Competition between FLS and DFR regulates the distribution of flavonols and proanthocyanidins in Rubus chingii Hu.

Rubus chingii Hu is a berry plant of the genus Rubus of the Rosaceae family, which has high nutritional and medicinal value and is rich in flavonoids. Flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) compete for the common substrate dihydroflavonols to regulate the metabolic flux of flavonoids. However, the competition between FLS and DFR based on enzyme is rarely reported. Here, we isolated and identified two FLS genes (RcFLS1 and RcFLS2) and one DFR gene (RcDFR) from Rubus chingii Hu. RcFLSs and RcDFR were highly expressed in stems, leaves, and flowers, although the flavonol accumulation in these organs was significantly higher than that of proanthocyanidins (PAs). The recombinant RcFLSs demonstrated bifunctional activities via hydroxylation and desaturation at the C-3α position having a lower Michaelis constant (Km) for dihydroflavonols than RcDFR. We also found that a low concentration of flavonols could significantly inhibit RcDFR activity. To investigate the competitive relationship between RcFLSs and RcDFR, we used a prokaryotic expression system (E. coli) to co-express these proteins. The transgenic cells expressing recombinant proteins were incubated with substrates, and the reaction products were analyzed. Furthermore, two transient expression systems (tobacco leaves and strawberry fruits) and a stable genetic system (Arabidopsis thaliana) were used to co-express these proteins in vivo. The results showed that RcFLS1 was dominant in the competition with RcDFR. Our results demonstrated that the competition between FLS and DFR regulated the metabolic flux distribution of flavonols and PAs, which will be of great significance for the molecular breeding of Rubus plants.

Optimization of the Biosynthesis of B-Ring Ortho-Hydroxy Lated Flavonoids Using the 4-Hydroxyphenylacetate 3-Hydroxylase Complex (HpaBC) of Escherichia coli.

Flavonoids are important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L-1, an induction temperature of 28 °C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caffeic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.

Functional analysis of flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylases from tea plant (Camellia sinensis), involved in the B-ring hydroxylation of flavonoids.

Flavonoids are major polyphenol compounds in plant secondary metabolism. The hydroxylation pattern of the B-ring of flavonoids is determined by the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). In this paper, one CsF3'H and two CsF3'5'Hs (CsF3'5'Ha and CsF3'5'Hb) were isolated. The phylogenetic tree results showed that F3'H and F3'5'Hs belong to the CYP75B and CYP75A, respectively. The Expression pattern analysis showed that the expression of CsF3'5'Ha and CsF3'5'Hb in the bud and 1st leaf were higher than other tissues. However, the CsF3'H had the highest expression in the 4th and mature leaf. The correlation analysis showed that the expression of CsF3'5'Hs is positively associated with the concentration of B-trihydroxylated catechins, and the expression of CsF3'H is positively associated with the Q contentration. Heterologous expression of these genes in yeast showed that CsF3'H and CsF3'5'Ha can catalyze flavanones, flavonols and flavanonols to the corresponding 3', 4' or 3', 4', 5'-hydroxylated compounds, for which the optimum substrate is naringenin. The enzyme of CsF3'5'Hb can only catalyze flavonols (including K and Q) and flavanonols (DHK and DHQ), of which the highest activities in catalyzing are DHK. Interestingly, The experiment of site-directed mutagenesis suggested that two novel sites near the C-terminal were discovered impacting on the activity of the CsF3'5'H. These results provide a significantly molecular basis on the accumulation B-ring hydroxylation of flavonoids in tea plant.