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.     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.     

Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species

Red colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. There has been no report so far to find anthocyanins and betacyanins existing together within the same plant. This curious phenomenon has been examined from genetic and evolutionary perspectives, however nothing is known at the molecular level about the mutual exclusion of anthocyanins and betacyanins in higher plants. Here, we show that spinach (Spinacia oleracea) and pokeweed (Phytolacca americana), which are both members of the Caryophyllales, have functional anthocyanidin synthases (ANSs). The ability of ANSs of the Caryophyllales to oxidize trans-leucocyanidin to cyanidin is comparable to that of ANSs in anthocyanin-producing plants. Expression profiles reveal that, in spinach, dihydroflavonol 4-reductase (DFR) and ANS are not expressed in most tissues and organs, except seeds, in which ANS may contribute to proanthocyanidin synthesis. One possible explanation for the lack of anthocyanins in the Caryophyllales is the suppression or limited expression of the DFR and ANS.     

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.     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Precursors and genetic control of anthocyanin synthesis in Matthiola incana R. Br.

After application of dihydroflavonols, naringenin, or suitable substituted chalcones, anthocyanins were synthesized in three genetically defined acyanic lines of Matthiola incana, indicating that the corresponding genetic block concerns the synthesis of the chalcone-flavanone intermediate. Independent of the precursors used, only cyanidin derivatives were produced. This supports the hypothesis that the oxygenation pattern of the B ring in anthocyanin formation is determined at a stage of a C₁₅ intermediate. In addition to the gene responsible for the oxygenation of the 3 position, the genes responsible for the glycosylation in the 3 and 5 positions of the anthocyanin molecule, and those responsible for the acylation with various hydroxycinnamic acids can still exert their influence. Two further genetically defined lines containing flavonol glycosides were not able to synthesize anthocyanins with any of the precursors tested. Their genetic blocks are assumed to be localized after dihydroflavonol synthesis but before anthocyanin formation.     

Characterization of uridine-diphosphate dependent flavonoid glucosyltransferase from Oryza sativa

We cloned a uridine-diphosphate dependent glycosyltransferase RUGT-10 from Oryza sativa. The recombinant enzyme was expressed by glutathione-S transferase gene fusion system in Escherichia coli. RUGT10 showed different regioselectivity depending on the structures of substrates (e.g. flavanone, flavonol, and flavone). Apparently, flavanone such as naringenin and eriodictyol gave one 7-O-glucoside while flavone and flavonol gave more than two products with preferential glucosylation position of hydroxyl group at C-3 position.     

Application of Pseudomonas fluorescens to Blackberry under Field Conditions Improves Fruit Quality by Modifying Flavonoid Metabolism

Application of a plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens N21.4, to roots of blackberries (Rubus sp.) is part of an optimised cultivation practice to improve yields and quality of fruit throughout the year in this important fruit crop. Blackberries are especially rich in flavonoids and therefore offer potential benefits for human health in prevention or amelioration of chronic diseases. However, the phenylpropanoid pathway and its regulation during ripening have not been studied in detail, in this species. PGPR may trigger flavonoid biosynthesis as part of an induced systemic response (ISR) given the important role of this pathway in plant defence, to cause increased levels of flavonoids in the fruit. We have identified structural genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways catalysing the conversion of phenylalanine to the final products including flavonols, anthocyanins and catechins from blackberry, and regulatory genes likely involved in controlling the activity of pathway branches. We have also measured the major flavonols, anthocyanins and catechins at three stages during ripening. Our results demonstrate the coordinated expression of flavonoid biosynthetic genes with the accumulation of anthocyanins, catechins, and flavonols in developing fruits of blackberry. Elicitation of blackberry plants by treatment of roots with P.fluorescens N21.4, caused increased expression of some flavonoid biosynthetic genes and an accompanying increase in the concentration of selected flavonoids in fruits. Our data demonstrate the physiological mechanisms involved in the improvement of fruit quality by PGPR under field conditions, and highlight some of the genetic targets of elicitation by beneficial bacteria.     

二氢黄酮醇 4 还原酶在花青素合成中的功能及调控研究进展

植物色素主要有花青素、类胡萝卜素和生物碱类色素三大类,其中花青素是决定大部分被子植物组织或器官颜色的重要色素。花青素通过类黄酮途径合成,该途径是生物学上研究较多且较为清楚的代谢途径之一。近年来的研究表明,在该途径中除了查尔酮合成酶(chalcone synthase,CHS)、查尔酮异构酶(chalcone isomerase,CHI)和黄烷酮-3-羟化酶(flavanone-3-hydrolase,F3H)起着关键作用外,二氢黄酮醇-4-还原酶(dihydroflavonol 4-reductase,DFR)对花青素的合成也至关重要。DFR可催化3种二氢黄酮醇和2种黄烷酮生成5种不同的花青素前体,且DFR基因家族不同成员对各个底物的催化效率不同,因此它在一定程度上决定着植物中花青素的种类和含量,从而影响植物组织或器官的颜色。该文对近年来国内外有关DFR在花青素合成过程中的生物学功能与调控,包括DFR的特征、作用机制和系统进化以及环境、转录因子和一些结构基因与DFR的关系等方面的研究进展进行了综述,以期为DFR今后的研究和利用基因工程改变植物组织或器官的颜色提供理论依据。     

Activation of flavonoid biosynthesis by solar radiation in bilberry (<Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> L.) leaves

The effect of solar radiation on flavonoid biosynthesis was studied in bilberry (Vaccinium myrtillus L.) leaves. Expression of flavonoid pathway genes of bilberry was studied in the upper leaves of bilberry, exposed to direct sunlight, in the shaded leaves growing lower in the same plants and in fruits. Bilberry-specific digoxigenin–dUTP-labeled cDNA fragments of five genes from the general phenylpropanoid pathway coding phenylalanine ammonia-lyase and from the flavonoid pathway coding chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase were used as probes in gene expression analysis. Anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids from the leaves and fruits were identified and quantified using high-performance liquid chromatography combined with a diode array detector. An increase in the expression of the studied flavonoid pathway genes was observed in leaves growing under direct sun exposure. Also, the concentrations of anthocyanins, catechins, flavonols and hydroxycinnamic acids were higher in the leaves exposed to direct sunlight. However, the concentration of polymeric procyanidins was lower in sun-exposed leaves, whereas that of prodelphinidins was slightly increased. The results give further support for the protective role of flavonoids and hydroxy cinnamic acids against high solar radiation in plants. Also, the roles of different flavonoid compounds as a defense against stress caused by sun exposure is discussed.Abbreviations ANS Anthocyanidin synthase - CHS Chalcone synthase - DFR Dihydroflavonol 4-reductase - F3H Flavanone 3-hydroxylase - GPD Glyceraldehyde-3-phosphate dehydrogenase - PAL Phenylalanine ammonia-lyase     

Maize <Emphasis Type="Italic">Lc</Emphasis> transcription factor enhances biosynthesis of anthocyanins,distinct proanthocyanidins and phenylpropanoids in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Here, we report on metabolic engineering of the flavonoid biosynthetic pathways in apple (Malus domestica Borkh.) by overexpression of the maize (Zea mays L.) leaf colour (Lc) regulatory gene. The Lc gene was transferred into the M. domestica cultivar Holsteiner Cox via Agrobacterium tumefaciens-mediated transformation which resulted in enhanced anthocyanin accumulation in regenerated shoots. Five independent Lc lines were investigated for integration of Lc into the plant genome by Southern blot and PCR analyses. The Lc-transgenic lines contained one or two Lc gene copies and showed increased mRNA levels for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3 beta-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductases (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). HPLC-DAD and LC-MS analyses revealed higher levels of the anthocyanin idaein (12-fold), the flavan 3-ol epicatechin (14-fold), and especially the isomeric catechin (41-fold), and some distinct dimeric proanthocyanidins (7 to 134-fold) in leaf tissues of Lc-transgenic lines. The levels of phenylpropanoids and their derivatives were only slightly increased. Thus, Lc overexpression in Malus domestica resulted in enhanced biosynthesis of specific flavonoid classes, which play important roles in both phytopathology and human health.     

Engineering of flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway

Flower color was modified in forsythia (Forsythia x intermedia cv Spring Glory) by inducing anthocyanin synthesis in petals through sequential Agrobacterium-mediated transformation with dihydroflavonol 4-reductase from Antirrhinum majus (AmDFR) and anthocyanidin synthase from Matthiola incana (MiANS) genes. This is the second report of flower color modification of an ornamental shrub after rose, and the first time an ANS gene is used for this purpose. Double transformants (AmDFR+MiANS) displayed a novel bronze-orange petal color, caused by the de novo accumulation of cyanidin-derived anthocyanins over the carotenoid yellow background of wild type (wt), and intense pigmentation of vegetative organs. Transformation with single genes (either AmDFR or MiANS) produced no change in flower color, showing a multistep control of late anthocyanin pathway in petals of forsythia. Analysis of relevant late flavonoid pathway genes – an endogenous flavonoid glycosyltransferase (FiFGT) and transformed DFR and ANS genes – showed appropriate expression in flower organs. Functional characterization of FiFGT expressed in E. coli revealed its ability to metabolize both flavonols and anthocyanidin substrates, a prerequisite for effective anthocyanin accumulation in petals of plants transformed with constructs leading to anthocyanidin synthesis. Biochemical analyses of flavonoid compounds in petals and leaves showed that, besides anthocyanin induction in petals of double transformants, the accumulation pattern of flavan-3-ols was quantitatively and qualitatively modified in petals and leaves of transformants, in agreement with the most recent model proposed for flavan-3-ol synthesis. On the other hand, phenylpropanoid, flavone and flavonol pools were not quantitatively affected, indicating a tight regulation of early flavonoid pathway.     

Biosynthesis of natural flavanones in Saccharomyces cerevisiae

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.