Functional study and sequence variation of the SrUGT85C2 genes in Stevia rebaudiana

Stevia rebaudiana Bertoni is recognized as the third major source of natural sweetness due to its leaves being rich in high-sweetness, low-calorie steviol glycosides (SGs). A crucial enzyme in the metabolic glycosylation grid of SGs, uridine diphosphate glycosyltransferase SrUGT85C2 catalyzes the glycosylation of the C13-hydroxyl of the steviol, creating a β-D-glucoside link and 13-steviol monoside. However, the key residues of the SrUGT85C2 enzyme and how SrUGT85C2 affects the accumulation of SGs in S. rebaudiana remain unclear. In this study, cloning and functional analysis of SrUGT85C2 gene sequences were performed in 10 different S. rebaudiana genotypes with divergent SG compositions. After sequences analysis, eleven variants of this gene were identified. The mutants N05-5 and N02-6, for which no catalytic function was detected, caused changes in substrate binding and pocket conformation due to single amino acid substitutions, thereby hindering the approach of the reaction group. Molecular docking analysis suggested that mutants N02-1, N02-5 and 023 − 3 increase the efficiency of steviol conversion, potentially by forming additional hydrogen bonds. Because steviol-19-O-glucoside (19-SMG) and UDP-glucose (UDPG) overlap spatially, mutants 11-14-5 and N01-1 prevent C19-glycosylation. Molecular docking-based site-directed mutagenesis verified that steviol and 19-SMG catalytic activity were enhanced by the mutant proteins K25H and E418G to 164.1% and 203.6% of the SrUGT85C2 protein, respectively. In addition, the expression analysis of SrUGT85C2 in each genotypes showed that the expression levels of the gene varied significantly among genotypes, with high expression observed in GP. K25 and E418 were also found to be effective positive mutation sites for SrUGT85C2 in this work, offering a theoretical foundation for the production of premium S. rebaudiana materials and the effective microbial synthesis of optimal steviol glycosides.