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EP1021551A1 - Genes d'hydroxylase d'acide gras provenant de vegetaux - Google Patents

Genes d'hydroxylase d'acide gras provenant de vegetaux

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Publication number
EP1021551A1
EP1021551A1 EP98947736A EP98947736A EP1021551A1 EP 1021551 A1 EP1021551 A1 EP 1021551A1 EP 98947736 A EP98947736 A EP 98947736A EP 98947736 A EP98947736 A EP 98947736A EP 1021551 A1 EP1021551 A1 EP 1021551A1
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EP
European Patent Office
Prior art keywords
fatty acid
plant
acid
hydroxylase
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98947736A
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German (de)
English (en)
Inventor
Nathalie Tijet
Franck Pinot
Irene Benveniste
Renaud Le Bouquin
Christian Helvig
Yannick Batard
Francisco Cabello-Huartado
Daniele Werck-Reichhart
Jean-Pierre Salaun
Francis Durst
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Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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Publication of EP1021551A1 publication Critical patent/EP1021551A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition

Definitions

  • the present invention relates to the identification of plant fatty acid hydroxylase genes; their use in genetic engineering and the modification of the fatty acid content of a cell, preferably a transfected cell; and products thereof, such as nucleic acids, recombinant vectors, polypeptides, host cells, transgenic plants, plant products with altered hydroxylated fatty acid content.
  • cytochrome P450 The level of cytochrome P450 in most plants is significantly increased by exposure to various xenobiotics, endobiotic substrates, fungal infections, light irradiation, wounding of the tissues and subsequent aging.
  • a synthetic plant hormone, 2, 4-dichloro-phenoxy acetic acid (2,4-D) increased the spectrophotometrically detectable amount of cytochrome P450 in Jerusalem artichoke tuber tissues (Adele et al., 1981).
  • cytochrome P450 content and, more specifically, the activities of the lauric acid in-chain hydroxylase and omega-hydroxylase were substantially induced by phenobarbitol in various plants (Sala ⁇ n et al., 1981; 1982).
  • Clofibrate ethyl 2-[4-chlorophenoxy]-2-methylpropanoate
  • Clofibrate is a hypolipidemic drug causing a proliferation of mitochondria, smooth endoplasmic reticulum, and peroxisomes in mammalian liver.
  • Induction of cytochrome P450 and, more specifically, lauric acid omega-hydroxylase activity was observed in liver tissue from clofibrate treated test animals (Gibson et al., 1982). Similar effects are elicited by di-(2-ethylhexyl)- phthalate (DEHP), a widely used industrial plasticizer.
  • DEHP di-(2-ethylhexyl)- phthalate
  • cytochrome P450 has allowed the purification of plant fatty acid hydroxylases from enriched sources, determination of a protein sequence from a plant fatty acid hydroxylase, and cloning of a family of hydroxylases as disclosed herein. The identity of the cloned genes as fatty acid hydroxylases is confirmed by functional assay.
  • An object of the invention is to provide cytochrome P450 genes encoding plant fatty acid hydroxylases.
  • plant genes for terminal (omega or ⁇ ) hydroxylases having a peptide sequence which is a unique signature of plant fatty acid omega-hydroxylases for example, cytochrome P450 subtype CYP94
  • an in-chain ( ⁇ -1, ⁇ -2, ⁇ -3, and ⁇ -4 ) hydroxylase for example, cytochrome P450 subtype CYP81
  • Yet another object of the invention is to provide products derived from the plant fatty acid hydroxylase genes. Such products include, for example, nucleic acids, polypeptides, host cells, and transgenic plants.
  • a further object of the invention is to provide processes of making and using the plant fatty acid hydroxylase genes.
  • genetic engineering allows making structural and functional variants of the plant fatty acid hydroxylases using the disclosed nucleotide and amino acid sequences.
  • plant products with altered hydroxylated fatty acid content are obtained by producing plants with a transgene that affects fatty acid metabolism.
  • nucleic acids e.g., DNA, RNA, variants thereof
  • recombinant polynucleotides comprised of the nucleic acids (e.g., recombinant and expression vectors)
  • polypeptides encoded by the nucleic acids e.g., enzymes with fatty acid hydroxylase activity
  • host cells e.g., bacteria, yeast, plant
  • whole plants containing wild-type/mutant genes or wild-type/mutant gene products
  • a second embodiment of the invention is a process of making recombinant polypeptide by expressing a plant fatty acid hydroxylase gene sequence.
  • the polypeptide may be isolated from a host cell expressing the gene sequence and used as an enzyme in an industrial process, or the polypeptide may act within a host cell or plant to hydroxylate suitable fatty acid substrates.
  • Variant plant fatty acid hydroxylases could be produced by genetic mutation. Random or site-directed mutation, domain shuffling, rational design based on structural contacts between enzyme and substrate, and correlation between protein structure-enzyme activity are examples of methods to produce variant genes and their cognate proteins. Variant plant fatty acid hydroxylases could be selected for desirable properties such as, for example, modification of substrate specificity.
  • Suitable examples of such modifications include hydroxylation of shorter or longer fatty acid chains, or fatty acids with odd carbon numbers; hydroxylation of FA with in-chain hydroxy or epoxy groups; hydroxylation of thia-FA, which is a FA having a methylene group replaced by a sulfur atom; hydroxylation of an ether-FA , which is a FA having a methylene group replaced by an oxygen atom; hydroxylation of modified fatty acids such as esters or amides, instead of the usual fatty acids with a free carboxylic group.
  • Other desirable properties for selection are substrate affinity, modification of the rate of catalysis, enzyme lability or stability, and cofactor requirements.
  • metabolites of the fatty acid hydroxylases and plant fractions enriched for such metabolites are provided.
  • Processes of making transfected host cells and transgenic plants are provided to increase or decrease specific fatty acid hydroxylases using the disclosed sequences and expression vectors.
  • Such transfected host cells and transgenic plants provide a useful starting source for obtaining the desired metabolites from enriched or depleted fractions.
  • Hydroxylated fatty acids are preferably produced as storage lipids in transgenic seeds.
  • hydroxylated fatty acids are generally present in minor amounts in the phospholipid fractions (cellular membranes) of plants.
  • Overexpression of plant fatty acid hydroxylase genes in a transgenic plant can elevate the content of hydroxylated fatty acids in triglycerides of the transgenic plant.
  • the use of seed-specific promoters may allow accumulation of high amounts of hydroxylated fatty acids as storage lipids.
  • the accumulated fatty acids can be recovered by extracting oil from the transgenic seed and isolating the fatty acids stored therein.
  • a fourth embodiment of the invention are processes providing for identification of additional fatty acid hydroxylase genes by hybridization (e.g., low or high stringency), nucleic acid amplification (e.g., LCR, PCR), or by screening databases using the omega-FA hydroxylase signature defined hereinbelow, and making fatty acid hydroxylase variants by chemical modification of the enzyme or genetic mutagenesis of the hydroxylase sequence (e.g., point mutation, deletion, insertion, domain shuffling).
  • hybridization e.g., low or high stringency
  • nucleic acid amplification e.g., LCR, PCR
  • screening databases e.g., the omega-FA hydroxylase signature defined hereinbelow
  • making fatty acid hydroxylase variants by chemical modification of the enzyme or genetic mutagenesis of the hydroxylase sequence (e.g., point mutation, deletion, insertion, domain shuffling).
  • the fatty acid substrate may be, for example, capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), oleic (C18:l), linoleic (C18:2 and enantiomers (9E, 12Z) and (9Z, 12E)), and linolenic (C18:3) fatty acid.
  • Capric, lauric, and myristic fatty acids are considered medium- chain fatty acids
  • palmitic, oleic, linoleic, and linolenic fatty acids are considered long-chain fatty acids.
  • Hydroxylated and epoxidated fatty acids produced by the invention will provide oils with novel properties that may be used for the manufacture of lubricants, anti-slip agents, plasticizers, coating agents, detergents, and surfactants.
  • omega-hydroxylated fatty acids Apart from industrial considerations such as mass production as storage lipids in seeds, there are other reasons to over or under produce omega-hydroxylated fatty acids in plants.
  • the involvement of the omega hydroxylases in cuticle synthesis suggests that manipulation of the expression of these genes will affect the resistance of plants towards drought, or attack by insects and other pathogens.
  • hydroxylated fatty acids are per se activators (elicitors) which trigger the mechanisms of plant defense against pathogens.
  • Figure 1 Nucleotide sequence of CYP94A1 (Clone A) and deduced protein translation (SEQ ID NOS:3-4, respectively). Nucleotides of the open reading frame are shown in capital letters. The typical heme- binding domain, which constitutes the P450 signature, is underlined.
  • FIG. 2 Carbon monoxide difference spectrum of yeast microsomes expressing CYP94A1 (Clone A). Microsomes (10 mg protein/ml) prepared as described by Pompon et al. (1996) were diluted 5- fold, reduced with a few grains of sodium dithionite, and divided into two cuvettes. A baseline was recorded between 400 and 500 nm using a Shimadzu MP2000 double-beam spectrophotometer. Carbon monoxide was bubbled into the sample cuvette and the P450-CO complex spectrum was recorded. Based on a millimolar absorbance of 91 /cm, the amount of
  • CYP94A1 was 176 pmole/mg protein.
  • Figure 3 Chemical structure of sulfur-containing lauric acid analogs and sulfoxide metabolites.
  • Figure 4 Radiochromatogram of the reaction products formed from capric (C10:0), lauric (C12:0), myristic (C14:0), and palmitic (C16:0) acids by CYP94A1 (Clone A). After incubation of microsomes from transformed yeast with fatty acids, the reaction mixtures were extracted as described and analyzed by TLC. A: in the presence of NADPH; B: in the absence of NADPH; and S: residual substrate.
  • Figure 5 Radiochromatogram of the reaction products formed from oleic (C18:l), linoleic (C18:2), and linolenic (C18:3) acids by CYP94A1 (Clone A). After incubation of microsomes from transformed yeast with unsaturated fatty acids, the reaction mixtures were extracted as described and analyzed by TLC. A: in the presence of NADPH; B: in the absence of NADPH (control); and S: residual substrate.
  • Figure 6 Radiochromatogram of the reaction products formed from 9Z, 12E-octadecadienoic (C18:2-9E,12Z); 9E, 12Z-octadecadienoic (C18:2-9Z,12E); 8-propylsulfinyloctanoic (8S-LAU); and 10- methylsulfinyldecanoic (lOS-LAU) acids by CYP94A1 (Clone A). After incubation of microsomes from transformed yeast with unsaturated fatty acids, the reaction mixtures were extracted as described and analyzed by TLC (C18:2-9E,12Z and C18:2-9Z,12E) or HPLC (8S-LAU and lOS-LAU). A: in the presence of NADPH; B: in the absence of NADPH (control); and
  • Figure 7 Nucleotide sequence of VAGH811 (incomplete at 5' end), also called Clone B (complete cDNA encoding CYP94A2), and deduced protein translation ( ⁇ -MAH or CYP94A2) (SEQ ID NOS:5-6, respectively).
  • the consensus heme-binding domain which constitutes the
  • Figure 8 Carbon monoxide difference spectrum of yeast microsomes expressing ⁇ -MAH (Clone B). Microsomes (10 mg protein/ml) prepared as described by Pompon et al. (1996) were diluted 5-fold, reduced with a few grains of sodium dithionite, and divided into two cuvettes. A baseline was recorded between 400 and 500 nm using a Shimadzu MP2000 double-beam spectrophotometer. Carbon monoxide was bubbled into the sample cuvette and the P450-CO complex spectrum was recorded. Based on a millimolar absorbance of 91/cm, the amount of ⁇ -MAH was 80 pmole/ml microsomes.
  • Figure 9 Radiochromatogram of the reaction product formed from capric acid by ⁇ -MAH. After incubation with 14 C capric acid, the reaction mixture was extracted as described and analyzed by TLC. A: in presence of NADPH, B: in absence of NADPH.
  • Figure 10 Radiochromatogram of the reaction product formed from lauric acid by ⁇ -MAH. After incubation with 14 C lauric acid, the reaction mixture was extracted as described and analyzed by TLC. A: in presence of NADPH, B: in absence of NADPH.
  • Figure 11 Radiochromatogram of the reaction product formed from myristic acid by ⁇ -MAH. After incubation with l C myristic acid, the reaction mixture was extracted as described and analyzed by TLC.
  • Figure 13 Radiochromatogram of the reaction product formed from stearic acid by ⁇ -MAH. After incubation with l4 C stearic acid, the reaction mixture was extracted as described and analyzed by TLC. A: in presence of NADPH, B: in absence of NADPH.
  • Figure 14 Radiochromatogram of the reaction product formed from oleic acid by ⁇ -MAH. After incubation with 14 C oleic acid, the reaction mixture was extracted as described and analyzed by TLC. A: in presence of NADPH, B: in absence of NADPH.
  • Figure 15 Nucleotide sequence of CYP94A3 (Clone C) and deduced protein translation (SEQ ID NOS:7-8, respectively); compared to Clone B, nine nucleotides were missing at the 5' end.
  • Figure 16 Carbon monoxide difference spectrum of yeast microsomes expressing CYP94A3 (Clone C). Microsomes (10 mg protein/ml) prepared as described by Pompon et al. (1996) were diluted 5- fold, reduced with a few grains of sodium dithionite, and divided into two cuvettes. A baseline was recorded between 400 and 500 nm using a Shimadzu MP2000 double-beam spectrophotometer. Carbon monoxide was bubbled into the sample cuvette and the P450-CO complex spectrum was recorded.
  • FIG. 17 Radiochromatogram of the reaction products formed from capric (C10:0) and lauric (C12:0) acids by CYP94A3 (Clone C). After incubation of microsomes from transformed yeast with fatty acids, the reaction mixtures were extracted as described and analyzed by TLC. A: in the presence of NADPH; B: in the absence of NADPH
  • Figure 18 Radiochromatogram of the reaction products formed from myristic (C14:0) and lauric (C16:0) acids by CYP94A3 (Clone C). After incubation of microsomes from transformed yeast with fatty acids, the reaction mixtures were extracted as described and analyzed by
  • TLC in the presence of NADPH; B: in the absence of NADPH (control); and S: residual substrate.
  • Figure 19 Radiochromatogram of the reaction products formed from oleic (C18:l) and linoleic (C18:2) acids by CYP94A3 (Clone C). After incubation of microsomes from transformed yeast with fatty acids, the reaction mixtures were extracted as described and analyzed by TLC. A: in the presence of NADPH; B: in the absence of NADPH (control); and S: residual substrate.
  • Figure 20 Nucleotide sequence of CYP81B1 (Clone D) and deduced protein translation (SEQ ID NOS: 15- 16, respectively). The consensus heme-binding domain which constitutes the P450 signature is underlined.
  • FIG 21 Carbon monoxide difference spectrum of yeast microsomes expressing CYP81B1 (Clone D). Microsomes (10 mg protein/ml) prepared as described by Pompon et al. (1996) were diluted 5- fold, reduced with a few grains of sodium dithionite, and divided into two cuvettes. A baseline was recorded between 400 and 500 nm using a Shimadzu MP2000 double-beam spectrophotometer. Carbon monoxide was bubbled into the sample cuvette and the P450-CO complex spectrum was recorded. Based on a millimolar absorbance of 91 /cm, the amount of CYP81B1 was 202 pmoles/mg protein.
  • Figure 23 Radio-HPLC analysis of the metabolites obtained after 45 min of incubation at 27°C of 100 ⁇ M C12:0(a), and C14:0(b) with microsomes of transgenic yeast (0.1 mg protein) and 600mM NADPH.
  • Figure 24 Nucleotide sequence of CYP94A4 (Clone E) and deduced protein translation (SEQ ID NOS: 9-10, respectively) prepared from tobacco mosaic virus-infected tobacco leaves.
  • Figure 25 Nucleotide sequence of CYP94A5 (Clone F) and deduced protein translation (SEQ ID NOS: 11-12, respectively) prepared from tobacco mosaic virus-infected tobacco leaves.
  • Figure 26 Nucleotide sequence of CYP94A6 (Clone G) and deduced protein translation (SEQ ID NOS: 13-14, respectively) prepared from tobacco mosaic virus-infected tobacco leaves.
  • FA oxidation not been isolated to date because these membrane-bound enzymes are generally present in tissues at very low concentrations.
  • cytochrome P450 products encoded by the CYP4 gene family mainly fatty acid hydroxylases
  • cDNAs have been isolated and sequenced from mammalian and insect cDNA libraries.
  • Oxygenated FA from plants are mainly found in polar lipids such as triglycerides and phospholipids, and as monomers in polymeric layers.
  • Cutins and suberins are polymers mainly composed of hydroxylated fatty acids, especially omega-hydroxylated fatty acids. They protect plants against water loss, chemical penetration, attack by pathogens (e.g., microbes, insects), and other environmental stresses. Some of these are potent inducers of fungal cutinase and some show anti-fungal properties. These contrasting and apparently opposite effects may be due to the great diversity of defense mechanisms found in plants, and also to strategies developed by the fungi to infect the plant host. On the other hand, and as reported in mammals, hydroxylated fatty acids may play a role in responding to various stresses by giving rise to reactions similar to inflammatory processes, as a defense mechanism. Moreover, the presence of large amounts of hydroxy derivatives of the C18 family in the plant stigma suggests that they may play a role in recognition of the stigma by pollen.
  • Long-chain fatty acid omega and in-chain hydroxylases may play an important role in the synthesis of plant cuticles by generating hydroxy functions which appear essential to polymerization of constitutive cutin monomers.
  • Cuticle monomers are often present as complex mixtures with species-specific profiles.
  • epoxidated derivatives are also found as monomers of cuticles in a few plant species.
  • vicinal diol derivatives resulting from chemical and enzymatic ring-opening of epoxides have not been detected in cuticles from the C16 fatty acid family, suggesting that an epoxide function is not essential for polymerization of the cuticle matrix.
  • Fatty acids and their derivatives are subjected to many types of oxidation reactions including hydroxylation, epoxidation, dehydration and reduction.
  • cytochrome P450 Several forms of cytochrome P450 are suspected of being involved in these reactions.
  • previous studies have demonstrated that at least three distinct P450 isoforms are present in microsomes from various plant species when incubated with a model substrate such as lauric acid.
  • a model substrate such as lauric acid.
  • An interesting feature is that these P450 systems catalyze alternatively hydroxylation and epoxidation of unsaturated laurate analogs with a regio-specificity strongly dependent on the position and stereo-specificity dependent on the configuration of the double bond in the aliphatic chain.
  • Cytochrome P450-dependent reactions are involved in oxidation of fatty acids and derivatives in plants.
  • the reactions are grouped below according to type of reaction and the position of the carbon attacked. Some examples of induction by chemicals and inactivation by suicide substrates of the cytochrome P450 activities under consideration are discussed.
  • a lauric acid omega-hydroxylase ( ⁇ -LAH), producing exclusively 12-hydroxylauric acid, has been described in Pisum sativum, Vicia sativa and other leguminosae.
  • the microsomal fraction from clofibrate-treated V. sativa seedlings also catalyzed the omega-hydroxylation of capric (C10:0) and myristic (C14:0) acids.
  • a free carboxyl group appears essential for the binding of substrates to the enzyme. Induction and inhibition studies suggest that a single cytochrome P450 is capable of omega-hydroxylating these fatty acids.
  • IC-LAH lauric acid in-chain hydroxylase
  • the lauric acid ( ⁇ -l)-hydroxylase (( ⁇ -l)-LAH) from wheat generates a mixture of monohydroxylaurate in the proportion of 65%, 31% and 4% for 11 -hydroxy, 10-hydroxy and 9- hydroxylaurate, respectively.
  • Capric and myristic acids were also converted to ( ⁇ -1) and ( ⁇ -2) hydroxylated products. Additional minor metabolites hydroxylated at ( ⁇ -3) and ( ⁇ -4) were also detected when myristic acid was the substrate.
  • the length of FA (CIO to C 14) incubated no omega-hydroxylated products were detected.
  • results from our laboratory suggest that the ( ⁇ -l)-LAH from wheat catalyzes the hydroxylation of the herbicide diclofop.
  • Biosynthesis of plant cuticles involves distinct P450 systems.
  • the in-chain hydroxylation of omega-hydroxypalmitic acid by V. faba microsomes gives rise to 9 (or 10),16-dihydroxypalmitic acid.
  • the reactions have been attributed to a cytochrome P450 which differs from those involved in omega-hydroxylation of palmitic acid by effective reversal by light of CO inhibition.
  • V. sativa microsomes The capability of V. sativa microsomes to catalyze the oxidation of two sulphur-containing lauric acid analogs has been examined.
  • Two sulphides synthesized in radiolabeled form, (1- 14 C) 10- methylsulphinyldecanoic acid (lOS-LAU) and (1- 14 C)8- propylsuphinyloctanoic acid (8S-LAU) were incubated with V. sativa microsomes under conditions promoting either P450 or peroxidase reactions.
  • both 8- and 10-thio fatty acids were actively converted to the sulphoxide by at least two distinct membrane bound enzymes.
  • Cytochrome P450 activities from plants are induced by light, UV-irradiation, wounding, ripening, fungal infection, elicitors, endogenous compounds and numerous chemicals, including safeners, herbicides, drugs and pollutants.
  • Clofibrate is a well known hypolipidemic drug which induces peroxisome proliferation in both mammals and plants.
  • Clofibrate and related arylphenoxy compounds such as 2,4- dichlorophenoxy-acetic acid (2,4-D), which selectively induce fatty acid omega-hydroxylase activity, have little or no effect on the activity of IC- LAH from H. tuberosus tubers and ( ⁇ -l)-LAH from wheat seedlings.
  • V. sativa microsomes contain exclusively fatty acid omega-hydroxylases.
  • Cytochrome P450 content was increased to about 0.5 nmole/mg, one of the highest levels so far recorded in plants.
  • the relative amounts of 11-, 10- and 9-hydroxylaurates formed remained unchanged under all conditions.
  • the ( ⁇ -l)-oleic acid hydroxylase activity was induced in treated seedlings to the same extent as ( ⁇ -l)-LAH, although these P450-dependent reactions were supported by distinct isoforms.
  • cytochrome P450 induction in plant systems remains unknown but most of the P450 inducers active in mammals are also effective in induction of plant P450.
  • hypolipidemic drugs such as clofibrate
  • certain physiological conditions involves transcriptional activation of the genes which was mediated by receptors (peroxisome proliferator-activated receptors).
  • receptors peroxisome proliferator-activated receptors
  • Mechanism-based inhibitors (suicide substrates) containing a terminal acetylene are potent irreversible inhibitors of both plant and mammalian fatty acid omega-hydroxylases. Pre-incubation of microsomes from clofibrate-treated V. sativa seedlings with 11-dodecynoic acid (11-
  • microsomal proteins which correlated well with diacid formation and inactivation of ⁇ - LAHs, increased as a function of incubation time and concentration of (1- 14 C)l l-dodecynoic acid. Based on these results, two potential inhibitors targeted to inactivate the omega-hydroxylation of oleic acid were synthesized. Incubation of microsomes from V.
  • the terminal olefin 11 -dodecenoic acid inactivates a P450 from wheat which catalyzes mainly oxidation of the internal carbon ( ⁇ -1) of laurate.
  • P450 inactivation by a terminal olefin proceeds via an oxidative attack on the internal carbon ( ⁇ -1) of the double bond leaving a terminal methylene radical free to alkylate the heme unit.
  • the plant ⁇ -LAH which exclusively attacks the external position, catalyzed the formation of the 11- 12 epoxide without any measurable loss of activity.
  • Acetylenic derivatives of lauric acid are also potent inactivators of ( ⁇ -l)-LAH from wheat.
  • At least three distinct roles for the plant fatty acid hydroxylases of the present invention are foreseeable: cutin and suberin synthesis, rapid catabolism of free fatty acids (i.e., detoxification), and synthesis of signaling molecules.
  • a plant with desirable characteristics may result from the modification of cutin and suberin production.
  • a null mutant or a hypomorph would be a slow growing plant relative to the wildtype plant. Wounding or other types of stress lead to the activation of phospholipases, drastic liberation of fatty acids, and an oxidative burst (Low et al., 1996).
  • elicitors activate phospholipases in plants (see Chandra et al., 1996) whose activation will lead to liberation of free fatty acids.
  • P450s involved in epoxidation of unsaturated fatty acids may also be involved in resistance to disease via the production of hydroxylated and epoxidated fatty acids which have been shown to inhibit the growth of pathogens (i.e., synthesis of signaling molecules). If the role of oxygenated fatty acids in fungal infections is considered, contradictory effects seem apparent, because reports indicate that certain monomers from cutin (i.e., dihydroxy fatty acids and 9,10,18- trihydoxystearic acid) are potent inducers of the cutinase of several pathogenic fungi. Schweizer et al.
  • Plastics that can be produced from hydroxylated fatty acids are polyurethanes and polyesters (Weber at al., 1994). It should be noted that cutin itself is a bioplastic constituted almost entirely of oxyfatty acids. Omega hydroxylation is required for the chain-elongation reaction, and in-chain hydroxylation and/or epoxidation is required for reticulation. Plants can be engineered to produce C12 fatty acids by transforming them with the acyl-ACP thioesterase from Umbellularia califomica which is specific for lauroyl-ACP. Arabidopsis thaliana transformed with this gene produce up to 25% laurate.
  • CYP81B1 was expressed in yeast, and a systematic exploration of its function revealed that this enzyme specifically catalyzes the hydroxylation of medium chain saturated fatty acids, namely capric (C10:0), lauric (C12:0) and myristic (C14:0) acids.
  • the same metabolites were obtained with transgenic yeast and plant microsomes: a mixture of ⁇ -1 to ⁇ -5 monohydroxylated products was observed.
  • the three fatty acids were metabolized with high and similar efficiencies, the major position of attack depending on chain length. When lauric acid was the substrate, turnover was
  • Table 1 Summary of reactions with fatty acids catalyzed by a plant cytochrome P450.
  • Phaseolus aureus lauric (C12:0) ⁇ -OH Phaseolus vulgaris lauric (C 12:0) ⁇ -OH Vicia sativa C10:0-C14:0 ⁇ -OH
  • Helianthus annuus C 12:0 ( ⁇ -2), ( ⁇ -3) (mainly) or ( ⁇ -4)-OH Zea mays Tulipa fosteriana Amaryllis belladonna Spinacia oleracea 18-OH-C 18:1 -CoA 9, 10-epoxy- 18-OH-C 18 :0-CoA Euphorbia lagascae linoleyl-PC 12,13-epoxy-C18:l ⁇ 9
  • CYP73A1 is the cinnamate 4-hydroxylase which catalyzes the first committed oxidation reaction in the general phenylpropanoid pathway, leading to lignins, flavonoids, defense molecules, anti-UV protectants etc.
  • This enzyme was purified to homogeneity using Triton XI 14 phase partitioning, and cloned using a specific antibody raised against the pure protein (Teutsch et al, 1993). Data suggest that the induction of cinnamate 4-hydroxylase activity primarily results from gene activation. Time-course experiments were performed after wounding and aminopyrine treatment. The timing of the induced changes in activity, protein and transcripts confirms that C4H induction results primarily from an increase in CYP73A1 mRNA both in wounded and aminopyrine treated tissues. However, post- transcriptional mechanisms might also contribute to the regulation of C4H activity.
  • CYP76B1 is an alkoxycoumarin O-dealkylase (Batard et al, 1995), whose true physiological function remains unknown.
  • the protein was purified by the same method employed for CYP73A1, and the gene was cloned using cytochrome P450 primers deduced from the microsequenced peptide (Batard et al, 1998). Determination of the steady-state level of CYP76B1 transcripts after slicing tuber tissues and aging them in water, alone or in the presence of various chemicals, showed that the expression of this P450 was not responsive to mechanical stress, but was strongly induced by chemical treatments. Therefore, CYP76B1 appears to be a good potential marker of chemical stress and of environmental pollution.
  • Clofibrate stimulates efficiently (20-fold) the cytochrome P450-catalyzed activities of lauric and oleic acid omega-hydroxylation in microsomes of Vicia sativa seedlings.
  • DEHP and 2,4-D have a similar stimulating effect on the lauric acid omega-hydroxylase in the same material.
  • the inventors isolated cDNAs coding for plant fatty acid omega-hydroxylases. After expression in yeast, the omega hydroxylase substrate specificities were characterized: CYP94A1 omega-hydroxylates fatty acids with different chain length (CIO to C18) and different degrees of unsaturation (C18:l, C18:2, C18:3).
  • RNAs from clofibrate-treated Vicia sativa seedlings revealed a very rapid (after 20 min) and large accumulation of the CYP94A1 transcripts, suggesting the involvement of a clofibrate receptor in the signal transduction.
  • a promoter sequence of CYP94A1 was isolated. A search for key regulator elements is currently in progress.
  • a study of peroxisome proliferation in Vicia sativa in response to clofibrate, at the level of Acyl CoA oxidase transcripts has been initiated.
  • CYP4A Mammalian ⁇ -hydroxy lating enzymes have been extensively studied (Simpson 1997), and they have been classified in the CYP4A family. CYP4A are known to be involved in the metabolism of arachidonic acid leading to the formation of physiologically important metabolites. They also participate in the catabolism of fatty acids (Gibson 1989). In plants, fatty acid ⁇ -hydroxylases are implicated in the biosynthesis of cuticle (Kolattukudy 1981). Moreover, ⁇ -hydroxy fatty acids have been recently reported to play an important role in plant defense mechanisms
  • Arylphenoxy compounds such as the hypolipidemic drug clofibrate and the herbicide 2,4-D are members of a class of chemicals known to induce fatty acid hydroxylase activities and proliferation of peroxisomes.
  • long-chain fatty acid omega-hydroxylases are believed to play a crucial role in synthesis of cuticles protecting plants from the outer environment.
  • CYP94A1 which is the first P450-dependent fatty acid omega-hydroxylase cloned from a plant was isolated by tagging of the P450 apoprotein with a radiolabeled mechanism-based inhibitor. The functional expression of this novel P450 in S.
  • CYP94A1 fatty acids
  • Clone A encodes the saturated and unsaturated fatty acid (FA) omega-hydroxylase, a microsomal cytochrome P450-dependent hydroxylase which catalyzes the transformation of capric (C10:0), lauric (C12:0), myristic (C14:0) palmitic (C16:0), oleic (C18:l), linoleic (C18:2 and enantiomers (9E,12Z); (9Z,12E)) and linolenic (C18:3) acids into their corresponding omega-hydroxy acids.
  • FA saturated and unsaturated fatty acid
  • CYP94A1 (Clone A) (Weissbart et al., 1992; Pinot et al., 1992, 1993; Helvig et al., 1997). Isolation of CYP94A1 (Clone A)
  • peptides were transferred to a nylon membrane (IMMOBILONTM) and sequenced by the Edman degradation method. Four peptides were sequenced. Only two showed homology to P450s and were subsequently found in the deduced amino acid sequence of clone A (SEQ ID NO:4).
  • the first peptide contained the 18-20 amino acid hydrophobic domain which is typical of the membrane anchor found in all microsomal P450s. After isolation of the clone, it was confirmed that this peptide corresponds to the N-terminal amino acid sequence of the enzyme:
  • the second peptide had the sequence LMNLYPPVPMMNAKEVVVXVLLXQ.
  • a computer search with this peptide against all known cytochrome P450 enzymes showed partial homology of the beginning with a domain which is found at about 130 residues from the C-terminus in several P450s of family CYP4, the family containing the mammalian fatty acid omega-hydroxylases: peptide p3, rat CYP4A1, rat CYP4B1 and rat CYP4A3.
  • This primer was used in association with an oligo (dT) primer to produce a probe of 661 bp by RT-PCR on total RNA from clofibrate-treated V. sativa seedlings.
  • Conditions were the following: denaturation for 5 min at 93°C; followed by 30 cycles of denaturation for 1 min at 93°C, 2 min hybridization at 48°C, and 3 min elongation at 72°C; and terminated by 10 min elongation at 72°C.
  • a ⁇ ZAP cDNA library prepared from poly(A) RNA from 48 hour clofibrate-treated V. sativa seedlings, following the manufacturer's instructions (Stratagene), was screened at high stringency using the 650 bp probe. The probe was random labeled using ( ⁇ - 32 dCTP) and hybridized for 24 hours at 65°C in 5 x SSC, 0.5% SDS, 5 x Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA, 2 mM EDTA, and 50 mM sodium phosphate, pH 6.0.
  • VAGH111 was isolated, sequenced, and found to encode a new cytochrome P450, CYP94A1 ( Figure 1)- Heterologous Expression in Yeast
  • CYP94A1 (Clone A) was assessed by functional expression in genetically engineered yeast.
  • the coding sequence of Clone A (SEQ ID NO:3) was PCR cloned into expression vector pYeDP60 using the BamRl and EcoRI restriction sites as follows.
  • Yeast strain WAT 11 transformed with pYeDP60 harboring CYP94A1 (Clone A) was grown and induced according to Pompon et al. (1996).
  • a culture was started from one isolated colony. After growth, cells were centrifuged 10 min at 7500g at 4°C. The pellet was washed with TEK (2 ml TEK g cells), and centrifuged 10 min at 7500g at 4°C. The pellet was resuspended in 1ml TES and glass beads were added up to liquid surface level. Cells were broken by manual shaking for 5 min in the cold room using a 30 ml conical Falcon plastic tube with 0.5 mm diameter glass beads.
  • TEK Tris-HCl 50mM pH 7.5; EDTA ImM; KC1 lOOmM TES: Tris-HCl 50mM pH 7.5; EDTA ImM; sorbitol 600mM
  • TEG Tris-HCl 50mM pH 7.5; EDTA ImM; glycerol 20%
  • the reaction was initiated by adding NADPH at 27°C and stopped after 10 min incubation with 0.2 ml acetonitrile-acetic acid (99.8/0.2, v/v). After extraction with 2 x 600 ⁇ l diethyl ether, the organic phase was spotted on silica thin-layer plates and developed in a mixture of diethyl ether-light petroleum (b.p. 40-60°C)-formic acid (70/30/1, v/v/v for CIO to C16 substrates, and 50/50/1 for C18 substrates). Plates were scanned with a Berthold thin-layer scanner. For precise rate measurements, radioactive spots were scraped into counting vials and product formation was quantified by liquid scintillation. All the reactions products identified in these experiments have been identified by GC/MS spectroscopy. The activity of CYP94A1 (Clone A) with different fatty acid substrates are shown in Table 2.
  • Oleic acid (Cl 8:1) 14.3 ⁇ 0.5 38.7 ⁇ 4.3 -
  • Linoleic acid (C18:2) 9.1 ⁇ 0.9 47.0 ⁇ 1.9 -
  • Linolenic acid (C18:3) 24.6 ⁇ 2.3 70.0 + 3.5 -
  • the TLC radiochromatograms are shown in Figure 4 (capric, lauric, myristic, and palmitic acids), Figure 5 (oleic, linoleic, and linolenic acids), and Figure 6 (C18:2-9£,12Z; C18:2-9Z,12£; 8S-LAU and lOS-LAU acids).
  • Figure 4 capric, lauric, myristic, and palmitic acids
  • Figure 5 oleic, linoleic, and linolenic acids
  • Figure 6 C18:2-9£,12Z; C18:2-9Z,12£; 8S-LAU and lOS-LAU acids.
  • NADPH NADPH
  • B hydroxylase is inactive
  • Clone B encodes ⁇ -MAH (omega-myristic acid hydroxylase), a microsomal cytochrome P450-dependent hydroxylase which catalyzes the transformation of myristic (C14:0) acid into 14-hydroxytetradecanoic acid (i.e., the terminal methyl was hydroxylated).
  • ⁇ -MAH omega-myristic acid hydroxylase
  • C14:0 myristic acid hydroxylase
  • 14-hydroxytetradecanoic acid i.e., the terminal methyl was hydroxylated
  • the clone was obtained by screening a ⁇ ZAP cDNA library prepared from clofibrate-treated Vicia sativa seedlings with Clone A, as follows.
  • DNA fragment (starting at position 1201 in Figure 1) produced by RT-PCR during the isolation of Clone A. The fragment was 32 P-radiolabeled by random priming. Hybridization was at 55°C overnight in 5 x SSC, 0.5% SDS, 5 x Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA, 2 mM EDTA, and 100 mM sodium phosphate, pH 6.0. After hybridization, blots were washed twice with 2 x SSC, 0.1% SDS at room temperature for 15 min, and twice with 0.2 x SSC, 0.1% SDS at 45°C for 30 min.
  • VAGH811 One clone (1437 bp) that was isolated, VAGH811, was sequenced and found to encode a new cytochrome P450 ( Figure 7).
  • the clone was incomplete at the 5' end, but the sequence of the incomplete clone was used to synthesize a primer for 5 '-RACE with poly(A) RNA -from 96 hour clofibrate-treated Vicia sativa seedlings to obtain the complete coding sequence of VAG811, named Clone B hereinafter ( Figure 7).
  • CYP94A2 Catalytic activity of CYP94A2 (Clone B) was assessed by functional expression in genetically engineered yeast, as described hereinabove for clone A.
  • the coding sequence of CYP94A2 (Clone B, SEQ ID NO:5) was PCR cloned into expression vector pYeDP60 using the Smal and Sacl restriction sites as follows (peptide sequences contained in SEQ ID NO:6).
  • Sacl Boehringer HiFiTM polymerase was used according to manufacturer's instructions and the amplified sequence was verified to avoid polymerase-generated errors.
  • Yeast strain WAT 11 (Urban et al., 1990) was transformed according to Schiestl and Gietz (1989).
  • TLC radiochromatograms are shown in Figure 9 (capric acid), Figure 10 (lauric acid), Figure 11 (myristic acid), Figure 12 (palmitic acid), Figure 13 (stearic acid), and Figure 14 (oleic acid).
  • NADPH hydroxylase is active
  • B hydroxylase is inactive
  • CYP94A3 encodes a cytochrome P450-dependent hydroxylase which catalyzes the methyl terminal oxidation of capric (C10:0), lauric (C12:0), and myristic (C14:0) acids and to a lesser extent the omega- hydroxylation of palmitic (C16:0), oleic (C18:l), and linoleic (C18:2) acids. Isolation of CYP94A3 (Clone C)
  • Clone C was obtained by screening a ⁇ Zap cDNA library prepared from clofibrate-treated Vicia sativa seedlings with a 3' terminal cDNA fragment (300 bp) of CYP94A2 (Clone B) (from the sequence coding for the heme binding domain to the poly A tail) at high stringency. Hybridization was for 24 hours at 65°C in 5 x SSC, 0.5% SDS, 5 x
  • CYP94A2 For heterologous expression, the first nine nucleotides of CYP94A2 were added in front of the incomplete sequence of CYP94A3 (Clone C). The full-length cDNA was isolated since and found to be identical to the one used in these activity experiments.
  • the coding sequence of CYP94A3 (Clone C, SEQ ID NO:7) was PCR cloned into expression vector pYeDP60 using the Smal and Sacl restriction sites as follows (peptide sequences contained in SEQ ID NO:8).
  • Boehringer HiFiTMpolymerase was used according to manufacturer's instructions and the amplified sequence was verified for polymerase-generated errors.
  • Yeast strain WATl l Urban et al., 1990
  • Schiestl and Gietz (1989) Characterization of CYP94A3 (Clone C)
  • Microsomes Yeast strain WAT11 transformed with pYeDP60 harboring CYP94A3 (Clone C) was grown and induced as described for the preceding clones. Microsomes can be stored at -20°C for several weeks without loss of activity. WAT11 cells transformed with pYeDP60 expression vector only were subjected to the same procedure for control experiments.
  • Measurement of P450 Microsomes were diluted 5-fold with TEG and P450 was measured (Figure 2) with the method of Omura and Sato (1964) using a mmolar absorbance coefficient of 91 cm 'mM "1 .
  • Measurement of Activity Enzymatic activities were measured as previously described as described hereinabove for the preceding clones. The activity of CYP94A3 (Clone C) with different fatty acid substrates are shown in Table 4.
  • Linoleic acid (C18:2) 1.6
  • In-chain Hydroxylase (IC-LAH) of capric, lauric, and myristic acids Clone D encodes a microsomal P450 from Helianthus tuberosus (Jerusalem artichoke), catalyzing the ⁇ -2, ⁇ -3 and ⁇ -4 hydroxylation of capric (C10:0), lauric (C12:0), and myristic (C14:0) acids.
  • the major metabolite is the ⁇ -3-hydroxylated compound.
  • Polyclonal antibodies raised against this P450-enriched fraction were used to screen a ⁇ ZAPII cDNA library prepared from H. tuberosus tuber tissues sliced and aged 24 hour in presence of 20 mM of aminopyrine. Positive clones (56) were isolated and tested for the presence of a P450 consensus sequence using the PCR technique previously described by Meijer et al.
  • PCR fragments of expected size were obtained from 15 clones, labeled and hybridized with total RNA prepared from dormant, wounded or aminopyrine-treated tuber tissues.
  • One of the 15 clones corresponded to a 2.2 kb transcript almost undetectable in dormant and wounded tuber, but induced by aminopyrine. Sequencing of its insert showed that it coded for a
  • CYP81B1 Catalytic activity of CYP81B1 (Clone D) was assessed by functional expression in yeast.
  • a genetically engineered yeast strain providing a suitable environment for plant P450 expression (membrane structures and presence of a plant P450 reductase) was used for this purpose.
  • This strain WATl l, the expression vector, subcloning of the coding sequence, yeast growth, transformation, and preparation of yeast microsomes are described in Pompon et al. (1996).
  • Figure 21 was about 202 pmoles/mg protein (i.e., about 1% of the microsomal protein). Catalytic activity was tested with more than 20 potential radiolabeled substrates including aromatic compounds, sterols, herbicides, and fatty acids. Fatty acid metabolism was assayed as described by Sala ⁇ n et al. (1981).
  • Apparent Vmax and Km of the reaction were determined in the case of capric and lauric acids.
  • the reaction proceeds with an enzyme turnover of 41 ⁇ 0.8 min "1 and Km of 903 ⁇ 168 nM in the case of capric acid, and with an enzyme turnover of 30.7 ⁇ 1.4 min "1 and Km of 788 ⁇ 400 nM in the case of lauric acid.
  • TLC profiles in Figure 22 show that CYP81B1 (Clone D) codes for a P450 catalyzing formation of the same metabolites.
  • the ⁇ -2, ⁇ -3, ⁇ -4 hydroxylated metabolites are generated from the three fatty acid substrates (capric, lauric, and myristic acids) in the same proportions as in plant microsomes.
  • An additional minor product is detected after incubation of lauric acid with the yeast-expressed enzyme; the structure of this metabolite is currently being investigated.
  • the presence and proportions of the three metabolites were confirmed by HPLC (Figure 23).
  • CYP94A4 encodes a cytochrome P450-dependent hydroxylase which catalyzes the methyl terminal oxidation of capric (C10:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), oleic (C18:l), linoleic (C18:2), and linolenic (C18:3) acids, with very contrasted efficiencies. Highest activity is with C14:0 and C12:0. Isolation of CYP94A4 (clone E)
  • Clone E was obtained by screening a lambda-Zap cDNA library prepared from TMV-infected tobacco leaves (Dr M. Legrand; IBMP France) with CYP94A1, A2 and A3 as follows:
  • Fifteen clones greater than 1500 pb were isolated and sequenced. Ten of these clones were full-length and were found to encode for a new cytochrome P450 of the CYP94 family, which was named CYP94A4 ( Figure 24).
  • CYP94A4 Catalytic activity of CYP94A4 (Clone E) was assessed by functional expression in a ad hoc engineered yeast, as described hereinabove for the preceding clones.
  • the coding sequence of Clone E (SEQ ID NO:9) was PCR cloned into expression vector pYeDP60 using the BamHl restriction site as follows: Sense primers (BamHl) A4 M M I D L E L
  • Microsomes were diluted 5-fold with TEG and P450 measured (Figure 2) with the method of Omura and Sato (1964) using a mmolar absorbance coefficient of 91. cm “1 . mM "1 .
  • Linoleic acid (Cl 8:2) 4.5 47.9 0.094
  • CYP94A5 encodes a cytochrome P450-dependent hydroxylase which catalyzes the methyl terminal oxidation of lauric (C12:0), myristic (C14:0), palmitic (C16:0), oleic (C18:l), linoleic (C18:2), and linolenic (C18:3) acids, with very contrasted efficiencies. Highest activity is with C14:0 and C18:2.
  • Clone F was obtained by screening a lambda-Zap cDNA library prepared from TMV-infected tobacco leaves (Dr M. Legrand; IBMP France) with CYP94A1, A2 and A3 as follows.
  • a ⁇ ZAP cDNA library prepared from poly(A + ) RNAs from tobacco ⁇ Nicotiana tabacum var. Samsun NN) leaves infected for 48h with TMV, was screened at low stringency using a mixture of the coding sequences for CYP94A1, A2 and A3 as probe. The probe was 32 P- radiolabeled by random priming.
  • Fifteen clones greater than 1500 pb were isolated and sequenced. Two full-length clones were found to encode for a new cytochrome P450 of CYP94 family, which was termed CYP94A5 ( Figure 25 ).
  • Catalytic activity of CYP94A5 was assessed by functional expression in a ad hoc engineered yeast as described for the preceding clones.
  • the coding sequence of Clone F (SEQ ID NO:l l) was PCR cloned into vector pYeDP60 using the BamHl restriction site as follows (peptide sequences contained in SEQ ID NO: 12).
  • Antisense primers (Kpnl) A4 GAA AGG AAC GGT ACG GAT ATT TGA A4 E R N G T D I stop
  • Boehringer HIFITM polymerase was used according to manufacturer's instructions and the amplified sequence was verified for polymerase errors.
  • Yeast strain WATl l (Urban et al. 1990) was transformed according to
  • Yeast (strain WATl l) transformed with pYeDP ⁇ O harboring clone F was grown and induced as described hereinabove for the preceding clones. Untransformed WATl l cells were subjected to the same procedure for control experiments.
  • Microsomes were diluted 5-fold with TEG and P450 measured (Figure 2) with the method of Omura and Sato (1964) using a mmolar absorbance coefficient of 91.cm “ '.mM “1 .
  • Oleic acid (Cl 8:1) 1.7 36.5 0.046
  • Linoleic acid (Cl 8:2) 4.3 36.5 0.12
  • Table 8 The relative efficiency of 94A4 versus 94A5 was compared.
  • CYP94A6 encodes a cytochrome P450.
  • the catalytic activity is presently being assessed. It is expected that it will show fatty acid hydroxylase activity since it displays the characteristic signature sequence for this class of enzymes.
  • Clone G was obtained by screening a lambda-Zap cDNA library prepared from TMV-infected tobacco leaves (Dr M. Legrand; IBMP France) with CYP94A1, A2 and A3 as follows.
  • CYP94A6 One uncomplete clone was found to code for a new cytochrome P450 of the CYP94 family, which was named CYP94A6.
  • the complete sequence for CYP94A6 was obtained by performing inverse-PCR on genomic tobacco ⁇ Nicotiana tabacum var. Samsun NN) DNA, using the Ndel restriction site ACATAT at position 594 ( Figure 26 ) and sequence specific primers.
  • CYP94A6 has been expressed in yeast as described hereinabove, and the protein produced has been detected by Western blotting. Catalytic activity of Clone G is being assessed by functional expression in a ad hoc engineered yeast. Reformatting of Clone G
  • Boehringer HIFITM polymerase was used according to manufacturer's instructions and the amplified sequence was verified for polymerase errors.
  • Yeast strain WATl l (Urban et al. 1990) was transformed according to Schiestl and Gietz (1989).
  • Yeast (strain WATl l) transformed with pYeDP60 harboring clone G was grown and induced as described hereinabove. Untransformed WATl 1 cells were subjected to the same procedure for control experiments.
  • Microsomes were diluted 5-fold with TEG and P450 measured (Figure 2) with the method of Omura and Sato (1964) using a mmolar absorbance coefficient of 91.cm “ '.mM “1 .
  • Tobacco ⁇ Nicotiana tabacum L. var Xanthi was transformed with the open REPETITION open reading frames of clones E (CYP94A4), F (CYPA5) and G (CYPA6), in sense and antisense orientation.
  • the coding sequences were cloned into pFB8, a custom built vector from our Institute
  • CYP94A4, CYPA5, CYPA6 coding sequences The coding sequences were PCR cloned into vector pFB8 using the BamHl and Kpnl restriction sites indicated in bold type as follows (peptide and nucleic acid sequences contained in SEQ ID NOS:4 and 3, respectively, for 94 A4, and SEQ ID NOS: 13 and 12, respectively, for 94A5/94A6 )
  • Plants transformed in both sense and antisense directions are growing at present with seeds for TI expected shortly.
  • the inventors have identified a peptide sequence (SEQ ID NO:2), marked by a double underline in Figures 1, 7, 15, 24, 25 and 26, which is a unique signature found in all plant fatty acid omega- hydroxylases characterized so far:
  • (AVS) means one of A, V or S;
  • TVS means on eof T, V, or S;
  • LIV means one of L, I or V.
  • This signature sequence is present in CYP86A1 (SEQ ID NO:l), CYP86A5, CYP94A1, CYP94A2, CYP94A3, CYP94A4, CYP94A5, and CYP94A6. All but CYP94A6 (characterization under way) have omega-hydroxylase activity. This signature sequence is not present in CYP81B1, the in-chain hydroxylase, as evidenced by sequence alignment.
  • a scan of all plant genes in Genbank for this signature sequence retrieved 12 sequences, all of which are cytochromes P450 isofoms. Some of the sequences are redundant because they originate from different laboratories recloning the same genes.

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Abstract

Il a été identifié plusieurs hydroxylases d'acide gras dépendantes du cytochrome P450 provenant de différentes sources végétales grâce à des techniques de clonage de recombinaison, ces hydroxylases ayant été ensuite caractérisées structurellement et fonctionnellement. Ces clones représentent de nouvelles hydroxylases végétales qui sont actives une fois exprimées dans un système hétérologue de levure. Ces enzymes hydroxylases, qui hydroxylent des substrats d'acide gras à des positions différentes et bien définies dans des substrats d'acide de longueurs de chaîne variées, catalysent l'époxydation d'acides gras naturels et synthétiques, portant une double liaison sur le site d'attaque.
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