Journal of Industrial Microbiology & Biotechnology

, Volume 38, Issue 9, pp 1193–1202 | Cite as

Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

  • Ronald E. Hector
  • Bruce S. Dien
  • Michael A. Cotta
  • Nasib Qureshi
Original Paper

Abstract

Saccharomyces’ physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04–0.17 h−1. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17–0.31 g/l/h, with ethanol yields 0.18–0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13–17% more ethanol than the parental control strains because of their ability to ferment xylose.

Keywords

Xylose Bioethanol Switchgrass Saccharomyces Industrial yeast 

References

  1. 1.
    Amberg BC, Burke DJ, Strathern JN (2005) Methods in yeast genetics: a Cold Spring Harbor laboratory course manual, 2005th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  2. 2.
    Baganz F, Hayes A, Marren D, Gardner DC, Oliver SG (1997) Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae. Yeast 13:1563–1573. doi:10.1002/(SICI)1097-0061(199712)13:16<1563:AID-YEA240>3.0.CO;2-6 PubMedCrossRefGoogle Scholar
  3. 3.
    Bailey RB, Benitez T, Woodward A (1982) Saccharomyces cerevisiae mutants resistant to catabolite repression—use in cheese whey hydrolysate fermentation. Appl Environ Microbiol 44:631–639PubMedGoogle Scholar
  4. 4.
    Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110:119–122. doi:10.1016/0378-1119(92)90454-W PubMedCrossRefGoogle Scholar
  5. 5.
    Chu BC, Lee H (2007) Genetic improvement of Saccharomyces cerevisiae for xylose fermentation. Biotechnol Adv 25:425–441. doi:10.1016/j.biotechadv.2007.04.001 PubMedCrossRefGoogle Scholar
  6. 6.
    Dien BS, Nichols NN, O’Bryan PJ, Iten LB, Bothast RJ (2004) Enhancement of ethanol yield from the corn dry grind process by conversion of the kernel fiber fraction. In: Nelson WM (ed) Agricultural Applications in Green Chemistry, ACS, Washington, DC, pp 63–77Google Scholar
  7. 7.
    Gibbons WR, Westby CA (1986) Effects of inoculum size on solid-phase fermentation of fodder beets for fuel ethanol production. Appl Environ Microbiol 52:960–962PubMedGoogle Scholar
  8. 8.
    Gietz D, Woods RA (2002) Transformation of yeasts by the lithium acetate/single-stranded carrier/polyethylene glycol method. Methods Enzymol 350:87–96PubMedCrossRefGoogle Scholar
  9. 9.
    Girio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S, Bogel-Lukasik R (2010) Hemicelluloses for fuel ethanol: a review. Bioresour Technol 101:4775–4800. doi:10.1016/j.biortech.2010.01.088 PubMedCrossRefGoogle Scholar
  10. 10.
    Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937–953. doi:10.1007/s00253-006-0827-2 PubMedCrossRefGoogle Scholar
  11. 11.
    Hahn-Hägerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF (2007) Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 108:147–177. doi:10.1007/10_2007_062 PubMedGoogle Scholar
  12. 12.
    Jojima T, Omumasaba CA, Inui M, Yukawa H (2010) Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook. Appl Microbiol Biotechnol 85:471–480. doi:10.1007/s00253-009-2292-1 PubMedCrossRefGoogle Scholar
  13. 13.
    Karhumaa K, Fromanger R, Hahn-Hägerdal B, Gorwa-Grauslund MF (2007) High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae. Appl Microbiol Biotechnol 73:1039–1046. doi:10.1007/s00253-006-0575-3 PubMedCrossRefGoogle Scholar
  14. 14.
    Keating JD, Robinson J, Bothast RJ, Saddler JN, Mansfield SD (2004) Characterization of a unique ethanologenic yeast capable of fermenting galactose. Enz Microb Technol 35:242–253. doi:10.1016/j.enzmictec.2004.04.015 CrossRefGoogle Scholar
  15. 15.
    Keating JD, Robinson J, Cotta MA, Saddler JN, Mansfield SD (2004) An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars. J Ind Microbiol Biotechnol 31:235–244. doi:10.1007/s10295-004-0145-6 PubMedCrossRefGoogle Scholar
  16. 16.
    Keating JD, Panganiban C, Mansfield SD (2006) Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol Bioeng 93:1196–1206. doi:10.1002/bit.20838 PubMedCrossRefGoogle Scholar
  17. 17.
    Krahulec S, Petschacher B, Wallner M, Longus K, Klimacek M, Nidetzky B (2010) Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization. Microbial Cell Factories 9. doi:10.1186/1475-2859-9-16
  18. 18.
    Li X, Kim TH, Nghiem NP (2010) Bioethanol production from corn stover using aqueous ammonia pretreatment and two-phase simultaneous saccharification and fermentation (TPSSF). Bioresour Technol 101:5910–5916PubMedCrossRefGoogle Scholar
  19. 19.
    Lin Y, Tanaka S (2006) Ethanol fermentation from biomass resources: current state and prospects. Appl Microbiol Biotechnol 69:627–642. doi:10.1007/s00253-005-0229-x PubMedCrossRefGoogle Scholar
  20. 20.
    Matsushika A, Inoue H, Kodaki T, Sawayama S (2009) Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives. Appl Microbiol Biotechnol 84:37–53. doi:10.1007/s00253-009-2101-x PubMedCrossRefGoogle Scholar
  21. 21.
    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 100:2392–2398. doi:10.1016/j.biortech.2008.11.047 PubMedCrossRefGoogle Scholar
  22. 22.
    Meinander NQ, Boels I, Hahn-Hägerdal B (1999) Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1. Bioresour Technol 68:79–87. doi:10.1016/S0960-8524(98)00085-6 CrossRefGoogle Scholar
  23. 23.
    Mohagheghi A, Tucker M, Grohmann K, Wyman C (1992) High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol. Appl Biochem Biotechnol 33:67–81. doi:10.1007/bf02950778 CrossRefGoogle Scholar
  24. 24.
    Ranatunga TD, Jervis J, Helm RF, McMillan JD, Hatzis C (1997) Toxicity of hardwood extractives toward Saccharomyces cerevisiae glucose fermentation. Biotechnol Lett 19:1125–1127. doi:10.1023/a:1018400912828 CrossRefGoogle Scholar
  25. 25.
    Renewable Fuels Association (2010) 2010 Ethanol industry outlook. http://www.ethanolrfa.org/pages/annual-industry-outlook/
  26. 26.
    Saha BC (2003) Hemicellulose bioconversion. J Ind Microbiol Biotechnol 30:279–291. doi:10.1007/s10295-003-0049-x PubMedCrossRefGoogle Scholar
  27. 27.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  28. 28.
    Sonderegger M, Sauer U (2003) Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Appl Environ Microbiol 69:1990–1998. doi:10.1128/Aem.69.4.1990-1998.2003 PubMedCrossRefGoogle Scholar
  29. 29.
    Sonderegger M, Jeppsson M, Larsson C, Gorwa-Grauslund MF, Boles E, Olsson L, Spencer-Martins I, Hahn-Hägerdal B, Sauer U (2004) Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng 87:90–98. doi:10.1002/bit.20094 PubMedCrossRefGoogle Scholar
  30. 30.
    Strathern JN, Klar AJ, Hicks JB, Abraham JA, Ivy JM, Nasmyth KA, McGill C (1982) Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell 31:183–192. doi:10.1016/0092-8674(82)90418-4 PubMedCrossRefGoogle Scholar
  31. 31.
    Van Vleet JH, Jeffries TW (2009) Yeast metabolic engineering for hemicellulosic ethanol production. Curr Opin Biotechnol 20:300–306. doi:10.1016/j.copbio.2009.06.001 PubMedCrossRefGoogle Scholar
  32. 32.
    Voth WP, Richards JD, Shaw JM, Stillman DJ (2001) Yeast vectors for integration at the HO locus. Nucleic Acids Res 29:E59. doi:10.1093/nar/29.12.e59 PubMedCrossRefGoogle Scholar
  33. 33.
    Wahlbom CF, van Zyl WH, Jonsson LJ, Hahn-Hägerdal B, Otero RR (2003) Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054. FEMS Yeast Res 3:319–326. doi:10.1016/S1567-1356(02)00206-4 PubMedCrossRefGoogle Scholar
  34. 34.
    Walker ME, Gardner JM, Vystavelova A, McBryde C, de Barros Lopes M, Jiranek V (2003) Application of the reuseable, KanMX selectable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences. FEMS Yeast Res 4:339–347. doi:10.1016/s1567-1356(03)00161-2 PubMedCrossRefGoogle Scholar
  35. 35.
    Zhu J, Zhu W, O’Bryan PJ, Dien BS, Tian S, Gleisner R, Pan X (2010) Ethanol production from SPORL-pretreated lodgepole pine: preliminary evaluation of mass balance and process energy efficiency. Appl Microbiol Biotechnol 86:1355–1365. doi:10.1007/s00253-009-2408-7 PubMedCrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology (outside the USA) 2010

Authors and Affiliations

  • Ronald E. Hector
    • 1
  • Bruce S. Dien
    • 1
  • Michael A. Cotta
    • 1
  • Nasib Qureshi
    • 1
  1. 1.U.S. Department of AgricultureBioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research ServicePeoriaUSA

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