Cross-Talking Between Two Fungal Type I Iterative Polyketide Synthases

The diverse resorcylic acid lactone (RAL) family of fungal polyketides has been shown to exhibit a broad spectrum of biological activities, such as growth stimulation, anticancer and treatment of neurodegenerative diseases. The biosynthetic gene cluster of hypothemycin, a nanomolar inhibitor towards a subset of protein kinases, has been identified recently. Similar to other members in RALs, it is also biosynthesized by two megasynthases, Hpm8 (HR-PKS) and Hpm3 (NR-PKS), functioning collaboratively. The entire enzymatic activities of these two megasynthases were reconstituted in vitro to produce the intermediate trans-7',8'-dehydrozearalenol (DHZ), with both of them expressed heterologously from Saccharomyces cerevisiae. The in vitro isotope feeding experiments showed that Hpm8 provides a hexaketide intermediate, which is transferred to Hpm3 for another three rounds of extension in polyketide backbone. Interestingly, Hpm3 (S121A) mutant showed no communication with Hpm8 demonstrated that the N-terminal domain of Hpm3 may function as an ACP-acyl: ACP transferase to initiate its biosynthesis. No cross-talking between Hpm8 and fully functional NR-PKSs from biosynthetic pathways of other RALs, zearalenone or radicicol, was observed. This result implied that the specific protein-protein recognition and interaction were further required for successful transferring of intermediate between HR- and NR-PKSs. Additionally, the results from in vitro assays with mixture of Hpm8 and different hybrids of NR-PKSs further demonstrated that the N-terminal domain of NRPKSs should be responsible for protein-protein recognition and intermediate transferring in the biosynthesis of RALs.