Cross‐Module Enoylreduction in the Azalomycin F Polyketide Synthase

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end‐product, has become a cornerstone of knowledge‐based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross‐modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly‐line catalysis. A switchable enoylreductase (ER) domain borrowed by neighboring module in macrolide antibiotic Azlomycin F biosynthesis shows modular polyketide synthase (PKS)’s versatility that was previously unknown. This internal ER domain not only serves as an unusual switch in an iterative module for enoylreduction but also functions surprisingly in a cross‐module mode during polyketide chain elongation. This discovery further expands our understanding of the wonderful nature of PKS.