Oenococcus oeni苹果酸-乳酸发酵关键酶基因在酿酒酵母中的转化与表达  

Cloning of Two Key Enzyme Genes from Oenococcus oeni for Malolactic Fermentation and Their Expressions in Saccharomyces cerevisiae

作　　者：李华[1] 刘延琳[1] 蒋思欣[2] 何秀萍[2] 

机构地区：[1]西北农林科技大学葡萄酒学院,杨凌712100 [2]中国科学院微生物研究所,北京100080

出　　处：《农业生物技术学报》2006年第4期606-611,共6页Journal of Agricultural Biotechnology

基　　金：陕西省自然科学基金项目;西北农林科技大学青年骨干支持计划资助。

摘　　要：利用西北农林科技大学葡萄酒学院筛选出的酒酒球菌(Oenococcusoeni)优良菌系SD-2a的包含苹果酸-乳酸酶基因(mleA)和苹果酸通透酶基因(mleP)约2.6kb的基因片段,并以PGK1强启动子和ADH1终止子为调控元件,以酵母-大肠杆菌穿梭质粒YEp352为载体,构建了重组表达质粒pYELmleAP,转化酿酒酵母(Saccharomycescerevisiae)YS58,筛选获得酵母转化子YS58/pYELmleAP。斑点杂交检测表明目的基因mleA转化到受体菌中,SDS-PAGE检测表明获得的转化子表达了苹果酸-乳酸酶的目的蛋白。对酵母转化子培养上清进行HPLC检测,采用t检验差异显著性分析,结果表明mleA基因在受体菌中进行了功能性表达。获得的酵母转化子在添加L-苹果酸的SD/-Ura培养基中培养4d,培养液中的L-苹果酸含量降低,培养液上清中L-苹果酸的剩余含量比空载体转化子极显著降低,苹果酸降低19.33%～19.42%。对照未检出乳酸的生成,供试的转化子L-乳酸生成量为1249～1368mg/L,与对照差异显著。The key enzymes for malolactic fermentation （MLF） are malolactic enzyme and malate permease, coded by mleA gene and mleP gene respectively. The role locus about 2.6 kb containing mleA and mleP genes, coming from an excellent Oenococcus oeni strain of SD-2a screened by College of Enology, NAFU, in China, was used together with PGK 1 promoter and ADH1 terminator were ligated and inserted into YEp352 （yeast-Escherichia coil shuttle plasmid）, which was named pYELmleAP. Yeast transformants were screened on SD/-Ura. Malolactic enzyme gene, one of the target genes, was detected in the yeast transformants by Dot blotting hybridization. The target protein of malolactic enzyme was expressed by SDS-PAGE detecting. After transformants were cultured in media containing L-malate for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. The results indicated that the functional expression of mleA gene was achieved in recombinants S. cerevisiae, turning L-malic acid into L-lactic acid. L-malate contents of the transformants showed extra significant difference with the control ones in t test, while L-lactic contents of the transformants showed significant difference with the control ones. In the culture supernatants adding L-malate, 1249-1368 mg/L L-lactic acids were detected and the comparative drop rates of L-malate were 19.33%-19.42%, while no L-lactic was detected from control transformants.

关 键 词：酒酒球菌 苹果酸-乳酸酶基因 苹果酸通透酶基因 重组表达质粒 表达 酿酒酵母 

分 类 号：S188[农业科学—农业基础科学]