Catalytic Versatility of FnsA: Substrate-dependent Activity of KR and DH Domains in Naringenin Biosynthesis

FnsA, a fungal nonribosomal peptide-polyketide synthase hybrid (NRPS-PKS), catalyzes the conversion of p-hydroxybenzoic acid (p-HBA) or p-coumaric acid (p-CA) into naringenin. The ketoreductase (KR) and dehydratase (DH) within FnsAPKS are pivotal in this process. Here, we demonstrate that FnsAKR and FnsADH exhibit distinct functional states (active or inactive) during the naringenin biosynthesis. Through point mutation and precursor feeding experiments in Saccharomyces cerevisiae, we show that both domains are indispensable when p-HBA is utilized but remain nonfunctional with p-CA. Phylogenetic analysis reveals that FnsAKR and FnsADH form independent clades, highlighting their unique evolutionary trajectories. Remarkably, mutations in these domains modestly enhance the naringenin yield, whereas their deletion reduces production. By augmenting malonyl-CoA availability in S. cerevisiae, fed-batch fermentation achieved a naringenin titer of 85.7 mg/L. This study unveils a novel mechanism of substrate-responsive domain activation, expanding our understanding of catalytic plasticity in fungal PKS systems.