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

摘要

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.