Glucosylation of Isoflavonoids in Engineered Escherichia coli

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-alpha-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4' and 7 hydroxyl groups, but not at the 5th hydroxyl group of the A-ring, resulting in the production of genistein 4'-O-beta-D-glucoside, genistein 7-O-beta-D-glucoside (genistin), genistein 4', 7-O-beta-D-diglucoside, biochanin A-7-O-beta-D-glucoside (sissotrin), daidzein 4'-O-beta-D-glucoside, daidzein 7-O-beta-D-glucoside (daidzin), daidzein 4', 7-O-beta-D-diglucoside, and formononetin 7-O-beta-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/Delta pgi Delta zwf Delta ushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 mu M of supplemented isoflavonoids, without any additional UDP-alpha-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.