<jats:title>Abstract</jats:title><jats:p>The nine‐membered indolactam antibiotics belong to a small group of antibiotics showing broad biological activities. However, the in vivo genetic engineering of compounds of this type has not been performed. Here we report the identification of a single gene cluster responsible for the biosynthesis of methylpendolmycin and pendolmycin, two members of this family of antibiotics, from the deep sea bacterium <jats:italic>Marinactinospora thermotolerans</jats:italic> SCSIO 00652. Bioinformatics analysis and gene inactivation, coupled with metabolite characterization, reveal that MpnB, a nonribosomal peptide synthetase, MpnC, a cytochrome P450, and MpnD, a prenyltransferase, are sufficient to catalyze the biosynthesis of the two antibiotics from <jats:sc>L</jats:sc>‐Ile (or <jats:sc>L</jats:sc>‐Val), <jats:sc>L</jats:sc>‐Trp, and methionine. MpnD is the first identified enzyme that transfers a C5 prenyl unit in a reverse manner to the C‐7 position of a Trp‐derived natural product.