酒类酒球菌mleP基因的克隆及其在酿酒酵母中的表达  

Cloning of mleP Gene from Oenococcus oeni and Expression in Saccharomyces cerevisiae

作　　者：蒋思欣[1] 刘延琳[2] 何秀萍[1] 郭雪娜[1] 张博润[1] 

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

出　　处：《微生物学报》2004年第4期465-468,共4页Acta Microbiologica Sinica

摘　　要：苹果酸通透酶具有协助苹果酸 乳酸发酵 (MLF)的重要功能。以酒类酒球菌 (Oenococcusoeni)优良菌系Oenococcus Lee SD 2a的总DNA为模板 ,用PCR方法克隆到苹果酸通透酶基因mleP ,构建了重组质粒pBMmleP。序列分析表明克隆到的基因序列与已报道的序列同源性为 99%。为使目的基因在酿酒酵母中表达 ,以大肠杆菌 酿酒酵母穿梭质粒YEp35 2为载体 ,以PGK1强启动子和ADH1终止子为调控元件 ,构建了重组表达质粒YEpmleP ,并转化酿酒酵母 (Saccharomycescerevisiae)YS5 8。酵母转化子用含有亮氨酸、组氨酸和色氨酸的YNB平板筛选鉴定。获得的转化子在添加了L 苹果酸 (5g L)的培养基中培养 4d ;取培养液上清用HPLC检测 ,结果显示重组转化子YSP的培养液中L 苹果酸剩余含量均低于空载体转化子YS35 2 。The malate permease, encoded by mleP gene, is important for assisting malolactic fermentation (MLF). In this paper, mleP gene was amplified by PCR from Oenococcus Lee SD 2a, an excellent Oenococcus oeni strain screened in China. The amplified DNA fragment, about 0 95kb, was inserted into pBluscript M13 to construct recombinant plasmid pBMmleP. Sequence analysis showed that it had 99% identity with the reported mleP gene. In order to be expressed properly in Saccharomyces cerevisiae , the mleP gene from pBMmleP, PGK1 promoter from plasmid pVC727 and ADH 1 terminator from plasmid pEA 1 were ligated and inserted into YEp352(yeast Escherichia coli shuttle vector). The resulted plasmid was named YEpmleP. Yeast transformants were screened on YNB medium containing leucine, histidine and tryptophan. After transformants were cultured in media containing L malate (5g/L) for 4d, the culture supernatant was collected and L malate content was detected by HPLC. The results showed that L malate residual content in culture supernatant of recombinant YSP was lower than that of control transformant YS352. Hence, the transporting ability of L malate in S. cerevisiae recombinant was improved.

关 键 词：酒类酒球菌 基因 克隆 酿酒酵母 表达 苹果酸通透酶基因 

分 类 号：TS261[轻工技术与工程—发酵工程]