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

Abstract

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 (k cat = 2.97 s -1 , K M = 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.

Genome-wide identification of UDP-glycosyltransferases in the tea plant (Camellia sinensis) and their biochemical and physiological functions

“…Likely, an uncharacterised C -glycosyltransferase and sulfotransferase in C. sinensis play a role in the production of the flavone C -glycoside Chafuroside. Although UGT708 enzymes are involved in the production of C -glucosides in many other plants ( Putkaradze et al., 2021 ; Putkaradze et al., 2023 ), members of other UGT classes are probably responsible for their formation in the tea plant, because UGT708 genes have not been identified in the tea plant genome ( Figure 3 ). Flavonoid C -glycosyltransferase, such as those of the flavone apigenin, have already been identified in other plants as well as sulfotransferases that catalyse the addition of a sulphate to position 7 of the flavonoid skeleton ( Hashiguchi et al., 2014 ; Sasaki et al., 2015 ; He et al., 2019 ; Mashima et al., 2019 ).…”