Efficient O-glycosylation of Triterpenes Enabled by Protein Engineering of Plant Glycosyltransferase UGT74AC1

Triterpene O-glycosylation has attracted significant interest from the pharmaceutical industry as a valuable means for drug design and development. Plant glycosyltransferases, which catalyze this glycosylation reaction, play the key step in preparing structure diverse and valuable triterpene glycosides. However, this class of enzymes usually suffered from low catalytic efficiency. To address this problem, triterpene glycosyltransferase UGT74AC1 from Siraitia grosvenorii was chosen and its crystal structure was solved and employed as the molecular basis to implement directed evolution and sequence/structure-based engineering. Several resultant UGT variants exhibit a 102- to 104-fold improvement in catalytic efficiency for triterpene glycosylation. Specially, one variant exhibited up to 4.17×104-fold increase in catalytic efficiency towards mogrol and 1.53×104-fold increase to UDP-glucose, respectively. Moreover, the mutants also displayed extended substrate promiscuity compared with wild type enzyme and cons...