David Hopwood

Lecture 1

(DH1)

Isolation of microbes from soil:fungi, actinomycetes, other bacteria (left);

streptomycetes (right)

Bioactive compounds from microbes (2002)

Antibiotics Other Total

Actinomycetes 7900* 1220 9120

Other bacteria 1400 240 1640

Fungi 2600 1540 4140

Total 11,900 3000 14,900

*70% from Streptomyces spp.

Diminishing returns in finding useful natural products

ActinomycetesOther bacteriaFungi

Antibiotic producers aredifferentiating microbes

Penicilliumnotatum

(penicillin)

Aspergillusterreus

(lovastatin)

Penicillins Cephalosporins

Griseofulvin

Cyclosporin Lovastatin

Valuablefungal

metabolites

Myxococcus

Sorangium(epothilone)

Stigmatella

Somemyxobacteria

Myxobacterial metabolites

Epothilone

Ambruticin

Valuablemyxobacterial

metabolites

Et

Me

MeMe

Me

CO 2H

O

OH

OH

O

EE

ES

S

S

R

R

S

SR

R

R

A Streptomyces colony on an agar plate

Streptomyces: scanning EM

Young

vegetative

hyphae

Transition

stage:

most

antibiotic

production

Aerial

hyphae,

young spores

Mature

spores

Apoptosis:nutrientrelease

Feeding

Antibioticproduction

Reproduction

Fatal attraction

Needs for new antibioticsNeeds for new antibiotics•Overcome acquired resistance:

Staphylococcus aureus (MRSA)Vancomycin-resistant EnterococcusMDR and XDR Mycobacterium tuberculosisGram-negative respiratory pathogens

•Less toxic anti-viral or anti-cancer agents

•Immunosuppressants, cholesterol lowerers…

How to find new antibiotics

•Novel natural products

•Chemical synthesis combichem

•Genetics, genetic engineering

Filamentous fungi:gene replacements, genomics

Myxobacteria:transduction, transposon libraries,gene replacements, genomics…

Streptomyces:plasmid-mediated conjugation, protoplast fusion,autonomous and integrating plasmid and phagecloning vectors, gene replacements, transposon libraries, genomics…

Genetics of antibiotic producers

Class Replicon Example

Low copy number autonomous plasmid SCP2* pRM5

High copy number autonomous plasmid pIJ101 pIJ699

Single copy number integrating plasmid pSAM2 None

pPM927 pSET152

Single copy number integrating phage ΦC31 KC515

Streptomyces cloning vectors

Streptomyces plasmid SCP2

Streptomyces phage C31

Streptomyces mycelium and protoplasts, light microscope

Streptomyces mycelium and protoplasts, electron microscope

The Streptomyces coelicolor genome

1958

First Streptomyces coelicolor linkage map

1965

First antibiotic gene(later named act)

1990 1990

antibioticbaldwhite

1993

Http://jic-bioinfo.bbsrc.ac.uk/streptomyces - then click “ScoDB II”

(325 clones)

LH arm = 1.5 Mb

RH arm = 2.3 Mb

Core = 4.9 Mb

7825 ORFs(55 pseudogenes)

63 tRNA genes6 rRNA operons

72.12% G+C

9 May 2002

Isolation of antibioticbiosynthetic genes

act mutant of Streptomyces coelicolor(Brian Rudd, 1976)

act mutants of Streptomyces coelicolor

The first act clone(Francisco Malpartida, 1984)

The act genes of Streptomyces coelicolor

Actinorhodin

Tailoring steps Regulationresistance

Chain assembly(PKS)

Actinorhodin(S. coelicolor)

Medermycin(S. AM-7161)

Mederhodin

First‘hybrid’

antibiotic(1985)

Manipulation of polyketide biosynthesis

Some actinomycete antibiotics(Polyketides)

Medicine Agriculture

Application Examples Application Examples

Anti-bacterial ErythromycinTetracyclinesRifamycin

Livestockrearing

MonensinTylosinVirginiamycin

Anti-cancer Adriamycin Anti-parasitic Avermectin

Immuno-suppression

FK 506 Fungicide PolyoxinKasugamycin

Antifungal Candicidin Herbicide Bialaphos

Polyketides

Palmitic acid

6-MSACyanidin

Erythromycin

Oxytetracycline

Brevetoxin

Aflatoxin

COOHA fatty acid

Variables in polyketides

OH

O

RStarterO

Side chains

Extender

Chainlength

(‘Combinatorial biosynthesis’)

OH

Reduction level

KR/DH/ER

OH OH

Chirality

Type II PKSact (simple)

Type Imodular PKS

ery (complex)

The act and ery PKS gene clusters

*

*

CLFKS

AT ACP

AT

ACPKS

KR AT

ACPKS

KR

AT ACPKS AT ACPKS KR

DH ER AT

ACPKS

KR AT

ACPKS

KR

TE

O

O

O

OH

OH

OH

Module 1Module 2

Module 3Module 6Module 4

Module 5LM

DEBS1 DEBS 2 DEBS3

6dEB

SO

SO

HO

HO

SO

HO

HO

HO

SO

O

HO

HO

O

SO

HO

HO

O

SO

HO

HO

HO

O

HO

SO

HO

1

TE

The DEBS paradigm for complex polyketide biosynthesis

Discovery of ‘cryptic’ secondary metabolites

‘Secondary metabolic’ gene clusters in Streptomyces coelicolor

3 antibiotics (type II PK, modular PK, NRP)

4 siderophores (2 NRP, 2 other)

3 pigments (type II PK, chalcone, carotenoid)

2 complex lipids (unsaturated FA, hopanoid)

2 signalling molecules (terpenoid, -butyrolactone)

8 other (2 modular PK, 1 NRP, 2 chalcones, 2 terpenoid, 1 deoxysugar)

PK = polyketide, NRP = non-ribosomal peptide, FA = fatty acid

Total length ~ 375 kb ~ 4.5% of the genome

S. coelicolor v. S. avermitilis

Class S. coelicolor S. avermitilis

Type I PK 3 8

Type II PK 2 (1) 3 (1)

NRP 4 6

Carotenoid 1 1

Desferrioxamine 1 1

Chalcone 3 (1) 1

Others 9 4

Red = similar gene clusters

Enediynes

PKS

Zazopoulos et al. (2003)Nature Biotech. 21:187

Discoveries/year

Cumulativediscoveries

Watve et al. (2001) Arch. Microbiol. 176:386 “How many antibiotics are produced by the genus Streptomyces?”

500

current effort level

Increased effort

2003

Total may be 150,000!

“Therefore, by genic manipulation of the cell we have a means for obtaining, in quantities sufficient for study, many of the metabolic products of the living organism that would be otherwise undetectable”

Albert Kelner (1949)

Improvement of productivity

Regulation

+ -

Environmental factors

Cofactoravailability

Shunt products

Feedbackinhibition

Pathway genes

Substrate Product

Uptake Export

Competing pathways

Synthesis

Undesiredsubstrate

Some targets for influencing antibiotic productivity

Need for functionalgenomics

Many genes with cumulative effects!

22,000 survivors of mutagenesis

Pick 11 best strains and fuse protoplasts

Pick 7 best from 1000 progeny and fuse protoplasts

Screen 1000 progeny: 2 as good as the best from1 million cultures screened over 20 year

[Zhang, y. et al. (2002) Nature 415: 644]

Increased productivity of S. fradiae for tylosin(24,000 colonies screened over 1 year)