During biosynthesis by multi-modular trans-AT polyketide synthases (PKSs), polyketide structural space can be expanded by conversion of initially-formed electrophilic beta-ketones into beta-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase (HMGS) cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT PKS. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for beta-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation, identifies a metabolite bearing a second beta-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin beta-branching programming. Furthermore, imperfections in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.

Xcalibur, 2.1