(667b) Phenotypic and Multi-Omic Approaches to Address Molecular Bottlenecks in the Fermentation of Lignocellulose Into Ethanol by Saccharomyces Cerevisiae

While cellulosic ethanol is being looked to for relief of the global energy demand, a number of molecular bottlenecks currently exist that prevent the efficient bioconversion of lignocellulose into ethanol. For example, it is well known that:  1) native Saccharomyces cerevisiae yeast strains cannot sufficiently ferment xylose, and 2) side products generated from pretreatment, including Ammonia Fiber Expansion (AFEX™) and alkaline hydrogen peroxide (AHP), of plant biomass illicit a cellular stress response, which further limits fermentation productivity. At the Great Lakes Bioenergy Research Center, we have taken a multi-comparative approach to facilitate the discovery and understanding of molecular bottlenecks in the fermentation of lignocellulosic hydrolysates by yeast. Through multi-phenotypic and bioinformatic analysis of 111 natural and domesticated isolates, we have identified a wild S. cerevisiae strain that maintains rapid growth and cell viability in a variety of distinctly prepared hydrolysates. Following directed engineering of a xylose metabolism pathway, we performed directed evolution that yielded mutants able to ferment 2 to 3-fold more xylose from AFEX™ corn stover hydrolysate (ACSH) than unevolved parents. Furthermore, we employed temporal profiling of gene expression levels during ACSH fermentations, which identified differences in cell physiology between evolved and unevolved strains. Analysis of extracellular metabolite, amino acid and metal concentrations additionally identified limiting nutrients during fermentation. Coupled with comparative genome resequencing of parental and evolved strains, this suite of Omic data is being integrated in metabolic network models to identify and understand genetic differences that impact xylose fermentation in stress-inducing lignocellulosic hydrolysates.