Abstract:

Cellulosic ethanol, as a clean and renewable green energy, has promising application prospects. However, the fermentation process of Saccharomyces cerevisiae using lignocellulose to produce ethanol is susceptible to various stresses，thus improving the stress tolerance of fermenting microorganisms has become an essential topic in the field of biorefinery. In this work, we attempted to engineer cells to use the output of the internal redox-sensitive biosensor Yap1 to drive intracellular metabolic reactions（including ROS metabolism and aldehyde degradation）, which will improve the tolerance of S. cerevisiae to reactive oxygen species and aldehyde inhibitors. Firstly, we analyzed the sequences of several native endogenous promoters（P_TRR1, P_TRX2, and P_MET16）in S. cerevisiae that can be regulated by the redox-sensitive biosensor Yapl and their response strengths to typical inhibitors in lignocellulosic hydrolysates. Secondly, we designed redox-sensitive genetic parts in order to improve the stress resistance of S. cerevisiae by combining the promoters with corresponding resistance genes. Finally, we tried to integrate the better performing GP-CTT and GP-ADH in tandem to construct a dual-genetic parts system, which was named GP-AC. The integration greatly improved the ability of S. cerevisiae to cope with multiple complex stresses, and the cell death rate decreased by 69.6% under the coexistence of aldehyde and oxidation stress. Compared with GP-CTT, the specific growth rate, glucose consumption rate and ethanol production rate of GP-AC increased by 64.2%, 60.1%, and 58.9%, respectively. At the same time, the catalase activity of the recombinant strain increased by 40.2% compared with the single genetic part. In conclusion, this study systematically improved the stress tolerance of S. cerevisiae by rationally designing the genetic circuit of redox-sensitive genetic parts and strengthening the intracellular vital antioxidant enzymes and aldehyde degradation pathways. It provides new insights for rationally designing and constructing feedback genetic circuits to dynamically improve yeast robustness.

Key words: redox-potential, genetic parts, Saccharomyces cerevisiae, stress tolerance, lignocellulose, fuel ethanol