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Peter Beyer - One of the best experts on this subject based on the ideXlab platform.
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structure of Phytoene desaturase provides insights into herbicide binding and reaction mechanisms involved in carotene desaturation
Structure, 2017Co-Authors: Anton Brausemann, Peter Beyer, Julian Koschmieder, Sandro Ghisla, Sandra Gemmecker, Oliver EinsleAbstract:Cyanobacteria and plants synthesize carotenoids via a poly-cis pathway starting with Phytoene, a membrane-bound C40 hydrocarbon. Phytoene desaturase (PDS) introduces two double bonds and concomitantly isomerizes two neighboring double bonds from trans to cis. PDS assembles into homo-tetramers that interact monotopically with membranes. A long hydrophobic tunnel is proposed to function in the sequential binding of Phytoene and the electron acceptor plastoquinone. The herbicidal inhibitor norflurazon binds at a plastoquinone site thereby blocking reoxidation of FADred. Comparison with the sequence-dissimilar bacterial carotene desaturase CRTI reveals substantial similarities in the overall protein fold, defining both as members of the GR2 family of flavoproteins. However, the PDS active center architecture is unprecedented: no functional groups are found in the immediate flavin vicinity that might participate in dehydrogenation and isomerization. This suggests that the isoalloxazine moiety is sufficient for catalysis. Despite mechanistic differences, an ancient evolutionary relation of PDS and CRTI is apparent.
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Kinetic scheme of the substrate channeling model and dynamic modeling of PDS reaction time courses.
2017Co-Authors: Julian Koschmieder, Mirjam Fehling-kaschek, Patrick Schaub, Sandro Ghisla, Anton Brausemann, Jens Timmer, Peter BeyerAbstract:(A) Substrate channeling model, accounting for substrate channeling between PDS homotetramers. Symbols are as given in Fig 4A. Two species of phytofluene, i.e. phytofluene fates, coexist. Left; nascent phytofluene (pf*) that is produced from Phytoene (p) can be restricted in its diffusion into the membrane residing in a microdomain in proximity to the PDS homotetramer, as indicated by the bent arrow. It can be channeled into a second PDS subunit of the homotetramer containing FADox, allowing rapid conversion to ζ-carotene (z) with the rate constant kpf*. Right; pf* can alternatively diffuse into PDS-distant membrane areas with rate constant kdiff, this defining the species pf. From there it can be taken up by another monomeric PDS subunit and be converted into ζ-carotene (z) with rate constant kpf. Rate constant kage represents enzyme inactivation which refers to both the reduced and oxidized enzyme states. (B-G) Dynamic modeling of reaction time courses of Phytoene and phytofluene conversion by PDS. Reaction time courses were conducted with 1.3 nmol Phytoene (p low; B), and 3.7 nmol Phytoene (p high; C). In addition, liposomes containing 5.2 nmol phytofluene were used (pf; D). The observables are given as data points (black, Phytoene, p; red, phytofluene, pf; blue, ζ-carotene, z). The model fit, represented by lines, is based on Eqs 1 and 6–10 with simultaneous parameter estimation for all three reaction time courses. Shadowed areas indicate one standard deviation as estimated by the error model (see Methods). Measurements were carried out in triplicate. (E) Prediction of the amount of oxidized, active PDS (ox) and reduced PDS (red) over time, indicating a rapid decrease in oxidized and reduced PDS levels due to enzyme inactivation. (F,G) Deduced carotene fluxes through the different sub-processes labeled with their rate constants (see Fig 4). Note the different scaling in F and G. Flux predictions are based on the Phytoene conversion reaction time course “p high” (C).
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Plant-type Phytoene desaturase: Functional evaluation of structural implications
2017Co-Authors: Julian Koschmieder, Mirjam Fehling-kaschek, Patrick Schaub, Sandro Ghisla, Anton Brausemann, Jens Timmer, Peter BeyerAbstract:Phytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-Phytoene, yielding 9,15,9'-tri-cis-ζ-carotene via the intermediate 9,15-di-cis-phytofluene. We present the necessary data to scrutinize functional implications inferred from the recently resolved crystal structure of Oryza sativa PDS in a complex with norflurazon. Using dynamic mathematical modeling of reaction time courses, we support the relevance of homotetrameric assembly of the enzyme observed in crystallo by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations are compatible with an ordered ping-pong bi-bi kinetic mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. The mutagenesis of a conserved arginine that forms a hydrogen bond with norflurazon, the latter competing with plastoquinone, corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. This mutagenesis also supports a “flavin only” mechanism of carotene desaturation not requiring charged residues in the active site. Evidence for the role of the central 15-cis double bond of Phytoene in determining regio-specificity of carotene desaturation is presented.
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Kinetic scheme of the monomeric model and dynamic modeling of PDS reaction time courses.
2017Co-Authors: Julian Koschmieder, Mirjam Fehling-kaschek, Patrick Schaub, Sandro Ghisla, Anton Brausemann, Jens Timmer, Peter BeyerAbstract:(A) Monomeric model. PDS monomeric subunits (orange and blue rectangles) within the homotetramer are assumed to work independently. Orange/blue color denotes reduced/oxidized half sides of Phytoene (p), phytofluene (pf) and ζ-carotene (z) and the respective redox state of the PDS-bound FAD. The overall reaction comprises the three main processes Phytoene desaturation (i), phytofluene desaturation (ii) and plastoquinone reduction (iii) with the rate constants kp, kpf and krox, respectively. Each rate constant encompasses the three equilibria represented by the reaction arrows associated to each of the three main processes which are highlighted by shadowed areas: association-dissociation of enzyme and substrate, desaturation-saturation of substrate and dissociation-association of enzyme and product. All hydrophobic carotene substrates and DPQ (Q) are soluble in the hydrophobic core of liposomal membranes. Progressive inactivation of PDS by denaturation (iv) is a process to be considered. (B-D) Reaction time courses of Phytoene and phytofluene conversion by PDS. Reaction time courses were initiated [p] = 3.7 nmol (p high; B), [p] = 1.3 nmol (p low; C) and [pf] = 5.2 nmol (pf; D). The observables are given as data points (black, Phytoene, p; red, phytofluene, pf; blue, ζ-carotene, pf), the model fit (obtained with model I; ODE 1–5) is represented by lines. The modeling was either based on simultaneous parameter estimation for all three reaction time courses (solid lines) or on simultaneous estimation of kp, krox and kage and individual estimation of kpf (dashed line). Measurements were carried out in triplicate.
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On the structure and function of the Phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase. PLoS One 2012; 7: e39550. doi: 10.1371/journal.pone
2016Co-Authors: Patrick Schaub, Salim Al-babili, Ra Gemmecker, Pierre Poussin-courmontagne, Justine Mailliot, Alastair G. Mcewen, Ro Ghisla, Jean Cavarelli, Peter BeyerAbstract:CRTI-type Phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from Phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate Phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition o
Norihiko Misawa - One of the best experts on this subject based on the ideXlab platform.
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enrichment of carotenoids in flaxseed linum usitatissimum by metabolic engineering with introduction of bacterial Phytoene synthase gene crtb
Journal of Bioscience and Bioengineering, 2008Co-Authors: Masaki Fujisawa, Mio Watanabe, Songkang Choi, Maki Teramoto, Kanji Ohyama, Norihiko MisawaAbstract:Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of α-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the Phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which Phytoene, α-carotene, and β-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4–156.3 μg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of Phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then Phytoene was consecutively decomposed to the downstream metabolites α-carotene, β-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.
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enrichment of carotenoids in flaxseed linum usitatissimum by metabolic engineering with introduction of bacterial Phytoene synthase gene crtb
Journal of Bioscience and Bioengineering, 2008Co-Authors: Masaki Fujisawa, Mio Watanabe, Songkang Choi, Maki Teramoto, Kanji Ohyama, Norihiko MisawaAbstract:Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of alpha-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the Phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which Phytoene, alpha-carotene, and beta-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4-156.3 microg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of Phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then Phytoene was consecutively decomposed to the downstream metabolites alpha-carotene, beta-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.
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the cyanobacterium gloeobacter violaceus pcc 7421 uses bacterial type Phytoene desaturase in carotenoid biosynthesis
FEBS Letters, 2005Co-Authors: Tohru Tsuchiya, Norihiko Misawa, Takashi Maoka, Shinichi Takaichi, Hideaki Miyashita, Mamoru MimuroAbstract:Carotenoid composition and its biosynthetic pathway in the cyanobacterium Gloeobacter violaceus PCC 7421 were investigated. β-Carotene and (2S,2′S)-oscillol 2,2′-di(α-l-fucoside), and echinenone were major and minor carotenoids, respectively. We identified two unique genes for carotenoid biosynthesis using in vivo functional complementation experiments. In Gloeobacter, a bacterial-type Phytoene desaturase (CrtI), rather than plant-type desaturases (CrtP and CrtQ), produced lycopene. This is the first demonstration of an oxygenic photosynthetic organism utilizing bacterial-type Phytoene desaturase. We also revealed that echinenone synthesis is catalyzed by CrtW rather than CrtO. These findings indicated that Gloeobacter retains ancestral properties of carotenoid biosynthesis.
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Evaluation of transgenic tomato plants expressing an additional Phytoene synthase in a fruit-specific manner.
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Paul D. Fraser, Norihiko Misawa, Wolfgang Schuch, Susanne Römer, Cathie A. Shipton, Philippa B. Mills, Joy W. Kiano, Rachel Drake, Peter M. BramleyAbstract:Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato Phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas Phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total Phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional Phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into Phytoene) were not significantly altered by the presence of the bacterial Phytoene synthase.
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elevation of the provitamin a content of transgenic tomato plants
Nature Biotechnology, 2000Co-Authors: Susanne Römer, Norihiko Misawa, Paul D. Fraser, Wolfgang Schuch, Cathie A. Shipton, Joy W. Kiano, Peter M. BramleyAbstract:Tomato products are the principal dietary sources of lycopene and major source of beta-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme Phytoene desaturase, which converts Phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the beta-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for Phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.
Gerhard Sandmann - One of the best experts on this subject based on the ideXlab platform.
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structures of new c41 carotenoids produced using recombinant escherichia coli expressing genes encoding isopentenyl pyrophosphate methyltransferase and carotenoid biosynthetic enzymes
Tetrahedron Letters, 2020Co-Authors: Chiharu Takagi, Gerhard Sandmann, Jurgen Breitenbach, Momoka Abe, Yumi Kaneko, Akane Sasaki, Ayumi Ito, Yuka Sakemi, Takashi Maoka, Kazutoshi ShindoAbstract:Abstract The production of methylated C41-C43 15-cis-Phytoene, lycopene, β-carotene, and zeaxanthin using recombinant Escherichia coli that express the genes encoding isopentenyl pyrophosphate, C4-methyltransferase, and carotenoid biosynthetic enzymes, as well as the detection of their chemical structures using high-pressure liquid chromatography-mass spectroscopy (HPLC-MS) analyses have been previously reported. However, the positions of the methyl groups were not fully elucidated due to multiple possible sites that could be methylated. In this study, we have isolated the methylated carotenoids using chromatographic methods and unambiguously determined their structures as 16-methyl derivatives of 15-cis-Phytoene, lycopene, β-carotene, and zeaxanthin via detailed MS, 1D, and 2D NMR analyses. These compounds were all identified as novel C41 carotenoids.
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carotenoid synthesis and Phytoene synthase activity during mating of blakeslea trispora
Phytochemistry, 2012Co-Authors: Jurgen Breitenbach, Paul D. Fraser, Gerhard SandmannAbstract:Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (-) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into Phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of Phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, Phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The Phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional Phytoene synthase and lycopene cyclase.
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transformation of the green alga haematococcus pluvialis with a Phytoene desaturase for accelerated astaxanthin biosynthesis
Applied and Environmental Microbiology, 2006Co-Authors: Jens Steinbrenner, Gerhard SandmannAbstract:Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme Phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified Phytoene desaturase was still active in conversion of Phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified Phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and Phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified Phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.
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cdna cloning and expression of carotenogenic genes during flower development in gentiana lutea
Plant Molecular Biology, 2002Co-Authors: Changfu Zhu, Saburo Yamamura, Masashiro Nishihara, Hiroyuki Koiwa, Gerhard SandmannAbstract:All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a Phytoene synthase, a Phytoene desaturase and a ζ-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the Phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), Phytoene desaturase (pds) and ζ-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.
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bleaching herbicide norflurazon inhibits Phytoene desaturase by competition with the cofactors
Journal of Agricultural and Food Chemistry, 2001Co-Authors: Jurgen Breitenbach, Changfu Zhu, Gerhard SandmannAbstract:Cofactor requirement was determined for the heterologous expressed Phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is depen...
Paul D. Fraser - One of the best experts on this subject based on the ideXlab platform.
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carotenoid synthesis and Phytoene synthase activity during mating of blakeslea trispora
Phytochemistry, 2012Co-Authors: Jurgen Breitenbach, Paul D. Fraser, Gerhard SandmannAbstract:Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (-) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into Phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of Phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, Phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The Phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional Phytoene synthase and lycopene cyclase.
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Evaluation of transgenic tomato plants expressing an additional Phytoene synthase in a fruit-specific manner.
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Paul D. Fraser, Norihiko Misawa, Wolfgang Schuch, Susanne Römer, Cathie A. Shipton, Philippa B. Mills, Joy W. Kiano, Rachel Drake, Peter M. BramleyAbstract:Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato Phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas Phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total Phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional Phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into Phytoene) were not significantly altered by the presence of the bacterial Phytoene synthase.
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elevation of the provitamin a content of transgenic tomato plants
Nature Biotechnology, 2000Co-Authors: Susanne Römer, Norihiko Misawa, Paul D. Fraser, Wolfgang Schuch, Cathie A. Shipton, Joy W. Kiano, Peter M. BramleyAbstract:Tomato products are the principal dietary sources of lycopene and major source of beta-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme Phytoene desaturase, which converts Phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the beta-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for Phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.
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Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit.
Plant molecular biology, 1999Co-Authors: Paul D. Fraser, Mark R. Truesdale, Wolfgang Schuch, Joy W. Kiano, Peter M. BramleyAbstract:The characteristic yellow fruit phenotype of the r,r mutant and Psy-1 (Phytoene synthase-1) antisense tomatoes is due to a mutated or down-regulated Phytoene synthase protein, respectively, resulting in the virtual absence of carotenoids. Based on detailed carotenoid determinations Psy-1 appeared to barely contribute to the formation of carotenoids in chloroplast-containing tissues. Despite the virtual absence of carotenoids in ripe fruit the formation of Phytoene in vitro was detected in fruit of both mutants. When [14C]isopentenyl pyrophosphate (IPP) was used as the substrate for Phytoene synthase a reduction (e.g. r,r mutant, 5-fold) in the formation of Phytoene was observed with an accumulation (e.g. r,r mutant, 2-fold) of the immediate precursor geranylgeranyl pyrophosphate (GGPP). Contrastingly, reduced Phytoene synthase activity was not detected when [3H]GGPP was used as the substrate. The profile of Phytoene formation during ripening was also different in the down-regulated mutants compared to the wild-type. Using specific primers, RT-PCR analysis detected the presence of Psy-2 transcripts in the down-regulated mutants and wild-type throughout fruit development and ripening. These data were supported by the detection of Phytoene synthase protein on western blots. Both GGPP formation and Phytoene desaturation were elevated in these mutants. Therefore, it appears that despite the absence of carotenoids in ripe fruit, both the mutants have the enzymic capability to synthesize carotenoids in this tissue. Implications of the data with respect to the regulation of carotenoid formation and the channelling of prenyl lipid precursors in tomato (and its potential manipulation) are discussed.
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Expression of an active Phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme
Biochimica et biophysica acta, 1998Co-Authors: Ute Neudert, Paul D. Fraser, Isabel M Martı́nez-férez, Gerhard SandmannAbstract:Abstract The crtB gene encoding Phytoene synthase from the carotenogenic enterobacterium Erwinia uredovora was overexpressed to about 20% of the total cellular protein in Escherichia coli. Formation of the active Phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in E. coli, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the Phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes crtE and idi, encoding the Erwinia GGPP synthase and Rhodobacter isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to Phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The Phytoene formed in vitro was present in both a 15-cis and all-trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn2+ or Mg2+. The Km value for GGPP was determined as 41 μM. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The I50 value for the latter inhibitor was 15 μM. Lineweaver–Burk plots showed constant Km values in the presence or absence of squalestatin.
Peter M. Bramley - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of transgenic tomato plants expressing an additional Phytoene synthase in a fruit-specific manner.
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Paul D. Fraser, Norihiko Misawa, Wolfgang Schuch, Susanne Römer, Cathie A. Shipton, Philippa B. Mills, Joy W. Kiano, Rachel Drake, Peter M. BramleyAbstract:Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato Phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas Phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total Phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional Phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into Phytoene) were not significantly altered by the presence of the bacterial Phytoene synthase.
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elevation of the provitamin a content of transgenic tomato plants
Nature Biotechnology, 2000Co-Authors: Susanne Römer, Norihiko Misawa, Paul D. Fraser, Wolfgang Schuch, Cathie A. Shipton, Joy W. Kiano, Peter M. BramleyAbstract:Tomato products are the principal dietary sources of lycopene and major source of beta-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme Phytoene desaturase, which converts Phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the beta-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for Phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.
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Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit.
Plant molecular biology, 1999Co-Authors: Paul D. Fraser, Mark R. Truesdale, Wolfgang Schuch, Joy W. Kiano, Peter M. BramleyAbstract:The characteristic yellow fruit phenotype of the r,r mutant and Psy-1 (Phytoene synthase-1) antisense tomatoes is due to a mutated or down-regulated Phytoene synthase protein, respectively, resulting in the virtual absence of carotenoids. Based on detailed carotenoid determinations Psy-1 appeared to barely contribute to the formation of carotenoids in chloroplast-containing tissues. Despite the virtual absence of carotenoids in ripe fruit the formation of Phytoene in vitro was detected in fruit of both mutants. When [14C]isopentenyl pyrophosphate (IPP) was used as the substrate for Phytoene synthase a reduction (e.g. r,r mutant, 5-fold) in the formation of Phytoene was observed with an accumulation (e.g. r,r mutant, 2-fold) of the immediate precursor geranylgeranyl pyrophosphate (GGPP). Contrastingly, reduced Phytoene synthase activity was not detected when [3H]GGPP was used as the substrate. The profile of Phytoene formation during ripening was also different in the down-regulated mutants compared to the wild-type. Using specific primers, RT-PCR analysis detected the presence of Psy-2 transcripts in the down-regulated mutants and wild-type throughout fruit development and ripening. These data were supported by the detection of Phytoene synthase protein on western blots. Both GGPP formation and Phytoene desaturation were elevated in these mutants. Therefore, it appears that despite the absence of carotenoids in ripe fruit, both the mutants have the enzymic capability to synthesize carotenoids in this tissue. Implications of the data with respect to the regulation of carotenoid formation and the channelling of prenyl lipid precursors in tomato (and its potential manipulation) are discussed.
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Constitutive expression of a fruit Phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway
The Plant Journal, 1995Co-Authors: Rupert G. Fray, Paul D. Fraser, Peter M. Bramley, Andrew Wallace, Daniel Valero, Peter Hedden, Donald GriersonAbstract:Tomato plants transformed with a copy of the fruit-expressed Phytoene synthase cDNA under control of the CaMV 35S promoter showed ectopic production of carotenoids. High expressers were reduced in stature. The dwarf character was inherited with an inverse relationship between expression of Phytoene synthase and plant height. Severely affected plants also showed reduced chlorophyll content in young leaves. These dwarfs showed a 30-fold reduction in levels of gibberellin A1 (GA1) and growth was partially restored by treatment with exogenous GA3. Qualitative and quantitative changes in carotenoids were also found. It is proposed that the dwarf phenotype results from the over-production of Phytoene synthase, which converts geranylgeranyl diphosphate to Phytoene and thereby diverts this intermediate away from the gibberellin (GA) and phytol biosynthetic pathways.
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Carotenoid Biosynthesis during Tomato Fruit Development (Evidence for Tissue-Specific Gene Expression)
Plant Physiology, 1994Co-Authors: Paul D. Fraser, Mark R. Truesdale, Colin Roger Bird, Wolfgang Schuch, Peter M. BramleyAbstract:Tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) fruit, at five stages of development, have been analyzed for their carotenoid and chlorophyll (Chl) contents, in vitro activities of Phytoene synthase, Phytoene desaturase, and lycopene cyclase, as well as expression of the Phytoene synthase (Psy) and Phytoene desaturase (Pds) genes. During ripening, the total carotenoids increased with a concomitant decrease in Chl. Although the highest carotenoid content (consisting mainly of lycopene and [beta]-carotene) was found in ripe fruit, the greatest carotenogenic enzymic activities were found in green fruit. Phytoene synthase was located in the plastid stroma, whereas the metabolism of Phytoene was associated with plastid membranes during all stages of fruit development. The in vitro products of Phytoene desaturation altered from being predominantly phytofluence and [zeta]-carotene in chloroplasts to becoming mainly lycopene in chromoplasts. The expression of Psy was detected in breaker and ripe fruit, as well as flowers, but was not detectable by northern blot analysis in leaves or green fruits. The Pds gene transcript was barely detectable in green fruit and leaves but was expressed in flowers and breaker fruit. These results suggest that transcription of Psy and Pds is regulated developmentally, with expression being considerably elevated in chromoplast-containing tissues. Antiserum to the Synechococcus Phytoene synthase cross-reacted with Phytoene synthase of green fruit only on western blots and not with the enzyme from ripe fruit. In contrast, a monoclonal antibody to the Psy gene product only cross-reacted with Phytoene synthase from ripe fruit. The enzymes from green and ripe fruit had different molecular masses of 42 and 38 kD, respectively. The absence of detectable Psy and Pds mRNA in green tissues using northern blot analyses, despite high levels of Phytoene synthase and desaturase activity, lends support to the hypothesis of divergent genes encoding these enzymes.
