Investigation of Domain–Domain Interaction between Acyl Carrier Protein and Thioesterase in Modular Polyketide Synthases

Modular polyketide synthases are multidomain assembly line enzymes that biosynthesize complex polyketide natural products. The thioesterase (TE) domain plays a critical role in product release by catalyzing the hydrolysis or lactonization of the polyketide intermediate tethered to the acyl carrier protein (ACP). Here, we report that, contrary to the prevailing assumption that TE recognizes only the polyketide moiety, efficient product release requires a specific interaction between the TE domain and the ACP domain. In vitro enzyme assays combined with molecular dynamics simulations revealed a potential mechanism of the ACP–TE interaction, in which loop I of ACP exhibits strong interaction with the TE in mediomycin biosynthesis. Comparative analysis of TE single-domain and ACP–TE didomain insertions in various polyketide biosynthetic pathways showed that ACP–TE didomain insertion generally results in higher product titers in the engineered pathways. Our results illustrate the crucial contribution of ACP–TE interaction for proper product release in polyketide biosynthesis and will guide rational engineering of modular polyketide synthases.