Identification of gene targets improving inhibitor resistance of Saccharomyces cerevisiae for efficient lignocellulosic biofuel production through inverse metabolic engineering

Lignocellulosic biomass has the potential to contribute substantially to future global energy demands. However, inefficient bioconversion of solubilized plant cell wall materials (hydrolyzates) to biofuels has hindered commercial-scale processes. One major problem is the toxicity of cellulosic hydrolysates to fermenting microorganisms.  Cellulosic hydrolysates usually contain mixed fermentation inhibitors (e.g. weak acids, furan aldehydes, and phenolics) with distinct toxicity mechanisms, making it challenging to develop resistant microbial strains by rational metabolic engineering. This study aimed to improve the resistance of the yeast Saccharomyces cerevisiae to mixed inhibitors in cellulosic hydrolysates for efficient utilization of cellulosic sugars (e.g. xylose and glucose) through inverse metabolic engineering.

Specifically, genome-wide scanning of gene targets was performed by constructing and screening a genomic DNA library in a parent yeast strain containing an optimized xylose-fermenting pathway. The approach successfully identified a novel gene target, whose overexpression substantially improved cell growth and consumption rates of xylose or glucose in the presence of toxic levels of acetic acid, furfural and vanillin. Additionally, comparative RNA-seq transcriptomic analysis showed that global transcriptional reprogramming was triggered in the resistant strain. Multiple transcriptional factors (TF) relevant to regulating the mixed inhibitor resistance were determined through deep analysis of the RNA-seq data, and these TFs are being tested as candidate perturbation targets to further improve the inhibitor resistance phenotype. Results in this study will contribute to developing a robust yeast platform for economically viable production of lignocellulosic biofuels/chemicals, and also help advance the fundamental understanding of the mechanisms of inhibitor resistance in yeast.