Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herbCurculigo orchioidesGaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production inYarrowia lipolyticawas improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield fromC.orchioidesroots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.