Synthetic pathways for aromatics

production from biobased feedstock

Harald Ruijssenaars

BIRD Engineering

Delft-Schiedam, The Netherlands

BIRD Engineering

Contract research company in Delft, The Netherlands• fermentation / medium optimization services

• strain development services (yeast; bacteria)

• non-food market (biofuels, bio-based chemicals)

Collaboration / Customers• (a.o.) Mascoma, Amyris, DSM, Tate&Lyle, Nedalco, Applikon, Heineken

• Delft University of Technology

• Knowledge networks: Kluyver Centre, BE-Basic, CLIB (Germany), SIM (USA)

Substituted (hydroxy-)aromatics

•many & diverse applications, e.g., in plastics (LCP‟s),

resins, fibers

•fossil-based

•often difficult to synthesize chemically

•improved / novel functionality with biotech OH

R

OH

Rresins

polymers

specialties

........

Biocatalytic production of aromatics - bioconversion

1. product toxicity

2. product formation

3. feedstock utilization

Product toxicity aromatics

Challenge for bioproduction:

•hydrophobic molecules (“solvents”)

•accumulation in cell membrane: cell death

OH

R

Solvent tolerant host: Pseudomonas putida S12

Grows in presence of:

•2nd phase of toluene (7.2 mM in water phase)

•2nd phase of 1-octanol (4.2 mM)

•benzene (mutant up to 25 mM – near saturation)

•butanol (up to 6 %)

Volkers et al., Env. Microbiol. Rep. (2010) 456-60

Rühl et al., AEM 75 (2009) 4653-6

Synthetic pathways for aromatics synthesis in P. putida S12

Native pathways: aromatic amino acid synthesis

L-phenylalanine and L-tyrosine as „base compounds‟

for production of non-native („synthetic‟) aromatics

L-phe L-tyr

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

Product formation

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

phenolL-tyr phenol lyase

(Pantoea agglomerans)

Wierckx et al., J. Bacteriol. 2008

Product formation

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

t-cinnamate

L-phe/L-tyr

ammonia lyase

(Rhodosporidium

toruloides)

Nijkamp et al., Appl. Microbiol. Biotechnol. 2005

Product formation

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

p-coumarate

L-phe/L-tyr

ammonia lyase

(Rhodosporidium

toruloides)

p-coumaroyl-CoA

p-hydroxybenzaldehyde

p-hydroxybenzoate

protocatechuatePyr + oxaloacetate

fcs

ech

vdh

pobA

Product formation

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

p-coumarate

L-phe/L-tyr

ammonia lyase

(Rhodosporidium

toruloides)

p-coumaroyl-CoA

p-hydroxybenzaldehyde

p-hydroxybenzoate

fcs

ech

vdh

Verhoef et al., J. Biotechnol. 2007

Product formation

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

p-coumarate

L-phe/L-tyr

ammonia lyase

(Rhodosporidium

toruloides)

Nijkamp et al., Appl. Microbiol. Biotechnol. 2007

Product formation

L-tyr

shikimate pathway

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

p-coumarate

L-phe/L-tyr

ammonia lyase

(Rhodosporidium

toruloides)

Verhoef et al., AEM. 2009

phenolic acid

decarboxylase

(Lactobacillus

plantarum)

p-hydroxystyrene

CO2

Product formation

0

1

2

3

4

5

6

7

8

S12TPL S12TPL1 S12TPL2 S12TPL3

aro

ma

tics y

ield

(%

, C

-mo

l)

L-tyr

C-source

chorismate

ED

PPPGAP

E4P

DAHP

L-phe

prephenate

PEPPyr

TCA

Product formation – synthetic network improvement

targeted and random

mutagenesis

screening and

selection

improved flux to L-tyr?

transcriptomics and

flux analysis

Wierckx et al., J. Bacteriol. 2008

Wierckx et al., J. Biotechnol. 2009

uptake

shikimate pathway

aromatic products

Feedstock use

Biobased feedstock

•multiple components: LC hydrolysate -> glc, xyl, ara, other sugars

•inhibitors: acetate, furaldehydes, aromatics

Synthetic biology: optimize efficient feedstock use

minimize effects inhibitors

WT P. putida S12

glucose 35.4 % +

xylose 20.2 % -

arabinose 2.4 % -

uronic acid 2.5 % +

org. acids (Ac-, formate) 4.8 % +

furaldehydes (HMF, furfural) 0.7 % -

aromatics (lignin) 19.2 % +/-

Oxidative / phosphorylative xylose utilization

xylose

xylonate

2-ketoglutarate

TCA

cycle

xylulose

xylulose-5-P

PPP

oxidative

pathway

phosphorylative

pathway

xylonate

2-keto-3-deoxyxylonate

2-ketoglutaric semialdehyde2-ketoglutarateTCA

cycle

NAD+NADH

XylD

XylX

XylA

xylose

periplasm

out

in

xylose

PQQ+ PQQH

Gcdxylonolactone xylonate

Gnl

Meijnen et al., AEM 75

(2009) 2784-2791

Xylose utilization - 1Oxidative xylose pathway Caulobacter crescentus

xylose xylonolactone

NAD+ NADH

XylB XylC

(h-1) Yxs (%)

xylXABCD 0.21 53

xylXAD 0.21 57

Hybrid Caulobacter-Pseudomonas pathway

Phosphorylative xylose pathway E. coli

xylose xylulose xylulose-5-P PPP

ATP ADP

XylA XylB

Meijnen et al. AEM 74 (2008) 5031-7

(h-1) Yxs (%)

P. putida S12xylAB 0.01 10

P. putida S12xylAB2 0.35 67yield

growth

rate

Strain improvement via

evolutionary selection

Xylose utilization - 2

glucose

non-ox. PPP GAP

ED

PEP Pyr TCA

Molecular basis improved xylose utilization phenotype?

G6P 6PG

KDPGRu5P

Ri5P Xu5P

F6P

ox. PPP CO2

catabolismanabolism

xylose

xylulose xylose

catabolism

phosphorylative xylose metabolism requires

extensive “rewiring” of the metabolic network!

non-ox. PPP GAP

ED

PEP Pyr TCA

Transcriptomics evolved xylose utilizing phenotype

G6P 6PG

KDPGRu5P

Ri5P Xu5P

F6P

ox. PPP CO2

xylose

xylulose xylose

*

Meijnen et al. in preparation

* mutated glc transporter

edd

eda

pgi

pgl

zwf-1

zwf-2

non-ox. PPP GAP

ED

PEP Pyr

Transcriptomics evolved xylose utilizing phenotype

G6P 6PG

KDPGRu5P

Ri5P Xu5P

F6P

ox. PPP CO2

xylose

xylulose xylose

*

Meijnen et al. in preparation

edd

eda

pgi

pgl

zwf-1

zwf-2

Pyr AcCoA

isocitrate

KGA succinate

malate

OAA

glyoxylate

2 CO2

reduced CO2 formation:

increased biomass yield

replenish Ri5P:

maintain PPP

non-ox. PPP GAP

ED

PEP Pyr

Transcriptomics evolved xylose utilizing phenotype

G6P 6PG

KDPGRu5P

Ri5P Xu5P

F6P

ox. PPP CO2

xylose

xylulose xylose

*

Meijnen et al. in preparation

edd

eda

pgi

pgl

zwf-1

zwf-2

Pyr AcCoA

isocitrate

KGA succinate

malate

OAA

glyoxylate

2 CO2

NADHNAD+

NA

DH

NA

D+

Furaldehyde metabolism

furfural hydroxymethyl

furfural (HMF)

Degradation products of pentoses (furfural) or hexoses (HMF)

Toxic fermentation inhibitors / carbon loss

No (genetic) characterization of catabolic pathways

Few microorganisms known to degrade furaldehydes

Wierckx et al. (2010) Microb

Biotechnol 3: 336-343

Furaldehyde metabolism

Novel furfural / HMF degrading bacterium isolated: Cupriavidus basilensis HMF14

• Gram- bacterium

• Mesophilic (<38 ºC), neutrophilic aerobe

• Growth on HMF, furfural, aromatics, NO sugars

• PHA production

Identify HMF-furfural degradation genes by transposon mutagenesis

Wierckx et al. (2010) Microb

Biotechnol 3: 336-343

Furaldehyde metabolic pathways of C. basilensis HMF14

Transposon mutagenesis

• Random insertions in genome: 14.000 clones

• Screen for HMF- or furfural- mutants: 25 clones

• Identify transposition loci: 8 genes in 2 clusters

HMF HMF + furfural

Koopman et al. (2010)

PNAS 107: 4919-4924

Furaldehyde metabolic pathways of C. basilensis HMF14

functional analysis of gene clusters

HmfH

(or generic

dehydrogenase)

HmfD HmfABC

HmfE

+ H2Ospontaneous

or lactonase

TCA

cycle

furfural

2-ketoglutarate

+ H2O

Koopman et al. (2010)

PNAS 107: 4919-4924

Furaldehyde metabolic pathways of C. basilensis HMF14

functional analysis of gene clusters

HmfH

(or generic

dehydrogenase)

HmfH

HmfFG

CO2

HMF

Koopman et al. (2010)

PNAS 107: 4919-4924

Furaldehyde metabolic pathways of C. basilensis HMF14

functional analysis of gene clusters

HmfH

(or generic

dehydrogenase)

HmfD HmfABC

HmfE

+ H2Ospontaneous

or lactonase

TCA

cycle

HmfH

(or generic

dehydrogenase)

HmfH

HmfFG

CO2

2-ketoglutarate

furfural

HMF

+ H2O

Koopman et al. (2010)

PNAS 107: 4919-4924

Furaldehyde metabolism in P. putida S12

μ (h-1) Yxs (%)

hmfABCDE 0.30 51 growth on furfural

hmfABCDE + hmfFGH 0.23 40 growth on HMF (and furfural)

WT P. putida S12 engineered P. putida S12

glucose 35.4 % + +

xylose 20.2 % - +

arabinose 2.4 % - +

uronic acid 2.5 % + +

org. acids (Ac-, formate) 4.8 % + +

furaldehydes (HMF, furfural) 0.7 % - +

aromatics (lignin) 19.2 % +/-+/-

Koopman et al. (2010)

PNAS 107: 4919-4924

Summary and conclusions - 1

Synthetic pathways constructed in P. putida S12 for:

•production of various aromatic products

•combined heterologous / endogenous activities / gene deletions

•utilization of xylose (+ arabinose)

•oxidative / phosphorylative

•utilization (detoxification) of furaldehydes

•novel pathway / genes isolated and characterized from

environmental isolate

Synthetic pathways may be:

•complete heterologous pathways

•hybrid heterologous / endogenous pathways

•“short-circuited” endogenous pathways

Synthetic pathways commonly need optimization

•improve metabolic flux towards (unnatural) product

•“rewiring” primary metabolic network

System-wide disturbance: optimization requires system-wide approach

•targeted / rational: extensive systems biology input (still underdeveloped)

•semi-targeted / random; classical strain improvement / evolutionary selection

combined with system-wide analysis: pragmatic

Summary and conclusions - 2

Acknowledgements

Nick Wierckx

Jean-Paul Meijnen

Frank Koopman

Suzanne Verhoef

TU Dortmund

Lars Blank, Andreas Schmid

TUDelft

Han de Winde