielsen, M. Workman,

J. C. Frisvad, Sci. Rep. 2016, 6, 35112.

208 Bibliography

[196] E. B. Lillehoj, A. Ciegler, Bacteriol. Proc. 1971, A-25.

[197] E. B. Lillehoj, A. Ciegler, Can. J. Microbiol. 1972, 18, 193–197.

[198] J. J. Talbot, J. Houbraken, J. C. Frisvad, R. A. Samson, S. E. Kidd, J. Pitt,

S. Lindsay, J. A. Beatty, V. R. Barrs, PloS ONE 2017, 12, e0181660.

[199] Y. Liu, T. Kurtán, C. Yun Wang, W. Han Lin, R. Orfali, W. E. Müller, G. Daletos,

P. Proksch, J. Antibiot. 2016, 69, 702–706.

[200] W. A. Ayer, P. A. Craw, K. Nozawa, Can. J. Chem. 1991, 69, 189–191.

[201] S.-B. Hong, H.-D. Shin, J. Hong, J. C. Frisvad, P. V. Nielsen, J. Varga, R. A.

Samson, Antonie van Leeuwenhoek 2008, 93, 87–98.

[202] J. C. Frisvad, J. Smedsgaard, T. O. Larsen, R. A. Samson, Stud. Mycol. 2004,

49, 201–204.

[203] D. Weisleder, E. B. Lillehoj, Tetrahedron Lett. 1971, 12, 4705–4706.

[204] N. K. Singh, A. Blachowicz, J. Romsdahl, C. Wang, T. Torok, K. Venkateswaran,

Genome Announc. 2017, 5, DOI \url10.1128/genomeA.01602-16.

[205] H. M. Geertz-Hansen, N. Blom, A. M. Feist, S. Brunak, T. N. Petersen, Proteins

2014, 82, 1819–1828.

[206] H. Leisch, K. Morley, P. C. K. Lau, Chem. Rev. 2011, 111, 4165–4222.

[207] W. Fang, S. Ji, N. Jiang, W. Wang, G. Y. Zhao, S. Zhang, H. M. Ge, Q. Xu,

A. H. Zhang, Y. L. Zhang, et al., Nat. Commun. 2012, 3, 1039.

[208] K. Piontek, M. Antorini, T. Choinowski, J. Biol. Chem. 2002, 277, 37663–37669.

[209] E. Ong, W. B. Pollock, M. Smith, Gene 1997, 196, 113–119.

[210] C. I. Grove, M. J. Di Maso, F. A. Jaipuri, M. B. Kim, J. T. Shaw, Org. Lett. 2012,

14, 4338–4341.

http://dx.doi.org/\url10.1128/genomeA.01602-16

209

[211] Y. Kashiwagi, H. Ono, T. Osa, Chem. Lett. 1993, 22, 81–84.

[212] M. Choquer, K. L. Dekkers, H.-Q. Chen, L. Cao, P. P. Ueng, M. E. Daub, K.-R.

Chung, Mol. Plant Microbe Interact. 2005, 18, 468–476.

[213] K.-R. Chung, H.-L. Liao, Microbiology (Reading Engl.) 2008, 154, 3556–3566.

[214] H.-L. Liao, K.-R. Chung, Mol. Plant Microbe Interact. 2008, 21, 469–479.

[215] N. Vaghefi, J. R. Kikkert, M. D. Bolton, L. E. Hanson, G. A. Secor,

S. J. Pethybridge, Fungal Ecol. 2017, 26, 125–134.

[216] B. D. Wingfield, D. K. Berger, E. T. Steenkamp, H.-J. Lim, T. A. Duong, B. H.

Bluhm, Z. W. de Beer, L. de Vos, G. Fourie, K. Naidoo, et al., IMA Fungus 2017,

8, 385–396.

[217] R. Chand, C. Pal, V. Singh, M. Kumar, V. K. Singh, P. Chowdappa, C. Pallem,

Curr. Sci. 2015, 109, 2103–2110.

[218] J. K. Hane, R. G. T. Lowe, P. S. Solomon, K.-C. Tan, C. L. Schoch, J. W. Spatafora,

P. W. Crous, C. Kodira, B. W. Birren, J. E. Galagan, et al., Plant Cell 2007, 19,

3347–3368.

[219] H. Yang, Y. Wang, Z. Zhang, R. Yan, Du Zhu, Genome Announc. 2014, 2,

e00011–14.

[220] C. Camacho, G. Coulouris, V. Avagyan, N. Ma, J. Papadopoulos, K. Bealer,

T. L. Madden, BMC Bioinformatics 2009, 10, 421.

[221] H. Deng, R. Gao, X. Liao, Y. Cai, Gene 2016, 580, 67–72.

[222] Y.-H. Chooi, G. Zhang, J. Hu, M. J. Muria–Gonzalez, P. N. Tran, A. Pettitt, A. G.

Maier, R. A. Barrow, P. S. Solomon, Environ. Microbiol. 2017, 19, 1975–1986.

[223] S. L. Yang, K.-R. Chung, Fungal Biol. 2010, 114, 64–73.

210 Bibliography

[224] V. Swart, B. G. Crampton, J. B. Ridenour, B. H. Bluhm, N. A. Olivier, J. J. M.

Meyer, D. K. Berger, Mol. Plant Microbe Interact. 2017, 30, 710–724.

[225] H. Deng, R. Gao, X. Liao, Y. Cai, J. Biotechnol. 2017, 259, 228–234.

[226] K. L. Dekkers, B.-J. You, V. S. Gowda, H.-L. Liao, M.-H. Lee, H.-J. Bau, P. P. Ueng,

K.-R. Chung, Fungal Genet. Biol. 2007, 44, 444–454.

[227] G. Assante, R. Locci, L. Camarda, L. Merlini, G. Nasini, Phytochemistry 1977,

16, 243–247.

[228] A. O. Fajola, Physiol. Plant Pathol. 1978, 13, 157–164.

[229] W.-B. Shim, L. D. Dunkle, Physiol. Mol. Plant Pathol. 2002, 61, 237–248.

[230] H. Kim, J. B. Ridenour, L. D. Dunkle, B. H. Bluhm, PLoS Pathog. 2011, 7,

e1002113.

[231] S. Obermaier, Report on the identification of split biosynthetic gene clusters

responsible for the formation of dimeric naphthopyrones in the genus

Aspergillus. Manuscript in preparation. (Collab.: L. Fürtges), 2018.

[232] A. Amnuaykanjanasin, M. E. Daub, Fungal Genet. Biol. 2009, 46, 146–158.

[233] S. Omura, Y. Iwai, K. Hinotozawa, Y. Takahashi, J. Kato, A. Nakagawa, A.

Hirano, H. Shimizu, K. Haneda, J. Antibiot. 1982, 35, 645–652.

[234] K. K. Chexal, C. Fouweather, J. S. E. Holker, T. J. Simpson, K. Young, J. Chem.

Soc. Perkin Trans. 1 1974, 1584–1593.

[235] W. Schmid, Ann. Chem. Pharm. 1855, 93, 83–88.

[236] A. G. McInnes, D. G. Smith, J. A. Walter, L. C. Vining, J. L. C. Wright, J. Chem.

Soc. Chem. Commun. 1975, 66–68.

211

[237] J. S. E. Holker, R. D. Lapper, T. J. Simpson, J. Chem. Soc. Perkin Trans. 1 1974,

2135–2140.

[238] E. Bardshiri, T. J. Simpson, J. Chem. Soc. Chem. Commun. 1981, 195–196.

[239] E. Bardshiri, C. R. McIntyre, T. J. Simpson, R. N. Moore, L. A. Trimble, J. C.

Vederas, J. Chem. Soc. Chem. Commun. 1984, 1404–1406.

[240] Y.-M. Chiang, E. Szewczyk, A. D. Davidson, R. Entwistle, N. P. Keller, C. C. C.

Wang, B. R. Oakley, Appl. Environ. Microbiol. 2010, 76, 2067–2074.

[241] J. F. Sanchez, R. Entwistle, J.-H. Hung, J. Yaegashi, S. Jain, Y.-M. Chiang,

C. C. C. Wang, B. R. Oakley, J. Am. Chem. Soc. 2011, 133, 4010–4017.

[242] B. Franck, F. Hüper, D. Gröger, D. Erge, Chem. Ber. 1968, 101, 1954–1969.

[243] I. Kurobane, L. C. Vining, A. McInnes, Tetrahedron Lett. 1978, 19, 4633–4636.

[244] R. Andersen, G. Buechi, B. Kobbe, A. L. Demain, J. Org. Chem. 1977, 42,

352–353.

[245] B. Proksa, D. Uhrín, T. Liptaj, M. Šturdíková, Phytochemistry 1998, 48,

1161–1164.

[246] P. S. Steyn, Tetrahedron 1970, 26, 51–57.

[247] J. W. Cornforth, G. Ryback, P. M. Robinson, D. Park, J. Chem. Soc. C 1971,

2786–2788.

[248] T. Wezeman, S. Bräse, K.-S. Masters, Nat. Prod. Rep. 2015, 32, 6–28.

[249] G. Liu, L. Zhang, X. Wei, G. Zou, Y. Qin, L. Ma, J. Li, H. Zheng, S. Wang,

C. Wang, et al., PloS ONE 2013, 8, e55185.

212 Bibliography

[250] C. L. Schardl, C. A. Young, U. Hesse, S. G. Amyotte, K. Andreeva, P. J. Calie,

D. J. Fleetwood, D. C. Haws, N. Moore, B. Oeser, et al., PLoS Genet. 2013, 9,

e1003323.

[251] N. D. Fedorova, N. Khaldi, V. S. Joardar, R. Maiti, P. Amedeo, M. J. Anderson,

J. Crabtree, J. C. Silva, J. H. Badger, A. Albarraq, et al., PLoS Genet. 2008, 4,

e1000046.

[252] Y. Kusuya, K. Sakai, K. Kamei, H. Takahashi, T. Yaguchi, Genome Announc.

2016, 4, e01568–15.

[253] M. Andrew, R. Barua, S. M. Short, L. M. Kohn, PloS ONE 2012, 7, e29943.

[254] D. Gröger, D. Erge, B. Franck, U. Ohnsorge, H. Flasch, F. Hüper, Chem. Ber.

1968, 101, 1970–1978.

[255] J. W. Bok, Y.-M. Chiang, E. Szewczyk, Y. Reyes-Dominguez, A. D. Davidson,

J. F. Sanchez, H.-C. Lo, K. Watanabe, J. Strauss, B. R. Oakley, et al., Nat. Chem.

Biol. 2009, 5, 462–464.

[256] L. Neubauer, J. Dopstadt, H.-U. Humpf, P. Tudzynski, Fungal Biol. Biotechnol.

2016, 3, 77.

[257] X. Xu, L. Liu, F. Zhang, W. Wang, J. Li, L. Guo, Y. Che, G. Liu, ChemBioChem

2014, 15, 284–292.

[258] S. Griffiths, C. H. Mesarich, B. Saccomanno, A. Vaisberg, P. J. G. M. de Wit,

R. Cox, J. Collemare, Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 6851–6856.

[259] K. Throckmorton, F. Y. Lim, D. P. Kontoyiannis, W. Zheng, N. P. Keller, Environ.

Microbiol. 2016, 18, 246–259.

[260] M. T. Nielsen, J. B. Nielsen, D. C. Anyaogu, D. C. Anyaogu, D. K. Holm, K. F.

Nielsen, T. O. Larsen, U. H. Mortensen, PloS ONE 2013, 8, e72871.

213

[261] I. Fujii, Y. Ebizuka, U. Sankawa, J. Biotechnol. 1988, 103, 878–883.

[262] L. Fürtges, D. Conradt, M. A. Schätzle, S. K. Singh, N. Kraševec, T. L. Rižner,

M. Müller, S. M. Husain, ChemBioChem 2017, 18, 77–80.

[263] D. Conradt, Dissertation, Albert-Ludwigs-Universität Freiburg, Freiburg, 2017.

[264] N. Saha, A. Mondal, K. Witte, S. K. Singh, M. Müller, S. M. Husain, Chem. Eur.

J. 2018, 24, 1283–1286.

[265] S. Steimle, Diplomarbeit, Albert-Ludwigs-Universität Freiburg, Freiburg, 2017.

[266] L. Kong, W. Zhang, Y. H. Chooi, L. Wang, B. Cao, Z. Deng, Y. Chu, D. You, Cell

Chem. Biol. 2016, 23, 508–516.

[267] K. C. Nicolaou, C. D. Papageorgiou, J. L. Piper, R. K. Chadha, Angew. Chem. Int.

Ed. 2005, 44, 5846–5851.

[268] B. B. Snider, X. Gao, J. Org. Chem. 2005, 70, 6863–6869.

[269] B. H. Howard, H. Raistrick, Biochem. J. 1954, 56, 56–65.

[270] H.-L. Li, X.-M. Li, H. Liu, L.-H. Meng, B.-G. Wang, Mar. Drugs 2016, 14, 223.

[271] A. Dieu, M. Millot, Y. Champavier, L. Mambu, V. Chaleix, V. Sol, V. Gloaguen,

Planta Med. 2014, 80, 931–935.

[272] K.-S. Masters, S. Bräse, Chem. Rev. 2012, 112, 3717–3776.

[273] J. E. Davis, Chem. Ind. 1956, 178–179.

[274] K. M. Henry, C. A. Townsend, J. Am. Chem. Soc. 2005, 127, 3300–3309.

[275] K. M. Henry, C. A. Townsend, J. Am. Chem. Soc. 2005, 127, 3724–3733.

[276] J. Cudlín, N. Steinerová, P. Sedmera, J. Vokoun, Collect. Czech. Chem. Commun.

1978, 43, 1808–1810.

214 Bibliography

[277] J. Cudlín, N. Steinerová, J. Mateju, M. Blumauerová, Z. Vanek, Collect. Czech.

Chem. Commun. 1978, 43, 1803–1807.

[278] V. Y. Fain, B. E. Zaitsev, M. A. Ryabov, Chem. Nat. Compd. 2005, 41, 146–152.

[279] M. P. Marzocchi, A. R. Mantini, M. Casu, G. Smulevich, J. Chem. Phys. 1998,

108, 534–549.

[280] A. M. d. R. Marinho, E. Rodrigues-Filho, M. d. L. R. Moitinho, L. S. Santos, J.

Braz. Chem. Soc. 2005, 16, 280–283.

[281] T. Sassa, Agr. Biol. Chem. 1991, 55, 95–99.

[282] T. Sassa, H. Kachi, M. Nukina, Y. Suzuki, J. Antibiot. 1985, 38, 439–441.

[283] H. Kachi, T. Sassa, Agr. Biol. Chem. 1986, 50, 1669–1671.

[284] K. Horiguchi, Y. Suzuki, T. Sassa, Agr. Biol. Chem. 1989, 53, 2141–2145.

[285] D. Rönsberg, A. Debbab, A. Mándi, V. Vasylyeva, P. Böhler, B. Stork, L. Engelke,

A. Hamacher, R. Sawadogo, M. Diederich, et al., J. Org. Chem. 2013, 78,

12409–12425.

[286] A. R. Ola, A. Debbab, A. H. Aly, A. Mandi, I. Zerfass, A. Hamacher, M. U.

Kassack, H. Brötz-Oesterhelt, T. Kurtan, P. Proksch, Tetrahedron Lett. 2014, 55,

1020–1023.

[287] L. Jahn, T. Schafhauser, D. Wibberg, C. Rückert, A. Winkler, A. Kulik, T. Weber,

L. Flor, K.-H. van Pée, J. Kalinowski, et al., J. Biotechnol. 2017, 257, 233–239.

[288] R. Jadulco, G. Brauers, R. A. Edrada, R. Ebel, V. Wray, Sudarsono, P. Proksch, J.

Nat. Prod. 2002, 65, 730–733.

[289] A. Plemenitaš, M. Žakeu-Mavric, R. Komel, J. Steroid Biochem. 1988, 29,

371–372.

215

[290] T. Lanišnik Rižner, M. Žakelj-Mavric, A. Plemenitaš, M. Zorko, J. Steroid

Biochem. Mol. Biol. 1996, 59, 205–214.

[291] A. Cassetta, T. Büdefeld, T. L. Rizner, K. Kristan, J. Stojan, D. Lamba, Acta

Crystallogr. Sect. F: Struct. Biol. Cryst. Commun. 2005, 61, 1032–1034.

[292] T. Lanišnik Rižner, J. Stojan, J. Adamski, Mol. Cell. Endocrinol. 2001, 171,

193–198.

[293] T. Lanišnik Rižner, G. Moeller, H. H. Thole, M. Žakelj-Mavric, J. Adamski, T.

Lanišnik Rižner, M. Žakelj-Mavric, J. Adamski, Biochem. J. 1999, 337, 425–431.

[294] T. Lanišnik Rižner, J. Stojan, J. Adamski, T. Lanišnik Rižner, J. Stojan, J.

Adamski, Chem. Biol. Interact. 2001, 130-132, 793–803.

[295] S. Gao, Y. Li, J. Gao, Y. Suo, K. Fu, Y. Li, J. Chen, BMC Genomics 2014, 15, 627.

[296] R. A. Ohm, N. Feau, B. Henrissat, C. L. Schoch, B. A. Horwitz, K. W. Barry,

B. J. Condon, A. C. Copeland, B. Dhillon, F. Glaser, et al., PLoS Pathog. 2012,

8, e1003037.

[297] L. Liu, Z. Zhang, C.-L. Shao, C.-Y. Wang, Front. Microbiol. 2017, 8, 1685.

[298] N. Li, J. Xu, X. Li, P. Zhang, Fitoterapia 2013, 84, 85–88.

[299] Y. Henry, A. Guissani, L. Gilles, Biochimie 1982, 63, 841–845.

[300] H. D. Youn, K. J. Kim, J. S. Maeng, Y. H. Han, I. B. Jeong, G. Jeong, S. O. Kang,

Y. C. Hah, Microbiology (Reading Engl.) 1995, 141, 393–398.

[301] P. Baldrian, FEMS Microbiol. Rev. 2006, 30, 215–242.

[302] R. D. Sjoblad, R. D. Minard, J.-M. Bollag, Pestic. Biochem. Physiol. 1976, 6,

457–463.

[303] R. D. Sjoblad, J.-M. Bollag, Appl. Environ. Microbiol. 1977, 33, 906–910.

216 Bibliography

[304] M. A. Schätzle, S. Flemming, S. M. Husain, M. Richter, S. Günther, M. Müller,

Angew. Chem. Int. Ed. 2012, 51, 2643–2646.

[305] S. Husain, M. Müller, Synlett 2017, 28, 2360–2372.

[306] M. Kearse, R. Moir, A. Wilson, S. Stones-Havas, M. Cheung, S. Sturrock, S.

Buxton, A. Cooper, S. Markowitz, C. Duran, et al., Bioinformatics 2012, 28,

1647–1649.

[307] S. Obermaier, HPLC-MS method for the detection of naphthopyrones. (Collab.:

L. Fürtges), 2017.

[308] K. Tamura, J. Dudley, M. Nei, S. Kumar, Mol. Biol. Evol. 2007, 24, 1596–1599.

[309] S. Kumar, G. Stecher, K. Tamura, Mol. Biol. Evol. 2016, 33, 1870–1874.

[310] R. S. Bojja, R. L. Cerny, R. H. Proctor, L. Du, J. Agric. Food Chem. 2004, 52,

2855–2860.

[311] R. A. E. Butchko, R. D. Plattner, R. H. Proctor, J. Agric. Food Chem. 2006, 54,

9398–9404.

[312] R. H. Proctor, R. D. Plattner, A. E. Desjardins, M. Busman, R. A. E. Butchko, J.

Agric. Food Chem. 2006, 54, 2424–2430.

[313] C. Ouzounis, C. Sander, FEBS Lett. 1991, 279, 73–78.

[314] D. Blankenberg, J. Taylor, I. Schenck, J. He, Y. Zhang, M. Ghent,

N. Veeraraghavan, I. Albert, W. Miller, K. D. Makova, et al., Genome Res. 2007,

17, 960–964.

[315] C. J. A. Sigrist, E. de Castro, L. Cerutti, B. A. Cuche, N. Hulo, A. Bridge,

L. Bougueleret, I. Xenarios, Nucleic Acids Res. 2013, 41, D344–7.

217

[316] A. Krogh, B. Larsson, G. von Heijne, E. L. Sonnhammer, J. Mol. Biol. 2001, 305,

567–580.

[317] P. J. A. Cock, B. A. Grüning, K. Paszkiewicz, L. Pritchard, PeerJ 2013, 1, e167.

[318] P. J. A. Cock, J. M. Chilton, B. Grüning, J. E. Johnson, N. Soranzo, GigaScience

2015, 4, 39.

[319] H. Nielsen, J. Engelbrecht, S. Brunak, G. von Heijne, Protein Eng. 1997, 10,

1–6.

[320] J. D. Bendtsen, H. Nielsen, G. von Heijne, S. Brunak, J. Mol. Biol. 2004, 340,

783–795.

[321] E. I. Solomon, U. M. Sundaram, T. E. Machonkin, Chem. Rev. 1996, 96,

2563–2606.

List of Publications

The following list contains all publications accepted by journals as of January 1st, 2018.

Journal Articles

• Phylogenetic Studies, Gene Cluster Analysis, and Enzymatic Reaction Support

Anthrahydroquinone Reduction as the Physiological Function of Fungal

17β -Hydroxysteroid Dehydrogenase, L. Fürtges, D. Conradt, M. A. Schätzle, S.

K. Singh, N. Kraševec, T. Lanišnik Rižner, M. Müller, S. M. Husain, ChemBioChem

2017, 18, 77–80.

Posters

• Identification of biocatalysts in fungal genomes for the selective synthesis of biaryls:

L. Fürtges, M. Müller, Synthetic Biology of Antibiotic Production II, Sant Feliu de

Guixols (Spain), 30.08.–04.09.2014.


L. Fürtges, M. Müller, 34th REGIO Symposium 2014, Sornetan (Switzerland),

08.09.–10.09.2014.


L. Fürtges, M. Müller, Tag der Forschung, Freiburg, 03.07.2015.

Appendix

1 Bioinformatics: data and results

1.1 Galaxy workflows

The internet bioinformatics platform Galaxy was used for the analysis of fungal genomes

with the aim to identify enzymes responsible for regio- and stereoselective oxidative

phenol coupling. The platform offers the possibility to create workflows that can be

used as standard procedures on a huge number of samples. As two different coupling

enzymes (laccase, CYP enzyme) have been identified in this work, workflows were

developed to identify members of these enzyme classes in the genomes of homodimeric

natural product producers.

The workflows are designed to identify all CYP enzymes and laccases encoded by a

fungal genome. The most important bioinformatics tool used in the workflow is called

fuzzpro and searches for PROSITE motifs in a given dataset.[314,315] PROSITE motifs

are conserved amino acid sequences identified in certain enzyme classes that are typical

for members of this class (i.e., heme binding motif in CYP enzymes). Enzymes of a

certain class should contain this motif, but also fulfill other standards typical for the

class.

222 Appendix

Using fuzzpro as the core tool can cause some problems; the genomes that are

screened with the workflow must be annotated. The tool uses protein sequences as

input and the genes of the annotated genome of interest have to be translated before

being analyzed. An incorrect annotation will consequently complicate the identification

of coupling enzymes with the designed workflow. A PROSITE search can also be carried

out on the DNA sequence level, using the tool fuzztran.[314] This tool works fine with

procaryotes, because their genes do not contain introns, but it cannot be used for

eucaryotes. A motif that is interrupted by an intron will not be recognized and the

enzyme will not be identified as putative phenol coupling enzyme.

Cytochrome P450 Finder

The workflow is designed to identify all CYP enzymes encoded by a fungal genome.

The workflow uses the proteome of a fungal strain of interest. The annotation has to

be carried out separately by using tools like AUGUSTUS. The proteins are filtered by

size in a first screening step. CYP enzyme are known to be around 500 amino acids

long. Enzymes bigger than 600 amino acids are excluded from the analysis.

The remaining enzymes are analyzed with TMHMM 2.0 to identify transmembrane

regions.[316,317] The majority of fungal CYP enzymes belongs to class II of CYP enzymes.

Members of this class are bound to the membrane of the endoplasmatic reticulum (ER)

with a membrane anchor.[124] The transmembrane region identified by TMHMM 2.0

is limited to the first 70 amino acids of each protein. If the transmembrane domain

identified by TMHMM 2.0 is not in this window, the sequence is discarded.

In parallel, the protein sequences are screened by fuzzpro for the CYP enzyme heme

binding motif (PS00086, [FW]-[SGNH]-x-[GD]-F-[RKHPT]-P-C-[LIVMFAP]-[GAD]).

Enzymes that do not contain this motif between the amino acids 340 and 510 are

Bioinformatics: data and results 223

discarded. Additionally, the protein sequences are screened for the less specific

E-x-x-R-motif. This motif has to be identified between the amino acids 330 and

480.

Enzymes containing the heme binding motif at the defined position in the analyzed

sequence and enzymes containing the E-x-x-R motif at the definded position, are

compared. The enzymes containing both motifs at the correct positions are then

compared with the enzymes that have passed the TMHMM 2.0 analysis. Enzymes that

contain a transmembrane domain in the first 70 amino acids of their sequence and

additionally carry the heme binding and the E-x-x-R motif are considered CYP enzymes.

The enzymes are then compared with the non- redundant protein database of NCBI

via NCBI BLAST+ blastp and analyzed with the NCBI BLAST+ rpsblast tool.[220,318]

The first comparison yields homologues of the identified CYP enzymes, which can

be used to deduce the function the new CYP enzyme. The second analysis identifies

conserved domains and therefore checks if all identified enzymes match the sequence

requirements of the CDD for CYP enzymes.

The workflow was tested with ascomycetes’ enzymes from the UniProtKB containing

the PROSITE motif PS00086. A total of 116 amino acid sequences of fungal CYP

enzymes with an average length of 551 +/− 149 amino acids were used for the test

(Table 1). The workflow identified 103 sequences containing the PROSITE motif

PS00086 and 98 sequences containing both the E-x-x-R motif and PS00086 in the

defined intervall. 68 sequences contain the heme binding motif PS00086 and a

transmembrane domain in the defined intervals. A total of 67 sequences contain

all three motifs.

224 Appendix

Table1.

UniProtK

Bidentifiers

ofsequencesused

totest

theG

alaxyw

orkflow.

Un

iProtKB

identifi

erFu

nction

Proteinn

ame

Strainn

ame

Q12732

averantinhydroxylase

AflG

Aspergillusparasiticus

ATCC

56775O

13345O

-methylsterigm

atocystinoxidoreductase

AflQ


ATCC

56775Q

2UPB

1–

AclC

Aspergillusoryzae

ATCC

42149Q

2UPC

5–

AclO

Aspergillusoryzae

ATCC

42149D

4AY62–

AR

B_01131

Arthroderma

benhamiae

ATCC

MYA

-4681Q

2UPB

7–

AclB

Aspergillusoryzae

ATCC

42149Q

5ATG9

–A

pdEEm

ericellanidulans

ATCC

38163Q

2UPC

4–

AclL

Aspergillusoryzae

ATCC

42149Q

6UEH

4–

AflU


ATCC

56775Q

9UW

95versicolorin

Bdesaturase

AflL


ATCC

56775S0D

PM1

–A

pf7Fusarium

fujikuroiCB

S195.34

A9JPE2

–A

tmQ

Aspergillusflavus

Q6U

EF4–

AflV


ATCC

56775A

0A097ZPE4

–A

ndKEm

ericellavariicolor

Q5A

R55

–A

sqLEm

ericellanidulans

ATCC

38163S0D

S17–

Apf8

FusariumfujikuroiC

BS

195.34Q

5AR

27–

AusI

Emericella

nidulansATC

C38163

Q0C

S60bim

odularacetylaranotin

synthesisprotein

AtaTC

Aspergillusterreus

FGSC

A1156

Q5A

R32

–A

usGEm

ericellanidulans

ATCC

38163Q

9Y8G7

bifunctionalcytochrome

P450/C

YP505Fusarium

oxysporumN

AD

PH-P450

reductaseQ

2UN

A2

bifunctionalcytochrome

P450/C

YP505A3

Aspergillusoryzae

ATCC

42149N

AD

PH-P450

reductaseA

1CLZ3

–C

csGAspergillus

clavatusATC

C1007

A8C

7R4

inactiveC

loAClaviceps

fusiformis

Q2PBY6

elymoclavine

oxidaseC

loAClaviceps

purpureaM

1WEN

7elym

oclavineoxidase

CloA

Clavicepspurpurea

20.1Q

09736lanosterol14-α

demethylase

Erg11Schizosaccharom

ycespom

beATC

C24843

P30607–

CYP52A

2Candida

tropicalisP16141

–C

YP52A4

Candidam

altosaP10613

lanosterol14-αdem

ethylaseErg11

Candidaalbicans

ATCC

MYA

-2876Q

12664eburicol14-α

demethylase

CYP51

Penicilliumitalicum

P10614lanosterol14-α

demethylase

Erg11Saccharom

ycescerevisiae

ATCC

204508Q

9Y757–

CYP52A

12D

ebaryomyces

hanseniiP30612

–C

YP52C1

Candidatropicalis

P43083–

CYP52E1

Candidaapicola

P17549benzoate

4-monooxygenase

BphA

Aspergillusniger

P30608–

CYP52A

6Candida

tropicalisP30611

–C

YP52B1

Candidatropicalis

Q12585

–C

YP52D1

Candidam

altosa


Tabl

e1.

Uni

Prot

KB

iden

tifie

rsof

sequ

ence

sus

edto

test

the

Gal

axy

wor

kflow

.U

niP

rotK

Bid

enti

fier

Fun

ctio

nPr

otei

nn

ame

Stra

inn

ame

Q12

573

–C

YP52

E2Ca

ndid

aap

icol

aP2

1595

–D

IT2

Sacc

haro

myc

esce

revi

siae

ATC

C20

4508

Q4W

R22

–C

YP50

51C

1As

perg

illus

fum

igat

usAT

CC

MYA

-460

9P5

0859

lano

ster

ol14

-αde

met

hyla

seEr

g11

Cand

ida

glab

rata

ATC

C20

01P1

6496

–C

YP52

A3-

ACa

ndid

am

alto

saP3

0609

–C

YP52

A7

Cand

ida

trop

ical

isQ

1258

8–

CYP

52A

10Ca

ndid

am

alto

saQ

759W

0la

nost

erol

14-α

dem

ethy

lase

Erg1

1As

hbya

goss

ypii

ATC

C10

895

O14

442

ebur

icol

14-α

dem

ethy

lase

CYP

51U

ncin

ula

neca

tor

P106

15–

CYP

52A

1Ca

ndid

atr

opic

alis

P244

58–

CYP

52A

3-B

Cand

ida

mal

tosa

Q12

586

–C

YP52

A9

Cand

ida

mal

tosa

Q9Y

758

–C

YP52

A9

Deb

aryo

myc

esha

nsen

iiQ

1258

7–

CYP

52C

2Ca

ndid

am

alto

saQ

1258

1–

CYP

52A

5Ca

ndid

am

alto

saP1

4263

lano

ster

ol14

-αde

met

hyla

seEr

g11

Cand

ida

trop

ical

isP3

0610

–C

YP52

A8

Cand

ida

trop

ical

isQ

1258

9–

CYP

52A

11Ca

ndid

am

alto

saQ

4WR

17–

CYP

5051

A1

Aspe

rgill

usfu

mig

atus

ATC

CM

YA-4

609

Q4W

R18

–C

YP50

51B

1As

perg

illus

fum

igat

usAT

CC

MYA

-460

9E9

FCP5

–D

txS2

Met

arhi

zium

robe

rtsi

iATC

CM

YA-3

075

Q4W

Z65

–Ea

sMAs

perg

illus

fum

igat

usAT

CC

MYA

-460

9O

1382

0–

Erg5

Schi

zosa

ccha

rom

yces

pom

beAT

CC

2484

3S4

W28

3–

EqxH

Fusa

rium

hete

rosp

orum

P547

81–

Erg5

Sacc

haro

myc

esce

revi

siae

ATC

C20

4508

S0E2

Y2gi

bber

ellin

13-h

ydro

xyla

seP4

50-3

Fusa

rium

fujik

uroi

CB

S19

5.34

S0E9

M0

gibb

erel

lin20

-hyd

roxy

lase

P450

-2Fu

sari

umfu

jikur

oiC

BS

195.

34S0

E2U

7gi

bber

ellin

(GA

)14

synt

hase

P450

-1Fu

sari

umfu

jikur

oiC

BS

195.

34B

8NYW

9–

AFL

A_1

1481

0As

perg

illus

flavu

sAT

CC

2000

26Q

4WA

Z6m

ulti

func

tion

alcy

toch

rom

eP4

50m

onoo

xyge

nase

Af5

10As

perg

illus

fum

igat

usAT

CC

MYA

-460

9Q

4WA

X0

fum

itre

mor

gin

Cm

onoo

xyge

nase

Ftm

P450

-3As

perg

illus

fum

igat

usAT

CC

MYA

-460

9W

7MLD

1–

FUS8

Fusa

rium

vert

icill

ioid

esFG

SC76

00S0

AR

X1

–Fs

dHFu

sari

umhe

tero

spor

umA

1DA

65fu

mit

rem

orgi

nC

mon

ooxy

gena

seFt

mP4

50-3

Aspe

rgill

usfis

cher

ianu

sAT

CC

1020

Q9H

GE0

bifu

ncti

onal

cyto

chro

me

P450

/FU

M6

Fusa

rium

vert

icill

ioid

esFG

SC76

00N

AD

PH-P

450

redu

ctas

eS0

EE84

–FU

S8Fu

sari

umfu

jikur

oiC

BS

195.

34B

9WZX

6fu

mit

rem

orgi

nC

mon

ooxy

gena

seFt

mP4

50-3

Aspe

rgill

usfu

mig

atus

E9R

CR

4gl

ioto

xin

bios

ynth

esis

GliC

Aspe

rgill

usfu

mig

atus

D7P

I20

desm

ethy

l-de

hydr

ogri

seof

ulvi

nA

synt

hase

Gsf

FPe

nici

llium

aeth

iopi

cum

226 Appendix

Table1.

UniProtK

Bidentifiers

ofsequencesused

totest

theG

alaxyw

orkflow.

Un

iProtKB

identifi

erFu

nction

Proteinn

ame

Strainn

ame

P0CT93

O-m

ethylsterigmatocystin

oxidoreductaseO

rdAAspergillus

flavusA

2R6G

9ochratoxin

biosynthesisO

ta3Aspergillus

nigerC

BS

513.88Q

6F5E4aphidicolin

synthasePbP405-1

Pleosporabetae

Q6F5E2

aphidicolan-16-β-olhydroxylase

PbP450-2Pleospora

betaeQ

9C450

paxillinesynthase

PaxQPenicillium

paxilliB

8NJH

2leporin

Csynthase

LepHAspergillus

flavusATC

C200026

I7LRH

3lactone

synthasein

mycophenolic

acidbiosynthesis

MpaD

EPenicillium

brevicompactum

Q12599

–C

YP55A3

Fusariumlichenicola

P23295N

AD

Pnitrous

oxide-forming

nitricoxide

reductaseC

YP55A1

Fusariumoxysporum

A0A

0D9M

RV9

O-m


oxidoreductaseO

rdAAspergillus

flavusATC

CM

YA-384

B8N

HY4

O-m


oxidoreductaseO

rdAAspergillus

flavusATC

C200026

A0A

1R3R

GJ7

–A

SPCA

DR

AFT_517149

Aspergilluscarbonarius

ITEM5010

Q12645

pisatindem

ethylasePD

AT9Fusarium

solanisubsp.pisi

P38364pisatin

demethylase

PDA

6-1Fusarium

solanisubsp.pisi

B0Y6R

2Psi-producing

oxygenaseA

PpoAAspergillus

fumigatus

CB

S144.89

G5EB

19Psi-producing

oxygenaseA

PpoAAspergillus

nidulansATC

C38163

Q6R

ET3Psi-producing

oxygenaseA

PpoAAspergillus

nidulansQ

4WPX

2Psi-producing

oxygenaseA

PpoAAspergillus

fumigatus

ATCC

MYA

-4609A

4D9R

2pyripyropene

Abiosynthesis

Pyr3Aspergillus

fumigatus

ATCC

MYA

-4609Q

4WB

01azaspirene

oxidasePsoD

Aspergillusfum

igatusATC

CM

YA-4609

B3FW

T9radicicolbiosynthesis

Rdc4

Metacordyceps

chlamydosporia

B6H

JU3

glandicolinA

reductaseR

oqOPenicillium

rubensATC

C28089

B6H

JU5

roquefortineC

synthaseR

oqRPenicillium

rubensATC

C28089

A0A

1B4X

BI1

sordarinbiosynthesis

SdnQSordaria

araneosaA

0A1B

4XB

I3sordarin

biosynthesisSdnT

Sordariaaraneosa

A0A

1B4X

BH

0sordarin

biosynthesisSdnE

Sordariaaraneosa

G3Y420

7-deacetoxyyanuthoneA

hydroxylaseYanH

Aspergillusniger

ATCC

1015Q

6Q881

sirodesmin

PLbiosynthesis

SirCLeptosphaeria

maculans

Q12608

sterigmatocystin

biosynthesisStcB

Aspergillusnidulans

ATCC

38163Q

12609sterigm

atocystinbiosynthesis

StcFAspergillus

nidulansATC

C38163

Q00707

versicolorinB

desaturaseStcL

Aspergillusnidulans

ATCC

38163A

0JJT9N

-hydroxylaseofpretenellin

BTenB

Beauveriabassiana

M1W

080epipolythiodioxopiperazine

biosynthesisTcpC

Clavicepspurpurea

20.1Q

0C8A

1terrenoid

oxidaseTrt6

Aspergillusterreus

FGSC

A1156

O13317

trichotheceneC

-15hydroxylase

TRI11

Fusariumsporotrichioides

Q12612

trichodienehydroxylase

TRI4

Fusariumsporotrichioides

B8M

9J6stipitaldehyde

hydroxylaseTropD

Talaromyces

stipitatusATC

C10500

B8N

M64

S-deoxyustiloxinH

synthaseU

stCAspergillus

flavusATC

C200026

D7PH

Z6viridicatum

toxinbiosynthesis

VrtE

Penicilliumaethiopicum


Laccase Finder

This workflow is designed to identify all laccases encoded by a fungal genome. The

input for the workflow is the proteome of the investigated fungus. The workflow does

not include an annotation step, which provides the needed proteome. The annotation

has to be done separately using tools such as AUGUSTUS. The first filter step of the

workflow excludes enzymes with a length of more than 700 amino acids.

The remaining enzymes are screened for signal peptide sequences with the tool

SignalP 3.0.[319,320] The majority of fungal laccases is secreted and contains a signal

peptide sequence.[321] The identified signal peptide sequences have to be located at

the N-terminus in the first 70 amino acids. Enzymes without a signal peptide sequence

in the defined interval are excluded.

Additionally, the sequences (length < 700 amino acids) are screened for different

sequence motifs. The tool fuzzpro searches for PROSITE motifs of MCO of type I and

type II (Table 2). Enzymes containing one PROSITE motif are compared with enzymes

containing a signal peptide sequence in the defined interval. The workflow therefore

yields two output datasets: firstly, the enzymes containing a signal peptide sequence

in the defined interval that contain the PROSITE motif PS00079 as well. Secondly,

enzymes containing a signal peptide sequence and the PROSITE motif PS00080.

Table 2. Sequence motifs used by fuzzpro.

Sequence motif Sequence

PS00079 G-x-[FYW]-x-[LIVMFYW]-x-[CST]-x-PR-K-x(2)-S-x-LFH-G-[LM]-MCO type I x(3)-[LIVMFYW]PS00080 H-C-H-x(3)-H-x(3)-[AG]-[LM]MCO type II

228 Appendix

24 amino acid sequences of fungal laccases were used to test the workflow (Table 3).

The amino acid sequences were downloaded from UniProtKB and only contain entries

that are marked “curated” and “reviewed”. The selected sequences have an average

length of 606 +/− 43 amino acids, the longest with 677 and the shortest with 454

amino acids. The workflow identified 22 sequences with the PROSITE motif PS00080

and 24 with the the PROSITE motif PS00079. 22 sequences contain a signal peptide

that was identified by SignalP 3.0.


Tabl

e3.

Uni

Prot

KB

iden

tifie

rsof

sequ

ence

sus

edto

test

the

Gal

axy

wor

kflow

.U

niP

rotK

Bid

enti

fier

Fun

ctio

nPr

otei

nn

ame

Stra

inn

ame

E9R

BR

0D

HN

-mel

anin

synt

hase

Abr

2As

perg

illus

fum

igat

usAT

CC

MYA

-460

9Q

4WZB

41,

8-D

HN

synt

hase

Abr

1As

perg

illus

fum

igat

usAT

CC

MYA

-460

9I1

RF6

4–

Aur

L2Fu

sari

umgr

amin

earu

mAT

CC

MYA

-462

0P7

8591

iron

tran

spor

tm

ulti

copp

erox

idas

eFE

T3Ca

ndid

aal

bica

nsP3

8993

iron

tran

spor

tm

ulti

copp

erox

idas

eFE

T3Sa

ccha

rom

yces

cere

visi

aeAT

CC

2045

08P4

3561

iron

tran

spor

tm

ulti

copp

erox

idas

eFE

T5Sa

ccha

rom

yces

cere

visi

aeAT

CC

2045

08Q

0992

0ir

ontr

ansp

ort

mul

tico

pper

oxid

ase

Fio1

Schi

zosa

ccha

rom

yces

pom

beAT

CC

2484

3Q

96W

T3ir

ontr

ansp

ort

mul

tico

pper

oxid

ase

FET3

Cand

ida

glab

rata

ATC

C20

01Q

6CII

3ir

ontr

ansp

ort

mul

tico

pper

oxid

ase

FET3

Klu

yver

omyc

esla

ctis

ATC

C85

85Q

0CC

X6

dihy

drog

eodi

nox

idas

eG

edJ

Aspe

rgill

uste

rreu

sFG

SCA

1156

I1R

F62

auro

fusa

rin

bios

ynth

esis

GIP

1Fu

sari

umgr

amin

earu

mAT

CC

MYA

-462

0Q

0439

9–

GM

C1

Sacc

haro

myc

esce

revi

siae

ATC

C20

4508

J5JH

35be

nzen

etet

rold

imer

izin

gen

zym

eO

pS5

Beau

veri

aba

ssia

naA

RSE

F28

60Q

1257

0lig

nin

degr

adat

ion

Lcc1

Botr

yoti

nia

fuck

elia

naP0

6811

ligni

nde

grad

atio

nLa

ccN

euro

spor

acr

assa

ATC

C24

698

Q96

WM

9lig

nin

degr

adat

ion

Lcc2

Botr

yoti

nia

fuck

elia

naP7

8722

ligni

nde

grad

atio

nLA

C2

Podo

spor

aan

seri

naD

4APX

3–

AR

B_0

5828

Arth

rode

rma

benh

amia

eAT

CC

MYA

-468

1Q

0396

6lig

nin

degr

adat

ion

LAC

-1Cr

ypho

nect

ria

para

siti

caQ

70KY

3lig

nin

degr

adat

ion

LAC

1M

elan

ocar

pus

albo

myc

esQ

96U

M2

ligni

nde

grad

atio

nLc

c3Bo

tryo

tini

afu

ckel

iana

P174

89co

nidi

algr

een

pigm

ent

synt

hase

Lacc

ase-

1As

perg

illus

nidu

lans

ATC

C38

163

Q0D

1P3

–Te

rEAs

perg

illus

terr

eus

FGSC

A11

56Q

4WQ

Y8m

onom

ethy

lsul

foch

rin

cycl

ase

TpcJ

Aspe

rgill

usfu

mig

atus

ATC

CM

YA-4

609

230 Appendix

1.2 Biosynthetic gene clusters - Identifiers

Table 4. Protein identifiers of the putative xanthomegnin (33) biosynthetic genecluster in T. rubrum.

Name Protein identifier Function

XtrA XP_003235798 NR-PKSXtrB XP_003235797 FMOXtrC XP_003235796 SDRXtrD XP_003235795 FAS proteinXtrE XP_003235794 laccaseXtrF XP_003235793 O-MTXtrG XP_003235792 transporterXtrH XP_003235791 transcription factorXtrI XP_003235790 FAD/FMN-binding protein

Table 5. Protein identifiers of the putative xanthoepocin (74) biosynthetic genecluster in P. arizonense.

Name Protein identifier Function

XpaA OGE57635 NR-PKSXpaF OGE57961 O-MTXpaK OGE58450 NADH-binding proteinXpaB OGE57647 FMOXpaE OGE58328 laccaseXpaD OGE58471 FAS proteinXpaL OGE58256 hypothetical proteinXpaM OGE58115 transcription factorXpaI OGE58232 FAD/FMN-binding proteinXpaH OGE58398 transcription factorXpaG OGE58057 transporter


Tabl

e6.

Prot

ein

iden

tifie

rsof

the

pery

lene

quin

one

bios

ynth

etic

gene

clus

ters

ofC.

nico

tian

ae,C

.bet

icol

a,C.

zein

a,C.

zeae

-may

dis,

P.no

doru

mSN

15,S

hira

iasp

.Sl

f14,

and

E.am

pelin

a.C

.nic

otia

nae

C.b

etic

ola

C.z

eina

C.z

eae-

may

dis

P.no

doru

mSN

15Sh

irai

aSp

.Sl

f14

E.am

peli

na

CnC

TB1

AAT

6968

2Cb

CTB

1X

P_02

3460

065

CzC

TB1

PKR

9844

8Cz

mC

TB1

Cer

zm1|

4294

9El

cAEA

T837

82H

ypA

AIW

0065

8Pe

aAEl

sam

p1|2

6856

4Cn

CTB

2A

BK

6418

0Cb

CTB

2X

P_02

3460

064

CzC

TB2

PKR

9844

9Cz

mC

TB2

Cer

zm1|

4297

8El

cDEA

T837

78H

ypD

AIW

0066

1Pe

aDEl

sam

p1|2

6855

7Cn

CTB

3A

BC

7959

1Cb

CTB

3X

P_02

3460

058

CzC

TB3

PKR

9847

5Cz

mC

TB3

Cer

zm1|

4333

0El

cBEA

T837

80H

ypB

AIW

0065

9Pe

aBEl

sam

p1|3

0464

5Cn

CTB

4A

BK

6418

1Cb

CTB

4X

P_02

3460

063

CzC

TB4

PKR

9847

6Cz

mC

TB4

Cer

zm1|

4330

7Cn

CTB

5A

BK

6418

2Cb

CTB

5X

P_02

3460

059

CzC

TB5

PKR

9844

7Cz

mC

TB5

Cer

zm1|

4332

5El

cEEA

T837

76H

ypE

AIW

0066

5Pe

aEEl

sam

p1|4

3113

CnC

TB6

AB

K64

183

CbC

TB6

XP_

0234

6006

2Cz

CTB

6PK

R98

477

Czm

CTB

6C

erzm

1|43

281

CnC

TB7

AB

K64

184

CbC

TB7

XP_

0234

6006

0Cz

CTB

7PK

R98

490

Czm

CTB

7C

erzm

1|43

304

CnC

TB8

AB

K64

185

CbC

TB8

XP_

0234

6006

1Cz

CTB

8PK

R98

474

Czm

CTB

8C

erzm

1|98

426

CbAT

R1

XP_

0234

5882

0Cz

Atr

1PK

R98

472

Czm

ATR

1C

erzm

1|43

271

Ccb

FX

P_02

3458

817

Ccz

FPK

R98

444

Ccz

mF

Cer

zm1|

6866

0El

cFEA

T837

83H

ypF

AIW

0065

7Pe

aFEl

sam

p1|4

3118

Ccb

GX

P_02

3458

816

Ccz

GPK

R98

471

Ccz

mG

Cer

zm1|

1126

19El

cGEA

T837

84H

ypG

AIW

0065

6Pe

aGEl

sam

p1|4

3111

ElcC

EAT8

3779

Hyp

CA

IW00

660

PeaC

Elsa

mp1|4

3175

ElcR

EAT8

3777

Hyp

RA

IW00

663

PeaR

Elsa

mp1|4

3130

Hyp

HA

IW00

664

PeaH

Elsa

mp1|2

6856

0

E.fa

wce

ttii

C.b

etic

ola

C.z

eina

C.z

eae-

may

dis

P.no

doru

mSN

15Sh

irai

aSp

.Sl

f14

E.am

peli

na

EfTS

F1A

BZ0

1831

CbTS

F1X

P_02

3450

905

CzTS

F1PK

R97

989

Czm

TSF1

Cer

zm1|

6681

64Pn

TSF1

EAT8

0399

Hyp

AA

IW00

658

EaTS

F1El

sam

p1|3

6214

EfR

DT1

AB

Z018

30Cb

RD

T1X

P_02

3450

906

CzR

DT1

PKR

9799

0Cz

mR

DT1

Cer

zm1|

5940

6Pn

RD

T1EA

T804

02Sh

RD

T1A

IW00

661

EaR

DT1

Elsa

mp1|2

0469

7Ef

PRF1

AB

Z018

33Cb

PRF1

XP_

0234

5091

1Cz

PRF1

PKR

9801

3Cz

mPR

F1C

erzm

1|12

1290

PnPR

F1EA

T803

91Sh

PRF1

AIW

0065

9Ea

PRF1

Elsa

mp1|2

6775

0Ef

OX

R1

AB

Z018

32Ef

PKS1

AB

U63

483

CbPK

S1X

P_02

3450

912

CzPK

S1PK

R98

014

Czm

PKS1

Cer

zm1|

6816

1Pn

EfPK

S1EA

T803

93Sh

EfPK

S1A

IW00

665

EaEf

PKS1

Elsa

mp1|3

0387

5Ef

HP4

AB

Z820

12Ef

HP3

AB

Z820

11Ef

HP2

AB

Z820

10Ea

HP2

Elsa

mp1|2

6776

4Ef

HP1

AB

Z820

09Ef

ECT1

AB

Z820

08

232 Appendix

1.3 Cluster sequence comparisons

Table 7. Genes of the putative biosynthetic gene cluster of vioxanthin (3) inT. rubrum, P. citreonigrum and A. ochraceus.

T. rubrum P. citreonigrum A. ochraceus Sequence Identity [%] Putative FunctionT. rubrum vs. P. citreonigrumT. rubrum vs. A. ochraceusP. citreonigrum vs. A. ochraceus

XtrA VpcA VaoA 68.8 NR-PKS64.973.8

XtrB VpcB VaoB 70.6 FMO73.576.8

XtrC VpcC VaoC 78.2 SDR75.885.4

XtrD VpcD VaoD 57.0 FAS protein54.865.5

XtrE VpcE VaoE 79.6 laccase79.083.4

XtrF VpcF VaoF 72.5 O-MT67.074.1

XtrG VpcG VaoG 64.6 MFS transporter64.774.4

XtrH VpcH VaoH 55.9 transcription factor32.929.8

XtrI – – – FAD/FMN-binding protein

– VpcJ VaoJ 61.7 hemerythrin-like protein


1.4 PT-domain analysis

Table 8. Accession numbers of the 55 NR-PKSs used in the construction of thePT-domain tree.[151]

No.

Gro

up

Cyc

l.A

cces

sion

no.

Stra

inn

ame

Prot

ein

nam

ePr

odu

cts

1IV

C4-

C9

AA

A81

586

Aspe

rgill

usni

dula

nsA.

nidu

lans

PKSS

Tst

erig

mat

ocys

tin

2IV

C4-

C9

Q12

397

Aspe

rgill

usni

dula

nsFG

SCA

4A.

nidu

lans

StcA

ster

igm

atoc

ysti

n3

IVC

4-C

9A

CH

7291

2As

perg

illus

ochr

aceo

rose

usA.

ochr

aceo

rose

usA

flCafl

atox

in(2

0)4

IVC

4-C

9A

AZ9

5017

Myc

osph

aere

llapi

niM

.pin

iPK

SAafl

atox

in(2

0)5

IVC

4-C

9Q

1205

3As

perg

illus

para

siti

cus

A.pa

rasi

ticu

sPk

sAafl

atox

in(2

0)6

IVC

4-C

9B

AE7

1314

Aspe

rgill

usor

yzae

A.or

yzae

PKSA

aflat

oxin

(20)

7IV

C4-

C9

AA

S925

37Le

ptos

phae

ria

mac

ulan

sL.

mac

ulan

sPK

S1si

rode

smin

PL8

IVC

4-C

9C

CE6

7070

Fusa

rium

fujik

uroi

F.fu

jikur

oiFs

r1fu

saru

bin

(61)

9IV

C4-

C9

XP_

0030

3992

9N

ectr

iaha

emat

ococ

cam

pVI

77-1

3-4

N.h

aem

atoc

occa

PKS1

bost

ryco

idin

,fusa

rubi

n(6

1)10

IVC

4-C

9A

AT69

682

Cerc

ospo

rani

coti

anae

C.ni

coti

anae

CTB

1ce

rcos

pori

n(4

0)11

III

C2-

C7

CA

B92

399

Fusa

rium

fujik

uroi

F.fu

jikur

oiPK

S4bi

kave

rin

(82)

12II

IC

2-C

7A

AU

1063

3F.

gram

inea

rum

F.gr

amin

earu

mPK

S12

auro

fusa

rin

(30)

13II

IC

2-C

7Q

0314

9As

perg

illus

nidu

lans

FGSC

A4

A.ni

dula

nsW

AYW

A1,

naph

thop

yron

e14

III

C2-

C7

EHA

2852

7A.

nige

rAT

CC

1015

A.ni

ger

Alb

AYW

A1,

dim

eric

naph

tho-

γ-p

yron

es15

III

C2-

C7

AA

C39

471

Aspe

rgill

usfu

mig

atus

A.fu

mig

atus

Alb

1na

phth

opyr

ones

16II

IC

2-C

7ED

P552

64As

perg

illus

fum

igat

usA

1163

A.fu

mig

atus

PksP

THN

17II

C2-

C7

AA

N75

188

Exop

hial

ale

cani

i-cor

niE.

leca

nii-c

orni

PKS1

THN

18II

C2-

C7

AA

D31

436

Exop

hial

ade

rmat

itid

isE.

derm

atit

idis

PKS1

THN

19II

C2-

C7

AB

U63

483

Elsi

noe

faw

cett

iiE.

faw

cett

iiPK

S1el

sino

chro

mes

(41)

20II

C2-

C7

BA

D22

832

Bipo

lari

sor

yzae

B.or

yzae

PKS1

tetr

ahyd

roxy

naph

thal

ene

(TH

N)

21II

C2-

C7

AA

N59

953

Gla

rea

lozo

yens

isG

.loz

oyen

sis

PKS1

THN

22II

C2-

C7

AA

O60

166

Cera

tocy

stis

resi

nife

raC.

resi

nife

raPK

S1TH

N23

IIC

2-C

7A

AD

3878

6N

odul

ispo

rium

sp.

ATC

C74

245

Nod

ulis

pori

umsp

.PK

S1TH

N24

IIC

2-C

7A

BD

4752

2O

phio

stom

api

ceae

O.p

icea

ePK

SATH

N25

IIC

2-C

7B

AA

1895

6Co

lleto

tric

hum

lage

nari

aC.

lage

nari

umPK

S1TH

N26

IIC

2-C

7C

AM

3547

1So

rdar

iam

acro

spor

aS.

mac

rosp

ora

PKS

THN

27V

C6-

C11

XP_

0012

1707

2As

perg

illus

terr

eus

NIH

2624

A.te

rreu

sA

CA

Sem

odin

(83)

28V

C6-

C11

XP_

6577

54As

perg

illus

nidu

lans

FGSC

A4

A.ni

dula

nsM

dpG

atro

chry

sone

carb

oxyl

icac

id(7

8)29

VC

6-C

11X

P_74

6435

Aspe

rgill

usfu

mig

atus

Af2

93A.

fum

igat

usEn

cAen

docr

ocin

(87)

30V

C1-

C6

AD

I249

53Pe

nici

llium

aeth

iopi

cum

P.ae

thio

picu

mG

sfA

gris

eofu

lvin

31V

C2-

C7

XP_

6646

75As

perg

illus

nidu

lans

FGSC

A4

A.ni

dula

nsPk

gAde

hydr

ocit

reoi

soco

umar

in,c

itre

isoc

oum

arin

,al

tern

ario

l32

VC

6-C

11A

DI2

4926

Peni

cilli

umae

thio

picu

mP.

aeth

iopi

cum

Vrt

Avi

ridi

catu

mto

xin

33V

C6-

C11

XP_

0013

9470

5A.

nige

rC

BS

513.

88A.

nige

rA

daA

TAN

-161

2,B

MS-

1925

4834

VC

6-C

11X

P_66

3604

Aspe

rgill

usni

dula

nsFG

SCA

4A.

nidu

lans

Apt

Aas

pert

heci

n35

IC

2-C

7A

CD

3976

2H

ypom

yces

subi

culo

sus

H.s

ubic

ulos

usH

pm3

hypo

cem

ycin

36I

C2-

C7

AB

B90

282

F.gr

amin

earu

mF.

gram

inea

rum

PKS1

3ze

aral

enon

e37

IC

2-C

7A

CM

4240

3Ch

aeto

miu

mch

iver

sii

C.ch

iver

siiR

AD

S2ra

dici

col

38I

C3-

C8

AC

D39

770

Met

acor

dyce

psch

lam

ydos

pori

aM

.chl

amyd

ospo

ria

RD

C1

radi

cico

l39

IIC

2-C

7A

GC

9532

1As

perg

illus

terr

eus

A.te

rreu

sA

tCU

RS2

10,1

1-de

hydr

ocur

vula

rin

40I

C2-

C7

XP_

6811

78As

perg

illus

nidu

lans

FGSC

A4

A.ni

dula

nsO

rsA

orse

llini

cac

id(2

1)41

VII

IC

2-C

7A

FL91

703

Arm

illar

iam

elle

aA.

mel

lea

Arm

Bor

selli

nic

acid

(21)

234 Appendix

Table8.

Accession

numbers

ofthe55

NR

-PKSs

usedin

theconstruction

ofthePT-dom

aintree. [151]

No.

Grou

pC

ycl.A

ccessionn

o.Strain

nam

eProtein

nam

eProdu

cts

42V

IC

2-C7

XP_681652

Aspergillusnidulans

FGSC

A4

A.nidulansA

usA3,5-dim

ethylorsellinicacid,austinol,

dehydroaustinol43

VI

C2-C

7A

DY00130

Penicilliumbrevicom

pactumP.brevicom

pactumM

paC5-m

ethylorsellinicacid,m

ycophenolicacid

44V

IC

2-C7

XP_664052

Aspergillusnidulans

FGSC

A4

A.nidulansPkbA

3-methylorsellinic

acid,cichorine45

VII

C2-C

7X

P_660990Aspergillus

nidulansFG

SCA

4A.nidulans

PkiA2,4-dihydroxy-3-m

ethyl-6-(2-oxoundecyl)benzaldehyde

46V

IIC

2-C7

XP_658127

Aspergillusnidulans

FGSC

A4

A.nidulansPkdA

2-ethyl-4,6-dihydroxy-3,5-dim

ethylbenzaldehyde47

VII

C2-C

7B

AD

44749M

onascuspurpureus

M.purpureus

PksCT

citrinin48

VII

C2-C

7C

AN

87161Sarocladium

strictumS.strictum

MO

S3-m

ethylorcinaldehyde49

VII

C2-C

7X

P_658638Aspergillus

nidulansFG

SCA

4A.nidulans

AfoE

asperfuranone50

VII

C2-C

7X

P_659636Aspergillus

nidulansFG

SCA

4A.nidulans

PkhA2,4-dihydroxy-6-[(3E,5E,7E)-2-oxonona-3,5,7-trienyl]benzaldehyde

51V

IIC

2-C7

XP_660834

Aspergillusnidulans

FGSC

A4

A.nidulansPkfA

orsellinaldehyde52

VII

C2-C

7X

P_001212610Aspergillus

terreusN

IH2624

A.terreusATEG

_03432citrinin

53V

IIC

2-C7

AN

ID_07903

Aspergillusnidulans

FGSC

A4

A.nidulansPkeA

2,4-dihydroxy-3-methyl-6-

54V

IIC

2-C7

EHA

28237A.niger

ATCC

1015A.niger

AzaA

azanigeroneA

55V

IIC

2-C7

AG

N71604

Monascus

pilosusM

.pilosusPK

S5rubropunctatin,(2-oxopropyl)benzaldehyde

Sequences 235

2 Sequences

2.1 Primers

Table 9. Primers used for cloning of vavH.

Plasmid Primer Sequence

pKGH2 vavHpKGH2iFWD CAGACATCACCGTTTATGGAGCGTCATTTCvavHpKGH2iREV CGGCATCTACTGTTTCTAAGGATGGCTGTT

pSUC vavHpSUCiFWD GCCCTCCCACGCTAGATGGAGCGTCATTTCACvavHpSUCiREV AGTGGATCCTTGTACCTAAGGATGGCTGTTAAATG

Table 10. Primers used for cloning of PCV_5588.

Plasmid Primer Sequence

pET28b PCV5588pET28binF AGAAGGAGATATACCATGTCTGACAAAGCGCCPCV5588pET28binR CGAATTCGGATCCCGCGGGTTCCAAC

pESC-His pESC-5588-infFWD ACTTTTAAGCTAATTACAGACTGTTTCGCpESC-5588-infREV TAGGTAGCTATGATCAATGCCCAAGGTPCV_5588EcoRI TTGGGAATTCAAAAAATGTCTGACAAAGCGCCAACGPCV_5588SpeI TGACTAGTTTATTAATGATGATGATGCGGGTTCCAACC

Table 11. Primers to generate knock-outs of vaoA.

Primer Sequence

P1.5_PKS GTGTTAGCTTGACCGGATACP2.5_PKS GGCCTGAGTGGCCATCGAATTCATCGATGAGATAGAAGCTCGACP3.1_PKS GAGGCCATCTAGGCCATCAAGCGGGAACAATCAACCGAACTGP4.8_PKS GAAGGCAAACCAGGATAAGC

Table 12. Primers to generate knock-outs of vaoE.

Primer Sequence

P1.3_LAC GAGGTCGATTCTGGAAGCATP2.1_LAC GGCCTGAGTGGCCATCGAATTCGTGCGAAATTGAACCATGCGP3.3_LAC GAGGCCATCTAGGCCATCAAGCAGCGACTATAGAAACGGCCTP4.1_LAC GTCAGAATATTGTGCGCCGT

236 Appendix

Table 13. Primers to generate pFC333 plasmids for the knock-out of vaoE.

Primer Sequence

CRISPR-P1.333 CGCTGAGGGTTTAATGCGTAAGCTCCCTAATTGGCCCRISPR-P.vaoE ACTCGTTTCGTCCTCACGGACTCATCAGTTGACTCGGTGATGTCRISPR-P.vaoE GAGGACGAAACGAGTAAGCTCGTCTTGACTCTGACATGGGAGCTGTTTTAGAGCRISPR-P4.333 GGCTGAGGTCTTAATGAGCCAAGAGCGGATTCCTCA

Table 14. Primers to construct the expression plasmid pSUC.

Primer Sequence

pSUC-TcgrA-FWD GCTAGCTACGTACAAGGATCCACTAGTACAGCAGApSUC-TcgrA-REV TTGAATCGCGCATTGTTCGAATGATTCATGACGTATATTCACCpSUC-PsucA-FWD CGCTGAGGGTTTAATTAAGATCACGTGGATGGCCTpSUC-PsucA-REV TTGTACGTAGCTAGCGTGGGAGGGCCTTGGAG

Sequences 237

Tabl

e15

.Pr

imer

sus

edfo

rcl

onin

gof

vaoE

.Pl

asm

idPr

imer

Sequ

ence

pET1

9bva

oEpE

T19b

infF

WD

GACG

ACGA

CAAG

CATA

TGAA

CCTC

TCAT

GTCG

CCA

vaoE

pET1

9bin

fREV

AGCA

GCCG

GATC

CTCT

CATG

GCCC

TTTG

TCGG

CAT

vaoE

SPpE

T19b

infF

WD

GACG

ACGA

CAAG

CATA

TGGT

GGTG

AAAG

AAGA

GTT

pET2

8bva

oEpE

T28C

HFW

DAG

GAGA

TATA

CCAT

GATG

AACC

TCTC

ATGT

CGCC

vaoE

pET2

8CH

REV

GCTC

GAAT

TCGG

ATCT

GGCC

CTTT

GTCG

GCAT

Cva

oEpE

T28C

HSP

FAG

GAGA

TATA

CCAT

GATG

GTGG

TGAA

AGAA

GAGT

Tva

oEpE

T28N

HFW

DGT

CGGG

ATCC

GAAT

TCTA

ATGA

ACCT

CTCA

TGTC

GCva

oEpE

T28N

HR

EVGA

CGGA

GCTC

GAAT

TCTC

ATGG

CCCT

TTGT

CGGC

pET2

8B-C

His

-FW

DGA

TCCG

AATT

CGAG

CTCC

GTC

pET2

8b-C

His

-REV

CATG

GTAT

ATCT

CCTT

CTTA

ApE

T28b

-NH

is-F

WD

AATT

CGAG

CTCC

GTCG

ACAA

GpE

T28b

-NH

is-R

EVAA

TTCG

GATC

CCGA

CCCA

TTT

pESC

-His

pESC

-H-L

AC

infF

TTGA

AAAT

TCGA

ATTC

ATGA

ACCT

CTCA

TGTC

GCC

pESC

-H-L

AC

infR

AGTG

AGGG

TTGA

ATTC

TCAT

GGCC

CTTT

GTCG

GCpK

GH

2va

oEpK

GH

2iFW

DCA

GACA

TCAC

CGTT

TATG

AACC

TCTC

ATGT

Cva

oEpK

GH

2iR

EVCG

GCAT

CTAC

TGTT

TTCA

TGGC

CCTT

TGTC

SSgl

aApK

GH

2_P1

CAGA

CATC

ACCG

TTTA

TGTC

GTTC

CGAT

CTCT

ACTC

GCCC

TGAG

CGGC

CTCG

TCTG

CAC

SSgl

aApK

GH

2_va

oECT

CTTC

TTTC

ACCA

CGCG

CTTG

GAAA

TCAC

ATTT

GCCA

ACCC

TGTG

CAGA

CGAG

GCCG

SSm

coA

pKG

H2_

P1CA

GACA

TCAC

CGTT

TATG

TATC

CCTT

TCAA

TTCG

GACT

GGCC

TTAT

TGGC

ATCA

GTCT

TCSS

mco

ApK

GH

2_va

oECT

CTTC

TTTC

ACCA

CATT

GCTG

CCAT

TGGC

CCAT

GAGA

GCAG

AGAG

AAGA

CTGA

TGCC

AASS

vaoE

_FW

DGT

GGTG

AAAG

AAGA

GTTG

ACT

pMA

171

LAC

-5U

TRfw

dTT

AGCG

GCCG

CATA

GAGG

TCGA

TTCT

GGAA

GCAT

LAC

-5U

TRre

vTG

GCGA

CATG

AGAG

GTTC

ATGG

TGTG

CGAA

ATTG

AACC

ATGC

GLA

C-3

UTR

fwd

TATG

CGGC

CGCT

AATT

TCCA

GGAG

AGGA

GAAA

CGG

LAC

-3U

TRre

vTA

TGCG

GCCG

CTAA

TCTC

TTGC

GGAG

AATT

TGCC

pMA

-LA

Cin

fFAG

TTCT

AGAG

CGGC

CGCA

TAGA

GGTC

GATT

CTGG

AAG

pMA

-LA

Cin

fRAC

CGCG

GTGG

CGGC

CGCT

AATT

TCCA

GGAG

AGGA

GAA

pSU

Cva

oEpS

UC

iFW

DGC

CCTC

CCAC

GCTA

GATG

AACC

TCTC

ATGT

vaoE

pSU

CiR

EVAG

TGGA

TCCT

TGTA

CCTA

TCAT

GGCC

CTTT

GTCG

238 Appendix

2.2 Gene cluster sequences

Putative vioxanthin biosynthetic gene cluster sequences

Penicillium citreonigrum ATCC® 42743™

>vpcA

ATGGCGAATCAACTTCAAATCTATCTCTTTGGAGACCAAACACACGAGATTTCTGCCAAGCTACAGGCCCT

CCTCCATTCTAAGAGCTCGCCCATCTTACAGTCATTTTTTGATGAAGTCCACCACAAGCTTCGTGCAGAGA

TCGGACAGCTACCAGCTCGAGACAAGGAGATCTTCCCTCGTTTCTCTAGCATTGCGGAGATCCTGGCGTGG

AGAAAGAGACAGGACAAATACATCCAGGCAATCGAGAATGCCTTGACTTGCACCTATCAATTAGCCTACTT

TATTAGGTTTGTCGTCCCCCCCCCCAAAAAGAACGGCAACCCACGTAACAATATTGACCATGTGGTTGTCT

ATAGCCAACACGATGGATTTGAGAAATCCTATCCTTCGCCCGCAGAGACTTTAGTATCTGGTTTTTGCACT

GGCGCCCTCTCCGTGGCTGCCATCAGTTGCTGCAAGAGCGTCTCAGAGCTCCTTCCTGTTGCCGTCCAGAC

TGTGTCGATTGCGTTCCGTACTGGGCTCTGCACTTTGAACACTGCCAAGGCAGTGGAGCCGTCGGAAGGCA

ACTGGTCGATGGTGATCTCGGGAAGTTCGCCTGAGGATGTGGCGATAAGACTGCAAAAGTTTTCCCAATCC

AGACACCTTCCTCTCACCTCCCGGCCTTGGATCAGCGCCCATGCTGAGGATTGGTTGACCATCAGTGGAAC

GCCCGTCGTACTGGCCAGTCTTCGCCGCTCCGAGGAATTCGCCGGCATTCATTCACGATCGATCTCTGTTT

ACGCTCCTTATCACGCTCCGCACCTCTTCTCAAAGACCGATATTGAGGCGATCTTGGAAAAGACCCCCACA

GACGTTTGGGAGCGGTACCGTGGAATCTTCCCAGTTGTTTCGAGCACCACTGGCTCTTTGGTTCAGGCGGG

GAATTTCTACTGTCGATTAGAGGCAGCACTGGCTCAGATCCTTATCGAGCCTGTCAGATGGCCTGAGTTCA

CACAAGGAGTCGCATCCGCCCTCACTTCAACCGGGGCACCGCAATTCAAGATTATTCCGGTTGCTACGAAC

GCAGAAGCGACACTGTCTGCTGCTCTGAGCTCGAAGGTTGAGGCATCCAGTGCTCCCACAATTGATATAGA

GCCCCATACTAGCACTGATGAAAGCCAGGCCGACGTGACTGCTCAGGGACGATCGAAAATTGCCATCATTG

GCATGTCTGGGAGATATCCCAGCGCTGAGAACAACGAGGAATTCTGGGATCTCATTTTCAAGGGACTGGAT

GTCCATAAGGTCGTTCCTGATCTTCACTGGAGCGCGCGGACTCATGTGGATCCTACCGGCAAGAAGAAGAA

CACCAGCGCAACTCCCTATGGCTGCTGGCTCGAGCATCCTGCGGCGTTCGATGCCACGTTCTTTAGCATGT

CTCCTCGCGAAGCACCCCAGGTCGATCCTGCCCAAAGAATTGCGTTGATGACTGCGTATGAAGCGATAGAA

Sequences 239

CAAGCTGGGATTGTTCCAGATGCAACGCCGTCGACTCGCAAAGACCGAGTTGGTGTTTTCTACGGTGTTAC

CAGCAACGACTGGATGGAGACGAACAGTGCTCAGAATATCGATACCTACTTCATTCCTGGTGGTAATCGAG

CCTTTATCCCAGGACGCATCAACTACTTCTTCAAGTTTAGCGGGCCCAGCTACAGCGTCGATACTGCGTGC

TCGTCCAGTTTGGCATCGATCCATATCGCCTGCAACGCCTTGTGGAGAGGTGATATTGACACTGCCATTGC

TGGCGGGACCAACATCCTTACAAACCCTGATTTCACAGCTGGCCTTGACCGCGGGCACTTCCTTTCCCGCA

CGGGCAACTGCAAGACCTTTGACGACGGAGCTGATGGATACTGCCGGGGTGAAGGCGTTGGCACTATCATA

CTCAAGCGGTTGGAGGATGCAATTGCTGACAAGGATCCCATCCAAGGTCTGATACTTGGAACCTACACCAA

CCATTCAGCGGAGGCTGAATCCATTACCCGTCCCCACGTCGGAGCCCAGAGAGACATCTTCAGTAAGATCC

TGAGCAATAGTGGAGTCGACCCCTACAGTGTCAGCTACGTCGAAATGCACGGCACTGGCACCCAGGCCGGT

GATGCCAGAGAGATGTCGTCCGTTCTGGATACATTCGCTCCTGCAAACGCTCGTCAGTATCGTCAGGCTGA

TCAAGCACTGCATCTTGGCTCGGTCAAGGCCAACATCGGTCACGGTGAGGCGGCCTCGGGAGTGAGCGCTC

TCATCAAGGTTCTGCTTATGATGAAAAACAATACCATCCCTCCCCACTGTGGCATCAAGACAAAGATCAAC

TCCAAGTTCCCAGTGGATCTCGAGGCCAGGAATGTCCATATTGCTAAGGAGGCCAAACCCTGGAACCGCGT

GAACGGACAGCCTCGCAGGGTTTTCGTCAACAACTTCAGCGCTGCGGGTGGTAATTCCGCCGTGCTGCTTG

AAGACAAGCCTGTCGAGCTTCCAGTCAAAGGAACCGATCACCGCACATCTCATGTGGTTGCCGTCTCTGCC

AAGTCCCCAGCTGCAATTGCCAGAAATTTGAATTCGTTGATCGCCCATATTGACTCGCACCGCCAGGAGCC

TCTGTTCCTCCCTCAGCTCTCCTACACTACGACCGCCCGCCGTACTCACCATCTTCACCGAGTAATGGTTG

CCGGTTCGACCATTGACGACATCAAACGTCACTTGGAGGCTGCTGCTGCCCGCGGTGATGGTGCGACCAGA

CCCAAGTCTGCACCAAAATTAACCTTCGCATTCACCGGCCAGGGCGCCCAGTATGTTGGCATGGCTAAGCA

GCTCTATCAATCTTTAAGCCAGTTCCAGACTGATATCCGCCACTTTGATGAGCTTGGCCAAGCCTTGGGCT

TCCCGAGCTTCAAGGGAATGATCGACGACGAGTCGGGCATGGACATCAAAGAGTTCTCCCCGGTATCTGTG

CAGCTCGCCAGCGTCTGCATGCAAATGGCTTTGTCCAGACTCCTCATCTCTTGGAATATCATCCCTGAGGC

TGTTGTCGGTCACAGCCTTGGCGAGTACGCCGCACTCAATGTTGCTGGTGTCCTTTCTGACGCTGACACCA

TTTTCCTTGTCGGAAAGCGTGCTCAGCTGTTGGAGCAGCTCTGCACACTTGGCACGCATAGTATGCTTGTG

GTCAAGGCCGCCCTGTCGACGGTCAAGTCTGCCCTTGCCCTTGAGGAGACGGAGTTTGAGATTGCATGCCT

240 Appendix

CAATGCTCCAGAGGAGACGGTGCTTGCGGGACCAAACGAGAAAATCGAGGCTCTCCAGGAGACTTTGAGCA

AGAAGGGTCTGAAGTCGAAGACTCTCTCTGTACCTTTCGCCTATCACTCCTCTCAGGTAGATGCTATCTTG

GAAGATTTCGAGACCCTCGCTAAGGGCGCGACATTCCGCAAGCCTGCGATCCCGGTCATCTCTCCTCTTCT

CGCTGAGGTGATTACCAATGCTGGTGTCATTGGACCAGAGTATCTCAGACGCCATTGCCGTGAGACCGTCA

ATTTCCTCGACGCTATCCGATCCGCAAAGGACGATGGCACACTGAAGGATAGATCGTTTGTGGTTGAGATC

GGACCCCATTCGATCGTCACTGGACTGGTGAAGGCAATCCTCGGATCTGCCATCACCGCAGAACCAACGCT

CCAGCGCAACCGTGATACTTGGAAGGTGCTCACAGAGACCATCGCCTCTTTGTATTCTGCTGGTATAGACC

TCCGCTGGCACGAGTACCATCGCGATTTCGAGGCCTCTCACAAAGTCCTGGAGCTCCCAACGTACAATTGG

GTGCTCAAGGAATACTGGATGCAATACGTTCATGACTGGTCCTTGCGCAAGGGTGATCCACCTGTGGTCGC

TCCGTCGGTCACGCCGCTTGCGTCTACAACAATCCACACTGTGTTACTGGATACTATCGACAAGGTCACCG

TCGAGTCAGATATGTCGCGTCCTGATTTCAATCCTCTTGTGCAGGGTCACGAGGTTGATGGAGTCCCACTG

TGTACACCGTCTGTCTACGCCGACATTGCTCTTACAGTTGGCAAGTACCTTCTTGACCGCTATCACCCCGA

CATTCAAGAGCGCCTTGTCGATGTTGCCCACATGGAAGTCCTGAAGGCTCTGATCGCCAAGCCAGGGGGTC

CTCAGCCATTGCGCACATTCATCACTGCCGACTGGGCCGCTAAGAAAGCGAAGGCCCGCTTCTGTTCATTT

GACAAGAAGGGTGAACCAACAGTGGAGCATGCCAACTGCGAAATCCACTTCACGGATCGATCTCGCGTCAA

GAAACTGGAGGCTACTGCTCAAGAAGTCAAGGCGCGCATGGCAGCCATGCGCAAGACCCTGGAGAGTGGTG

AGACTCAAAGATTCAATCGCGTCATGGTGTACAAGATGATTAGTCCTCTTGCGAAGTTCCATAAGGATTAT

CGCCCAATCGACGAGGTGGTCATGGATAGTGACACCCGTGAATGTGCCAGCCGTGTGACCTTCAATGGTGT

TGAGGCCGCTGGAAATTTCCACACTCATCCGGCCTATATCGACGGTCTCACGCAAGCCGGCGGGTTCATCC

TGAACTGCAACGACAGTAACGATTTGGACGTTGAAGTCTTCGTCAACCACGGATGGGAGTCTCTGCAGCTC

TACGAGCCTCTTAGTAAAGAAATGACCTACAGGACATTCTGCCGGATGTTCGAGAAGGACAACCGGAAGTT

CCAGGGTGACGTGATTGTGTTCGATGAAAGCGACAAGATTGTGGCCAGCTATAAGGGCATTGTGTTCCAAG

GTGTTCCTCGTAGGGTGCTCCGTTACTTCTTGAAACCTGACGCTCCTAATGCTCGCGCGGTGCCACAACCG

AAGATCAACGGGAAGCCTGCAAGTCCAGTTGTGACTGCGCCAAAGGTTTCTGCGCCAGCAGCTCCTGCGGT

CATCAAGGCCGCCCCTGTTAAGATTGAACCTGCTTTGAAGGTCATTTCAGAGGAAAGCGGCGTTGCCTTCA

Sequences 241

GTGATTTGACTGACAACTGTGTCTTCTCAGACATTGGAATTGACTCCCTCCTCTCTTTGGTCATCACGAGC

AGATTTCGTGAGGAGCTCAACCTTGATCTAGAACTGGATGACATCTTCAGCACGTACCCGTCCGTGAAGGA

ACTCAAGAAATTCCTTGCCAAGCAGACAGGTTTCGCGGAGTCGGTAGCGGTGGAGGCACCTCAGCCTAATG

TCGTTACAAAAGCCACTGAGATCCTGGGCCAGGCACCTGCACTGTTGAACACGGCGGCGGTTGAGGTCGCT

ACCACCAGCGTTCAGGTTGGCGCTGACTTCGACGCTGCTCTTGCTGTCGTTTCTGAAGAGAGTGGTGTTGC

CATCTCAGAACTGACGGATGACTGCATCTTTTCCGACATTGGCATTGATTCTCTGTTGTCCCTGGTCATCG

TCAGCCGCTTCCGAGAAGAGCTAGATATGGACATTGAGATTGACTCCGTCTTTACGGACTATCCGACAGTC

AAAGATCTGAAGAGCTTGTTTGGACGAGGCGAGTCAGAGAGCACGACCCGCACCCCATCGGTCAGATTTGA

TAGTTCTGATCAGGAGGCGACTCCTTCGGCTACCTCCGCAGATTCCGACTTTGACCAAGACATAGGATGTC

TGAAGCAAGAACGCGCCCCTGTACCCGCGGCAACCTCTGTCCTCTTGCAAGGCATACCCAAGTTTGCTGAG

AAGACCCTGTTCCTCTTCCCCGACGGTGCCGGCTCTGCAACGTCGTACTCGGGAATTCCCAAGGTTGGCTC

CAAGGTCGCCATCATTGGTCTCAACTCTCCTTACTACAAAATTCCCGAGCACTTCAAATGCACGGTGGATG

ATCTCATCGACAGCTACATCACTGAAGTCCGCCGGCGGCAGCCAAATGGCCCATTCAACTTGGGTGGATGG

TCGGCAGGAGGCATTCTCGCTTATCGCGCGACTCAGAAGCTGATCGCGGCCGGGGAGACTGTTGACAATCT

CGTCCTGATCGATTCACCTGTTCCGAGGGGTCTTGACAAGCTGCCGCAGCATTTCTACGATTATTGTAACA

AGCTCCAGTTGTTTGGTCACGGTACAGGATCTGCAAAAACCTCCGCCAAGGCACCCGAATGGCTGATTCCC

CATTTCAATGCGACCATTGATACTTTGCATGACTACCACGCCACACCTCTCCCTGCCGCCAAGTCTCCAAG

GACATCAATTATCTGGGCCAGTGAGAGCGTGATGGACGGTGTGAAGGTCCCCAAGATGCCACCGCATCCGG

ACGACACCGAAGGTATGAAGTTCTTGACCGAGAATCGCACCGACTTTTCAGCGGCCGGATGGCAAGAGCTG

TTCCCGGGGGGGGAAATTATTCTAGATCGGACTGAGGGTGCAAATCATTTTTCGATGATGAGAGGAGAGCA

TGGTCCCAAACTGGCTTCTTTCATCTCTGGAGCGCTTGCATGA

>vpcB

ATGTCTGACAAAGCGCCAACGGTTGCGGTGTTGGGCTTGGGGGCTCTTGGCCTCGTTGCCATGAAGAACAT

GCTTGAGGAGGGCTTCAAGGTTACGGGATTTGATAGGAATCCATACGTTGGCGGTCTTTGGCACTACAATG

AGGGCGGCAATATCTCGGTGCTTGAAAACACGCCCGATTTCCCTACGGCAGCACACATGGCCAAATACTTG

242 Appendix

GTCTCCTATGCTGAGCATTTTGGTCTGATGCCCAATACTCGCTTGAACACCAGTATCCACCGCGCCGAATG

GAGTGAAAAGAAGAGAAAATGGGAAGTGGAGAGCTCACAGGTCGGCAGCAACGATCGGGTAATCGAAGATT

TTGACAAAGTGATATACGCGATGGGACCCGACCAGATCCCCAATGTCCCCAAAATTCCAGGTATCGAGAAG

TTCAAGGGAGATATAACTCATTCGATAGGTTTCAAGAAGCCTCATCAATGGGCTGGAAAGCGAGTCTTGGT

TGTAGGATTCGGTAATACCGCTGCAGACATTGCGGGCGTTCTCGTCGACGTTGCTGAGAAGGTTTACTTGT

CTCACCGCCATGGCGCGATTGTGTTGCCTCGTTGGGTGGACGGCAAGCCCGTGGACCACCAAAGGACCTAC

CGCAAGGGTTATCTACTGGGACTGATGTCCCGATACACTCCTAACCTGTGGATGAGAACAATGGACAGCTT

CATTCTGGGCATGAGAAACCGGATCTACGATTTGAAACCCGAATGGCGTCTCGACCCTGCCCCGTCGTTCA

ATCAGCAGCGACCCATTGTCAGCGATAATCTAATTGATAATCTCTCCAAGGGTCTGATTACGTCACTGTGC

CCGATCAAAGAGGTTCTCGATGCGATGACGGTCGAACTGACCGATGGAATCAAGGTGGAGGTCGATTCCAT

AATCTGGTGTACTGGATACACGGTGGATTATTCGATACTCGGGAAGAATAACCCAACCCTCTACAACGCGA

CAGAGAGCTTCCAGGTTGCCAACGGTCGCAAAGCGCCCCGTCTCTACCAGAATATCATCTCGCTGGAGCAT

CCCGAGTCTCTTGCTTTTATGGGCAACCTGTCGTTCATGAACCCCGCTTTTCTTATGTTCGATCTTGCCAG

CATGGCTCTCGCGCAGGTCTGGAAGGGTGCCTCCCCGCTACCGTCGAAAGCAGAAATGAATCGCCATGTGG

ACGAGCAGCACAAATGGATCGCTAGCTTGGCCAGCAACGGCCCCGTGACGCCTGGTCTGGTCAAGGGTATC

GACTGGATGGAGTGGGTGGATGAAGCTGCCGGACTTAAAATGGCTGAGAACTTGGGCTATGGCACAAAAGG

CTGGTACTTCTGGCTGACGGATCGCGAGTTCTGCAACATGGTCATGGACGGCCTCCTGCTGCCTTTCCAGT

ACCGACTCTTCGAGGGCGGCAAGAGAAAGCGCTGGGAGGGAGCGAGAGACGCCATCATCAAGGTGAACAAG

GAACTGAGGGCGAAGGGTTGGAACCCGTAA

>vpcC

ATGGCGATCGAGCCGAACGAAGCCCCTCTTCAGCACACGGAGTTGTTCGATGTCAGTGACTGCGTTGCAGT

TGTCACTGGAGGAGGCACCGGTATGGGTTTGATGATGGCCAAGGCTCTCGAGGCCAACGGCGCCAAGGTGT

ACATTCTGGGTCGGCGACTGGAAGTCCTCCAGGAAGCTGCAAAGCAATCGACATACGGAAACCTCTGCCCG

CTCCAATGCAACATCACCTCCAAAGAAGACCTGCAGAACGCAGTCGACCATATCACCAACGAGGATGGCTA

CGTGAACTTGGTTGTGAACAACGCTGGCATTTCCACCCCGAACCTGGGCTCGCAGAGAAGCCGCCCCAACG

Sequences 243

CGAAATGGGATGTCTCAGCTATGCGCAACTACTGGTTCAACAAGCCTTCCTTCGAGGACTATGCCAAGGTC

CTTGAAGCGAACACCACAGCTCCGCTGATGGTGACATTTGCCTTCCTGGAGCTCCTAGACAAAGGCAACCA

GGTGCGCGGCGAGCAGGCGAAGGAGAAAGGAAGCAAGGACTTCATCAGAAGCCAGGTGATCATGGTGAGCA

GCGTGGGCGGATTTGGCAGAGACAACTCGGCATTCATATACGGCGCGAGCAAGGCAGGCACGACGCAGATG

ACGAAGAATCTGTCCACGTATTTGATCCCCTGGAAGATCAGAGCGAACGTTATCGCACCGGGCTATTTTCA

CACTGAAATGACGGAAGACTTTTACAAGAGCACTGGAGGACGACTGCCAGCCTCAATGGCTCCCGAAGAGC

GTTTTGGTGACATGCAGGAGATTGGAGGGACCGTTCTCTACCTTGCATCGAAAGCGGGGGCCTACTGCAAT

GGCTCTGTCATGCTGGCTGATGGGGGCTATCTCGGAAACCACCCTAGCGCCTATTGA

>vpcD

ATGGAGCGCAAGAGACTCAGGCGCGTCCTTCACAAGGCGACGAGAGCGACGGTGGTGGCGGCCACCGTCGC

GCTCGGAGTCCAAGTATTCCTGCGCAATTCACAGAAAGATGTGAAGCCTCTCGGTGCGCTAGAAGATGAGG

CTATCGCCGCGGACTCGTCATTAGTGACAGTCTGGGAGCTTATCAGGAACGACACGAGAGTCTCCACGTTT

GCAAATATTCTTGGAGAATTCAAAGATATCGTCGGGGGTCTCAATGCGCCAAAGGCCAAGTTTACGGTGTA

CGCCCCAACGAACGAAGCGTTCGAGAAGGAGACGTTCGCCTGGGATCTACCCTCGTTCTACTGGCTGTATG

TGGTCGGTTACCACATGGGACCAGGCGCTTTCTCCATGGAAGATCTATCAAAGATGAACACAGCTCCATCG

TTCATCTTCGCCGACATCTTCCAGAACTACCGCCAGCGCATCAGTACGCAGAGTGAGGGGGGACGCTTCTC

GTTCAATCACAAAGCAAAATATGTCACGCCTGACATTGATGGAGTGAATGGATACGTACATCACATTGACC

GCGTTCTCATGCTTCCTGAATCCACCTCAGACCTCCTGCGCGAGGACCCCGATTTCAGCATGTTCCGAAAG

GGACTTATCCAGACCGATGTCGCCGTTGTCGTCAACGACACTTCCTCTCATGTCGGACAGACGGTGTTTGC

GCCGTCCAATACCGCGTTCAAGAAGCTAGGCTCTAAGGTCATCAAGTTCTTATTCAGCCCATACGGAAAGC

AATACCTGAAGGCTTTGCTTGAATACCATGTCGTGGCTAACCGCACGTTGTTTACCGATATCTACTTCCAG

GCCAATGGACAAGGGCAAATTCCACTAACGGAGGGATCGACTCTCGATCTTCCGACGCTACATCCCAAGCA

CAATCTTAGCATCTCCATCAACATGGACGGTTCCCGCAGCTGCCTGAAGATCAACGAGGTCAGAATAGCGC

AGGCTGATATCGTGGTCATGGATGGCGTCGTTCATAAAATTGACAACATCCTACTACCACCTAAGCGTGCT

CAAGAAGAAGTTGAGTCCCAGCAAGAGTCGTGGCTAGATTCGCTTCTGCGGTGGACAGCTGGGTATTCGAG

244 Appendix

GTTAGACATTGAGGAGCTGATGGAGAGGTTGCAGCCGTTCATCGATGACTCTGTCTTGTAG

>vpcE

ATGCATCTGCCCTCGTATTGTCGGATTCTCTTACTCTCTGCCCTGGCAGAGGCCGTGTCCGGTAAGATGGT

GAGAGAGGAACTGACTTTGGCGTGGGAGCGGAGGGCTCCAAATGGCCAGCCCAGAGATGTGATCACGATGA

ATGGGCAGTTTCCAGGACCTACGTTTGTTTGGGATGAAGATGATGATGTTGAGGTCATTGTACATAACAAA

ATGCCTTTCAACACGACCATCCACTGGCATGGGCTGATGATGCTGAATACCCCGTGGTCAGATGGCGTGCC

AGGCCTTACTCAGAAGCCTATTGAGATGGGCCAGTCATTTATCTATCGCTTTAAAGCTTCTCCTTCTGGAA

CGCACTGGTGGCACTCGCACACGCGGACAACAATGTTCGATGGATTATATGGTGCCCTTTATATAAGACCC

AAGCCCAGCAACCCAGCGCCTTGGTCACTGATTAGTAGTGATCCGAAGGATATTGCAGCCATGCAGAGGGC

CGTAGCAAATCCACATGTCATAATGGTTTCCGACTGGACTCAATTCAAGTCTTGGGAATACATGGACGCTC

AAGCGGCATCAGGGTATACGATCTTTTGTGTAGACAGCCTTTTGATAAACGGGAAAGGGAGTGTATACTGT

CCAGGTGAAGATTTCTTGGTCAACCATACGAGCAACTATATGAAGTGGGCTATTTACCCGGGCCACGTCAA

CGACAAAGGATGCCTTCCGTTCGTAAAGTCCACTGAGAATAAATATCTTGCCGATGGAAGGCCAGAGGCTA

TCCCATTACACCTGCAAAAGGGATGTGTCTCGTCGGAGGGCGACCGTGCAGTCATCGAGGTCGACCCTGCC

GATGAATGGGTCAGCCTTAACTTTGTCGACGGAGCCACCTTCAAGACGCCTATCTTCTCCATCGATGAGCA

CCAGATGTGGGTATATGAGGCAGACGGGCACTTTATCGAGCCCCGATTGGTTGACACGATCAAATTCTACG

CGGGCGAGCGCTACTCCGTCATGGTCAAGCTGAACAAGCAACCAAGAGATTACACAATCCGTGTCACCGAC

AGCGGGTTAACTCAAATTATTGCTTCGTATGCGACTTTGAGATACAAAGGTGGCCGCCAGGATCTTGGAGA

AACGCAGGGAATCATCAGTTACGGTGGCTTGAATACCACTGCTGTTATCACCCTCGATCGAGACTACCTGC

CGCCGTTTCCTCCCAATCCGCCAGCAGCACACGCCGATTCTATCCATCTCCTCGAGACGCACCGGTGGCAC

AGTGCCTGGCAATATACCATGACCGGCGGAGGAATGTACCAAGAAGATCGCAGCGCTTACGAGCCGCTTCT

CTACAATCCCTATTCTGCCGATGCTATGAACGAGAGCCTCGTCATCCGAACCAAGAATAATACCTGGGTCG

ATCTGGTGATCCAGGTCGGCTCCCTTCCAGAGCAACCACAAGAGTTCCCACACATCATGCACAAGCACACT

GGGAAGACGTGGCAGATTGGTGCTGGCGAGGGACACTGGAACTACTCCACAGTAGATGAGGCGATGCTGGC

CGAGCCAACAAAGTTCAATCTCAAGAACCCGAATTACAGAGACACTTTCATCACTTCCTTCGATGGGCCGT

Sequences 245

CTTGGATTGTGCTGCGATACCATTCGAACAACCCTGGTCCGTGGCTCATGCACTGCCACTTTGAGATACAT

TTGGGTGGTGGAATGGCCGTAGCAATCCTGGACGGTGTGGATGCTTGGCCAGATGTCCCGCCGGAATACAG

CGCGGATCAACGAGGCTTTTACCTCGATGATGGGCGGCAGCACACTGGATCGGAATCAGAGAAGCACGGCA

ATGGTGAGCCAGAAGCTGAGAACAAAGCCGACGAAGCTGCCTTGACTGGGGAGATTGCGAAGATTATCGAG

GGCCACAAGGGTCACCGTTTAGGATCGAAGAAAAATACCTGGCCATTCTCTCGCGCTACCCTTGCTACCTC

GATTGAGGCCTCAGTTTCCTTCTGA

>vpcF

ATGGCTCCAGCACTCGTCGCCGAACCCATCTCTTCCACAAAGCCGGTCGCCGCCGAAGAAAAGGCGACGGT

TCAGTCTACCCTCCAGGAGCTTGCGGATCAAGTCCAGCAGAATGCAAGGACCATCACTACTTTTCTTCGCT

CCTATGGACACCCATCACCTTCATTTGAACGGGATGCACCTACATCTACACTTCCTCCGTCTGCTCCAACC

GAAATCCGTGCGGCACGACTGGCCCTGATGGGAGCTGCTCTCAAGGCTTTCCAGTTGGCCGCTGGTCCTAG

TGAATATTTACCTAACTTGGCTGTTGGGTATCAATACACCGCATGTCTACGCTGGTTGACTCATTTCGATA

TTTTCCATCTGACTCCTCTCGCCGGATCCATTACATACAGCGAATTGGCTGCATCCGCAAAGGTCTCCACA

AAGCAGCTAAAAACCGTCGCGCGCATGGCTATGACGAACAACCTCTTTTGCGAGCCGGAGCCCAACACCAT

TGCACACACTGCTACATCTGCCCTTTTCGTGACGAACCAGAGTTTCCATGATTGGGCGTCTTTCATGTGCG

AGGCATCAGTTCCCATGGCCTCGAAATTGGTCGAAGCCTCTGAGAAGTGGCCTGGAAGCGAGGAGAAGAAC

CAAACCGCTTATAACATCGCTTTCGACACTGATATGCCATTCTTTGACCACTTGGCAACCCTGCCTGAGAA

GACGAAGCAGTTTGCGAGCTACATGAAGAACGTTCAGAACAGCGAAGGAACAGCTATCGGTTACCTTGTGC

ATGGATTTGACTGGGCCAGCTTGGGCAAGGCCACTGTCGTTGATGTTGGGGGCTCCACCGGTGCTGCAAGC

ATTGCCTTAGCCAATGCTTTCTCAGAGCTCAACTTCGTCGTGCAGGATCTTCCCGAAAATGCCACCGACGG

ACAAGTCGCCCTCGCTGCACAGCCAGGCAACATTTCCTCCCGCATCTTCTTCCAGGGCCACAACTTCTTCG

AAGCGCAGCCCTTCAAAGGCGCTGACGTCTACCTTCTGCGCATGATCCTACATGACTGGCCCCTGCGGGAA

GCCACGACGATCCTCAAGAACCTTGTACCTGCCTTGAAGAAGACCTCGCGCATCATCATCATGGACACTGT

TCTGCCCCGACCGGGTTCAATCCCATCTGTGCAGGAACGTCTCCTAAGAGCCAGAGACATGACTATGTTGC

AGGCGTTCAACAGTCTGGAGAGAGACCTAGAGGACTGGAAGGATTTGCTGCAGGGTGTAGATGAGAGACTG

246 Appendix

CGTATCGCCAATGTTGTGCAGCCCGTTGGGAGCGTCATGTCTGCATTGGAAATCGTGTTTGAGCTTTGA

>vpcG

ATGGCCATGAGGACATCCGACACACCCCTCTCTGCGAAGCCAGAGCAGTCTTTAAAGGTTGAAAAGGGAGA

TGCAGCTCCCGAGAAGCCAGCTCCTCTGAGTGAGGATGGCGGTGAATCGGGATCTCGGCTGGCGTTGATCA

TGGCCGCTATCCTTCTCGCCATGTTTCTTGTTGCGCTGGACCGAACGATTATCGCAACCGCTGTCCCCACT

ATCGCTAATGAATTTAACACCTTGAACGATATCAGTTGGTACGCGAGCGCTTACTTGATCACCAGCTGCGC

GACTCAGCTTCTGTGGGGACGTATCTACACGTTCTACAATACCAAGCTCGTCTTCCTAATTGCAATCTTGA

TCTTCGAAGTCGGATCTGCCCTGTGCGGTGGAGCTCCCAACTCCACCGCGTTCATCCTTGGCAGAGCCATT

GCTGGTATTGGATCAGCCGGCATTTTCTCAGGTGCGACTGTCATCATCACTCAGATAATTCCGCTGCAGAA

ACGTCCCATGTATATCGGATTCATGGGCTCTACCTTTGGCGTTGCTTCCATAGTTGGACCTTTGCTGGGAG

GAGCCTTTACTGATAGGGTGACTTGGAGGTGGTGTTTCTACATCAATCTTCCCATTGGTGGTTTTACTCTT

GCTGTTCTCGTCTTCTTCCTCCACGTCCCCGCGAACAAGAATTCAACGACATTGTCTCGCCAGCTGATCCG

ACTCGATCCTCTGGGCACCCTCCTCTTCCTGCCAGGAGTCATCAGCTTCCTCCTAGCCCTCCAATGGGGCG

GCAACAACTACGCCTGGAGCAACGGCCGGATCATTGCTCTTTTTGTTGTCGCTGGCGTTCTGTCCATTGCC

TTCAGCATCGTGCAAATCTGGCGCCAGGAAGATGCCACCATCCCTCCTCGCATTCTCCGCCAGCGAAGCGT

CTTCTTTGGTGCCATCTTCGCGCTCTGCATCGGTGGAGGGATGATATCCATGCTCTACACCTTGGCTCTCT

GGTTCCAAACCATCAAGGGTACCACGGCTGTCCATTCCGGTATTGACACCATCCCCATGGTCTTGTCGCTC

GTGCTTGGCTCCATCATCTCAGGATCTATCATCACAAGAACGGGATACTACGTTCCCTTCATGTACCTGAG

CACAATCCTGGTGTCCGTAGGGTCGGGCTTGATCACCACCTTCAAATTGTCTACTGGTCACTCAGCCTGGA

TCGGATACCAAGTCCTGTTCGGTTTTGGATTAGGTGTGGGTATGCAACAGCCAAGCATGGCTGCCCAGACG

GTGCTAGAATGGAAGGATGTCTCTACCGGTGTGGCCATTATGTTCTTCATGCAGTCCCTCGGCGGTTCAGT

CTTCATCTGTATTGGCCAGGCACTCTTCACCAACTACATCACGGAAAATCTCTCCACAATCCCTGGTATTG

ACATGAAGAAGATACTCGCCACAGGTTCTGTGGATTTGGCCAAGGTCGTGCCCGCCGATAAGCTGACGGAA

GTTCTGACCTACTACAACGAGGGATTGCGGAGGGCGTTTATTGTCGTCGTCGCGGTATCGTGCTTGATGAT

CCTGCCCACTCTTGGAATGGAGTGGAAGTCCGTGAAGGCCAAGCGTGAGGCTATGGCTAAAGCGATGGCTC

Sequences 247

AGGCAAAGGATCAACCTCAGTCTCCGGCTAAGACTGAGACAGACGCTCAACCTGACGCTCAAGCGGAAGAG

AAAGCTGAAGAGAAGGCCGATGCTTGA

>vpcH

ATGGTCGTGAGGCATCCCGCCAATGCGGGCTTTGTGCAAACGTGCATGCTTGTGCTGCTCGGACTCGGTGC

GGTCAGGGTCCAATCTCCAAGCGCTCGTATAGACTCGGAATATCAATACTCCTATACCAGGAATATCCATC

CATTGGACGAGGCTACCTCGGAGAACTCCACAGGCAAGCTGGATTTGGGCGCTGCGGCTGGAGAAACAATC

GAAGCGCACTGCGAGGAGGCTCTCCAGGCGCATGGGATCAAGCTTGACGAAGACCGTCTGCTGACCGACAA

GGCACCTTTTGATTTGTTGCCGACCCCTTCCTTGTCATCTCCCGATGGGCAGATGATAGTCAAGCGAGGAG

AATCCCGCTACGTAGAAAATTCTCTATGGAAAGGGCTGACGGACGAATTGCAAGATTCTGGTGATCCGGCC

GATGATTCAGATGACGAGTCGTCGCAAATCATGGAGACGAGATCCGTGAGCGGCCGGTTTCTTCTCATCGC

TGATTCGCGACCAGTGGTGTTGTACACCCTCCACCCGGCGGTGACACAGCTCTTTCAGCTTTGGCAAGTCT

TCCTCAATAATGTTGATCCCATTGTCAAGCTGTTTCATGCTCCCTCCGTTCAGCGGATGGTCCTGGAAGCT

GCGTCGGATCTTGGAAGCATTTCCAGAAGTACCGAGGCGTTGCTGTTCGCCATCTACATCTCCGCAATCAC

TTCCATGGATGAGGAGTCGTGCCAGAAAGTACTTTACAAGAGCAAATCCGACCTCGTGGTGCGCTTCTCGA

AAGCAGCTGAGCAGGCACTGGTCAATGCCGACTTCCTCAAATCGACAAACATTGTCGTTCTACAAGCGCTG

ACCTTGTACTTGCTTGCAACTAGTCGTCTGAATGATCCCCAGTCGCAATGGCTGCTCACGGGGCTTGCCAA

CCGTATCGGTCAGGCGATGGGCCTCCATCGTGAGGTGAGCCTTGGCAGCTTCTCGGCCTTTGAACGCGAGA

TCCGTCGACGGCTATGGTGGCAGATTCTCATCATGGATAGTCGCGCCGCCCAGCTCTCCGGTGTGGCGGTT

GACGCGCATTCATATCTCTGCTGGGACACGAAACGTCCCCTCAATTTGAACGATAGCGACCTATCACCTTC

GATGCGAGAACTACCTCCTGAGTATGAAGGGCCGACCGAAATGCTTTTTTGCTCAATTCGGTTCGAGATCG

GGGACTGCATGCGCCAATTGAAGGCAATCGAGAACGACCAGCCCAGAGCCAGCAGCGCCATACGGATGGCC

GAGGAGGCACGGGCGATCGACGCGCTGGAGACGCGTCTCGACCAAGGATACCTTAGAAAATGCGATTCGTC

TATCCCATTGCATCTGCTCGCCATCTATCTTGGACGATCGTCAGTCTGTCAGTTGCGGTTGTCGACTCACC

ACCCACAGATGTATCTGGACAAGGGCGCGGGCTTGTCCCAGGAAGAAAGGGACCGACTTTTCTTCCTGGGG

TTACAGATTCTTACCTACGAAAATCTGGTTTACTCCAACAAGAGCCTAGCGCCATATCTCTGGCACGTTTC

248 Appendix

TATCAACTTTCCGTTTGAAGCCTTCATTCTCATTCTAACGGAATTACTCACACGGAATGATTGCGAAATCA

TCGACCGAGCGTGGACGAAGGTCAACCAGGCGTATGAGAATCACCCGGAACTAATCACAGAGTCGAGGACA

AACGCGTTGTTCTTTGCCGTGGGCAGCCTCACCCTCCGCGCATGGGAGGTGCGAGTCACAGCTACTCGCAA

CCAACAGCTTCTCTATCAGCCGGTAGAGCCATCCTCCATCAGTGACCTCCGGGCACTCCGAAGGGGGTTTC

AAGTGCGATCATCGATGAACGCGGCGGATGGCTTCAACTCGACAGCACCAGCCGGATACCTGACCGCAGGT

GACACGATGAGCTTAGCAGAGACAGGGCCGGAGGGCGTTGTGCCGGTTCCTACTGACATGTCCTATATGGA

CTGGCAGTACTGGCAAGCACTCCTGGACGGACGAGTGGAACATCTCAACGACCGTTTATAA

>vpcJ

ATGTCATCAGCACGCGCAATCCGCGCCAGTAGCACCATTGCCTCTCGCCGGTTGGTCGCAAGTCGCTCTCT

TCATCTTTCAACTCCTTGCAAGCTAGGCCTCCAGCGGCAGCTTCAGCAATCGCTCTCCACGTCTCTCAAGA

TGCAACAAGCGGAGCAGCAACGCCAGGTTTGGGCAGACTCCCCTTTCTCTCTCATCACCAGTACTGGTGTC

GAAGCCAGGCCCGAGATCCCGCAGGACCATTATGCCCGTGAGTTCGCGCGCAGCATGGCGGGGATTCACAA

CGTACTCCTCCGCGCACTCAACGCTTCTTACAACCAGTGCCTCTCAATCTCTCCCGGTGATGAGGCGCGAG

ACTTTTTCATCTTCAACCAAGCCTTTTACACGATGCTGCAAAGCCACCACGATATGGAAGAGGAGTCTCTC

TTCCCGGCGATTGGGAAAGTGAGCGGCAACCCGGACGCCATGGCCGTCAATGTCCGCGAACATGCCGACTT

CGAAAAGGAACTCCTGCAATTCAAAAATTATATCTTTGAGACGGACCCAAAGGACTACGATGGTCCGCAGA

TGAAGTCCCTGATCGACCGCCTCGGCCCCCTGCTCCAGAAGCACCTGCACAACGAGATTTCCACGCTGTTG

GATCTCCACGTAGTTGGCAGCGCTGCGCTCAGGGGCGTCTTCAGTAACGCCGAAAGAGGCACCTCGGGAGG

AATGCACGATCTGTTTAAGCACATTCCGTTCACGCTGGTCTGTGCGGACAATACCTTCGAGCTGGACGGTC

AAGTGGACCCGCAATTTTTGCCCAACTTTGCGAAGCAGTTGCTCAAATTGACCTTTGGATGGCGGTACTCA

GGGGCGTGGCGTTTTGCACCCTGTGATTTCTCAGGAAACCCTCGTCCTCTGTCACTTCCCAATGCTTCGAA

TCTGTGA

Aspergillus ochraceus DSM 2499

>vaoA

Sequences 249

ATGGTGGATCAACTACACGTCTATCTCTTCGGGGACCAGACGTTTGAGGTCTCCACTAAACTTCCGCCACT

CCTTCATTCTAAGGCCACGCCACTGCTGGAGGCGTTCTTCGAGCAAGCGTACCACGCGCTTCGATCTGAAA

TTGCACAGCTCCCCGCTCGAGACAGAGACACCTACCCTCGCTTCTCGAGCATCGCAGAGCTTCTAGCGTGG

AGAAATCGACAGGAAAAGCTCCATCAGCCCATCGAGACGACCTTGACTTGTATCTACCAGTTAGCCCAATT

TATCAGGTTTGTTGCGTTCCCAAAAAAGGGCCACTCGGTTGCCCTCTGCAGTTGTCCAATTCATTGTCTGA

CCATGGCTAGTGAGCATGACGGATTTGGACGGGCCTATCCTTCCCCAACGGAGACGTGTGTGTCCGGTCTA

TGCACAGGTGCGCTGGCTGCGGCCGCAGTCAGTTGCTGCAACACCGTTTCGGAGCTTCTTCCAGTTGCCGT

CCAGACCGTTCTCGTGGCACTTCGCACAGGACTTTGCGCGTTGAACGCCGCCAAGGCTGTCGATCAGTCCG

AGGGAAATTGGTCGATGGTGCTTGTTGGCCTTTCGCAACAGGAGGTTTCGGACCGGCTACAGGAACTTTCC

CACTCGCACCGCCTTCCCGTCACTTCCCAGCCCTATATCAGCGCCTACGCGGAGAATTGGCTGACAATCAG

TGGACCGCCTCGCGTACTCGGTCTTCTCCGTGGTTTGGAGAACTTCACAACGGTTCATCCACGCACAATTC

CCGTTTTTGCTCCATATCATGCCTCACATTTGTTCACGAAGGCGGATATTGAGGCTATCTTGTCGACGACG

GCTGACGGTGTCTGGGGCCGTTATAGTGGGGTTCTACCAGTCGTTTCGAGCACCACCGGCAGTTTTATCGA

AGGGGATAACCTTCGACGTCGCTTAGAAGCTGCGCTGTTTCAAATCCTCTTGGAGCCTGTCCAATGGGACC

GGCTCACACAGGGGGTAGCCTCCGTCCTCTCTTCAAGTGGTGCATCCCAGTTCACGATCAGCCCAGTTGGC

ACAAATGCTGACACTGCTCTCGGGGTTGCTCTCAACTCCACAGCGGGGTTGTCTAATGGAGGCATCCCAGA

GAGAGCTCCTCGTCACGTTGAGAGTGATCAGGGTAAGCCCACCGGTTCGACAGGTGGCTCAAAAATCGCCA

TCATTGGCATGTCGGGCCGATACCCCAGCGCCGAGAACAACGAAGAATTCTGGGATCTGATTTTCCAAGGA

CTGGACGTGCATAAAGTCGTCCCCGATCTTCACTGGAGCGCACAGACGCATGTGGATTTGACAGGAAAGAA

GAAGAACACAAGTGCGACGCCGTACGGCTGCTGGCTGGAATCCCCTGCCGCGTTCGATGCCCCGTTCTTCA

ACATGTCTCCCCGCGAAGCACCACAGGTAGACCCGGCGCAAAGGATCGCCTTGATGACCGCTTATGAAGCC

ATTGAACAAGCTGGCATTGTCCCCGACGCAACGCCGTCGACTCGCAAGGATCGCGTGGGTGTCTTCTACGG

CGTGACCAGTAACGATTGGATGGAGACCAATAGCGCGCAGAACATCGACACCTACTTCATCCCGGGCGGTA

ATCGTGCATTCATCCCAGGCCGTCTCAACTACTTCTTCAAGTTTAGCGGTCCAAGCTACAGCGTGGACACG

GCCTGTTCGTCGAGTTTGGCATCTCTCCATGTGGCCTGCAACGCCTTGTGGCGAGGCGATATAGATACTGC

250 Appendix

AATTGCCGGTGGAACCAACGTTTTGACCAACCCAGACTTCACGGCCGGTCTGGATAGAGGACACTTTCTGT

CCCGGACGGGAAACTGCAAGACCTTTGATGATGGCGCCGACGGATACTGCCGAGGCGAAGGAGTTGGCACC

GTCATCCTGAAGCGGCTCGAAGATGCAGTGGCTGACAAGGACCCAATCCATGGCCTGATTCTCGGAACGTA

CACAAACCATTCCGCGGAGGCAGAATCCATTACGCGTCCCCATGTCGGAGCCCAGAGAGACATCTTCCAAA

AGATCTTGAATAGCTGCGGAGTTGACCCTTACAGCGTCAGCTATGTGGAGATGCATGGGACCGGTACCCAA

GCGGGTGATGCCAGAGAGATGTCGTCCGTCCTGGATACCTTCGCTCCCACGAATACCCGCCACCAACGGCA

GCCCGACCAGGCCCTTCATCTCGGCTCGGTGAAAGCCAACATCGGACATGGCGAGGCGGCCTCTGGCGTAA

GCGCCCTCACCAAGGTTCTGCTCATGATGCGAAACAATACCATACCTCCCCACTGCGGCATCAAAACAAAG

ATCAACTCTAAGTTCCCGTCAGACATGGAGGCGAGAAATGTGCTTATTGCAAGGGAGCCGATTCCGTGGGA

ACGCGCTTGTAACGAGCCGCGCAGGGCATTCGTGAACAACTTCAGCGCCGCAGGAGGCAATTCGGCGGTGC

TGCTTGAAGACAAGCCTGACGAAGTCGAAATCGAGGGAACCGATCCCCGGACAACCCATTTGGTTGCCGTT

TCCGCCAGGTCCTCAGCTTCCATGGCGGCGAACCTGAAGTCACTGCTGGCGTACATTGAAGGGCAGCAGGA

TAAGCCACTATTCCTGCCCCAGCTGTCGTATACTACGACCGCTAGGCGCATGCACCATCTCCATCGAGTCA

TGCTTGCTGGGTCAACAACCGAGGAAATCAAGCATCATCTGGAAGCAGCAATCTCCCGCGGCGATGGCGCA

ACGAGACCGAAGTCGCCTCCGAAAATGGTCTTTGCCTTTACAGGCCAGGGAGCGCAGTATGTTGGCATGGC

CCGAACGCTCTACCGAAGTTTCCGCCAGTTCCAGGATGATATATCCCGCTTTGATGAGCTGAGCAAGGCAT

TGGGCTTTTCAGGTTTCAAGGACATCATCGTGGATGAGTCGGGGAGAGACATCCAGGAGTTCTCCCCAGTA

GCGGTGCAGTTAGCCACCGTCTGCATGCAAATGGCATTGACCAGGCTGTTGATATCGTGGAATGTGACGCC

TGATGCCGTTGTCGGCCACAGCATTGGGGAGTATGCCGCCCTGAACGTTGCCAAGGTTCTCTCAGACGCAG

ATACGATTTTCTTGGTGGGCAGGCGCGCGCAATTACTGGAAGAGCGATGCACCCTTGGCACACATAGCATG

CTGGTCGTCAAGGCCGGCCTGTCGGAGGTTCAAACTGCTCTTGCCGGAGCAACTCATGAGATTGCATGCGT

CAATGCTCCAGAGGAGACTGTTCTCGCAGGACCGAACGAGCAGGTCGAAGAGCTGCAGCAGGTATTGAGCG

GCAAAGCATTGAAATCCAAAATCCTTTCCGTTCCTTTCGCCTATCACTCTTCTCAAGTCGACCCCATCCTA

GGCGATTTGGAAGACGTCGCAGGCGGAGCAACGTTCCACCAACCGACGATCCCAGTTATATCCCCACTCCT

GACCACCGTCATTGAGAATGCCGGCATTGTTGGACCAGAGTATCTGAGACGCCATTGTCGTGAGACTGTCA

Sequences 251

ATTTTGCAGATGCTCTTCAGTCCGCAAAGGCCTATGGAACAATCAAGGACGGGTCCTTCGTTGTCGAGATC

GGACCACATAGCATCGTCACCGGAATGGTGAAAGCGACCCTCGGGCCTGCGGTGGCCGCCGCAGCAACACT

CCAGCGCAACAGAGATGATTGGAAAGCGTTGACCGATATGCTCTCGTCTCTGTACCGTGCTGGCATTGATA

TTCGCTGGCACGAGTATCACCGGGACTTTGCAGCGGCTCACAAAGTCTTGGAGCTGCCGGCATACAAATGG

AACCTCAAGGATTACTGGATGCAATATACCCATGACTGGTCCTTGCGCAAGGGTGACCCACCTCTCGTCGC

CCCATCGGTTGCACCCCTCGCATCTACGACGATTCACAATGTGGTGGAGGAGAGCATCGATGCCATCACCG

TGGAGTCAGATATGGCACGGGCCGATTTCAATCCTCTGGTACAAGGCCATGAGGTAGATGGGGTTCCGCTG

TGTACACCGTCTGTCTACGCCGATATCGCGCTTACCGTTGGCAAATATCTGCTCCAGCGCTATCACCCTGA

AATGGAAGAATCCCTGGTTGATGTTTCCAACATGACTGTCATGAAGGCCCTCATTGCAAAACCGGAGGGCC

CCCAACCGCTGCGCACATTTGTGACGGTGGACTGGGCAGCTAAGAGAGCGAAGGGCAGATTCTGTTCTTTT

GACCTCATGCAGAAGAAGGGTCAGCCAACTGTAGAGCATGCCAACTGCGAGATTCACTTCACCGACAGATC

TCGATTGGAGCAGCTGGAACTAACGGCTCACGAGAAAAAGGCACGGATGGCGAGTATGCGCAAGGACCTGG

AGAATGGTCCGACACAGCGGTTCAACCGAGCCATGGTGTACAAGATGATCAGTCCACTCGCTCAGTTCCAC

AAGGACTATCGGCCTCTTGATGAGGTTGTCATGGATAGCAACACTTATGAGTGCGCCAGTCGGGTGAGTTT

CAAGGGCGTTGAAACTGAGGGCACGTTTCATACCCACCCAGCTTACCTGGACGGTCTCACCCAAGCTGGTG

GGTTTGTTATGAACTGCAACGACAGTAATGATCTAGATGTGGAGGTCTTCGTCAATCATGGATGGGAGTCC

TTGCAGGTCTACGAGCCTCTCAGTAAAGATCAAACCTATTCGACCTTCTGTCAAATGTCCGAGAGGGATTC

AAGAAAATATCAAGGAGACGTGACGGTTTTCGATGAAAGCTGGAGGGTTGTCGCCAGCTACAAAGGTATTG

TGTTCCAAGGCGTTCCACGAAGGGTCCTCCGCTTCTTCTTGACCGTCGACGGGGTCAAACCACCTCGTGGT

CAGCCGCAACAGAAGGGCAACACAGAACCAGCACGGTCCGCCGCAACCGTATCCAAACCTTCCATGCCCTC

GGTCCCTGCTGTCAGCAGCAAGACCACAGGAGTCAATAGCCCAGCCCCTGCCAGCCGGCCAAAGCCTTCAG

TTGCTGAGCCGGCTTTGAAGATCATCTCAGAAGAAAGCGGTCTCTCCCTGGGTGATTTGACAGATGACTGC

GTCTTCGCTGACATTGGGATCGATTCCCTTCTCTCCTTGGTCATCACGAGCCGGTTCCGTGAGGAACTCAA

TTTGGACATGGAGTTTGATGATATCTTTACCAGATACACGTCCGTGAAGGAACTGAACAGCTTCCTCAGAC

AATACGGCGGGGGGGAAGATCCAGCCCCTTCAGACAGCATATCTGAGCAGCCTCGTCCCGTCAGAGATGCA

252 Appendix

GTGCCTGAAATCCTGGGGCAAGCACCCGAATTTTCCGCCACGCAGGCCGTCCCCGATACCGGCCTGTACGT

CGGCGCCGTCTTTGATGCCGCCCTTGGTATCATCTCTGAAGAAAGTGGTATGGCTGTCTCGGAGTTGACGA

ATGACTGCGTATTCGCCGACATTGGCATTGACTCGCTTTTGTCGCTTGTCATCGTCAGCCGCTTCCGTGAG

GAGTTAGATCTAGACATCGACATGGATTCCGTCTTTATCGACTATCCGACAGTACAAGAGTTGAAGACATT

GTTCAAGCCGGCAGCCTCTGAATCTGAATACACCTGCCGGACTCCATCCGAACAACTCGACCGGTCGGATC

AAGACATCTCTTCTGGAAATCGCTCCTCGGCAACTTCTCAGGGCTCGGACTCCGAGCAGGATGCCGCTGAC

CTGAAGGAGGAACAAGCTACTGTGCCGGCCGCGACTTCCGTGCTCTTGCAAGGAATCCCCAAGTTTGCCGA

AAAGATCTTGTTTCTTTTCCCCGACGGCGCTGGTTCGGCAACGTCCTACTCTGGAATCCCAAGGTTAGGGT

CGACTGTGGCGGTGATCGGCCTCAACTCTCCTTATTACAAGATCCCGGAGCTCTTCAAGTGCACCTTGGAC

GCCCTTATCGACAGCTACATCACTGAGGTCCGCCGCCGCCAACCCCACGGGCCGTACCACCTCGGTGGCTG

GTCCGCCGGGGGCATCCTTGCCTATCGCGCCGCGCAGAAACTAATCAACGCCGGGGAGACGGTGCACAATC

TGCTGCTGATCGACTCACCCGTGCCCAAGGGTCTTGACAAGCTTCCCCAGCATTTCTACGACTACTGCAAC

AAGCTGCAACTATTTGGTCAGCCGACCACCTCAGGGTCTGAGCGCGCAGCAGCAGCCGCCAAACCTCCAGC

ATGGCTAGTCCCCCATTTCAATGCCACGATCGACACGCTGCACGATTACTTCGCCACGCCTCTACCGGTCG

GCAAGGCTCCGCGCACGTCGATCATCTGGGCGTGCGAGAGTGTCATGGACGGGAAGAATGTGCCCAAAATG

CCACCGCATCCGGACGATACCGAGGGCATGAAGTTCTTGACGGAGACTCGCACCGACTTTTCGGCCAGCGG

ATGGGAGGCTTTGTTTCCCGGTGGCACAATCATTTTAGATCGAACGGAGGGGTTGAATCATTTCTCGATGA

TGAGACGAGAACACGGGGCTAAGTTGGCGGAGTTCATTTCCCAGGCTCTGGCATGA

>vaoB

ATGTCCGAGAAAAGCCCGTCAGTTGCCGTTCTGGGCTTAGGAGCGCTTGGCCTCGTGGCGATGAAGAACAT

GCTTGAGGAGGGATTCAATGTCACCGGGTTTGACAGAAACCCATACGTTGGCGGCCTCTGGCACTACAACG

AGGGCTCTACCATCTCAGTGCTCCAAAGCACCGTCTCCAATGGATCAAAGCATAGGGGCTGCTTTACAGAC

TTCCCATTTCCAGACGACACTCCTGATTTCCCTCACGCAACAGATATGGCCAAATACTTGGTTTCGTACGC

CGAGCATTTTGGTCTTATGCCCCATGCACGCCTTAGCATCAATATCCACCGTGCAGAGTTCTCAGAGAAGA

CGAACAAATGGGAGGTGGAAATATCGCCAGTCGCTGGCGGACCCCGGGTGGTCGAAAAGTTCGATAAAGTG

Sequences 253

ATCTATGCCATGGGACCAGACCAGGTTCCCAATATCCCCAAGGTGCCAGGGATCGAGAAATTTAAAGGAAA

TATCACTCACTCTATTGGATTCAAAGAACCGGAGGACTGGACCAGAAAGAGGGTCTTAGTAGTTGGATTCG

GGAATACAGCAGCGGACATTGCGGGAGTCCTGGTGGGTGTTGCCAAGCACGTCTACCTGTCGCACCGCGGA

GGTGCCATCGTGCTCCCTCGATGGGTGGATGGGAAGCCGGTTGACCATGTCCGGACGTACCGAAAAGGGTT

TCTGCTAGGCTTGCTCTCACGGTACGCCCCGGGCCTGTGGAAAAAGACCGTGGACAAAGTCATTCTAGGGC

TACGCAATCGGCTATACGACTTGAAGCCTGAATGGCGGCTAGACCCGGCACCGTCCTTCAATCAGCAGAGG

CCAATCGTTAGCGACAACCTGGTGGATAATCTTTCGAAGGGACTCATTACGTCCGTGTATCCGATTAAGGA

AATCCTTGACGATGCGACGGTCGAATTTAGTGATGGCACCAAGGTAGAAGTCGATGCCATCATCTGGTGCA

CTGGATATACCATCGATTACTCCATGCTTGGAAAAGCCAACCCTACCCTCTACCATGAAATGGAGGGAATC

CCAGTTGCCAATAATCGAAAGATGCCCCGTCTCTACCAGAATGTCATATCACTTGAGCATCCCGAGTCCCT

CGCCTTCATGGGCAACCTGTCATTCATGAATCCGGCCTTCCTGATGTTTGACATTGCTAGCATGGCTCTCG

CCCAAGTTTGGAAAGGAAGGTCACCTCTACCCTCGAAGGCGGAAATGAGGCGCTCAGTGGACGAGCAGCAC

AAATGGATCGCAAGCCTCGCCAACAATGGCCCGGTGACGCCTGGCCTGGTCAGCGGGGTTGACTGGCTGGA

GTGGGTCGACCAGGCGGCCGGTTTGGGGCTGCAAGAAAACCTCGGCTACGGCATTAAGGGCTGGTATTTTT

GGCTTACCGATCACGAGCTCTGCAACATGATTATGGATGGTCTGCTGCTGCCGTTTCATTACCGACTGTTC

GATGCCGGCAAACGGAAGCCATGGAAAGAGGCTCGCGAATCGATCATCAAGGTCAATCGGGAGCTGAGGGA

GAAAAATTGGTACCCCTAG

>vaoC

ATGACATTGAAAGCCTCAGAGGTTCCCCTTCAGAACAATGAGCTGTTCGACGTCAGCGACTGTGTCGCGGT

CATCACCGGCGGTGGAACCGGAATGGGGTTGATGATGGCGAAAGCGCTGGAAGCCAACGGTGCCAAAGTCT

TCATTCTCGGACGACGGCTGGAGATCCTCCAAGAGGCAGCCAAACAATCTACATTCGGCAACATCTGCCCC

GTTCAATGCAACATCACCTCCAAGGACGACCTCCAAACCGCGGTCGATTACATCGCCAACGAGGACGGCTA

CGTCAACCTCCTCGTGAACAATGCAGGCATTGCGACGCCGAATCTCGGACCACACGCCTCGCGCCCTAATG

CAAAGTGGGACGTCTCCGCGGTGCGGGAGTACTGGTTCGACAAGTCGTTTGCGGACTACGCCAAGGTCCTG

GAGGCGAATACGATCGCACCCCTGCAAGTATCATTTGCGTTCCTGGAGCTGCTAGACCAAGGAAACAAGGT

254 Appendix

GCGCGCCGAGCAGGCCAAGGCGAAAGGGCGGAAGGACTACGTGCGCAGCCAGGTCGTGATGCTCAGCAGTG

TCGGTGGTTTCGGACGAGACAATTCGGCATTTATCTACGGAGCCAGCAAGGCGGGCACGACGCAGATGACC

AAGAACCTGTCGACGTATCTGATTCCGTGGAAGATCAGAGCGAACGTCATTGCGCCTGGATATTTCCATAC

AGAGATGACGGAAGGCTTCTACAAGAGCACTGGGGGAAAACTGCCTGCTTCCATGGCACCGGAGGAGCGAT

TTGGTGACGTCCAGGAGATCGGAGGAACGATCCTCTACCTAGCTTCCAAAGCCGGGGCGTACTGCAACGGC

TCTGTGATGTTGGCTGACGGCGGATATTTGGGTAATCATCCCAGCGCCTATTGA

>vaoD

ATGAGGGTGAATTGGAAAAGATGCAGACAAATCGTTCGCTGGGTGACGAGGGCGACAGTGGTAGTTGCGAC

CATCGCGTTAGGCATCCAGTTCTTCATGAGGAATCTACTCCAGGGGGAAGTTCAACCACTGCGCATACCGG

AACCCACTGCTATTGACAACAGTTCCCCCTCGATGACGGTCTGGGAGTTGATCCGGAACGACAACCGGACC

TCGACCTTTGCAAACATCTTGGGGGAGTTCACCCATATCGTGGCCGGGTTGAATGCGCCCAAGGCTAAATT

TACCGTCTTCGTCCCCACGAACGAGGCATTCGAACAAGAAACGTTTGAATGGAATCTCCCTTCCTTCTACT

GGATGTACCTGGTCGGCTACCATATGGGACCGGGCGCCTTCAGCCGGGACGTGCTGTCACGCATGAAGACG

GCGCCGTCGTTTGTTTTCGCGGACATTTACCAGAAATATCGCCAGCGCATCAGTCTTCAGAATCTATCGGA

CCGCTTCTCATTCAACTATAGGGCGCGCTATGCTACCGCGGACAGCTCGCAGGCCGGAGTGAATGGTTACG

TCCACCATGTCGACCGCGTGCTCATGCTTCCAGAATCGACCTCCGACCTCCTCAGGGACGACCCCGACTTT

TCCACGCTGCGCGAGGGTCTCACCCTGACAAACGTCGCGGTGACCATCAACGACACCTCCACCCATGTGGG

CCAGACGCTGTTTGCTCCATCCAATGCCGCGTTCGACAAGTTGGGGCCCAAGACTAAGCAGTTTCTCTTCA

GTTCCGGTGGACGGCAATATCTGAAAGCTCTACTCGAGTACCACGTCGTGGCCAACCGCACGATGTTTACG

GACGTCTATTTTCAAGAGAGTGGACAGGGAGAGGTTCCGCTCCAGATGGGATCGACACTCGATCTCCCTAC

GCTGGTGCCGGGATACAATCTCAGCATCTCCATCGACGCGGGCGATTCCTCCCGTTCCTCGTCCCCTCTAA

TCAACAACGAAGTCAGAATAGCGCAGCCGGACCTCGTCGTCATGGATGGCGTGGTTCACAAACTAGATACG

GTTCTACTGCCACCACGGTCGCCTCGAGATGATACGGAGTGGCAACAACAGACTTGGTTTGGTTCGCTGGT

GCACTGGGCAACAAGTAGTTCGGGGGTAAATGTTGGGGAGTTGGTGAGGCGACTCGAGGAGTTCGTTGACG

TCGCTGGGACGTAG

Sequences 255

>vaoE

ATGAACCTCTCATGTCGCCACCAGGCCGCCTTGCTTTTTGCCCTGGCTGGGTTCGTCTCCGCCGCGGTGGT

GAAAGAAGAGTTGACTCTGACATGGGAGCTGGGAGCGCCCAACGGACAGGCCAGGGAGATGATTATGATGA

ATGGCCAATTCCCGGGACCGACTTTTACGTGGGATGAGGATGATGATGTTGAGGTGATTGTACACAATCAA

ATGCCGTTCAACACCACAATCCACTGGCATGGGCTTATGATGCAGGATACACCGTGGTCCGATGGTGTACC

GGGCCTTACGCAAAAACCTATCGAAGCGAATCAGTCGTTCATTTATCGCTTCAAAGCGTCGCCACCGGGGA

CTCATTGGTGGCACTCCCACTCTCGCACCACATTGCTTGACGGGTTGTATGGCGCGTTGTACATAAGACCC

AAACCGACCAACCCAGCTCCCTGGGCGCTCATCAGTAACGATCCCCGGGATGTCGAAGCAATGCAGAGGGC

CGTCGCCAACCCTCAGGTTATTGTGGTCTCTGACTGGACTCAGTTCAAGTCATGGGAATATATGGACGCCC

AGGAAGCATCGGGATACGCCATCTTCTGCGTCGACAGCCTCCTTATCAACGGAAAGGGAAGTGTTTACTGT

CCCGGTGAGGATTTCTTGGTCAATCATACGAGTACCTATATGAAGTGGGCGATTTACCCAGGCCACGTCAA

CGACAAAGGATGTCTACCATTCGTGAGATCGACCGAAAACAAATACCTCGCCGATGGCCGACCGGAGGCGA

TCCCGTTGCATCTTCAACGGGGCTGCGTTCCAGCAACGGGCGAGCAAGCGATCATCGAGGTGGATCCCGCA

GCAGAATGGGTGAGCCTGAACTTCGTGGACGCGTCGACGTTCAAGACGCCAGTCTTCTCCATCGACGCGCA

TCAGATGTGGGTGTACGAGGCGGACGGGCACTTCATCGAGCCTCGACTGGTGGACACCGTCAAGTTCTACG

CCGGCGAACGCTACTCGGTGATGGTCAAGTTGGACAAGAAACCACGAGACTACACGATCCGGGTGATGGAC

ACCGGGTTGACTCAGATCATCGGCTCTTACGCCACCCTCAGATATAAGGGTGGTAGTCGCGACGACGCTGC

AGAGACTCAGGCCGTCATTAATTATGGGGGATTGAACACCACTGCGGTTGTGACGCTCGACCGAGACCACC

TCCCACCCTATCCTCCCAACCCCCCGGCAGCACACTCCGACGCGCAGCACCTATTAAACACGCATCGGTGG

CACAACGCATGGCAATACACCATGACGGGTGGGGGGATGTACACGGAGGACCGAAGCGCATACGCTCCGCT

GTTATACCACCCGAACTCTGTCGACGCGATGCACGAGAGCCTGGTCATCCGGACCAAGAACGGCACCTGGG

TGGACTTGGTGATCCAGGTCGGATCGCTGCCCGAACAGCCCCAGGAGTTCCCGCACGTCATGCACAAACAC

ACGGGCAAGACGTGGCAAATCGGGGCCGGTGAGGGCATCTGGAATTACACCTCGGTGGACGAGGCGATCCG

CGCCGAACCCACCAAGTTTAACCTCAAGAATCCTAATTTCAGGGACACGTTCATCACCTCCTTTGACGGGC

CGTCCTGGGTCGTGCTGCGATATCAAGTCACTAATCCCGGGCCGTGGCTCATGCACTGCCATTTCGAAATT

256 Appendix

CATTTGGGCGGTGGTATGGCTGTTGCCATCTTGGATGGGGTAGATGCGTGGCCCGAGATTCCGCCAGAATA

CGCACCGGATCAAAATGGTTTCTATCTTGATGCCGACAAAGGGCCATGA

>vaoF

ATGGCTCTGACAAACGAGGCTGCTGAACCCGCGTCACCCGCACACGACGCCGGCTCCCAGGAAGAGCACCC

CCGCCCATCTAATAGGACAACCTCGGAGTCCTTGCTGGAGGATCTTGCTACCAGAATTCAGGAGAATGCGG

GTCGCATAAGTTCGTTCCTGCGAGGCAATGGCCACTCTTTACCGTCGTTTGACGTGGACGCCCCTACCGCC

ACATTGCCAGCCTCGGCTCCAACCGAGATCCACGCCGCAAGACAGGCGCTGATGGAGGCTGCTTTGCAGAC

GTTCCAGTTGGCGGCGGGTCCCAGCGAGTATCTGCCACACCTGGCGGTTGGGTATCAATATGTCACCTGTT

TGCGATGGTTAACCCATTTTGGAATCTTCGCTCGGGTTCCCCTCACCGGTAGCGTCCCATATAGCGAGATC

GCAGCGTCCGCGAATGTCTCTGAAAGCCAGTTGAAGACGGTGGCCCGGATGGCCATGACGAATCATGTCTT

CTGTGAGCCGGAGCCGAATTCCATTGCGCACACCGCGACCTCTGCCCTACTAGTGACCAACCCGATGTTCC

ACGACTGGGCTTCGTTTATGTGTGAAGCATCGGTTCCGATGGCGTCGAAGTTGGTCGAAGCCTCCGAGAGG

TGGCCGGGAAGCGTGGAAAAGAATCAGACGGCATACAACGTTGCTTTTGACACTGATCTTCCTTTCTTCGA

TCACCTCGCCACTTTGCCGAAAAGATCAAAGCAATTCGCGAACTATATGAGGAACGTCCAGAATAGCCAAG

GCACGGCAATTCATCATCTTTTGCAGGGCTATGATTGGGCGGCCTTGGGTGAAGCAACAGTCGTTGATGTC

GGGGGCTCCACTTGTGCCGCCAGTATCGCACTCGCCCGTGCATTTCCAAACCTCAATTTCATCGTGCAGGA

CCTGCCAGAAAACGCCGCAAACGCGCAAGCGGCGCTTGCTGAACAGCCGTCGAGCATTGCACCCCGGATCT

CCTTCCAGGCCCACAATTTCTTCGAAGACCAGCCCTATAAAAACGCCGATGTCTATCTCCTGCGCATGATC

CTGCATGACTGGCCCATGCGCGAAGCTACGACTATTCTCAAGCGCCTCCTTCCAGCGCTGAAGAAGTCCTC

GCGCATCATCATCATGGACACGGTTCTCCCGCGACCGGGCTCAATCCCGTCGGCAGAGGAGCGTCTGCTTC

GCGCAAGAGACATGACGATGCTGCAGGCTTTCAACAGCCTGGAGAGAGATCTCGATGATTGGAAGGAATTG

CTGAAGGGCGTCGACGAGAGACTCTCTCTTGTAAACGTGGTGCAACCGGCTGGGAGCGTGATGTCGGCCAT

GGAAGTTGCTTTCGAAGCGTGA

>vaoG

ATGACCGCAAGCACCGACACACCACCAGCTGCGGAAGCAAATCCGTCGACGAAGGCCTCCAATGGCGAGAC

Sequences 257

CACCCAAAATGAGAACAACCACGCTTCAGGATCTCGTCTGGCGCTGATTATGACAGCAATTCTCTTAGCCA

TGTTTCTTGTTGCTCTGGACCGTACAATTATTGCAACCGCTGTTCCGCGCATCGCAAATCAGTTCCACGCC

TTGAACGACATTGGCTGGTACGCGGGCGCCTACCTGATCACCAGCGCTGCCACTCAATTGCTGTGGGGGCG

CATCTATACTTTCTACAACACCAAGCTCGTTTTCATCATTGCCGTGGTTATCTTCGAGGTCGGTTCTGCGC

TTTGCGGTGGAGCTCCCAACTCGAACGCGTTTATTGTCGGCCGCGCAATCGCAGGGAGTGGTTCGGCAGGC

ATTTTCTCGGGGGCGACGGTCATCATCACCCAGATCATTCCTCTTGCGAAACGACCCATGTATATCGGCTT

TATGGGCTCGACCTTTGGCGTTGCCTCAATTGCTGGGCCGCTCGTGGGAGGTGCATTCACGGACAGAGTAA

CCTGGAGATGGTGTTTCTATATTAATCTTCCCATTGGCGGCTTTACCCTCGCGATTCTCGCCTTCTTCCTC

CGTGTTCCCAAAGTCACGAACTCTGATCCGTTGTCTCGTCAGTTGATCCGTCTCGATCCTTTGGGCACGCT

TGTGTTCTTGCCGGGAATCATCTGTTTCCTCCTCGCTCTCCAATGGGGCGGGAACGACTATCCGTGGTCAA

GCGGCCGGATCATCGCGCTGTTCGTGGTCGCTGGCGTTCTCATTATTGCGTTCGCGATCATCCAGGTCTGG

CGCCAGGAAGATGCGACTATTCCACCGCGCATCATCCGCCAACGTAGCGTCTTTTTCGGTGCCATTTTCGC

ACTCTGTATCGGAGGAGGCATGATTTCCATGCTCTACACGCTGGCCTTATGGTTTCAAGCCACCAAGGGAA

CCTCAGCGGTTCAGGCCGGCATTGACACTATTCCCGTCGTGCTGTCCCTGGTGGTCGGATCCATTTTATCG

GGCGCCATGATTTCCCGCATGGGATACTACGTTCCCTTCATGTACCTCTCGAACGTGCTCACGTCTGTCGG

CTCTGGCTTGATCACAACATTCTCATCATCTACTGGTCATTCCGCGTGGATCGGGTACCAGGTCCTGTATG

GCCTGGGACTCGGTGTCGGCATGCAACAACCCAGCATGGCCGCGCAGACGGTGCTGCAGTGGAAAGACGTC

TCCATCGGCGTGTCCATCATGTTCTTCATGCAATCGCTCGGGGGCTCGATCTTCAACTGCATCAGCCAGGC

ACTGTTTACGAACTACATTCGTGCGCACCTCGCCGCGATTCCTGGTCTGGATGCGGAGAAGGTCCTGGCCA

CAGGTGCTGCAGAGCTGTCTAAAGTTATTCCTGAAGACAGGTTACCGCAGGTGGTCGAAATTTATAACGAG

GGCTTACGGAGGGCGTTCATCGTTGTGCTCGCCGTGTCCTGCTTGACCATTTTGCCCGCCCTCGGTATGGA

GTGGCGATCGGTTAAGCAAAAGCGGGAACAGATGAAGCAAGAGCATCAACCCGATACTAAGGATGAGCCCA

GTACTAAAAATGAGGCCTGA

>vaoH

ATGGCGGACAGCCAGCCACTGGGCCCGGGCTCCACAATAACAACGCCGTCATCGCAGGATCCGAAACCACC

258 Appendix

AAAGTCGCACTCGTGCGTCACTTGTCAGCGGAGAAAAGTCAAGTGCAGCAGGCAACAACCGTGTGCTGGAT

GCACCAAAAATGGGGTGCAGTGTGTGTACCGCTCGCCCCAACCGCCTCGGCGGCGGAAGAGGAACAATCCT

GAAGCCATTCTGGCGGCGCGTCTAGAAAGGTACGAAGAAGTGCTGAGGCAGAACAGCATCGATCCAGAGGC

TTGCTCGCCTACGCGTGCAGTGCCCTCGCCGGGCCATGGGGTGGCGAATTCAACGCCGGTGGTTTCTGTCG

CCGAGCTAGCTAATCCAAGGGGGCCTCTGTCGGCTCCTCGCACATCCAATCCCAGTCATGCTGAGTCGCGG

AGGTTGGTTTCCAAAAAGGGAAGCTCGGTATATCTTGATAGCAATCTATGGACGGGTGTGCGCGACGAGGA

GTCGTCGTCTTCTGCCGACTCTGACCAGTCCGGCCAAAACGACAATTCCCCGGCCCTTGATGATGAATCAG

GTTTGATCTTCGGTCCGCCGGGACGGACGACCCTCAGCTCCATCCATCCAAACCCGGTGCATTAATTCAAG

CTGTGGCAGACGTTTCTCGAGAACGTTAATCCACTCATAAAAATTGTGCACGCACCAGTTGTGCAGCAGCA

GGTCTTGGAGGCCGCTGGAGACCTGCACGCAGTCAGCAAAGAGATGGAGGCGCTGATGTTCTCCATCTACT

GTATTTCAGTGATTTCCCTGGGCGAGGACGAGGTAAAGCGTTCCTATGGCGAGTCGCGTGCCACCCTGATC

TCGAGGTACAAGCACAGTACGGAAACGGCCCTTTGCAATGCCGGGCTGCTAAAGACATCGAACATGGTGTT

GTTGCAGGCCTTCTTGCTATATCTGTTTTCCCTGCGCCTGCTGTCCGACCCGCACACACTGTGGTCCTTCT

GCGGCATAGCTATGCGCATCGCCCAGCGCATCGGCCTTCACAAGGACGGCTCTCATCTCGGACTGTCCGCC

TACGAGACAGAAATGCGACGGCGCCTCTGGCTCCAACTCGTTATCTTTGATGCCACTGCTGGTTATTTCTC

GGGGTGCGGCTCGGCATTCTTACCCCCTCCTACATCCGAAACGACACTGCCCCCTCTCAACATCAACGACA

GCAACCTTGACCCTAACATGACGGAGCCGCCACGGGAATACACCGGGCCGACGGAGATGGTGTTCTGCCTC

GTCCGGCATGAGTTTGGCGAATGGCTACGCCGCCGCGCCGGGATGAGGTCTGGGTTCGATGGACATTGGGG

GTTCCTTCGGTCTTCGTCCGTGCCGTTATCAGAAAAGGATGCGGCCATCGACGAGCTGGAGGAGAAGTTCG

AGAACAAGTTTCTAAGACACTGTGATGTCTCAATCCCACTGCACTTCATCACCGTGATGATGGCACGGTCG

GTGGTATGCATGATTCGCCTCGCCGCGCACCACCCGCGACAATACACCGGTACCAGCATGGTCGGTGTCGG

TGACGGTGACGGTGCCGCCGCACCCAACCCGACACAGCGCACCCTCCAATTATCCGAGGCGGAGCAAGACC

GCATCTTCACCACCTGCGTCCAGGTCGCCGAGCACTGCGATGCTGTGCAGACCAACCACGCCACCAAGAAA

TACCTCTGGCATGTGGACTACAACATTCAGTGGGACGCCCTGATTTACATGCTGTCTGAGCTGCGCGGGCG

CCGGGTCGAAGGCGCCGAAGTTGCCCATGCCTGGGTCCTTATCAACAGCATATGCGGCCGACACTATCGCC

Sequences 259

AGATGGGCCCGCGCGCACGTCGCAGCACATTGCACGTTGCCATCCGCAACCTTATCATCAAGGCCTGGAAA

GGCCATATCGTTACGTGTGAGCAGAGGGGTATCCCCTCGCAGCCGTGTCCTGACCTGGTCGCGCCATGGCT

GCAGCTGGGCCTGGGGGAGTTACGGACCCTAGCAGCTGCCCCTCCTCCGCCACTGTCTGTGCCGTTGCACC

AGCAAGAAGCCTCAAGCAGCAGGGTTTCATATTATGTAGAGAACACAGAACAGACCCTCGACAGCGAAAGC

TCCCTCGCGGGGAATACCAATGCCACCAGTCCACCGCTGCAACATTCCATCGCGCCGTTCCAGCAGCAGCA

AGGCCCTGGAACTGGAATTGGGGTAGGCACCGGCGCAGAAGAGATGGACTTCGTGGCCATGAGCGGAGTGC

CCGCGCTGGACGTCAGCCCCGTGAACTGGGAGCAGTGGGACGATCTGCTCCAGCAATTCCAACAGGAGTGT

TGGAGTGAGAATCCGCTCTTGAGCCAGACATAG

>vaoJ

ATGTCCAGCCCCATGCAAGAACAGCAAATATGGGCGAACGCCCCGTTCGCTCTCATCACGGACACCGGCAT

AGGCGCCCGCCCCGAAGTCCCCCGAGACCATTACGCCTACGAGCTGGCTCGCATGATGGCGCACATCCACA

ATCTACTCCTCCGCGCCCTCAACGCCTCGTATAACCAGTGTCTGTCGGTCCGCCCGGACACCCCCGAGACG

CGAGACTTCCTCGTCTTCAACCAGTGCCTGTACTCCATGCTGAAAAGCCACCATGACCAGGAAGAAGAGTC

CCTGTTTCCGGCGTTCGGCCAGGTCAGCGGCAATCCCGACGTGATGGCCGTCAACGTGCAGGAGCACAAGC

GCTTCGAGACGGAACTGCAGAATTTCCGAGATTATGTTTTCAACACGGACCCGGAGGTCTACAGTGGTGCG

CAGCTGAAGTCGCTCCTCGACTGTCTCGGCCCGTTGGTTCAGGAGCACCTGCACCACGAGATCGCGACGCT

GTTGGATCTCCATGTTGTGGACAGCAAGGCCCTCAAGGGGGTGTTCGGCATGGCCAGCAGGGGCTCGTCGG

GCGAACCCCACGACATTTTCAAGGACATCCCCTTCACCCTTACATGTGAAGACATTACGTTGCAGCTAGAC

GGCAAGGTGACTCCCCCCTTTCTCGGAAGTTTTGCCCGGCAGTTGATCAAATGGGCTGTGGGTTGGAGATA

CCCTGGTGTATGGCGATTTGCCCCAAGCGATTTCTTGGGGAATCCGCGTCCCCTGTTATTTCCAAACCCGT

CGGATCTATGA

260 Appendix

Putative viriditoxin biosynthetic gene cluster sequences

Aspergillus viridinutans ATCC® 16901™, CBS 127.56, NRRL 4365

>vavA

ATGGCGCAACATCATCGAATTTACTTGTTTGGAGATCAAACATATGATGTCGATGCTCCCTTGCGGGAGCT

CTTACACCATTCAGACCCAATATTGACGTCTTTCTTCGAAAGGGCCTTGCAGGTTCTGCGCCACGAGGTCG

GTCAGCTACCCAGTGAGCTCCGTCATGAATTCCCTCGATTCTCCAGTATAGCCGACTTGATCTCGCGGCGC

CGGGACACTCGACTTCATCCTAGTCTTGAAATGGCTCTGGTCCTAATCTATCAGTTGGGGTCCTTCATTCG

GATTCATTCAGAAGGGGGACTGGAGTACCCTACGGCTTCTGATACTCACCTGTTAGGTCTCTGTACTGGGG

CCTTTGCTGCTGCGGCAGTTAGTTGCTCACGCTCCTTGGTCGAACTGCTTCCAGTCGCCGTACAAACGGTA

CTGGTCGCTTTCCGAACCGGTCTGTCTGCCGGCAGAATTGGAAACTGTATTGAGCCAAGTGCAACCGGCTC

CCGAGAATGGTCGATGCTGGTTGCTGGAATGGACAGTCAGACGGTCCAAGATGCTTTAGCAGAATTCTCTG

CCACGAAGTCCCTCCCTCGCATGTCTCGGCCTTATATCAGCGCATACGCTCCCAATGGTTTGACTGTCAGC

GGCCCTCCTTCGATCCTGGGTGAGCTGCGCCAATCGTCCGTATTCGCGCGTGTAGCCTGCAAATCGATCCC

GATACATGCGCCCTATCATGCTCCCTTGCTTTTCGCCAAGGACGACATTGACGAAATTGTCGACTCCACGA

TCAACGGCAACTGGGCATCCTATATTGTCCAACTGCCTGTGATATCAAGTGCCACCGGGCGCTTGATATGG

GCTGGCAACTTTCGTGCTCTGGTGCAGTCTGCAATTGAAGACGTGCTTCTTCAACCAATCCATTGGCTCAA

GGTTCAGGATGGTATTAAGACTATTCTGCCGTCTACATCCATGGTCAACTATGAGGTCATCCCCATGGCGA

CCCAGGCGGATCCTCTGCTGTCTCGGATCTTGCAACAACCGGTAATGAACCCAATCCCGGCCCTGTCGGAG

ACGCAGCAGGATCCACCGACGCCCAATAACAAGGCCAAAATTGCCATCGTCGGCATGTCTGGTCGATTCCC

AGGCGCCAGCAACGCGGAGGCCCTTTGGGAGCTCCTCTCACAAGGGCTGGATCTGTGCAAGGAGGTCCCTG

CCAACCGATGGAACGTCAAGACGCATGTTGACCCGACAGGCAAGCGTAAAAATACCAGTAAGATCCGTTGG

GGCTGCTGGCTGGATAATCCCGAACTATTCGACGCGCGCTTTTTCAACATGTCCCCTCGGGAAGCCCCGCA

AGTTGACCCGGCGCAGCGCATTGCTCTGCTCACCGCGTTTGAAGCCATCGAACAAGCTGGCATTGTTCCTG

GTCGTACGCCGTCTACGCAGCAGGATCGGGTTGGTGTCTTCTTTGGCACTACCAGCAATGACTGGTGCGAG

ACCAACAGCGGACAAGACATTGATACCTACTACATCCCCGGGGCCAATCGCGCATTCATCCCTGGGCGAAT

Sequences 261

CAACTACGTCTTCAAGTTTAGCGGACCGAGTTATAGCACCGACACTGCATGCAGCTCCAGCTTGGCGGCTA

TCCATCTGGCGTGCAACTCTCTGTGGCAGGGAGACATTGACACAGCTATTGCTGGTGGTACCAACTGTCTG

ACGAATCCTGACATGACCGCCGGTTTGGACCGTGGTCACTTCCTATCACCAACGGGTAACTGCAAGACCTT

TGACGATACGGCAGACGGGTATTGTCGCGGCGAGGGCGTGGCTACAGTTGTGCTCAAGCGACTGGACGACG

CTCTGGCTGACCATGACCCTATTCTGGGATTGATTCTGGGCGCATATACCAACCACTCGGCCGAGGCAGAG

TCCATTACACGGCCTCATGTCGGCGCACAAAAGGCCATCTTCGAGCATATACTAAACCAGTCTGGTGTGCG

TCCGTACGATGTTGATTACATCGAGATGCATGGCACTGGCACTCAGGCAGGCGACTCCCGCGAGATGGCCT

CAGTGCTGGGGACATTTGCCCCACACGCAACGGGCCCTCAGAAGCGCCGAGACGAGCAGCCTCTATATCTT

GGCGCAGTCAAGTCCAATATCGGACACGGCGAGTCGGTGTCAGGTGTGACTGCGTTGGTCAAGGTGCTGCT

GATGATGCAGAAGAACATGATCCCACCCCATTGTGGCGTCAAGACACGGCTGAATCGAAAGTTTCCCTCAG

ACCTTGCCGAGCGCAATGTGCATATCCTTTTTCAGGCCAGTGTGTGGCCACGGCGTCATGACCAGCGAGGC

CGGCGTGCCTTTATCAACAATTTTAGCGCTGCTGGTGGGAACAGCTCGATCTTGGTAGAGGATGCGCCGGT

GCCTACTCCACTAAATTGTAGCGATCCCCGCTCCTCCCATGTGGTCACTCTGTCGGCCAAATGCCCCTCCT

CGCTCAAAGGCAACGTCGAATCCCTCTTGAGCTATCTGGGAGGCCTGTCAAGCAGCGACGATCTGGGAGCC

ACTCTTTCCCAGCTTTCGTATACTACGACAGCCCGCCGCATCCATCATCCGCATCGCGTTGCAGTGACAGG

CTCAACGATAGCAGATATCACTTCCAATCTGCAAGTGGTCTTGGACGAAGGATCGGCATTTCCCCGTTCAG

GACCATGTCCCACAGCCATCTTCACCTTCACCGGTCAAGGTGCACAATACCCTGGCATGGGTGAACAGCTG

TTCAAGACGCTCTCACTGTTCCGCTCTGAGCTACGCCGCTTCGACCGACTTGCCCAAGGCATGGGATTCCC

GTCCTTTTTACCCATCTTTCTCGCGGGGACAGGAAGTCGACTGGATGATTTCACTCCCACGGTTGTCCAGC

TCGCCAACACCTGCATGCAGATTGCACTGGCCCGCCTTTGGACCTCATGGGGGATCAAACCTTCAGCAGTC

GTTGGCCACAGCCTCGGCGAATATGCCGCACTCAATACCGCAGGAGTGCTGTCCGACGCAGACACTATCTT

CCTCGTTGGCCGTCGAGCACAGCTGCTCGAGGAGAAATGTCAGAGGGGAACACATGCAATGCTTGCGGTCC

TCGCACCAACTTCCTTGATCAATCCTGTCTTGCTGGACAGCAATGTGGAGATTGCCTGTATCAATGCGCCC

GAGGAGACGGTGCTCGCTGGTCCAGCCGCAGAGATTGCAGCTATACGGCAGAAGCTGACGGCGCAGAATGT

CAAATGTACCCTACTTCAGGTGCCTTTTGCATTTCACTCTTCGCAAGTGGATCCAATCCTAAAGGACTTCG

262 Appendix

AACAGTCCGCGAAAGGGGTCACCTTCCGCGCACCGTCTATCCCAGTGATCAGTCCCTTATTGGGAACCGCC

ATTGCTGATCAGGGCACTTTCAGTCCGGAATATTTGGCACGGCATTGCCGTGAGCCCGTCAACATGCTACA

GGGTTTGCAGACGGCATCTGCGGCAAACATTATCAGATCAAAGAGTATTGTGATCGAGTTTGGACCCCATC

CAGTGGTTTCCGGCATGGTCAAATCGTGCCTAGGCAACGAAATCACTTGCCTATCCACCCTCCGCCGCAAC

AAAGACCCTTGGACCATCCTAGCGGATAGTGTCGCTGCTCTGTATAGGGCTGGCGCGGACATCAACTGGTC

CGAATACCATCGTGATTTCAGTGCGGCGCAACGCGTGCTACCTCTACCCTCGTATTCCTGGGACTTGAAAC

CGTACTGGATTGAATACAGGAATGATTGGGCCCTGTACAAGGGTGATCTTGTTCCCGGTGGCAATGTTGCT

CCTCTAAAGGCAGTCCAAGCTCCTCCCTCCCCTTCTCCTCCGCCGACGACAACCACGCTCCATCGTCTAGT

GGAAGAGAAGGCGGATCCGGAGAAATTTGTGATCATCTACGAATCTGACGTTTCGCGCCCGGATTTGAATC

CTCTTGTCCAGGGGCATAAGGTCGAAGGCTTAGGATTGTGTACACCGTCAGTCTACGCGGACATTGGGTTC

ACACTGGGCAATTATTTCCTTGATCGATTTTCCCAGCAGCTTAAAGGAGGGGATGGCGACAAAGTGGTCGA

TGTCACTGACATGGTGATTGAAAAGGCCTTGATCGCAGAGAATGCTGGTCCCCAGTTGCTGCGAGTTGCGG

CCGACTTAAATTGGTCGTCCAAAGAGGCTGCTGTCAGATACTACAGTGTTGATCTGAATCACGTCGAGACC

GTACAACATGCCCACTGTTGCGTCCGCTTCAGTGACAAGTCCACCTTTAACTCTCTCGCCAAGGGCGTGTC

CACCCTCCGAACACACATTGGAACCATGCGCGAAAACTCCTGCAAAGGGGCAATCTTCCGTTTCAATGGAC

CAATGGCGTACAACATGGTCCAGGCGTTGGCGGAATTCCACCCCGATTATCGGTGTATCGATGAAACGATC

CTTGACAACGAGACTTTAGAAGCTGCAAGTATGGTCAGCTTTGGGGATGTCAAGAAGGGCGGCACTTTCCA

CACGCATCCTGGATTCATTGATGGGTTGACACAGTCTGGTGGCTTTGTCATGAACGCCAACACCAAAACGA

ATTTGGGGGTGGAGGTTTTCGTGAACCATGGCTGGGATTCGTTCCAGCTATATGAGAGTGTCACGGATGAT

CGGCAGTACCAGACATATGTGCAGATGAAACCTGCTGAATCAAGCCAGTGGAAGGGCGACGTCGTCATCCT

CAGCGGTGATCGGCTCATTGGGAGCGTCAGAGGGTTGACGTTGCAAGGTGTCCCGCGAAGAATCTTACGAT

ACATCCTCAAGGGCAGCGCAAAACCTAGCAAAGCCAGCCGGACACATTCTGTTCCAATCAAAGCTCCTCCT

CAGCCCGTGGACAAGAGCTCCGAAAGGAAAAATGAGCCTCCTGATCGTGAAAAGGTCTCCTCTGCAGCATC

AGTAGCAGGACCTGTTGCTGCTATCAAGCCTCAAACCCATAATTCAGCACTGGCTCAGGCTCTTCGCATCA

TTTCTGAGCAGAGCAATGTTCCGATTTCCGAATTGACAGACGATGCGGCATTTGCAGACATTGGCATTGAC

Sequences 263

TCTCTCCTGGCGCTGACCATCACCTCTGCTTTCAGCGAAGACCTTGATCTGGATTTGGATTCCTCATTCTT

CATCGAGCATCCCACGGCGGCCGACCTCAAGAAATTTTTTGGCAGCTGCTCTTCCAGCTCGCCGGACCCTG

AAGTGGTTGTCCCCGCTGTTATTCCTCAAGTCGAGTCTCGAAGCATGGTGCAACAGCCGGTGGCAGTTCCA

TCGACAGACCATGGTGTTGTCACTGATATGGTGAGGCAAGGACTACAAACCAACCCAGACACCAGTAGCAT

TTTCCAGTCGGCTGTTCGCATTATTGCAGACGAAAGCGGCGTCAGCCCTGCCGATCTCACTGGGGAGACCG

TATTCGCGGACATTGGTATTGACTCTTTGCTGGCACTGGTCATTGGCAGTCGACTTAGCGAAGAGCTCGGG

CTGGACACTGACGCAGAGTCCTTCTTCCAGAACTGTGTGACCTTGCAAGATCTGCAGAACTTTCTTCAAGA

TTCCAGCGGATCGCCTTCTTTGCAAGTAAATTCCCAACCTAGCGCTATACAGACCACGATTGTCTCTCCAC

CTGTAAAGCAAGCTGCTTTTCCTCAGGTCTCGCAGATTTCACCTGATATGACGGCGCTCCAGACTCCAGCC

GAGTCGGAACCATCACCGCCGATGGTAGCTCAATCTACCCCGTCAAGTGAGCCAGGTCCCTCATTCCTAGA

GGTCCTGCAGATAATCAGCGAGGAGAGCGGCGTGAACGTCACCGATCTTTCCGACGAGACGGTGTTTGCGG

ATATCGGCGTGGATTCGCTGCTCGCTTTGGTGATTGGGAGCCGAATCCGAGAGGAACTTCTATGGGACCTG

GATGTCGAGTCGATGCTGATTAATTATCCCACGGTCGGAGATCTGAAATCGCATATGCTAGGAGGAAGGTC

TGTGTATCAAGCGGACCAGAATCTGTCCACTTCCTCTATTTCCACTCTCTCGTCTTCATTTCCTTCTAGTG

ACAGCGTCAGCTCGGCCGCTGAGACGCCCCTGTCCGAGTTGTCCGCTTCTGAGCTGGAACCCGGCATTCCA

GACGCATCATCTGTCGTCCTGCAAGGCTCGTCCCGGAATGCGGAGAAGATCCTGTTCCTCTTTCCCGACGG

CTCAGGATCAGCCACCTCCTACGCCTCTCTGCCACGTATCGCAGCTAATATTGCCGTCATTGGTCTCAACT

CGCCCTTCCTTAAGAAAGATGTCGAAGTGCACTGCACCATAGACGAGCTTGTGGGAAGTTATCTGAAAGAG

ATCCGCACCCGCCAGCCCACCGGACCCTATCACTTCGCCGGCTGGTCCGCGGGTGGTATTCTGGCGTACCG

GGCCGCTCAGATGCTCCTCGCTCGCGGGGAGCAAGTTCCCAGCTTAGTCCTGCTGGATGCTCCCCCGCCGA

TGGGACTGGGGAAGCTCCCGCAGCACTTTTTCGACCATTGTGACGCGATGGGCATCTTCGGCCAGGAGGGC

AAAGCTCCGGAATGGCTTATTCCACATTTCAAGAAAACCAATGCCATTCTCAGTGGGTACTACGCGACGCC

CTTTGCGGCAGGTCGGGGCCCCGGCAGGACAGGAATCATCTGGGCAAGTCAGAGCGTCTTTGATACCAAAG

GATGCGCTCGACCGGCACCGCATCCTGAGGACACGGAGGATATGAAGTTCCTCACTGAGGCACGGACCGAT

TTCTCAGCGGGGATCTGGGGGTCATTGTTCCCTGGAGCTCAGGTGGTTGTGGATAAGGTAGATGGGGCGGA

264 Appendix

TCATTTCTCCATGATGGTAAGCACATCCTGTACTTTGATCTGCTGTAGATTGTTCGATTGA

>vavB

ATGGCAGAGTCTACCGGTTCAACACCCCAGAAAGACCAGCCGCAGGTCAAGTTACGATCGGCCTGCGACCG

GTGCTCATCCAACAAAGTCAAGTGTAGTCAGGAAAGACCAGAGTGTCAGCGATGTCGTTCGCTCAATCTAC

CCTGCCACTACAGTCGGTCATTGCGGATGGGGAAGCCTCCTCGATCTCGGCAGCGATCCATTAGAGCTTGC

TGCCATCATGACCATCGCGAGAATTCGGCCAGTCTCGAACATTATAAGCCGGCCGCTGCTGCTGCTGCTGC

TGCTGCCACAACAGACGCTGCTGCCATTCCCAAGTCCATGCTCAGCGATCCATTGCCCTCGCGAGACAACG

AGGTGATGGGCACACTGGTGCATAGCTGGCCGGACGAGTTCGACTATCAGTCGATGCTGAATGGTGTCGAT

CCCTCTCTCCAGCCACTCTCCCCGGACCTTTCCGACATACTGGGAGACCCGACAGCCATAGCGCCAGGTCA

CAGTCACCCGCGGTTATCAGACGGCCCCGGCACCCTGTCTGATTTGTCAAAGCAACAAGCCGAGTCGATGT

CCAAGATGCTGCAATCCACACTCCACAATGCAATGTACGAAAGGCTAACTCCAGTCAGCCTCACTGGGTTC

ACCTTCCCCGCCACAGGAATGAACTCGGACAATCAGCATGTTATTGAACCCGCGGTCTCCACGGGTCCGAG

TCCCCCATCCCCGTCCGACCATCAGTGTGTCCGCCGGGCCATGGAGACTCTGGACTGTCTGTATCGAGTCT

GCTCCTCAGCCAACTCATCTGATGCTGGCTTCGGCAAGCCAAGCGTCGATGACATCCTCCAGGTTAACGGC

GAGGCGATGGAGGCCGCCGCGACCCTCCTGGCATGTCCGTGCACCAAGGACTTCTGCCTCCTCATCATTCT

CGCCATAATCCCGTGCAAGGTGCTTGCCGGGTACCAGGCCGTTGTCAACATGCGCGACCCACATATTCAGG

TCCCAGCAGGCGAGGCAACTATCTGTCGTCCGATTGCCGTCGGAGCCTATACCCTAGATGAACACGCTAGC

CGCATGGTGATCATCCAGGTAGTTCTCGCCAAGCTGCGCGAGGTGAGCAGGTTCGTCCAGACCTATATCGA

CACATTCTGCTTCGATGCTAGCAAGAACCGCCAGGGTCACTGTGGACTGGTATACCGGACCCTGGGGTTGT

TTATCCAAAGCCGGCTAGGTACCACCACGGACGGGCTGCGGGAGAGACTGTCAGCCTTGGTGGGAGACGGG

AGATCTGAATTCAAAGGAGAATCATGCCGGTATGGG

>vavC

ATGGCTGCTGCCGTGGAAAAGCAGCCCCTGGACGCGCGACAGGAGAAGCAGCCACCACCATCAGCGCATCC

GTCAGAAAAACCTCAAGGTGTTGTCGAATCCAAACCAGGTCCTCCAGGTCCTGCAGGCCCTCCAGGTGGCC

CCGGTGGTGGAAAACCCTTTGGACCTGGCATGGGTCCTGCCGTCGAGTATCCCACAGGTCCCAAACTCTAC

Sequences 265

TCGATCATCATCTCCCTTTATCTTGCTGGCTTTCTCACCGCGCTGGATCGCACCATCATCGTAAACGCCAT

TCCCCATATCACGAACGAGTTCGACTCTATCAACGATGTCGGCTGGTACGGTAGCGCATATCTCCTGACGT

TTTGCGCGTTTCAGCTGCTTTTTGGCAAGATCTACTCGTTCTACAATCCAAAATGGGTGTTTTTGACCGCC

GTGGTCATCTTCGAAATCGGGTCTGCTATCTGCGGTGCCGCGCCCAATTCGACAGTCCTCATCGTTGGTCG

CGCAATCGCCGGCCTGGGATCGTCGGGGATCTTTGGTGGCAGTGTAATCATCACCTTCTTCACCGTCCCAT

TGCACATGCGACCCATCTACTCGGGCATTGTCAGCGTCATCTTCGCCATCGCATCCGTCACAGGTCCCCTG

ATTGGTGGTGGCTTCACCGAACATGTTACCTGGCGCTGGTGCTTCTACATCAACCTGCCCATTGGCGCAGT

GACTATAGTGGTACTCATCCTGGTGCTCCAGATGCCGCCTGCGCGCAAGGCAGGCACCCCGGTACGCGAGC

AGTTTCTGCAGATGGATCCCCTGGGAAATCTATGCCTGATCCCTGGCGTGGTGTGCCTGCTACTGGCGCTG

CAATGGGGAGGGGCGACCTATGCCTGGAATAATGGACGCATTATCGCGCTTTTAGTTCTGGCAGGAGTATT

GCTGCTCGCCTTTGTTGGGATTCAGATCTGGCTTCAGGAGAAAGCCACCATTCCTCCCCGGATCATCAAAC

AGCGCAGCATCGCAGCCGGAGTTGTCTTCACTCTCATGGTCACTGCGGCGATGATGACGTTCACCTACTAC

CTGCCAATTTGGTTTCAGGCCATCAAGTCCGCGTCGCCAGTCCACTCGGGTGTCATGATGTTACCCACCGT

CATCTCATCGGCGGTCGCCAGTCTCATAGCTGGTTTCCTGATCAATCGACTCGGCTACTACACGCCATTCA

TGATTGGCGGATCAGTGCTTATGTCTCTTGGGGCCGGTCTGCTCACAACATTCACCCCCAACATTAGCGAA

GGCAAATGGATCGGCTACCAGATCCTCTGGGCGGTTGGATGCGGAATGAGCATCCAACAAGCCTCTTTGGC

CGCTCAAGCCGTCCTCGCTCGCCCAGATGCTCCCATCGGTATCTCCTTGATTTTCTTCGCGCAATCCTTGG

GTGGTTCGGTGTTCCTGTCTGTGGACCAGGCCATATACAGCAACAAACTGTCTGCCAACCTGGGTGATATC

GCCAACGTGACCGACAGCGGAGTGACGAGTATCCAAGATCGCGTGAGCCCCCAGGACCTGCCACGATTTTT

GAGCGGATACAATGGCGCGGTAATGGACGTGTTTCGCGTCGCTTTGGCGGCGTCCTGCGCGTGTGTTGTGG

CGTCTGCTCTTATGGAGTGGAAGAGCGTAAAGGCTCAGAAAGGAGGCCCAGGAGGACCTGGTGGCCCTGGT

GGCCCTGGTGGCCCTGGATCAGGGCAACCCGGAATGGGTCCTGGGAAGGGACCGTCAACTGCCAATGGCGC

AACAACCATGAAGCCGAACGGTGATGCTAGCACTCAGGTT

>vavD

ATGGCTAGTGCACAGCAGGTTGACGTCGTTGTCGTAGGAGCTGGCTTCGGAGGGTTATGGATAACTCATAC

266 Appendix

ACTTCGAAATGCCGGGCTGAATGTCGTCTGCGTCGAAAAAGCCCCCCAGGCCGGCGGCGTCTGGTATTGGA

ACTGCTATCCGGGGGCTCGAGTCGACAGTCGATACCCCGTTTACCAGTATACCGATCCGGAGCTCTGCCAG

GACTGGACCTGGAGCGAGTTGTTGCCCGGATACGAGGAGCTCCGGCGGTACATTGCGTACGTGGTGGAAAA

GTGGCAGCTGAACGGATACATACAGTATAACACCACGGTGACAGGGGCGCAATTCGACGAGTCGGCTCACC

AGTGGACGGTCGAATGCGTCGGGGCCGATGGTTCCCCCGTAGCGATCCGCAGTCGCTGGTTCATCCTCGCT

CTCGGCTTCGCCACCAAGCCGTATATCCCCGATATCCCAGGTCTGAACACCTTCGCCGGTCGATGTTTCCA

TTCCGCCGCCTGGCCGCAGGATGGGATCGACCTGGATGGTAAGAGGGTGGCCGTTGTGGGCACGGGAGCCA

GCGCAGTCCAGATCATCCAGACTATTGCCGAGCGGGTGGGCCATTTGACCGTCTACCAGCGCACGCCGTGC

ACTGCGGTCCCGGCCCGGCAGCGCCCCCTCAGCCCCGAGGAGCAGCAGCGGCTGAAAGACAGCGGCGAGTT

GGCCGCCCTGCTGCGACAGGCCAAGTACGAAAAGTTTGGCGGACAGGACGTTGGGTTCGTCCCGCGTCGGT

GGCACGAGGATAACGAACAGCAGCGGCGGGAGGTGTTCGAGGCAGCCTGGGAAAAGGGCGGTTTCCACCCT

TTACTGTCGACATACTTTGACGTCTTCGTGGATGAGGAGGTCAATAGGGCCGCGTGGCGGTTCTGGGCCGA

AAAGACCCGCGAACGGATCAAGGACCCGAGATACAAGGATTTGCTGGCTCCCCTGGAGGCAGCCCATTTGT

TCGGGGGAAAGCGCACGCCGTTCGAGATCAACTATTATGAGGCATTCAACCGAGACAACGTCGAATTGATT

GACAGCAAGGCCTCCCCGATTGTTGAGTTCACCGAGGACGGCATTGTCACGCAGGAGGAAGGCCTCAAACG

GCTCGATGTGATTGTGCTCGCAACAGGATTCGACACCAACACAGGAGCATTGACCGCAATCTCCATCCGGG

ACACGGCAGGGAGGACGCTCAAGGACCGCTGGCAGGATCCAGAAACAGGAGTCAGGACCAATCTGGGTCTG

TCCACCAGCAAGTTCCCCAACATGTTCTTTTTCTATGGGCCACAGGCGCCGACGGCATTTTCCAACGGGCC

GGCCTGCATCGAGCTGCAGGGGGAGTTTGTCGAACGACTCATCCTGGACATGACAGCCAGAGGAATAACAA

GAGTTGAAAACACAGTGGATGCTGAGATGGAGTGGAAGGGCTTGACGCAGCGTCTGTGGGACCGATTTGTT

TTCTCGTCGACGACTTCGTCGTACTATACCGGGGCGAATATTCCGGGCAAGAAGCGGGAGCCGTTGAACTT

TTTCGGAGGGTTCCCCAGCTACCACCAGGCGCTGACCGGCTGTCGTGAGAATGATTATCAGGGTTACAAAA

TGGCGTCTCTCATGGAGAAGATACAGTCTGCCCCTGCTGCGTTGGAGGTTAAGGCTCCCAGTGTTGTCGAA

ACACCTGTCTTGGCGACGTTACCCGTACAAGCTAAATAG

>vavE

Sequences 267

ATGGCATCGAACTCGCTCCACTCGACGGATCTCTTCGACCTGACCGGCTGCATTGCCGTCGTCACCGGTGG

TGGCACAGGTCTAGGACTCATGATGGCTAAGGCCCTCGAAGCCAACGGAGCCAAGGTGTACATAATCGGAC

GACGATTGGAAGTCCTACAGACAGCTGCCAAAGAAGCCAAATTCAACAACATCCATCCTCTCCAGTGCAGC

ATCACCTCACACGCCGAGCTCCAGTCCGCCGTGGACCACATCACCGCCCAAGATGGCTACATCAATCTCCT

CATCAACAACGCCGGCATCTCAACGCCGAACTTGGGCGCACATAACACCCGACCCACGCCCAAATGGGACA

TCGCCAAGGTGCGGGACTACTGGTTCCAAAAGTCCTTCGAGGACTATGCGGCCGTGTTCGAGACCAACACC

ACCGCCACGCTGATGGTGACGTTTGCCTTTCTCGAGTTATTGGACAAGGGAAATAAGGTGCGTGCTGGGGC

AGGTTCCCCCGTTGCGCAGGGCAATGGCAACGGCATTGCCAATGCTGGAACCAGCTGCCAGTATATCCGCA

GCCAGGTGGTCACGGTCAGCAGCGTCGGCGGGTTCGGGCGGGACAACTCGGCCTTCATATATGGGGCCAGC

AAAGCGGGGACAACCCATATGATGAAGAATCTCGCGACGTATCTAGCGCCGTGGAGAATACGCGTGAACAT

CGTTGCGCCGGGGTGTATGTTCTTTCCTTCTTTATCAGATTTGATGACGAGTGCGCGATTTGCTGACTGA

>vavF

ATGCTCTCCATTAAGCAAGTTATCCTAGGTATCGCCTCTGTCTATCCCTTTACCGGGTATGACCCTCTATC

TCCTGTAACGGTACTCGACCCCGGCTCTAACGTCACCTACAACGGCATCCGCACAAGCACCTATGTCGAAG

AATTTCTCAACATCCAGTATGCCCACGACACAAGCGGAAGCCATCGCTTTTCTCCCCCAAGGCCCTATACC

CCCAGCGCAAACAGCATAATCAACGCCACTTTCCCAGGAGCTGCATGCCCTCAGCCGCACATCCCCCTCCC

CGCGGACCCATACACAGTCCTCGCCAACGTCTCAGAAGACTGTTTGACTCTCCGCGTCGCACGGCCAGCAG

GCACAGGCGCGACGCAAAATGCGCACAAGCTCCCCGTGATGGTCTTTGTCTACGGCGGCGGCAGTACCGTG

GGAACTGTCTACGACGGCTCCTACGACCCAGTCGGTCTTATCAGACGAGCCGTTGACCTTGGGACGCCGAT

GGTGTATGTAGCTATGAATTATCGACTGAATTGTACACCTTACCACATTATCCTGTTCGGTTTCGCGGATT

TGCCTGCTCTACGGGCCTCCAACTCGACCAACGTCGGTCTGCGGGATCAGCGCCTCGCGCTTGACTGGGTC

AGGAGAAACATTGCGTTGTTTAGCGGGGATCCAGACAATGTGACGCTGTTTGGAGAGGACGCTGGTGCGGT

GTTTGCGAGCCTGCATATGCTGGCATCTTCGGATGATGAGAGTCTGCCAGTCCATCGGGTCATTGCCCAGA

GTGGTGCGGTTACCTCCCTTGCTGGTGTAACCGGGGATACTAGTGCGCGTAATTCTCTGGATGTTGCGAGG

AAGATTGGCTGTCTTGATTCATCTGGGACTGCTTCGGAAGAAATAAATGCTACCGCCGTGGTACAGTGTCT

268 Appendix

GCGAGAACAACCCCTGGAGCTGCTGGTGAACAAGACGTTCGACGTGGCATACAAAGTGAGTCCGGGAGATG

GTTTCGGGGCCTTTATACCCACGGTTGACGGCACAATCCTGCCTTCTGCTCCATCGGGGTTGATGGCAGCT

GGTCACCTCCCGAATAAGAGCATTCCACTCATCATCGGCTGGAATCGCGACGAGGCCTCGCTCCACGTCCC

AACCAGCATAACGACCCAATCGGACATTTCTAACCTCCTAGCTGGCAATTATCCGTCTCTCACTGCGTCGA

GTATTGCAGACTTGCTCGCCCTTTACCCGGAGTCGGACTATCAAGTCCAAAAGGAGGAGAAGGGAGCACAA

ATGACCCCGGCTTGGCATGCCGCATCGGCCCTCGTGCGTGATCTGACAGTGACCTGTCCCTCACTATACCA

AGCCGCGTCACTATTCCAGCATGCCCGGATCTCAGTAGAGGAACCGGAGATCTATCTCTACGAACTGCAAC

ACACGCCATTCGCGGCGACTCTCGCGCAACAGGGAAAGGCGTATCTGGGCGTCGTCCACTTCTCAGACGTG

CCGTATGTTTTCAACAACCTACAGAGTAGATACTATATCTCCGACCCGGCTGAGTTGGCGCTCGCGGCTCG

GGTGAGCGGAAGCTGGGCTGAATTCGCATCGGGACGGAGACCGGGGAGCAATAGGACTTTGGGGGAGTGGG

AGGTTGCCTTTTCCTCGTCAACCGATAGGGTACCAAGTCTAGAAACAGCGAGATACAGAGCGATTGGAGGG

AAAGATGACGGGATGCGACAGGTCGGAAAGAAGCTGGCGCGGAGATGTGAGACGATCAACCGATTGGCGGG

GGAGCTGAAGACT

>vavG

ATGACGAAGACCCCTGCCCTCGAGGTCTTTGCCGATCAGATCAGTGCTGCGGCCAAAGTCATCACAGCCTT

CTGTGCCTCCACCGGCCATCCACATCCATCCTTTGATGATACCCAACTCAATGGCCTCGATACGCTACCCT

CGTCTGCACCAGCTGAAATTCAGGAGGCTCGACAGGTCATTCTCGAGAGCGCGTATCGACTCCAACAGCTC

ATCATCGAGCCCAGCCAGTACCTGGCACGATTAGCAGTCTACCCTCAGCATCTTGCCGCAATTCAATGGCT

CTGTCACTTCAAGATCCCCTCTCTCGTCCCGCATGAGGGCACCATCTCCTACCCGGATCTCGCGGCCGCGG

CCCACGTACCACTGCACCAGCTCAAGAGCATCACCCGCATGGCCATCACGGGGAATTTCCTTGGGGAGCCA

GCCTTAAACCAGGTTGCGCACAACAAGACATCCGCGCGTTTCGTATCAAACCCGTCCCTGTGCGACTGGGC

GCTGTTCCTGGCTGAGGACTCGGCACCGATTGCGCCCAAGCTGGTCGAGGCGACGCAGAAATGGGGCGAGA

CAACGAAGAAGACAGAGACCGCGTTTAACCTGACCCTGAATACGGACCTGCCCTTTTTCGACTATCTCTCG

AGCTCTCCCGAGTTTACCCAGCGGTTTTCTGCGTACATGAAGAACGTGGCCAAGGGCGAGGCTACAAACAT

CAAGCACTTGGTTCAGGGATACGACTGGGCAAGTCTGGATAATGCCACTGTGGTGGATGTTGGAGGCTCCA

Sequences 269

GCGGCCACGCCAGCATTGCTCTGGCCGAAACCTACCCGCAGCTGAGATTCATCGTCCAAGACCTTCCCATG

GTCATTGAAACTTCTCAATCCCAGGTCCCTGACGCCGTGCGCTCGCGAATTACATTCCAGAGCCACGATTT

CTTCACCCCACAGCCCGTGCGCGACGCGGATGTGTACCTGCTCCGTATGATCCTGCACGACTGGCCGCGGG

AAGAAGCCCAAAAGATCCTGTCTCACGTGGCTGCGGCCCTTCGGCCTGGCGCTCGCATCATTGTGATGGAC

ACTGTCCTTCCCAATCCCGGGAGTGTACCGGTTAGCGAGGAGGCGCTGCTGCGTGTGCGGGATATGACGAT

GATGCAGACGTTCAACAGCCATGAGCGGGGGATGGATGAGTGGGAGGAACTGGTGCACGCGGCAGATTCGA

AATTGAACATCACCCGGTCTATCCAGCCTGTGGGTAGTGCGATGACCGTCTTGGAGGTTGGCCTGGGCCAG

>vavH

ATGGAGCGTCATTTCACAATACTGTTCCTGTTCTTGAGCCATTTGCTCACTGCCTTTGCCGCGCATGTCAA

ACGCGAACTGACTCTGACCTGGGAAGAAGGTGCTCCCAATGGCCAAAGTCGCCAAATGATCAGGACAAATG

GCCAGTTTCCCTCGCCTACTTTGATTTTCGACGAAGGCGACGATGTGGAAATCGTCGTCCACAATCATATG

CACCAGAATACCACCATTCATTGGCATGGAATTCTGATGCAAGATACCCCGTGGTCCGATGGTGTTCCGGG

TCTGAGCCAAACGCCTATTGAGCCCGGCGAGAGCTATGTCTATCGCTTCACTGCATATCCTCCCGGTCAAT

ACTGGTACCATTCACATTCGCGAGCCACCTTGTTGGATGGTCTCTACGGGGCGCTCTTTATCCGACGGAAG

CCAGGGACTCCAGGACCGTGGGCAATGATCAGCGAAGATCCAGTTGAGATTCAAGCCATGGAACGCGCTGC

CAATGATCCCCGGATTATCATGCTCTCAGACTGGGATTACTACAACTCATCCCAATACAAGGAGGCTGACG

CAAACAGCAATCTCCAGATATTCTGCGTCGATAGTATTTTGATCAACGGAAAAGGGAGCGTGTATTGCCCT

GGTCATCAATGGCTCATTGACAAGCAAATTCCGTTCATGCACAAGAGCTGGCCAAATGATACCATCACCGA

CAAGGGTTGCTTCCCATTCGTTCCTTCCACTGAAGGCCCGTGGCTTAAGGATGGCAACGTCTCCGCCATTC

CTCCAGGGTTACAAGAGGGCTGCGTTCCCTACACCGGTCCCACGGAGACTATCGAGGTCGATGCCCGCGAT

CGATGGGTCAGTATCAACTGGATTGGAGGCTCGACCTTCAAAACCCTGCAGCCCACGATCGATGAGCACGA

GATGTGGATCTATGAGGTCGATGGCCATTACATCGAACCTCGCCGCGCAGACACGTTCCTGATCTGGGCCG

GGGAGCGATACTCGGCTCTGGTCCGGCTCGACAAGAAACCAATGGACTACTCCATCCGCGTGCCTGACGGT

GGCTACTCGCAGATGATCGCAGCCTTTGGGATCTTACGGTACAAGAACGGCGACCCGGACGCTCGTCGGCC

TCCCGATCGGTTCGGCGTCACGACCATCTCGCAGCCCTACTTCGACTACAACGCCTGGCCGACCCGCGACG

270 Appendix

GGATCGGCTTTCTGGACAAATTCGATCTGCCGCCCTGGCCGCCCAAGGTTCCCTTCACCGGCGACGGCGAC

GCAATGCACGTCCTGTACCTAGGCAAAGCCAACTCGACCTGGGAATTCACCCTGAGTGGCAAGAAGAAGTA

CCCCTCGGACCGGTCGGCGTACCATCCTGTGCTGTACAACGTCAACTCTCCCCAGGCTCGCGATGACGACC

TGATCATCCGCACTCAGAATGGGACCTGGCAGGATATCGTCCTGCAGGTGGGTCACTCGCCCCTCTGGCCG

GTTGACTTCCCGCACGCCGTGCACAAGCATGCCAATAAGTTCTGGCGACTGGGTTCTGGTCAGGGATTGTG

GAACTACTCCTCGGTCGGCGAGGCCGTCGCTGATCATCCAGAGAACTTCAATCTGGTCAACCCACCCTACC

GTGATACCTTCCTGACAGAGTTCACAGGGACGATGTGGGTGGTGCTACGGTACCAGGTGACTAGTCCGGGA

GCGTGGTTGCTGCACTGTCACTTTGAGATGCATCTCGACAACGGCATGGCCATGGCCATTCTGGATGGAGT

CGACAAGTGGCCCGAAGTGCCGGATGAGTATGCCCTGGGTTCCCATGGATTCCGTGTGGAGGACAAGAATG

AGCTGCAGAACACGTTCTTCCAGCAGGTGATTGACCGGCTAGTCAGCCAGGTGTCTGCGTGGGTGTTGGGG

GCTGCAGTCGTAGCATTATGTATAGCGGTTTGGGCTGTTGTCCGACTCATAAGATGGAGAAAGGAGAGGCA

ACCGATCACGATCAACTATAGTCCATTAGCATCTGGGCCCATGGTTAGTCAAGATGACAAGGACGGTCTGT

TGTCATTTAACAGCCATCCT

Cladosporium cladosporioides CBS 112388

>vccA

ATGTCAAGGCCATATATAAGCGCATATGCATCTGGCGGAGTCACCATCAGTGGTCCGCCATCAGTTCTGGC

AGAGCTTCGAAACACACCTGGGCTGTCTAAACTGCGTGCTAAAGACGTTCCAATACACGCACCATATCACT

CATCAGCAATATTCAATCAATGTGATGTAGAGACAATTCTGAGTTCTTCGTTAATCGATCTGGCCTCTCGC

GCGACTCATGTTCCAATTCTATCAACCGGTACCGGACGACTGGTCTGGGCAGGCACTCTTCGAGCCGCAAT

ACAGTCCGCCCTGCAGGATGTACTCCTTCGTCCGATAAGCTGGGAAAATATGTCATGTGGAATAAGCACCT

GTCTTCAGTCCATAGATCCAAGTGAGGTGGAGGTGATCCCAATCGCCACCTTGGCCGGCCCACTGCTCTGT

CGCTCAGTACAGGTCGCAAAAAGCCAGATCCCAGCTACAATCGATCCAAAGAACGATGTCATGAACGAAGC

ACAAAGTCAAATTGCAGAGGCTATGGACCGAGCCAAAATTGCCATTGTGGGCATGTCCGGTCGTTTTCCAG

GCGCTGAGAATGTCGATTCTCTCTGGGAGCTTTTGATGGCCGGCCGTGATATGTGCAAAGAGGTACCACCC

Sequences 271

ACCCGGTGGAACGTTGACACTCACGTTGATCCCACTGGTAAACGGAAGAATACCAGCAAGATCCGATGGGG

CTGCTGGCTCGACAACCCGGATATGTTTGACGCGCGGTTTTTCAACATGTCTCCGCGAGAGGCGCCGCAGG

TTGATCCGGCCCAGCGAATTGCGCTCCTCACTGCGTACGAAGCTATTGAGCAAGCTGGTATCGTTCCAGGG

AGAACGCCGTCAACGCAGGAAGATCGAGTGGGCGTCTTCTTCGGTACGACCAGCAATGACTGGTGCGAGAG

CAACAGCGGGCAAGACATCGACACGTATTACATTCCGGGCGCCAACCGTGCCTTCATCCCAGGCCGAATCA

ACTACGTGTTCAAGTTCAGCGGGCCCAGCTATAGCATCGACACTGCATGTAGTTCGAGCCTGTCCGCGCTT

CATGTAGCATGTAATGCCCTCTGGCATGGAGATATCGACACTGCAATTGCGGGTGGCACTAATGTCCTCAC

GAACCCCGATATGACTGCCGGCCTGGACAGAGGCCACTTCCTTTCCGCAACTGGTAACTGCAAGACGTTTG

ATGACACCGCTGATGGGTACTGCCGTGGTGAAGGCGTAGCGACCGTCGTCCTCAAGCGCATGGATGACGCT

ATTGCAGACAAAGATCCAATCCTAGGTGTGATCCGTGGCGTATATACCAACCACTCTGCAGAAGCTGAGTC

AATCACACGGCCTCATGTCGGCGCCCAGAAAGCCATTTTCCAACATGTCTTGAATCACTCGGGTATTCGAC

CCCAGGATATCAGTTACATTGAGATGCACGGAACTGGAACCCAGGCAGGAGACATGCGAGAGATGACCTCC

GTGCTTGATACCTTTAGCCCGCAGTACCCAGGAGCAGTCCAGCGAGAAAAGCCGTTATATCTGGGGGCCGT

GAAATCAAACATCGGACATGGAGAGTCTGTTTCGGGGGTGACAGCCCTTGTCAAGGTGATAATGATGATGC

AGAACAACACTATACCTCCCCATCGCGGAGTCCACACGCGCTTAAATCGGAGGTTTCCCTCAAACCTCGAT

GAACGAAATGTTCATATTGCATTCCAGGCGACTGAGTGGCCCCGCGGGCAGACTCCCCGACGAGCGTTTAT

CAACAATTTCAGTGCCGCTGGGGGGAATAGCTCAGTTCTAGTAGAGGACCCACCACTGATACTGAAGGAAG

AGGGTTCTGATCCTAGGTCATCTCATGTTATTGCAGTGTCCGCTAAATCACCTTTGGCATTAAGGAAGAAC

CTAGAGTCTATGCGTCGATATGCGATGTCAGAACATACAGAAAAATCTCTATGTGAGCTGTCTTATACCAC

AACAGCTCGACGCATTCACCACTCGCATCGGTTGATGTTTGCTGGGTCATCTCTAGGGGATATTCTGCGTG

AGATGGACAGCAAGTTAGCGATTAAAGAACCACTCAGTCCTTGCGCACCACTTCCATCGGTCATTTTCACC

TTCACCGGCCAAGGCGCACAATACCCGGGAATGGGTCAAGTCTTTTTTAATAACTTCTCCGTGTTCCGGTC

TGATCTCTGCCGCCTTGACGATTTGGCCCAAAAGCTTGGATTTCCGACATTTCTCCCGATTTTCTCAGCAA

GTACCCATGCCAGACTGGATGGTTTCACACCCACTGTGGTCCAGCTTGCCAATACGTGCATGCAGCTTGCA

CTCACCAGGCTCTGGGTGTCGTGGGGTATTCGTCCGTCGGCAGTAGTCGGTCACAGCATTGGGGAGTACGC

272 Appendix

AGCGCTGAACACAGCGGGCGTCCTGTCCGACGCGGACACGGTTTATTTGGTAGGCAAAAGAGCCCAGCTGC

TCGAGGAGAAGTGCAACCGAGGGTCACACACTATGCTGGCAGCGCTGGCCTCTTTCGAAAAGGTGTCACGT

CTACTTGATAGCGCACCGTGTGAGGTTGCGTGTATCAACGGACCCGAGGAGATCGTTCTCGCTGGACCGCG

TTCGCACATGACAGATATCCAAAAGATCCTCGTGGCGCATTCAATTAGATGCACCATGCTGCAAGTCCCAT

TTGCATTCCATTCGTCCCAGGTGGATCCAATTCTGCAAGACTTCGAGTCTGCAATCGAAGGCGTTACCTTC

CATAAACCAACTATCCCGGTCATTAGTCCACTCCTTGGTGATGTTGTGACAGAAACTGGGACCTTCAACCC

AAACTATCTGGCACGCCATTGCCGGGAGCCAGTGAACATACTACAAGCACTTCGCCAAGCCAGCACAATGA

ATCTTGTCCATGACAGCAGCGTAGTCATGGAGTTTGGACCACATCCTGTCGTATCAGGCATGGTGAAATCA

ACGCTGGGGAACAGCATCAAGGCACTTCCCACTCTGCAACGAAACCGAAACACCTGGGAAGTACTCACGGA

GAGCGTGTCAACACTATACTGTATGGGATTCGACATCAACTGGACCGAGTACCATCGAGATTTTCCATCAT

CGCAGCGTGTCTTGCGACTCCCATCGTACTCCTGGGATCTGAAGTCGTACTGGATTCCGTACCGGAATGAT

TGGACTCTGTACAAGGGCGATATTGTGCCTGAATCAAGCATCGCGCTGCCAACCCACCAAAACAAGCCACA

CACTACATCGCCGAAACAGCAAGCACCGACACCGATCCTGGAGACGACAACATTACACCGGATTGTGGATG

AGAAGTCCACCGAAGGGACGTTTTCAATCACATGCGAGTCAGATGTATCCCGACCAGACCTCAGCCCTCTG

GTTCAGGGCCATAAGGTCGAAGGGATCGGACTTTGTACACCGTCCGTTTATGCTGATATAGGATTCACGCT

GGGAAATTACCTTCTAGATCGTTTCCCAACTCGATTCGGACCGGATACTAAAGTTGTGGATGTCACGGACA

TGGTGATTGAAAAGGCTCTTATGCCGTTGAATGCGGGACCACAATTACTGCGAGTCACGGCTTCATTAATC

TGGTCCGAGAAAGAGGCTTCTGTCCGGTTCTACAGCGTGGATGAAAATCACGCCGAAACAGTACAACATTC

CCATTGCCGCATTAAATTCAGCGACCGTTCCACGTACCAAGCCTATCAAGAGCAAATCTCCGCCGTTAAGG

CTCGTATGTTTGAGATGAAGGCCAACTCCTCATCGGGTAGAACCTACCGATTCAACGGACCAATGGCATAC

AATATGGTGCAGGCGTTGGCGGAATTCCACCCGGATTACCGGTGTATTGACGAGACGATTCTCGACAACGA

GACACTCGAAGCAGCCTGTACAGTCAGCTTCGGGAATGTCAAGAAGGAGGGTGTATTCCACACACATCCTG

GCTATATAGATGGACTCACGCAGTCGGGCGGGTTTGTGATGAACGCTAACGACAAGACTAATCTCGGAGTA

GAAGTGTTCGTTAATCATGGGTGGGACTCGTTCCAGTTGTACGAGCCTGTCACTGATGATCGTTCGTATCA

GACTCATGTTCGGATGAGGCCGGCGGAGTCGAATCAGTGGAAGGGTGATGTGGTCGTTCTAAGTGGGGAGA

Sequences 273

ATTTGGTCGCTTGTGTTCGAGGATTGACGATCCAAGGAGTACCCAGACGAGTCCTGCGGTATATCCTGCAA

AGCAGTGCAAAAACCACACAGACAGCCACTTCGAGTGTGCCTGCCCCGTCTCAAGCTCCGGTGATGGTGCC

ACAGATTGTCCAAGTACCAAAAGCTAAGCCTATCTGCCAAATTTCCGGGACCCTGACAGAGGCTCTCCGGA

TTATTTGTGAACAAAGTGGTGTGCCTCTAGCAGAGCTCACGGATGATGCAACTTTCGCGAACATCGGCGTA

GACTCTCTCCTAGCGCTGACTATCACAAGTGCATTCGTTGAGGAGCTGGGTTTAGACGTCGATTCTTCCTT

GTTCATGGACTATCCTACTGTGGCGGACCTGAAGCGGTTCTTCGACAAGATCAACACGCAGCATGCTCCGG

CACCAGCCCCGGTATCAGACGCGCCAAAGCAATTACAACCAAGCAGTAGCCCAGTTGCATCTGCTACTCCG

TCTGCACCCATCCATGGCAGATCGAAATTTGAATCAGTTCTTAACATCCTTACCGAGGAAAGTGGTGTTGA

AATGGCAGGTCTTCCGGACTCTACTGCGCTTGCAGACATAGGTATCGATTCGCTCTTGTCCCTGGTAGTCA

CGAGCCGGCTGAACGATGAGTTAGAGCTAGATGTGTCGTCTGAAGACTTCAATGACTGTCTGACTATCCGG

GATCTCAAGGCACATTTCATGTCCAACAACTCCGACAATGGTTCGTCTGCGGTTCTTACTCCTCAGCCATC

TCGGGACTCCGCACTCCCTGAGCGCACGAGACCTAGGGTCGCTGATACAAGCGATGAGGAGGATGCACCGG

TTTCAGCAAATGAATTCACAACCAGTCCCCGCTCTACATCTAAGTATATGGCTGTGCTCAACATAATTTCC

GAAGAAAGCGGCATGGCAATCGAAGACTTCACCGACAATGTAATGTTCGCAGATATCGGAATAGACTCGCT

GCTGTCCTTGGTCATTGGAGGCAGAATACGGGAAGAGCTATCTTTCGACCTCGAGGTGGACTCTCTTTTCG

TGGACCACCCAGATGTCAAGGGACTGAAGTCATTTTTCGGATTTGAGAGCAACAAGACGGCGACAAATCCA

ACTGCGAGTCAATCGTCTTCGTCCATTTCAAGCGGCACTTCGGTCTTCGATACATCACCTTCTCCCACAGA

CTTAGACATCCTAACTCCAGAATCCAGCCTCTCACAAGAGGAGTTCGAGCAACCGCTCACAATAGCAACAA

AGCCACTTCCACCCGCAACTTCAGTCACTCTGCAGGGTTTACCCTCCAAGGCACACAAGATACTTTTCCTT

TTCCCAGATGGCTCTGGCTCAGCAACATCATACGCGAAACTCCCCCGACTCGGTGCGGACGTCGCCATTAT

CGGCCTGAACTCACCCTACCTGATGGACGGCGCCAACATGACCTGCACCTTCGACGAGCTCGTTACACTGT

ACCTAACAGAAATCCAGCGACGTCAACCCGCAGGCCCATACCACTTGGGCGGCTGGTCCGCCGGTGGCATT

CTCGCTTACCGCGCTGCGCAAATCCTCCAAAAAGCCGCCGCCAACCCCCAGAAACCAGTAGTAGAATCCTT

GCTCCTCCTCGACTCTCCACCACCAACAGGGCTCGGCAAGCTCCCCAAACATTTCTTCGACTACTGTGACC

AAATTGGCATTTTCGGGCAAGGGACAGCCAAGGCCCCGGAGTGGCTGATCACCCATTTCCAGGGCACGAAC

274 Appendix

TCCGTTCTGCACGAATACCACGCCACGCCGTTCTCATTCGGTACAGCACCCAGAACTGGGATCATCTGGGC

TTCGCAGACAGTGTTCGAGACGAGGGCCGTGGCGCCCCCACCTGTACGTCCTGACGATACGGAGGACATGA

AGTTTCTGACGGAGCGACGGACAGATTTCTCGGCCGGGCCTTGGGGACATATGTTTCCTGGTACAGAGGTA

TTGATTGAGACGGCCTATGGGGCGGATCATTTTAGTTTGCTGGTGAGTCTTCTCTTCCGTAAT

>vccB

CTGAAATTGTCTATGTGGCCGGTCTGTACTGATCAACTCCAAGTCTCCCCTCCTCTTATCGATAGGTTCGT

CTGGTTTATAAGTAGCGCTTGCTCCGCTAGTAAGTCGCCAGTCAAACCCTATCTCAACATGTCGTGGCAGG

GTCATGAAGGCCCCAAGGTGAAGCTGCGGTCGGCATGCGACCGGTGCTCTGCGAACAAGGTCAAATGCACT

CAGGAGAAGCCCGAATGCGAGCGTTGTCGTCTTCTGAGCTTGCCTTGCAACTATAGCCGTTCGATGCGGAT

AGGGAAACCACCCAAATCCCGACAGCGTGGATTGTCCAATATTGACCCGAAGACTCTCATGGGCGGGACTG

TTACCAAAAAGCTCAGGCCGTGTCCGAGCGCGCCAGAGTCAGCGTGTAGAGGTTCATTCGAAGATGGAGAT

GGGGGCCCGTGGACAGAAACTATGACATTCGAGGAGATGTTATCCCGTCCCTCCCCACCTCCCTTTGCTGG

ACCGTCACACAATTCAAACCGTCCTACGAACATGGCTTCTACGAACCAAGATCAGTACTATCACGACAAAG

GGAAACATGGCGAAACAATGGATGAGATGCTGCAGACGCTGGTCCCAGACTCAGTGCAGTTCATAGAGTTC

CCCAACACAGCCCGCGAAGACCAGAAACAACATCCAGAACTCAGGTCGGAAGAAGAGTATAGTGATTATAG

ATCCAAGTCTCTCTTCGAGGAAGGCTTGGCACGTATCGCACCGGATTGTGCTGGGGGGGTTATGGACGTCT

TATATGGCGAGGAGGCCTTAGTGCAGATGCCCAATCTGCACTCGAGTACGCATGACGGAAGCTCAAACACC

CATGTTACTTCGTCCCACAACTGTACGAGAGCCGTGATGGAGAATCTAGCCAAGCTATACCAAGTATGCGC

ACCTGCTGGAGTAGAGAATGGTTCCCACCCCACGACCGACCAAGTACTCAAAGCAAACAGCGACGCCATGA

AGGACGCTGCGGATCTTTTGGCGTGCCCGTGTGCCAAGGACTTCTGCTTTCCCATCATACTTGGCATCACA

GCATGCAGAGTTCTGGCCTGGTATCAGGTCGTGATCGACATGTATGACCCGGAGATTCCCATGGCCACGAT

GCCAACGGCACGCGAGGACATCAAGCACTGTCCGATTGCATTCGGAGCGTATCAGCTGGATGAGGAGGTGA

GCCAAGCAATGACCAGTCAATTCGTTCTACGAAACCTCCGTGCAATGACTCGATTTGTCAAGACCTATGTG

GAGAACTTCTGCTCTGATATCAATAAGAACCGGCCAGGAAGCTGTAGCCTCATCTACCGCTCCCTGGGCAC

TTTTATGCAGACGCGGCTTGGAAATACCATTGAGCAACTGGAGGATCGGTTGGCTGCATTTGATGGCGAGT

Sequences 275

ATACAAAGAACATCGGATAG

>vccC

ATGGCAGACGAGCAAAAGACCCCCTTGGAGAGCGGCCAGCAACCAGCAGTCGCACAACACACATCGACCGC

TGAGCTGCAGACAGAAAAGCCCGGCCAGATGAACGGTAATGGAACAGCAGACAAGCCAGGCCCTCCAGGAG

GCAAACCGTTTGGGCCTGGCATGGGCCCGCCTATACAGTATCCCACTGGATTCAAGCTCTACTCGATCATG

ACCGGGCTCTACCTCGCGAGCTTTCTCACGGCTTTGGATCGTACCGTGTTGGTGGTCGCCATTCCCCAAAT

TACCGACCATTTCAACTCGATTGACGATATCGGTTGGTACGGCAGTGCATATCTGCTCACCTTTTGCGCCT

TCCAGCTGTTGTTTGGCAAAATTTACTCGTTCTACAATCCCAAATGGGTCTTTCTGTCAGCTGTTTTGATC

TTCGAGATCGGATCGGCCATTTGCGGTGCTGCTCCCAACTCCACTGCCTTGATCATCGGTCGTGCGATCGC

GGGTCTCGGGTCCTCAGGGATTTTTGGTGGAAGTGTCATTATCACCTTCTTCACTGTCCCCTTGCATCAGC

GACCGATCTACACTGGCATCGCCGGCGTCATATTCGCGTTGGCCTCCTCGGTCGGACCGCTCATCGGTGGT

GGATTCACCAACAACGTGTCCTGGCGGTGGTGCTTCTATATCAACCTACCCGTTGGAGCCCTGACCGTGGT

GACTATTCTGCTGTTCTTGAACCTGCCACCCGCTCGTAAGGCCGGGACACCTCTCCGTGAACAGTTACTGC

AAATGGACCCACTGGGTAACCTTTGCTTAATCCCTGGTATCATTTGCCTTCTGCTTGCCATCCAATGGGGT

GGCTCAACATATGCGTGGAGCAATGGTCGGATAGTTGCACTGTTGGTTTTGGCTGGTGTCCTCTTAATCGC

CTTTGTGGGAGTCCAGCTGTGGCTGCAGGATAAGGGCACTATCCCTCCACGCGTGATGAAGCAGCGCAGCA

TTGCGGCTGGTATGGCATTCACGATCTGTGTGACCGCAGGCTTCATGTCTTTCAACTACTACCTCCCGATC

TGGTTCCAAGCAATCAAAAATGCGTCATCCTTCCACTCAGGTGTGATGATGCTGCCCACAGTAATCTCATC

AGGAGTAGCCAGCTTGGCCTGTGGATTCATCATTCATCGAGTTGGATACTACACGCCGTTTATGATCGGTG

GCTCTGTGCTCATGGCGATTGGCGCAGGTTTGCTCACCACGTTCACGCCCACCACAGAGCACCCGAAATGG

ATTGGCTATCAGGTTCTATGGGCATTAGGATGCGGAATGAGCATGCAGCAAGCCTCCCTGGCCGCCCAAAC

AGTGCTCCCGAAGCCTGACGCGCCAATTGGTATCTCGCTCATCTTCTTCTCACAATCATTAGGCGGCTCAA

TATTTCTAGCAGTGGACGATTCCATCTACAGCAACCGACTTGCGGCCAAACTGGGCAGCATTCCAAATCTG

CCTCAGTCAGCGCTGACAAACACGGGAGCCACCAATATTCGCAACTTGGTGGCACCTCAATATTTGGGTCG

TCTCCTTGGCGGCTACAATGACGCATTAATGGATGTCTTCCGGGTTGCGGTAGCCAGCAGTTGTGCGTGCG

276 Appendix

TGGTAGCTGCTGCCTTTATGGAATGGAAGAATGTCAGGGCGGCCAAGGCTGCTGGACCAGGTGGCCCAGGG

GGTCCAGGGGGTCCAGGGGGCCCAGGGGGTCCTGAAGGCCTGAGGGGAGGAAACAAGGTA

>vccD

ATGGCAAGTATGCAAGAAGTCGATGCGCTCGTGGTCGGTGCTGGATTCGGTGGCTTGTGGATGACAAACAG

ACTGAAAGAGGCCGCCCTAAATGTCCTCTGCGTTGAGAAAGCACCTCAAGCAGGCGGAGTCTGGTACTGGA

ACTGCTACCCTGGCGCCCGGGTAGACAGTCGCTACCCTGTCTATCAGTATTCCGACGAGTCGCTCTGTAAA

GACTGGAATTGGAGTGAACTTTTCCCAGGGTACGAAGAGATCCGCAAGTACCTCTCCTACGCGGTCGACAA

GTGGCAGCTAAACAGCCACATCCGGTACAATACAACAGTGACAGGTGCACGCTTCGACGAGTCGGACCACA

AATGGACCGTTGAAGGTATTAATGGCTCACATGGGACGATACGCATCCGGTGTCGTTGGTACATCCTCGCC

CTAGGGTTTGCCTCAAAACCATACATCCCCGACTTCGAGGGCCTGAACCGCTTCCAGGGTCCCTGCTTCCA

CTCCTCGGCGTGGCCACAGGAAGGAATCGACCTGAAAGACAGACGCGTCGCCGTCGTTGGCACAGGTGCAA

GCGCTGTGCAGATCATCCAGACCATTTCCAAAGAAGTCGGCCACTTGACCGTCTACCAGCGGACCCCGTGC

ACTGCCATGCCAATGAGACAGCAGTCCCTGACACCCGAATACCAGGACAATTTCAAAGCCTCCGGCGAGAT

GGCAGCCACGATGCGTCGTACAAAGTACGAGAGATTTGGTGGCCAAGACGTGCAGTTTGTTAGCCGTCGCT

GGCACGAAGATACCCCTGAGCAACGGCGTGCAGTGTTCGAGCAAGCCTGGCAGAAGGGCGGATTCCACCTG

CTCCTATCCACCTACTTCGAAGTCTTCGACGACGTGGAGGTGAACCACGCCGCATGGCGATTCTGGGCCGA

GAAGTCGCGCGAGCGCATCCATAACACCAAATACAAGGACATCCTAGCCCCGCTAGAAGCGGTCCACGCCT

TCGGAGGAAAACGTACCCCGTTCGAACAAGATTACTTCGAGGCCTTTAACCGTCGTAATGTAGATCTAATC

GATATGAAAGCGTCACCAATTCTCTCCTTTGCGGAGAAAGGCATCATAACCCAGAACGAGGGCTTGCAGGA

ATTTGATGTGATCATTCTAGCGACCGGCTTCGACACAAACACTGGCGCCCTAACATCCATCCATATCCAAG

ACACAGATGGCATCCTACTCAAGGATCGTTGGAGCTACGACGGCGTTAGGACGACCTTCGGGATGTCGACT

AGCAAGTTCCCCAACATGTTTTTCTTCTACGGGCCGCAGGCACCGACAGCTTTCTCGAATGGACCCTCGTG

CATTGAACTACAGGGCGAGTTCGTCGAGGAGCTGATCCTCGATATGATCGGGAAAGGTGTAACGCGCGTCG

ACACGACTAGTGAGGCCGAGAAGAGGTGGAAGGAGTCGACTTTGTCGCTGTGGAACCAATTTGTGTTTTCT

TCTACAAAGGGATTCTATACGGGGGAGAATATCCCGGGGAAGAAAGCTGAGCCGTTGAATTGGTTCGGGGG

Sequences 277

CTTTCCTCGCTATAGGAAGGCGTTGACGGAGTGTCGCGACGGCGGGTATAAGGAATATTCTCTTCAGTCTT

TGCCTAAAGTTCCGGATCCAGAGCACAGGGGGTTGATTGATAAGGTGGCCGTTGTTACATCGGCCCAGCCG

GTGGGAGCG

>vccE

ATGGATGGAAAAGCATTAACCGCATCGAGCCTGTTCGACACCACTGGTCGTGTGGCCGTGATAACAGGCGG

AGGAACAGGGCTTGGGCTGATGATGGCGAAGGCCCTCGAGGCCAACGGGGCTAAAGTCTACATACTCGGGC

GACGGCTCGAGCCCTTACAAGAAGCAGCCAAGCAGTCGACCCATGGCAACATCCACCCGGTCCAGTGCAGC

GTTACCTCGCATGCTGACCTTCAAGGCGTCGTCGACCACATCGCCGCCAAGGATGGGTATATCAACCTCCT

GGTGAATAACGCTGGAATCTCCACACCGAACCTGGGGCCCCATGCCACACGACCGACGCCGAAATGGGACA

TATCCAAAGTGCGTGACTACTGGTTTCACAAGTCCTTCGCGGACTACGCGGCGGTCTTCGAGACCAACACC

ACCGCTTCGCTGATGGTGACTTTCGCTTTTCTGGAGCTCTTGGACAAAGGCAACAAGAAGAGTGAGGAGGA

GGCAAAGGCTTCCCAGGCGCGCAATGGAGCCGGCAAAGCGCGCATCGAGTACGTGCGAAGCCAGGTGGTGA

CGTTGAGCAGCGTGGGCGGATTCGGTAGAGACAATTCCGCGTTCATCTACGGTGCGAGTAAGGCGGCCGCG

ACACATATGATGAAGAATCTAGCCACATATCTGGCCCCGTGGAAGATCAGAGTTAATATCATTGCCCCTGG

ATATTTCAACACCGACATGATGGGCAACTTCTACAAAGCAACTGGAGGTCGACTGCCTGCTTCACTGGCCC

CAGAAGAGAGGTTTGGGGATGCGCAGGAGATCGGGGGAACCATTGTGTATTTGGCATCAAAAGCTGGGGCG

TACTGTAATGGGATGGTTCTACTTGTGGATGGCGGTTATGTGTCGAATAAGCCAAGTTCATAT

>vccF

ATGTTTATGACCCAGATCGTGTTCGGGATAGCCCCGACCCTTCTCAAGACCTTCTCCCACCTCACTGCCCT

CGATCTCTGGCGACCATCCGCACCCTACGTGTTCGATCCTGTCACGAGTAGCACCTACCTAGGGACTATAG

CTGATGGGGTCGAGGAGTTCCTTGGGATCTTCTACGGCCAAGACACGGGTGGATCGAACCGTTTCGCACCC

CCAAAGCCCTATATTCCCTCCCGCCACAGTTTCATTAATGCGAGCACGGCGGGCGCCGCATGTCCCCAGCC

CTATGTTCCTCTGCCAGCCGATCCATATACCGTTCTCACCAATGTATCAGAGGACTGTCTCAGCCTGCGCG

TTGCGCGACCAGAAAATACGAAGTCTACTGCGAAGCTGCCCGTGATGCCTTCAGTTTTTGGACATGCCTTC

TCGGACGCTCTTCTAAAGTCCAAGTCCACGAATCTGGCTATGCAAGACCAACGTCTTGGGATCGAATGGAT

278 Appendix

CAAGAATCATATTTCTGCGTTCGGAGGCGATCCAGACAATATCACCCTCTTCGGAGAAGACGAGGGTGCAA

CGTACATCGCTCTTCACATTCTCTCAAACCATGAAGTGCCATTTCATAGAGCAATCTTGCAGAGTGGAGCC

GCCATAACGCATCACGATGTCAACGGGAATAGATCCGCGAGGAACTTCGCGGCCGTCGCAGCCAGGTGCAA

TTGTCTCTCTGATGGTAACCGAGACGTAGACTCCCAAGACACAGTTGACTGTCTCCGACAAGTTCCTATGG

AAGATCTAGTCAACGCAACGTTTAAAGTCGCGCACTCTGTTGATCCCGTGAACGGGTTCCGCGCATTGTAC

GTCCTCTTACACTTTCCCCTCTCACAAATGCAAGCAAGACTAACAGTCCCAGTATGCCCGCCAGGCCAAGT

ACCAGCCAATATATCTATCCTAGCAGGATGGACGCGCGACGAAAGCTCCATGTCCGTCCCAACGAGTATCC

GCACCGCAGCCGACGCTGCGTCCTTCATCTCAACCCAATTTCCGTTATTGAACGCCTCAACTATCCACCAC

TTTCTCACATCTCTCTATCCCGAATCGGACTTTACCACCAACAGTCCATCTTCAGCGGAGAAAGTTACGCC

CGCCTGGCGCGCGACATCTGCTCTCCACAGAGACCTTACCCTTACGTGCCCGACTATCTTCCAAGCATGGT

CCCTGCGCCTCTCATCCAACTGTACAACTCCGGTGTATCTGTACGAACTGCGTCAAAGTCCCTTCGCGACA

GCTCTCAACAACAGTGGTGTGGGATACTTGGGCATCGTACATTTCTCGGATGTACCGTATGTCTTTAATGA

GTTGGAAAGAACATACTACATTACTGACCCGGAGGAGAATAAGCTGGCTCAGAGGATGAGCGCGAGCTGGA

CTGCTTTTGCAAGCGGTGCTTTCCCTCTTTGTGAACGGTCTGAGAGATCATTGGGGAGATGGGAGGAGGCG

TATGGGGGGGACAGGGTTTGTAGGGATCGAATGCCAGAGCATGTACGGGTGAAAGGTATTGGGGATAATGG

CAACCAGGATGATGGGGATGAGATAGGGAAGCTTATGGGAAGGTGTGGGTTTATTAATCGGTTGGAGTACT

AG

>vccG

ATGTTGGACTCTGGTCATTTCCAAGAAACAACGTCGATTACTATGGCAGAGGAAACCAAGCTGACTCCCCT

GGAGACCTTCGCACAGGCAATCAGTGCCTCTGCGAAGACTATTGCAACTTACTGCAGAGACTCCGGTCATC

CTCAACTGTCCGACGATAATTCTAGCGGCCTCACTGGGGATGTTCTCCCCCCTTCCGCACCACAGGCAGTC

ACCGCCGCCAGACAGACCATCTTGGAGGCATCGTACCGGCTACAGCAATTGGTCACTGAGCCTAGCCAATA

CCTGCCGCGACTGACCGTTTACCACCTGGCTGCCTTACGCTGGCTGTGCCATTTCAGAATCCCGGAGCTCA

TCCCCGTACAAGGCACCAGGACATACTATGAGCTGGCTACAGAAGCCAAAGTTCCTCTTCATCAACTGCAG

AGCATTGCAAGAATGGCAATTACTGGGAGCTTTCTCCGAGAGCCGGAGCCCAATATCGTCGCCCACAGCAG

Sequences 279

GACGTCAGCCCATTTTGTTGAGAATCCTTCGCTCCGTGACTGGACACTATTCCTGGCAGAGGATACCGCGC

CCATGGCGATGAAGCTTGTTGAGGCGACTGAAAAGTGGGGAGACACGAGGAGCAAGACAGAGACGGCCTTT

AACCTGGCGCTGGGCACGGATCTGGCTTTCTTCAAGTATCTTTCCAGCAACCCGCAGTTCACCCAGAAATT

CTCGGGATATATGAAAAATGTCACAGCGAGCGAGGGTACTAGCATCAAACATCTCGTCAACGGATTCGACT

GGGCGAGCCTCGGAAATGCGATCGTGGTTGATGTTGGCGGTTCCACTGGTCATGCAAGCATTGCTCTCGCG

GAATCATTCCCCGATCTGAAATTCATCGTGCAAGACCTGCCCATGGTGACATCTACCTCGAAGGACAATCG

CGAAAAGACCCCTCTCCCAGAGACGGTTGCTTCCCGCATCTCCTTCGAGAGCCATGACTTCTTCAAGCCTC

AGCCGGTGCAGAATGCGGATGTTTATCTTCTTCGCATGATTCTGCATGACTGGTCATTCAAAGAAGCAGGC

GAGATCCTTGCCAATCTAGTACCGTCCGTCAAGCAGGGTGCTCGGATCCTCATTATGGACACTGTGCTTCC

CCGTCATGGTACTGTCCCCGTAACTGAGGAAGCGCTACTTCGTGTGAGAGATATGACGATGATGGAGACAT

TCAACAGCCATGAGCGGGAGATTGACGAGTGGAAGGACCTGATCCAGGGGGTGCATACTGGGCTTCGGGTG

CAGCAGGTCATTCAGCCGGCGGGGAGCTCAATGGCGATCATTGAGGTTGTTCGGGGA

>vccH

ATGCCGGCCTATCTTTTACTGCTGGCCTGCAATGTATTGCTCGTCCTGGGCGCCCATGTCCAACGCGAGCT

AGTGCTCACCTGGGAAGAGGGCGCCCCCAACGGCCAATCTCGGCAGATGATCAAGACAAATGAAGGCAGCC

GAAAGCCAGGCACCGCTGGACCCTGGGCAATGATTAGCGAAGATCCCGAGGACATTGCTGCCATGGAGCGC

GCAAGCAACAATCCTCACATCATGATGCTTTCTGACTGGGACTACTACAACTCGACCCAGTATAAAGAAGC

AGATGCAAACAGCCGACTGCAGATCTTTTGCGTCGATAGTATTCTTCTCAACGGCAAAGGGAGCGTCTATT

GTCCTGGTCATCAATGGCTCATTGACAAGCAGATCCCGTTCATGCACAAGAGCTGGCCGAATGACACTATC

ACAGACAAAGGGTGCTTCCCATTCGTACCATCGACAGAAGGTCCGTGGCTCGCAGACGGGAACGTGTCCGC

CATCCCGCCCGGTCTCCAGGAAGGCTGTGTTCCCTACTCCGGTCCAACAGAGGTAATCGAGGTTGACCCCG

CAGACCGCTGGGCGAGTGTGAACTGGATCGGAGGCTCGACATTCAAGACCCTCCAGCCAACAATCGATGAG

CACGAAATGTGGATCTATGAAGTCGACGGCCATTACATCGAGCCTCGCCGGGCAGACACCTTCCTGATTTG

GGCTGGAGAGCGATATTCAGCCATGATCAGACTGGATAAGAAGCCCATGGACTACTCGATCCGCGTCCCAG

ATGGTGGATATTCCCAGATGATCGCAGCATTCGGGATCCTCCGATACAAAAATGGAGACCCCAACGCCCGT

280 Appendix

CAGAAACCAGACCGATTTGGTGTCACGACTATATCTAAGCCCTATTTCGATTACAACGCTTGGCCGATGCG

AGATGCTGTTTTCCTAGACAAGCTTGACCTGCCTCCTTGGCCGAGAAAAGTTCCAGCTGCCCACGGAGACG

ATATGCATGTGTTGTACTTGGGCAAGGCTAACTCCACTTGGGAATTTACTCTGAGTGGGAAGAAAAAATAT

CCCCCAGACCGCTCAGCCTACGAACCCCTTCTTTACAACGTCAATTCCGAGCAGGCTCATGATGACGATTT

GATCATCCGAACCCAGAACGGCACCTGGCAAGACATCGTACTGCAGGTAGGTCATTCACCATTATGGCCAG

TCGACTTCCCGCACGCGGTCCATAAACACGCGAACAAATACTGGCGGATTGGAGGGGGGCAAGGCCTGTGG

AACTATTCCTCGGTAGAAGAAGCAATGGCCGACCAGCCCGAAAGCTTTAACATGGTGAATCCCCCTTACCG

AGACACGTTCCTGACAGAGTTCACGGGAGCGATGTGGGTTGTGCTGCGGTACCAGGTTACGTCGCCAGGCG

CGTGGCTGTTACACTGCCATTTCGAGATGCATCTGGATAATGGAATGGCAATGGCTATCCTAGACGGTGTA

GATAAGTGGCCCCACGTTCCGCCGGAGTATACGCAGGGTTTCCATGGGTTTCGGGAACACGAACTGCCAGG

CCCAGCGGGGTTTTGGGGTCTTGTATCGAAGATTCTGCGGCCGGAGAGTTTGGTATGGGCGGGAGGAGCTG

CGGTTGTTCTACTGTCGCTTTTCATCGGGGGTCTGTGGCGTCTGTGGCAACGAAGGATGCAAGGAACATAT

TATATTCTGTCCCAAGAAGATGAGAGGGATCGTTTCTCAATGGACAAGGAAGCATGGAAGTCTGAGGAGAC

TAAGCGGATG

Putative xanthone biosynthetic gene cluster sequences

Monilinia fructicola

>mfr1

ATGTCCGTACCTTCTCAGACCTCTGTCCTCGTTATAGGTGGTGGTCCAGCTGGGTCATACGCTGCTTCTCT

GCTGGGACGAGAAGGTGTCGACGTTGTGCTTCTTGAAGCAGACAAGTTTCCAAGATACCATGTTGGAGAGA

GTATGCTTGCTTCTATGCGTTTCTTCCTCAGATTCATCGATCTCGAGAAAACATTTGATGAGCATGGATTT

GAGAAAAAGTTTGGAGCAACATTCAAGATCACTAGCAAGAAGGAAGCCTATACCGACTTCGCCGCTTCTCT

TGGAAAAGGAGGTCATTCATGGAATGTAGTGCGTTCCGAGTCGGACGAGCTACTGTTCAAACACGCTGGAA

AGAGCGGTGCCAAAACTTTCGATCAGACAAAGGTAGACTCGCTCCAGTTCGAGCCATATCCCCATGATGGA

Sequences 281

TTCACCTCAGAGGATCGTCTTGCCAACCCAGGACGCCCTGTATCAGCGGCTTGGTCTCGCAAAGATGGCAC

CAGCGGTTCCATCAAATTCGACTACCTCATCGATGGCAGCGGCCGTAACGGTATTATTTCTACAAAGTACT

TGAAGAACCGTCGATTCAATGAAGGTTTGAAGAATATTGCAATCTGGAGTTATTGGAAGGGTGCAAAACGC

TATAAGCAAGGCGAGGAAAATGAGAACTCCCCTTTCTTTGAGGCTCTTAATGATGGCAGTGGTTGGGTCTG

GGCTATACCTCTCCACAACGGTACACTCTCGGTCGGTATCGCTGCTCGCCAGGATTTCTTTTTTGCAAGGA

AGAAGGCGTCTGAACTGGAAGGCAAAGCTTTCTATACCGACTACCTCAACCTAGTGCCAGGTATTCAAGCT

ATGTTGAAGGATGCTGAGATTGTATCAGACCTCAAACAAGCATCTGATTGGTCGTACAGCGCTTCTGCCTA

TGCTGGTCCTCACTTCCGTGTGATTGGCGATGCTGGTTGCTTCGTTGATCCATACTTCTCTTCTGGTGTTC

ATTTGGCTCTGACTTCTGGACTTTCTGCCGCTATCTCTGTGCAGGCAGCTCGAAGAGGCCAGGCTGATGAG

CGCAGCGCTGCTAAATGGCATACAACGAAGGTCTCCGAAGGGTATACACGCTTCTTGTTGTTGGTCATGAC

AGTCTTGCGGCAATTGAGAATGAAGGAGGCACATCTCATTACAACAGAGCAGGAAGAAGGGTTTGATATGG

CTTTCAAAAAAATCCAACCCGTTATCCAAGGTGTCGCTGACACGGAAACGAGTGATGCACGAGTTCAGAAG

AATGCCGCCGAGGCAGTCGATTTCTCGCTTGAATCTTTTGAGGTCACCCCCGAGAAGCAGCGCGCTGTCAT

TGATAAGATTGAGAAAGCTCAGGCAGCACCCGAGACTCTTGAGAAACTCACACCTGAAGAGGTTCACATTT

TGGGTGGCATCGTAACTCGAACTTTCGAGCGCGAGAAGGACGAGTTGAATTTGACTAGCTTTACTGGTGAC

GTTATCGAGGGATTGTCCGCGAACCTAGTTCGCGGTGACTTGGGACTCATCAGAAAGGATGCAAAGCCGGC

TACACCTGAGGCTACTGCTATCGTAGGAGTGCCAGCTGTTGAAAATATCAAGTCGGTGGCG

>mfrA

ATGACACCATCGGACTCAACAAGTACTTCTTTAGTAGAGCCTTCCAAAATGAAAGTTGGTTATTTCAGTAA

CGAATTCCCACACGATGACTTACGAGATCTTATTCGCCGCCTTCGAGTTCACAGTAAAGATTTGCGACACA

CTACCCTAGCAAGATTCATAGATGAGGCAACTTTAGCTATTCGCGAAGAAGTGCGGTTATTACCAGCCTCC

TTAAGAAAACTAGTTCAGCCGTTCGAGACTGTGTTTAATTTTGTCGACCAAACTACTCTCCGTAGTGGTCC

TCTTGGAGGTGCTATAGATGGCGTTCTACTCTGCGCTGTTCAGATAGCGACGCTCATTGGGTTTTACGAAA

CTGGTTCCGAGGAAGAATTTGACATCCACTGCCTCGCTGGTTTAGGCACTGGCCTTATCTCCACAGCTGCA

GTTTCTTTGTCCCCCACACCTCGCCAAATATCAGAGAATGGTCCCAAAGAAAGTTGGGCTTATGTTGTGCC

282 Appendix

AAATGTTGCTGCTAGCGATGTACAGAAGGAGCTTGATGCAATCCATGTGACTGAAAAAACACCCACGGCGA

GTAAAGTATTTATCAGTGCTCTTAATCAAACTTCCGTGACAGTTAGCGGGCCACCAGCAAGACTTCAGCAT

ATTTTTTACAAATCAGACTTCCTCCGGGACTGCAAGTTTGTTGCTTTGCCAGTATTTGCAGGGCTATGTCA

TGCAAAGCATGTCTACCACGAGGACCATGTTGAAAGAGTGATTCAAACTCCATCTATGGACCTATTGAATG

CCAAGTATTTTCCGCGAGTGCCGGTTTACTCAACCAGCACAGGGACGCCATTCAAGGCAAAAAGTGCCAAA

GGGTTATTCCATGATATCATTAAAGAAGTCTTGACCGAGGCAATTCGTTGGGATAATGTCATTCAAGGAAT

CATCCAACGAGCTAAGGATACTGCTGCTTCCGAATGCCAAGTTCTTGTCTTTCGAACATCTCTACCTGTTC

ACGATCTCTTCGCAGCTTTTGACGCGGACCCAGTCGAGCTCAAGGCAACAACATTAGATTTGGTACCATGG

ATTGCGAAACCAGATGCTCGACCAAAAGTGCCCAGAGGGACTGCACAGTCAAAAATTGCCATTGTTGGCAT

GTCTTGCAGGATGCCAGGAGGTGCGACAGATACCGAAAAATTCTGGGAGATCTTGGAGAAGGGCTTAGACG

TACATCGAAAAATTCCTGCCGATAGGTTCGACGTGGACACACATTTCGATCCAAACGGCAAGCGTTTGAAC

GCCAGCCATACTGCTTATGGTTGCTTCATCGACGAACCAGGTCTCTTCGACGCCCCTTTTTTCAACATGTC

TCCTCGTGAAGCTCTGCAAACAGACCCAATGCAAAGACTAGCATTAGTCACTGCCTACGAAGCTTTAGAGA

GATCTGGTTACGTGGCTAATCGAACTGCTTCCACTGACCTTCATCGTATCGGAACCTTCTATGGACAAGCT

AGCGATGATTATCGTGAAGTAAATACTGCCCAGGAGATCGGTACCTATTTCATAACAGGTGGTTGTCGTGC

ATTCGGCCCAGGGCGCATTAACTACTTCTTTAAATTCTCGGGACCAAGTTACAGTATTGACACGGCGTGTT

CATCAGGGTTAGCGACAATACAGACAGCATGCACCTCACTCTGGAACGGCGATACGGATATGGCGGTTGCC

GGAGGAATGAACGTTCTTTCTAACTCTGATGCGTTCGCTGGGCTAAGCCAGGGTCATTTCCTGACCAAGAC

GCCCAACGCATGCAAAACATGGGATGCTGAAGCTGATGGTTACTGTCGAGCTGATGGTATTGCCTCGATCG

TTATGAAAAGACTCGAAGATGCCGAAGCAGATAACGATAACATCATCGGTGTTATTCTTGGTGCGGGTACA

AATCACTCAGCCGATGCCATCTCGATTACACACCCACACGCTGGTGCGCAAGCGTACCTCACAAGACAGGT

GATAAGTCAGGCGGGTGTTGATCCTCTGGATGTCAGCTTCGTAGAAATGCATGGCACCGGCACGCAGGCTG

GTGATGCGCAAGAGATTGTATCTGTTTGTGACGTTTTTGCACCTCTTACCAGACGTCGTAATTCAAAGCAA

CCTCTGCACATTGGATCAGTCAAAGCAAACGTGGGCCATGGTGAGGCTGTTGCTGGTCCAACTGCTCTTCT

GAAAGTCTTGCTTATGCTTGAGAAAGAGACAATTCCACCGCACGTTGGAATCAAGAACAGGATTAATCCGG

Sequences 283

GTTTTCCTAAGGACCTAGATAAGCGAAACTTGCATATTCCCTACGAGAAACAACATTGGCCTAGAGTTCCT

GGACAGAAGCGCGTTGCTGTTGTGAACAACTTCAGTGCAGCCGGTGGTAATTCGACTCTTGCTATTGAAGA

AGGGCCATATCGTGAGGCGAGTGAAGTTGTAGATCCTCGCTCAGCCCATCTCATCGTCGTATCAGCTAAGA

GCAAAGTATCTCTCAAGGGTAACATCCAAAGGCTGATTGCTTATTTGGATGCAAACCCTAAAGTCTCCCTG

GAGAACCTAGCCTACACAACTACGGCTCGACGTCACCATCATAATCACCGAGTTGCTGTAAATTCATCGGA

TGTAGCTCATTTGAAGAAGCAACTTGGTTCTGCGTTGGGATCAGTTGATACTCATAAGCCCATTCCAGCAA

CTGGTCCTCCCCCGGTAGTCTTTTCATTTACCGGACAAGGAGCATCACATAGATCTATGAGCTTGGAGCTC

TTCCGTGATTCTCCTTTCTTCAAGGAACAGCTACTGCACCTTGATTCTATCTCGCAGGGCCAAGGTTTCCC

ATCGTTTATCCCTGCTATCGATGGCAGCCATCCTCATGATTATACACATCCACCAGTCATCTCTCAAGTAG

CACTTTGCTGTATAGAAATCGCGCTTGCTAAGTATTGGGAGTCCCTCGGTGTGAAGCCTGACGTTGTTGTT

GGACACAGCCTGGGAGAATACGCTGCACTCCATGTTGCCGGCGTCTTATCGGCTAGTGATACCATATTTCT

GGTTGGGAAAAGGGCACAGTTGCTTGAGGAGAAATGCCAAATTGGAACCCACAAGATGCTTGCGGTTCGAG

CTTCGCTTTCTCAGATACAAGAGAACGCTGGTACCAAGCCTTTTGAAGTCGCATGCATAAATGGTCCCAAG

GATACAGTACTAAGTGGCACTCGTCAAGAGATCGACGAAGTATCAGAGGTCTTACAAGGTGTTGGCTTTAA

GTGTTTCAGCTTGGACGTTGCATTTGCATTTCATTCTGAGCAGACAGATCCTATTCTGGAAGACTTTGAAG

AGGTTGCAAATAGCGGGATTTTATTCCATGCACCTAGCCTTCCAATCATCTCACCTCTCCTTGGCAAAGTT

GTTTTTGATGAAAAGACAATCAATGGAAACTACATGCGACGAGCTACTCGGGAAGCCGTTAACTTTCAATT

GGCAATTGAAGTAGCACAAAAGGTGTCGATAATCGATGAGTCCATGGTTTGGATTGAAATCGGCCCACATC

CTGTATGTATAGGATTTGTAAAGTCGATTCTGCCATCCGTGAATGTCGCAGTTCCTTCAATTCGTAGAGGA

GAGGACAACTGGCAGAGCTTGTCGCAGAGCTTAGGGCAATTACATTGCGCTGGCGTGGAGGTTAGCTGGCA

AGAGTTCCATCGACCATTCGAGCAAAGCCTGCGCCTCCTAGATTTACCCACGTATGCTTGGAACGACAAGA

ACTACTGGATTCAATATAACGGTGACTGGGCACTCACAAAAGGAAACACCTTCTACGATGCAGAAAAGGCC

ACAGCAAAGACGATCCAGGCATCTCCTGTGGCCAAATCAGTTTCCACTCTGAGAACTTCGACTGTCCAATC

GATCATCGAAGAGACCTTTTCTGGATCTACTGGTAGAGTTGTAATGCAGTCAGATTTAATGCAGCCAGATT

TCTTGGCTGCTGCATGGGGACATCAAATGAACAACTGCGGCGTGGTGACATCATCAATCCATGCAGATATA

284 Appendix

GCATTTACCCTTGGACAATATCTCTTGAAGAAATTGCGGCCCAAGTCAAAGAATATTCACATGAATGTAGC

CAACCTGGAAGTACTCAAGGGTCTGGTTGCGAAGAAAGATACCACAACACCACAGATTATCCAGGTTTCGA

TTGAAGTAAAGGATATTGACTCCGGGGTGGCTGATCTGACATGGTATAATGTCACAAATGGGGATATTTAT

GAACAATTTGCCAGTGCCAACATCTACTTTGGTAACGCTGATACTGCGCTCGCAAGCTGGGTCCCAATGAC

GCATCTTGTCCAAGGTCGAATCGAGACCCTCGAGCGTCTCGCAGAAGAGGGAGTAGCTAACAAGCTTTCGC

ATAATATGGCGTATCTTCTCTTTGCTAGTAACCTGGTCGACTATGCAGACAAATACCGGGGGATGCAATCA

GTGGTTCTGCACGGATTCGAAGCATTTGCAGACATCACTCTCACAACAGAAAAAGGAGGCGTATGGACCAT

CCCTCCTTACTTCATTGATAGCGTGGCTCATCTCGCGGGATTCATCATGAATGTTTCGGACGCTGGTGATA

TCAAAAATAATTTCTGTGTTACACCTGGCTGGGGATCATTGCAATTTGCTAAACCACTCGTTGCAGGTGGC

AAATATCGATCGTATGTTAAGATGATCCCAACGGCTGAGGATCCAAACATTTACCATGGTGATGTTTTTAT

TCTCCAGAATAATGTTATCATTGGTATGTGTGGAGCTATAAAGTTCCGTCGTTATCCACGCCTACTCCTGA

ATCGCTTTTTCTCTGCCCCCGACGAGAGCGAATCTAAGCATGGCGCTGCTGCTGCAGTACCAGCAAAAGTG

CAATCTTCCAAGGCAGCGGAGAAGTCGGTGGCGCCAAAATCTGTAATTATCCCAGAGCCAGCATCAGCTCC

GGTTCCCACCCCTCAAGCAGCATTGGAGCCAGTCAGGGACGCTCCTACCAAAATAGAGTCAGTCGTTCCCA

CTGATGTAGCAGTTGAGGACTCGAATAGTACCGCCGTCAAAGCCATGGTGCTTGTGGCTGCTGAAGCAGGA

ATTGATGTTTCCGAGCTTCAAGACGACGTGAATTTCGCGACTCTCGGTGTTGATAGTCTCATGAGTCTTGT

TATAGCCGAGAAATTCCGTGATGAGCTTGGTGTTACAGTCAACGGAAGTTTGTTTTTAGAATATCCAACCG

TGGGCGACTTGCGGGCTTGGTTAGTAGAATACTACAGC

>mfrB

ATGGCGCCAGGACAAGGTGGCTACCGCCAGATTAACAAAGCTCTTAATATTTGTGTCTTTGAAGATTATCT

AGATGGACAACAAACATATTTGCCAAAACTACAGGATGTGGAACAGCTCAGCCCGAGAGTGATTCGTGTTC

TTGGGCAAAATCCAGGCAAATTTACTCTTCAAGGAACAAATACATATATCGTTGGAACGGGAGAGAAACGT

CTAATCATCGATACCGGTCAGGGCATCCCAGACTGGGCTGATCTTATTTCCTCTACACTAACCGAGAACAA

AAGCTCACTGTCCCATGTGTTCCTCACGCATTGGCATGGTGATCACACGAAGGGCGTTCCTGATTTGCTTC

GAATGTATCCAGATCTTTCTTCATCGATTTACAAGCATACACCGAGTAAAACTCAACAACCTATAGTAGAC

Sequences 285

GGCCAAATTTTCAAAGTTGAAGGCGCTACTATCCGCGCAGTACACTCTCCTGGTCACTCGAAAGATCATAT

GTGCTTTATTCTTGAGGAGGAGCAAGCCATGTTCACTGGAGACAATATTTTAGGACATGGTACCGCCGCGG

TAGAGCATCTCAGTACCTGGATGGCTGCTTTACGCAAGATGCAGACCTATAATTGTGTAAAAGGCTATCCA

GCCCATGGTACTGTTGTCGAGGACCTACAGGCCAAAATCGGAATTGAATTATCGCAAAAGATACGACGCGA

ACGACAAGTTTTACAGAATCTGGAGAAGAGCAAACGCAGGGAAAGAGCTTCTGGTGGACGTGGCAAAGGAA

GTGTTACGGTCAAGGAACTGGTCACTGCTATTTATGGGAGTAAGATAGATGATGAGCTCCGTGAGATGGCG

TTGGAGCCTTTAATGGAGGAAGTTCTGCGGAAATTGTCTGAGGATGGGCTTGTAGCGTTTGAAATGAGGGC

AGCGGTTAAGAAGTGGTTTTCAATTGAAATGATA

>mfrC

ATGGCTCCGAAATCCACCTCTAATCCACCGCCCGTTGGGGTACAAGCTACTGCTGTTGCTACAGGATCTTT

CCTGACAGTTACAGGTGCTATGGTCTGTCTCACCACAGTGGTAGTCCCCGTCTTTCTCGACACCGACACCG

AGTCCATTCATTTACTCCGCCATTGGACTCGTCTCTACCACTACGGTCATATCTACATGCCTGCAGTATGT

ATCGGAACTGTAGGACTATATGGATACTCAGTATTGAGTAAGCGGGCTTCAAAAAGCCAGGAATGGGCTAT

TTACGCCGTAGCTGCTGCGATAACAATTACTATGGTACCATTCACATGGATCTTCATGGCGCCTACCAACA

ATATTTTGTTTGATTGGGAAAAATTGGCGACAGCTGAAACTTCAGTGGTGGAGCTGAATATCGTACAAGAG

CTTGTGGTGAAATGGGCTTGGTTGCATCTTGCTCGTTCAGTATTTCCCTTGATCGGTGTGATTCTAGGTTT

CAAGGGGGTTCTGCAGGAGCGTCATCTACAGCGAATCTCGGGG

>mfrD

ATGGCAATCATTCCACAGCCCAAAGTTTCCAATCCACATGGCTCGAATAAAGTTCGATACCTCTGCCTTAC

GATTTGTGGCTACCGTAAGCCGGGTATGAGTGAAGAGGACTATCGCAACCATATGGTCAATGTATCAGCAC

CTCTCACAAAAGATCTTATGGTCAAATATGGTGTTAAAAGGTGGACTCAGATTCACAATCAAACAGCTACC

CGAGCGTTGATGTCACAACTTTTTGATCCGCAAATGTGTAATGTTGCCGACTTTGACTGCTTTAGTCAGGT

AGTTTTCGAAAACCTAGAGGACTACAAGCGCATGAAGCAAGATCCATGGTATAAGGAGCATTTGATAGGAG

ATCATGAGAAGTTCGCAGACACTAAGCGAAGCCAAATGACTATCGGGTGGATCGAAGAATTTGTGAGAGAC

GGGCAGGTTGTCGATGGATTTGAAGTGAAGCATACTTCATCAAGATTAAACAAGTATTTTTATGCATTGAT

286 Appendix

AGCTGGTTTAATTGTGCTAGGTGGACTAGGTTGGAGGAGTGTTCAAATCATGGGGCTGAATGCCTTGAAGG

GATCTCTT

>mfrF

ATGCATGTCGCGCACACACCTTTATCAGCATCATTCGTTAACAACCCCTCTTTACTGGATGCTGCAATGTT

CCTTGCCGAGTGTGCGACACCAGCCGTACTGCAAACCCAATCACATACGCTAGTGCCGAGGCAAATCAATG

TCGCGAGCCAATCGACGAAATTTCGGCGCCAGCAATCAGCTTATCTGACTCTTGCGGGGGGACTGAATGCA

GAGGACTCCGTTGTTGATAACCTCATGCAGTTGAACTGGGCAAAGATTGGCAATTCTGCGATCGCTAAAGG

AGCCCATGTTGTTGAGGTGGGCGCCGAATCAACAACCACTGCGCGCAGTCTGACTGCTTTGAATCCAGCCC

TCCACTTCCAAATGCAGCTCGATGCTCCTAAAGACAGGGATATATACATGGAGAAACAACGTGAAGAAACG

TTTGAGCAGGAGCTTGGTAGTCCAGAGCCACGAATCACAATCACCCATAGACCAATAGGTGTGGGGTATCC

ACAAACAGCCACTGATGCTGCTGTCTATATTCTACATCTGCCTGAAGAGCCATCCGCAACTTTAGCTGAGC

TGAAGGAACATCTCAATATCCTACGAGCTGGAAATGCTGTAATGTTAATCTTGACAGCAAGTCTCCTACCC

GAGCCTGGCAGCATACCTGACCCCGAAACTGAGGCTACAGTTCGCTCACGGGATCTAGCGTTACTGCACTT

GCAACTTACTACTACGGGTGAGATGGAAATGGTGGAGCTGCTTCGAATGATTGAGACGGTCAGCGATAATT

CGGGAAAGTTGGTCGTGATAAATAAGCTTCGCTCACGCAACAACATAGTCGTCGCATTGGCTGTTAAGTAT

GTTGACCATGGTCTGGGCCTATGGTCTGGGAATGATAATGGGGATAGAGTTGGTAATGGGTTTGGAAGCTT

GGCTGCCAATGAAGGA

>mfrH

ATGGCTATCTACGCAGTTCTTGGATCTACTGGCAACTGCGGTACAGCCCTCATCCAAAATCTTTTAACGCA

GCCGAAAGCAAAGATTAATGCCTATTGCCGAAACAAGTCTAAGCTTCTGCGACTTGTTCCTGAAGTTGGAG

ATAGTAAGCAGGTGGAGGTTTTTGAAGGCAGCATCCAGGATGAGAAGCTACTCGAAAATTGTTTGAAGGGA

TGTCGTGCCGTCTTTCTGGTCGTATCTACCAATGACAACGTTCCTGGGTGTCATCTCAGCCAAGACACAGC

AATCAGTGTCATTAATGCACTCAAGAGCCTCAAAAAATCAGGAGATGTGAAGATACCCAAGCTCGTTCTTC

TCTCGTCAGCAACTATCGATGACCACTTCTCACGCCACCTACCCTGGCTGTTGAGGCAAATCTTACTTCGG

TCTGCATCCCACGTCTATAATGATCTGAGAGAAACCGAGAAGCTTCTTCGTTCACAAGAAGATTGGTTGAC

Sequences 287

AACCATCTACATCAAACCGGGAGCGCTCTCTGTCGATAAACAACGGGGCCATGCTTTGAGCTTCACTGAGG

AGGGTAGTCCACTTTCGTATATGGACCTGGCAGCCGCTATGACAGAGGCTGCTAACGACAAGGAAGGACAG

TATGATTTGCGGAATGTGAGTGTGGTAAACACAAATGGCAGAGCGAGATTTCCAACAGGAACCCCAATGTG

TATTTTAACTGGACTTCTGAGACACTTTCTTCCCTTTTTGCATCCGTATTTACCGACAACAGGACCT

>mfrI

ATGACCACCCCATACATTCCATACCGCCTTGATGGCAAAGTAGCCCTCGTAACTGGCTCCGGACGGGGTAT

TGGTAAGGCTATGGCTATTGAGCTTGGCCGATTGGGTGCTAAGGTTGTTGTCAACTATGCAAATTCTGCGG

AATCTGCCGAAGAAACGGTTGCTGAGATCAAGGCTGTGGGATCTGACGCAGTTGCTTTCAAAGCCGACGTT

CGCCAGGTTCCCCAGACAGTCAAGCTATTCGACGACGCAGTTGCTCACTTTGGTGGGTTGGACATCGTGTG

TAGCAACTCGGGCGTGGTCAGCTTCGGACATTTGGGCGATGTGACTGAGGAAGAATTCGATCGTGTTTTCA

GCCTGAACACGCGCGGACAATTTTTTGTTGCACGTGAAGCCTATCGCCACCTCAATGAGGGTGGACGTATC

ATCTTGATGTCTTCCAATACCGCGAAAGACTTCACTGTACCTAAGCACTCACTGTACGCGGCTTCTAAAGG

TGCCATTGATTCTTTTGTCCGAGTCTTCTCCAAAGACTGTGGCCACAAAAAGATTACAGTCAATGCGGTAG

CACCCGGTGGCACAGTTACAGATATGTTCCATGCTGTATCTCAGCACTACATTCCCAATGGTGAAAAATAT

ACGGCTGAAGAGCGACAGCAGATGGCGGCGCATGCTTCACCATTGGTCCGAAATGGATATCCGATTGATAT

TGCAAGGGTTGTTTGCTTCTTGGCTAGCAACGAGGCCGAATGGGTTAACGGGAAAATCTTGACACTTGATG

GAGGCGCTGCA

>mfrJ

ATGGTTTCCGCTCTCCGAGATGAACCCCGTAGATCGGGTCCGGGGGTCGGAAGAATTCAAAAATTGACCTG

CAGCAATGCCACCCGCTCCAACTCAGTGAACTTCTTCAGTATGGCGAAACAACCTTCTTTCCAAGATATTA

TTGGATGCCAAGCGGCTCTTTACGAGTGGGCTGAGAGTTACGATACAAAAGACTGGGAACGTCTTTCGAAA

ATAGTTGCCCCTGTCCTCCGGATCGACTACCGCTCGTTCCTCAATATGCTCTGGGAAGAAATGCCCGCTGA

ACAATTCATAGCCATGGCTTCCGACCAAAATGTCCTAGGGAATAAGCGTCTCAAGACACAGCACTTGGTCG

GCGTAACAAAATGGGTCCAAACAAGCGAAAATGAAATTACCGGATATCATCAGATGCGAGTGGCTCACCAA

AGATATAAAGATGATGAGCTTAAGGAAGTCGAGATCAAAGGACACGCTCACGGCAAGGGCACAGTTTGGTA

288 Appendix

TCGTAGGGTCGATGAAGTATGGAAGTTCGCTGGAATTGAGCCTGACATTCGGTGGGGAGAATATGACCATG

AGAAGATTTTTGAGCGACATGAA

>mfrK

ATGACTGCCACCAAATCCTCACAAGTTCTTTTGTCCCTCGAGTATCCCGGCTTGGGTCGTCTAGATTCAAA

TCCGCCGGGAACTTTCGAGTTGACAAGCGAACTGCTCCAAAAGAACCATGAAAAGTACCACATGTACTTTC

GAGATGTCGCCGGCCATAATCATATTCCGCATTCTTTGCTGACCGTCCTCGCAATGGGGGGCGGGCATGAA

GAATTAAAGAGGGCATATGACGATGGATACGTTATTCAGCGTCCCCTAGCTCCCGCAAACCCTGAAATCTT

TAAGGAGTTGGGCGAAGAAGAGAAGTTCAAGGAGCATATGTATACATTATCCGAATATACCAACTTCTTAG

CTTTCTTTGAGCAAGAGATCGATGCCAAAGGGTGGAAAGCCATTCTCAATAAGTATTGCTTCGAACGCACT

ACTTTCGCTGATACCATGCTTTCCCAATTATACGAAGGCCTCTACCATCCTATTCTCCACCTCGGTTTCGG

CGTGGAATTTAATCAACCTAGCCTCATCGCCGAAGCTCTCGCTCACGCAGCGTCCCATGATCCAGGTCAAA

TTCAACCCTTCTTCCTAGAGGCCGAGGAGCTGGCATCTTCAGGCTCAGTCAAGCCTAAGCCACTTGTCGAG

CTATACGCTGAGATTCGCGCGAATCAAAAGATCCGGGAGGCCGCATGGCTGAAAGATGGGCCATTACGCGG

AAAGGGAGTTCTGAGCCGTGCTCACAATGAACTAGTCAAGATTGCGGCCCAATTTCAGGTGAAACCTGAGG

ACTTGGAACGCGGCACAGCGGAGATGATGAGCTGTGGTGCACTCACCAGCGCTGCCCAAAAAGCCGGGAAG

ACACGCAAGATTGACTTTTTCTATTTGCATATCGTCACATGCTCTATCTTCTTCAGCGTTTTTAATCAGCA

GGATTGGCTCAAAACAGAGGATAAGGTGCGTCTGCTTGAGTGGAAGGGACGCCTAGATCTTGTATGGTATG

CAGCCAATGGGGCGGCTGAGATGAACATCGAAAATTTGATCGACTATGAACCTTCTTTGAGCAAGGGATGG

GACTGGAGTGCTATATACAAAGGGTCAATCGATATTCATGATGATGGGCACGTCGTAAAATTCATCCGAGC

TCTGAAGCATGCCGAAGATACGGTGGAGCCTTTTGTGAAGAAAGAAGGTGCTGCTTCTTTCCCCGTCAATG

GCGATATATGGCTCAAAATCGCCCAAATCTGTTATGATTCTACTGCAACTATTGCACCAGATCTCCCGGGT

CAGTTATCGAAGAAATGGGTTTGGGGTGCTGGCTTCGAGCCACCGTGGTTCCAGATCCCAGACTTGCAAAA

CGCCGTC

>mfrN

ATGTCCTCAAACGGCACAACTTCAAATGGCAAGATCTTTGCCCAAGATAAAAAATTCTGGAATAACTACCT

Sequences 289

CAAAGGCCGACCCCGTGCTCCAGATATCTTCTTCGATCGTATATTCAATTACCACAAAGCCCAAGGGGGCG

AATTTGGCATAGCTCATGATGTAGGCGCTGGAAATGCACCTTACGCGAACAAATTACGGTCCAAATTTTCT

CACGTTATCGTCTCGGACATTGTTGCTAAGAACATCGAACTTGCTCAAGATCGCCTTGGAACTGATCAATA

CAGCTACCGAGTAGCAAAAGTGGAGGAAGCTGAAGACATACCCGCGGGGAGCGTGGATTTAGTCTTTGCGA

CAAATGTGATGCATTTTCCAGATCAAAAGGAAGCCATGAGTGCTATTTCGAAGCAGCTGAAGTCGGGCGGA

ACTTTCGTCTGTAGTGTATTTGGGCCTGCTCGTTTTGAAGATCCGGCATTACAAGACCTATGGGCACGAAT

TAGCCACGAGGGAGGTCGTGTACTTTTGAAAAATGCAGAACAGCCTGATCAGACTATCAAAATTATGGCCA

GAACTCAGGGCAACTACAACGTAGCTCCTCTTGATCCTGAATTCTTCCAGCAAGGTGCCACACGTGTGCAC

TTGAACATGAGAAATGGTGGAATTATTGAGCTTTTGCCTCCCGAAGAGGCACACAGAAATAAGGAACCCAA

CTACACTGGACCAGATGATGTCGAGGTATTCGAAGATGAGCAGGGGTGGAATTTTGAAACAGACTTGGAAG

GAGTCAAGGAGCATATTGGCTCGTTTCCATTCATCGAAGCAGATCCTACAGCTTTTACAGACCTGTTCAAG

GAGCTTGAGGTGCTAGTTGGCCAAAAGAAAGTTCAAGGTTATTGGCCTGCGAAAGTCATTCTAGCTACACG

GCGT

>mfrO

ATGTCTTCCACAAATGGTATCTCTCACGACCATGGAGACGGCGAAGGGTTCAAGTTCTTTGTGGATGGCGA

CAAAGAAGAATTCGATCCAGAAAAATGGACTGGACTCGATAATCAGTTGCCAAGACGTGGCAAAAATACGG

GAGTGAATGTGCTCATAGTGGGAGCAGGTCTCGCTGGATTGACGTGTGCATTGGAGTGCTGGAGAAAGGGA

CACAACGTTGTGGGAATCTTAGAGAGGAACAAAGGCCCAAATTATTCTGGAGATCTCATTTTCATTCAGCC

ATCTGCTGTAAAATTCATGCGCTACTGGCCCGACATGTGCCGAGATTTGGAGCAAGACAAAACTGATATTC

CGGAGTATTATCGGAAGCATAACGGCGAGCTCATCTATGGACCTTCGGAGCCTCACTTCAACGATCCTGAA

GACATAGCCCTACGGGAAAAGAATGGGATTCCTCACGTGGGGGCTTTGCAAATACGCAAGAAGTTCTATCG

AATGTTGTTACGACAAGTCGCTAAATTAGGCATCAAAGTCGAGTATGGGCAGCGGGTCGAGAAGTATTTCG

AAGATGAAGATGCTGGTCTGGGTGGTGTGGTGACTGAGAGTGGCTCTGTTAGGGTGGCCAACATAGTTGTT

GCTGCCGATTCTGCTAAGTCTAAATCTGAAATCCTCATTGCTGGTGAACACATGCCATCGGTATCTAGCGG

CATGTCCGTTTACCGTGCTTCATATCCAGGACATCTGGCAACGAAAGACCCGATTCTTCAAAAAAGATGGG

290 Appendix

GAGGCGACAGCTCTGTTTCTCATGAGCTTTGGCTGGGACCTGGCATGCATATTGGCCTCTACTTTTCACCT

GAATTTGTCGCTTTCGGAATCACACCGCGCAATAACTTCTTGGCAGAAGAGAGTGCCGAGGCAAAAGAGTC

ATGGGATCCTTCTGTTGATCCTGAGGAAGTTATTCAAGTCCTTAAAAGGGTACCTGACTGGGACGAAGCCA

TCATAGCTCTAGTAAAGAACGCACCCAAGGGATCCGTAATACATTGGCCGTTATTATGGCGCAATCTTCGC

CGTGAATGGACTTCCAAGGGTGGCCGTGTTGTACAGATTGGTGACTCTGCGCATTCCAATGTCCCAAGTTC

TGCTAGTGGTGGTACCCTAGCTCTTGAAGACGCTATAACATTAGCTTCTTGCCTGCAATTGGCAACATATG

AAAGTGGACCTAAAGCTGCGGGTCTTGGCGCCAAGATATATAACTTGCTTCGATGGCAACGAGTGAGTTGT

AATCAAAAGGTCGCATTTGTCAACTCGCAAGCCACCAATACAAAGAGTATGGACTGGGAAGCTATCAAACA

GGATCCAAAGAAGGTCCGCTTGAGGTTCTGCAAATGGTTTTTCAGACACGATCCTGAAGCCTATGTTTACG

AGAAGTACGGGCAAGCTTTTGCACATTTGGTTGACGGTACTGAGTTCCAGAATACCAATATACCTCCTGGC

CACAAGTTTGTACCTTGGACAGTTGAAGAAGTCCAAAGGGATATCAAAGAGGGCAAGAGGATAGAAGATTT

CTTGGATGGGGACTGGTCA

>mfrU

ATGCCTGCACCAGCCGAAGTTCAAGCAGCTACTCTCGAAAAATTTATTGCAGGATGGAAGGAATTCACCCC

CGAATCTTGGATGGCAACATGGTCTGAGGATTGCACTCAAAAGATGTTGCCTTTGTCATTGGGGGTACCTG

CTAGGTCGCGAACTGAGGTCTTGGGCATCTTACCAAAGCTCATTGGTATCTTGAAAAACTACAAGGTTGAT

ATCTACGAGATCGTGCATGATGCTCCCAGGGGGAAAGCAGTAATCTACGCAACTTCTTATGCGGACACGCC

ATTTGGCGATTTCAAGTGGACGAATGAATATGCTGTCTTCATTACTTTTACTGAGGACGGGACACAGGTCC

AGAAGTTCGAAGAGATGGTCGATACGGCATTCTATCAGGAATTCTTTCCCAAGTTCCTGCGATATATGGAG

CAACAGGGAGCTCCCGTTCAC

Sequences 291

292 Appendix

3 Analytics

3.1 Cytochrome P450 conversions

0

200

400

600

Abs

orpt

ion

[mA

U]

(a) Standards

0

100

200

300

Abs

orpt

ion

[mA

U]

(b) Conversions with S. cerevisiae pESC-His::PCV_6005

m− cresol

0

100

200

300

Abs

orpt

ion

[mA

U]

m−hydroxybenzylalcohol

0 10 20 30 40 50 600

100

200

300

Time [min]

Abs

orpt

ion

[mA

U]

toluhydroquinone

Figure 1. HPLC chromatograms of conversions with S. cerevisiaepESC-His::PCV_6005. Detection was carried out with DAD (λ 254.4 nm).A) Standards: m-cresol (58, blue), m-hydroxybenzylalcohol (59, green), andtoluhydrochinone (57, orange). The analysis was performed according to publishedparameters.[150]

Analytics 293

0

200

400

600

Abs

orpt

ion

[mA

U]

(a) Standards

0

100

200

300

Abs

orpt

ion

[mA

U]

(b) Conversions with S. cerevisiae pESC-His::PCV_6007

m− cresol

0

100

200

300

Abs

orpt

ion

[mA

U]

m−hydroxybenzylalcohol

0 10 20 30 40 50 600

100

200

300

Time [min]

Abs

orpt

ion

[mA

U]

toluhydroquinone

Figure 2. HPLC chromatograms of conversions with S. cerevisiaepESC-His::PCV_6007. Detection was carried out with DAD (λ 254.4 nm).Standards: m-cresol (58, blue), m-hydroxybenzylalcohol (59, green), andtoluhydrochinone (57, orange). The analysis was performed according to publishedparameters.[150]

294 Appendix

3.2 NMR spectra

[13C]-(R)-semi-Vioxanthin (4)

[ppm]

1210

86

42

[rel] 0.000.050.100.15

10-OH

9-OH

5-H

6-H

8-H

3-H

O-CH3 4-H

CH3

Figu

re3.

1H

NM

Rsp

ectr

umof

[13C

]-(R

)-se

mi-v

ioxa

nthi

n([

13C

]-4)

.C

DC

l 3,2

1°C

,400

MH

z.

Analytics 295

[ppm]

150

100

50

[rel] -01234

C1

C7C10

C9

C5a

C4a

C5

C9a

C8C6 C10a

C3

O-CH3

C4

CH3

Figu

re4.

13C

NM

Rsp

ectr

umof

[13C

]-(R

)-se

mi-v

ioxa

nthi

n(4

).C

DC

l 3,2

1°C

,100

MH

z.

296 Appendix

F2[ppm]

1210

86

42

F1 [ppm]150 100 50

10-OH

9-OH

5-H 6-H8-H

O-CH3

4-H

CH3

C1C7C10C9

C5a

C4a

C5 C9a

C8C6

C10a

C3

O-CH3

C4

CH3

Figure

5.H

MB

CN

MR

spectrumof

[ 13C]-(R

)-semi-vioxanthin

(4).C

DC

l3 ,21°C

,1H

NM

R400

MH

z,13C

NM

R100

MH

z.

Analytics 297

F2[ppm]

86

42

0

F1[ppm] 150100500

CH3

4-H

OCH3

3-H

8-H

6-H

5-H

9-H

C3

CH3

C4

OCH3

C6C8

C5 Fi

gure

6.H

SQC

NM

Rsp

ectr

umof

[13C

]-(R

)-se

mi-

viox

anth

in(4

).C

DC

l 3,2

1°C

,1H

NM

R40

0M

Hz,

13C

NM

R10

0M

Hz.

298 Appendix

F2[ppm]

150100

50

F1 [ppm]150 100 50 0

C1

C7C10

C9

C5a

C4a

C5

C9a

C8

C6C10a

O-CH3

C4

CH3

C3

Figure

7.IN

AD

EQU

ATEN

MR

spectrumof[ 13C

]-(R)-sem

i-vioxanthin(4).

CD

Cl3 ,21

°C,100

MH

z.

Analytics 299

[13C]-P-Pigmentosin (7)

[ppm]

1412

108

64

2

[rel] -0.00.51.01.5

10-OH

9-OH

8-H

5-H

3-H

4-H

O-CH3

CH3

Figu

re8.

1H

NM

Rsp

ectr

umof

[13C

]-P-

pigm

ento

sin

(7).

CD

Cl 3

,21

°C,4

00M

Hz.

300 Appendix

[ppm]

150100

50

[rel]0 5 10 15

CH3

C4

OCH3

C3

C8

C10a

C9a

C6

C5

C4a

C5a

C9

C7

C10

C1

Figure

9.13C

NM

Rspectrum

of[ 13C]-P-pigm

entosin(7).

CD

Cl3 ,21

°C,100

MH

z.

Analytics 301

F2[ppm]

1412

108

64

2

F1[ppm] 15010050

10-OH

9-OH

8-H5-H

3-H

OCH3 4-H

CH3

C1C10

C9C7

C5aC4a

C5C6

C8C10a

C3

OCH3

C4

CH3

Figu

re10

.H

MB

CN

MR

spec

trum

of[13

C]-

P-pi

gmen

tosi

n(7

).C

DC

l 3,

21°C

,1H

NM

R40

0M

Hz,

13C

NM

R10

0M

Hz.

302 Appendix

F2[ppm]

1412

108

64

2

F1 [ppm]150 100 50

C1C10

C9C7

C5aC4a

C5C6

C8C10a

C3

OCH3

C4

CH3

10-OH

9-OH

8-H5-H

3-H

OCH3

4-H

CH3

Figure

11.H

MB

CN

MR

spectrumof

[ 13C]-P-pigm

entosin(7).

CD

Cl3 ,

21°C

,1H

NM

R400

MH

z,13C

NM

R100

MH

z.

Analytics 303

F2[ppm]

86

42

F1[ppm] 15010050

C1C10

C9C7

C5aC4a

C5C6

C8C10a

C3

OCH3

C4

CH3

8-H5-H

3-H

OCH3

4-H

CH3

Figu

re12

.H

SQC

NM

Rsp

ectr

umof

[13C

]-P-

pigm

ento

sin

(7).

CD

Cl 3

,21

°C,

1H

NM

R40

0M

Hz,

13C

NM

R10

0M

Hz.

oxidative phenol coupling in ascomycetes: regioselectivity

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