酿酒酵母乙醇脱氢酶Ⅰ基因的超表达

Overexpression of Alcohol DehydrogenaseⅠ in Saccharomyces cerevisiae

摘要:

利用重叠延伸PCR融合磷酸甘油激酶(phosphoglycerate kinase, PGK)启动子和酿酒酵母乙醇脱氢酶基因Ⅰ(alcohol dehydrogenaseⅠ, adh1), 将该融合片段插入带有G418抗性基因(KanMX)和loxP位点的pUG6质粒中, 并在adh1基因下游插入细胞色素c (Cytochrome c transcription, CYC1)终止子, 构建了酿酒酵母整合表达载体pUPGKAT。TthⅢⅠ内切酶线性化后转化酿酒酵母乙醇脱氢酶基因Ⅱ(adh2 )敲除菌株YS2-△adh2。根据酿酒酵母同源重组机制, 使adh1基因增加1个拷贝, 且其中1个拷贝置于PGK强启动子下游, 利用PGK启动子的调控成功实现adh1基因的超表达。厌氧发酵试验表明该重组菌株YS2- △adh2-adh1的乙醇产量较出发菌株提高了8.84％。

Abstract:

To improve ethanol production in Saccharomyces cerevisiae, an integration plasmid pUPGKAT with PGK promoter (phosphoglycerate kinase promoter), adh1 gene (the coding sequences of alcohol dehydrogenaseⅠ) and CYC1 terminator (Cytochrome c transcription terminator) was constructed. Firstly, a fusion fragment composed of PGK promoter and adh1 gene was generated by over lap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA. Subsequently, CYC1 termi nator was amplified from pSH65 by PCR and ligated to the SpeⅠand SacⅡrestriction site of pUPGKA. To integrate PGK-adh1-CYC1 into S. cerevisiae genome, pUPGKAT was digested by TthⅢⅠand the linearized plasmid was used to transform S. cerevisiae YS2-△adh2 (adh2 disrupted strain) by lithium acetate method. The yeast mutant YS2-△adh2-adh1 which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed. Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84% higher ethanol production compared to YS2-△adh2.