Employing a combinatorial expression approach to characterize xylose utilization in Saccharomyces cerevisiae

Luke N. Latimer, Michael E. Lee, Daniel Medina-Cleghorn, Rebecca A. Kohnz, Daniel K. Nomura, John E. Dueber

Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed 1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; 2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; 3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH.