Advertisement

Applied Microbiology and Biotechnology

, Volume 76, Issue 3, pp 615–623 | Cite as

Effect of pyruvate dehydrogenase complex deficiency on l-lysine production with Corynebacterium glutamicum

  • Bastian Blombach
  • Mark E. Schreiner
  • Matthias Moch
  • Marco Oldiges
  • Bernhard J. EikmannsEmail author
Applied Genetics and Molecular Biotechnology

Abstract

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on l-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the l-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and l-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific l-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific l-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific l-lysine yield by 6 and 56%, respectively. In addition to l-lysine, significant amounts of pyruvate, l-alanine and l-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve l-lysine production by engineering the l-lysine biosynthetic pathway.

Keywords

Corynebacterium glutamicum l-lysine Pyruvate dehydrogenase complex l-lysine production 

Notes

Acknowledgement

We thank Brigitte Bathe for providing C. glutamicum DM1729 and Lothar Eggeling for providing plasmids pJC33, pJC40 and pJC50. The support of the Fachagentur Nachwachsende Rohstoffe of the BMVEL (grant 04NR004/22000404) is gratefully acknowledged.

References

  1. Bergmeyer HU (1983) Methods of enzymatic analysis, vol VI, 3rd edn. Verlag Chemie, Weinheim, pp 59–66Google Scholar
  2. Blombach B, Schreiner ME, Holatko J, Bartek T, Oldiges M, Eikmanns BJ (2007) l-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol, in pressGoogle Scholar
  3. Cremer J, Treptow C, Eggeling L, Sahm H (1988) Regulation of the enzymes of lysine biosynthesis in Corynebacterium glutamicum. J Gen Microbiol 134:3221–3229Google Scholar
  4. Cremer J, Eggeling L, Sahm H (1991) Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl Environ Microbiol 57:1746–1752Google Scholar
  5. de Graaf AA, Eggeling L, Sahm H (2001) Metabolic engineering for l-lysine production by Corynebacterium glutamicum. Adv Biochem Eng Biotechnol 73:9–29Google Scholar
  6. Eggeling L, Oberle S, Sahm H (1998) Improved l-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation. Appl Microbiol Biotechnol 49:24–30CrossRefGoogle Scholar
  7. Eikmanns BJ, Metzger M, Reinscheid D, Kircher M, Sahm H (1991) Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains. Appl Microbiol Biotechnol 34:617–622CrossRefGoogle Scholar
  8. 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 140:1817–1828CrossRefGoogle Scholar
  9. 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 7:291–301CrossRefGoogle Scholar
  10. Guest JR, Creaghan IT (1974) Further studies with lipoamide dehydrogenase mutants of Escherichia coli K12. J Gen Microbiol 81:237–245Google Scholar
  11. Hanahan D (1985) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefGoogle Scholar
  12. Ikeda M (2003) Amino acid production process. Adv Biochem Eng Biotechnol 79:1–35Google Scholar
  13. 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, in pressGoogle Scholar
  14. Kalinowski J, Cremer J, Bachmann B, Eggeling L, Sahm H, Puhler A (1991) Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum. Mol Microbiol 5:1197–1204CrossRefGoogle Scholar
  15. Kelle R, Hermann T, Bathe B (2005) l-lysine production: In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, pp 465–488Google Scholar
  16. Kimura E (2005) l-Glutamate production. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, pp 439–463Google Scholar
  17. Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivates: current status and prospects. Appl Microbiol Biotechnol 69:1–8CrossRefGoogle Scholar
  18. Liebl W (2005) Corynebacterium taxonomy. In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, pp 9–34Google Scholar
  19. Marx A, Hans S, Mockel B, Bathe B, de Graaf AA (2003) Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol 104:185–197CrossRefGoogle Scholar
  20. Menkel E, Thierbach G, Eggeling L, Sahm H (1989) Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate. Appl Environ Microbiol 55:684–688Google Scholar
  21. Nakayama K, Tanaka H, Hagino H, Kinoshita S (1966) Studies on lysine fermentation. Part V. Concerted feedback inhibition of aspartokinase and the absence of lysine inhibition on aspartic semialdehyde–pyruvate condensation in Micrococcus glutamicus. Agric Biol Chem 30:611–616Google Scholar
  22. Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K, Ikeda M (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new l-lysine-producing mutant. Appl Microbiol Biotechnol 58:217–223CrossRefGoogle Scholar
  23. 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 242:265–274CrossRefGoogle Scholar
  24. Petersen S, Mack C, de Graaf AA, Riedel C, Eikmanns BJ, Sahm H (2001) Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic mechanisms in vivo. Metab Eng 3:344–361CrossRefGoogle Scholar
  25. 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 3:295–300Google Scholar
  26. Pfefferle W, Möckel B, Bathe B, Marx A (2003) Biotechnological manufacture of lysine. Adv Biochem Eng Biotechnol 79:59–112Google Scholar
  27. 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 3:573–583Google Scholar
  28. Sahm H, Eggeling L, de Graaf AA (2000) Pathway analysis and metabolic engineering in Corynebacterium glutamicum. Biol Chem 381:899–910CrossRefGoogle Scholar
  29. Sambrook J, Russel DW, Irwin N, Janssen UA (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  30. 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 187:6005–6018CrossRefGoogle Scholar
  31. Schrumpf B, Schwarzer A, Kalinowski J, Pühler A, Eggeling L, Sahm H (1991) A functional split pathway for lysine biosynthesis in Corynebacterium glutamicum. J Bacteriol 173:4510–4516Google Scholar
  32. Schrumpf B, Eggeling L, Sahm H (1992) Isolation and prominent characteristics of an l-lysine hyperproducing strain of Corynebacterium glutamicum. Appl Microbiol Biotechnol 37:566–571CrossRefGoogle Scholar
  33. Shiio I, Miyajima R (1969) Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem (Tokyo) 5:849–859Google Scholar
  34. Shiio I, Ozaki H, Ujigawa-Takeda K (1982) Production of aspartic acid and lysine by citrate synthase mutants of Brevibacterium flavum. Agric Biol Chem 46:101–107Google Scholar
  35. Shiio I, Toride Y, Sugimoto S (1984) Production of lysine by pyruvate dehydrogenase mutants of Brevibacterium flavum. Agric Biol Chem 48:3091–3098Google Scholar
  36. Shimizu H, Hirasawa T (2007) Production of glutamate and glutamate-related amino acids: molecular mechanism analysis and metabolic engineering. In: Wendisch VF (ed) Amino acid biosynthesis—pathways, regulation and metabolic engineering. Springer, Heidelberg, Germany, in pressGoogle Scholar
  37. Sonntag K, Eggeling L, de Graaf AA, Sahm H (1993) Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum: quantification by 13C- and 1H-NMR spectroscopy. Eur J Biochem 213:1325–1331CrossRefGoogle Scholar
  38. 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 52:541–545CrossRefGoogle Scholar
  39. Wittmann C, Becker J (2007) The l-Lysine story: from metabolic pathways to industrial production. In: Wendisch VF (ed) Amino acid biosynthesis—pathways, regulation and metabolic engineering. Springer, Heidelberg, Germany, in pressGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Bastian Blombach
    • 1
  • Mark E. Schreiner
    • 1
  • Matthias Moch
    • 2
  • Marco Oldiges
    • 2
  • Bernhard J. Eikmanns
    • 1
    Email author
  1. 1.Institute of Microbiology and BiotechnologyUniversity of UlmUlmGermany
  2. 2.Institute of Biotechnology 2Research Center JülichJülichGermany

Personalised recommendations