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.