<scp>d</scp>‐Galactose‐Esterification of a Fungal Polyketide Catalyzed by a Carnitine Acyltransferase Domain

While sugar-containing natural products are commonly biosynthesized via glycosyltranferases using sugar-UDP as the electrophile, nature has evolved alternative strategies of glyco-modification to expand the diversity of natural products. Hydroxyl groups on sugars can serve as nucleophiles in the release of polyketide products from polyketide synthases. Herein, we demonstrate a highly reducing polyketide synthase (HRPKS) from the biocontrol fungus Trichoderma afroharzianum T22, which is terminated with a carnitine acyltransferase (cAT) domain, catalyzes the biosynthesis of a d-galactose esterified polyketide named as trichogalactin. Structure-guided enzymatic assays showed that the sugar nucleophile in the esterification reaction catalyzed by cAT is α-d-galactose-1-phosphate (Gal-1-P) instead of free d-galactose. The released product, trichogalactin phosphate, is subsequently dephosphorylated by a host alkaline phosphatase to complete the biosynthesis of trichogalactin. The cAT domain is highly specific for Gal-1-P and does not accept α-d-glucose-1-phosphate or α-d-mannose-1-phosphate. Our study expands the inventory of natural products from an agriculturally important fungus and demonstrates the potential of mining cAT-containing HRPKSs to discover new glyco-esterified natural products.