The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites − a reduction in chain length of 14 carbons relative to the 51-membered parental compounds − but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.

Exchange of polyketide synthase (PKS) modules by genetic engineering leads to efficient modification of polyketide stereochemistry.

During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase 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 polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.

The acyl transferase (AT) of module 12 of the stambomycin polyketide synthase (PKS) exhibits unusual substrate tolerance. This promiscuity was exploited to generate multiple new bioactive stambomycin macrolides by extender unit-based mutasynthesis.