Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene

Abstract

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Adachi E, Torigoe M, Sugiyama M, Nikawa J, Shimizu K (1998) Modification of metabolic pathways of Saccharomyces cerevisiae by the expression of lactate dehydrogenase and deletion of pyruvate decarboxylase genes for the lactic acid fermentation at low pH value. J Ferment Bioeng 86:284–289

  2. Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330

  3. Bianchi MM, Brambilla L, Protani F, Liu CL, Lievense J, Porro D (2001) Efficient homolactic fermentation by Kluyveromyces lactis strains defective in pyruvate utilization and transformed with the heterologous LDH gene. Appl Environ Microbiol 67:5621–5625

  4. Branduardi P, Sauer M, De Gioia L, Zampella G, Valli M, Mattanovich D, Porro D (2006) Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export. Microb Cell Fact 5:4

  5. Dequin S, Barre P (1994) Mixed lactic acid-alcoholic fermentation by Saccharomyces cerevisiae expressing the Lactobacillus casei L(+)-LDH. Biotechnology (N Y) 12:173–177

  6. Flikweert MT, Van Der Zanden L, Janssen WM, Steensma HY, Van Dijken JP, Pronk JT (1996) Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12:247–257

  7. Haber JE, Garvik B (1977) A new gene affecting the efficiency of mating-type interconversions in homothallic strains of Saccharomyces cerevisiae. Genetics 87:33–50

  8. Hofvendahl K, Hahn-Hägerdal B (2000) Factors affecting the fermentative lactic acid production from renewable resources. Enzyme Microb Technol 26:87–107

  9. Hohmann S (1991) Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae. J Bacteriol 173:7963–7969

  10. Hohmann S, Cederberg H (1990) Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5. Eur J Biochem 188:615–621

  11. Ishida N, Saitoh S, Tokuhiro K, Nagamori E, Matsuyama T, Kitamoto K, Takahashi H (2005) Efficient production of L-lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene. Appl Environ Microbiol 71:1964–1970

  12. Ishida N, Saitoh S, Onishi T, Tokuhiro K, Nagamori E, Kitamoto K, Takahashi H (2006) The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production. Biosci Biotechnol Biochem 70:1148–1153

  13. Leskovac V, Trivić S, Pericin D (2002) The three zinc-containing alcohol dehydrogenases from baker’s yeast, Saccharomyces cerevisiae. FEMS Yeast Res 2:481–494

  14. Manivasakam P, Weber SC, McElver J, Schiestl RH (1995) Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res 23:2799–2800

  15. Neilands JB (1955) Lactic dehydrogenase of heart muscle. In: Colowick SP, Kaplan NO (eds) Methods enzymol, vol 1. Academic, New York, pp 449–454

  16. Porro D, Brambilla L, Ranzi BM, Martegani E, Alberghina L (1995) Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid. Biotechnol Prog 11:294–298

  17. Racker E (1955) Alcohol dehydrogenase from baker’s yeast. In: Colowick SP, Kaplan NO (eds) Methods enzymol, vol 1. Academic, New York, pp 500–503

  18. Saitoh S, Ishida N, Onishi T, Tokuhiro K, Nagamori E, Kitamoto K, Takahashi H (2005) Genetically engineered wine yeast produces a high concentration of L-lactic acid of extremely high optical purity. Appl Environ Microbiol 71:2789–2792

  19. Skory CD (2003) Lactic acid production by Saccharomyces cerevisiae expressing a Rhizopus oryzae lactate dehydrogenase gene. J Ind Microbiol Biotechnol 30:22–27

  20. Tokuhiro K, Ishida N, Kondo A, Takahashi H (2008) Lactic fermentation of cellobiose by a yeast strain displaying b-glucosidase on the cell surface. Appl Microbiol Biotechnol 79:481–488

  21. Tsuji F (2002) Autocatalytic hydrolysis of amorphous-made polylactides: effects of L-lactide content, tacticity, and enantiomeric polymer blending. Polymer 43:1789–1796

  22. Van Dijken JP, Weusthuis RA, Pronk JT (1993) Kinetics of growth and sugar consumption in yeasts. Antonie Van Leeuwenhoek 63:343–352

  23. Van Maris AJ, Winkler AA, Porro D, Van Dijken JP, Pronk JT (2004) Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export. Appl Environ Microbiol 70:2898–2905

  24. Wee YJ, Kim JN, Ryu HW (2006) Biotechnological production of lactic acid and its recent applications. Food Technol Biotechnol 44:163–172

Download references

Acknowledgments

We thank Keiko Uemura for the technical assistance.

Author information

Correspondence to Kenro Tokuhiro.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tokuhiro, K., Ishida, N., Nagamori, E. et al. Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene. Appl Microbiol Biotechnol 82, 883–890 (2009). https://doi.org/10.1007/s00253-008-1831-5

Download citation

Keywords

  • Yeast
  • l-Lactate
  • ADH1
  • PDC1
  • Disruption
  • l-ldh