title: Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S-adenosyl-l-methionine.
abstract: 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 DH5α and C. glutamicum ATCC13032. In DH5α, 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 °C; SAM2-C and MetK have similar Kmfor ATP, but quite different Kmfor l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Han
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China. xwang@jiangnan.edu.cn.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, People's Republic of China. xwang@jiangnan.edu.cn.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D008716:Methionine Adenosyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012436:S-Adenosylmethionine / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D003062:Codon; D012436:S-Adenosylmethionine; D008716:Methionine Adenosyltransferase
keywords: 
  l-methionine; Corynebacterium glutamicum; Escherichia coli; SAM2; metK; methionine adenosyltransferase, S-adenosyl-l-methionine