Efficient Production of Orientin and Vitexin from Luteolin and Apigenin Using Coupled Catalysis of Glycosyltransferase and Sucrose Synthase

Abstract

Orientin and vitexin are flavone 8-C-glycosides that exhibit many biological characteristics. This study aimed to establish a two-enzyme-coupled catalytic strategy to enhance the biosynthesis of orientin and vitexin from apigenin and luteolin, respectively. The C-glucosyltransferase (TcCGT1) gene from Trollius chinensis was cloned and expressed in Escherichia coli BL21­(DE3). The optimal activity of TcCGT1 was achieved at pH 9.0 and 37 °C. TcCGT1 was relatively stable over the pH range of 7.0–10.0 at a temperature lower than 45 °C. The coupled catalytic strategy of TcCGT1 and different sucrose synthases was adopted to enhance the production of orientin and vitexin. By optimizing the coupling reaction conditions, orientin and vitexin production successfully achieved 2324.4 and 5524.1 mg/L with a yield of 91.4 and 89.3% (mol/mol), respectively. The coupled catalytic strategy proposed in this study might serve as a promising candidate for the large-scale production of orientin and vitexin in the future.

Enhancement of Rebaudioside M Production by Structure-Guided Engineering of Glycosyltransferase UGT76G1

“…It showed that the cascade reaction at 30 °C with pH 8.0 gave the best performance (Figure c,d). Since the supply of UDPG significantly affected the glycosylation reaction, , the concentrations of sucrose and UDP were also optimized. The yield of Reb M reached the maximum when increasing the sucrose concentration from 50 to 600 mM (Figure e).…”

Controllable Iterative β-Glucosylation from UDP-Glucose byBacillus cereusGlycosyltransferase GT1: Application for the Synthesis of Disaccharide-Modified Xenobiotics

“…To obtain better control over the glycosylated products formed from 15HCM than was possible by varying the donor/acceptor ratio with UDP-glucose, we turned to a coupled reaction of Bc GT1 and sucrose synthase, with the idea of supplying the UDP-glucose donor in situ from sucrose and UDP. − We considered that the sucrose synthase reaction might enable a constant steady-state level of UDP-glucose during the synthesis, contrary to the continuously changing conditions when UDP-glucose is used directly. We were pleased to discover conditions achieving selective production of the disaccharide-modified product at full conversion of the 15HCM substrate, as shown in Figure .…”

“…The permissive character of the sucrose synthase allows for different nucleotide-activated forms of d -glucose to be prepared from sucrose. ,, Combination with a suitable sugar nucleotide epimerase can expand the scope of glycosyl donors prepared in situ . , Other “reverse” glycosyltransferase reactions previously applied for in situ release of sugar nucleotide donors , can be used in a conceptually analogous manner. Difference should be noted between the idea of in situ sugar nucleotide synthesis for controlling the product selectivity in iterative glycosylation processes, which is new, and use of the same for a mere regeneration of the sugar nucleotide, which is well established for biocatalytic synthesis with glycosyltransferases. − …”

Whole-Cell Biocatalyst for Rubusoside Production in Saccharomyces cerevisiae

“…Huang et al used GmSUS from soybean to construct a UDPG regeneration platform, synthesizing a variety of glycosylated small molecules, in which the yield of geranioside was improved up to 99%. 44 More complex natural products like flavone C-glycosides 24 and triterpenoid glycosides 45 have also been efficiently synthesized through this method. For the production of SGs, Chen et al established SUS-UGT cascade reaction systems in E. coli for rebaudioside D 46 and rebaudioside E 47 tuberosum.…”

“…The SUSs catalyze a reversible reaction to produce UDPG and fructose from sucrose and UDP (Figure B). Therefore, the SUSs are usually coupled with specific UGTs in whole-cell catalysis to realize cascade reactions of glycosylation and UDPG regeneration. − …”