Applied Microbiology and Biotechnology

, Volume 91, Issue 4, pp 1239–1246 | Cite as

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

  • Jing-Jing Liu
  • Wen-Tao Ding
  • Guo-Chang Zhang
  • Jing-Yu Wang
Bioenergy and Biofuels


Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain’s performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.


Saccharomyces cerevisiae Ethanol fermentation Mutagenesis Meiotic recombination STE2 


  1. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314(5805):1565–1568. doi:10.1126/science.1131969 CrossRefGoogle Scholar
  2. Arifeen N, Wang R, Kookos I, Webb C, Koutinas AA (2007) Optimization and cost estimation of novel wheat biorefining for continuous production of fermentation feedstock. Biotechnol Prog 23(4):872–880. doi:10.1021/bp0700408 Google Scholar
  3. Basso LC, de Amorim HV, de Oliveira AJ, Lopes ML (2008) Yeast selection for fuel ethanol production in Brazil. FEMS Yeast Res 8(7):1155–1163. doi:10.1111/j.1567-1364.2008.00428.x CrossRefGoogle Scholar
  4. Bayrock DP, Michael Ingledew W (2001) Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology. J Ind Microbiol Biotechnol 27(2):87–93. doi:10.1038/sj/jim/7000167 CrossRefGoogle Scholar
  5. Bothast RJ, Schlicher MA (2005) Biotechnological processes for conversion of corn into ethanol. Appl Microbiol Biotechnol 67(1):19–25. doi:10.1007/s00253-004-1819-8 CrossRefGoogle Scholar
  6. Burkholder AC, Hartwell LH (1985) The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res 13(23):8463–8475CrossRefGoogle Scholar
  7. Dai M, Copley SD (2004) Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Appl Environ Microbiol 70(4):2391–2397CrossRefGoogle Scholar
  8. Demirbas A (2009) Political, economic and environmental impacts of biofuels: a review. Appl Energy 86:S108–S117. doi:10.1016/j.apenergy.2009.04.036 CrossRefGoogle Scholar
  9. Elliott B, Futcher B (1993) Stress resistance of yeast cells is largely independent of cell cycle phase. Yeast 9(1):33–42. doi:10.1002/yea.320090105 CrossRefGoogle Scholar
  10. Gerstein AC, Cleathero LA, Mandegar MA, Otto SP (2011) Haploids adapt faster than diploids across a range of environments. J Evol Biol 24(3):531–540. doi:10.1111/j.1420-9101.2010.02188.x CrossRefGoogle Scholar
  11. Giudici P, Solieri L, Pulvirenti AM, Cassanelli S (2005) Strategies and perspectives for genetic improvement of wine yeasts. Appl Microbiol Biotechnol 66(6):622–628. doi:10.1007/s00253-004-1784-2 CrossRefGoogle Scholar
  12. Glazunov AV, Boreiko AV, Esser A (1989) Relative competitiveness of haploid and diploid yeast cells growing in a mixed population. Mikrobiologiia 58(5):769–777Google Scholar
  13. Gong J, Zheng H, Wu Z, Chen T, Zhao X (2009) Genome shuffling: progress and applications for phenotype improvement. Biotechnol Adv 27(6):996–1005. doi:10.1016/j.biotechadv.2009.05.016 CrossRefGoogle Scholar
  14. Hahn-Hagerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74(5):937–953. doi:10.1007/s00253-006-0827-2 CrossRefGoogle Scholar
  15. Herskowitz I, Jensen RE (1991) Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 194:132–146CrossRefGoogle Scholar
  16. Hida H, Yamada T, Yamada Y (2007) Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Appl Microbiol Biotechnol 73(6):1387–1393. doi:10.1007/s00253-006-0613-1 CrossRefGoogle Scholar
  17. Hou L (2009) Novel methods of genome shuffling in Saccharomyces cerevisiae. Biotechnol Lett 31(5):671–677. doi:10.1007/s10529-009-9916-5 CrossRefGoogle Scholar
  18. Hou L (2010) Improved production of ethanol by novel genome shuffling in Saccharomyces cerevisiae. Appl Biochem Biotechnol 160(4):1084–1093. doi:10.1007/s12010-009-8552-9 CrossRefGoogle Scholar
  19. Hou L, Cao X, Wang C, Lu M (2009) Effect of overexpression of transcription factors on the fermentation properties of Saccharomyces cerevisiae industrial strains. Lett Appl Microbiol 49(1):14–19. doi:10.1111/j.1472-765X.2009.02615.x CrossRefGoogle Scholar
  20. Lam FH, Hartner FS, Fink GR, Stephanopoulos G (2010) Enhancing stress resistance and production phenotypes through transcriptome engineering. Methods Enzymol 470:509–532. doi:10.1016/S0076-6879(10)70020-3 CrossRefGoogle Scholar
  21. Lawrence CW (2004) Guide to yeast genetics and molecular and cell biology. Methods in enzymology Part A. Elesevier Academic Press, New York, p 194Google Scholar
  22. Li BZ, Cheng JS, Ding MZ, Yuan YJ (2010) Transcriptome analysis of differential responses of diploid and haploid yeast to ethanol stress. J Biotechnol 148(4):194–203. doi:10.1016/j.jbiotec.2010.06.013 CrossRefGoogle Scholar
  23. Matsushika A, Inoue H, Murakami K, Takimura O, Sawayama S (2009) Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae. Bioresour Technol 100(8):2392–2398. doi:10.1016/j.biortech.2008.11.047 CrossRefGoogle Scholar
  24. Nass R, Rao R (1999) The yeast endosomal Na+/H+ exchanger, Nhx1, confers osmotolerance following acute hypertonic shock. Microbiology 145(Pt 11):3221–3228Google Scholar
  25. Novotny C, Flieger M, Panos J, Dolezalova L (1992) Effect of growth rate on ethanol tolerance of Saccharomyces cerevisiae. Folia Microbiol (Praha) 37(1):43–46CrossRefGoogle Scholar
  26. Otte B, Grunwaldt E, Mahmoud O, Jennewein S (2009) Genome shuffling in Clostridium diolis DSM 15410 for improved 1,3-propanediol production. Appl Environ Microbiol 75(24):7610–7616. doi:10.1128/AEM.01774-09 CrossRefGoogle Scholar
  27. Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WP, Ryan CM, del Cardayre S (2002) Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol 20(7):707–712. doi:10.1038/nbt0702-707nbt0702-707 CrossRefGoogle Scholar
  28. Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, Frederick WJ Jr, Hallett JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinski T (2006) The path forward for biofuels and biomaterials. Science 311(5760):484–489. doi:10.1126/science.1114736 CrossRefGoogle Scholar
  29. Rautio JJ, Huuskonen A, Vuokko H, Vidgren V, Londesborough J (2007) Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression. Yeast 24(9):741–760. doi:10.1002/yea.1510 CrossRefGoogle Scholar
  30. Sambrook JaR D (2001) Molecular cloning: a laboratory manual (third edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  31. Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41CrossRefGoogle Scholar
  32. Stratford M, Anslow PA (1996) Comparison of the inhibitory action on Saccharomyces cerevisiae of weak-acid preservatives, uncouplers, and medium-chain fatty acids. FEMS Microbiol Lett 142(1):53–58CrossRefGoogle Scholar
  33. Wei P, Li Z, He P, Lin Y, Jiang N (2008) Genome shuffling in the ethanologenic yeast Candida krusei to improve acetic acid tolerance. Biotechnol Appl Biochem 49(Pt 2):113–120. doi:10.1042/BA20070072 CrossRefGoogle Scholar
  34. Zhang YX, Perry K, Vinci VA, Powell K, Stemmer WP, del Cardayre SB (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415(6872):644–646. doi:10.1038/415644a415644a CrossRefGoogle Scholar
  35. Zheng DQ, Wu XC, Wang PM, Chi XQ, Tao XL, Li P, Jiang XH, Zhao YH (2010) Drug resistance marker-aided genome shuffling to improve acetic acid tolerance in Saccharomyces cerevisiae. J Ind Microbiol Biotechnol. doi:10.1007/s10295-010-0784-8

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jing-Jing Liu
    • 1
  • Wen-Tao Ding
    • 1
  • Guo-Chang Zhang
    • 1
  • Jing-Yu Wang
    • 1
  1. 1.Department of Biochemical Engineering, School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina

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