酿酒酵母adh2和ald6双基因缺失突变株的构建

Construction of Saccharomyces cerevisiae Mutant Deficient in adh2 and ald6 Genes

酿酒酵母乙醇合成代谢过程中,阻断或削弱乙醛至乙酸代谢流不但能增强乙醇合成流,同时还能降低发酵乙酸含量。本研究以乙醇脱氢酶Ⅱ(adh2)基因缺陷型酿酒酵母YS2-Δadh2为出发菌株,应用长侧翼同源两步PCR(LFH-PCR)策略构建乙醛脱氢酶Ⅵ(ald6)基因敲除组件,转化酿酒酵母YS2-Δadh2敲除ald6基因,之后转入表达质粒pSH65到阳性克隆中,半乳糖诱导表达Cre重组酶切除Kanr基因筛选标记,最后,传代丢失质粒pSH65获得单倍体ald6基因缺失突变株。利用同样的敲除组件和技术再次敲除其等位基因,最终获得双基因缺失突变株YS2-△adh2-Δald6。发酵实验表明与出发菌株YS2相比,突变株乙酸合成量降低18%,乙醇最高产量提高12.5%。

The purpose of this investigation is to improve ethanol production and decrease acetate formation in Saccharomyces cerevisiae strain YS2-Aadh2. The strain YS2-Aadh2 with deleted alcohol dehydrogenase Ⅱ （adh2） gene was isolated in our lab with higher ethanol production than that of the strain YS2. The acet- aldehyde dehydrogenase Ⅵ （ald6） gene encoded a cytosolic acetaldehyde dehydrogenase, a key enzyme of the pyruvate dehydrogenase （PDH） bypass, transfers acetaldehyde to acetate. To disrupt ald6 gene of the strain YS2-△adh2, ald6 gene targeting cassettes were synthesized by long flanking homology PCR （LFH-PCR） and then were transformed into YS2-△adh2 mutants by LiAc/SS Carrier DNA/PEG method. Positive transformants were selected with G418 and further confirmed by PCR. Once correctly integrated into the genome, the selective marker was rescued by transforming the plasmid pSH65 into the positive transformants and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure. We named the ald6 gene knocked-out strain as YS2-△adh2-△ald6 which has a 12.5% higher ethanol production and a 18% lower acetate formation compared to the strain YS2.