Aromatic Compounds from Sugar

Jan de Bont

28th Symposium on Biotechnology for Fuels and Chemicals April 30 - May 3, 2006, Nashville, Tennessee

Aromatic Compounds from Sugar Jan Wery

Harald Ruijssenaars

Hendrik Ballerstedt

Rita Volkers

Karin Nijkamp

Nick Wierckx

Maaike Westerhof

Luaine Bandounas

Suzanne Verhoef

Jean-Paul Meijnen

Frank Koopman

Corjan van der Berg

Jan-Harm Urbanus

Louise Heerema

Hugo van Buijsen

Dorien Wijte

Marijke Mol

Nicole van Luijk

Jan de Bont

TNO

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B-Basic

Summary

H2N CH C

CH2

OH

O

OH

O

H

HO

H

HO

H

OH

OHHH

OH

OH

R

SUGARTYROSINE

Target compounds

Solvent-tolerant Pseudomonas putida as production host for hydroxylated aromatics

Hydroxy-aromatics in many cases are chemically difficult to synthesize- Compounds find many applications.- For instance as monomers in the production of various polymers including liquid crystal polymers.- Prices of biologically-produced compounds may go down to $5/kg or less.

Overproduction of tyrosine

Genomics approach

Introduction of genes

Downstreamprocessing

PURE AROMATICCOMPOUND

Often Dirty

Chemistry

Toxicity Issues

Pseudomonas putida S12

• Able to grow on many compounds including glucose,

also in the presence of a separate phase of either

toluene or octanol

• Toluene and octanol are very toxic for any normal

microorganism

Efflux Pump in Pseudomonas putida S12

Solvent tolerance of Pseudomonads: A new degree of freedom in biocatalysis Jan Wery and Jan de Bont, Pseudomonas, Volume 3, Edited By J-L Ramos

Kluwer Academic / Plenum Publishers, New York, 2004.

OUT

IN

MEMBRANE

Efflux system

removes many,

chemically unrelated

compounds

Hydrolysate from Wheat Straw

• Acid pretreatment

• pH adjusted to 7

• Cellulase hydrolysis

• Supernatant used as growth medium

Compounds Present in Wheat Straw Hydrolysate

Compound Concentration (g/l)

cellobiose 1.7

glucose 26.9

xylose 6.8

arabinose 1.3

5-hydroxymethylfurfural 0.1

furfural 0.6

acetic acid 2.7

Growth in Wheat Straw Hydrolysate

0.0

1.5

3.0

4.5

0 20 40 60 80

Time (h)

CO

2 (

%)

E. Coli

P. Putida S12

Strategy:

• Compare wild type under various growth conditions

• Create diversity in solvent tolerant phenotypes by directed

evolution

• Assess cellular response of various phenotypes by

proteomics and transcriptomics

• Pinpoint relevant mechanisms

Mechanisms Other than Efflux System

and

Regulatory Networks?

Effect of Exposure to Toluene as Analyzed by

Comparative Proteomics

Chemostat experiments under 4 conditions:

•Either carbon or nitrogen limitation

•With or without 5 mM toluene

Technology:

2-D Fluorescence Difference in Gel Electrophoresis

(DIGE)

Advantages:

•Effective way to overcome gel to gel variation because

each protein spot has its own internal standard

•High sensitivity due to fluorescent labeling

Cy3 Cy5

Cy5

Cy3

Effect Toluene on Yield

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.2 0.4 0.6 0.8

Dilution Rate (1/h)

Pro

tein

(g

/l)

0

0.4

0.8

1.2

1.6

Glu

cose

(g

/l)

P. putida S12 was grown in a glucose-limited chemostat

in the absence or presence of 6.2 mM toluene

Presence

Absence

Protein

Protein

Glucose

Glucose

Compensation required for H+ leakage and pumping activity

H+

H+ leakage

+

H+

ATPase

--

H+

Solvent pump

+

Isocitrate dehydrogenase

2-ketoglutarate dehydrogenase

Succinyl-CoA synthetase

Succinate dehydrogenase

Fumarase

TCA cycle

H+ H+

+

Rita Volkers et al; In Press

Production of Aromatic Compounds

Efflux system

Overproduction of an aromatic

amino acid

Aromatic

product

SUGAR

AROMATIC PRODUCT

P. Putida S12

General Approach

• Introduce relevant gene(s)

• Create mutants in random procedures

• Screen for producing mutants

• Proteomics and Transcriptomics analyses of mutants

• Targeted knocking out, or overexpressing of genes

• Cultivate selected mutants in fed-batch and remove

product during fermentation (in situ product recovery)

Target Compounds

Via phenylalanine:

• Cinnamic acid

• 1 other compound

Via tyrosine:

• Phenol

• 4 other compounds

Mutagenesis and High-throughput Screening

Transcriptomics

Affymetrix NimbleExpress Custom Array

based on P. putida KT2440

Sequence of P. putida S12 available soon

O

H

HO

H

HO

H

OH

OHH

H

OH

SUGAR

Overproduction of phenylalaninein P. putida S12 Introduction of PAL

NH2

O OH O OH

PHENYLALANINE CINNAMIC ACID

Karin Nijkamp et al.; Appl Microbiol Biotechnol (2005) 69: 170-177

O

HO OH

HO OH

HO

glucose

H2N

O

OH

phenylalanine

HO

HO

HO

O

O-

shikimate

NH2

OHO

HO

tyrosine

O

HO

cinnamic acid

O

HO

cinnamic acid

PAL

6 enzymes

2 enzymes

13 transport-associated

proteins

50 genes > 1.8 upregulated

16 unknown function

22 clearly related to the process

12 unclear relation

P. putida S12TPL

Glucose

Phenol

Phenol

Tyrosine

Tyrosine phenol lyase

Efflux system

Product recovery

• Introduction of tpl enables phenol production.

• Optimization is necessary.

Phenol Production in P. putida S12

Generation of a Phenol-producing Strain

0

200

400

600

800

1000

1200

1400

1600

0 10 20Time (hrs)

μM

ph

en

ol

S12TPLS12TPL1S12TPL2S12TPL3S12Tn1

NTG mutagenesis

fluoro-tyrosine selection

Transposon mutagenesis

aroF-1 overexpression

tpl overexpression

NTG mutagenesis

fluoro-phenylalanine selection

Negative control

Nick Wierckx et al. Appl. Environm. Microbiol. (2005) 71: 8221-8227

Chemostat culture: at steady state, add 1 mM tyrosine pulse

400

450

500

550

-30 70 170 270

T (min)

μM

ph

en

ol

0

0.2

0.4

0.6

0.8

1

1.2

OD

600

μM fenol

OD600

Phenol Production After a Tyrosine Pulse

+ 1 mM tyrosine

tyrosine, phenylalanine

glucose

dahp

3-dehydroshikimate

shikimate

phenylalanine

3-dehydroquinate

tyrosine

phenol

degradation via

protocatechuate

degradation via

homogentisate

phenol

Primary metabolism

Green: up-regulated

Red: down-regulated

Summary Transcriptomics Results

Interpretation of Transcriptomics Results

• Many hits in relevant pathways

• Results obtained for 7 aromatic compounds produced

• Results obtained for mutants generated independently

• Results from proteomics

Combining these results allow for selection of

relevant genes

Phenol Toxicity

and

Recovery of Phenol During Fermentations

• 5 mM phenol in the fermentor

completely inhibits production.

Fed-batch Phenol Production

0

1

2

3

4

5

6

7

0 10 20 30Time (hrs)

CD

W (

g/l

)

Ph

en

ol

(mM

)

0

2

4

6

8

10

12

14

16

18

20

Am

mo

nia

(m

M)

NH4+

Phenol

CDW

Fermentor

Glucose feed

Culture

Phenol Toxicity

0

1

2

3

4

0 2 4 6 8 10

T (h)

OD

600

0 mM

3 mM

6 mM

9 mM

12 mM

Growing cells of P. putida S12TPL3 in the presence of phenol.

Phenol Inhibition at Enzyme Level

0

100

200

300

400

0 5 10 15T (min)

μM

Py

ruv

ate

0 mM phenol

0.25 mM phenol

1 mM phenol

Effect of phenol on activity in cell extract from P. putida

S12TPL3 of tyrosine phenol lyase (tyrosine phenol) activity.

Extractive Recovery of Phenol

via

Octanol

Fed-batch with 2nd Phase (20%) of Octanol

• Phenol no longer

inhibits its production.

0

10

20

30

40

50

60

70

0 20 40 60 80 100

Time (hrs)

Ph

en

ol

(mM

)

Am

mo

nia

(m

M)

0

0.5

1

1.5

2

2.5

3

3.5

CO

2 (

%)

Phenol in Octanol

CO2 concentration

NH4+

Fermentor

Glucose feed

Culture

Octanol

Extractive Recovery of Phenol

via

Solvent-impregnated Resins (SIR’s)

Process Layout Involving SIR’s

Filter

Fermentor

Product

Recycle

aqueous

phase

Recycle

SIRS

SIR

Microstructure of a typical

macroporous polymer

SISCA versus Pertraction

• SISCA much larger area for extraction (particles vs fibres) = faster

extraction kinetics

• SISCA is potentially cheaper

SISCA = Extraction(/adsorption) + flotation

Status

•Principle proven

•Patent pending

Filter

Fermentor

Product

Recycle

aqueous

phase

Recycle

SIRS

SIR

Microstructure of a typical

macroporous polymer

SISCA versus Pertraction

• SISCA much larger area for extraction (particles vs fibres) = faster

extraction kinetics

• SISCA is potentially cheaper

SISCA = Extraction(/adsorption) + flotation

Status

•Principle proven

•Patent pending

Start End

How Solvent-impregnated Resins Operate

P. putida Fed-batch (2 L) Fermentations

0

2

4

6

8

10

0 20 40 60 80

Time (h)

Ph

en

ol in

aq

ueo

us p

hase (

mM

)

+50g Resin

+50g SIR

Control

+SIRs/Resin

Product release

Phenol Production?

• Production cost phenol via P. putida S12 5 $/kg

• Current phenol price 1.5 $/kg

Not Phenol

• Phenol is no option; just a model compound

• However, several 4-hydroxy-aromatic compounds

(produced via tyrosine) will be of interest

Goals in Time at TNO

Currently • Bioconversion of glucose into aromatic several compounds in the

host Pseudomonas putida S12 • Integration of production of aromatics and product recovery • Production of non-aromatics via other amino acids

Longer term: • Complex lignocellulosic biomass to aromatic compounds;

expansion of substrate profile of host - xylose, furfural - methanol obtained from biomass via syngas Collaborations: • Industrial partners and others