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Title: Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

item Hughes, Stephen
item Rich, Joseph
item Bischoff, Kenneth
item Hector, Ronald - Ron
item Qureshi, Nasib
item Saha, Badal
item Dien, Bruce
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: 2/19/2009
Publication Date: 8/1/2009
Citation: Hughes, S.R., Rich, J.O., Bischoff, K.M., Hector, R.E., Qureshi, N., Saha, B.C., Dien, B.S., Liu, S., Jackson Jr, J.S., Sterner, D.E., Butt, T.R., Labaer, J., Cotta, M.A. 2009. Automated yeast transformation protocol to engineer S. cerevisiae strains for cellulosic ethanol production with open reading frames that express proteins binding to xylose isomerase identified using robotic two-hybrid screen. Journal of the Association for Laboratory Automation. 8:200-212.

Interpretive Summary: Expressing genes from other microorganisms in the yeast Saccharomyces cerevisiae is important for constructing yeast strains that can convert all the sugars derived from agricultural residues into ethanol. Xylose isomerase is one of the important genes, but expressing its gene product in a fully active form in yeast is difficult. We used a "two-hybrid protein interaction" screen to find native yeast proteins that bind to the foreign xylose isomerase. Five yeast proteins were identified that interact with xylose isomerase, suggesting that they affect the function of this enzyme. This information will be used to improve the expression and activity of xylose isomerase expressed in yeast.

Technical Abstract: Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI), which catalyzes conversion of xylose to xylulose. In this study, an automated two-hybrid interaction protocol was used to find yeast genes encoding proteins that bind XI to identify potential targets for improving xylose utilization by S. cerevisiae. A pDEST32 vector re-engineered for TRP selection and containing the Gal4 binding domain fused with the Piromyces sp.E2 XI open reading frame (ORF) was used as bait with a library of LEU-selectable pOAD vectors containing the Gal4 activation domain in fusion with members of the S. cerevisiae genome ORF collection. Binding of a yeast ORF protein to XI activates two chromosomally located reporter genes in a PJ69-4 yeast strain to give selective growth. Five genes, including ADH1, were identified in the two-hybrid screen, suggesting the proteins encoded by these genes bind to XI. The effect of ADH1 over-expression was examined using the pSUMOduoHisADH1 vector in an automated protocol to transform eight previously identified yeast strains that showed anaerobic growth on xylose. One transformant consumed all available glucose, xylose, and arabinose during growth on wheat straw hydrolysate.