Effect Of Qverexpression Of Key Enzyme Genes On Stress Tolerance Of Saccharomyces Cerevisiae

Fuel ethanol production using renewable cellulosic substrates has attracted widespread attention. Yeast strains of Saccharomyces cerevisiae strains are exposed to various stress factors during fuel ethanol production, including high-gravity ethanol and inhibitors derived from cellulose hydrolysate, among which, acetic acid is one of the most important inhibitors released from the pretreatment process and exerts severe inhibition on yeast cell growth and ethanol fermentation. Development of stress-tolerant S. cerevisiae strains can not only improve cell growth and ethanol yield, reduce equipment investment, but also reduce the fermentation time and the operating costs.Our previous studies showed that zinc sulfate addition improved stress resistance of S. cerevisiae, and the proteomic data revealed that Sodlp, Grx5p and Ycr102cp were all up-regulated in yeast cells with zinc supplementation. However, the effect of overexpression of these key enzyme genes on yeast stress tolerance is still unexplored.Therefore, in this work, the effect of SOD1, GRX5 and YCR102C overexpression on acetic acid tolerance and ethanol fermentation using inhibitor containing lignocellulosic hydrolysates were studied. It was revealed that overexpression of GRX5, YCR102C in S. cerevisiae 4126 improved cell growth under various inhibitory conditions (e.g.5 g/L acetic acid,5 mM H2O2,10% ethanol and 42 ?) on agar plates, whereas the tolerance of SOD 1 overexpressing strain showed the dependency of zinc, growing better only in the presence of 0.03 g/L zinc under stressful conditions.Furthermore, the fermentation time was all shortened by the overerpression of SOD1, GRX5 and YCR102C in the presence of 5 g/L acetic acid. Meanwhile, the activity of SOD, CAT and GSH-PX were increased by the overexpression of GRX5 and YCR102C (50.7%,34.5%,158% and 6.3%,47.4%,16.0%, respectively). It was found that GRX5 and YCR102C overexpression affected the concents of intracellular metals, indicating remodeling of metal metabolism by elevated levels of these two genes.The effect of GRX5 and YCR102C overexpression on simulated hydrolysate was further studied. It was found that ethanol productivity of GRX5 and YCR102C overexpressing strains increased to 0.62 g/L/h and 0.52 g/L/h, respectively, when exposed to the mixture of formic acid, acetic acid, furfural and HMF, which was 61.4% and 33.3% higher than that from the reference strain. The recombinant strains also showed stronger ability of detoxification of furfural and 5-HMF. Overexpression of GRX5 shortened the fermentation time from 84 hrs to 48 hrs the presence of furfural, and from 96 hrs to 60 hrs for 5-HMF. In contrast, the fermentation time of 24 hrs was shortened for both furfural and 5-HMF by overexpression of YCR102C. When the transcription levels of several key genes between YCR102C overexpressing strain and control strain with empty plasmid under both acetic acid stress and control condition were examined, it was found that transcription of the regulatory gene STB5 was increased in both the control condition and in the presence of acetic acid, implying that the function of YCR102C is related to the transcript level of the key transcription factor STB5.Our results demonstrate that overexpression of SOD 1, GRX5 and YCR102C can be employed as a feasible strategy to develop yeast strains with improved ethanol production efficiency in the presence of inhibitors. These results provide basis for further exploration of the molecular mechanisms underlying improved stress tolerance by overexpression of functional genes, and development stress tolerant yeast strains to increase fuel ethanol production using cellulosic substrates.