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

Abstract

C-glycosylated flavones (CGFs) are promising candidates as anti-nociceptive compounds. The leaves of bamboo and related crops in the grass family are a largely unexploited bioresource with a wide array of CGFs. We report here pathway-specific enzymes including Cglycosyltransferases (CGTs) and P450 hydroxylases from cereal crops and bamboo species accumulating abundant CGFs. Mining of CGTs and engineering of P450s that decorate the flavonoid skeleton allowed the production of desired CGFs (with yield of 20-40 mg/L) in an Escherichia coli cell factory. We further explored the antinociceptive activity of major CGFs in mice models and identified isoorientin as the most potent, with both neuroanalgesic and antiinflammatory effects superior to clinical drugs such as rotundine and aspirin. Our discovery of the pain-alleviating flavonoids elicited from bamboo and crop leaves establishes this previously underutilized source, and sheds light on the pathway and pharmacological mechanisms of the compounds.

FlavonoidC‐Glycosyltransferases: Function, Evolutionary Relationship, Catalytic Mechanism and Protein Engineering

“…UGT708 members that scatter in cluster I and II catalyze the C ‐glycosylation of closed‐circular or open‐circular 2‐hydroxyflavanone tautomers, chalcones, or other structure‐like compounds with a common 2′,4′,6′‐trihydroxyacetophenone skeleton 43. Cluster I comprises thirty‐two UGT708 members from the monocotyledon, whereas cluster II contains eight dicotyledonous UGT708 members, especially the di‐ C ‐glycosyltransferases, indicating that plants acquired the C ‐glycosylatation capability before the split of monocotyledon and dicotyledon 43. UGT708 members should evolve from a common ancestral CGT and their capability to C ‐glycosylate 2‐hydroxyflavanones may be conserved in the plant kingdom 43.…”

“…Cluster I comprises thirty‐two UGT708 members from the monocotyledon, whereas cluster II contains eight dicotyledonous UGT708 members, especially the di‐ C ‐glycosyltransferases, indicating that plants acquired the C ‐glycosylatation capability before the split of monocotyledon and dicotyledon 43. UGT708 members should evolve from a common ancestral CGT and their capability to C ‐glycosylate 2‐hydroxyflavanones may be conserved in the plant kingdom 43. Cluster III members (TcCGT1 from Trollius chinensis and PIUGT43 from Pueraria lobata ) and cluster VI members (GtUF6CGT1 from Gentiana triflora and UGT84A57 from Eutrema japonicum ) are phylogenetically distant to UGT708 members, which directly catalyze the C ‐glycosylation of flavones or isoflavones 32, 34, 44, 45.…”

“…Subsequently, the closed‐circular 2‐hydroxyflavanone C ‐6 and C ‐8 glucosides undergo enzymatic or spontaneous dehydration to produce a mixture of flavanone C ‐6 and C ‐8 glucoside Wessely‐Moser isomers 49. Other group 2 flavonoid CGTs preferentially C ‐glycosylate the open‐circular 2‐hydroxyflavanone as well as phloretin (a member of chalcones) and 2′,4′,6′‐trihydroxyacetophenone‐like chemicals 43, 50–52. These flavonoid CGTs are UGT708 subfamily members from grass plants, including five from Oryza sativa , one from Glycine max , six from Phyllostachys species, four from Sorghum bicolor , six from Triticum aestivum , three from Z. mays , one from Brachypodium distachyon , three from Setaria italica , two from Fagopyrum esculentum , and one from Mangifera indica .…”

“…These flavonoid CGTs are UGT708 subfamily members from grass plants, including five from Oryza sativa , one from Glycine max , six from Phyllostachys species, four from Sorghum bicolor , six from Triticum aestivum , three from Z. mays , one from Brachypodium distachyon , three from Setaria italica , two from Fagopyrum esculentum , and one from Mangifera indica . These enzymes mainly utilize UDP‐Glc or UDP‐Ara as sugar donors 43. Intriguingly, MiCGT from M. indica is a trifunctional and promiscuous GT, which utilizes both UDP‐Glc and UDP‐Xyl as sugar donors to conduct C ‐, O ‐, and N ‐glycosylation of various natural and unnatural products 52.…”

“…3 and Tab. 1), including MiCGTb from M. indica , UGT708G1 from Fortunella crassifolia , UGT708G2 from Citrus unshiu , UGT708A40 from O. sativa , UGT708A11 from Z. mays , UGT708A8 from B. distachyon , and UGT708B4 from Glycyrrhiza glabra 36, 37, 43, 47. All these flavonoid CGTs catalyze the di‐ C ‐glycosylation of aglycones bearing a 2′,4′,6′‐trihydroxyacetophenone‐like structure.…”