Glycosylation in natural product metabolism and xenobiotic detoxification often leads to disaccharide-modified metabolites. The chemical synthesis of such glycosides typically separates the glycosylation steps in space and time. The option to perform the two-step glycosylation in one pot, and catalyzed by a single permissive enzyme, is interesting for a facile access to disaccharide-modified products. Here, we reveal the glycosyltransferase GT1 from Bacillus cereus (BcGT1; gene identifier: KT821092) for iterative O-β-glucosylation from uridine 5'-diphosphate (UDP)-glucose to form a β-linked disaccharide of different metabolites, including a C15 hydroxylated detoxification intermediate of the agricultural herbicide cinmethylin (15HCM). We identify thermodynamic and kinetic requirements for the selective formation of the disaccharide compared to the monosaccharide-modified 15HCM. As shown by NMR and high-resolution MS, β-cellobiosyl and β-gentiobiosyl groups are attached to the aglycone's O15 in a 2:1 ratio. Glucosylation reactions on methylumbelliferone and 4-nitrophenol involve reversible glycosyl transfer from and to UDP as well as UDP-glucose hydrolysis, both catalyzed by BcGT1. Collectively, this study delineates the iterative β-d-glucosylation of aglycones by BcGT1 and demonstrates applicability for the programmable one-pot synthesis of disaccharide-modified 15HCM.

Cinmethylin is a well-known benzyl-ether derivative of the natural terpene 1,4-cineole that is used industrially as a pre-emergence herbicide in grass weed control for crop protection. Cinmethylin detoxification in plants has not been reported, but in animals, it prominently involves hydroxylation at the benzylic C15 methyl group. Here, we show enzymatic β-glycosylation of synthetic 15-hydroxy-cinmethylin to prepare a putative phase II detoxification metabolite of the cinmethylin in plants. We examined eight Leloir glycosyltransferases for reactivity with 15-hydroxy cinmethylin and revealed the selective formation of 15-hydroxy cinmethylin β-d-glucoside from uridine 5'-diphosphate (UDP)-glucose by the UGT71E5 from safflower (Carthamus tinctorius). The UGT71E5 showed a specific activity of 431 mU/mg, about 300-fold higher than that of apple (Malus domestica) UGT71A15 that also performed the desired 15-hydroxy cinmethylin mono-glycosylation. Bacterial glycosyltransferases (OleD from Streptomyces antibioticus, 2.9 mU/mg; GT1 from Bacillus cereus, 60 mU/mg) produced mixtures of 15-hydroxy cinmethylin mono- and disaccharide glycosides. Using UDP-glucose recycling with sucrose synthase, 15-hydroxy cinmethylin conversion with UGT71E5 efficiently provided the β-mono-glucoside (≥95% yield; ∼9 mM) suitable for biological studies.