Evolutionary Reprogramming of Acyltransferase Domains in Polyene Macrolide Pathways

The evolution of type I polyketide synthase (T1PKS) assembly lines remains poorly understood. Through systematic mining of polyene biosynthetic gene clusters, we identified a novel eurocidin biosynthetic pathway capable of producing identical compounds with divergent loading module architectures, thereby capturing an evolutionary transitional state. Biochemical analysis revealed unprecedented functional reprogramming of acyltransferase (AT) domains, shifting substrate specificity from extender units (malonyl-CoA) to starter units (acyl-CoA). This paradigm shift enables direct initiation of polyketide chain assembly via AT-mediated loading of starter units, thereby elucidating the origin of extant AT-initiated assembly lines and establishing AT functional plasticity as a novel mechanism for polyketide structural diversification. Parallel evolution of ketosynthase (KS) domains through KSS→KSQ mutations further diversified initiation strategies. Applying this evolutionary insight, we engineered the candicidin pathway by replacing its native aromatic-starting bimodule with a starter-selective monomodule from eurocidin, generating aliphatic-starting analogs. This demonstrates that evolution-inspired AT reprogramming provides a rational framework for modifying polyketide starter units, expanding structural diversity, and enhancing therapeutic potential.