Oenococcusoeni苹果酸-乳酸酶基因克隆及其在酿酒酵母中的表达  

Cloning of Malo-lactic Enzyme Gene from Oenococcus oeniand Expression in Saccharomyces cerevisiae

作　　者：刘延琳[1] 蒋思欣[2] 何秀萍[2] 李华[1] 张博润[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：《中国生物工程杂志》2005年第12期50-55,共6页China Biotechnology

基　　金：陕西省自然科学基金;西北农林科技大学青年学术骨干支持计划资助项目

摘　　要：苹果酸-乳酸酶是苹果酸-乳酸发酵过程中负责苹果酸转化为乳酸的功能酶。在进行酒酒球菌SD2a的苹果酸-乳酸酶基因(mleA)克隆测序基础上,以PGK1强启动子和ADH1终止子为调控元件,以大肠杆菌-酵母菌穿梭质粒YEp352为载体,构建了重组表达质粒并转化酿酒酵母YS58。酵母转化子用SD/Ura平板筛选鉴定。斑点杂交检测表明目的基因mleA转化到受体菌中,SDSPAGE检测表明获得的转化子表达了约60kDa的目标蛋白。获得的转化子在添加了L苹果酸的培养基中培养4d;取培养液上清用HPLC检测L苹果酸及L乳酸含量,采用t检验进行差异显著性分析,结果表明mleA基因进行了功能性的表达,将L苹果酸转化成L乳酸,L苹果酸和L乳酸含量分别与对照差异极显著和显著,苹果酸的相对降低率平均为20.95%。在有选择压力条件下,重组质粒相对稳定,而在无选择压力条件下,传代培养10d后大约有65%的重组质粒丢失。Malo-lactic enzyme is the function enzyme to turn L-malic acid into L-lactic acid during malolactic fermentation （MLF）. mleA gene encoding for Malo-lactic enzyme from pLmleA, PGK1 promoter from plasmid pVC727-6 and ADH1 terminator from plasmid pEA-1 were ligated and inserted into YEp352, resulted the expression plasmid. Yeast transformants were screened on SD/-Ura （YNB medium containing leucine, histidine and tryptophan）. The target gene was detected in the yeast transformants by dot blotting hybridization. The target protein was expressed by SDS-PAGE detecting. After transformants were cultured in media containing L-malate for 4d, the culture supernatant was collected and L-malate and L-lactic acid content were detected by HPLC. The result indicated that functional expression of mleA gene was achieved in recombinant S. cerevisiae, turning L-malic acid into L-lactic acid. L-malate contents of the transformants show extra significant difference with the control ones in t test, while L-lactic contents of the transformants show significant difference with the control ones. Average amount of 106d-mg/L L-lactic acids were detected and the comparative average drop rates of L-malate were 20.95%, while no L-lactic was detected from control transformants. The yeast transformants containing the foreign gene of mleA were stable relatively under selective pressure, but about 65% of the recombined plasmid were lost in 10 days subculture.

关 键 词：酒酒球菌 苹果酸-乳酸酶基因 转化表达 

分 类 号：Q785[生物学—分子生物学] TQ921.7[轻工技术与工程—发酵工程]