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 K for ATP, but quite different K for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.

Ferulic acid production by metabolically engineered Escherichia coli

“…Additionally, the caffeic acid O -methyltransferase COMT consumes S -adenosyl- L -methionine (SAM), which is a ubiquitous intracellular methyl donor involved in a great number of reactions (Han et al 2016 ). Increasing the intracellular SAM supply is considered a generally useful approach for improving the production of natural products (Wang et al 2007 ).…”

Ferulic Acid production by metabolically engineered Escherichia coli

“…Meanwhile, the reaction catalysis by the caffeic acid O-methyltransferase, COMT, consumes S-Adenosyl-lmethionine (SAM), which is a ubiquitous intracellular methyl donor involved in a great number of reactions (Han et al, 2016). Increasing intracellular SAM level is proposed to be a generally useful tool for improving the production of natural products (Wang et al, 2007).…”

“…Increasing intracellular SAM level is proposed to be a generally useful tool for improving the production of natural products (Wang et al, 2007). Methionine adenosyltransferase /Sadenosylmethionine synthetase catalyzes the formation of SAM from L-methionine and ATP, and it has been found in E.coli, baker's yeast, Mycobacterium smegmatis, rat liver, bovine brain, and Saccharomyces cerevisiae (Han et al, 2016). Methionine adenosyltransferase is also worth trying in FA heterologous biosynthetic strain.…”

Knockout of the DAS gene increases S-adenosylmethionine production in Komagataella phaffii

“…SAM production, through the optimization of cultivation or gene co-expression [4], has been reported in many recombinant microbial species, such as Saccharomyces cerevisiae [5,6], Pichia pastoris (a.k.a. Komagataella phaffii) [7], Candida utilis [8], Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum [9] and Escherichia coli [10]. In recent years, Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, has been reported to be a promising host for high levels of SAM production [11].…”