Development and characterization of a robust industrial yeast for high solids softwood fermentation using adaptation, directed evolution, and transcriptome profiling

Abstract

Currently almost all the fuel ethanol produced in the United States is generated from processes using corn as the feedstock. To increase production of ethanol, other feedstocks must be developed as viable substrates for fermentation processes. Efficient ethanol fermentation from softwood biomass, which is produced in large quantities globally, at titers suitable for an industrial process is challenging for a variety of reasons. One challenge is the generation of inhibitory compounds from the biomass as it is pretreated prior to enzymatic digestion. These compounds are then present in the fermentation medium where they inhibit the activity of the fermenting organism. To overcome this bottleneck in softwood fermentation, we have developed a Saccharomyces cerevisiae yeast strain that is capable of producing ethanol from greater than 17.5% dry weight per volume of pretreated softwood at ethanol yields over 90% of the theoretical maximum. This strain was developed from an industrial corn ethanol yeast by adaptation and directed evolution in increasing concentrations of pretreated softwood. Isolates from the directed evolution experiments were screened for the inhibitor resistant phenotype and for the ability to ferment pretreated softwood at high solids concentrations. Use of a model inhibitor medium for inoculum preparation enhanced fermentation performance with high solids concentrations of pretreated pine. When the inoculum was prepared for fermentation without the inhibitors present in the medium, divergent phenotypes were observed. One set of isolates retained the ability to ferment high solids of pretreated pine regardless of the method used to prepare the inoculum, whereas another set of isolates displayed reduced performance when the inhibitors were removed during inoculum preparation. Transcriptome profiling was used to characterize the gene expression patterns of a robust strain capable of fermentation of high softwood solids after preparation in either media to a strain showing the divergent phenotype. Many expression differences were observed; including multiple changes not previously known to be related to inhibitor tolerance or high solids fermentation. Potential mechanisms by which the observed expression differences aid in the robust strains’ performance in high softwood solids fermentations are presented.