Investigation of two distinct flavone synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4'-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.     

Investigation of Two Distinct Flavone Synthases for Plant-Specific Flavone Biosynthesis in Saccharomyces cerevisiae

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4′-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.     

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Cloning of parsley flavone synthase I

A cDNA encoding flavone synthase I was amplified by RT-PCR from leaflets of Petroselinum crispum cv. Italian Giant seedlings and functionally expressed in yeast cells. The identity of the recombinant, 2-oxoglutarate-dependent enzyme was verified in assays converting (2S)-naringenin to apigenin.     

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins

The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.     

A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Expression of a soluble flavone synthase allows the biosynthesis of phytoestrogen derivatives in Escherichia coli

Flavones are plant secondary metabolites with potent pharmacological properties. We report the functional expression of FSI, a flavonoid 2-oxoglutarate-dependent dioxygenase-encoding flavone synthase from parsley in Escherichia coli. This expression allows the biosynthesis of various flavones from phenylpropanoid acids in recombinant E. coli strains simultaneously expressing five plant-specific flavone biosynthetic genes. The gene ensemble consists of 4CL-2 (4-coumarate:CoA ligase) and FSI (flavone synthase I) from parsley, chsA (chalcone synthase) and chiA (chalcone isomerase) from Petunia hybrida, and OMT1A (7-O-methyltransferase) from peppermint. After a 24-h cultivation, the recombinant E. coli produces significant amounts of apigenin (415 μg/l), luteolin (10 μg/l), and genkwanin (208 μg/l). The majority of the flavone products are excreted in the culture media; however, 25% is contained within the cells. The metabolic engineering strategy presented demonstrates that plant-specific flavones are successfully produced in E. coli for the first time by incorporating a soluble flavone synthase confined only in Apiaceae.     

Analysis of flavonoids and characterization of theOsFNS gene involved in flavone biosynthesis in Rice

The major flavonoids in rice leaves were analyzed via LC-MS/MS after their total flavonoid extracts were hydrolyzed. The most abundant flavones were apigenin, luteolin, and tricetin. Of these, tricetin was methylated at its 3′ and 5′-hydroxyl group to form tricin, which was probablyO-glycosylated. Both 3′-O-methylated luteolin and luteolin were found in theC-glycosylated form while apigenin wasC-glycosylated. We also cloned and characterizedOsFNS, which catalyzes the reaction from flavanone (naringenin) to flavone (apigenin). Analysis of the reaction product with recombinant OsFNS showed that it indeed converts naringenin to apigenin.     

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.     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Metabolic profiling of tryptophan-overproducing rice calli that express a feedback-insensitive alpha subunit of anthranilate synthase

The profile of aromatic metabolites in calli was compared between wild-type rice (Oryza sativa cv. Nipponbare) and tryptophan-overproducing transgenic rice lines that express a gene (OASA1D) for a feedback-insensitive alpha subunit of anthranilate synthase. Metabolic profiling by high-performance liquid chromatography coupled with photodiode array detection of ultraviolet absorbance revealed a total of 71 peaks in both wild-type and transgenic calli. Only a limited effect on the pattern of major aromatic compounds was observed in tryptophan-accumulating transgenic rice lines, with the exception of an approximately 80-fold increase in the amount of tryptophan. Expression of OASA1D induced relatively small changes in several minor metabolites. One of the minor metabolites whose abundance was increased by OASA1D expression was purified and identified as a previously unknown indole-alkaloid glucoside. The levels of free and conjugated forms of indole-3-acetic acid (IAA), a plant hormone derived from the tryptophan biosynthetic pathway, were determined separately by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The amounts of both free IAA and its conjugates were increased in the transgenic calli, suggesting that the activity of anthranilate synthase or the concentration of tryptophan (or both) is an important determinant of IAA biosynthesis.     

Twelve 6-oxygenated flavone sulphates from Lippia nodiflora andL. canescens

From the aerial parts of the maritime plantLippia nodiflora, 15 flavonoids, 3 flavone aglycones and 12 new flavone sulphates, have been isolated and identified. The new flavone sulphates are mono- and disulphates of nepetin, jaceosidin, hispidulin, 6-hydroxyluteolin and nodifloretin present as the sodium salts. These sulphates are the only flavone conjugates detected in this plant. Flavone trisulphates are additionally present in populations of this species from Malaysia and Saudi Arabia, but lack of plant material prevented their complete characterization. Analysis of the closely related speciesLippia canescens showed that it has the same flavonoid pattern. By contrast, a third speciesL. triphylla showed a flavonoid pattern lacking flavonoid sulphates, but characterized by the presence of 7-glucuronylglucosides of luteolin, diosmetin and apigenin. This is the first finding of flavonoid sulphates in the Verbenaceae.     

Combinatorial biosynthesis of flavones and flavonols in Escherichia coli

(2S)-Flavanones (naringenin and pinocembrin) are key intermediates in the flavonoid biosynthetic pathway in plants. Recombinant Escherichia coli cells containing four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:CoA ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-CoA carboxylase, have been established for efficient production of (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. Further introduction of the flavone synthase I gene from Petroselinum crispum under the control of the T7 promoter and the synthetic ribosome-binding sequence in pACYCDuet-1 caused the E. coli cells to produce flavones: apigenin (13 mg/l) from tyrosine and chrysin (9.4 mg/l) from phenylalanine. Introduction into the E. coli cells of the flavanone 3β-hydroxylase and flavonol synthase genes from the plant Citrus species led to production of flavonols: kaempferol (15.1 mg/l) from tyrosine and galangin (1.1 mg/l) from phenylalanine. The combinatorial biosynthesis of the flavones and flavonols in E. coli is promising for the construction of a library of various flavonoid compounds and un-natural flavonoids in bacteria.     

1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants

The initial step of the plastidic 2C-methyl-D-erythritol 4-phosphate (MEP) pathway that produces isopentenyl diphosphate is catalyzed by 1-deoxy-d-xylulose-5-phosphate synthase. To investigate whether or not 1-deoxy-d-xylulose-5-phosphate synthase catalyzes a limiting step in the MEP pathway in plants, we produced transgenic Arabidopsis plants that over- or underexpress this enzyme. Compared with non-transgenic wild-type plants, the transgenic plants accumulate different levels of various isoprenoids such as chlorophylls, tocopherols, carotenoids, abscisic acid, and gibberellins. Phenotypically, the transgenic plants had slight alterations in growth and germination rates. Because the levels of several plastidic isoprenoids correlate with changes in 1-deoxy-D-xylulose-5-phosphate synthase levels, we conclude that this enzyme catalyzes one of the rate-limiting steps of the MEP biosynthetic pathway. Furthermore, since the product of the MEP pathway is isopentenyl diphosphate, our results suggest that in plastids the pool of isopentenyl diphosphate is limiting to isprenoid production.     

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.     

Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biological synthesis of plant secondary metabolites has attracted increasing attention due to their proven or assumed beneficial properties and health-promoting effects. Phenylpropanoids are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. In this study, engineered Escherichia coli containing artificial phenylpropanoid biosynthetic pathways utilizing tyrosine as the initial precursor were established for production of plant-specific metabolites such as ferulic acid, naringenin, and resveratrol. The construction of the artificial pathway utilized tyrosine ammonia lyase and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis, cinnamate/4-coumarate:coenzyme A ligase from Streptomyces coelicolor, caffeic acid O-methyltransferase and chalcone synthase from Arabidopsis thaliana, and stilbene synthase from Arachis hypogaea.