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Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The GPD2 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P PGK -gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P PGK -GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (μ max) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.

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References

  1. Albers E, Larsson C, Lidén G, Niklasson C, Gustafsson L (1996) Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl Environ Microbiol 62:3187–3195

    PubMed  CAS  Google Scholar 

  2. André L, Hemming A, Adler L (1991) Osmoregulation in Saccharomyces cerevisiae. Studies on the osmotic induction of glycerol production and glycerol 3-phosphate dehydrogenase (NAD+). FEBS Lett 286:13–17

    Article  PubMed  Google Scholar 

  3. Ansell R, Granath K, Hohmann S, Thevelein JM, Adler L (1997) The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaption and redox regulation. EMBO J 16:2179–2187

    Article  PubMed  CAS  Google Scholar 

  4. Arnon DI, Allen MB, Whatley FR (1954) Photosynthesis by isolated chloroplasts. Nature 173:1132–1134

    Article  CAS  Google Scholar 

  5. Bakker BM, Overkamp KM, van Maris AJ, Kotter P, Luttik MA, van Dijken JP, Pronk JT (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol Rev 25:15–37

    Article  PubMed  CAS  Google Scholar 

  6. Björkqvist S, Ansell R, Adler L, Lidén G (1997) Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae. Appl Environ Microbiol 63:128–132

    PubMed  Google Scholar 

  7. Blomberg A, Adler L (1992) Physiology of osmotolerance in fungi. Adv Microb Physiol 33:146–212

    Google Scholar 

  8. Bro C, Regenberg B, Forster J, Nielsen J (2006) In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production. Metab Eng 8:102–111

    Article  PubMed  CAS  Google Scholar 

  9. Cronwright GR, Rohwer JM, Prior BA (2002) Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 68:4448–4456

    Article  PubMed  CAS  Google Scholar 

  10. Crow VL, Wittenberger CL (1979) Separation and properties of NAD+- and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenases from Streptococcus mutans. J Biol Chem 254:1134–1142

    PubMed  CAS  Google Scholar 

  11. De Vries S, van Witzenburg R, Grivell LA, Marres CAM (1992) Primary structure and import pathway of the rotenone-insensitive NADH-ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae. Eur J Biochem 203:587–592

    Article  PubMed  Google Scholar 

  12. Guadalupe Medina V, Almering MJ, van Maris AJ, Pronk JT (2010) Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor. Appl Environ Microbiol 76:190–195

    Article  PubMed  Google Scholar 

  13. Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524

    Article  PubMed  Google Scholar 

  14. Guo ZP, Zhang L, Ding ZY, Wang ZX, Shi GY (2009) Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production. Appl Microbiol Biotechnol 82:287–292

    Article  PubMed  CAS  Google Scholar 

  15. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    PubMed  CAS  Google Scholar 

  16. Iddar A, Valverde F, Serrano A, Soukri A (2002) Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum. Protein Expr Purif 25:519–526

    Article  PubMed  CAS  Google Scholar 

  17. Iddar A, Valverde F, Serrano A, Soukri A (2003) Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli. Mol Cell Biochem 247:195–203

    Article  PubMed  CAS  Google Scholar 

  18. Larsson K, Ansell R, Eriksson P, Adler L (1993) A gene encoding sn-glycerol-3-phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae. Mol Microbiol 10:1101–1111

    Article  PubMed  CAS  Google Scholar 

  19. Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J (2000) Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2:69–77

    Article  PubMed  CAS  Google Scholar 

  20. Nissen TL, Schulze U, Nielsen J, Villadsen J (1997) Flux distributions in anaerobic, glucose-limited continuous cultivations of Saccharomyces cerevisiae. Microbiology 143:203–218

    Article  PubMed  CAS  Google Scholar 

  21. Remize F, Barnavon L, Dequin S (2001) Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae. Metab Eng 3:301–312

    Article  PubMed  CAS  Google Scholar 

  22. Robinson HW, Hogden CG (1940) The biuret reaction in the determination of serum proteins. I. A study of the conditions necessary for the production of a stable color which bears a quantitative relationship to the protein. J Biol Chem 135:707–1940

    CAS  Google Scholar 

  23. Valadi H, Larsson C, Gustafsson L (1998) Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 50:434–439

    Article  PubMed  CAS  Google Scholar 

  24. Van Maris AJ, Abbott DA, Bellissimi E, Van den Brink J, Kuyper M, Luttik MA, Wisselink HW, Scheffers WA, Van Dijken JP, Pronk JT (2006) Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Anton Van Leeuwenhoek 90:391–418

    Article  CAS  Google Scholar 

  25. Verduyn C, Postma E, Scheffers WA, van Dijken JP (1990) Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J Gen Microbiol 136:395–403

    PubMed  CAS  Google Scholar 

  26. Verduyn C, Postma E, Scheffers WA, van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517

    Article  PubMed  CAS  Google Scholar 

  27. Verduyn C, Stouthamer AH, Scheffers WA, van Dijken JP (1991) A theoretical evaluation of growth yields of yeasts. Antonie van Leeuwenhoek 59:49–63

    Article  PubMed  CAS  Google Scholar 

  28. Verho RP, Richard PH, Jonson L, Sundqvist J, Londesborough, Penttilä M (2002) Identification of the first fungal NADP-GAPDH from Kluyveromyces lactis. Biochemistry 41:13833–13838

    Article  PubMed  CAS  Google Scholar 

  29. Wang Y, Shi WL, Liu XY, Shen Y, Bao XM, Bai FW, Qu YB (2004) Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae. Biotechnol Lett 26:885–890

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was financial supported by the Natural Science Foundation of China (20706024), the “863” Program (2006AA020101, 2007AA10Z359), Innovative Research Team of Jiangsu Province and China Postdoctoral Science Foundation funded project (200801361).

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Center for Bioresources and Bioenergy, School of Biotechnology, Jiangnan University, 214122, Wuxi, People’s Republic of China

    Zhong-peng Guo, Liang Zhang, Zhong-yang Ding, Zheng-Xiang Wang & Gui-Yang Shi

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  1. Zhong-peng Guo
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  2. Liang Zhang
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  4. Zheng-Xiang Wang
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Correspondence to Gui-Yang Shi.

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Guo, Zp., Zhang, L., Ding, Zy. et al. Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast. J Ind Microbiol Biotechnol 38, 935–943 (2011). https://doi.org/10.1007/s10295-010-0864-9

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  • DOI: https://doi.org/10.1007/s10295-010-0864-9

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