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Sueharu Horinouchi - One of the best experts on this subject based on the ideXlab platform.
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efficient production of 2s Flavanones by escherichia coli containing an artificial biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2005Co-Authors: Ikuo Miyahisa, Masafumi Kaneko, Hisashi Kawasaki, Nobutaka Funa, Yasuo Ohnishi, Hiroyuki Kojima, Sueharu HorinouchiAbstract: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.
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Efficient production of (2S)-Flavanones by Escherichia coli containing an artificial biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2005Co-Authors: Ikuo Miyahisa, Masafumi Kaneko, Hisashi Kawasaki, Nobutaka Funa, Yasuo Ohnishi, Hiroyuki Kojima, Sueharu HorinouchiAbstract: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. © Springer-Verlag 2005.
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production of plant specific Flavanones by escherichia coli containing an artificial gene cluster
Applied and Environmental Microbiology, 2003Co-Authors: Eui Il Hwang, Masafumi Kaneko, Yasuo Ohnishi, Sueharu HorinouchiAbstract:In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, and flavonoids. In addition to their roles in the structure and protection of the plant, phenylpropanoid compounds have an important effect on plant qualities, such as texture, flavor, color, and other characteristics (29). Recently, the flavonoid-derived plant natural products have drawn much attention because of their benefits to human health, such as antimicrobial, cancer chemopreventive, antioxidant, and antiasthmatic activities (6, 14). These phenylpropanoid and flavonoid biosynthetic enzymes are therefore attractive targets for metabolic engineering to provide enhancement or initiation of the production of economically desirable traits or compounds. The phenylpropanoid and flavonoid biosynthetic pathways and their regulation have been the subject of many studies (4, 5, 25, 29). Recent advances in the regulation of the pathways and the biochemistry of specific enzymes and enzyme complexes have opened up strategies for the enhancement of flavonoid compounds by modifying the pathways (4). The phenylpropanoid pathway in plants converts phenylalanine into naringenin chalcone (Fig. (Fig.1).1). As the first step, phenylalanine is deaminated to yield cinnamic acid by the action of phenylalanine ammonia lyase (PAL). Cinnamic acid is hydroxylated by cinnamate-4-hydroxylase (C4H) to 4-coumaric acid, which is then activated to 4-coumaroyl-coenzyme A (CoA) by the action of 4-coumarate:CoA ligase (4CL). Chalcone synthase (CHS) catalyzes the stepwise condensation of three acetate units from malonyl-CoA with 4-coumaroyl-CoA to yield naringenin chalcone, the precursor for a large number of flavonoids (29). Naringenin chalcone is converted to naringenin by chalcone isomerase or nonenzymatically in vitro (5, 19, 24, 25). Because some of the PALs show tyrosine ammonia lyase activity, tyrosine is also used as the precursor (2, 15, 22, 26). FIG. 1. Flavanone biosynthetic pathway in plants. The dashed arrows represent the expected flavanone biosynthetic pathway in E. coli containing the artificial gene cluster including PAL, 4CL, and CHS. TAL, tyrosine ammonia-lyase; CHI, chalcone isomerase. Production of Flavanones or flavonoids by genetically engineered bacteria has not been reported before, although the heterologous expression of phenylpropanoid biosynthetic enzymes in bacteria has been reported (1, 8, 16, 30). One of the barriers for the production of these compounds is the difficulty in expression of active C4H, which would not be efficiently expressed due to its instability and the lack of its specific cytochrome P-450 reductase in bacteria (8, 20). We have recently discovered a 4CL in the gram-positive filamentous bacterium Streptomyces coelicolor A3(2) that can activate cinnamic acid to cinnamoyl-CoA, as well as 4-coumaric acid to 4-coumaroyl-CoA (12). The use of the bacterial 4CL enzyme would bypass the C4H step for the production of pinocembrin chalcone from phenylalanine via the phenylpropanoid pathway (Fig. (Fig.1).1). On the basis of this idea, we constructed an artificial gene cluster containing PAL from a yeast, Rhodotorula rubra; 4CL from an actinomycete, S. coelicolor A3(2); and CHS from a licorice plant, Glycyrrhiza echinata, to produce a plant-specific flavanone, pinocembrin. As expected, the Escherichia coli cells containing the gene cluster produced pinocembrin. In addition, the E. coli cells produced naringenin, because the yeast PAL enzyme also used tyrosine as a substrate and yielded 4-coumaric acid, as is found for many PALs (2, 22, 26), and the 4CL enzyme used cinnamic acid and 4-coumaric acid to yield the corresponding CoA thioester compounds. This paper describes successful production of plant-specific Flavanones, pinocembrin and naringenin, in E. coli. Furthermore, we compared the yields of the Flavanones by E. coli cells that contained the gene clusters in different organizations.
Olivier Dangles - One of the best experts on this subject based on the ideXlab platform.
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a comprehensive review on Flavanones the major citrus polyphenols
Journal of Food Composition and Analysis, 2014Co-Authors: Muhammad Kamran Khan, Olivier DanglesAbstract:The consumption of Citrus fruits and juices has been widely investigated for its possible role in the prevention of cardiovascular disease and cancer. These beneficial effects are mainly attributed to Flavanones, the typical polyphenols of Citrus species. Major Flavanones in plant species include hesperetin, naringenin, eriodictyol, isosakuranetin and their respective glycosides. Hesperetin and its derivatives are characteristic Flavanones of sweet orange, tangelo, lemon and lime, while naringenin and its derivatives are those of grapefruit and sour orange. Advances in analytical techniques like ultra high performance liquid chromatography (UPLC) coupled with mass spectrometry has facilitated (a) the estimation of flavanone contents in other plant species and in humans after ingestion and (b) the determination of flavanone metabolites more rapidly and with greater efficiency. The present review will summarize the current knowledge about Flavanones from their occurrence in plants to the bioactivity of their metabolites in humans.
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A comprehensive review on Flavanones, the major citrus polyphenols
Journal of Food Composition and Analysis, 2014Co-Authors: Muhammad Kamran Khan, Olivier DanglesAbstract:The consumption of Citrus fruits and juices has been widely investigated for its possible role in the prevention of cardiovascular disease and cancer. These beneficial effects are mainly attributed to Flavanones, the typical polyphenols of Citrus species. Major Flavanones in plant species include hesperetin, naringenin, eriodictyol, isosakuranetin and their respective glycosides. Hesperetin and its derivatives are characteristic Flavanones of sweet orange, tangelo, lemon and lime, while naringenin and its derivatives are those of grapefruit and sour orange. Advances in analytical techniques like ultra high performance liquid chromatography (UPLC) coupled with mass spectrometry has facilitated (a) the estimation of flavanone contents in other plant species and in humans after ingestion and (b) the determination of flavanone metabolites more rapidly and with greater efficiency. The present review will summarize the current knowledge about Flavanones from their occurrence in plants to the bioactivity of their metabolites in humans. © 2013 Elsevier Inc.
Christine Morand - One of the best experts on this subject based on the ideXlab platform.
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Flavanones protect from arterial stiffness in postmenopausal women consuming grapefruit juice for 6 mo a randomized controlled crossover trial
The American Journal of Clinical Nutrition, 2015Co-Authors: Dragan Milenkovic, Andrzej Mazur, Veronique Habauzit, Marieanne Verny, Nicolas Barberchamoux, Claude Dubray, Christine MorandAbstract:BACKGROUND: The consumption of citrus fruits is associated with health benefits. However, clinical data regarding the effects of grapefruit flavanone consumption on vascular function are lacking. OBJECTIVE: The objective of the present study was to address the role of Flavanones in the long-term effects induced by grapefruit juice (GFJ) consumption on vascular function in healthy postmenopausal women. DESIGN: Forty-eight healthy postmenopausal women aged 50-65 y within 3-10 y since menopause, a body mass index (in kg/m(2)) of 19-30, and a waist size >88 cm completed this double-blind, randomized, controlled, crossover trial. These volunteers were randomly assigned to consume 340 mL GFJ/d, providing 210 mg naringenin glycosides, or a matched control drink without Flavanones for 6 mo each, with a 2-mo washout between beverages. The primary endpoint was the assessment of endothelial function in the brachial artery by using flow-mediated dilation. Blood pressure, arterial stiffness, and endothelial function in the peripheral arterial bed were also evaluated as indicators of vascular function. These measurements and blood collection for clinical biochemical markers were performed in overnight-fasted subjects before and after the 6-mo treatment periods. RESULTS: The mean ± SD carotid-femoral pulse wave velocity, which reflects central aortic stiffness, was statistically significantly lower after consumption of GFJ (7.36 ± 1.15 m/s) than after consumption of the matched control drink without Flavanones (7.70 ± 1.36 m/s), with a P value of 0.019 for the treatment effect. Endothelial function in macro- and microcirculation, blood pressure, anthropometric measures, glucose metabolism, and biomarkers of inflammation and oxidative stress were not affected by the intervention. CONCLUSIONS: Regular GFJ consumption by middle-aged, healthy postmenopausal women is beneficial for arterial stiffness. This effect may be related to Flavanones present in grapefruit. This trial was registered at clinicaltrials.gov as NCT01272167.
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citrus Flavanones what is their role in cardiovascular protection
Journal of Agricultural and Food Chemistry, 2012Co-Authors: Audrey Chanet, Claudine Manach, Dragan Milenkovic, Andrzej Mazur, Christine MorandAbstract:Flavanones, including hesperidin and naringin, are polyphenolic compounds highly and almost exclusively present in citrus. Epidemiological studies reported an inverse relationship between their intake and the risk of cardiovascular diseases. Clinical and experimental data further showed their antihypertensive, lipid-lowering, insulin-sensitizing, antioxidative, and anti-inflammatory properties, which could explain their antiatherogenic action in animal models. Although Flavanones may be promising compounds that are particularly active in cardiovascular disease prevention, clinical data are still scarce and most in vitro data have been obtained under nonphysiologically relevant conditions. Moreover, the mechanisms responsible for flavanone action are not fully elucidated. Therefore, further research is needed to better evaluate and understand the protective effects of Flavanones in cardiovascular diseases.
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differential effects of two citrus Flavanones on bone quality in senescent male rats in relation to their bioavailability and metabolism
Bone, 2011Co-Authors: Veronique Habauzit, Christine Morand, Sandra Maria Sacco, Angel Gilizquierdo, Anna Trzeciakiewicz, Denis Barron, Stephane Pinaud, Elisabeth Offord, Marienoelle HorcajadaAbstract:Abstract The effect of hesperidin (Hp) and naringin (Nar), two major citrus Flavanones, on the regulation of bone metabolism was examined in male senescent rats. Twenty -month -old gonad-intact male Wistar rats received a casein-based diet supplemented with or without either 0.5% hesperidin (Hp), 0.5% naringin (Nar) or a mix of both Flavanones (Hp + Nar, 0.25% each). After 3 months, daily Hp intake significantly improved femoral bone integrity as reflected by improvements in total and regional bone mineral densities (BMD) (9.7%–12.3% improvements, p 5-fold higher than that of hesperidin (1.44 μM) at equivalent doses (0.5%) and a linear reduction in plasma levels was observed upon co-administration (0.25% each) indicating absence of competition for their intestinal absorption sites and metabolism. The higher efficacy of Hp at a lower plasma concentration than naringin, as well as the identification of the major circulating metabolite of hesperidin (hesperetin-7-O-glucuronide) underlines the importance of flavanone bioavailability and metabolism in their biological efficacy and suggests a structure–function relationship in the mechanism of action of the active metabolites.
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Flavanone metabolism in healthy and tumor-bearing rats.
Biomedicine & Pharmacotherapy, 2006Co-Authors: Mathieu Silberberg, Angel Gil-izquierdo, Christian Rémésy, Lydie Combaret, Augustin Scalbert, Christine MorandAbstract:Abstract Flavanones, the main polyphenols of citrus fruits, are thought to contribute to the protective effects of these fruits against cardiovascular diseases and cancer. The metabolism of naringin (naringenin 7- O -neohesperidoside) is studied here in healthy (sham-operated, ShO) and tumor-bearing (TuB) rats. The tumor was induced by implanting Yoshida's sarcoma in hindlimb. Both groups received for 7 days a semi-synthetic diet containing 0.5% naringin in per feeding conditions. Flavanones were analyzed in plasma, liver, kidney and urine by tandem mass spectrometry. Naringenin conjugates (essentially glucuronides) accounted for up to 98% of the total Flavanones in plasma. Low amounts of hesperetin (4′- O -methyl naringenine) and isosakuranetin (3′-hydroxy-4′- O -methylnaringenin) were also detected in all biological samples and accounted for 2% of the total Flavanones in plasma. They were largely present as aglycones. The in vivo hydroxylation of Flavanones is described here for the first time. Total concentrations of naringenin metabolites reached 17.3 ± 2.7 μM in plasma 6 hours after the beginning of the meal in healthy rats and only 10.6 ± 1.3 μM in TuB rats. The nature of metabolites was similar in both healthy and TuB rats and in plasma, tissues and urine. The lower concentration of Flavanones in the TuB rats suggests that disease and more particularly cancer, may affect the bioavailability of flavonoids.
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Bioavailability in humans of the Flavanones hesperidin and narirutin after the ingestion of two doses of orange juice
European Journal of Clinical Nutrition, 2003Co-Authors: Claudine Manach, Angel Gil-izquierdo, Corinne Bouteloup-demange, Christine Morand, Christian RémésyAbstract:Objective : Flavanones are polyphenols specific of citrus fruits, where they are present in high amounts. Although citrus fruits and juices are widely consumed in the world, little information has been published on flavanone bioavailability in humans. The aim of the present study was to determine the nature of the circulating metabolites, the plasma kinetics and the urinary excretion patterns of the Flavanones, hesperidin and narirutin. Design : After an overnight fast, five healthy volunteers ingested 0.5 or 1 l of a commercial orange juice providing 444 mg/l hesperidin and 96.4 mg/l narirutin, together with a polyphenol-free breakfast. Blood was sampled at 10 different timepoints over a 24 h period. Urine was collected for 48 h, in five fractions. Results : Flavanones metabolites appeared in plasma 3 h after the juice ingestion, reached a peak between 5 and 7 h, then returned to baseline at 24 h. The peak plasma concentration of hesperetin was 0.46±0.07 µmol/l and 1.28±0.13 µmol/l after the 0.5 and 1 l intake, respectively. It was lower for naringenin: 0.20±0.04 µmol/l after the 1 l dose. The circulating forms of hesperetin were glucuronides (87%) and sulphoglucuronides (13%). For both Flavanones, the urinary excretion was nearly complete 24 h after the orange juice ingestion. The relative urinary excretion was similar for hesperetin and naringenin and did not depend on the dose: values ranged from 4.1±1.2 to 7.9±1.7% of the intake. Conclusion : In case of a moderate or high consumption of orange juice, Flavanones may represent an important part of the pool of total polyphenols present in plasma.
Masafumi Kaneko - One of the best experts on this subject based on the ideXlab platform.
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efficient production of 2s Flavanones by escherichia coli containing an artificial biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2005Co-Authors: Ikuo Miyahisa, Masafumi Kaneko, Hisashi Kawasaki, Nobutaka Funa, Yasuo Ohnishi, Hiroyuki Kojima, Sueharu HorinouchiAbstract: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.
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Efficient production of (2S)-Flavanones by Escherichia coli containing an artificial biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2005Co-Authors: Ikuo Miyahisa, Masafumi Kaneko, Hisashi Kawasaki, Nobutaka Funa, Yasuo Ohnishi, Hiroyuki Kojima, Sueharu HorinouchiAbstract: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. © Springer-Verlag 2005.
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production of plant specific Flavanones by escherichia coli containing an artificial gene cluster
Applied and Environmental Microbiology, 2003Co-Authors: Eui Il Hwang, Masafumi Kaneko, Yasuo Ohnishi, Sueharu HorinouchiAbstract:In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, and flavonoids. In addition to their roles in the structure and protection of the plant, phenylpropanoid compounds have an important effect on plant qualities, such as texture, flavor, color, and other characteristics (29). Recently, the flavonoid-derived plant natural products have drawn much attention because of their benefits to human health, such as antimicrobial, cancer chemopreventive, antioxidant, and antiasthmatic activities (6, 14). These phenylpropanoid and flavonoid biosynthetic enzymes are therefore attractive targets for metabolic engineering to provide enhancement or initiation of the production of economically desirable traits or compounds. The phenylpropanoid and flavonoid biosynthetic pathways and their regulation have been the subject of many studies (4, 5, 25, 29). Recent advances in the regulation of the pathways and the biochemistry of specific enzymes and enzyme complexes have opened up strategies for the enhancement of flavonoid compounds by modifying the pathways (4). The phenylpropanoid pathway in plants converts phenylalanine into naringenin chalcone (Fig. (Fig.1).1). As the first step, phenylalanine is deaminated to yield cinnamic acid by the action of phenylalanine ammonia lyase (PAL). Cinnamic acid is hydroxylated by cinnamate-4-hydroxylase (C4H) to 4-coumaric acid, which is then activated to 4-coumaroyl-coenzyme A (CoA) by the action of 4-coumarate:CoA ligase (4CL). Chalcone synthase (CHS) catalyzes the stepwise condensation of three acetate units from malonyl-CoA with 4-coumaroyl-CoA to yield naringenin chalcone, the precursor for a large number of flavonoids (29). Naringenin chalcone is converted to naringenin by chalcone isomerase or nonenzymatically in vitro (5, 19, 24, 25). Because some of the PALs show tyrosine ammonia lyase activity, tyrosine is also used as the precursor (2, 15, 22, 26). FIG. 1. Flavanone biosynthetic pathway in plants. The dashed arrows represent the expected flavanone biosynthetic pathway in E. coli containing the artificial gene cluster including PAL, 4CL, and CHS. TAL, tyrosine ammonia-lyase; CHI, chalcone isomerase. Production of Flavanones or flavonoids by genetically engineered bacteria has not been reported before, although the heterologous expression of phenylpropanoid biosynthetic enzymes in bacteria has been reported (1, 8, 16, 30). One of the barriers for the production of these compounds is the difficulty in expression of active C4H, which would not be efficiently expressed due to its instability and the lack of its specific cytochrome P-450 reductase in bacteria (8, 20). We have recently discovered a 4CL in the gram-positive filamentous bacterium Streptomyces coelicolor A3(2) that can activate cinnamic acid to cinnamoyl-CoA, as well as 4-coumaric acid to 4-coumaroyl-CoA (12). The use of the bacterial 4CL enzyme would bypass the C4H step for the production of pinocembrin chalcone from phenylalanine via the phenylpropanoid pathway (Fig. (Fig.1).1). On the basis of this idea, we constructed an artificial gene cluster containing PAL from a yeast, Rhodotorula rubra; 4CL from an actinomycete, S. coelicolor A3(2); and CHS from a licorice plant, Glycyrrhiza echinata, to produce a plant-specific flavanone, pinocembrin. As expected, the Escherichia coli cells containing the gene cluster produced pinocembrin. In addition, the E. coli cells produced naringenin, because the yeast PAL enzyme also used tyrosine as a substrate and yielded 4-coumaric acid, as is found for many PALs (2, 22, 26), and the 4CL enzyme used cinnamic acid and 4-coumaric acid to yield the corresponding CoA thioester compounds. This paper describes successful production of plant-specific Flavanones, pinocembrin and naringenin, in E. coli. Furthermore, we compared the yields of the Flavanones by E. coli cells that contained the gene clusters in different organizations.
Yasuo Ohnishi - One of the best experts on this subject based on the ideXlab platform.
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efficient production of 2s Flavanones by escherichia coli containing an artificial biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2005Co-Authors: Ikuo Miyahisa, Masafumi Kaneko, Hisashi Kawasaki, Nobutaka Funa, Yasuo Ohnishi, Hiroyuki Kojima, Sueharu HorinouchiAbstract: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.
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Efficient production of (2S)-Flavanones by Escherichia coli containing an artificial biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2005Co-Authors: Ikuo Miyahisa, Masafumi Kaneko, Hisashi Kawasaki, Nobutaka Funa, Yasuo Ohnishi, Hiroyuki Kojima, Sueharu HorinouchiAbstract: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. © Springer-Verlag 2005.
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production of plant specific Flavanones by escherichia coli containing an artificial gene cluster
Applied and Environmental Microbiology, 2003Co-Authors: Eui Il Hwang, Masafumi Kaneko, Yasuo Ohnishi, Sueharu HorinouchiAbstract:In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, and flavonoids. In addition to their roles in the structure and protection of the plant, phenylpropanoid compounds have an important effect on plant qualities, such as texture, flavor, color, and other characteristics (29). Recently, the flavonoid-derived plant natural products have drawn much attention because of their benefits to human health, such as antimicrobial, cancer chemopreventive, antioxidant, and antiasthmatic activities (6, 14). These phenylpropanoid and flavonoid biosynthetic enzymes are therefore attractive targets for metabolic engineering to provide enhancement or initiation of the production of economically desirable traits or compounds. The phenylpropanoid and flavonoid biosynthetic pathways and their regulation have been the subject of many studies (4, 5, 25, 29). Recent advances in the regulation of the pathways and the biochemistry of specific enzymes and enzyme complexes have opened up strategies for the enhancement of flavonoid compounds by modifying the pathways (4). The phenylpropanoid pathway in plants converts phenylalanine into naringenin chalcone (Fig. (Fig.1).1). As the first step, phenylalanine is deaminated to yield cinnamic acid by the action of phenylalanine ammonia lyase (PAL). Cinnamic acid is hydroxylated by cinnamate-4-hydroxylase (C4H) to 4-coumaric acid, which is then activated to 4-coumaroyl-coenzyme A (CoA) by the action of 4-coumarate:CoA ligase (4CL). Chalcone synthase (CHS) catalyzes the stepwise condensation of three acetate units from malonyl-CoA with 4-coumaroyl-CoA to yield naringenin chalcone, the precursor for a large number of flavonoids (29). Naringenin chalcone is converted to naringenin by chalcone isomerase or nonenzymatically in vitro (5, 19, 24, 25). Because some of the PALs show tyrosine ammonia lyase activity, tyrosine is also used as the precursor (2, 15, 22, 26). FIG. 1. Flavanone biosynthetic pathway in plants. The dashed arrows represent the expected flavanone biosynthetic pathway in E. coli containing the artificial gene cluster including PAL, 4CL, and CHS. TAL, tyrosine ammonia-lyase; CHI, chalcone isomerase. Production of Flavanones or flavonoids by genetically engineered bacteria has not been reported before, although the heterologous expression of phenylpropanoid biosynthetic enzymes in bacteria has been reported (1, 8, 16, 30). One of the barriers for the production of these compounds is the difficulty in expression of active C4H, which would not be efficiently expressed due to its instability and the lack of its specific cytochrome P-450 reductase in bacteria (8, 20). We have recently discovered a 4CL in the gram-positive filamentous bacterium Streptomyces coelicolor A3(2) that can activate cinnamic acid to cinnamoyl-CoA, as well as 4-coumaric acid to 4-coumaroyl-CoA (12). The use of the bacterial 4CL enzyme would bypass the C4H step for the production of pinocembrin chalcone from phenylalanine via the phenylpropanoid pathway (Fig. (Fig.1).1). On the basis of this idea, we constructed an artificial gene cluster containing PAL from a yeast, Rhodotorula rubra; 4CL from an actinomycete, S. coelicolor A3(2); and CHS from a licorice plant, Glycyrrhiza echinata, to produce a plant-specific flavanone, pinocembrin. As expected, the Escherichia coli cells containing the gene cluster produced pinocembrin. In addition, the E. coli cells produced naringenin, because the yeast PAL enzyme also used tyrosine as a substrate and yielded 4-coumaric acid, as is found for many PALs (2, 22, 26), and the 4CL enzyme used cinnamic acid and 4-coumaric acid to yield the corresponding CoA thioester compounds. This paper describes successful production of plant-specific Flavanones, pinocembrin and naringenin, in E. coli. Furthermore, we compared the yields of the Flavanones by E. coli cells that contained the gene clusters in different organizations.