Overexpression of methionine adenosyltransferase inCorynebacterium glutamicumfor production ofS‐adenosyl‐ l ‐methionine

Two genes encoding methionine adenosyltransferase,SAM2fromSaccharomyces cerevisiaeandmetKfromCorynebacterium glutamicum, were individually cloned into pDXW‐8, the shuttle vector betweenEscherichia coliandC. glutamicum, and overexpressed inE. coliDH5α andC. glutamicumATCC13032. In DH5α, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032,metKwas overexpressed, its product MetK showed the enzyme activity and could convertl‐methionine toS‐adenosyl‐l‐methionine (SAM). However, whenSAM2was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM froml‐methionine was observed. Reverse transcription PCR analysis and SDS‐PAGE showed thatSAM2was transcribed but not translated inC. glutamicum. Therefore,SAM2‐C, a mutantSAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convertl‐methionine to SAM. Finally,SAM2‐CandmetKwere individually overexpressed inE. coliBL21(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 similarKmfor ATP, but quite differentKmforl‐methionine. These results suggest that SAM2‐C and MetK can be useful for developingC. glutamicumto produce SAM.