Molecular basis for depsipeptide HDAC inhibitor combinatorial biosynthesis

Polyketides and nonribosomal peptides are important natural product classes with wide-ranging medical and agricultural applications. The analogous enzymatic logic employed by bacterial modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) enables the assembly of hybrid products. One important group of polyketide-nonribosomal peptide hybrids is exemplified by the HDAC-targeting drug romidepsin. This group is assembled by combinatorial biosynthesis involving fusion of a conserved Zn²⁺-binding pharmacophore to a variable peptide-based cap. Here, we use gene proximity searching to identify the FR-901375 biosynthetic gene cluster in Pseudomonas chlororaphis subsp. piscium DSM 21509. Comparison of the PKS-NRPS encoded by this gene cluster with those assembling related depsipeptide HDAC inhibitors suggests an unusual subunit docking modality enables interaction between the conserved pharmacophore and variable cap biosynthetic machineries. This hypothesis is validated using crosstalk assays, mutagenesis, AlphaFold predictions, and carbene footprinting, providing insight into the evolution of mechanisms for hybrid polyketide-nonribosomal peptide combinatorial biosynthesis.