<jats:title>ABSTRACT</jats:title> <jats:p> The peptidyl nucleoside arginomycin is active against Gram-positive bacteria and fungi but displays much lower toxicity to mice than its analog blasticidin S. It features a rare amino acid, β-methylarginine, which is attached to the deoxyhexose moiety via a 4′-aminoacyl bond. We here report cloning of the complete biosynthetic gene cluster for arginomycin from <jats:named-content content-type="genus-species">Streptomyces arginensis</jats:named-content> NRRL 15941. Among the 14 putative essential open reading frames, <jats:italic>argM</jats:italic> , encoding an aspartate aminotransferase (AAT), and adjacent <jats:italic>argN</jats:italic> , encoding an <jats:italic>S</jats:italic> -adenosyl methionine (SAM)-dependent methyltransferase, are coupled to catalyze arginine and yield β-methylarginine in <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> . Purified ArgM can transfer the α-amino group of <jats:sc>l</jats:sc> -arginine to α-ketoglutaric acid to give glutamate and thereby converts <jats:sc>l</jats:sc> -arginine to 5-guanidino-2-oxopentanoic acid, which is methylated at the C-3 position by ArgN to form 5-guanidino-3-methyl-2-oxopentanoic acid. Iteratively, ArgM specifically catalyzes transamination from the donor <jats:sc>l</jats:sc> -aspartate to the resulting 5-guanidino-3-methyl-2-oxopentanoic acid, generating β-methylarginine. The complete and concise biosynthetic pathway for the rare and bioactive amino acid revealed by this study may pave the way for the production of β-methylarginine either by enzymatic conversion or by engineered living cells.