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.     

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.     

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.     

The monomeric polypeptide comprises the functional flavanone 3beta-hydroxylase from Petunia hybrida

Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthesis of flavonoids, catechins and anthocyanidins. The enzyme had been partially purified from Petunia hybrida and proposed to be active as a dimer of roughly 75 kDa in size. More recently, the Petunia 3beta-hydroxylase was cloned and shown to be encoded in a 41655 Da polypeptide. In order to characterize the molecular composition, the enzyme was expressed in a highly active state in Escherichia coli and purified to apparent homogeneity. Size exclusion chromatographies of the pure, recombinant enzyme revealed that this flavanone 3beta-hydroxylase exists in functional monomeric and oligomeric forms. Protein cross-linking experiments employing a specific homobifunctional sulfhydryl group reagent or the photochemical activation of tryptophan residues confirmed the tendency of the enzyme to aggregate to oligomeric complexes in solution. Thorough equilibrium sedimentation analyses, however, revealed a molecular mass of 39. 2+/-12 kDa for the recombinant flavanone 3beta-hydroxylase. The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3beta-hydroxylase of P. hybrida, which may readily associate in vivo with other proteins.     

Flavanones structure-related inhibition on TPA-induced tumor promotion through suppression of extracellular signal-regulated protein kinases: involvement of prostaglandin E2 in anti-promotive process

Biological functions of flavanones have been studied extensively, however, the structure-related activities of flavanones on 12-o-tetradecanoylphorbol 13-acetate (TPA)-induced promotive effects are still unclear. In this study, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the most significant dose-dependent inhibition on TPA-induced proliferative effects among eight tested flavanones in NIH3T3 cells. TPA-induced mitogen activated protein kinases (MAPK) phosphorylation, ornithine decarboxylase (ODC), c-Jun, and cyclooxygenase 2 (COX-2) protein expressions in a time-dependent manner, and the maximal inductive time point is at 1 h for MAPK phosphorylation and 6 h for others. Flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the dose-dependent inhibition on TPA-stimulated MAPK phosphorylation, COX-2, ODC, c-Jun protein expressions. Induction of, prostaglandin E(2) (PGE(2)) production was detected in TPA-treated NIH3T3 cells, and flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone inhibited significantly PGE(2) production induced by TPA. Addition of PGE(2) reverses the inhibitory activities of flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone on TPA-induced proliferation. And, PD98059, a specific inhibitor of ERKs, inhibited TPA-induced MAPK phosphorylation, accompanied by decreasing COX-2, c-Jun, and ODC protein expression, and showed dose-dependent inhibition on TPA-induced proliferation in cells. These results demonstrated that PGE(2) is an important mediator in TPA-induced proliferation, and MAPK phosphorylation was located at the upstream of COX-2, c-Jun, and ODC gene expressions in TPA-induced responses. Furthermore, flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone (100 microM) suppressed TPA-induced colony formation associated with blocking MAPK phosphorylation, ODC, c-Jun, and COX-2 proteins expression. And, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone did not perform potent anti-radical activities among these eight tested compounds. In conclusion, this study provided molecular evidences to demonstrate that flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone were potent inhibitors on TPA-induced responses without notable cytotoxicity through suppression of PGE(2) production; and anti-radical activity of flavanones was not correlated with preventing the occurrence of tumor promotion. We proposed that blocking TPA-induced intracellular signaling responses might be involved in the anti-promotive mechanism of flavanones.     

Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.     

Multiple mutagenesis of P450 isoflavonoid synthase reveals a key active-site residue

The leguminous isoflavonoid skeleton is constructed by P450 2-hydroxyisoflavanone synthase (CYP93C). Two active-site residues of CYP93C2, Ser 310 and Lys 375, are critical for unusual aryl migration of the flavanone substrate. Leu 371 is located near the substrate in a homology model, and mutant proteins regarding this residue were expressed in recombinant yeast microsomes. The single mutant, L371V, yielded only inactive P420, but multiple mutants incorporating K375T restored the P450 fold: the S310T-L371V-K375T triple mutant showed four times higher P450 level than the wild type. L371V-K375T and S310T-L371V-K375T produced a mixture of major 3beta-hydroxyflavanone and minor flavone, and 100% flavone, respectively, from a flavanone. Thus, Leu 371 appeared to control the substrate accommodation in favor of hydrogen abstraction from C-3 of the flavanone molecule and contribute to the P450 fold under the presence of Lys 375, the residue responsible for aryl migration. The molecular evolution of CYP93 enzymes is discussed.     

Flavanone and 2'-OH flavanone inhibit metastasis of lung cancer cells via down-regulation of proteinases activities and MAPK pathway

Flavanones richly exist in citrus and have been well characterized to have various bioactive properties. However, the anti-metastasis properties of flavanones remain unclear. The anti-metastatic effects of six flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, naringin, and naringenin were investigated in lung cancer cells. Despite little influence on cell viability, flavanone and 2'-OH flavanone markedly inhibited the invasion, motility, and cell-matrix adhesion of A549 cells. This was associated with a reduced expression of matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) in treated cells. Treatment with flavanone and 2'-OH flavanone also potently attenuated the phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38(MAPK), as well as the activations of NF-kappaB and AP-1. The reduced expressions of MMP-2 and u-PA, as well as inhibition of cell invasion were obtained in the cultures treated with U0126 (ERK 1/2 inhibitor) and SB203580 (p38(MAPK) inhibitor). Thus, the inhibitory effects of flavanone and 2'-OH flavanone on the expression of MMP-2 and u-PA may be at least partly through inactivation of ERK 1/2 and p38(MAPK) signaling pathways. Finally, oral administration of flavanone and 2'-OH flavanone were evidenced by its inhibition on the metastasis of A549 cells and Lewis lung carcinoma (LLC) cells in vivo. In conclusion, flavanone and 2'-OH flavanone perturb the invasion and metastasis of lung cancer cells, thereby constituting an adjuvant treatment for metastasis control.     

Identification of flavanone metabolites in rat urine by combined gas–liquid chromatography and mass spectrometry

1. The metabolism of flavanone in the rat was studied after oral or intraperitoneal administration of the compound. Flavone and flav-3-ene together with five other unidentified minor metabolites were excreted in the urine. 2. The formation of flavanone metabolites was not suppressed by the administration of high doses of the antibacterial compounds aureomycin and phthaloylsulphathiazole. 3. No aromatic acids that could be attributed to ring cleavage of flavanone were detected. 4. Administration of 100 or 200mg of flavanone daily per rat caused some deaths during the 7-14-day period. 5. The application of combined gas-liquid chromatography/mass spectrometry and proton nuclear-magnetic-resonance spectroscopy to the separation and identification of the flavanone metabolites is described. 6. Measurement of the two major flavanone metabolites was carried out by gas-liquid chromatography.     

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.     

Enhanced flavonoid production in Streptomyces venezuelae via metabolic engineering

Metabolic engineering of plant-specific phenylpropanoid biosynthesis has attracted an increasing amount of attention recently, owing to the vast potential of flavonoids as nutraceuticals and pharmaceuticals. Recently, we have developed a recombinant Streptomyces venezuelae as a heterologous host for the production of flavonoids. In this study, we successfully improved flavonoid production by expressing two sets of genes predicted to be involved in malonate assimilation. The introduction of matB and matC encoding for malonyl-CoA synthetase and the putative dicarboxylate carrier protein, respectively, from Streptomyces coelicolor into the recombinant S. venezuelae strains expressing flavanone and flavone biosynthetic genes resulted in enhanced production of both flavonoids.     

The tumor-growth inhibitory activity of flavanone and 2′-OH flavanone <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> through induction of cell cycle arrest and suppression of cyclins and CDKs

Summary Natural products, including flavonoids, are suggested to be involved in the protective effects of fruits and vegetables against cancer. However, studies concerning the effect of flavonoids frequently lacked data regarding to flavanones. In this study, we investigated the inhibitory effect of flavanone compounds, including flavanone, 2′-OH flavanone, 4′-OH flavanone, 6-OH flavanone, naringin and naringenin, on cell growth of various cancer cells. We determined that flavanone and 2′-OH flavanone inhibited cell growth of A549, LLC, AGS, SK-Hepl and HA22T cancer cells, while other flavanones showed little or no inhibition. We evaluated growth-inhibitory activity of flavanone and 2′-OH flavanone against highly proliferative human lung cancer cells (A549) via anchorage-independent and -dependent colony formation assay, and further showed that treatment of flavanone resulted in a G1 cell cycle arrest with reduction of cyclin D, E and cyclin-dependent kinase (CDK) 2, while treatment of 2′-OH flavanone led to a G2/M phase accumulation with reduction of cyclin B, D and Cdc2. Moreover, we demonstrated the improvement effect of flavanone and 2′-OH flavanone with anti-cancer drug, doxorubicin, on A549 cells. Finally, flavanone and 2′-OH flavanone were evidenced by its inhibition on the growth of A549 and Lewis lung carcinoma cells in vivo. Yung-Chin Hsiao and Yih-Shou Hsieh are equally contributed to this work.     

Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirA_Pl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirA_Ec) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirA_Ec and the C terminus of BirA_Pl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.     

Eight new biflavonoids with lavandulyl units from the roots of Sophora flavescens and their inhibitory effect on PTP1B

In the course of studying the components from the roots of Sophora flavescens, eight new unusual biflavonoids consisting of a flavanone fused with a dihydrochalcone skeleton were isolated. These new chemical structures were elucidated by means of UV, IR, HRESIMS, NMR and ECD spectroscopic data and a comparison of experimental ECD spectra with calculated ECD spectra. Some compounds were subjected to an antidiabetic bioassay on human recombinant PTP1B inhibition, and showed strong inhibitory activity.     

茼蒿总黄酮提取及对羟自由基清除作用

采用超声波乙醇浸提法从茼蒿中提取黄酮类物质,对所提取的黄酮类物质进行验证,并用分光光度法测定含量,用茼蒿总黄酮对羟自由基清除作用进行试验。测得样品中总黄酮的含量C=0.169 0 mg/mL,回收率为101.3%,其纯度和产率均较高。该方法采用全物理过程,无任何污染,是提取茼蒿黄酮类物质的有效途径。茼蒿总黄酮提取液对Fenton体系产生的.OH自由基有很好的清除作用。     

Enzymic synthesis of an aromatic ring from acetate units. Partial purification and some properties of flavanone synthase from cell-suspension cultures of Petroselinum hortense.

Flavanone synthase was isolated and purified about 300-fold from fermenter-grown, light-induced cell suspension cultures of Petroselinum hortense. The enzyme catalyzed the formation of the flavanone naringenin from p-coumaroyl-CoA and malonyl-CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed. No coafactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl-CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30 degrees C, and at an ionic strength of 0.1--0.3 M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about 0.7 muM did not affect the enzyme activity. Apparent molecular weights of approx. 120 000, 50 000, and 70 000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl-CoA: flavonoid glycoside malonyl transferase. The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl-CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6-methylsalicylic acid synthesis from "acetate units", are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.     

Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster

For the fermentative production of plant-specific flavanones (naringenin, pinocembrin) by Escherichia coli, a plasmid was constructed which carried an artificial biosynthetic gene cluster, including PAL encoding a phenylalanine ammonia-lyase from a yeast, ScCCL encoding a cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor A3(2), CHS encoding a chalcone synthase from a licorice plant and CHI encoding a chalcone isomerase from the Pueraria plant. The recombinant E. coli cells produced (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. When the two subunit genes of acetyl-CoA carboxylase from Corynebacterium glutamicum were expressed under the control of the T7 promoter and the ribosome-binding sequence in the recombinant E. coli cells, the flavanone yields were greatly increased, probably because enhanced expression of acetyl-CoA carboxylase increased a pool of malonyl-CoA that was available for flavanone synthesis. Under cultural conditions where E. coli at a cell density of 50 g/l was incubated in the presence of 3 mM tyrosine or phenylalanine, the yields of naringenin and pinocembrin reached about 60 mg/l. The fermentative production of flavanones in E. coli is the first step in the construction of a library of flavonoid compounds and un-natural flavonoids in bacteria.