Exogenous γ-aminobutyric acid strengthens phenylpropanoid and nitrogen metabolism to enhance the contents of flavonoids, amino acids, and the derivatives in edamame.

γ-aminobutyric acid (GABA) has been reported to improve stress resistance in plants. Nonetheless, little is known about the effects of GABA on the nutritional quality and regulatory mechanisms of edamame. Therefore, we analyzed the flavonoid and amino acid (AA) metabolism and the effects of GABA on the nutrient content of edamame seeds through physiological and metabolomic analyses. Exogenous GABA increased endogenous GABA metabolism and GABA transaminase activity and enhanced the oxoglutarate content, which entered into nitrogen metabolism and increased the activity and expression of nitrogen metabolism-related enzymes, to accumulate AAs and bioactive peptides. Meanwhile, exogenous GABA induced the metabolism of flavonoids, including total flavonoids, anthocyanins, 6''-o-malonyglycitin, glycitin, ononin, cyanin, and ginkgetin, by increasing the activity and expression of flavonoid biosynthetic enzymes. This is the first study to reveal that GABA effectively improves the nutritional quality of edamame through the accumulation of AAs, bioactive peptides, isoflavones, anthocyanins, sugars, and organic acids.

Engineering the Entrance of a Flavonoid Glycosyltransferase Promotes the Glycosylation of Etoposide Aglycone.

Enzyme entrances, which function as the first molecular filters, influence substrate selectivity and enzymatic activity. Because of low binding affinities, engineering enzyme entrances that recognize non-natural substrates is a major challenge for artificial biocatalyst design. Here, the entrance of flavonoid glycosyltransferase UGT78D2 was engineered to promote the recognition of the aglycone of etoposide, a chemotherapeutic agent. We found that Q258, S446, R444, and R450, the key residues surrounding the substrate entrance, specifically guide the flux of etoposide aglycone, which has a high steric hindrance, into the active site; this activity was inferred to be determined by the entrance size and hydrophobic and electrostatic interactions. Engineering the coordination of Q258 and S446 to increase the entrance size and hydrophobic interaction between UGT78D2 and etoposide aglycone increased the affinity by 10.10-fold and the conversion by 10%. The entrance-engineering strategy applied in this study can improve the design of artificial biocatalysts.

The Advances and Challenges in Enzymatic C-glycosylation of Flavonoids in Plants.

Flavonoid glycosides play determinant roles in plants and have considerable potential for applications in medicine and biotechnology. Glycosyltransferases transfer a sugar moiety from uridine diphosphateactivated sugar molecules to an acceptor flavonoid via C-O and C-C linkages. Compared with O-glycosyl flavonoids, C-glycosyl flavonoids are more stable, resistant to glycosidase or acid hydrolysis, exhibit better pharmacological properties, and have received more attention. In this study, we discuss the mining of C-glycosyl flavones and the corresponding C-glycosyltransferases and evaluate the differences in structure and catalytic mechanisms between C-glycosyltransferase and O-glycosyltransferase. We conclude that promiscuity and specificity are key determinants for general flavonoid C-glycosyltransferase engineering and summarize the C-glycosyltransferase engineering strategy. A thorough understanding of the properties, catalytic mechanisms, and engineering of C-glycosyltransferases will be critical for future biotechnological applications in areas such as the production of desired C-glycosyl flavonoids for nutritional or medicinal use.

Flavonoid Metabolism in Tetrastigma hemsleyanum Diels et Gilg Based on Metabolome Analysis and Transcriptome Sequencing.

Tetrastigma hemsleyanum Diels et Gilg, known as a "plant antibiotic", possesses several attractive properties including anti-inflammatory, anti-tumor, and antioxidant effects, with its efficacy being attributed to flavonoids. However, the flavonoid biosynthesis of T. hemsleyanum has rarely been studied. In this study, we investigated the flavonoid metabolism of T. hemsleyanum through metabolome analysis and transcriptome sequencing. The metabolomic results showed differences in the flavonoids of the leaves and root tubers of T. hemsleyanum. A total of 22 flavonoids was detected, and the concentrations of most flavonoids in the leaves were higher than those in the root tubers. Transcriptome analysis revealed that differentially expressed genes (DEGs) in the leaves and root tubers were enriched in photosynthesis-antenna proteins. Pearson correlation analysis indicated that the expression levels of chalcone isomerase (CHI) and UDP-glycose flavonoid glycosyltransferase (UFGT) were highly correlated with the concentrations of most flavonoids. Further, this study found that the photosynthesis-antenna proteins essentially contributed to the difference in the flavonoids in T. hemsleyanum. The gene expressions and concentrations of the total flavonoids of leaves and root tubers in Hangzhou, Jinhua, Lishui, and Taizhou in Zhejiang Province, China, showed that CHI (CL6715.Contig1_All, Unigene19431_All, CL921.Contig4_All) and UFGT (CL11556.Contig3_All, CL11775.Contig1_All) were the potential key genes of accumulation of most flavonoids in T. hemsleyanum.

Isolation and characterization of a glycosyltransferase with specific catalytic activity towards flavonoids from Tripterygium wilfordii.

Flavonoids are important secondary metabolites that exist in many medicinal plants. Flavonoid glycosyltransferases can transfer sugar moieties to their parent rings, producing various flavonoid glycosides with significant pharmacological activities. Here, we report the molecular cloning of the O-glycosyltransferase TwUGT2 from Tripterygium wilfordii and its catalytic activity was explored by heterologous expression in E. coli. The results showed that TwUGT2 has specific glycosyltransferase activity towards C-3 and 7 hydroxyl groups of flavonoids, thereby converting quercetin and pinocembrin into isoquercitrin and pinocembrin 7-O-beta-D-glucoside, respectively. The identification of TwUGT2 will provide a useful molecular tool for synthetic biology and contribute to drug discovery.[Formula: see text].

Improved soluble bacterial expression and properties of the recombinant flavonoid glucosyltransferase UGT73G1 from Allium cepa.

Glycosylation of quercetin using flavonol-specific glycosyltransferases offers an alternate method for isoquercitrin production. Obtaining sufficient quantities of bioactive enzymes is an important prerequisite for highly effective biocatalysis and biotransformation. In this study, a codon-optimized gene for the flavonoid glucosyltransferase UGT73G1 from Allium cepa was heterologously expressed in the preferred prokaryotic expression host Escherichia coli. By combining expression as a fusion protein with 6-histidine tags with coexpression with molecular chaperones, increased soluble expression of UGT73G1 was achieved in E. coli. Two-terminal 6-histidine tags contributed more to the expression than molecular chaperones, as demonstrated by comparison of specific activities in crude extracts obtained from the recombinant E. coli strains. Studies of the catalytic properties of purified UGT73G1 indicated that its activity was significantly promoted by Mn2+ and Mg2+, while it was strongly inhibited by Cu2+. These expression strategies enhanced the solubility and activity of the overexpressed protein and enabled characterization of this plant-derived glucosyltransferase expressed in a prokaryotic host.