Condensation-independent intramodular translocation mechanism of the trans-AT polyketide synthase assembly line

Decarboxylative condensation drives chain elongation and translocation of polyketides and fatty acids. However, the mechanism by which nonelongating modules in trans-acyltransferase polyketide synthases (trans-AT PKSs) enable intramodular polyketide chain translocation without decarboxylation remains poorly understood. Here we elucidate a condensation-independent intramodular translocation mechanism in which KS⁰ within the nonelongating module operates as a transacylase, directly translocating the polyketide chain to its downstream cognate acyl carrier protein (ACP). Notably, the inherent demalonylation activity of trans-AT_HtmA7 facilitates efficient ACP recycling ensuring intramodular translocation. Structural modeling and site-directed mutagenesis studies uncover a conserved KS⁰-ACP binding mode that underpins intramodular translocation across diverse nonelongating modules. Additionally, the strict discrimination of polyketide intermediate by the nonelongating module highlights its critical role in maintaining biosynthetic precision and efficiency. These findings provide mechanistic insights into evolutionary adaptation and sophisticated crosstalk between catalytic domains within trans-AT PKS, illuminating how metabolic flux and fidelity are maintained and opening avenues for polyketide engineering.