In Vivo Mutagenesis of a Ketosynthase Domain Uncovers Productivity and Specificity Control in Modular Polyketide Synthases

Ketosynthase domains govern chain transfer and substrate selectivity in modular polyketide synthases (PKS), yet their functional tunability in native contexts remains poorly understood. We performed phylogenetically guided mutagenesis of the KS5 domain from the Streptomyces cinnamonensis monensin PKS and evaluated 72 variants in vivo across wild-type and reductive-loop-null backgrounds. This revealed discrete active-site motifs that control productivity, redox-state specificity, and extender-unit selection, functions traditionally ascribed to other PKS domains. AlphaFold3 structural mapping linked these motifs to substrate-tunnel and catalytic-core features, providing a mechanistic basis for the observed phenotypes. Our findings demonstrate that KS domains can be rationally re-tuned to overcome productivity bottlenecks and alter specificity in intact PKSs, offering a route to improved yields and expanded chemical diversity in engineered polyketides.