(165d) Systematic Analysis and Decoupling of Yeast Tolerance to Combined Inhibitors In Liginocellulose Hydrolytes

Inhibitors produced during lignocellulose pretreatment including furan derivatives, weak acids and phenolic compounds impose severely synergistic effects on Saccharomyces cerevisiae during ethanol fermentation. Tolerant yeast which could exhibit better characteristics in the presence of combined-inhibitors ((1.3 g/L furfural, 5.3 g/L acetic acid and 0.5 g/L phenol)) was obtained. The complex relationship between combined inhibitors and yeast cells was analyzed systematically on mRNA, proein and metabolite levels.

GC-TOF/MS based metabolomics investigation indicated that acetic acid played the most important role in the combined inhibitors. Tolerant yeast exhibited better tolerance to inhibitors in growth, fementation and metabolism, reflecting on less affected amino acid metabolism and carbon central metabolism. 2-DE and MALDI-TOF/MS based proteomic study suggested that oxidative, osmotic and unfolded protein stress responses were induced in yeast by combined inhibitors, but the mechanisms of stress response in parental and tolerant yeasts were different. Higher stress response and detoxification realated proteins were important for tolerant yeast to protect itself. And lower levels of amino acid and nucleotide metabolism related proteins and higher levels of energy related proteins were necessary for tolerant yeast to defend the inhibitors stresses. Glycolysis and gluconeogenesis related proteins were upregulated in parental yeas to produce more energy to resist the inhibitors stresses. Metabolomic and proteomic results indicated that protein degradation was increased by combined inhibitors, inducing oxidative stress and inhibiting growth and production of yeast.

Microarray based transcriptomics study found that RNA and protein synthesis related genes were repressed, while electron transport and membrane-associated energy conservation related genes were upregulated by combined inhibitors. It indicated that biosynthesis in yeast was inhibited, and more energy was needed to defend the inhibitors stresses. In addition, inhibitors caused protein degradation in cells, resulting in oxidative detoxification and autophagy. LC-MS/MS based lipidomic study revealed that the variation of chain length in PC and PI was induced by inhibitors. PE and PS were significantly different for parental and tolerant yeasts in response to combined inhibitors. Transcriptomics and lipidomics results indicated that PC synthesized was inhibited by inhibitors, resulting in increased membrane curvature and pore size, which facilitated the import of inhibitors to cells to affect normal metabolism.