Reconfiguring the Nonreducing Polyketide Synthase of <i>Monascus</i> Azaphilone Pigment Biosynthesis for Collaboration with A Highly Reducing Polyketide Synthase

Benzenediol lactones (BDL) and <i>Monascus</i> azaphilone pigments (MonAzP) are different classes of bioactive polyketides produced by distinct pathways. BDL are produced by highly reducing polyketide synthase (hrPKS) - nonreducing PKS (nrPKS) pairs where the starter acyl transferase (SAT) domain of the nrPKS recruits an advanced starter unit provided by the hrPKS. In contrast, the MonAzP nrPKS does not use a hrPKS partner: instead, its SAT domain selects acetyl-CoA as the priming unit. Here, we reconfigured the MonAzP nrPKS to collaborate in a dual-PKS system by replacing its SAT domain with that of a BDL nrPKS. This shifted the product spectrum from benzaldehydes to novel α-pyrones in the chimeric system. Our work highlights that SAT domain engineering may enforce noncognate hrPKS-nrPKS collaborations even for solitary nrPKSs. Nevertheless, the derailment of the native cyclization and product release routines indicates that domain compatibility must be considered when engineering to produce unnatural polyketides.