Systematic Analysis And Decoupling Of Yeast Tolerance To Combined Inhibitors In Lignocellulose Hydrolysates

Inhibitors produced during lignocellulose pretreatment including furan derivatives, weak acids and phenolic compounds impose severely synergistic effects on Saccharomyces cerevisiae during ethanol fermentation. Obtaining a stain that could tolerate combined inhibitors and deeply investigating the relationship between strain and inhibitors were challenges for lignocellulosic fermentation. In this study, a tolerant yeast which could exhibit better characteristics in the presence of combined-inhibitors (furfural, acetic acid and phenol) was obtained. The complex relationship between combined inhibitors and yeast cells was analyzed systematically on mRNA, protein and metabolite levels.The tolerant yeast which was obtained by UV mutagenesis and domestication exhibited 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). The lag phase of tolerant yeast shortened from 48 h to 4 h. The rates of growth, glucose consumption, and ethanol production increased 8.7, 3.3 and 3.3 fold, respectively. Furfural conversion time shortened from 40 h to 6 h. And ethanol production was 48.28 g/L, which did not change significantly compared to parental yeast.Analysis on metabolite levels indicated that acetic acid played the most important role in the combined inhibitors. And its influence on the growth and metabolism of yeast cells was enhanced by the presence of phenol and furfural. Tolerant yeast exhibited better tolerance to inhibitors, reflecting on less affected amino acid metabolism and carbon central metabolism. Study on protein levels 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 related 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.Study on transcriptional level 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. Research on phospholipids of cell membrane revealed that the variation of chain length in phosphatidylcholine (PC) and phosphatidylinositol (PI) was induced by inhibitors. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) were significantly different for parental and tolerant yeasts in response to combined inhibitors. Transcriptomic and lipidomic 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.