A hybrid assembly mechanism employs nonreducing polyketide synthase-like logic for partially reduced polyketide formation.

Fungal iterative type I polyketide synthases (iPKSs) are commonly classified into nonreducing (NR-), partially reducing (PR-), and highly reducing (HR-) polyketide synthases based on their assembly mechanisms and domain structures. These iPKSs have been considered functionally and evolutionarily distinct, characterized by clear boundaries. However, emerging genomic analyses suggest that the diversity of iPKSs in fungi is far from fully understood. Here, we describe the discovery and characterization of PbPKS1 from a marine-derived fungus Penicillium brocae HDN12-143, which exhibits an atypical domain organization arranged as KR-KS-AT-PT-ACP1-ACP2-CMeT-TE. Heterologous expression of PbPKS1 resulted in the production of two monohydroxybenzoic acids and two pyrones. In vivo and in vitro characterizations demonstrated that PbPKS1 has the capability to synthesize Cα-methylated partially reducing polyketides, yet involved a NR-PKS-like assembly mechanism, featuring a product template (PT) domain for aldol cyclization and a C-terminal thioesterase (TE) domain for product release. Phylogenetic analysis suggests that PbPKS1 belongs to a non-canonical PR-PKS (nPR-PKS) family, which is a minor grouping across the fungal kingdom, and possibly evolved from an NR-PKS through gene recombination. The discovery of nPR-PKS not only expands the diversity of iPKSs but also provides new insights into the evolutionary development of fungal iPKSs. The online version contains supplementary material available at 10.1007/s42995-025-00342-5.