Promiscuity Characteristics of Versatile Plant Glycosyltransferases for Natural Product Glycodiversification

Glycodiversification can optimize the properties of pharmaceutical compounds, and versatile glycosyltransferases (GTs) are the key enzymatic toolkits to achieve this goal. Plant GTs in the GT1 family (GT1-pGTs) have attracted much attention due to their promising substrate promiscuity, but previous investigations on GT1-pGTs were mainly conducted sporadically and without systematic phylogenetic comparisons. In this study, we exemplified the phylogeny-guided characterization of highly promiscuous GT1-pGTs from the contemporary surge of genomic information. All the available GT1-pGT sequences in the database were analyzed to explore the relationships between the substrate promiscuity and the phylogeny of GT1-pGTs. This systematic phylogenetic analysis directed us to choose 29 anonymous GT sequences from different evolutionary branches to probe their substrate promiscuity toward 10 aromatic compounds differing in chemical scaffolds. We found that promiscuous plant GTs (PPGTs) active toward ≥3 substrates were widely distributed in different clades but particularly enriched in the one containing the known promiscuous enzyme GuGT10. Ten highly promiscuous plant GTs were found to tolerate a wide spectrum (≥8) of substrates and inclusively catalyze the formation of O-, N-, and S-glycosidic bonds. The promiscuity of these 10 PPGTs was further tested using 15 sugar donors. Finally, we characterized FiGT2 that simultaneously exhibited pronounced promiscuity in terms of both the sugar acceptor and sugar donor. All in all, this study paves the way to unearth many more PPGTs and thus strengthen the enzymatic toolkit for the sustainable production of valuable glycosides through a synthetic biological approach.