Systems-Level Analysis Of Xylose Fermentation By Experimentally-Evolved Saccharomyces Cerevisiae

Experimental directed evolution and systems‐level analysis are powerful tools that can provide insights into the genetic regulation of biological and industrial processes. The fermentation of renewable plant biomass into cost‐effective biofuels is one such applied process. For the yeast biocatalyst, Saccharomyces cerevisiae , a major barrier to cellulosic ethanol production is its inability to rapidly ferment xylose from hemicellulose. To identify genetic factors regulating xylose fermentation, we performed directed evolution of an engineered S. cerevisiae strain on xylose and generated two evolved strains; GLBRCY127 was isolated by aerobic selection and a second was isolated from subsequent evolution of GLBRCY127 anaerobically. Metabolomic analyses between evolved and parent strains during the consumption of xylose from lab and cellulosic media suggested enhanced flux through the Pentose Phosphate Pathway and altered levels of TCA cycle intermediates. Genome sequencing and RNAseq analysis identified mutations in three genes that regulate mitochondrial function and signal transduction pathways. Importantly, deletion of these genes conferred xylose fermentation by the unevolved parental strain. This work identifies unexpected metabolic and physiological processes that limit cellulosic ethanol production and, when genetically modified, may help to realize a viable biofuels industry. Grant Funding Source : Supported by DOE BER Office of Science DE‐FC02‐07ER64494.