Mechanistic Dissection of the Loading Module of PKS: ACP Domain Engineering Enhances Natamycin Biosynthesis in Streptomyces gilvosporeus

Streptomyces gilvosporeus F607 governs natamycin biosynthesis via a modular polyketide synthase (PKS) system, which includes an atypical loading module (SgnS0) featuring unique functional domains: a carboxylic acid-CoA ligase (CoL), an acyl carrier protein (ACPL1), a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an ACPL2 configuration (CoL-ACPL1-KS^S-AT-ACPL2). Here, we resolve the catalytic logic of this initiation module and leverage its architectural features for the efficient synthesis of natamycin. An sgnS0 knockout strain S. gilvosporeus F607/ΔsgnS0 produces undetectable natamycin, suggesting that sgnS0 is indispensable for natamycin biosynthesis. We employed a combination of site-directed mutagenesis targeting the KS^S and AT domains of SgnS0, demonstrating that the AT domain specifically loads a malonyl group onto the ACP domain, followed by KS^S-mediated decarboxylation to generate acetyl-ACP intermediates. The SgnS0 enzyme presented a transacylation catalytic efficiency (k_cat/K_m = 0.59 ± 0.02 μM^–1·min^–1), while the CoL-deletion mutant SgnS0-AKAA showed a 30.5% reduction in transacylation catalytic efficiency (0.41 ± 0.01 μM^–1·min^–1). This indicates that, nonessential for core catalysis, the CoL domain acts as a structural modulator optimizing catalytic efficiency. Additionally, in vivo mutagenesis and in vitro enzymatic analysis identified both ACPL1 and ACPL2 as essential for biosynthetic function with dual inactivation abolishing natamycin production. Guided by these findings, we engineered the SgnS0 module incorporating tandem ACP architectures in vitro and in S. gilvosporeus F607. Systematic insertion of one or two ACPL2 copies downstream of the native ACPL2 domain of SgnS0 in vitro revealed that a three-ACP construct significantly enhanced the transacylation catalytic efficiency, achieving a 2.64-fold increase in k_cat/K_m. Furthermore, dual ACPL2 insertions occurring in S. gilvosporeus F607 yielded a natamycin titer of 9.5 g L^–1─representing a 160% improvement over the wild-type strain S. gilvosporeus F607. These findings provide a mechanistic basis for the ACP domain function in modular PKSs and highlight tandem ACP engineering as a powerful strategy to boost secondary metabolite production.