Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

Characterization of the Brassica napus flavonol synthase gene family reveals bifunctional flavonol synthases

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyses the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent-dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homoelogs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3 hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

Microbial Production of Diosmetin from Hesperetin in Engineered Escherichia coli with Flavone Synthase and Flavonol Synthase

In this study, diosmetin was synthesized with recombinant Escherichia coli expressing flavone synthase (FNS) or flavonol synthase (FLS). Forty-four FNS/FLS were selected from 40 different plants and their bioinformatic data, such as isoelectric point, instability index, grand average of hydropathicity, transmembrane structure, secondary structure, and conservative domain were analyzed with computer tools or software. Nine recombinant E. coli strains expressing FNS/FLS were constructed for diosmetin synthesis, and the products were detected through UPLC, LC-MS, and SDS-PAGE. Results showed that FNS/FLS from different sources were different in transmembrane structures, instability coefficients, and conservative regions. Among the nine recombinant E. coli strains, six recombinant E. coli strains were observed expected bands by SDS-PAGE, four recombinant E. coli strains were detected to have diosmetin with a molecular weight of 300.06 confirmed by LC-MS in broth, and the diosmetin concentration of a DE3/pAnFNS fermentation broth was the highest (39.6 mg/L). The enzyme expression and catalytic reaction were accordance with the results of bioinformatics analysis. In addition, the ratio of predicted intermediate product (4'-O-Methyl taxifolin) and final product (diosmetin) was significant different among FNS/FLS from different sources, although they are similar in physicochemical properties and structures. Therefore, the hypothesis that FNS/FLS catalyzed the synthesis of diosmetin from hesperetin by hydroxylation at the C-2 and C-3 positions respectively, 2-hydroxyhesperetin undergoes an elimination reaction and is converted to diosmetin, 3-hydroxyhesperetin (4'-O-Methyl taxifolin) was retained as a byproduct has been proposed.Key points:1) A possible biosynthesis pathway of diosmetin was proposed.2) Diosmetin was biosynthesized by expressing FNS and FLS in recombinant Escherichia coli strains.3) Enzyme selection in biosynthesis was guided with bioinformatic analysis.4) The speculation that simultaneous hydroxylation of the reaction occurs at both C-2 and C-3 positions of flavanones has been proposed.

Molecular cloning and expression characterization of flavonol synthase genes in peanut (Arachis hypogaea)

Flavonol is an important functional bioactive substance in peanut seeds, and plays important roles responding to abiotic stress. The flavonol content is closely related to the activity and regulation of gene expression patterns of flavonol synthase (FLS). In this study, eight FLS genes, AhFLSs were cloned and their expression characterization in different peanut organ and seedling under different abiotic stress were conducted. The results showed that the expressions levels of AhFLSs were differed in all assayed peanut organs and seedlings under abiotic stress treatments. Expression levels of AhFLS2, AhFLS3, AhFLS4, and AhFLS6 were higher than those of other AhFLSs. The flavonol contents of peanut organs and seedlings under different abiotic stress were also determined using high performance liquid chromatography (HPLC). Dried mature peanut seeds were the organ tissue with the highest flavonol content, and flavonol content increased with seed development. Under abiotic stress treatments, the types of flavonols induced differed among stress treatments. Correlation analysis results suggested that eight AhFLS genes may have different functions in peanut. Moreover, changes in the expression of the eight genes appear to has substrate preference. These results can lay the foundation for the study of improving nutritional value of peanut seed and resistance of peanut plant.

Evaluating Expression of Gene Encoding Flavonol Synthase in Green TrungDu and Purple TrungDu by Real time PCR and HPLC Technique

In plants, flavonol synthase (FLS) is a multifunctional enzyme that converts dihydroflavonol into flavonols and naringenin into dihydrokaempferol. FLS from tea has been shown to metabolize dihydroquercetin to quercetin. In this study, we conducted studying on the relationship between quercetin content and the expression level of FLS in two traditional tea cultivars of Vietnam, TrungDuxanh and TrungDutim tea grown in tea garden of Thai Nguyen University of Agriculture and Forestry. Tea shoots with one apical bud and two to three young leaves using as research materials which collected in September 2017. The using of HPLC technique did not detect the quercetin content from the these tea samples. The preliminary results of quantification of FLS gene expression by real time PCR showed the expression level of FLS in green Trung Du tea is higher than purple Trung Du, although the difference is not great. Thus, at the time of collecting, the expression of FLS in green Trung Du and purple Trung Du can not synthesize quercetin but synthesize other flavonols.

Expression of gene encoding flavonol synthase isolating from trung du xanh tea (Camellia sinensis var. macrophylla) in E. coli

Common flavonols in plants including quercetin, kaempferol and myricetin are synthesized from dihydroflavonols (dihydroquercetin-DHQ, dihydrokaempferol-DHK and dihydromyricetin-DHM) by flavonol synthase (FLS). In tea, FLS has been shown to metabolize dihydroquercetin to quercetin. The FLS gene was cloned and sequenced from the cultivated tea (Camellia sinensis var. macrophylla) in Thai Nguyen province. In this study, we presented the results of optimizing and designing an expression vector for recombinant FLS (recombinant FLS-rFLS). The FLS gene was ligated completely to the pET32a (+) vector, then expressed in E. coli Rosetta1 and Rosetta2 strain. Using 1mM IPTG to induce the expression of rFLS at 37oC, rFLS was obtained with 52.83 kDa in size and existed predominantly as insoluble form. E. coli Rosetta1 pET32a (+)_FLSproduces rFLS in the soluble fraction than E. coli Rosetta2 pET32a (+)_FLS. Next, E. coli Rosetta1 pET32a (+)_FLSwas optimized for expression at temperatures of 30oC, 23oC and 16oC (24 and 48 hours). After being induced for expression with 1mM IPTG in 48 hours and cultured at 16oC, E. coli Rosetta1 strain containing pET32a (+) FLS produced the largest amount of rFLS in the soluble form.