Mining and engineering of UDP-dependent glycosyltransferases from Dendrobium officinale for trilobatin biosynthesis.

The development of homologous food and medicine products often involves a trade-off between maintaining a pure and natural profile and optimizing flavor. The relatively monotonous taste of Dendrobium officinale has substantially limited its market expansion. Within D. officinale, phloretin and its glycosides are present, yet the natural sweetener trilobatin (phloretin-4'- O-glucoside) is absent. Different glycosylation positions often confer distinct taste properties on specific metabolites. Following melatonin treatment, there is a predominant enrichment of flavonoid pathways in D. officinale, accompanied by a notable decrease in phloretin levels, suggesting a possible upregulation of its glycosides. Subsequently, we annotate 135 uridine diphosphate-dependent glycosyltransferases (UGTs) in D. officinale and identify three phloretin UGTs, among which the enzymatic product trilobatin has been scarcely detected. Through functional modifications of these UGTs using AlphaFold and molecular docking, we have achieved a functional shift in DoUGT885 from solely producing phlorizin to trilobatin via mutation of the M2 fragment. The alteration of fragment M5 enables DoUGT019, which originally possessed multisite glycosylation functionality for phloretin, to primarily produce trilobatin as its main enzymatic product. The discovery of these key amino acid residues crucial for trilobatin formation serves as candidate insertion or substitution sequence fragments for gene editing, significantly advancing the creation of sweet D. officinale germplasm.