Carbon dioxide fixation - Anaplerotic key reaction for industrial/white biotechnology. Dr. Achim Marx
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1 Carbon dioxide fixation - Anaplerotic key reaction for industrial/white biotechnology Dr. Achim Marx
2 Evonik`s Chemicals Business Area creates new biobased business in its Science-to-Business Center in Marl near Essen
3 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... enable Evonik to use a flexible feedstock-concept for the use of fats, glycerol, lignocellulose, methanol, oils, and sugars Supplier BUSINESS Upstream Fermentation Downstream Catalysis Isolation Formulation 2006 Marx Customer... enable bioprocesses which are superior regarding ecology, safety, health, and quality Intellectual Property Science Seite 3
4 Biologists, 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 Supplier Upstream Fermentation Downstream Catalysis Isolation Formulation Customer 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 cell" must be optimized for carbon recovery! The profitability of biobased supply chains depends the yield of conversion of feedstock carbon into product carbon! Seite 4
5 Definition 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.... Seite 5
6 In order to design viable biobased supply chains we will develop new enzyme activities to establish synthetic pathways.... select robust microbial strains and optimize product resistance. Succinate CO 2 Metabolic network Lactate Microbial strain Metabolic network Pyruvate Acetate Citrate Oxaloacetate CO 2 Glucose Seite 6
7 Anaplerotic carboxylation replenishes citric acid cycle with C4-units Metabolic network Succinate CO 2 Lactate Acetate Citrate Pyruvate Oxaloacetate CO 2 Glucose Anaplerotic carboxylation Seite 7
8 Succinate 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 No. application WO2006/ JP US JP JP WO JP WO Affiliation Assignee Rice University, USA RITE, Japan KAIST, South Korea Mitsubishi, Japan Showa Denko, Japan Georgia University, USA Nippon Shokubai, Japan Ajinomoto, Japan Strain Escherichia coli Corynebacterium glutamicum Mannheimia succiniciproducens Escherichia coli Corynebacterium glutamicum Escherichia coli bacteria bacteria Seite 8
9 The net anaplerotic carboxylation depends on the difference between forward carboxylation and backward decarboxylation Pfefferle, W., Möckel, B., Bathe, B., Marx, A Biotechnological Manufacture of Lysine. pp In: Faurie, R., Thommel, J. (eds.), Advances in Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin Seite 9
10 Many enzyme activities participate in the carboxylation and decarboxylation bewteen pyruvate/pep and oxaloacetate/malate Pfefferle, W., Möckel, B., Bathe, B., Marx, A Biotechnological Manufacture of Lysine. pp In: Faurie, R., Thommel, J. (eds.), Advances in Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin Seite 10
11 Net anaplerotic carboxylation might be maximized by reducing decarboxylation and increasing carboxylation C 3 C 4 Pfefferle, W., Möckel, B., Bathe, B., Marx, A Biotechnological Manufacture of Lysine. pp In: Faurie, R., Thommel, J. (eds.), Advances in Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin Seite 11
12 β-alanine formation for the final synthesis of 3-hydroxypropionic acid production should be improved by enhanced pyruvate carboxylation and aspartate decarboxylation pyruvate carboxylase (Pyc) CO 2 aspartate decarboxylase (PanD) 3-hydroxypropionic 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 Feasibility of acrylic acid production by fermentation. Appl. Microbiol. Biotechnol. 67: DE-A and DE-A for pand /EC Seite 12
13 Extracellular conc. (mm) of β-alanine is increased for recombinant cells with overexpression of PanD or Pyc/PanD 25 ATCC enhanced PanD enhanced PanD and Pyc ATCC13032 pand C.g. DM1727 pand C.g. 0 beta-alanine β-alanine (mm) Georgi et al. Metabol. Eng. 7: 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 Mikrobiologische Herstellung von 3-Hydroxypropionsäure, Deutsche Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/ Seite 13
14 Extracellular conc. (mm) of α-alanine is increased for recombinant cells with overexpression of PanD or Pyc/PanD 25 ATCC enhanced PanD enhanced PanD and Pyc ATCC13032 pand C.g. DM1727 pand C.g. 0 alpha-alanine (mm) α-alanine (mm) Georgi et al. Metabol. Eng. 7: 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 Mikrobiologische Herstellung von 3-Hydroxypropionsäure, Deutsche Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/ Seite 14
15 Ratio of extracellular α- and β-alanine is hihger for overexpression of pyc/pand than for pand alone enhanced PanD beta/alpha x10 enhanced PanD and Pyc β-alanine/α-alanine ATCC13032 ATCC13032 pand C.g. DM1727 pand C.g. Georgi et al. Metabol. Eng. 7: 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 Mikrobiologische Herstellung von 3-Hydroxypropionsäure, Deutsche Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/ Seite 15
16 Extracellular conc. (mm) and ratio of α- and β-alanine for recombinant cells with PanD or Pyc/PanD alpha-alanine α-alanine (mm) (mm) beta-alanine β-alanine (mm) (mm) enh. PanD enhanced PanD and Pyc beta/alpha β-alanine/α-alanine x10 x10 ATCC13032 ATCC13032 pand C.g. DM1727 pand C.g. Georgi et al. Metabol. Eng. 7: for pyc (Pro-458->Ser) strain DM1727 and medium CG XII with 40 g/l glucose Marx, Deutsche A., Wendisch, Offenlegungsschrift V. F., Rittmann, D., Buchholz, S. DE ,2005,048,818 Mikrobiologische Herstellung A1 von and 3-Hydroxypropionsäure, WO 2007/ Deutsche Offenlegungsschrift DE 10,2005,048,818 A1 and WO 2007/ Seite 16
17 Overexpression 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 Biotechnological Manufacture of Lysine. pp In: Faurie, R., Thommel, J. (eds.), Advances in Biochemical Engineering/Biotechnology. Vol. 79: Microbial Production of L-Amino Acids. Springer Verlag, Berlin Seite 17
18 Summary 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 Seite 18
19 Acknowledgements 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. Seite 19
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