Engineering Saccharomyces Cerevisiae For Improved Inhibitor Tolerance

The inhibitory compounds generated during the pretreatment of lignocellulosic biomass limit the efficient commercial production of cellulosic ethanol. These inhibitors severely affected the fermentation process through their synergistic effects on important biological processes. Therefore, it is urgent to enhance the tolerance of Saccharomyces cerevisiae to inhibitors.In this study,the metabolomic analysis was performed on an adaptation process to the mixture of three representative inhibitors(furfural, acetic acid and phenol; FAP). For further selection, the growth sensitivity of the knockout mutants involved in the potential biomarkers was investigated in the presence of inhibitors. Accordingly, it was found that energy metabolism, alanine metabolism, glutamate synthesis and catabolism, and the synthesis of glycerol, proline or myo-inositol were important for resisting multiple inhibitors in S. cerevisiae. Subsequently, it was observed that the addition of proline or myo-inositol in medium rescued strain growth under FAP stress. The enhancement of proline or myo-inositol synthesis through overexpressing key gene PRO1 or INO1 also conferred the strain significantly higher FAP tolerance and the fermentation rate was increased by 2 times in the presence of inhibitors. Considering the feedback inhibition of proline on gene PRO1, the strategy of site-directed mutagenesis and random mutation were used to genetically modify the gene PRO1 simultaneously. The fermentation time of the recombinant strain BY4742/PRO1D154 N, G220 C was shortened to 30 h from 125 h in the presence of inhibitors. The proline contents in the different PRO1 mutants indicated that an appropriate proline level in yeast cells is important for its stress-protective effect. Furthermore, the detection of the intracellular reactive oxygen species(ROS) suggested the potential role of proline as a ROS scavenger in protecting cells from damage caused by FAP.Then, the exogenous tolerance modules were specifically selected to engineer the strain inhibitor tolerance. The GadC module(glutamate/GABA antiporter) was heterologously expressed in S.cerevisiae to construct the new proton pump. Through optimizing GadC expression under the control of different promoters, the fermentation rate was improved by 3 times under acetic acid stress. The metabolomic analysis suggested that the recombinant strain with GadC module has the lower demand of antioxidant and higher energy supply. On the other hand, an exogenous global regulatory factor IrrE was engineered in S.cerevisiae to regulate its inhibitor tolerance. The mutant IrrE library was constructed and then screened for improved tolerance to inhibitors. Accordingly, the fermentation rate of the mutant strain under FAP stress was increased by 4 times and its thermostability was also improved. Mutant analysis found that yeast cells expressing I103 T, E119 V, S133 R, L160 F, P162 S, R244 G, V299 A or A300 V mutant Irr E were more tolerant to multiple inhibitors than the strain BY4742/IrrE.In addition, the limitations of the xylose-fermenting yeast during the co-fermentation of glucose and xylose in the presence of inhibitors were also investigated through metabololomic analysis. It was found that the decreased tolerance of the xylose-fermenting yeast was associated its capability to buffer oxidative stress caused by inhibitors. Meanwhile, xylose catabolism and energy supply were showed to be important for xylose fermentation in the presence of inhibitors. These results provide valuable insights into the design and development of recombinant xylose-fermenting yeast with improved tolerance to multiple inhibitors.