Flavone Synthase Research Articles

Bivalent RNA interference to increase isoflavone biosynthesis in soybean (Glycine max)

In this work, a bivalent RNA interference (RNAi) plant-transformation vector was constructed to silence both the flavanone 3-hydroxylase (F3H) gene and the flavone synthase II (GmFNSII) gene in soybean (Glycine max). Two further unit RNAi vectors were constructed for each of these two genes. RNAi-mediated suppression of these genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes ATCC15834. Notably, the bivalent RNAi vector had a significantly higher effect for increasing isoflavone production compared with the two unit RNAi vectors. The study highlighted molecular methods that could be used to enhance isoflavone production in soybean and demonstrated the challenges associated with such metabolic engineering for the production of plant natural products.

Genetic mapping of semi-polar metabolites in pepper fruits (Capsicum sp.): towards unravelling the molecular regulation of flavonoid quantitative trait loci

Untargeted LCMS profiling of semi-polar metabolites followed by metabolite quantitative trait locus (mQTL) analysis was performed in ripe pepper fruits of 113 F2 plants derived from a cross between Capsicum annuum AC1979 (no. 19) and Capsicum chinense No. 4661 Selection (no. 18). The parental accessions were selected based on their variation in fruit morphological characteristics and fruit content of some target phytonutrients. Clear segregation of fruit colour and fruit metabolite profiles was observed in the F2 population. The F2 plants formed three clusters based on their metabolite profiles. Of the total of 542 metabolites, 52 could be annotated, including a range of flavonoids, such as flavone C-glycosides, flavonol O-glycosides and naringenin chalcone, as well as several phenylpropanoids, a capsaicin analogue, fatty acid derivatives and amino acid derivatives. Interval mapping revealed 279 mQTLs in total. Two mQTL hotspots were found on chromosome 9. These two chromosomal regions regulated the relative levels of 35 and 103 metabolites, respectively. Analysis also revealed an mQTL for a capsaicin analogue, located on chromosome 7. Confirmation of flavonoid mQTLs using a set of six flavonoid candidate gene markers and their corresponding expression data (expression QTLs) indicated the Ca-MYB12 transcription factor gene on chromosome 1 and the gene encoding flavone synthase (FS-2) on chromosome 6 as likely causative genes determining the variation in naringenin chalcone and flavone C-glycosides, respectively, in this population. The combination of large-scale metabolite profiling and QTL analysis provided valuable insight into the genomic regions and genes important for the production of (secondary) metabolites in pepper fruit. This will impact breeding strategies aimed at optimising the content of specific metabolites in pepper fruit.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9967-0) contains supplementary material, which is available to authorized users.

Flower colour and cytochromes P450

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers.

Flavonoids are one of the major plant pigments for flower colour. Not only coloured anthocyanins, but also co-pigment flavones or flavonols, accumulate in flowers. To study the regulation of early flavonoid biosynthesis, two R2R3-MYB transcription factors, GtMYBP3 and GtMYBP4, were identified from the petals of Japanese gentian (Gentiana triflora). Phylogenetic analysis showed that these two proteins belong to the subgroup 7 clade (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. Gt MYBP3 and Gt MYBP4 transcripts were detected specifically in young petals and correlated with the profiles of flavone accumulation. Transient expression assays showed that GtMYBP3 and GtMYBP4 enhanced the promoter activities of early biosynthetic genes, including flavone synthase II (FNSII) and flavonoid 3′-hydroxylase (F3′H), but not the late biosynthetic gene, flavonoid 3′,5′-hydroxylase (F3′5′H). GtMYBP3 also enhanced the promoter activity of the chalcone synthase (CHS) gene. In transgenic Arabidopsis, overexpression of Gt MYBP3 and Gt MYBP4 activated the expression of endogenous flavonol biosynthesis genes and led to increased flavonol accumulation in seedlings. In transgenic tobacco petals, overexpression of Gt MYBP3 and Gt MYBP4 caused decreased anthocyanin levels, resulting in pale flower colours. Gt MYBP4-expressing transgenic tobacco flowers also showed increased flavonols. As far as is known, this is the first functional characterization of R2R3-MYB transcription factors regulating early flavonoid biosynthesis in petals.

Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

BackgroundCyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds.ResultsEscherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds.ConclusionWe elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries.

Three 2-oxoglutarate-dependent dioxygenase activities of Equisetum arvense L. forming flavone and flavonol from ( 2S)-naringenin

Equisetum arvense L. ( Equisetaceae–horsetail) accumulates various flavones and flavonols in infertile shoot. Enzyme assays conducted with crude extracts of the green tissue revealed chalcone synthase activity and also three further activities assigned to flavonoid biosynthesis and identified as flavone synthase I, flavanone 3 β-hydroxylase and flavonol synthase. The latter three activities were characterized as soluble, 2-oxoglutarate-dependent dioxygenases by their typical cofactor requirements and peculiar inhibition. Notably, this is the first report of flavone synthase I which had been considered to be restricted solely to species of the Apiaceae from a distant plant taxon.

Flavonoid Production Is Effectively Regulated by RNAi Interference of Two Flavone Synthase Genes from Glycine max

Flavonoids are a group of secondary metabolites found in many higher plants. The multiple roles of their flavone subclass include protection against UV damage, regulation of auxin transport, and modulation of flower color. In soybean (Glycine max), flavone synthase II (FNS II) is the key enzyme responsible for flavone biosynthesis. Two FNS II genes from soybean cultivar Hefeng 47 were cloned according to basic local alignment search tool (BLAST) contexts using flavone synthase sequences reported in other species. These were named GmFNSII-1 and GmFNSII-2. Sequence alignments showed that the cDNA of GmFNSII-1 was identical to that of CYP93B16, whereas GmFNSII-2 was clearly distinct. Functional assays in yeast (Schizosaccharomyces pombe) suggested that these two enzymes could convert (2S)-naringenin into apigenin. The two GmFNSII genes had similar tissue-specific expression patterns, but GmFNSII-2 was significantly expressed in the roots after treatment with 0.4 M glucose. This demonstrates that the gene plays an important role in the response to defense signals in soybean. RNA interference-mediated suppression of those GmFNSII genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes (ATCC15834). Our study also highlights some of the challenges associated with metabolic engineering of plant natural products.

Biosynthesis of Plant-Specific Flavones and Flavonols in Streptomyces venezuelae

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.

CYP93G2 Is a Flavanone 2-Hydroxylase Required for C-Glycosylflavone Biosynthesis in Rice

C-Glycosylflavones are ubiquitous in the plant kingdom, and many of them have beneficial effects on human health. They are a special group of flavonoid glycosides in which the sugars are C-linked to the flavone skeleton. It has been long presumed that C-glycosylflavones have a different biosynthetic origin from O-glycosylflavonoids. In rice (Oryza sativa), a C-glucosyltransferase (OsCGT) that accepts 2-hydroxyflavanone substrates and a dehydratase activity that selectively converts C-glucosyl-2-hydroxyflavanones to 6C-glucosylflavones were recently described. In this study, we provide in vitro and in planta evidence that the rice P450 CYP93G2 protein encoded by Os06g01250 is a functional flavanone 2-hydroxylase. CYP93G2 is related to the CYP93B subfamily, which consists of dicot flavone synthase II enzymes. In the presence of NADPH, recombinant CYP93G2 converts naringenin and eriodictyol to the corresponding 2-hydroxyflavanones. In addition, CYP93G2 generates 2-hydroxyflavanones, which are modified by O-glycosylation in transgenic Arabidopsis (Arabidopsis thaliana). Coexpression of CYP93G2 and OsCGT in Arabidopsis resulted in the production of C-glucosyl-2-hydroxyflavanones in the dibenzoylmethane tautomeric form. The same structure was reported previously for the in vitro OsCGT reaction products. Thus, CYP93G2 generates 2-hydroxyflavanone substrates from flavanones for C-glucosylation by OsCGT in planta. Furthermore, knocking down Os06g01250 in rice (O. sativa subsp. japonica 'Zhonghua 11') preferentially depleted the accumulation of C-glycosylapigenin, C-glycosylluteolin, and C-glycosylchrysoeriol but did not affect the levels of tricin, which is frequently present as O-glycosides in cereals. Taken together, our work conclusively assigned CYP93G2 as the first enzyme that channels flavanones to C-glycosylflavone biosynthesis in rice.

Flavone synthase II (CYP93B16) from soybean (Glycine max L.)

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4'-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNSII was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.

A flavonol synthase gene GtFLS defines anther-specific flavonol accumulation in gentian

Flavonoids are important for male gametophyte development. Here we report on the flavonoid components and their biosynthetic regulation in gentian anthers. Among flavonoids, flavonols, including kaempferol, quercetin and isorhamnetin derivatives, accumulated abundantly in gentian anthers. However, flavones and anthocyanins, which are the main flavonoids accumulating in petals, were not detected. Northern blot analysis of nine flavonoid biosynthetic genes showed that the ‘early’ flavonoid biosynthetic genes were expressed in both anthers and petals, and that flavonol synthase (FLS) transcripts were restricted to anthers. In contrast, flavone synthase II (FNSII) and ‘late’ flavonoid biosynthetic genes were expressed specifically in gentian petals. To confirm anther-specific expression of FLS, the 5′-upstream region of FLS (GtFLSpro) was cloned by inverse PCR and fused to the uidA (GUS) reporter gene. Tobacco, Arabidopsis and gentian plants, transformed with the GtFLSproGUS construct, exhibited anther-specific GUS expression. Expression was observed in the tapetum and in pollen at late stages of anther development in transgenic plants. These results revealed that flavonol accumulation in gentian anthers was regulated by the spatial expression of GtFLS. Our results also suggest that anther-specific regulation of FLS is conserved among higher plants and the GtFLS promoter is useful for induction of specific gene expression in anthers.

Identification of flavone phytoalexins and a pathogen-inducible flavone synthase II gene (SbFNSII) in sorghum.

Following inoculation with the anthracnose pathogen Colletotrichum sublineolum, seedlings of the sorghum resistant cultivar SC748-5 showed more rapid and elevated accumulation of luteolin than the susceptible cultivar BTx623. On the other hand, apigenin was the major flavone detected in infected BTx623 seedlings. Luteolin was demonstrated to show stronger inhibition of spore germination of C. sublineolum than apigenin. Because of their pathogen-inducible and antifungal nature, both flavone aglycones are considered sorghum phytoalexins. The key enzyme responsible for flavone biosynthesis has not been characterized in monocots. A sorghum pathogen-inducible gene encoding a cytochrome P450 protein (CYP93G3) in the uncharacterized CYP93G subfamily was identified. Transgenic expression of the P450 gene in Arabidopsis demonstrated that the encoded protein is a functional flavone synthase (FNS) II in planta. The sorghum gene was then termed SbFNSII. It is a single-copy gene located on chromosome 2 and the first FNSII gene characterized in a monocot. Metabolite analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in precursor ion scan mode revealed the accumulation of 2-hydroxynaringenin and 2-hydroxyeriodictyol hexosides in the transgenic Arabidopsis plants. Hence, SbFNSII appears to share a similar catalytic mechanism with the licorice and Medicago truncatula FNSIIs (CYP93B subfamily) by converting flavanones to flavone through the formation of 2-hydroxyflavanones.

Biological Synthesis of 7-O-Methyl Apigenin from Naringenin Using Escherichia coli Expressing Two Genes

Within the secondary metabolite class of flavonoids, which consist of more than 10,000 known structures, flavones define one of the largest subgroups. The diverse function of flavones in plants as well as their various roles in the interaction with other organisms offers many potential applications including in human nutrition and pharmacology. We used two genes, flavone synthase (PFNS-1) that converts naringenin into apigenin and a flavone 7-O-methyltransferase gene (POMT-7) that converts apigenin into 7-O-methyl apigenin, to synthesize 7-O-methyl apigenenin from naringenin. The PFNS-1 gene was subcloned into the E. coli expression vector pGEX and POMT-7 was subcloned into the pRSF vector. Since both constructs contain different replication origins and selection markers, they were cotransformed into E. coli. Using E. coli transformants harboring both PFNS-1 and POMT-7, naringenin could be converted into 7-O-methyl apigenin, genkwanin.

포플러로부터 flavone synthase I 유전자의 클로닝 및 생화학적 특성

Poplar contains various flavonoids including naringenin, kaempferol, myricetin, apigenin, luteolin, rhamnetin, and quercetin. These flavonoids are synthesized from naringenin with various enzymes. However, none of genes from poplar involved in flavonoid biosynthesis have been biochemically characterized. We cloned PFNS I-1 from Populus deltoids by RT-PCR method. The open reading frame of PFNS I-1 consisted of 1,017-bp and it showed high similarity with other FNS genes. The purified recombinant PFNS I-1, expressed in Escherichia coli, catalyzed the reaction from flavanone (naringenin) to flavone (apigenin). The reaction of PFNS I-1 was enhanced by cofactors such as oxoglutarate, , ascorbate and catalase. Thus, it is concluded that PFNS N-1 encodes a flavone synthase I.

Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti

Flavonoids play critical roles in legume-rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti, flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non-nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula.

Unraveling the Biochemical Base of Dahlia Flower Coloration

Dahlia ( Dahlia variabilis) exists in a dazzling array of cultivars, showing red, orange, magenta, lilac, yellow and white flower color, which is exclusively based on the presence of flavonoids and biochemically related compounds. Red hues (red, orange, magenta, lilac) are a result of anthocyanin accumulation in varying concentration and composition, while a yellow color is based on the formation of 6′-deoxychalcones in the petals. Red dahlia pigments are all derived from pelargonidin and cyanidin. Delphinidin derivatives are not formed due to the absence of flavonoid 3′,5′-hydroxylase in dahlia petals, which provides an explanation for the lack of blue dahlia flowers. Orange, lilac and rose cultivars are characterized by a lower anthocyanin content compared to many red cultivars. We investigated 198 cultivars for the presence of flavonoid enzymes. The activities of chalcone isomerase (CHI), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (FHT), flavone synthase II (FNSII), flavonol synthase (FLS) and flavonoid 3′-hydroxylase (F3′H) were demonstrated in enzyme preparations of dahlia petals. CHI accepted 6′-hydroxychalcones as substrates, but did not catalyze the conversion of 6′-deoxychalcones to the corresponding flavanones. White cultivars were frequently characterized by the lack of DFR activity, whereas in many yellow cultivars neither FHT nor DFR activity could be shown.

Expression of Parsley Flavone Synthase I Establishes the Flavone Biosynthetic Pathway inArabidopsis thaliana

Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.

Characterization of flavone synthase I from rice

Flavones are synthesized from flavanones through the action of flavone synthases (FNSs). There are two FNSs, FNS I and II. FNS I is a soluble dioxygenase present in members of the Apiaceae family and FNS II is a membrane bound cytochrome P450 enzyme that has been identified in numerous plant species. In this study, we cloned OsFNS I-1 from rice by RTPCR, expressed it in E. coli, and purified the recombinant protein. By NMR analysis, we found that OsFNS I-1 converted the flavanone (2S)-naringenin into the flavone, apigenin. Moreover, we found that the cofactors oxoglutarate, FeSO(4), ascorbate and catalase are required for this reaction. OsFNS I-1 encodes a flavone synthase I. This is the first type I FNS I found outside of the Apiaceae family.

Synthesis of Unnatural Flavonoids and Stilbenes by Exploiting the Plant Biosynthetic Pathway in Escherichia coli

Flavonoids and stilbenes have attracted much attention as potential targets for nutraceuticals, cosmetics, and pharmaceuticals. We have developed a system for producing "unnatural" flavonoids and stilbenes in Escherichia coli. The artificial biosynthetic pathway included three steps. These included a substrate synthesis step for CoA esters synthesis from carboxylic acids by 4-coumarate:CoA ligase, a polyketide synthesis step for conversion of the CoA esters into flavanones by chalcone synthase and chalcone isomerase, and into stilbenes by stilbene synthase, and a modification step for modification of the flavanones by flavone synthase, flavanone 3beta-hydroxylase and flavonol synthase. Incubation of the recombinant E. coli with exogenously supplied carboxylic acids led to production of 87 polyketides, including 36 unnatural flavonoids and stilbenes. This system is promising for construction of a larger library by employing other polyketide synthases and modification enzymes.