Synthesis of biosynthetic precursors of red fluorescent proteins' chromophores

A method for the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones, the corresponding chromophore of green fluorescent protein (GFP) with acylaminoalkyl substituents at position 2 of imidazole core have been developed. These structures represent biosynthetic precursors of the chromophores of red fluorescent proteins. The method is based on masking of the dehydrotyrosine fragment with beta-hydroxytyrosine moiety The key stages of the synthesis include peptide coupling of beta-hydroxytyrosine with the N-acetylamino acid of choice, unmasking of dehydrotyrosine by O-acylation with subsequent elimination, and cyclization of the obtained derivatives of 3-acylaminocinnamic acid in basic media.     

Synthesis and electrochemical behaviour of β-halodehydroamino acid derivatives

Several new β,β-dihalo and β-halo-β-substituted dehydroalanines and dehydrodipeptides were synthesized by reacting the corresponding dehydroamino acid derivative with a N-halosuccinimide or in the case of β,β-di-iododehydroalanines with iodine. The results obtained confirmed that the stereochemical outcome of the halogenation reaction with β-substituted dehydroamino acids depends on the substrate. Thus, an increase Z-stereoselectivity was found when the β-phenyldehydroalanines were used as substrates and when these compounds were N-protected with 4-tolylsulfonyl or with carbamates. From this study, it is also possible to conclude that when N-iodosuccinimide was used as reagent a much higher Z-stereoselectivity is found. The electrochemical behaviour of the halogenated dehydroamino acids was studied by cyclic voltammetry. The results show a shift in the reduction peak to higher potentials of the β-halogenated dehydroamino acids when compared with the corresponding non-halogenated derivatives. As expected, the β,β-dihalodehydroalanines exhibit higher peak potentials than β-halo-β-substituted dehydroalanines and the bromo derivatives have lower peak potentials when compared with the corresponding iododehydroamino acids. Controlled potential electrolysis of several β-halo-β-substituted dehydroamino acids afforded the corresponding dehalogenated dehydroamino acids as mixtures of their E and Z-isomers. In all cases, the major isomer isolated results from dehalogenation without isomerization. These new results show that electrochemical reduction constitutes a valuable method for the synthesis of the E-isomer of β-substituted dehydroalanines.     

Chemosystematic investigations on phenolics from flowerheads of Central European taxa of Hieracium sensu lato (Asteraceae)

 HPLC-UV and HPLC-MS investigations of phenolic acids and flavonoids in flowerheads of 84 samples of 76 taxa belonging to 66 species of Hieracium resulted in the identification of three phenolic acids (chlorogenic acid, 3,5-dicaffeoyl quinic acid, 4,5-dicaffeoyl quinic acid) and six flavonoids (apigenin 4^′-O-β-D-glucuronide, isoetin 4^′-O-β-D-glucuronide, luteolin, luteolin 7-O-β-D-glucoside, luteolin 7-O-β-D-glucuronide, luteolin 4^′-O-β-D-glucoside). The contents of these secondary metabolites were quantified by HPLC using quercetin and cynarin as internal standards. In contrast to the previously investigated genera Leontodon and Crepis, cichoric acid and caffeoyl tartaric acid were not found in any of the investigated Hieracium taxa. Results of HPLC analyses revealed only a limited degree of qualitative variation between the different taxa, and luteolin 7-O-β-D-glucuronide and isoetin 4^′-O-β-D-glucuronide were the only compounds, which were not detectable in some of the investigated taxa. Quantitative patterns of phenolics differed markedly between particular taxa and Principal Component Analysis of the quantification results yielded separate clusters for the members of the subgenera Hieracium and Pilosella. Received January 23, 2001 Accepted October 11, 2001     

1β,7β-Dihydroxydehydroabietic acid,a new biotransformation product of dehydroabietic acid by Aspergillus niger

Microbial transformation of dehydroabietic acid by Aspergillus niger afforded the new derivative 1β,7β-dihydroxydehydroabietic acid and the known 1β-hydroxy and 7β-hydroxy derivatives. The structures were elucidated by spectroscopic methods. The compounds were assessed towards Gram (+) and Gram (−) bacteria and showed a weak antimicrobial effect. This revised version was published online in August 2006 with corrections to the Cover Date.     

Synthesis of bis-amino acid derivatives by Suzuki cross-coupling,Michael addition and substitution reactions

Several bis-amino acids were prepared using a bis-Suzuki coupling (compounds 4–8, 10), a sequential Michael addition and bis-Suzuki coupling (compounds 12, 13) and a Michael addition followed by a substitution reaction (compounds 18, 19). Thus, the pure stereoisomer of the methyl esters of N-(tert-butoxycarbonyl)-β-bromodehydroaminobutyric acid and dehydrophenylalanine and of N-benzyloxycarbonyl-β-bromodehydroaminobutyric acid were reacted with 1,4-phenylene-bis-boronic acid or 9,9-dioctyl-9H-fluorene-2,7-bis-boronic acid using modified Suzuki coupling conditions. The corresponding bis-dehydroamino acid derivatives were obtained in good to high yields maintaining the stereochemistry of the starting materials. This reaction was also applied successfully to a brominated dehydrodipeptide and 1,4-phenylene-bis-boronic acid showing that it could be used to create cross-links in peptide chains. An N,N-diacyldehydroalanine derivative was used in a sequential Michael addition and bis-Suzuki coupling giving a p-terphenyl bis-amino acid and a fluorenyl bis-amino acid in good yields. Two bis-α,β-diamino acids were obtained by a Michael addition of 1,2,4-triazole to the methyl esters of N-(4-toluenesulfonyl), N-(tert-butoxycarbonyl) dehydroamino acids followed by treatment with ethylenediamine.     

2-Oxo-2H-benzo[h]benzopyran as a new light sensitive protecting group for neurotransmitter amino acids

Aiming at the development of new benzopyran-based photocleavable protecting groups, novel chloromethylated and hydroxymethylated 2-oxo-2H-benzo[h]benzopyran derivatives bearing a methoxy substituent were designed and used in the synthesis of a series of fluorescent bioconjugates, by linking through an ester or urethane bond to several model neurotransmitter amino acids (glycine, alanine, β-alanine and γ-aminobutyric acid, GABA). The resulting fluorescent bioconjugates with emission in the visible range and high fluorescent quantum yields, were subjected to photocleavage reaction in methanol/HEPES buffer (80:20) solution at different wavelengths of irradiation (250, 300, 350 and 419 nm) and photocleavage kinetic data were obtained.     

Three new polyhydroxysteroids from the tropical starfish <Emphasis Type="Italic">Asteropsis carinifera</Emphasis>

Thirteen steroidal compounds including three new polyhydroxysteroids, (24R,25S)-24-methyl-5α-cholestane-3β,6α,8,15β,16β,26-hexaol, (22E,24R,25S)-24-methyl-5α-cholest-22-ene-3β,6α,8,15β,16β,26-hexaol, and (22E,24R,25S)-24-methyl-5α-cholest-22-ene-3β,4β,6α,8,15β,16β,26-heptaol, have been isolated along with ten previously known polyhydroxysteroids from the tropical starfish Asteropsis carinifera collected near the coast of Vietnam. The structures of the new compounds were elucidated by spectroscopic methods (mainly 2D NMR and ESI mass spectrometry).     

Inhibitory effects of arbutin-β-glycosides synthesized from enzymatic transglycosylation for melanogenesis

To develop a new skin whitening agent, arbutin-β-glycosides were synthesized and evaluated for their melanogenesis inhibitory activities. Three active compounds were synthesized via the transglycosylation reaction of Thermotoga neapolitana β-glucosidase and purified by recycling preparative HPLC. As compared with arbutin (IC₅₀ = 6 mM), the IC₅₀ values of these compounds were 8, 10, and 5 mM for β-d-glucopyranosyl-(1→6)-arbutin, β-d-glucopyranosyl-(1→4)-arbutin, and β-d-glucopyranosyl-(1→3)-arbutin, respectively. β-d-Glucosyl-(1→3)-arbutin also exerted the most profound inhibitory effects on melanin synthesis in B16F10 melanoma cells. Melanin synthesis was inhibited to a significant degree at 5 mM, at which concentration the melanin content was reduced to below 70% of that observed in the untreated cells. Consequently, β-d-glucopyranosyl-(1→3)-arbutin is a more effective depigmentation agent and is also less cytotoxic than the known melanogenesis inhibitor, arbutin.     

Distribution and expression characteristics of triterpenoids and OSC genes in white birch (<Emphasis Type="Italic">Betula platyphylla</Emphasis> suk.)

Betulin and oleanolic acids (pentacyclic triterpenoid secondary metabolites) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we detected the accumulation and the distribution characteristics of betulin and oleanolic acid in various organs of white birch at different ages. We also determined the expression of 4 OSC genes (LUS, β-AS, CAS1 and CAS2) involved in the triterpenoid synthesis pathways by real time RT-PCR. The result showed that the 1-year old birch can synthesize betulin and oleanolic acid. In addition, betulin and oleanolic acids were mainly distributed in the bark, while the content in the root skin and leaf was very low. The content of betulin and oleanolic acid in birch varied in different seasons. The content of betulin and oleanolic acid and their corresponding LUS and β-AS gene expression were very low in 1-year old birch. With increasing age of birch, betulin content was increased, while oleanolic acid was decreased. Similar changes were also observed for their corresponding synthesis genes LUS and β-AS. In the leaf of 1-year old plant, the highest expression of CAS1 and CAS2 occurred at end of September, while expression of LUS and the β-AS was low from June to October. In the stem skin,high expression of β-AS and the LUS genes occurred from the end of July to September. In the root, high expression of the β-AS gene was observed at the end of October. These results indicated that triterpenoid gene expression was similar to the triterpene accumulation. Expression of LUS gene and β-AS gene in birch with different ages were corresponding to the betulinic and oleanolic acid accumulation. Expression of CAS1 and CAS2 genes were elevated with increasing age of birch. This study provides molecular mechanisms of triterpenes synthesis in birch plants.     

A new β-galactosidase with a low temperature optimum isolated from the Antarctic <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. 20B: gene cloning,purification and characterization

A psychrotrophic bacterium producing a cold-adapted β-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F′ and screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing agar plates led to the isolation of β-galactosidase gene. The β-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted β-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS–PAGE, respectively. The β-galactosidase was optimally active at pH 6.0–8.0 and 25°C. P-nitrophenyl-β-d-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG—o-nitrophenyl-β-d-galactopyranoside). The Arthrobacter sp. 20B β-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na⁺, K⁺ and by 11% for Mn²⁺) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb²⁺, Zn²⁺, Cu²⁺).     

Biotransformation using <Emphasis Type="Italic">Mucor rouxii</Emphasis> for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30°C for 216 h with shaking at 120 rpm. Three new derivatives, 7β-hydroxy-3-oxo-olean-12-en-28-oic acid, 7β,21β-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3β,7β,21β-trihydroxyolean-12-en-28-oic acid, and one know compound, 21β-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21β-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21β-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.     

Enzyme analyses demonstrate that β-methylbutyric acid is converted to β-hydroxy-β-methylbutyric acid via the leucine catabolic pathway by Galactomyces reessii

Galactomyces reessii accomplishes the enzymatic transformation of β-methylbutyric acid (isovaleric acid) to β-hydroxy-β-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas β-methylbutyric acid hydroxylase, α-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, β-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to β-hydroxy-β-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to β-hydroxy-β-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into β-hydroxy-β-methylbutyric acid, thus demonstrating that β-methylbutyric acid is part of the leucine catabolic pathway. The rate of β-methylbutyric acid conversion to β-hydroxy-β-methylbutyric acid with cell-free extract was 0.013 μmol β-hydroxy-β-methylbutyric acid (mg protein)^–1 h^–1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 μmol β-hydroxy-β-methylbutyric acid (g cells)^–1 h^–1] was also observed with β-methylbutyric acid. The results indicate that β-methylbutyric acid is transformed to β-hydroxy-β-methylbutyric acid through the leucine catabolic pathway. Received: 18 July 1997 / Accepted: 12 November 1997     

Synthesis of disaccharide neu5Gcα(2–6)galNAcα as a spacer glycoside

The first synthesis of the Neu5Gc analogue of SiaT _n disaccharide, which can be detected in breast tumors by immunochemical methods, is reported. The regioselective sialylation of (3-trifluoroacetamidopropyl)-2-azido-2-deoxy-α-D-galactopyranoside with peracetate of the methyl ester ofN-acetoxyacetyl-neuraminic acid β-ethylthioglycoside in the presence ofN-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) resulted in the derivatives of α- and β-sialyl(2→6)galactosaminide in 39 and 32% yields, respectively. The catalytic hydrogenolysis of the azido group and subsequentN- andO-acetylation of the α-anomer gave the peracetate of trifluoroacetamidopropyl glycoside. Removal of the protective groups led to glycoside Neu5Gcα2→6GalNAcα-O(CH₂)₃NH₂. Using the Neu5Gc derivative with acetoxyacetyl groups at positions O9 and O4 as a donor increases the α-selectivity of sialylation to afford the α- and β-anomers in 69 and 8% yields, respectively.     

Spectroscopic Detection of Pseudo-Turns in Homodetic Cyclic Penta- and Hexapeptides Comprising β-Homoproline

β-Amino acids with side chains at C² and/or at C³ are of growing interest in drug design, as they may induce astonishing and unusual peptide conformations. Therefore it is of eminent importance to gather information on the consequences of β-amino acid incorporation on the three-dimensional structure of a peptide. This paper describes the synthesis and conformational analysis of cyclic penta- and hexapeptides comprising either (S)-Pro or (S)-β-Hpro. The conformational influence of the β-homoproline building block was analyzed by the combined application of CD, FT-IR and NMR. While the CD spectra of the proline containing peptides indicate the presence of inverse γ-turns and βII-turns, the CD spectra of the β-homoamino acid analogs are dominated by an unprecedented negative band near 205 nm associated with a pseudo-β-turn (Ψβ) or pseudo-γ-turn (Ψγ). These results were confirmed by FT-IR spectroscopy, which also indicates the formation of two internal hydrogen bonds in the cyclic peptides containing the β-homoproline. The conformations of the β-homoproline containing pentapeptides were additionally determined by NMR in combination with MD simulations in two different solvents. The conformation in trifluoroethanol (TFE) is characterized by a bifurcated hydrogen bond stabilizing a pseudo-γ-turn with β-homoproline in the central position, nested with a pseudo-β-turn with β-homoproline in the i+1 position. The combined CD/FT-IR studies clearly show that the replacement of proline by β-homoproline gives rise to a more flexible peptide backbone, and CD spectroscopy hints towards the presence of pseudo-β- or pseudo-γ-turns.     

β-Carbon stereoselectivity of N-carbamoyl-d-α-amino acid amidohydrolase for α,β-diastereomeric amino acids

N-Carbamoyl-d-α-amino acid amidohydrolase (d-carbamoylase) was found to distinguish stereochemistry not only at the α-carbon but also at the β-carbon of N-carbamoyl-d-α-amino acids. The enzyme selectively acted on one of the four stereoisomers of N-carbamoyl-α,β-diastereomeric amino acids. This simultaneous recognition of two chiral centers by d-carbamoylase was useful for the fine stereoselective synthesis of α,β-diastereomeric amino acids such as threonine, isoleucine, 3,4-methylenedioxyphenylserine and β-methylphenylalanine. The stereoselectivity for the β-carbon was influenced by the pH of the reaction mixture and by the bulk of the substituent at the β-carbon. Received: 18 June 1999 / Received revision: 30 July 1999 / Accepted: 6 August 1999     

PNU-91325 increases fatty acid synthesis from glucose and mitochondrial long chain fatty acid degradation: a comparative tracer-based metabolomics study with rosiglitazone and pioglitazone in HepG2 cells

The mitochondrial membrane protein termed “mitoNEET,” is a putative secondary target for insulin-sensitizing thiazolidinedione (TZD) compounds but its role in regulating metabolic flux is not known. PNU-91325 is a thiazolidinedione derivative which exhibits high binding affinity to mitoNEET and lowers cholesterol, fatty acid and blood glucose levels in animal models. In this study we report the stable isotope-based dynamic metabolic profiles (SIDMAP) of rosiglitazone, pioglitazone and PNU-91325 in a dose-matching, dose-escalating study. One and 10 μM concentrations 1 and 10 μM drug concentrations were introduced into HepG2 cells in the presence of either [1,2−¹³C₂]-D-glucose or [U−¹³C₁₈]stearate, GC/MS used to determine positional tracer incorporation (mass isotopomer analysis) into multiple metabolites produced by the Krebs and pentose cycles, de novo fatty acid synthesis, long chain fatty acid oxidation, chain shortening and elongation. Rosiglitazone and pioglitazone (10 μM) increased pentose synthesis from [U−¹³C₁₈]stearate by 127% and 185%, respectively, while PNU-91325 rather increased glutamate synthesis in the Krebs cycle by 113% as compared to control vehicle treated cells. PNU-91325 also increased stearate chain shortening into palmitate by 59%. Glucose tracer-derived de novo palmitate and stearate synthesis were increased by 1 and 10 μM rosiglitazone by 41% and 83%, respectively, and by 63% and 75% by PNU-91325. Stearate uptake was also increased by 10 μM PNU-91325 by 15.8%. We conclude that the entry of acetyl Co-A derived from long-chain fatty acid β-oxidation into the mitochondria is facilitated by the mitoNEET ligand PNU-91325, which increases glucose-derived long chain fatty acid synthesis and breakdown via β-oxidation and anaplerosis in the mitochondria.     

Characterization of Surfactin-like Cyclic Depsipeptides Synthesized by Bacillus pumilus from Ascidian Halocynthia aurantium

A marine bacterium (KMM 1364), identified as Bacillus pumilus, was isolated from the surface of ascidian Halocynthia aurantium. Structural analysis revealed that the strain KMM 1364 produced a mixture of lipopeptide surfactin analogs with major components with molecular masses of 1035, 1049, 1063, and 1077. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portions of the compounds. Structurally, these lipopeptides differ from surfactin in the substitution of the valine residue in position 4 by leucine, and have been isolated as two carboxy-terminal variants, with valine or isoleucine in position 7. As constituents of the lipophilic part of the peptides, only β-hydroxy-C₁₅-, β-hydroxy-C₁₆-, and a high amount of β-hydroxy-C₁₇ fatty acid were determined.     

Homologue expression of a β-xylosidase from native <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. β-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-β-xylanase activity. This work reports the partial characterization of a purified β-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding β-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. β-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein⁻¹ at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol⁻¹, and at 50°C optimum stability was shown at pH 4.0–5.0. β-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), β-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn²⁺. Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.     

Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between <Emphasis Type="Italic">C. reticulata</Emphasis> and <Emphasis Type="Italic">C. limon</Emphasis>: the case of pulp carotenoid biosynthesis pathway

Allopolyploidy is known to induce novel patterns of gene expression and often gives rise to new phenotypes. Here we report on the first attempt to relate phenotypic inheritance in an allotetraploid somatic hybrid with gene expression. Carotenoid compounds in the fruit pulp of the two parental species and the hybrid were evaluated quantitatively by HPLC. Only very low levels of β-carotene and β-cryptoxanthin were observed in Citrus limon, while β-cryptoxanthin was a major component of C. reticulata, which also displayed high levels of phytoene, phytofluene, β-carotene, lutein, zeaxantin and violaxanthin. Total carotenoid content in mandarin juice sacs was 60 times greater than that in lemon. The allotetraploid hybrid produced all the same compounds as mandarin but at very low levels. Transgressive concentration of abscisic acid (ABA) was observed in the somatic hybrid. Real-time RT-PCR of total RNA from juice sacs was used to study expression of seven genes (CitDxs, CitPsy, CitPds, CitZds, CitLcy-b, CitChx-b, and CitZep) of the carotenoid biosynthetic pathway and two genes (CitNced1 and CitNced2) involved in abscisic acid synthesis from carotenoid. Gene expression was significantly higher for mandarin than lemon for seven of the nine genes analyzed. Lemon under expression was partially dominant in the somatic hybrid for three upstream steps of the biosynthetic pathway, particularly for CitDxs. Transgressive over expression was observed for the two CitNced genes. A limitation of the upstream steps of the pathway and a downstream higher consumption of carotenoids may explain the phenotype of the somatic hybrid.