Engineering a Fungal Non-Reducing Polyketide Synthase with an Apparently Inactive Product-Template Domain Reveals Insights into the Catalytic Reprogramming

Fungal iterative non-reducing polyketide synthases (NR-PKS) contain a unique product template (PT) domain for aromatic cyclization. Among them, some NR-PKSs, such as the sorbicillin NR-PKS (SorB), have an apparently inactive PT. It is unknown what role such PT plays in NR-PKS programming. In this study, the PT domain of SorB was first dissected and engineered. Removal of the PT domain from SorB did not change the product profile, but the yield decreased. Meanwhile, a significantly lower transcriptional level of the ketoacyl synthase (KS) domain was observed in the knockout mutant (UvSorB∆PT). Phylogenetic tree analysis and multiple sequence alignments revealed this PT belongs to group I (C2–C7, monocyclic ring), and mutations were found at catalytic dyad sites when compared with functional fungal PTs. However, mutating these residues back to the conserved ones did not give rise to products corresponding to a functional PT, but rendered the NR-PKS unproductive. Likewise, removal of the C-methyltransferase (CMT) domain from SorB destroyed the polyketide production. Furthermore, in an attempt to alter the methylation pattern, mutations of the key substrate-binding sites of the CMT domain were made. Site-directed mutations of the C-MT led to cessation of the polyketide production. This reveals CMT is vulnerable to engineering in a collaborating NR-PKS (SorB). These results provide additional insights for catalytic reprogramming in fungal NR-PKS.