Aromatic Compounds from Sugar · H ydr oxy-ar omati cs i n many cases ar e chemi cal l y di f f i...
Transcript of Aromatic Compounds from Sugar · H ydr oxy-ar omati cs i n many cases ar e chemi cal l y di f f i...
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Aromatic Compounds from Sugar
Jan de Bont
28th Symposium on Biotechnology for Fuels and Chemicals April 30 - May 3, 2006, Nashville, Tennessee
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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
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TNO
TNO is a knowledge organisation in The Netherlands
for companies, government bodies and public
organisations. The work of some 5,000 employees is to develop and
apply knowledge. We provide contract research and
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patents and specialist software. We test and certify
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Any further information at www.tno.nl
Or Christian Koolloos present at this meeting
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SPONSORS OF WORK PRESENTED
B-Basic
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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
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Toxicity Issues
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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
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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.
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OUT
IN
MEMBRANE
Efflux system
removes many,
chemically unrelated
compounds
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Hydrolysate from Wheat Straw
• Acid pretreatment
• pH adjusted to 7
• Cellulase hydrolysis
• Supernatant used as growth medium
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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
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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
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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?
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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
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Cy3 Cy5
Cy5
Cy3
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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
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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
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Production of Aromatic Compounds
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Efflux system
Overproduction of an aromatic
amino acid
Aromatic
product
SUGAR
AROMATIC PRODUCT
P. Putida S12
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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)
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Target Compounds
Via phenylalanine:
• Cinnamic acid
• 1 other compound
Via tyrosine:
• Phenol
• 4 other compounds
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Mutagenesis and High-throughput Screening
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Transcriptomics
Affymetrix NimbleExpress Custom Array
based on P. putida KT2440
Sequence of P. putida S12 available soon
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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
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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
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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
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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
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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
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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
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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
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Phenol Toxicity
and
Recovery of Phenol During Fermentations
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• 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
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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.
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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.
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Extractive Recovery of Phenol
via
Octanol
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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
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Extractive Recovery of Phenol
via
Solvent-impregnated Resins (SIR’s)
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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
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Start End
How Solvent-impregnated Resins Operate
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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
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Phenol Production?
• Production cost phenol via P. putida S12 5 $/kg
• Current phenol price 1.5 $/kg
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Not Phenol
• Phenol is no option; just a model compound
• However, several 4-hydroxy-aromatic compounds
(produced via tyrosine) will be of interest
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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