<jats:title>ABSTRACT</jats:title> <jats:p> The epiphyte <jats:italic>Pseudomonas syringae</jats:italic> pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen <jats:italic>Pseudomonas syringae</jats:italic> pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of <jats:italic>P. syringae</jats:italic> pv. glycinea. The MeArg biosynthesis gene cluster codes for the <jats:italic>S</jats:italic> -adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into <jats:italic>Escherichia coli</jats:italic> resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in <jats:italic>E. coli</jats:italic> as a His-tagged protein and functionally characterized ( <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> , 7 mM; <jats:italic>k</jats:italic> <jats:sub>cat</jats:sub> , 85 min <jats:sup>−1</jats:sup> ). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.