Probing and Engineering Key Residues for Bis-C-glycosylation and Promiscuity of a C-Glycosyltransferase

C -Glycosyltransferases (CGTs) are powerful tools for the C -glycosylation of natural and unnatural products. However, CGTs able to catalyze bis- C -glycosylation are very rare and the key amino acids of which have not been uncovered. Here, we discovered a C -glycosyltransferase MiCGTb from Mangifera indica that has the capacity for bis- C -glycosylation. Further studies on active-site motifs revealed that I152 of MiCGTb was the critical amino acid residue for the second C -glycosylation and its S60/V100/T104 residues were pivotal for bis- C -glycosylation activity. Moreover, we developed a panel of variants with acceptor and donor promiscuity by site-directed mutagenesis. Among these variants, a mutant MiCGT-E152L displayed a broader acceptor scope for bis- C -glycosylation, and three mutants of MiCGTb exhibited sugar donor promiscuity toward structurally varied α- d - and β- l -glycosyl donors. Our work provides insights into the pivotal amino acid residues of CGTs for bis- C -glycosylation and biocatalytic tools to efficiently produce structurally diverse bis- C -glycosides with two identical or different sugar moieties in drug discovery.