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Applied Microbiology and Biotechnology

, Volume 99, Issue 11, pp 4679–4689 | Cite as

Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation

  • Yota Tsuge
  • Shogo Yamamoto
  • Naoto Kato
  • Masako Suda
  • Alain A. Vertès
  • Hideaki Yukawa
  • Masayuki InuiEmail author
Biotechnological products and process engineering

Abstract

We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale.

Keywords

Corynebacterium glutamicum Oxygen deprivation D-Lactic acid Metabolic engineering Glycolytic enzyme 

Notes

Acknowledgments

This work was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO).

Conflict of interest

We declare that we have no conflict of interest.

References

  1. Arai K, Hishida A, Ishiyama M, Kamata T, Uchikoba H, Fushinobu S, Matsuzawa H, Taguchi H (2002) An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution. Protein Eng 15:35–41Google Scholar
  2. Bailey JE, Sburlati A, Hatzimanikatis V, Lee K, Renner WA, Tsai PS (1996) Inverse metabolic engineering: a strategy for directed genetic engineering of useful phenotypes. Biotechnol Bioeng 52:109–121CrossRefPubMedGoogle Scholar
  3. Becker J, Zelder O, Häfner S, Schröder H, Wittmann C (2011) From zero to hero—design-based systems metabolic engineering of Corynebacterium glutamicum for D-lysine production. Metab Eng 13:159–168Google Scholar
  4. Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, Eikmanns BJ (2011) Corynebacterium glutamicum tailored for efficient isobutanol production. Appl Environ Microbiol 77:3300–3310CrossRefPubMedCentralPubMedGoogle Scholar
  5. Emmerling M, Bailey JE, Sauer U (2000) Altered regulation of pyruvate kinase or co-overexpression of phosphofructokinase increases glycolytic fluxes in resting Escherichia coli. Biotechnol Bioeng 67:623–627CrossRefPubMedGoogle Scholar
  6. Fukushima K, Chang YH, Kimura Y (2007) Enhanced stereocomplex formation of poly(L-lactic acid) and poly (D-lactic acid) in the presence of stereoblock poly(lactic acid). Macromol Biosci 7:829–835Google Scholar
  7. Grabar TB, Zhou S, Shanmugam KT, Yomano LP, Ingram LO (2006) Methylglyoxal bypass identified as source of chiral contamination in L and D-lactate fermentations by recombinant Escherichia coli. Biotechnol Lett 28:1527–1535Google Scholar
  8. Hasegawa S, Suda M, Uematsu K, Natsuma Y, Hiraga K, Jojima T, Inui M, Yukawa H (2013) Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions. Appl Environ Microbiol 79:1250–1257CrossRefPubMedCentralPubMedGoogle Scholar
  9. Ikada Y, Jamshidi K, Tsuji H, Hyon S (1987) Stereocomplex formation between enantiomeric poly(lactides). Macromolecules 20:904–906CrossRefGoogle Scholar
  10. Inui M, Murakami S, Okino S, Kawaguchi H, Vertès AA, Yukawa H (2004a) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196CrossRefPubMedGoogle Scholar
  11. Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H (2004b) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254CrossRefPubMedGoogle Scholar
  12. Inui M, Suda M, Okino S, Nonaka H, Puskas LG, Vertès AA, Yukawa H (2007) Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions. Microbiology 153:2491–2504CrossRefPubMedGoogle Scholar
  13. Ishida N, Suzuki T, Tokuhiro K, Nagamori E, Onishi T, Saitoh S, Kitamoto K, Takahashi H (2006) D-lactic acid production by metabolically engineered Saccharomyces cerevisiae. J Biosci Bioeng 101:172–177Google Scholar
  14. Jojima T, Fujii M, Mori E, Inui M, Yukawa H (2010) Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid L-alanine under oxygen deprivation. Appl Microbiol Biotechnol 87:159–165Google Scholar
  15. Joshi DS, Singhvi MS, Khire JM, Gokhale DV (2010) Strain improvement of Lactobacillus lactis for D-lactic acid production. Biotechnol Lett 32:517–520Google Scholar
  16. Karjomaa S, Suortti T, Lempiainen R, Selin J, Itavaara M (1998) Microbial degradation of poly-(L-lactic acid) oligomers. Polym Degrad Stab 59:333–336Google Scholar
  17. Kinoshita S (1985) Glutamic acid bacteria. In: Demain AL, Solomon NA (eds) Biology of industrial microorganisms. Benjamin Cummings, London, pp 115–146Google Scholar
  18. Litsanov B, Brocker M, Bott M (2012) Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate. Appl Environ Microbiol 78:3325–3337CrossRefPubMedCentralPubMedGoogle Scholar
  19. Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KL (2009) Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 11:262–273CrossRefPubMedGoogle Scholar
  20. Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480CrossRefPubMedGoogle Scholar
  21. Okino S, Suda M, Fujikura K, Inui M, Yukawa H (2008a) Production of D-lactic acid by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 78:449–454CrossRefPubMedGoogle Scholar
  22. Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H (2008b) An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain. Appl Microbiol Biotechnol 81:459–464CrossRefPubMedGoogle Scholar
  23. Papagianni M, Avramidis N (2011) Lactococcus lactis as a cell factory: a twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation. Enzym Microb Technol 49:197–202CrossRefGoogle Scholar
  24. Pearce AK, Crimmins K, Groussac E, Hewlins MJ, Dickinson JR, Francois J, Booth IR, Brown AJ (2001) Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions. Microbiology 147:391–401PubMedGoogle Scholar
  25. Radoš DX, Turner DL, Fonseca LL, Carvalho AL, Blombach B, Eikmanns B, Neves AR, Santos H (2014) Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum. Appl Environ Microbiol 80:3015–3024CrossRefPubMedCentralPubMedGoogle Scholar
  26. Sambrook J, Fritsh E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold spring harbor laboratory press. Cold Spring Harbor, New YorkGoogle Scholar
  27. Solem C, Koebmann BJ, Jensen PR (2003) Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363. J Bacteriol 185:1564–1571CrossRefPubMedCentralPubMedGoogle Scholar
  28. Solem C, Koebmann BJ, Jensen PR (2008) Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis. IET Syst Biol 2:64–72CrossRefPubMedGoogle Scholar
  29. Stephanopoulos G, Vallino JJ (1991) Network rigidity and metabolic engineering in metabolite overproduction. Science 252:1675–1681CrossRefPubMedGoogle Scholar
  30. Suzuki N, Okayama S, Nonaka H, Tsuge Y, Inui M, Yukawa H (2005) Large-scale engineering of the Corynebacterium glutamicum genome. Appl Environ Microbiol 71:3369–3372CrossRefPubMedCentralPubMedGoogle Scholar
  31. Tsuge Y, Yamamoto S, Suda M, Inui M, Yukawa H (2013) Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum D-lactate productivity under oxygen deprivation. Appl Microbiol Biotechnol 97:6693–6703Google Scholar
  32. Vertès AA, Inui M, Kobayashi M, Kurusu Y, Yukawa H (1993) Presence of mrr- and mcr-like restriction systems in coryneform bacteria. Res Microbiol 144:181–185CrossRefPubMedGoogle Scholar
  33. Vertès AA, Inui M, Yukawa H (2013) The biotechnological potential of Corynebacterium glutamicum, from umami to chemurgy. In: Yukawa H, Inui M (eds) Corynebacterium glutamicum: biology and biotechnology. Springer, New York, pp 1–49CrossRefGoogle Scholar
  34. Wang Q, Ingram LO, Shanmugam KT (2011a) Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose. Proc Natl Acad Sci U S A 108:18920–18925Google Scholar
  35. Wang L, Zhao B, Li F, Xu K, Ma C, Tao F, Li Q, Xu P (2011b) Highly efficient production of D-lactate by Sporolactobacillus sp. CASD with simultaneous enzymatic hydrolysis of peanut meal. Appl Microbiol Biotechnol 89:1009–1017Google Scholar
  36. Yamamoto S, Gunji W, Suzuki H, Toda H, Suda M, Jojima T, Inui M, Yukawa H (2012) Overexpression of glycolytic genes enhances Corynebacterium glutamicum glucose metabolism and alanine production under oxygen-deprived conditions. Appl Environ Microbiol 78:4447–4457CrossRefPubMedCentralPubMedGoogle Scholar
  37. Yamamoto S, Suda M, Niimi S, Inui M, Yukawa H (2013) Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation. Biotechnol Bioeng 110:2938–2948CrossRefPubMedGoogle Scholar
  38. Yukawa H, Omumasaba CA, Nonaka H, Kos P, Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vertès AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058CrossRefPubMedGoogle Scholar
  39. Zhou L, Niu DD, Tian KM, Chen XZ, Prior BA, Shen W, Shi GY, Singh S, Wang ZX (2012) Genetically switched D-lactate production in Escherichia coli. Metab Eng 14:560–568CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yota Tsuge
    • 1
  • Shogo Yamamoto
    • 1
  • Naoto Kato
    • 1
  • Masako Suda
    • 1
  • Alain A. Vertès
    • 1
  • Hideaki Yukawa
    • 1
  • Masayuki Inui
    • 1
    • 2
    Email author
  1. 1.Research Institute of Innovative Technology for the Earth (RITE)KizugawaJapan
  2. 2.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan

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