Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion
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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.
KeywordsXylose Bioethanol Switchgrass Saccharomyces Industrial yeast
We thank Dr. Kenneth Vogel for the generous gift of switchgrass and Loren Iten for its pretreatment, Dr. Cletus Kurtzman for recommending the yeast strains and Katherine Card and Tricia Windgassen for their excellent technical assistance throughout this study. We would also like to thank Patricia O’Bryan for her help evaluating extracellular metabolite concentrations and assistance with switchgrass fermentations.
- 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.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
- 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
- 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
- 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
- 25.Renewable Fuels Association (2010) 2010 Ethanol industry outlook. http://www.ethanolrfa.org/pages/annual-industry-outlook/
- 27.Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
- 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
- 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.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