Sequence mining yields 18 phloretin C‐glycosyltransferases from plants for the efficient biocatalytic synthesis of nothofagin and phloretin‐di‐C‐glycoside

C-glycosyltransferases (C-GTs) offer selective and efficient synthesis of natural product C-glycosides under mild reaction conditions. In contrast, the chemical synthesis of these C-glycosides is challenging and environmentally harmful. The rare occurrence of C-glycosylated compounds in Nature, despite their stability, suggests that their biosynthetic enzymes, C-GTs, might be scarce. Indeed, the number of characterized C-GTs is remarkably lower than O-GTs. Therefore, discovery efforts are crucial for expanding our knowledge of these enzymes and their efficient application in biocatalytic processes. This study aimed to identify new C-GTs based on their primary sequence. 18 new C-GTs were discovered, 10 of which yielded full conversion of phloretin to its glucosides. Phloretin is a dihydrochalcone natural product, with its mono-C-glucoside, nothofagin, having various health-promoting effects. Several of these enzymes enabled highly selective production of either nothofagin (UGT708A60 and UGT708F2) or phloretin-di-C-glycoside (UGT708D9 and UGT708B8). Molecular docking simulations, based on structural models of selected enzymes, showed productive binding modes for the best phloretin C-GTs, UGT708F2 and UGT708A60. Moreover, we characterized UGT708A60 as a highly efficient phloretin mono-C glycosyltransferase (kcat = 2.97 s‒1, KM = 0.1 μM) active in non-buffered, dilute sodium hydroxide (0.1–1 mM). We further investigated UGT708A60 as an efficient biocatalyst for the bioproduction of nothofagin.