ADH7启动子精细调控表达MSN2酿酒酵母菌株对糠醛耐受的研究

Study on furfural tolerance of recombinant Saccharomyces cerevisiae strain with fine-tune expression of MSN2 under ADH7 promoter control

【目的】构建自我精细调控表达应激转录调控基因MSN2的酿酒酵母(Saccharomyces cerevisiae)基因工程菌株,提高其对糠醛的耐受能力。【方法】以酿酒酵母BY4742基因组DNA为模板,采用PCR技术扩增获得ADH7启动子、CYC1终止子以及MSN2编码框序列,以p UG6质粒为载体构建含ADH7p-MSN2-CYC1t表达盒的重组表达质粒p UG6-AM。通过醋酸锂法,将线性化后的质粒p UG6-AM转入酿酒酵母BY4742,筛选阳性转化子,初步分析其对糠醛的耐受能力,采用荧光定量PCR技术检测MSN2基因及其调控代表基因的转录变化。【结果】构建了在ADH7启动子控制下表达MSN2的酿酒酵母基因工程菌株AM01,该菌株对糠醛耐受能力明显增强,MSN2基因的转录得到了自我精细调控,并提高了其调控基因的转录水平。【结论】以糠醛诱导表达基因的启动子精细调控应激转录调控基因MSN2的转录表达,既可提高酿酒酵母工程菌株对糠醛的耐受能力,又能避免其持续高效表达带来的副作用。

[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 ADHTp-MSN2-CYClt 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.