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

item Hughes, Stephen
item Hector, Ronald - Ron
item Rich, Joseph
item Qureshi, Nasib
item Bischoff, Kenneth
item Dien, Bruce
item Saha, Badal
item Liu, Siqing
item Jackson Jr, John
item Cotta, Michael

Submitted to: Journal of the Association for Laboratory Automation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2009
Publication Date: 8/1/2009
Citation: Hughes, S.R., Hector, R.E., Rich, J.O., Qureshi, N., Bischoff, K.M., Dien, B.S., Saha, B.C., Liu, S., Jackson Jr, 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 the Association for Laboratory Automation. 8:190-199.

Interpretive Summary: Cellulosic ethanol production will need strains of yeasts engineered for the utilization of all available sugars derived from biomass. One gene that will be required to use the pentose sugars is xylose isomerase, but auxiliary genes that complement xylose isomerase have not previously been identified. In the present study, the xylose isomerase gene was inserted into a yeast strain, and then a high-throughput screening method was used to identify potential auxiliary genes. Seven auxiliary genes were identified that helped the strain grow anaerobically on xylose, one of the major sugars in lignocellulosic biomass. These results will be valuable to researchers developing new strains of yeast that will allow complete fermentation of released sugars from lignocellulosic biomass for cellulosic ethanol production.

Technical Abstract: Engineering the industrial ethanologen Saccharomyces cerevisiae to utilize 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 over-expression 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 XI gene. The resulting 6113 diploid strains containing the XI gene and a different yeast gene open reading frame (ORF) were screened for growth on xylose in anaerobic solid 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 wheat straw alkaline hydrolysate. All successfully utilized glucose and xylose, consuming most of the glucose and a small amount of the xylose. Transforming the strains with an additional vector expressing xylulokinase (XKS) 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.