Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S-adenosyl-l-methionine
Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S-adenosyl-l-methionine
复制标题
谷氨酸棒杆菌中甲硫氨酸腺苷转移酶的过表达用于生产 S-腺苷-L-甲硫氨酸
DOI：
10.1002/bab.1425
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发表时间：
2016-09-01
影响因子：
2.8
通讯作者：
Wang, Xiaoyuan
中科院分区：
文献类型：
--
作者：
Han, Guoqiang;Hu, Xiaoqing;Wang, Xiaoyuan
将编码蛋氨酸腺苷转移酶的两个基因（酿酒酵母的SAM2和谷氨棒状杆菌的metK）分别克隆到大肠杆菌与谷氨酰胺间穿梭载体pDXW-8中，并在大肠杆菌DH5和谷氨酰胺ATCC13032中过表达。在DH5中，这两个基因都过表达，它们的蛋白产物显示出蛋氨酸腺苷转移酶的活性。在ATCC13032中，metK过表达，其产物metK具有酶活性，可将l-蛋氨酸转化为s -腺苷基-l-蛋氨酸（SAM）。然而，当SAM2在ATCC13032中过表达时，既没有观察到酶活性，也没有观察到l-蛋氨酸对SAM的转化。反转录PCR和SDS-PAGE分析表明，SAM2在谷氨酸中只被转录而未被翻译。因此，通过密码子优化构建SAM2突变体SAM2- c，并在ATCC13032中过表达；它转录和翻译良好，能将l-蛋氨酸转化为SAM。最后，在大肠杆菌BL21（DE3）中分别过表达SAM2-C和metK，并对其产物SAM2-C和metK进行纯化和表征。两种酶的最佳活性均在pH 8.5和35℃；SAM2-C和MetK对ATP的K-m相似，但对l-蛋氨酸的K-m差异很大。这些结果表明SAM2-C和MetK可能对谷氨酸酵母产生SAM有帮助。(C) 2015国际生物化学与分子生物学联合会
Two genes encoding methionine adenosyltransferase, SAM2 from Saccharomyces cerevisiae and metK from Corynebacterium glutamicum, were individually cloned into pDXW-8, the shuttle vector between Escherichia coli and C. glutamicum, and overexpressed in E. coli DH5 and C. glutamicum ATCC13032. In DH5, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032, metK was overexpressed, its product MetK showed the enzyme activity and could convert l-methionine to S-adenosyl-l-methionine (SAM). However, when SAM2 was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM from l-methionine was observed. Reverse transcription PCR analysis and SDS-PAGE showed that SAM2 was transcribed but not translated in C. glutamicum. Therefore, SAM2-C, a mutant SAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convert l-methionine to SAM. Finally, SAM2-C and metK were individually overexpressed in E. coli BL21(DE3), and their products SAM2-C and MetK were purified and characterized. The optimum activity for both enzymes was found at pH 8.5 and 35 degrees C; SAM2-C and MetK have similar K-m for ATP, but quite different K-m for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM. (C) 2015 International Union of Biochemistry and Molecular Biology, Inc.