Metabolic engineering of anthocyanin biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3beta-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat

Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H(+)-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.     

A survey of anthocyanins in fruits of some angiosperms,I

The anthocyanin pigments in the fruits of fifty-two species belonging to seventeen families of angiosperms were investigated paper-chromatographicallly. They were identified as cyanidin 3-monoglucoside, pelargonidin 3-monoglucoside, cyanidin 3-rutinoside, pelargonidin 3-rutinoside, cyanidin 3-xylosylglucoside, cyanidin 3-xylosylgalactoside, delphinidin 3-xylosylglucoside and delphinidin 3-sophorosido-5-monoglucoside. Of those anthocyanins detected, the most common was cyanidin 3-monoglucoside. In general, the plants belonging to a certain genus contained the same anthocyanin.     

Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

Anthocyanins in fruits of Vaccinium Japonicum

Cyanidin-3-arabinoside (54%) and pelargonidin-3-arabinoside (39%) were the main anthocyanins isolated from berries of Vaccinium japonicum. In addition smaller amounts of 3-galactosides of cyanidin (5%) and pelargonidin (2%) were found. The total anthocyanin content in the fruit averaged 113 mg/100 g fresh fruit. This is the first report of pelargonidin derivatives in the genus Vaccinium.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

RELATION OF FLOWER COLOR TO OPTICAL-DENSITY SPECTRA OF INTACT TISSUE AND OF ANTHOCYANIN EXTRACTS

Data from measurements of optical density of intact tissue and of anthocyanins in extracts resolved on cellulose thin layer plates were compared with visual evaluations of color quality and intensity in poinsettia, rose, and snapdragon. Visual evaluation was in good agreement with both instrumental and chemical determinations. However, the number or kinds of anthocyanins present could not be predicted from the visual evaluation or from the spectra of the fresh tissue. Data from the resolved extracts did not provide a basis for predicting the optical-density spectrum or the color of the intact tissue. In addition to the genetic factors which have been shown to control (1) the type of anthocyanin, (2) the amount of anthocyanin, and (3) the distribution of anthocyanins within the flower, we suggest another group of genes which apparently affect color through control of structural modification of individual anthocyanins in the living cell through shift in pH, metal chelation, and/or copigmentation. Such genes are apparently responsible for the modification of red color within the Wh Wh genotype of poinsettias containing both pelargonidin and cyanidin glycosides and for a very similar pink color in a snapdragon and a rose, each containing a single anthocyanin, a pelargonidin glycoside, and a cyanidin glycoside, respectively.     

Study on Degradation of Flavonols in Mutants of PoppyPapaver somniferumL.

We studied flavonol-degrading activity of cell-free extracts from petals of the flower color and structure mutants. The relationship between degradation of flavonols (kaempferol, quercetin, and myricetin) and biosynthesis of anthocyanins has been revealed. The white-flower mutant proved to have the highest flavonol-degrading activity toward all substrates, particularly quercetin. The mutations inhibiting synthesis of pelargonidin, an anthocyanin, provide for synthesis of various amounts of cyanidin in the petals. The flavonol-degrading activity considerably increases proportionally to the content of cyanidin. A similar relationship has been revealed in the mutants synthesizing both cyanidin and pelargonidin. The plants accumulating considerable amounts of pelargonidin in their petals have accordingly higher flavonol-degrading activity and predominantly hydrolyze kaempferol. The plants forming additional pods in their flower (pistillody) have higher flavonol-degrading activity as compared to the anther-in-petal and doubleness mutants     

Optimization of the extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc produced during traditional wine processing and characterization of the extracts

The extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc generated during traditional wine processing was optimized using response surface methodology (RSM). A face-centered cube design (FCD) consisting of 17 experimental runs, including five replicates at the center point, was used to investigate the effects of the three variables (solid-liquid ratio, time, and temperature) on anthocyanin extraction, and the results showed that the relationship between the three variables and the total anthocyanin content followed a quadratic model (R2=0.8853). In addition, the RSM analysis predicted that the optimum conditions for extraction consisted of a solid-liquid ratio of 20, a time of 60min, and a temperature of 60 degrees C. Verification tests performed under these optimum conditions gave 34.7+/-1.4mg/100g of anthocyanin, which was close to predicted value of 37.2mg/100g. Additionally, analysis of water extracts prepared using the predicted optimum conditions revealed that the carbohydrates (sugar and pectin) in Bokbunja marc underwent significant variation toward the formation of by-products (glycerol and uronic acids) during yeast fermentation, and that the amount of anthocyanin produced was reduced 10-fold when compared to the original extraction. Further, the results of HPLC-PDA-MS/MS analysis of the anthocyanins extracted from Bokbunja marc revealed the presence of six anthocyanin components, which were tentatively identified as cyanidin 3-O-sambubioside, cyanidin 3-O-xylosylrutinoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-rutinoside, delphinidin 3-O-rutinoside-?, and delphinidin 3-O-glucuronide.     

Anthocyanin content of Tulipa species and cultivars and its impact on tepal colours

Flowers of tulips (17 species and 25 cultivars) were subjected to qualitative and relative quantitative examination for anthocyanins. Altogether five anthocyanins were identified as the 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) of delphinidin (1), cyanidin (2) and pelargonidin (3), and the 3-O-[6″-O-(2‴-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside] of cyanidin (4) and pelargonidin (5). The pigments 1–5 represented 7%, 43%, 12%, 2% and 31%, respectively, of the total anthocyanin amount in the tepals of the Tulipa species, and 20%, 37%, 30%, 6% and 4%, respectively, in the cultivar tepals. Nearly 50% of the samples contained acetylated anthocyanins. The colours of the freeze-dried tepals described by the CIELab coordinates, hue angle (h_ab), saturation (C*), and lightness (L*) together with the anthocyanin content were subjected to multivariate analysis. All tepals classified with hue angles described as “blue nuances” were from cultivars. They contained 1 as the major anthocyanin, and no or just traces of pelargonidin derivatives. The species and cultivars having “magenta nuances” showed similar anthocyanin content with increased relative proportions of 2 at the expense of 1. Orange coloured tepals were to a large extent correlated with high relative proportions of the pelargonidin derivatives, 3 and 5. Acetylation of anthocyanins furnished a weak colour effect opposite to the bluing effect previously reported for anthocyanins with aromatic acyl groups. All six species belonging to the section Eichleres (subgenus Tulipa) were after principal component analysis grouped closely together. They were characterized by high concentrations of the pelargonidin derivatives 3 and 5, and orange petal nuances. However, within section Tulipa (subgenus Tulipa), considerable anthocyanin variation was observed. Species in the subgenus Eriostemones were generally characterized by the two anthocyanins 1 and 2, and no pelargonidin derivatives.     

Human tumor cell growth inhibition by nontoxic anthocyanidins, the pigments in fruits and vegetables

Anthocyanidins, the aglycones of anthocyanins, impart brilliant colors in many fruits and vegetables. The widespread consumption of diets rich in anthocyanin and anthocyanidins prompted us to determine their inhibitory effects on human cancer cell proliferation. Five anthocyanidins, cyanidin (1), delphinidin (2), pelargonidin (3), petunidin (4) and malvidin (5), and four anthocyanins, cyanidin-3-glucoside, cyanidin-3-galactoside, delphinidin-3-galactoside and pelargonidin-3-galactoside were tested for cell proliferation inhibitory activity against human cancer cell lines, AGS (stomach), HCT-116 (colon), MCF-7 (breast), NCI H460 (lung), and SF-268 (Central Nervous System, CNS) at 12.5-200 microg/mL concentrations. The viability of cells after exposure to anthocyanins and anthocyanidins was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric methods. The anthocyanins assayed did not inhibit cell proliferation of cell lines tested at 200 microg/mL. However, anthocyanidins showed cell proliferation inhibitory activity. Malvidin inhibited AGS, HCT-116, NCI-H460, MCF-7 and SF-268 cell growth by 69, 75.7, 67.7, 74.7 and 40.5%, respectively, at 200 microg/mL. Similarly, pelargonidin inhibited AGS, HCT-116, NCI H460, MCF-7 and SF-268 cell growth by 64, 63, 62, 63 and 34%, respectively, at 200 microg/mL. At 200 microg/mL, cyanidin, delphinidin and petunidin inhibited the breast cancer cell growth by 47, 66 and 53%, respectively. This is the first report of tumor cell proliferation inhibitory activity by anthocyanidins.     

Fractionation and chemistry of ethyl acetate-soluble thearubigins from black tea

Sephadex LH-20 chromatography was used to fractionate purified ethyl acetate-soluble thearubigins, prepared from an aqueous ethanolic extract of black tea. Three subfractions were so produced, each having a MW of about 1500 and each being degradable into cyanidin, delphinidin, gallic acid, the same two flavan-3-ols, and the same two flavan-3-ol gallates, though in different yield. Some evidence for the presence of benzotropolone moieties in at least one of the subfractions was obtained. Overall the ethyl acetate-soluble thearubigins are viewed as pentameric flavan-3-ols/flavan-3-ol gallates, containing both hydrolysable and non-hydrolysable interflavanoid links, as well as benzotropolone units, rather than as polymeric proanthocyanidins, a term previously used for all thearubigin subgroups.     

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.     

Structure of the acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase gene in carnations and its disruption by transposable elements in some varieties

The pink, red and crimson petal colors of carnations (Dianthus caryophyllus) are produced by anthocyanins. The anthocyanins, pelargonidin and cyanidin can be modified by two glucoses at the 3 and 5 positions, and by a single malic acid. Petal color variation can result from failure of such modification, for example, the lack of a glucose at the 5 position is responsible for the color variants of some commercial varieties. With respect to this variation, modification by 5-O-glucosyltransferase plays the most important role in glucosylation at the 5 position. Recently, we identified a novel acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase (AA5GT), that uses acyl-glucoses, but not UDP-glucose, as the glucose donor. Although we showed that loss of AA5GT expression was responsible for loss of glucosylation at the 5 position of anthocyanin in some varieties, the cause of this repression of AA5GT expression could not be determined. Here, we have succeeded in isolating the AA5GT gene and found that it consists of 12 exons and 11 introns. In carnation varieties lacking a glucose at the 5 position, we identified the insertion of two different retrotransposons, Ty1dic1 and Retdic1, into AA5GT. Ty1dic1, which belongs to the class I long terminal repeat (LTR)-retrotransposons of Ty1/copia families, was inserted into exon 10. Retdic1, which includes a long interspersed nuclear element (LINE)-like sequence, was inserted into intron 5. Thus, insertion of either Ty1dic1 or Retdic1 can disrupt AA5GT and result in the lack of glucosylation at the 5 position in anthocyanins.     

High pressure liquid chromatographic analysis of flavonoid chemical markers in petals from Gerbera flowers as an adjunct for cultivar and germplasm identification

Flavonoids present in petals from Gerbera flowers were resolved and quantitated by high pressure liquid chromatography (HPLC). The anthocyanins isolated from 18 cultivars, ranging in color from orange through lavender, were pelargonidin and cyanidin 3-malonylglucosides accompanied by smaller amounts of pelargonidin and cyanidin 3-glucosides. Related flavonoid copigments were apigenin and luteolin 4′-glucosides and 7-glucosides, apigenin 7-malonylglucoside, kaempferol and quercetin 3-glucosides, 4′-glucosides and 3-malonylglucosides. Both qualitative and quantitative differences in these flavonoid chemical markers distinguished cultivars with very similar colors. Malonyl esters of anthocyanins are easily degraded by HCl and conventional extraction and purification procedures were adjusted to preserve their natural state.     

Characterization of dihydroflavonol 4-reductases for recombinant plant pigment biosynthesis applications

Anthocyanins are colorful plant pigments with promising applications as pharmaceuticals and colorants. In order to engineer efficient pigment biosynthesis in Escherichia coli, the activities of various dihydroflavonol 4-reductases (DFRs) were characterized for the three primary dihydroflavonol substrates. The biochemical assays demonstrated variable DFR activities for dihydroflavonol with one B-ring hydroxyl group, the precursor of pelargonidin derivatives. In contrast, dihydroflavonols with two and three B-ring hydroxylation were metabolized with comparable efficiency. Furthermore, the catalysis of DFR for the secondary substrates, flavanones, also depended on the number of B-ring hydroxyl groups. Engineering the expression of the DFR clones together with plant-specific 4-coumaroyl:CoA ligase, chalcone synthase, chalcone isomerase, and flavanone 3-hydroxylase in E. coli resulted in the synthesis of pelargonidin at various levels, from p-coumaric acids. The identification of a robust DFR from this study can also be used for engineering recombinant synthesis of other bioactive flavonoids, such as flavan-3-ols.     

Genetic control of UDP-glucose: anthocyanin 5-O-glucosyltransferase from flowers of Matthiola incana R.Br.

In flower extracts of defined genotypes of Matthiola incana, an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5-diphosphoglucose (UDPGlc) to the 5-hydroxyl group of pelargonidin and cyanidin 3-glycosides and acylated derivatives. The best substrate for 5-glucosylation is the 3-xylosylglucoside acylated with p-coumarate, followed by the 3-xylosylglucoside and by the acylated (p-coumarate) 3-glucoside. The 3-glucoside itself is a very poor substrate. Besides UDPGlc, thymine 5-diphosphoglucose is a suitable glucosyl-donor, but with a reduced reaction rate (42%). The anthocyanin 5-O-glucosyltransferase exhibits a pH optimum at 7.5 and is generally inhibited by divalent ions and by ethylenediaminetetraacetic acid and p-chloromercuribenzoate. Investigations on different genotypes showed that the 5-O-glucosyltransferase activity is clearly controlled by the gene l. In confirmation of earlier chemogenetic work, enzyme activity is only present in lines with the wild-type allele l⁺. The anthocyanin 5-O-glucosyltransferase activity is strictly correlated with the formation of 5-glucosylated anthocyanins during bud development.Abbreviations Cg 3,5-T-cyanidin 3-sambubioside-5-glucoside - EDTA ethylene diaminetetraacetic acid - 5GT UDP-glucose: anthocyanin 5-O-glucosyltransferase - 3GT UDP-glucose: anthocyanidin/flavonol 3-O-glucosyltransferase - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - UDPGlc uridine 5-diphospho-glucose