Production of flavonoid  O -diglycoside naringin via sequential glycosylation in engineered  Schizosaccharomyces pombe  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 α1,2-rhamnosyltransferase (1,2RhaT), producing NRGI as the final product. In this study, we introduced three α1,2-rhamnosyl-glucoside synthesis-related enzymes-7- 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.
Kubomura A, Ohashi T, Misaki R, Fujiyama K