Abstract:[Objective] The aim of this study is to improve furfural tolerance of Saccharomyces cerevisiae by constructing its recombinant strain with fine-tune expression of MSN2 under a self-regulated promoter control. [Methods] The ADH7 promoter, coding region of MSN2, and CYC1 terminator sequences from genomic DNA of the S. cerevisiae strain BY4742 were obtained by PCR amplification, and then were ligated into the pUG6 plasmid resulting in a recombinant plasmid pUG6-AM containing the ADH7p-MSN2-CYC1t cassette. The recombinant plasmid was transformed into the S. cerevisiae BY4742 strain by the LiAc/SS-DNA/PEG transformation method after linearization, and then positive transformants were screened. Tolerability of a selected recombinant yeast strain to furfural was compared to its control, and transcription responses of MSN2 and its representative regulons between this recombinant strain and its control under both furfural stress and normal conditions were further comparatively studied. [Results] A recombinant S. cerevisiae strain (named as AM01) with fine-tune expression of MSN2 under the ADH7 promoter control was successfully constructed. This recombinant strain showed significantly improved tolerance to furfural, and MSN2 displayed fine-tune transcription response to furfural stress and then positively affected transcription responses of its regulons. [Conclusion] Fine-tune expression of MSN2 controlled by a self-regulated promoter from a gene induced by furfural not only improved furfural tolerance of the genetically engineered S. cerevisiae strain, but also avoided side-effects due to its constitutive overexpression under the strong promoter control.
