高山被孢霉亚甲基四氢叶酸脱氢酶的克隆、表达和功能鉴定  

Cloning,Expression and Function Analysis of Methylenetetrahydrofolate Dehydrogenase from Mortierella alpina

作　　者：王鸿超[1] 张陈[1] 陈殿宁 乔菊园 陈海琴[1] 顾震南[1] 张灏[1] 陈卫[1] 陈永泉[1] 

机构地区：[1]江南大学食品学院,无锡214122

出　　处：《中国生物工程杂志》2016年第11期23-29,共7页China Biotechnology

基　　金：国家自然科学基金青年科学基金资助项目(31400038)

摘　　要：叶酸代谢途径中的亚甲基四氢叶酸脱氢酶(MTHFD)可将5,10-亚甲基四氢叶酸氧化为5,10-甲炔基四氢叶酸,此过程会生成NADH或NADPH。对高山被孢霉中的MTHFD基因进行克隆、表达和功能鉴定,可进一步阐明脂质合成所需还原力NADPH的来源。首先对MTHFD序列进行分析,并以p ET28a(+)质粒为载体构建了MTHFD的表达载体,然后转化至大肠杆菌BL21中进行诱导表达。进一步利用Ni金属螯合层析纯化目的蛋白,采用比色法分析酶反应产物,表明纯化蛋白质具有MTHFD活性。高山被孢霉MTHFD对NAD^+和NADP^+均具有催化能力,但更偏好于将NADP^+转化为NADPH。最后对高山被孢霉进行发酵培养,发现MTHFD的转录水平在脂质开始积累后发生了明显的上调,表明MTHFD在高山被孢霉脂质合成过程中发挥重要作用,很可能是脂质合成所需NADPH的关键来源。这为对高山被孢霉进行分子改造,使之成为高产各种多不饱和脂肪酸的细胞工程提供了理论依据。In folate metabolism, 5, 10-methylenetetrahydrofolate is oxidized to 5, 10-methenyltetrahydrofolate by the methylenetetrahydrofolate dehydrogenase（ MTHFD） with the production of NADH or NADPH. To clarify the alternative NADPH sources in fatty acid synthesis,the heterologous expression vector for MTHFD was constructed under the skeleton of plasmid p ET28a（ ＋） and expressed in BL21 gold strain of E.coli. The enzymatic activity was investigated by VIS-UV spectroscopy. The M. alpina MTHFD can both catalyze NAD^＋and NADP^＋,and is preferred to convert NADP^＋to NADPH. The transcript level of MTHFD is upregulated by nitrogen exhaustion,when M. alpina starts to accumulate lipids. This indicate the relationship between MTHFD and lipid metabolism is thus of major importance,and MTHFD may be an alternative NADPH source in fatty acid synthesis. The range of target candidates for genetic manipulation in M. alpina for obtaining strains with increased amounts of lipids were extended.

关 键 词：功能鉴定 亚甲基四氢叶酸脱氢酶 NADPH 脂质合成 高山被孢霉 

分 类 号：Q813[生物学—生物工程]