| Summary: | Streptomycetes are soil-dwelling multicellular microorganisms famous for their unprecedented ability to synthesize numerous bioactive natural products (NPs). In addition to their rich arsenal of secondary metabolites, <i>Streptomyces</i> are characterized by complex morphological differentiation. Mostly, industrial production of NPs is done by submerged fermentation, where streptomycetes grow as a vegetative mycelium forming pellets. Often, suboptimal growth peculiarities are the major bottleneck for industrial exploitation. In this work, we employed genetic engineering approaches to improve the production of moenomycins (Mm) in <i>Streptomyces ghanaensis</i>, the only known natural direct inhibitors of bacterial peptidoglycan glycosyltransferses. We showed that in vivo elimination of binding sites for the pleiotropic regulator AdpA in the <i>oriC</i> region strongly influences growth and positively correlates with Mm accumulation. Additionally, a marker- and “scar”-less deletion of <i>moeH5</i>, encoding an amidotransferase from the Mm gene cluster, significantly narrows down the Mm production spectrum. Strikingly, antibiotic titers were strongly enhanced by the elimination of the pleiotropic regulatory gene <i>wblA</i>, involved in the late steps of morphogenesis. Altogether, we generated Mm overproducers with optimized growth parameters, which are useful for further genome engineering and chemoenzymatic generation of novel Mm derivatives. Analogously, such a scheme can be applied to other <i>Streptomyces</i> spp.
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