Doping In Vivo Alkylation in <i>E. coli</i> by Introducing the Direct Sulfurylation Pathway of <i>S. cerevisiae</i>

Methylation and alkylation are important techniques used for the synthesis and derivatisation of small molecules and natural products. Application of S-adenosylmethionine (SAM)-dependent methyltransferases (MTs) in biotechnological hosts such as Escherichia coli lowers the environmental impact of alkylation compared to chemical synthesis and facilitates regio- and chemoselective alkyl chain transfer. Here, we address the limiting factor for SAM synthesis, methionine supply, to accelerate in vivo methylation activity. Introduction of the direct sulfurylation pathway, consisting of O-acetylhomoserine sulfhydrolase (ScOAHS) and O-acetyltransferase (ScMET2), from S. cerevisiae into E. coli and supplementation with methanethiol or the corresponding disulfide improves atom-economic methylation activity in three different MT reactions. Up to 17-fold increase of conversion compared to the sole expression of the MT and incorporation of up to 79 % of the thiol compound added were achieved. Promiscuity of ScOAHS allowed in vivo production of methionine analogues from organic thiols. Further co-overproduction of a methionine adenosyltransferase yielded SAM analogues which were further transferred by MTs onto different substrates. For methylation of non-physiological substrates, conversion rates up to 73 % were achieved, with an isolated yield of 41 % for N-methyl-2,5-aminonitrophenol. The here described technique enables E. coli to become a biotechnological host for improved methylation and selective alkylation reactions.