C‐Glycosyltransferases catalyzing the formation of di‐C‐glucosyl flavonoids in citrus plants

Abstract

Citrus plants accumulate many kinds of flavonoids, including di-C-glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di-C-glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C-glycosyltransferases (CGTs) FcCGT (UGT708G1) and CuCGT (UGT708G2) as the primary enzymes involved in the biosynthesis of di-C-glucosyl flavonoids in the citrus plants kumquat (Fortunella crassifolia) and satsuma mandarin (Citrus unshiu), respectively. The amino acid sequences of these CGTs were 98% identical, indicating that CGT genes are highly conserved in the citrus family. The recombinant enzymes FcCGT and CuCGT utilized 2-hydroxyflavanones, dihydrochalcone, and their mono-C-glucosides as sugar acceptors and produced corresponding di-C-glucosides. The K and k values of FcCGT toward phloretin were <0.5 μm and 12.0 sec , and those toward nothofagin (3'-C-glucosylphloretin) were 14.4 μm and 5.3 sec , respectively; these values are comparable with those of other glycosyltransferases reported to date. Transcripts of both CGT genes were found to concentrate in various plant organs, and particularly in leaves. Our results suggest that di-C-glucosyl flavonoid biosynthesis proceeds via a single enzyme using either 2-hydroxyflavanones or phloretin as a substrate in citrus plants. In addition, Escherichia coli cells expressing CGT genes were found to be capable of producing di-C-glucosyl flavonoids, which is promising for commercial production of these valuable compounds.

Pathway-specific enzymes from bamboo and crop leaves biosynthesize anti-nociceptive C-glycosylated flavones

“…Unlike O-glycosylation that terminates flavone biosynthesis, Cglycosylation occurs on unstable intermediates prior to the final scaffold formation. Except for PiUGT43 25 , GtUF6CGT1 26 , and TcCGT1 27 , all characterized CGTs to date belong to the UGT708 family [15][16][17][18]22,23 , indicating that the UGT708 family may be specialized for C-glycosylation of phenylpropanoids and flavonoids. Through this study, we have significantly expanded the knowledge on monocot UGT708A family by not only tripling the number of known CGTs from current 12 (2 monocot, 7 dicot UGT708, and 3 non-UGT708 CGTs) to 38, but also by revealing a group of hitherto unknown group that could recognize UDP-Ara (Figs.…”

“…The in planta CGF biosynthesis ( Fig. 1) has been so far partially established in a few monocotyledons and dicotyledonous plants [15][16][17][18][19][20][21][22][23] . The pathway is understood to involve upstream precursor biosynthesis and several downstream modification enzymes including cytochrome P450s and glycosyltransferases.…”

Molecular and Structural Characterization of a Promiscuous C‐Glycosyltransferase from Trollius chinensis

“…Recently, the enzymatic synthesis of flavonoid C ‐glycosides by plant CGTs has attracted considerable interest (Supporting Information, Scheme S1) . The 2‐hydroxyflavanone CGTs, including OsCGT from Oryza sativa , FeCGTa and FeCGTb from Fagopyrum esculentum , UGT708D1 from Glycine max , FcCGT from Fortunella crassifolia , and CuCGT from Citrus unshiu , utilize the open‐circular form of 2‐hydroxyflavanones as substrates to produce flavone C ‐glycosides after dehydration. The bifunctional glycosyltransferase (GT) UGT708A6 from Zea mays uses the closed‐circular form of 2‐hydroxyflavanones as substrates .…”

Ectopic expression of citrus UDP-GLUCOSYL TRANSFERASE gene enhances anthocyanin and proanthocyanidins contents and confers high light tolerance in Arabidopsis

“…Additionally, citrus UGTs genes are involved in metabolism [34] and expressed during growth and development [35]. The citrus UGTs are involved in flavonoids biosynthesis [35,36] and stimulates the anthocyanin biosynthesis in citrus [37]. Based on our (Additional file 2) and published transcriptome data [38], we have selected UGTs genes and confirmed that the UGT78D3 gene was differentially expressed in citrus specie (Citrus sinensis) by quantitative real time PCR (qRT-PCR) (Additional file 1).…”