Coordinated Action of NRPS, Baeyer-Villiger Monooxygenase, and Methyltransferase Ensures the Economical Biosynthesis of Bohemamines in Streptomyces sp. CB02009

<p> Bohemamines (BHMs) are bacterial alkaloids containing a pyrrolizidine core with two unprecedented methyl groups. Herein we report the activation of BHMs biosynthesis in <i>Streptomyces </i>sp. CB02009 using a ribosome engineering approach. Identification and characterization of the <i>bhm</i> gene cluster reveals a coordinated action of nonribosomal peptide synthetase BhmJ, Baeyer-villiger monooxygenase BhmK and methyltransferase BhmG for BHMs biosynthesis. BhmG is responsible for the C-methylation on C-7, while the C-9 methyl group is from a non-proteinogenic amino acid (2<i>S</i>,5<i>S</i>)-5-methylproline, required for BHMs production in three model <i>Streptomyces </i>hosts. Our study shed light on the intricate interaction of BhmJ/BhmK/BhmG for the economical biosynthesis of BHMs in their native producer, and also unraveled that BhmJ and BhmK are competent biocatalysts in <i>S</i><i>treptomyce </i><i>albus</i>.</p>