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.

Saccharides in straw hydrolysate decrease cell membrane damage by phenolics by inducing the formation of extracellular matrix in yeast.

The bioconversion of rice straw into ethanol can alleviate the energy crisis and solve problems related to waste treatment. In this study, the effect of soluble polysaccharides (SPs) produced during rice straw saccharification on the formation of extracellular matrices (EMs) by the yeast Saccharomyces cerevisiae was investigated. SPs were characterized by high-performance liquid chromatography (HPLC) and fourier transform infrared spectroscopy (FT-IR). SPs reduced the inhibition of alcohol dehydrogenase activity by phenolic acids (PAs) and regulated the intracellular redox state, resulting in higher ethanol production. The results of flow cytometry, confocal laser scanning microscopy, and atomic force microscopy indicated that PAs changed microbial morphology and caused damage in microbial cell membranes. The protective effect of SPs against cell membrane damage could be attributed to the synthesis of polysaccharide-dependent extracellular matrix, which maintained cellular integrity even under phenolic acid stress. These findings provide new strategies to improve pretreatment and saccharification processes.

Extraction of Flavonoids from the Saccharification of Rice Straw Is an Integrated Process for Straw Utilization.

To date, bioethanol is not economically competitive. One strategy to overcome this limitation is co-producing ethanol and high value-added products as an integrated process. The results of this study demonstrated that flavonoids could be extracted from rice straw, and the flavonoids apigenin and kaempferol were detected by HPLC. Compared with untreated straw, ball-milling slightly increased the total amount of flavonoids and antioxidant activity measured by ABTS, DPPH, and FRAP assays. The saccharification step in the bioconversion of straw strongly affected the extraction of flavonoids from straw. The residue obtained after saccharification of ball-milled straw for glucose production was more suitable for flavonoid extraction than untreated and ball-milled straw. The yield of flavonoids from the residue was 1.51-fold higher than that from untreated straw. The antioxidant activity of flavonoids derived from the residue was similar to that of flavonoid-rich biomasses such as rice bran and wheat bran. More importantly, saccharification may significantly affect the conditions of flavonoid extraction. In this respect, treatment with cellulase may reduce the extraction time from 2.0 to 0.5 h and the extraction temperature from 80 to 30 °C. Therefore, saccharification in the bioconversion of straw may be considered as an enzyme pretreatment step for the efficient extraction of flavonoids from straw, serving as a sustainable process for straw utilization.

Molecular Evidence for Catechin Synthesis and Accumulation in Tea Buds (Camellia sinensis).

Early spring buds of the Camellia sinensis variety Shuchazao were separated into two parts, including the shoot tip (ST) and non-expanded young leaves (YL), in which the synthesis and accumulation of catechins in the two parts were assessed by high-performance liquid chromatography (HPLC), p-dimethylaminocinnamaldehyde (DMACA) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and in situ hybridization. HPLC showed that (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) amounts in YL were increased significantly by 74.0 and 71.8%, respectively. The results of DMACA staining indicated that catechins in buds accumulated mainly in mesophyll cells and the bud shaft of YL. Meanwhile, qRT-PCR demonstrated that the relative expression levels of genes related to flavonoid metabolism, including CsPAL1, CsC4H1, CsC4H2, CsCHS2, CsF3'5'H1, CsDFR1, CsDFR2, and CsANR1, were significantly higher in YL than in the ST. In situ hybridization revealed that CsDFR1, CsDFR2, CsLAR, and CsANR1 were expressed in leaf primordia and YL but not in the apical meristem. These findings highlight the synthesis and accumulation patterns of catechins in different parts of the ST in C. sinensis, providing a theoretical basis for the assessment of synthesis, accumulation, and transfer patterns of catechins in tea plants.

Functional Analysis of an Uridine Diphosphate Glycosyltransferase Involved in the Biosynthesis of Polyphenolic Glucoside in Tea Plants (Camellia sinensis).

Polyphenols are one of the largest groups of compounds that confer benefits to the health of plants and humans. Flavonol glycosides are a major ingredient of polyphenols in Camellia sinensis. Flavonol-3-O-glycosides are characteristic astringent taste compounds in tea infusion. A polyphenolic glycosyltransferase (CsUGT72AM1) belonging to cluster IIIb was isolated from the tea plant. The full-length cDNA of CsUGT72AM1 is 1416 bp. It encodes 472 amino acids with a calculated molecular mass of 50.92 kDa and an isoelectric point of 5.21. The recombinant CsUGT72AM1 protein was expressed in Escherichia coli and exhibited catalytic activity toward multiple flavonoids and coniferyl aldehyde. The enzyme assay indicated that rCsUGT72AM1 could perform glycosidation of flavonols or coniferyl aldehyde in vitro to form 3-O-glucoside or 4-O-glucoside, respectively. Interestingly, this enzyme also had activities and performed multisite glycosidation toward flavanones. The consistent products were confirmed to be naringenin-7-O-glucoside and -4'-O-glucoside by the nuclear magnetism assay. In addition, in the enzyme assay with cyanidin as the substrate, the results suggested that the glycosylated activity of CsUGT72AM1 was remarkably inhibited by a high concentration of anthocyanins. The above results indicate that CsUGT72AM1 may be involved in the metabolism of flavonol, flavanone, anthocyanin, and lignin.

Inhibitory effects of phenolic compounds of rice straw formed by saccharification during ethanol fermentation by Pichia stipitis.

In this study, it was found that the type of phenolic acids derived from rice straw was the major factor affecting ethanol fermentation by Pichia stipitis. The aim of this study was to investigate the inhibitory effect of phenolic acids on ethanol fermentation with rice straw. Different cellulases produced different ratios of free phenolic acids to soluble conjugated phenolic acids, resulting in different fermentation efficiencies. Free phenolic acids exhibited much higher inhibitory effect than conjugated phenolic acids. The flow cytometry results indicated that the damage to cell membranes was the primary mechanism of inhibition of ethanol fermentation by phenolic acids. The removal of free phenolic acids from the hydrolysates increased ethanol productivity by 2.0-fold, indicating that the free phenolic acids would be the major inhibitors formed during saccharification. The integrated process for ethanol and phenolic acids may constitute a new strategy for the production of low-cost ethanol.