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Corynebacterium glutamicum tailored for high-yield L-valine production

  • Applied Genetics and Molecular Biotechnology
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Abstract

We recently engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of L-valine from glucose by inactivation of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. Based on the first generation of pyruvate-dehydrogenase-complex-deficient C. glutamicum strains, a second generation of high-yield L-valine producers was constructed by successive deletion of the genes encoding pyruvate:quinone oxidoreductase, phosphoglucose isomerase, and pyruvate carboxylase and overexpression of ilvBNCE. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 410 mM (48 g/l) L-valine, showed a maximum yield of 0.75 to 0.86 mol/mol (0.49 to 0.56 g/g) of glucose in the production phase and, in contrast to the first generation strains, excreted neither pyruvate nor any other by-product tested.

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References

  • Bartek T, Blombach B, Zönnchen E, Makus P, Wahl A, Lang S, Eikmanns BJ, Oldiges M (2008a) Importance of NADPH supply for improved L-valine formation based on stoichiometric modelling. Submitted to AMB

  • Bartek T, Makus P, Klein B, Lang S, Oldiges M (2008b) Influence of L-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses. Bioprocess Biosyst Eng DOI https://doi.org/10.1007/s00449-008-0202-z

    Article  CAS  Google Scholar 

  • Becker J, Klopprogge C, Zelder O, Heinzle E, Wittmann C (2005) Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71:8587–8596

    Article  CAS  Google Scholar 

  • Bergmeyer HU (1983) Methods of enzymatic analysis vol. VI, 3rd edn. Verlag Chemie, Weinheim, pp 59–66

    Google Scholar 

  • Blombach B, Schreiner ME, Moch M, Oldiges M, Eikmanns BJ (2007a) Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum. Appl Microbiol Biotechnol 76:615–623

    Article  CAS  Google Scholar 

  • Blombach B, Schreiner ME, Holatko J, Bartek T, Oldiges M, Eikmanns BJ (2007b) L-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol 73:2079–2084

    Article  CAS  Google Scholar 

  • Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145

    Article  CAS  Google Scholar 

  • Eggeling L (2001) Amino acids. In: Ratledge C, Kristiansen B (eds) Basic biotechnology. Cambridge University Press, London, pp 281–303

    Google Scholar 

  • Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC, Boca Raton

    Book  Google Scholar 

  • Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke KU, Sahm H (1994) Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology 140:1817–1828

    Article  CAS  Google Scholar 

  • Eikmanns BJ, Rittmann D, Sahm H (1995) Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177:774–782

    Article  CAS  Google Scholar 

  • Elisakova V, Patek M, Holatko J, Nesvera J, Leyval D, Goergen JL, Delaunay S (2005) Feedback-resistant acetohydroxyacid synthase increases valine production in Corynebacterium glutamicum. Appl Environ Microbiol 71:207–213

    Article  CAS  Google Scholar 

  • Georgi T, Rittmann D, Wendisch VF (2005) Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. Metab Eng 7:291–301

    Article  CAS  Google Scholar 

  • Gourdon P, Baucher MF, Lindley ND, Guyonvarch A (2000) Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism. Appl Environ Microbiol 66:2981–2987

    Article  CAS  Google Scholar 

  • Hanahan D (1985) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580

    Article  Google Scholar 

  • Kabus A, Georgi T, Wendisch VF, Bott M (2007) Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine. Appl Microbiol Biotechnol 75:47–53

    Article  CAS  Google Scholar 

  • Lange C, Rittmann D, Wendisch VF, Bott M, Sahm H (2003) Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine. Appl Environ Microbiol 69:2521–2532

    Article  CAS  Google Scholar 

  • Leuchtenberger W (1996) Amino acids—technical production and use. In: Rehm H-J, Reed G, Pühler A, Stadler P (eds) Biotechnology, vol. vol. 6. VCH, Weinheim, pp 465–502

    Chapter  Google Scholar 

  • Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivates: current status and prospects. Appl Microbiol Biotechnol 69:1–8

    Article  CAS  Google Scholar 

  • Liebl W (1991) The genus Corynebacterium—nonmedical. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH (eds) The Prokaryotes, vol. 2, Springer, New York, pp 1157–1171

    Google Scholar 

  • Marienhagen J, Kennerknecht N, Sahm H, Eggeling L (2005) Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum. J Bacteriol 187:7639–7646

    Article  CAS  Google Scholar 

  • Marx A, de Graaf AA, Wiechert W, Eggeling L, Sahm H (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49:111–129

    Article  CAS  Google Scholar 

  • Marx A, Striegel K, de Graaf AA, Eggeling L (1997) Response of central metabolism of Corynebacterium glutamicum to different flux burdens. Biotechnol Bioeng 56:168–180

    Article  CAS  Google Scholar 

  • Marx A, Eikmanns BJ, Sahm H, de Graaf AA, Eggeling L (1999) Response of central metabolism of Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase. Metab Eng 1:35–48

    Article  CAS  Google Scholar 

  • Marx A, Hans S, Mockel B, Bathe B, de Graaf AA (2003) Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol 104:185–197

    Article  CAS  Google Scholar 

  • Moritz B, Striegel K, de Graaf AA, Sahm H (2000) Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose pathway flux in vivo. Eur J Biochem 267:3442–3452

    Article  CAS  Google Scholar 

  • Ohnishi J, Katahira R, Mitsuhashi S, Kakita S, Ikeda M (2005) A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum. FEMS Microbiol Lett 242:265–274

    Article  CAS  Google Scholar 

  • Park JH, Lee KH, Kim TY, Lee SY (2007) Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico knockout simulation. Proc Natl Acad Sci U S A 104:7797–7802

    Article  CAS  Google Scholar 

  • Patek M (2007) Branched-chain amino acids. In: Steinbüchel A (ed) Microbiol monographs, vol. 5, Springer, Berlin/Heidelberg, pp 129–162

    Google Scholar 

  • Peters-Wendisch PG, Kreutzer C, Kalinowski J, Pátek M, Sahm H, Eikmanns BJ (1998) Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene. Microbiology 144:915–927

    Article  CAS  Google Scholar 

  • Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Mockel B, Sahm H, Eikmanns BJ (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol 3:295–300

    CAS  Google Scholar 

  • Radmacher E, Vaitsikova A, Burger U, Krumbach K, Sahm H, Eggeling L (2002) Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum. Appl Environ Microbiol 68:2246–2250

    Article  CAS  Google Scholar 

  • Riedel C, Rittmann D, Dangel P, Möckel B, Sahm H, Eikmanns BJ (2001) Characterization, expression, and inactivation of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production. J Mol Microbiol Biotechnol 3:573–583

    CAS  PubMed  Google Scholar 

  • Ruklisha M, Paegle L, Denina I (2007) L-Valine biosynthesis during batch and fed-batch cultivations of Corynebacterium glutamicum: relationship between changes in bacterial growth rate and intracellular metabolism. Process Biochem 42:634–640

    Article  CAS  Google Scholar 

  • Sahm H, Eggeling L (1999) D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction. Appl Environ Microbiol 65:1973–1979

    Article  CAS  Google Scholar 

  • Sambrook J, Russel DW, Irwin N, Janssen UA (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the E. coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    Article  Google Scholar 

  • Schreiner ME, Fiur D, Holátko J, Pátek M, Eikmanns BJ (2005) E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum: Molecular analysis of the gene and phylogenetic aspects. J Bacteriol 187:6005–6018

    Article  CAS  Google Scholar 

  • Schreiner ME, Riedel C, Holatko J, Patek M, Eikmanns BJ (2006) Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects. J Bacteriol 188:1341–1350

    Article  CAS  Google Scholar 

  • Takors R, Bathe B, Rieping M, Hans S, Kelle R, Huthmacher K (2007) Systems biology for industrial strains and fermentation processes—example: amino acids. J Biotechnol 129:181–190

    Article  CAS  Google Scholar 

  • van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogenic plasmid DNA. Appl Microbiol Biotechnol 52:541–545

    Article  Google Scholar 

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Acknowledgement

We thank Lothar Eggeling for providing plasmids pJC4ilvBNC, pJC4ilvBNCD, and pJC4ilvBNCE and Brigitte Bathe (Evonik Degussa) for providing plasmid pK18mobsacB pgidel. We are grateful to Andreas Karau and Robert Gerstmeir (Degussa AG) for valuable discussions. We thank Konstanze Fleischer for technical assistance. The support of the Fachagentur Nachwachsende Rohstoffe of the BMVEL (grant 04NR004/22000404) is gratefully acknowledged.

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Correspondence to Bernhard J. Eikmanns.

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Blombach, B., Schreiner, M.E., Bartek, T. et al. Corynebacterium glutamicum tailored for high-yield L-valine production. Appl Microbiol Biotechnol 79, 471–479 (2008). https://doi.org/10.1007/s00253-008-1444-z

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