Identification of a flavonoid C-glycosyltransferase from fern species Stenoloma chusanum and the application in synthesizing flavonoid C-glycosides in Escherichia coli

Abstract

Background Flavonoid C-glycosides have many beneficial effects and are widely used in food and medicine. However, plants contain a limited number of flavonoid C-glycosides, and it is challenging to create these substances chemically. Results To screen more robust C-glycosyltransferases (CGTs) for the biosynthesis of flavonoid C-glycosides, one CGT enzyme from Stenoloma chusanum (ScCGT1) was characterized. Biochemical analyses revealed that ScCGT1 showed the C-glycosylation activity for phloretin, 2-hydroxynaringenin, and 2-hydroxyeriodictyol. Structure modeling and mutagenesis experiments indicated that the glycosylation of ScCGT1 may be initiated by the synergistic action of conserved residue His26 and Asp14. The P164T mutation increased C-glycosylation activity by forming a hydrogen bond with the sugar donor. Furthermore, when using phloretin as a substrate, the extracellular nothofagin production obtained from the Escherichia coli strain ScCGT1-P164T reached 38 mg/L, which was 2.3-fold higher than that of the wild-type strain. Finally, it is proved that the coupling catalysis of CjFNS I/F2H and ScCGT1-P164T could convert naringenin into vitexin and isovitexin. Conclusion This is the first time that C-glycosyltransferase has been characterized from fern species and provides a candidate gene and strategy for the efficient production of bioactive C-glycosides using enzyme catalysis and metabolic engineering.

Insights into the Unusual Activity of a Novel Homospermidine Synthase with a Promising Application to Produce Spermidine

“…Determination of Kinetic Parameters. Determination of the kinetic parameters of Pf HSS with dual substrates using Ni et al 22 as a reference. Briefly, the enzyme-catalyzed reaction was set up with different substrates containing 1 mM 1,3-diaminopropane and various concentrations of putrescine (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM) or 2 mM putrescine and various concentrations of 1,3-diaminopropane (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM).…”