17-05: Phenotypic and multiomic approaches to understanding molecular bottlenecks in the fermentation of lignocellulosic biomass 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 cost-effective 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 cellular stress responses, which further limit fermentation productivity. At the Great Lakes Bioenergy Research Center, we have taken a multi-comparative approach to discover and understand the molecular bottlenecks in the fermentation of lignocellulosic hydrolysates by yeast. Through multi-phenotypic and bioinformatic analysis of 300 natural isolates, we have identified a wild S. cerevisiae strain that maintains cell viability and rapid growth in a variety of hydrolysates. Following directed engineering of bacterial or fungal xylose metabolism pathways, 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 intracellular metabolite and gene expression levels during fermentation of ACSH and standard lab media, identifying bottlenecks in flux through the Pentose Phosphate Pathway. Analysis of extracellular metabolite, amino acid and metal concentrations additionally revealed limiting nutrients during fermentation. Coupled with comparative genome resequencing of parental and evolved strains, this suite of Omic data is being integrated in regulatory and metabolic network models to identify and understand genetic differences that impact the fermentation of xylose in stress-inducing lignocellulosic hydrolysates.