Automated Yeast Mating Protocol Using Open Reading Frames from Saccharomyces cerevisiae Genome to Improve Yeast Strains for Cellulosic Ethanol Production

Stephen R. Hughes, Ronald E. Hector, Joseph O. Rich, Nasib Qureshi, Kenneth M. Bischoff, Bruce S. Dien, Badal C. Saha, Siqing Liu, Elby J. Cox, John S. Jackson, David E. Sterner, Tauseef R. Butt, Joshua Labaer, Michael A. Cotta

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

Engineering the industrial ethanologen Saccharomyces cerevisiae to use pentose sugars from lignocellulosic biomass is critical for commercializing cellulosic fuel ethanol production. Approaches to engineer pentose-fermenting yeasts have required expression of additional genes. We implemented a high-throughput strategy to improve anaerobic growth on xylose and rate of ethanol production by evaluating overexpression of each native S. cerevisiae gene from a collection of haploid PJ69–4 MATa strains expressing the gene open reading frames (ORFs) mated to a haploid PJ69–4 MATalpha strain expressing the Piromyces sp.E2 xylose isomerase (XI) gene. The resulting 6113 diploid strains containing the XI gene and a different yeast gene ORF were screened for growth on xylose in anaerobic plate cultures using an integrated robotic workcell. Nine unique strains were isolated; two were found to no longer grow on glucose; seven were further evaluated for fermentation of alkaline peroxide pretreated enzymatically saccharified wheat straw hydrolysate. All successfully used glucose and xylose, consuming most of the glucose and a small amount of the xylose. Transforming the strains with an additional vector expressing xylulokinase gene did not improve anaerobic growth on xylose but improved glucose use and ethanol production on the hydrolysate, with three strains giving maximum ethanol production ≥ 14.0 g L−1.

Original languageEnglish (US)
Pages (from-to)190-199
Number of pages10
JournalJournal of Laboratory Automation
Volume14
Issue number4
DOIs
StatePublished - Aug 2009

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Keywords

  • ORFs from Saccharomyces cerevisiae genome
  • cellulosic ethanol production
  • high-throughput screen for anaerobic growth on xylose
  • laboratory automation for metabolic engineering of yeast

ASJC Scopus subject areas

  • Computer Science Applications
  • Medical Laboratory Technology

Cite this

Hughes, S. R., Hector, R. E., Rich, J. O., Qureshi, N., Bischoff, K. M., Dien, B. S., Saha, B. C., Liu, S., Cox, E. J., Jackson, J. S., Sterner, D. E., Butt, T. R., Labaer, J., & Cotta, M. A. (2009). Automated Yeast Mating Protocol Using Open Reading Frames from Saccharomyces cerevisiae Genome to Improve Yeast Strains for Cellulosic Ethanol Production. Journal of Laboratory Automation, 14(4), 190-199. https://doi.org/10.1016/j.jala.2008.12.009