Submitted to: Molecular Genetics and Genomics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/17/2009
Publication Date: 6/11/2009
Citation: Liu, Z., Menggen, M., Song, M.J. 2009. Evolutionarily Engineered Ethanologenic Yeast Detoxifies Lignocellulosic Biomass Conversion Inhibitors by Reprogrammed Pathways. Molecular Genetics and Genomics. 282(3):233-244. Interpretive Summary: In this research, we discovered reprogrammed pathways by tolerant ethanologenic yeast that allow the yeast to in situ detoxify biomass conversion inhibitors while producing ethanol. Inhibitory compounds generated during hydrolysis pretreatment of lignocellulosic biomass inhibit cell growth and interfere with subsequent fermentation of fermentative microbes. Currently, there is no tolerant yeast available for cellulosic ethanol production at industrial scale. Development of tolerant yeast to overcome inhibitor stress is needed for a sustainable biomass-to-ethanol industry. Using a tolerant yeast strain developed at ARS, we identified numerous genes, new elements, and alternative pathways responsible for the detoxification of the inhibitors and ethanol production. The tolerant yeast can be applied for two-stage fermentation using hydrolysates without additional inhibitor removing treatment such as overliming. Thus, processing procedures can be simplified and cost reduced. Knowledge obtained from this study contributes understanding of mechanisms of stress tolerance and guides future more robust strain development in bioenergy field.
Technical Abstract: Lignocellulosic biomass conversion inhibitors furfural and HMF inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol; while its parental contro1 Y-12632 failed to function until 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and AW4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF in return provided sufficient NAD(Pt for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress.