Unveiling phenylpropanoid regulation: the role of DzMYB activator and repressor in durian (Durio zibethinus) fruit.

KEY MESSAGE: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.

Evaluation of Polyphenols Synthesized in Mature Seeds of Common Bean (Phaseolus vulgaris L.) Advanced Mutant Lines.

This study aimed to investigate the availability of flavonoids, anthocyanins, and phenolic acids in mutant bean seeds, focusing on M7 mutant lines, and their corresponding initial and local cultivars. HPLC-DAD-MS/MS and HPLC-MS/MS were used to analyze twenty-eight genotypes of common bean. The obtained results suggest that the mutations resulted in four newly synthesized anthocyanins in the mutant bean seeds, namely, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside, and petunidin 3-O-glucoside, in 20 accessions with colored seed shapes out of the total of 28. Importantly, the initial cultivar with white seeds, as well as the mutant white seeds, did not contain anthocyanins. The mutant lines were classified into groups based on their colors as novel qualitative characteristics. Five phenolic acids were further quantified: ferulic, p-coumaric, caffeic, sinapic, and traces of chlorogenic acids. Flavonoids were represented by epicatechin, quercetin, and luteolin, and their concentrations in the mutant genotypes were several-fold superior compared to those of the initial cultivar. All mutant lines exhibited higher concentrations of phenolic acids and flavonoids. These findings contribute to the understanding of the genetics and biochemistry of phenolic accumulation and anthocyanin production in common bean seeds, which is relevant to health benefits and might have implications for common bean breeding programs and food security efforts.

Integrated metabolomic and transcriptomic analysis reveals the regulatory mechanisms of flavonoid and alkaloid biosynthesis in the new and old leaves of Murraya tetramera Huang.

BACKGROUND: Murraya tetramera Huang is a traditional Chinese woody medicine. Its leaves contain flavonoids, alkaloids, and other active compounds, which have anti-inflammatory and analgesic effects, as well as hypoglycemic and lipid-lowering effects, and anti-tumor effects. There are significant differences in the content of flavonoids and alkaloids in leaves during different growth cycles, but the synthesis mechanism is still unclear. RESULTS: In April 2021, new leaves (one month old) and old leaves (one and a half years old) of M. tetramera were used as experimental materials to systematically analyze the changes in differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) with transcriptomics and metabolomics technology. This was done to identify the signaling pathways of flavonoid and alkaloid synthesis. The results showed that the contents of total alkaloids and flavonoids in old leaves were significantly higher than those in new leaves. Thirteen flavonoid compounds, three isoflavone compounds, and nineteen alkaloid compounds were identified, and 125 and 48 DEGs related to flavonoid and alkaloid synthesis were found, respectively. By constructing the KEGG (Kyoto Encyclopedia of Genes and Genomes) network of DEGs and DAMs, it was shown that the molecular mechanism of flavonoid biosynthesis in M. tetramera mainly focuses on the "flavonoid biosynthetic pathway" and the "flavonoid and flavonol biosynthetic pathway". Among them, p-Coumaryl alcohol, Sinapyl alcohol, Phloretin, and Isoquercitrin were significantly accumulated in old leaves, the up-regulated expression of CCR (cinnamoyl-CoA reductase) might promote the accumulation of p-Coumaryl alcohol, upregulation of F5H (ferulate-5-hydroxylase) might promote Sinapyl alcohol accumulation. Alkaloids, including indole alkaloids, pyridine alkaloids, imidazole alkaloids, and quinoline alkaloids, were significantly accumulated in old leaves, and a total of 29 genes were associated with these substances. CONCLUSIONS: These data are helpful to better understand the biosynthesis of flavonoids and alkaloids in M. tetramera and provide a scientific basis for the development of medicinal components in M. tetramera.

Integrated Metabolomics and Transcriptomics Analysis of Flavonoid Biosynthesis Pathway in Polygonatum cyrtonema Hua.

Flavonoids, a class of phenolic compounds, are one of the main functional components and have a wide range of molecular structures and biological activities in Polygonatum. A few of them, including homoisoflavonoids, chalcones, isoflavones, and flavones, were identified in Polygonatum and displayed a wide range of powerful biological activities, such as anti-cancer, anti-viral, and blood sugar regulation. However, few studies have systematically been published on the flavonoid biosynthesis pathway in Polygonatum cyrtonema Hua. Therefore, in the present study, a combined transcriptome and metabolome analysis was performed on the leaf, stem, rhizome, and root tissues of P. cyrtonema to uncover the synthesis pathway of flavonoids and to identify key regulatory genes. Flavonoid-targeted metabolomics detected a total of 65 active substances from four different tissues, among which 49 substances were first study to identify in Polygonatum, and 38 substances were flavonoids. A total of 19 differentially accumulated metabolites (DAMs) (five flavonols, three flavones, two dihydrochalcones, two flavanones, one flavanol, five phenylpropanoids, and one coumarin) were finally screened by KEGG enrichment analysis. Transcriptome analysis indicated that a total of 222 unigenes encoding 28 enzymes were annotated into three flavonoid biosynthesis pathways, which were "phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "flavone and flavonol biosynthesis". The combined analysis of the metabolome and transcriptome revealed that 37 differentially expressed genes (DEGs) encoding 11 enzymes (C4H, PAL, 4CL, CHS, CHI, F3H, DFR, LAR, ANR, FNS, FLS) and 19 DAMs were more likely to be regulated in the flavonoid biosynthesis pathway. The expression of 11 DEGs was validated by qRT-PCR, resulting in good agreement with the RNA-Seq. Our studies provide a theoretical basis for further elucidating the flavonoid biosynthesis pathway in Polygonatum.

Puerarin-A Promising Flavonoid: Biosynthesis, Extraction Methods, Analytical Techniques, and Biological Effects.

Flavonoids, a variety of plant secondary metabolites, are known for their diverse biological activities. Isoflavones are a subgroup of flavonoids that have gained attention for their potential health benefits. Puerarin is one of the bioactive isoflavones found in the Kudzu root and Pueraria genus, which is widely used in alternative Chinese medicine, and has been found to be effective in treating chronic conditions like cardiovascular diseases, liver diseases, gastric diseases, respiratory diseases, diabetes, Alzheimer's disease, and cancer. Puerarin has been extensively researched and used in both scientific and clinical studies over the past few years. The purpose of this review is to provide an up-to-date exploration of puerarin biosynthesis, the most common extraction methods, analytical techniques, and biological effects, which have the potential to provide a new perspective for medical and pharmaceutical research and development.

Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi.

Highly efficient bioconversion of icariin to icaritin by whole-cell catalysis.

BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.

Identification of genes associated with biosynthesis of bioactive flavonoids and taxoids in Taxus cuspidata Sieb. et Zucc. plantlets exposed to UV-B radiation.

UV-B radiation is a typical environmental stressor that can promote phytochemical accumulation in plants. Taxus species are highly appreciated due to the existence of bioactive taxoids (especially paclitaxel) and flavonoids. However, the effect of UV-B radiation on taxoid and flavonoid biosynthesis in Taxus cuspidata Sieb. et Zucc. is largely unknown. In the present work, the accumulation of taxoids and flavonoids in T. cuspidata plantlets was significantly induced by 12 and 24 h of UV-B radiation (3 W/m2), and a large number of significantly differentially expressed genes were obtained via transcriptomic analysis. The significant up-regulation of antioxidant enzyme- and flavonoid biosynthesis-related genes (phenylalanine ammonia lyase 1, chalcone synthase 2, flavonol synthase 1, and flavonoid 3', 5'-hydroxylase 2), suggested that UV-B might cause the oxidative stress thus promoting flavonoid accumulation in T. cuspidata. Moreover, the expression of some genes related to jasmonate metabolism and taxoid biosynthesis (taxadiene synthase, baccatin III-3-amino 3-phenylpropanoyltransferase 1, taxadiene-5α-hydroxylase, and ethylene response factors 15) was significantly activated, which indicated that UV-B might initiate jasmonate signaling pathway that contributed to taxoid enhancement in T. cuspidata. Additionally, the identification of some up-regulated genes involved in lignin biosynthesis pathway indicated that the lignification process in T. cuspidata might be stimulated for defense against UV-B radiation. Overall, our findings provided a better understanding of some potential key genes associated with flavonoid and taxoid biosynthesis in T. cuspidata exposed to UV-B radiation.

Cool Temperature Enhances Growth, Ferulic Acid and Flavonoid Biosynthesis While Inhibiting Polysaccharide Biosynthesis in Angelica sinensis.

Angelica sinensis, a perennial herb that produces ferulic acid and phthalides for the treatment of cardio-cerebrovascular diseases, prefers growing at an altitude of 1800-3000 m. Geographical models have predicted that high altitude, cool temperature and sunshade play determining roles in geo-authentic formation. Although the roles of altitude and light in yield and quality have been investigated, the role of temperature in regulating growth, metabolites biosynthesis and gene expression is still unclear. In this study, growth characteristics, metabolites contents and related genes expression were investigated by exposing A. sinensis to cooler (15 °C) and normal temperatures (22 °C). The results showed that plant biomass, the contents of ferulic acid and flavonoids and the expression levels of genes related to the biosynthesis of ferulic acid (PAL1, 4CLL4, 4CLL9, C3H, HCT, CCOAMT and CCR) and flavonoids (CHS and CHI) were enhanced at 15 °C compared to 22 °C. The contents of ligustilide and volatile oils exhibited slight increases, while polysaccharide contents decreased in response to cooler temperature. Based on gene expression levels, ferulic acid biosynthesis probably depends on the CCOAMT pathway and not the COMT pathway. It can be concluded that cool temperature enhances plant growth, ferulic acid and flavonoid accumulation but inhibits polysaccharide biosynthesis in A. sinensis. These findings authenticate that cool temperature plays a determining role in the formation of geo-authentic and also provide a strong foundation for regulating metabolites production of A. sinensis.

Homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of transgenic tobacco.

Flavonoids and lignin consist of a large number of secondarymetabolites which are derived from the phenylpropanoid pathway, and they act as a significant role in plant growth, development, and stress response. However, few reports have documented that how different subbranches of phenylpropanoid metablolic pathway mutually interact. In Arabidopsis, AtCPC (AtCAPRICE) is known to play a negative role in anthocyanin accumulation. Nonetheless, whether AtCPC could control the biosynthesis of lignin is largely unknown. Additionally, whether the RrFLS and RrANR, flavonol synthase and anthocyanidin reductase, from Rosa rugosa regulate different branches of phenylpropanoid pathway is unclear. Here, we performed a series of transgenic experiments with short life cycle tobacco and RNA-Seq analysis. Finally, a series of assays related to biological, physiological, and phenotypic characteristics were undertaken. Our results indicated that ectopic expression of AtCPC in tobacco not only decreased the flavonoid compound accumulation, but also up-regulated several lignin biosynthetic genes, and significantly increased the accumulation of lignin. Our results also revealed that although they respectively improved the flavonol and proanthocyanidin contents, the overexpression of RrFLS and RrANR plays positive roles in lignin biosynthesis in transgenic tobacco plants. Our findings provide a novel insight into the mechanism underlying homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of plants.

Flavonoids naringenin chalcone, naringenin, dihydrotricin, and tricin are lignin monomers in papyrus.

Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the ß-ether linkages, indicating that at least a fraction of each was integrated into the lignin as ß-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3' and C-5' positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4'-O-ß bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon-carbon linkages, including 3'- or 5'-ß linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value.

Overexpression of chalcone isomerase A gene in Astragalus trigonus for stimulating apigenin.

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

Chalcone Scaffolds, Bioprecursors of Flavonoids: Chemistry, Bioactivities, and Pharmacokinetics.

Chalcones are secondary metabolites belonging to the flavonoid (C6-C3-C6 system) family that are ubiquitous in edible and medicinal plants, and they are bioprecursors of plant flavonoids. Chalcones and their natural derivatives are important intermediates of the flavonoid biosynthetic pathway. Plants containing chalcones have been used in traditional medicines since antiquity. Chalcones are basically α,ß-unsaturated ketones that exert great diversity in pharmacological activities such as antioxidant, anticancer, antimicrobial, antiviral, antitubercular, antiplasmodial, antileishmanial, immunosuppressive, anti-inflammatory, and so on. This review provides an insight into the chemistry, biosynthesis, and occurrence of chalcones from natural sources, particularly dietary and medicinal plants. Furthermore, the pharmacological, pharmacokinetics, and toxicological aspects of naturally occurring chalcone derivatives are also discussed herein. In view of having tremendous pharmacological potential, chalcone scaffolds/chalcone derivatives and bioflavonoids after subtle chemical modification could serve as a reliable platform for natural products-based drug discovery toward promising drug lead molecules/drug candidates.

Chemical Elicitors-Induced Variation in Cellular Biomass, Biosynthesis of Secondary Cell Products, and Antioxidant System in Callus Cultures of Fagonia indica.

Fagonia indica is a rich source of pharmacologically active compounds. The variation in the metabolites of interest is one of the major issues in wild plants due to different environmental factors. The addition of chemical elicitors is one of the effective strategies to trigger the biosynthetic pathways for the release of a higher quantity of bioactive compounds. Therefore, this study was designed to investigate the effects of chemical elicitors, aluminum chloride (AlCl3) and cadmium chloride (CdCl2), on the biosynthesis of secondary metabolites, biomass, and the antioxidant system in callus cultures of F. indica. Among various treatments applied, AlCl3 (0.1 mM concentration) improved the highest in biomass accumulation (fresh weight (FW): 404.72 g/L) as compared to the control (FW: 269.85 g/L). The exposure of cultures to AlCl3 (0.01 mM) enhanced the accumulation of secondary metabolites, and the total phenolic contents (TPCs: 7.74 mg/g DW) and total flavonoid contents (TFCs: 1.07 mg/g DW) were higher than those of cultures exposed to CdCl2 (0.01 mM) with content levels (TPC: 5.60 and TFC: 0.97 mg/g) as compared to the control (TPC: 4.16 and TFC: 0.42 mg/g DW). Likewise, AlCl3 and CdCl2 also promoted the free radical scavenging activity (FRSA; 89.4% and 90%, respectively) at a concentration of 0.01 mM, as compared to the control (65.48%). For instance, the quantification of metabolites via high-performance liquid chromatography (HPLC) revealed an optimum production of myricetin (1.20 mg/g), apigenin (0.83 mg/g), isorhamnetin (0.70 mg/g), and kaempferol (0.64 mg/g). Cultures grown in the presence of AlCl3 triggered higher quantities of secondary metabolites than those grown in the presence of CdCl2 (0.79, 0.74, 0.57, and 0.67 mg/g). Moreover, AlCl3 at 0.1 mM enhanced the biosynthesis of superoxide dismutase (SOD: 0.08 nM/min/mg-FW) and peroxidase enzymes (POD: 2.37 nM/min/mg-FW), while CdCl2 resulted in an SOD activity up to 0.06 nM/min/mg-FW and POD: 2.72 nM/min/mg-FW. From these results, it is clear that AlCl3 is a better elicitor in terms of a higher and uniform productivity of biomass, secondary cell products, and antioxidant enzymes compared to CdCl2 and the control. It is possible to scale the current strategy to a bioreactor for a higher productivity of metabolites of interest for various pharmaceutical industries.

Toward understanding of the methoxylated flavonoid biosynthesis pathway in Dracocephalum kotschyi Boiss.

Nowadays, with the development and advancement of next-generation sequencing technologies, a new path has been provided for transcriptomic studies. In this study, the transcriptome of Dracocephalum kotschyi Boiss., as an endemic and endangered plant which is contained a large amount of valuable secondary metabolites with antioxidant and anticancer properties, was sequenced. Then functional annotation and gene ontology analysis for 165,597 assembled transcripts were performed, most were associated with the metabolic pathways. This might be because there are various active biochemical pathways in this plant. Furthermore, after comprehensive transcript annotation, the putative genes involved in the main metabolic pathways of D. kotschyi were identified. Then, the biosynthetic pathway of its valuable methoxylated flavones was proposed. Finally, the accumulations of important methoxylated-flavone metabolites in three different tissues were quantified by HPLC. The relative expression of the genes involved in the proposed pathway was investigated by qRT-PCR, which indicated high expression levels in the bud tissue. The present results may lead to the design strategies to preserve the genetic diversity of endangered D. kotschyi plants and apply the new methods for engineering its valuable methoxylated-flavones pathway.

Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review.

Flavonoids belong to a class of plant secondary metabolites that have a polyphenol structure. Flavonoids show extensive biological activity, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, and antibacterial properties, so they are widely used in the food, pharmaceutical, and nutraceutical industries. However, traditional sources of flavonoids are no longer sufficient to meet current demands. In recent years, with the clarification of the biosynthetic pathway of flavonoids and the development of synthetic biology, it has become possible to use synthetic metabolic engineering methods with microorganisms as hosts to produce flavonoids. This article mainly reviews the biosynthetic pathways of flavonoids and the development of microbial expression systems for the production of flavonoids in order to provide a useful reference for further research on synthetic metabolic engineering of flavonoids. Meanwhile, the application of co-culture systems in the biosynthesis of flavonoids is emphasized in this review.

Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae.

Flavonols are health-promoting bioactive compounds important for plant defense and human nutrition. Quercetin (Q) and kaempferol (K) biosynthesis have been studied extensively while little is known about myricetin (M) biosynthesis. The roles of flavonol synthases (FLSs) and flavonoid 3',5'-hydroxylase (F3'5'H) in M biosynthesis in Morella rubra, a member of the Myricaceae rich in M-based flavonols, were investigated. The level of MrFLS transcripts alone did not correlate well with the accumulation of M-based flavonols. However, combined transcript data for MrFLS1 and MrF3'5'H showed a good correlation with the accumulation of M-based flavonols in different tissues of M. rubra. Recombinant MrFLS1 and MrFLS2 proteins showed strong activity with dihydroquercetin (DHQ), dihydrokaempferol (DHK), and dihydromyricetin (DHM) as substrates, while recombinant MrF3'5'H protein preferred converting K to M, amongst a range of substrates. Tobacco (Nicotiana tabacum) overexpressing 35S::MrFLSs produced elevated levels of K-based and Q-based flavonols without affecting M-based flavonol levels, while tobacco overexpressing 35S::MrF3'5'H accumulated significantly higher levels of M-based flavonols. We conclude that M accumulation in M. rubra is affected by gene expression and enzyme specificity of FLS and F3'5'H as well as substrate availability. In the metabolic grid of flavonol biosynthesis, the strong activity of MrF3'5'H with K as substrate additionally promotes metabolic flux towards M in M. rubra.

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.

Effect of Light and Dark on the Phenolic Compound Accumulation in Tartary Buckwheat Hairy Roots Overexpressing ZmLC.

Fagopyrum tataricum 'Hokkai T10' is a buckwheat cultivar capable of producing large amounts of phenolic compounds, including flavonoids (anthocyanins), phenolic acids, and catechin, which have antioxidant, anticancer, and anti-inflammatory properties. In the present study, we revealed that the maize transcription factor Lc increased the accumulation of phenolic compounds, including sinapic acid, 4-hydroxybenzonate, t-cinnamic acid, and rutin, in Hokkai T10 hairy roots cultured under long-photoperiod (16 h light and 8 h dark) conditions. The transcription factor upregulated phenylpropanoid and flavonoid biosynthesis pathway genes, yielding total phenolic contents reaching 27.0 ± 3.30 mg g-1 dry weight, 163% greater than the total flavonoid content produced by a GUS-overexpressing line (control). In contrast, when cultured under continuous darkness, the phenolic accumulation was not significantly different between the ZmLC-overexpressing hairy roots and the control. These findings suggest that the transcription factor (ZmLC) activity may be light-responsive in the ZmLC-overexpressing hairy roots of F. tataricum, triggering activation of the phenylpropanoid and flavonoid biosynthesis pathways. Further studies are required on the optimization of light intensity in ZmLC-overexpressing hairy roots of F. tataricum to enhance the production of phenolic compounds.

Metabolomics and transcriptome analysis of the biosynthesis mechanism of flavonoids in the seeds of Euryale ferox Salisb at different developmental stages.

Flavonoids belong to polyphenolic compounds, which are widely distributed in plants and have rich functions. Euryale ferox Salisb is an important medicinal and edible homologous plant, and flavonoids are its main functional substances. However, the biosynthesis mechanism of flavonoids in E. ferox is still poorly understood. To explore the dynamic changes of flavonoid biosynthesis during the development of E. ferox seeds, the targeted flavonoid metabolome was determined. A total of 129 kinds of flavonoid metabolites were characterized in the seeds of E. ferox, including 11 flavanones, 8 dihydroflavanols, 16 flavanols, 29 flavones, 3 isoflavones, 12 anthocyanins, 29 flavonols, 6 flavonoid carbonosides, 3 chalcones and 13 proanthocyanidins. The relative content of flavonoid metabolites accumulated continuously during the development of E. ferox seeds, and reached the highest at T30. In transcriptome, the expression of key genes in the flavonoid pathway, such as PAL, CHS, F3H, FLS, ANS, was highest in T30, which was consistent with the trend of metabolites. Six candidate transcription factors (R2R3MYBs and bHLHs) may affect the biosynthesis of flavonoids by regulating the expression of structural genes. Furthermore, transcriptome analysis and exogenous ABA and SA treatment demonstrated that ABA (PYR1, PP2Cs, SnRK2s) and SA (NPR1) are involved in the positive regulation of flavonoid biosynthesis. This study clarified the differential changes of flavonoid metabolites during the development of E. ferox seeds, confirmed that ABA and SA promote the synthesis of flavonoids, and found key candidate genes that are involved in the regulation of ABA and SA in the positive regulation of flavonoid biosynthesis.