The activity annotation of peach glycosyltransferase PpUGT78B based on engineering bacterial anthocyanin biosynthesis

Glycosylation modification allows the formation of anthocyanin from anthocyanidin, which enhances the stability of anthocyanins and improves fruit coloration and anthocyanin availability as a human functional component. Flavonoid glycosyltransferases (UFGT) are responsible for catalyzing anthocyanidin glycosylation. In the present study, to better clarify peach (Prunus persica L.) UFGT (PpUGT78B) function, an engineering bacterial system was constructed, which used the anthocyanidin synthase (ANS) gene for producing cyanidin with the incorporation of (+)-catechin precursors and further synthesized cyanidin-3-O-glucoside (C3G) with UFGT co-expression. In addition, it was found that expression of fusion proteins with ANS and UFGT could improve C3G production by about 15 %-20 % in engineering bacterial systems. Furthermore, combining the molecular modeling prediction and targeted mutagenesis, this engineering bacterial system linked some residues in PpUGT78B to glycosylation capacity, which involved F210, L148, Q393, G391, and H230, whose mutation resulted in reduced enzyme activity or even loss and also involved F203 and S29 whose mutation resulted in the increased catalytic activity. Subsequently, a natural mutation of PpUGT78B was detected by analyzing 109 peach genome re-sequencing data, and two residue mutants (E82D, V276F) were found in two peach varieties. Further, these two natural mutation sites were confirmed to reduce PpUGT78B activity in engineering bacterial systems. This study demonstrates the effectiveness of the engineering bacteria system in anthocyanin biosynthesis. It offers valuable insights into the functional and structural roles of PpUGT78B, advancing our understanding of anthocyanin glycosylation.