Production of flavonoid <i>O</i>-diglycoside naringin via sequential glycosylation in engineered <i>Schizosaccharomyces pombe</i> as a whole-cell biocatalyst

Some functions of flavonoid glycosides are determined by the glycan structure or enhanced by increasing the attached sugars. Therefore, engineering the glycan moiety can modify the functions of the flavonoid. We attempted to construct a biotransformation method for producing a flavonoid diglycoside, naringin (NRGI) as a model target, using engineered yeast. Naringenin (NRG) glycosylation is initiated by the regiospecific 7-O-glucosyltransferase (7-O-GlcT) to form naringenin-7-O-glucoside (N7G), followed by further rhamnosylation via the branching-type α-1,2-rhamnosyltransferase (1,2RhaT), producing NRGI as the final product. In this study, we introduced three α-1,2-rhamnosyl-glucoside synthesis-related enzymes—-O-GlcT from Arabidopsis thaliana (AtGT-2), 1,2RhaT from Citrus maxima (Cm1,2RhaT), and UDP-Rha synthase from A. thaliana (AtRHM2)—into the fission yeast Schizosaccharomyces pombe. To improve the titer of NRGI, we examined the effects of biotransformation medium composition, initial cell concentration, and cell-permeabilizing reagents. Consequently, we successfully constructed a biotransformation method for producing a flavonoid diglycoside from the aglycone via sequential glycosylation using a single recombinant yeast. Using the optimized biotransformation method, we produced 23.1±1.1 mg l-1 of NRGI, 8.0±0.4 % molar conversion, from 136 mg l-1 of NRG in 24 h cultivation. This study demonstrated the first example of flavonoid O-diglycoside production in engineered S. pombe via sequential glycosylation by uridine diphosphate sugar-dependent glycosyltransferases (UGTs) under optimized production conditions.