Domain Swapping Reveals Direction-Dependent Compatibility of Fungal Polyketide Synthase–Nonribosomal Peptide Synthetase Off-Loading Domains

Abstract Two fungal natural products, hirsutellone and didymellamide, are proposed to be biosynthesized from closely related β-ketoamide intermediates despite producing structurally distinct cyclic products. This unusual biosynthetic relationship raises the question of whether the corresponding polyketide synthase–nonribosomal peptide synthetase (PKS–NRPS) off-loading domains are functionally interchangeable. Here, we identified and characterized HirA, a PKS–NRPS involved in hirsutellone biosynthesis, and used it together with AsolS, a PKS–NRPS involved in didymellamide biosynthesis, as a model system to investigate the compatibility of fungal PKS–NRPS off-loading domains. Heterologous expression in Aspergillus oryzae established that HirA produces a β-ketoamide intermediate possessing a nonaketide polyketide chain closely related to that generated by AsolS. Reciprocal domain swapping revealed asymmetric compatibility: artificial fusion of the HirA-derived reductase domain to the AsolS assembly line supported productive processing of the AsolS-derived intermediate, whereas reciprocal fusion of the Dieckmann cyclase domain yielded the C18-derived product at a low level but did not produce the expected tetramic acid derivative. These results demonstrate that off-loading activity can be retained following domain swapping between distinct fungal PKS–NRPS systems, but the extent of productive processing is strongly direction-dependent. Taken together, our findings identify direction-dependent compatibility and fusion-site dependence as important constraints for engineering fungal PKS–NRPS off-loading domains.