Production of flavonol and flavone 6-C-glucosides by bioconversion in Escherichia coli expressing a C-glucosyltransferase from wasabi (Eutrema japonicum).

OBJECTIVES: To produce flavonol and flavone 6-C-glucosides by bioconversion using recombinant Escherichia coli expressing a C-glucosyltransferase from wasabi (WjGT1). RESULTS: Escherichia coli expressing WjGT1 (Ec-WjGT1) converted flavones (apigenin and luteolin) and flavonols (quercetin and kaempferol) into their 6-C-glucosides in M9 minimal media supplemented with glucose, and released these products into the culture media. Ec-WjGT1 system also converts a flavanone (naringenin) into its C-glucoside at a conversion rate of 60% in 6 h. For scale-up production, apigenin, kaempferol, and quercetin were sequentially fed into the Ec-WjGT1 system at concentrations of 20-50 µM every 15-60 min, and the system was then able to produce isovitexin, kaempferol 6-C-glucoside, and quercetin 6-C-glucoside at an 89-99% conversion rate. CONCLUSIONS: The Ec-WjGT1 system quickly and easily produces flavone and flavonol 6-C-glucosides at high conversion rates when using sequential administration to avoid precipitation of substrates.

Identification and Characterization of Apigenin 6-C-Glucosyltransferase Involved in Biosynthesis of Isosaponarin in Wasabi (Eutrema japonicum).

Wasabi (Eutrema japonicum) is a perennial plant native to Japan that is used as a spice because it contains isothiocyanates. It also contains an isosaponarin, 4'-O-glucosyl-6-C-glucosyl apigenin, in its leaves, which has received increasing attention in recent years for its bioactivity, such as its promotion of type-I collagen production. However, its biosynthetic enzymes have not been clarified. In this study, we partially purified a C-glucosyltransferase (CGT) involved in isosaponarin biosynthesis from wasabi leaves and identified the gene coding for it (WjGT1). The encoded protein was similar to UGT84 enzymes and was named UGT84A57. The recombinant enzyme of WjGT1 expressed in Escherichia coli showed C-glucosylation activity toward the 6-position of flavones such as apigenin and luteolin. The enzyme also showed significant activity toward flavonols, but trace or no activity toward flavone 4'-O-glucosides, suggesting that isosaponarin biosynthesis in wasabi plants would proceed by 6-C-glucosylation of apigenin, followed by its 4'-O-glucosylation. Interestingly, the enzyme showed no activity against sinapic acid or p-coumaric acid, which are usually the main substrates of UGT84 enzymes. The accumulation of WjGT1 transcripts was observed mainly in the leaves and flowers of wasabi, in which C-glucosylflavones were accumulated. Molecular phylogenetic analysis suggested that WjGT1 acquired C-glycosylation activity independently from other reported CGTs after the differentiation of the family Brassicaceae.