Carbon dioxide fixation - Anaplerotic key reaction for industrial/white biotechnology - Dr. Achim Marx 19.02.08
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Carbon dioxide fixation - Anaplerotic key reaction for industrial/white biotechnology Dr. Achim Marx 19.02.08
Evonik`s Chemicals Business Area creates new biobased business in its Science-to-Business Center in Marl near Essen 19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08
Mission of the Science-to-
Business Center Biotechnology
• Establish "factory in a cell" in order to ...
• ... reduce number of unit operations of
our production processes
• ... ensure production cost leadership for
Evonik's bulk-chemicals
• ... ensure the use of carbon feedstock
with non-volatile and low price
BUSINESS
BUSINESS
©
© 2006
2006 Marx
Marx
• ... enable Evonik to use a flexible
feedstock-concept for the use of fats, Supplier
Supplier
Upstream
Upstream
Fermentation
Downstream
Downstream
Catalysis
Isolation
Isolation
Formulation Customer
Customer
Fermentation Catalysis Formulation
glycerol, lignocellulose, methanol, oils, and
sugars
• ... enable bioprocesses which are superior
regarding ecology, safety, health, and Intellectual
quality Property
Science
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 3Biologists, Chemists, and Engineers
will jointly establish
biobased supply chains
• The event "Industrielle Nutzung Nachwachsender Rohstoffe -
Chemie, Biotechnologie, Verfahrenstechnik" covers all aspects
which are essential to establish biobased supply chains
Upstream
Upstream Downstream
Downstream Isolation
Isolation
Supplier
Supplier Customer
Customer
Fermentation
Fermentation Catalysis
Catalysis Formulation
Formulation
• Contributions by Evonik address
• "Rohstoffauswahl - Verfügbarkeit und Logistik" (Presentation by T. Böhland)
• "Chemische und biotechnologische Reaktionstechnik"
(Presentation by A. Marx et al. and Poster 30 by B.A. Kaup et al.)
The "factory in a The profitability of biobased supply
cell" must be chains depends the yield of
optimized for conversion of feedstock carbon
carbon recovery ! into product carbon !
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 4Definition of the biologist's term
"anaplerotic carboxylation"
• In Google on February 17, 2008:
• On German: "Anaplerotische Carboxylierung"
241 entries
• On Englich: "Anaplerotic carboxylation"
5110 entries
• H. L. Kornberg on this topic in his 2006 contribution:
"Anaplerotische Sequenzen im mikrobiologischen Stoffwechsel"
... Neben den katabolischen und anabolischen Bahnen, auf
welchen den zentralen Bahnen des Stoffwechsels Nahrungsstoffe
zugeführt bzw. entzogen werden, gibt es Enzymsequenzen, die für
den Ersatz von Zwischenstufen sorgen, die von den zentralen
Bahnen während der Biosynthese entfernt wurden. Diese
Nachschubbahnen werden als anaplerotische Sequenzen
bezeichnet. ...
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 5In order to design viable biobased
supply chains we will ...
... develop new enzyme
activities to establish
synthetic pathways.
Metabolic network
... select robust microbial CO2
strains and optimize product Succinate
resistance.
Lactate
Microbial strain Metabolic Acetate
network
Pyruvate
Citrate
Oxaloacetate CO2 Glucose
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 6Anaplerotic carboxylation replenishes
citric acid cycle with C4-units
Metabolic network
CO2
Succinate
Lactate
Acetate
Pyruvate
Citrate
Oxaloacetate CO2 Glucose
Anaplerotic carboxylation
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 7Succinate prod. has been optimized with a
major focus on anaplerotic carboxylation ...
... and represents a platform technology which might be customized for
the production of different C4-intermediates for the chemical industry
Patent
PatentNo.
application Affiliation
Assignee Strain
WO2006/034156 Rice University, USA Escherichia coli
JP2006-197821 RITE, Japan Corynebacterium glutamicum
US2007042477 KAIST, South Korea Mannheimia succiniciproducens
JP2006238843 Mitsubishi, Japan Escherichia coli
JP2005333886 Showa Denko, Japan Corynebacterium glutamicum
WO2003070913 Georgia University, USA Escherichia coli
JP2005211041 Nippon Shokubai, Japan bacteria
WO2005113745 Ajinomoto, Japan bacteria
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 8The net anaplerotic carboxylation depends on the difference between forward carboxylation and backward decarboxylation Pfefferle, W., Möckel, B., Bathe, B., Marx, A. 2003. Biotechnological Manufacture of Lysine. pp. 59-112. In: Faurie, R., Thommel, J. (eds.), Advances in Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin 19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 9
Many enzyme activities participate in the carboxylation and decarboxylation bewteen pyruvate/PEP and oxaloacetate/malate Pfefferle, W., Möckel, B., Bathe, B., Marx, A. 2003. Biotechnological Manufacture of Lysine. pp. 59-112. In: Faurie, R., Thommel, J. (eds.), Advances in Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin 19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 10
Net anaplerotic carboxylation might be
maximized by reducing decarboxylation
and increasing carboxylation
C3
C4
Pfefferle, W., Möckel, B., Bathe, B., Marx, A. 2003. Biotechnological Manufacture of Lysine. pp. 59-112. In: Faurie, R., Thommel, J. (eds.), Advances in
Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 11β-Alanine formation for the final
synthesis of 3-hydroxypropionic acid
production should be improved ...
... by enhanced pyruvate carboxylation and aspartate decarboxylation
pyruvate
carboxylase
(Pyc)
CO2
aspartate
decarboxylase
(PanD)
3-hydroxy-
propionic acid ...
... is beside succinate one of the 12 biobased building blocks
as defined by Department of Energy, US
Straathof, A. J. J., Sie, S., Franco, T. T., van der Wielen, L. A. M. 2005. Feasibility of acrylic acid production by fermentation.
Appl. Microbiol. Biotechnol. 67: 727-734 DE-A-198 55 313 and DE-A-198 55 314 for panD /EC 4.1.1.11
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 12Extracellular conc. (mM) of β-alanine is
increased for recombinant cells with
overexpression of PanD or Pyc/PanD
25 ATCC13032
pVWEx1
20
ATCC13032
15 pVWEx1
enhanced
PanD and Pyc
PanD
panD C.g.
enhanced
10
DM1727
pVWEx1
5 panD C.g.
0
beta-alanine (mM)
β-alanine (mM)
Georgi et al. Metabol. Eng. 7: 291-301 for pyc (Pro-458->Ser) strain DM1727 and medium CG XII with 40 g/L glucose
Marx, A., Wendisch, V. F., Rittmann, D., Buchholz, S. 2007. Mikrobiologische Herstellung von 3-Hydroxypropionsäure, Deutsche
Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/042494
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 13Extracellular conc. (mM) of α-alanine is
increased for recombinant cells with
overexpression of PanD or Pyc/PanD
25 ATCC13032
pVWEx1
20
PanD and Pyc
ATCC13032
enhanced
15 pVWEx1
panD C.g.
10
enhanced
DM1727
PanD
pVWEx1
5 panD C.g.
0
alpha-alanine (mM)
α-alanine (mM)
Georgi et al. Metabol. Eng. 7: 291-301 for pyc (Pro-458->Ser) strain DM1727 and medium CG XII with 40 g/L glucose
Marx, A., Wendisch, V. F., Rittmann, D., Buchholz, S. 2007. Mikrobiologische Herstellung von 3-Hydroxypropionsäure, Deutsche
Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/042494
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 14Ratio of extracellular α- and β-alanine is
hihger for overexpression of pyc/panD
than for panD alone
40
. ATCC13032
pVWEx1
35
.
30
.
PanD and Pyc
ATCC13032
enhanced
25
. pVWEx1
20
. panD C.g.
15
. DM1727
enhanced
10
. pVWEx1
PanD
panD C.g.
0.5
0
beta/alpha x10
β-alanine/α-alanine
Georgi et al. Metabol. Eng. 7: 291-301 for pyc (Pro-458->Ser) strain DM1727 and medium CG XII with 40 g/L glucose
Marx, A., Wendisch, V. F., Rittmann, D., Buchholz, S. 2007. Mikrobiologische Herstellung von 3-Hydroxypropionsäure, Deutsche
Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/042494
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 15Extracellular conc. (mM) and ratio of
α- and β-alanine for recombinant cells
with PanD or Pyc/PanD
40
ATCC13032
35 pVWEx1
enhanced PanD and Pyc
30
25 ATCC13032
20 pVWEx1
panD C.g.
15
enh. PanD
DM1727
10
pVWEx1
5 panD C.g.
0
alpha-alanine
α-alanine beta-alanine β-alanine/α-alanine
β-alanine beta/alpha x10 x10
(mM)
(mM) (mM)
(mM)
Georgi et al. Metabol. Eng. 7: 291-301 for pyc (Pro-458->Ser) strain DM1727 and medium CG XII with 40 g/L glucose
Deutsche
Marx, A., Wendisch,Offenlegungsschrift
V. F., Rittmann, D., Buchholz, S.DE
2007.10,2005,048,818 A1von
Mikrobiologische Herstellung and WO 2007/042494
3-Hydroxypropionsäure, Deutsche
Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/042494
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 16Overexpression of pyc/panD led to
decreased α-alanine and
increased β-alanine formation
α-alanine
β-alanine
Pfefferle, W., Möckel, B., Bathe, B., Marx, A. 2003. Biotechnological Manufacture of Lysine. pp. 59-112. In: Faurie, R., Thommel, J. (eds.), Advances in
Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 17Summary and Outlook
• Metabolic Engineering of anaplerotic carboxylation is nowadays
already state-of-the-art in amino and succinic acid production
• The concept has been adapted successfully for the production of
β-alanine; metabolomics available; optimization to be done
• Recently new carbon dioxide capturing paths have been
discovered; they serve as a source of enzymes for the
establishment of synthetic pathway
• In future Industrial/White Biotechnology will enable the production
of biobased bulk chemicals; the extend to which we can optimize
anaplerotic carboxylation will siginificantly determine profitability
19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 18Acknowledgements L. Li, L. Neumann, J. Obuch, M. Pötter, T. Haas, S. Buchholz Evonik`s Chemicals Business Area V. F. Wendisch Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster D. Rittmann, H. Sahm, M. Oldiges, C. Wandrey Institut für Biotechnologie, Forschungszentrum Jülich B. Alber, T. Erb, I. A. Berg, D. Kockelkorn, H. Pondlekova, G. Fuchs Mikrobiologie, Institut für Biologie II, Universität Freiburg B. A. Kaup, D. Holtmann, M. Schilling, M. Etschmann, M.-A. Mirata, P. Gebhart, D. Sell, J. Schrader DECHEMA e.V. We thank Land Nordrhein-Westfalen, Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV), Bundesministerium für Bildung und Forschung (BMBF), Europäische Union, Fachagentur Nachwachsende Rohstoffe and Projekträger Jülich for financial support. 19.02.08 | Industrielle Nutzung Nachwachsender Rohstoffe, DECHEMA Haus, Frankfurt, 18.-19.02.08 Seite | 19
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