Lignocellulosic biomass has been identified as the prime renewable source to produce biofuels and value-added chemicals. Metabolic engineering has recently expanded the substrate range of Saccharomyces cerevisiae to lignocellulosic sugars. However, a key limitation yet to overcome is the low resistance of the microorganism to fermentation inhibitors in cellulosic hydrolysates, which hinders efficient bioconversion processes. Cellulosic hydrolysates usually contain mixed fermentation inhibitors (e.g. weak acids, furan aldehydes, and phenolics) with distinct toxicity mechanisms, rather than a single inhibitor, making it more challenging to develop resistant strains. While prior research presented important information on mechanisms for resistance in yeast to some individual inhibitors, it remains unclear how to rationally engineer resistance to mixed inhibitors due to the limited understanding of relevant gene regulatory networks. The objective of the study was to improve the resistance of S. cerevisiae to the mixed fermentation inhibitors in cellulosic hydrolysates for efficient utilization of cellulosic sugars through inverse metabolic engineering. We chose a parent strain containing an optimized xylose assimilating pathway so as to evaluate the improvements in both xylose and glucose fermentations with mixed inhibitors. Specifically, we applied a genomic library approach and successfully identified a novel gene target, whose overexpression substantially improved cell growth and glucose/xylose consumption rates with the presence of acetic acid (2 g/L), furfural (1.5 g/l) and vanillin (0.3 g/L). Batch fermentation experiments demonstrated that the resistant strain had a specific sugar consumption rate four times higher than the control with the presence of the mixed inhibitors. Comparative RNA-seq transcriptomic analysis showed that global transcriptional reprogramming has been triggered. With a deep analysis of the data, a list of transcriptional factors (TF) relevant to regulating the mixed inhibitor resistance was generated. These TFs are being tested as candidate perturbation targets to further improve the resistance phenotype. Results in this study will contribute to the development of a robust yeast platform for economically viable bioconversion of lignocellulosic biomass, and also help advance the fundamental understanding of the mechanisms for the mixed inhibitor resistance in yeast and provide new insights for rational metabolic engineering.

Improve resistance of Saccharomyces cerevisiae to mixed fermentation inhibitors through inverse metabolic engineering for harnessing lignocellulosic biomass

249th National Meeting (2015)

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