Orientin and vitexin production by a one-pot enzymatic cascade of a glycosyltransferase and sucrose synthase

Orientin and vitexin, important components of bamboo-leaf extracts, are C-glycosylflavones which exhibit a number of interesting biological properties. In this work, we developed an efficient biocatalytic cascade for orientin and vitexin production consisting of Trollius chinensis C-glycosyltransferase (TcCGT) and Glycine max sucrose synthase (GmSUS). In order to relieve the bottleneck of the biocatalytic cascade, the biocatalytic efficiency, reaction condition compatibilities and the ratio of the enzymes were determined. We found that the specific activity of TcCGT was significantly influenced by enzyme dose and Triton X-100 or Tween 20 (0.2%). Coculture of BL21-TcCGT-Co and BL21-GmSUS-Co affected the catalytic efficiency of TcCGT and GmSUS, and the maximum orientin production rate reached 47 mu M/min at the inoculation ratio of 9:1. The optimal pH and temperature for the biocatalytic cascade were pH 7.5 and 30 degrees C, respectively. Moreover, the high dose of the enzymes can improve the tolerance of biocatalytic cascade to substrate inhibition in the one-pot reaction. By using a fed-batch strategy, maximal titers of orientin and vitexin reached 7090 mg/L with a corresponding molar conversion of 98.7% and 5050 mg/L with a corresponding molar conversion of 97.3%, respectively, which is the highest titer reported to date. Therefore, the method described herein for efficient production of orientin and vitexin by modulating catalytic efficiencies of enzymes can be widely used for the C-glycosylation of flavonoids.

Automated summary

A highly efficient biocatalytic cascade for orientin and vitexin production was developed in this study, utilizing Trollius chinensis C-glycosyltransferase (TcCGT) and Glycine max sucrose synthase (GmSUS). By modulating the catalytic efficiencies of enzymes, high titers of orientin and vitexin were achieved, with maximal titers reaching 7090 mg/L and 5050 mg/L, respectively. The method described herein can be widely used for C-glycosylation of flavonoids.