JP2001519164A - Plant fatty acid hydroxylase gene - Google Patents

Abstract

  (57) [Summary] A plurality of cytochrome P450-dependent fatty acid hydroxylases collected from various plant resources were identified by recombinant cloning technology, and the structures and functions of these hydroxylases were characterized. These clones are representative novel plant hydroxylases that show activity when expressed in heterologous yeast systems. These hydroxylase enzymes hydroxylate fatty acid substrates at various well-defined positions in acid substrates of various lengths. This hydroxylase exerts a catalytic effect in the epoxidation reaction of natural fatty acids and synthetic fatty acids having a double bond at the attack position.

Description

DETAILED DESCRIPTION OF THE INVENTION

 (Background of the Invention)

FIELD OF THE INVENTION The present invention relates to the use of plant fatty acid hydroxylase genes in the identification of plant fatty acid hydroxylase genes, genetic engineering and modification of fatty acid content of cells, preferably of transgenic cells, and nucleic acids, Recombinant vectors, polypeptides,
The present invention relates to host cells, transgenic plants, and plant products having altered contents of hydroxylated fatty acids.

2. Description of the Related Art Some plant microsomes have the thermal hydroxylation of fatty acid substrates or intramolecular (in)
-chain) There are two laurate hydroxylases that catalyze hydroxylation. These two hydroxylases are both cytochrome P-450 enzymes, but these hydroxylases do not coexist in the same plant (
Salaun et al., 1982).

[0003] Lauric acid is hydroxylated by intramolecular hydroxylases in jerusalem artichoke tubers, tulip bulbs, corn seedlings, and several other plants to produce ω-2, ω-3, and ω-4. Produces a mixture of monohydroxylaurate. In other plants, mainly leguminosae, laurate omega hydroxylase is present, and the hydroxylase hydroxylates the terminal methyl group of the fatty acid substrate. Although these two effects were found in separate P450 species, they did not co-exist in the twelve plant species analyzed (Salaun et al., 1982).

[0004] In many plants, the concentration of cytochrome P450 has been significantly increased when exposed to various xenobiotics or endogenous organisms, infecting fungi, irradiating with light or damaging tissues. The synthetic plant hormone 2,4-dichloro-phenoxyacetic acid (2,4
-D) increased the amount of cytochrome P450 contained in tuber tissue of Jerusalem artichoke within a concentration range detectable spectroscopically (Adele et al., 1981). Similarly, in a variety of plants, cytochrome P450 content, and more particularly the effects of lauric acid intrachain hydroxylase and omega hydroxylase, were substantially induced by phenobarbitol (Salaun et al., 1981; 1982). ).

[0005] Clofibrate (ethyl 2- [4-chlorophenoxy] -2-methylpropanoate) is a lipid-lowering agent that acts to proliferate peroxisomes in mitochondria, endoplasmic reticulum, and mammalian liver. The action of cytochrome P450, more specifically the action of lauric omega hydroxylase, was observed in liver tissue collected from test animals treated with clofibrate (Gibson et al., 1982). A similar effect is induced by di- (2-ethylhexyl) -phthalate (DEHP), which is widely used as an industrial plasticizer.

[0006] As disclosed herein, by inducing cytochrome P450, purification of plant fatty acid hydroxylases from enriched resources, sequencing of proteins collected from plant fatty acid hydroxylases, and the hydroxylase family Cloning is now possible. The fact that the cloned gene is fatty acid hydroxylase itself has been confirmed by evaluating its function.

SUMMARY OF THE INVENTION An object of the present invention is to provide a cytochrome encoding a plant fatty acid hydroxylase.
To provide a P450 gene. In particular, the invention relates to plant fatty acid omega hydroxylases (eg, cytochrome P450, subtype CYP94) and intrachain (ω-1, ω-2, ω-3, and ω-4) hydroxylases (cytochrome P450, subtype CY
P81), a plant gene of terminal (omega or ω) hydroxylase having a unique peptide sequence.

It is still another object of the present invention to provide a plant fatty acid hydroxylase gene derivative product. Such products include, for example, nucleic acids, polypeptides, host cells, and transgenic plants.

It is still another object of the present invention to provide a process for producing a plant fatty acid hydroxylase gene and using the same. In particular, the nucleotide and amino acid sequences disclosed herein can be used to create structural and functional variants of plant fatty acid hydroxylases by genetic engineering. Furthermore, plants containing transgenes that affect the metabolism of fatty acids are produced, thereby obtaining plant products with altered hydroxylated fatty acids.

[0010] In one embodiment of the invention, nucleic acids (eg, DNA, RNA, and variants thereof), recombinant polynucleotides composed of nucleic acids (eg, recombinant vectors and expression vectors), polypeptides encoded by the nucleic acids (Eg, an enzyme having fatty acid hydroxylase activity), host cells (eg, bacteria, yeast, plants) containing the molecule, and wild species or mutations generated by using the fatty acid hydroxylase sequences disclosed herein. Whole plants, including bodies (or wild species or mutant gene products) are provided.

[0011] The second aspect of the present invention is a step of producing a 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 industrial processes or to hydroxylate a suitable fatty acid substrate in the host cell or plant. The polypeptide can act.

[0012] A variant plant fatty acid hydroxylase can be made by mutation of a gene. Examples of methods for producing mutant genes and their homologous proteins include random or site-directed mutation, domain shuffling, rational design based on structural contact between enzyme and substrate, and correlation between protein structure and enzyme action. Can be mentioned. The mutant plant fatty acid hydroxylase can be selected for the desired properties at that time, eg, modification of the substrate specificity. Suitable examples of such modifications include hydroxylation of short or long fatty acid chains or fatty acids having an odd number of carbon atoms, hydroxylation of fatty acids having a hydroxy or epoxy group in the molecular chain, thia fatty acids (sulfur atom Hydroxylation of a fatty acid having a substituted methylene group, hydroxylation of an ether fatty acid (a fatty acid having a methylene group substituted with an oxygen atom), (not a normal fatty acid having a free carboxyl group)
Hydroxylation of modified fatty acids such as esters or amides may be mentioned.
Other preferred properties to be selected include affinity for the substrate, modification of the catalytic rate, enzyme instability or stability, and requirements as cofactors.

In a third aspect, the invention provides metabolites of fatty acid hydroxylases and fractions enriched in such metabolites. The present invention also provides a process for producing a transgenic host cell and a transgenic plant that increase or decrease a specific fatty acid hydroxylase using the sequence and the expression vector disclosed in the present invention. Such transgenic host cells and transgenic plants provide a useful starting resource for obtaining the desired metabolic effects from the enriched or depleted fraction. Preferably, the hydroxylated fatty acids are made as conserved lipids in the transgenic seed.

For example, hydroxylated fatty acids are usually present in small amounts in the phospholipid fraction (cell membrane) of plants. Overexpression of the plant fatty acid hydroxylase gene in transgenic plants can increase the content of hydroxylated fatty acids present in triglycerides in transgenic plants. The use of a seed-specific promoter allows large amounts of hydroxylated fatty acids to accumulate as stored lipids. The accumulated fatty acids can be recovered by extracting the oil from the transgenic seed and isolating the fatty acids stored therein.

[0015] A fourth aspect of the present invention is to provide a database by hybridization (eg, a combination of high and low stringency) or amplification of nucleic acids (eg, LCR, PCR) or under the heading of omega fatty acid hydroxylase as defined below. To identify additional fatty acid hydroxylase genes and chemically modify the enzyme or gene mutagenesis of the hydroxylase sequence (eg, point mutation, deletion, insertion,
Domain shuffling) to produce a fatty acid hydroxylase mutant.

The fatty acid substrate includes, for example, capric fatty acid (C10: 0), lauric fatty acid (C12: 0)
), Myristic fatty acids (C14: 0), parimitin fatty acids (C16: 0), oleic fatty acids (C18: 1), linoleic fatty acids (C18: 2) and their enantiomers (9E, 122) and (9Z, 12E),
And linolenic fatty acids (C18: 3). Caprine, lauric, and myristic fatty acids are medium-chain fatty acids, palmitic, oleic, linoleic, and linolenic fatty acids are long-chain fatty acids.

The hydroxylated and epoxidized fatty acids made according to the present invention provide useful oils with new properties to manufacturers of lubricants, antislip agents, plasticizers, coatings, detergents, and surfactants. I do.

[0018] As a preservative lipid in seeds, omega hydroxylated fatty acids in plants may be over-produced or under-produced. Apart from industrial production reasons such as mass production, there are reasons for this. Omega hydroxylase is involved in epidermis synthesis, which indicates that the manipulation of expression of these genes affects the resistance of plants to sunshine or the resistance to insects and other pathogens. Furthermore, hydroxylated fatty acids essentially act as activators (triggers) that elicit a defense mechanism against plant pathogens.

DETAILED DESCRIPTION OF THE INVENTION Plants are characterized by the presence of distinct isomeric forms of cytochrome P450 (Salaun and Helvig, 1995). Some of these isomers may have different structures,
It is likely that the species is specific to organs and species. Other isomers, such as CYP73 (CA4H = cinnamic acid 4-hydroxylase), are widely distributed in the plant kingdom. As is well known in mammals, many forms of cytochrome P-450 are involved in the oxidation of medium and long chain fatty acids (FA) present in plants. is there. Interestingly, there are some similarities between mammals and plants in the induction of catalytic mechanisms and enzymatic activities by various xenobiotics. However, plant P450s involved in fatty acid oxidation have not been isolated to date. The reason is that these membrane-bound enzymes are generally present at very low concentrations in tissues. In the past decade, several cytochrome P450 products encoded by a family of CPY4 genes (primarily fatty acid hydroxylases) have been purified and cDNAs have been isolated therefrom and their cDNAs from mammalian and insect cDNA libraries. The sequence has been determined. To date, more than 50 plant P450-encoded cDNAs have been sequenced. However, none of them corresponded to CYP4 family genes collected from mammals and insects.

The biological role and substrate specificity of cytochrome P450 isoforms involved in fatty acid and eicosanoid oxidation are poorly understood. Oxygenated fatty acids from plants are found primarily in polar lipids such as triglycerides and phospholipids and monomers in the polymer layer.

[0021] Cornocytes and cork are polymers composed primarily of hydroxylated fatty acids, especially omega-hydroxylated fatty acids. These substances can cause water loss, chemical penetration,
Protects plants from attack by pathogens (eg, microorganisms, insects, etc.) and other environmental stresses. Among these are also strong inducers of fungal cutinase,
Some exhibit antifungal properties. In contrast, the apparently opposite effect is that the defense mechanisms found in plants are very diverse, but this is because fungi are needed to facilitate infection of plant hosts. Is considered to be a developed strategy.

On the other hand, as already reported for mammals, hydroxylated fatty acids evoke a response similar to the stimulation process. This is thought to be due to responding to various stresses and playing a role as a defense mechanism. In addition, large amounts of C18 group hydroxyl derivatives are present on the stigma of plant flowers. This suggests that the hydroxylated fatty acid plays a role in recognizing symptoms caused by pollen.

Omega hydroxylases and long-chain hydroxylases of long-chain fatty acids generate hydroxyl groups which are considered to be essential components for the polymerization of the constituent keratinocytes in the synthesis of plant epidermis. Conceivable. This is thought to be an important role of the hydroxylase. Epidermal monomers often exist as complex mixtures, and the complex compounds have a species-specific profile. Epoxidized derivatives in addition to hydroxylated fatty acids have also been found as epidermal monomers in several plant species. In addition, epoxidants are ring-opened by chemical and enzymatic reactions, thereby producing vicinal diol derivatives from the C16 fatty acid family, but these derivatives have not yet been detected in the epidermis of plants. This means
It suggests that epoxy groups may not be essential for the polymerization of the skin matrix. In this case, by introducing an internal hydroxyl group, cytochrome P450
It was suggested that the direct hydroxylation reaction mechanism catalyzed by dependent fatty acids is involved in the reaction.

[0024] Fatty acids and derivatives thereof may be subjected to hydroxylation, epoxidation, dehydration and reduction, etc.
Various oxidation reactions were performed. The results suggested that some forms of cytochrome P450 might be involved in these reactions. For example, in a previous study, when incubated with a model substrate such as lauric acid,
At least three different P450 isoforms have been demonstrated to be present in microsomes collected from various plant species. Either of these P450 systems may be unsaturated laurate analogs (which have site specificity, which site specificity is strongly dependent on regiospecificity and stereospecificity, and furthermore the regiospecificity and stereospecificity It is interesting to have a catalytic effect on the hydroxylation and epoxidation (depending on the shape of the double bond in the aliphatic chain).

[0025] Cytochrome P450-dependent reactions are involved in the oxidation of fatty acids and their derivatives in plants. The reaction is categorized below according to the type of reaction and the location of the carbon atom to be attacked, and several examples are given for the chemical induction and inactivation by suicide substrates of the cytochrome P450 activity currently under consideration. Will be described.

[0026] Lauric omega hydroxylase (ω-LAH), which exclusively produces 12-hydroxylauric acid, has been described in Pisum sativum, Vicia sativa and other Leguminosae. In addition to laurate hydroxylation, microsomal fractions collected from clofibrate-treated V. sativa seedlings also catalyze the omega hydroxylation of capric (C10: 0) and myristic (C14: 0). Was. Thus, free carboxyl groups seem to be essential for the binding of the enzyme to the substrate. The evoked response and initiation response were also examined. The result is that a single cytochrome P450
It suggests that these fatty acids can be omega hydroxylated.

[0027] To explore the catalyst capability of the omega-LAH, a series of ^(1-14 C) radiolabeled with unsaturated lauric acid itself (7-, 8-, 9- and 10-dodecene acid) Then, the cells were incubated with microsomal fractions collected from clofibrate-treated V. sativa seedlings. In the presence of O ₂ and NADPH, this subcellular fraction was capable of catalyzing the omega oxidation of the lauric acids themselves. The cis and trans forms of these four intramolecularly unsaturated analogs have led to 12-hydroxylation with approximately equal efficiency. It is also noteworthy that the oxidation of the allyl group (ie, the 12-hydroxylation of 10-dodecenoate) occurred while retaining the stereochemical structure of the double bond, and that no allyl group transfer was observed. is there. In contrast, terminal olefins (11-dodeceneic acid) were epoxidized by an enzyme-forming reaction. When microsomes were incubated in the presence of CO, an anti-cytochrome P450 reductase antibody and a suicide substrate, the production of each metabolite was inhibited to a similar extent. This is a single
It suggests that the P-450 isoenzyme can omegahydroxylate unsaturated analogs with lauric acid, double bonds or 1,4-pentadiene motifs, and epoxidize terminal olefins (11-dodesenic acid). The fact that ω-LAH activity was not inhibited by oleic acid (C18: 1) at ten times the concentration of laurate suggests that the activity is unique to short and medium chain fatty acids. ing.

In an earlier study, Soliday and Kolattukudy demonstrated that palmitic acid (C16: 0) could be omega hydroxylated by microsome fractions collected from V. faba. In addition, the inhibition of the reaction by CO suggests the involvement of cytochrome P-450 monooxygenase. However, the inhibitory effect of CO was not reversed by light irradiation. Recently, taken from seedlings dark etiolated Vicia sativa, (1- ¹⁴ C) oleic acid (Z9- octadecenoic acid), (1- ¹⁴ C) 9,10- epoxy stearate or (1- ¹⁴ C) 9 Microsomes incubated with 10,10-dihydroxystearic acid were found to have catalytic activity in the NADPH-dependent formation of hydroxylated metabolites. When the chemical structures of these compounds were determined by GC-MS analysis, 18-hydroxyoleic acid and 18-hydroxy-9,10-
It was found to be epoxy stearic acid and 9,10,18-trihydroxystearic acid. The reaction was inhibited by CO. The inhibition of the reaction was thought to be partially reversible by light, and all three reactions were inhibited by antibodies cultured against NADPH-cytochrome P450 reductase collected from Jerusalem artichoke. Linoleic acid inhibits the hydroxylation reaction of oleic acid, and conversely, oleic acid inhibits the hydroxylation reaction of linoleic acid. Thus, both inhibition reactions occurred competitively, suggesting that a single P450 may be involved in the oxidation of oleic acid and linoleic acid (C18: 2).

In microsomes collected from Jerusalem artichoke tubers (Helianthus tuberosus), lauric acid intramolecular hydroxylase (1C-LAH) is hydroxylated at carbon 10, 9, and 8 in a ratio of 24:63:13, respectively. It catalyzes the reaction. By this effect, an activity not detected in dormant tuber tissue was induced by scratching the stem and then exposing it to the drug. Several other plant species catalyze this type of reaction, such as corn and tulip. In wheat seedlings, lauric acid is mainly converted to 11-hydroxy derivatives. Lauric acid collected from wheat (ω-1
) -Hydroxylase ((ω-1) -LAH) provides a mixture of the corresponding monohydroxylaurate at 65%, 31% and 4% for 11-hydroxy, 10-hydroxy and 9-hydroxylaurate respectively Generate at the rate of Capric and myristic acids were also converted to (ω-1) and (ω-2) hydroxylated products. In addition, when myristic acid was used as a substrate, a small amount of metabolites hydroxylated at positions (ω-3) and (ω-4) were also detected. Whatever the length of the fatty acids (C10-C14) to incubate, no omega hydroxylated products were detected. In addition, the results obtained in our laboratory show that (ω-1) -L
AH was suggested to catalyze the hydroxylation of the herbicide diclofop.

A variety of different P450 systems are involved in the biosynthesis of plant epidermis. Intramolecular hydroxylation of omega hydroxypalmitic acid by V. faba microsomes produces 9 (or 10), 16-dihydroxypalmitic acid. This reaction is inhibited by CO, but this effect can be effectively reversed by light. By this method, the cytochrome P-450 is considered to be a different cyclotome P450 from the cyclotome P450 that induces omega hydroxylation of palmitic acid.

The most abundant components found in the cuticle of wheat cereals are omega-hydroxylated oleic acid and 9,10-epoxystearic acid. Surprisingly, the microsomal fraction collected from dark yellowed wheat sprouts (Triticum aesrivum L.)
Incubation with ^1-14 C) oleic acid, 18, 17 and 16-hydroxy oleic acid was formed and could be identified this with GC-MS. The production ratio of these products was 1.4: 4.6: 4, respectively. The dependence of these hydroxylation reactions on O ₂ and NADPH and the reversal of the inhibitory effect of CO by light demonstrated the involvement of cytochrome P-450. This reaction was selectively inhibited by a reaction inhibitor based on a properly designed reaction mechanism (see below). On the other hand, the hydroxylation of lauric and cinnamic acids was not affected by the reaction inhibitors.

The catalytic capacity of V. sativa microsomes for the oxidation reaction of two sulfur-containing lauric acid analogs was examined. Radiolabelled two synthetic sulfide (1- ¹⁴ C) i.e. 10 methylsulfinyl decanoic acid (10S-LAU) and (1- ¹⁴ C) δ- propyl sulfinyl octane acid (8S-LAU), P- Incubated with V. sativa microsomes under conditions that promoted the 450 or peroxidase reaction. In addition to the expected oxidation by peroxidase, at least two different membrane-bound enzymes allow 8- and
10-thio fatty acids could be actively converted to sulfoxides. The reaction requires NADPH, and the inhibitor 11-dodecic acid (11-DDYA) was used to target the effect of ω-LAH (see below), which mechanically inhibits the reaction. As a result, it was found that the inhibitor reversed the inhibitory effect by CO and the inactivation effect by NADPH-dependent reaction. From this, it can be considered that the sulfoxylation reaction of 10S-LAU and 8S-LAU showed that these inhibitors or a similar P450, hydroxylated lauric acid, showed a catalytic effect on the reaction. For the characterization of the second membrane-bound enzyme (which seems to be NADPH dependent) this could not be done satisfactorily. However, beta-mercapto-methanol present in the culture medium had no effect on sulfoxylation of 8S-LAU or 10S-LAU. This suggests that peroxidase present in these membranes did not participate in the reaction.

A salient property of living microorganisms is their ability to elicit the activity of P450 monooxygenase in response to chemical or physical stress. The activity of cytochrome P-450 collected from plants can be measured by light irradiation, ultraviolet irradiation, cuts, ripening, fungal infection, use of inducers, endogenous compounds and safeners, herbicides, pharmaceuticals, and many chemicals including contaminants. Triggered by the use of a class.

The plant P450 system involved in the omega hydroxylation of lauric acid (ω-LAH) and oleic acid (ω-OAH) is triggered by clofibrate in a dose-dependent manner. Clofibrate is a well-known lipid-lowering agent that induces the growth of peroxisomes in mammals and plants. Clofibrate and related2,
Arylphenoxy compounds that selectively induce fatty acid omega hydroxylase activity, such as 4-dichlorophenoxyacetic acid (2,4-D), were obtained from 1C-LAH collected from tubers of H. tuberosus and wheat seedlings ( It has little or no effect on the activity of ω-1) -LAH. The microsomes of V. sativa contain fatty acid omega hydroxylase exclusively. Exposure to these xenobiotics results in an omega hydroxylase activity of approximately 20-fold with phenobarbital and 30-50 with clofibrate.
Although there is a significant increase of more than 2-fold, intra-chain hydroxylated fatty acids have not been detected in microsomes collected from untreated Vicia seedlings or Vicia seedlings treated with clofibrate or phenobarbital. There is no. It is noteworthy that in a mammalian system, clofibrate selectively induces omega hydroxylase, while phenobarbital enhances (ω-1) -hydroxylation of lauric acid. Treatment with naphthalic anhydride (NA) or phenobarbital (PB) stimulates (ω-1) -LAH activity of shoot microsomes of dark yellowed wheat. Coating the seeds with NA, a detoxifier, increased (ω-1) -LAH activity 4.5-fold and P-450 content 1.5-fold. On the other hand, the activity of cinnamate hydroxylase (CA4H; P450 involved in lignin synthesis) was reduced by the coating operation. The herbicide metabolism activity of diclohop allyl hydroxylase (DIAH) was stimulated 4-fold by the coating operation. When the seedlings were aged on a 5 mM PB solution, (ω-1) -LAH and DIA
It was observed that the stimulating effect of H was significantly enhanced. Seeds were first coated with NA and then aged with PB, resulting in a synergistic stimulatory effect of (ω-1) -LAH and DIAH (increased 20-fold). On the other hand, CA4H activity was strongly suppressed by this operation. Cyclotome P450 content increased to about 0.5 nmol / mg, the highest level ever recorded in a plant. The relative amounts of 11-, 10- and 9-hydroxylaurate formed were unchanged under all conditions. Similarly, (ω-1)
-Oleic acid hydroxylase activity was induced to the same extent as (ω-1) -LAH in the treated seedlings. However, these P450-dependent reactions were promoted by
Each was a different isomer.

Experiments with a wide range of drugs have shown that these drugs induce 1C-LAH activity in tubers and bulbs collected from Jerusalem artichoke, tulip and corn seedlings. Jerusalem artichoke tuber slices were injured in the presence of 25 mM MnCl ₂ and 20 mM aminopyrine. By this operation, the activity was induced above untreated levels. However, exposing the tissue to 8 mM phenobarbital further enhanced the activity.

[0036] The mechanism of induction of cytochrome P450 in plant systems has not yet been elucidated. However, most of the P450 inducers in mammals were also effective at inducing plant P450. According to recent experimental results, when a lipid-lowering agent such as clofibrate induces murine liver cytochrome P450-dependent fatty acid hydroxylase, the receptor (peroxisome proliferating agent) has an effect on the induction of the reaction and certain physiological conditions. (Activated receptor) was found to be involved in the transcriptional activation of genes. These facts suggest that perturbation of lipid metabolism occurs and that this perturbation is a common cause of fatty acid hydroxylase induction by peroxisome proliferators.

[0037] Mechanistic inhibitors containing terminal acetylene (suicide substrates) act as potent irreversible inhibitors against fatty acid omega hydroxylases in both plants and mammals. Microsomes were collected from clofibrate-treated V. sativa seedlings and
Pre-cultured with 11-dodecic acid (11-DDYA) and NADPH. This resulted in a pseudo-primary loss of omega hydroxylated laurate (K = 150 μM, half-life = 2).
.4 minutes). The apparent rate constant of the inactivation reaction by 11-DDYA was 4.3-4.8 × 10 ⁻³ / sec. When cultured microsomes obtained from V.sativa with ^{(1- 14 C) 11-DDYA} , is a major metabolite 1,12 decane diacid obtained. It is considered that the 1,12-decandioic acid was formed by adding water to the ketene intermediate. It is thought that this ketene also acts on the nucleophilic residue at the active site to selectively chemically label two types of protein bands (about 50 kDa). Labeling of microsomal proteins (diacid formation and ω-LA
Good correlation between inactivation of H is obtained), the culture time and ^(1-14 C)
Progressed as a function of 11-dodecic acid concentration. Based on these results, two potential inhibitors were synthesized targeting the inactivation of the oleic acid omega hydroxylation reaction. Microsomes having terminal acetylene groups collected from V. sativa, ie, (Z) 9-octadecene-17-ic acid (17-ODNYA) and the corresponding (Z) 9,10
Incubation of -epoxyoctadecane-17-inoic acid (17-EODNYA) resulted in a pseudo-primary loss reaction of oleic acid omega hydroxylation (apparent K values of 60 μM and 50 μM, respectively). The calculated half-life of the enzyme activity was 6 minutes and 8 minutes, respectively, for the saturating concentrations of 17-ODNYA and 17-EODNYA. Interestingly, the fact that these suicide substrates inhibit the omega hydroxylation of oleic acid, its epoxidized and diol derivatives and of linoleic acid to the same extent.

To purify the plant cytochrome P450 protein and determine its sequence, P450
The method of selectively covalently binding a plant cytochrome P450 protein with a mechanistic inhibitor labeled with an apoprotein is an effective means of tracking the labeled protein in the purification step.

The terminal olefin, 11-dodeceneic acid, has the function of inactivating P450 collected from wheat, which mainly has a catalytic effect on the oxidation reaction of internal carbon (ω-1).
As suggested by Ortiz de Montellano and co-workers, the inactivation of P450 by terminal olefins allows free heme units to be freely converted via oxidative attack on the internal carbon (ω-1) of the double bond. Changes to a free terminal methylene residue that can be alkylated. In contrast, the plant ω-LAH, which exclusively attacks the external position, effectively functioned as a catalyst for the reaction of producing 11-12 epoxides without significant loss of activity. In addition, acetylene derivatives of lauric acid were also collected from wheat (ω-1) -L
Acts as a strong inactivator for AH. When microsomes collected from dark-yellow wheat seedlings were cultured with 10-dodecinic acid (10-DDYA), a marked inhibitory effect on lauric acid hydroxylation was observed. The inhibitory effect varied depending on time and inhibitor concentration. This is a characteristic phenomenon of mechanistic inhibitors. A kinetic study of the pseudo-first-order reaction on the inhibition of (ω-1) -LAH was performed to determine the half-life and apparent inhibition constant. As a result, the half-life was 3 minutes, and the apparent inhibition constant K was 14 μM. Microsomes with terminal acetylene groups, ie, 11-dodecic acid (11
-DDYA) gave the same results as above.

Further, using a substrate analog exhibiting an acetylenic function at position (ω-1),
It irreversibly inhibited oleate hydroxylase (ω-1) -OAH (mainly oxidizing the position of ω-1) collected from wheat. Microsomes cis-9-octadecene-16-
Incubation with formic acid (16-ODNYA) inhibited oleic acid hydroxylation but not lauric acid hydroxylation. From these results, it is highly likely that at least two different P450 enzymes are involved in the oxidation reaction of oleic acid and lauric acid.

Therefore, it is considered that internal acetylene exerts a high destructive effect on P450, thereby catalyzing intramolecular oxidation. The mechanism of inactivation has not yet been fully elucidated. However, the fact that the chemical structure of the unstable epoxidized acetylene undergoes rearrangement has already been estimated in a reaction mechanism in which ketene is generated from a terminal acetylene group, and the possibility that this reaction is involved cannot be denied.

Compared with the results of studies on mammalian fatty acid hydroxylases, the catalytic mechanism and substrate specificity of plant fatty acid hydroxylases (Table 1) have not yet been fully elucidated. To date, the only P450s isolated and used for cloning are the two P450s that have a catalytic effect on the dehydration of fatty acid peroxides. Oxidized allene synthase can play an important role as an enzyme in plant development by controlling various stages of production by generating precursors of jasmonic acid. In this regard, it is also of interest to understand the physiological role of RPP taken from guayule gum particles, which have a clear catalytic effect on similar reactions. On the other hand, there is also evidence that the long chain fatty acid (omega and intrachain) hydroxylases play an important role in the biosynthesis of plant epidermis by generating terminal and internal hydroxyl groups. These hydroxyl groups appear to be essential for the polymerization reaction of the keratin monomer.

For the plant fatty acid hydroxylase of the present invention, at least three different roles can be expected. Synthesis of keratinocytes and cork, rapid catabolism of free fatty acids (
Detoxification) and the synthesis of signaling molecules.

As described above, it is expected that the activity of plant fatty acid hydroxylase can be controlled by changing the synthesis reaction of keratinocytes and cork. Therefore, it is thought that by modifying the conditions for producing keratinocytes and cork, plants having desired characteristics (for example, resistance to drought or chemical penetration) can be obtained. For example,
We can imagine that a null mutant or a lower-morphic plant grows slower than a wild-type plant.

When a cut or other type of stress is applied, phospholipases are activated, fatty acids are rapidly released, and oxidative rupture proceeds (Low et al., 1996). Several reports have shown that inducers activate phospholipase in plants (Chandra et al., 1).
996). When the phospholipase is activated, free fatty acids are released. By analogy with the role of P450 fatty acid hydroxylases in animals, the role of rapidly catabolizing these free fatty acids may be a role. Recent data (Tijet et al., 1
998) show that CYP94A1 is strongly induced after 2-3 minutes in plant tissue exposed to clofibrate, and that this effect lasts up to 400 times after 2-3 hours. Clofibrate is an agent that induces the proliferation of peroxisomes in animals and plants (Palma et al., 1991). Peroxisome proliferation is strongly associated with oxidative rupture.

The results of Schweizer et al. (1996ab) indicate that omega hydroxy C16: 0 and C18: 1 fatty acids are resistance inducers. Due to their similarity to the arachidonate cascade, other omega-hydroxylated fatty acids may be involved in the transmission of stress signals. In addition, as omega hydroxylation and omega-1 hydroxylation progress, the production of molecules such as pheromones that enhance insect attraction for pollination increases. This means that many insect pheromones are known as omega and (omega-1)
-Based on the fact that they are (or are derived from) hydroxy fatty acids or alkanes (see Engels et al. [1997]).

In addition, P450s involved in the epoxidation of unsaturated fatty acids produce hydroxylated fatty acids and epoxidized fatty acids, which have been shown to inhibit the growth of pathogens (
That is, by synthesizing the signal transmitting molecule), it may be involved in the resistance to the disease. Considering the role of oxygenated fatty acids in fungal infections, certain monomers taken from keratinocytes (ie, dihydroxy fatty acids and 9,10,18-trihydroxystearic acid) are responsible for the potent activity of some pathogenic fungal cutinases. It is clear that some reports have shown that this is a potent inducer and that this report contradicts this fact. The results of Schweizer et al. (1996ab) show that monomers of keratinocytes, especially the omega hydroxy form, induce resistance of barley to Erysiphe graminis. In this case, the best effect is exhibited for 9,10,18-trihydroxystearic acid. Pinot et al. (1993) acted on 9,10-epoxynase (but not P450), and then moved the 18 positions to P450 (CYP94A1, CYP94A4, CYP94A5 and CYP86A1).
Has a catalytic effect on this reaction) and further opening of the epoxy ring by epoxide hydrolase indicated that this compound was formed. Blee et al. (1993) showed that the activity of epoxygenases and epoxidases was extremely strong, so that P450-catalyzed omega hydroxylation was the rate-limiting factor. This gene is probably under the control of a pathogen-responsive promoter, but overexpression of this gene may increase resistance.

[0048] Oils produced by altering the activity of plant fatty acid hydroxylases may exhibit different properties than oils produced by wild-type plants. This means that the lubricant,
It is believed to be useful in the production of anti-slip agents, plasticizers, coatings, detergents, and surfactants. For example, hydroxylated fatty acids having 10-14 carbon atoms (derived from capric, lauric, or myristic acids) are believed to provide the basis for new detergents and plasticizers. Plastics that can be made from hydroxylated fatty acids are polyurethanes and polyesters (Weber et al., 1994). It is noteworthy that keratinocytes themselves are bioplastics made almost entirely from oxy fatty acids.

The molecular chain extension reaction requires omega hydroxylation, and the network formation reaction requires hydroxylation and / or epoxidation within the molecular chain.
Plants can be designed to produce C12 fatty acids by transformation with aryl-ACP thioesterase from Umbellularia california, which is unique to lauroyl-ACP. Arabidopsis thaliana transformed with this gene,
Created up to 25% laurate. Further transformation of these plants with CYP94A1 would produce large amounts of omega hydroxylauric acid. Similarly, plants designed to produce C14 fatty acids and then transformed with CYP94A2 would produce large amounts of omega hydroxymyristate. To date, the properties of hydroxylated fatty acids and their industrial use have not yet been fully elucidated. The lack of commercial use of such fatty acids is because these hydroxylated fatty acids do not accumulate in plants under normal conditions. The use of the plant fatty acid hydroxylase of the present invention enables mass production of such compounds. Chemical synthesis of omega hydroxylated fatty acids is difficult and expensive. This means that producing omega hydroxylated fatty acids in plants can provide significant economic benefits.

With respect to relatively higher plants, some have completed ω-fatty acid hydroxylase and intrachain fatty acid hydroxylase characterization. In microsomes taken from the cuticle elements of Helianthus tuberosus, omega-2 laurate, against omega-3 and omega-4-hydroxylation, closely related, the aminopyrine and MnCl ₂ with inducible a cytochrome P450 each other One or more are used as catalysts. Antibodies against the P450-enriched fraction, purified from Mn ^++- induced tissues, were used to isolate cDNA and sequence these enzymes. By screening an expression library of cDNAs collected from aminopyrine-treated keratinocytes, a cDNA (CYP81B1) corresponding to aminopyrine-induced transcripts could be identified. As a result of expressing CYP81B1 in yeast and systematically examining its function, this enzyme was found to be particularly useful for middle-chain saturated fatty acids, namely, capric acid (C10: 0) and lauric acid (C
12: 0) and myristic acid (C14: 0) were found to have a catalytic effect on the hydroxylation reaction. The same metabolite, ie, an ω-1-ω-5 monohydroxylation product, was obtained in the transfected yeast and plant microsomes. These three fatty acids could be metabolized with similarly high efficiency, and the main attack position was different depending on the length of the molecular chain. When lauric acid was used as the substrate, the turnover was 30.7 ± 1.4 / min and the apparent K _{m, app} was 788 ± 400 nM. No metabolic reactions were detected with long-chain fatty acids, aromatic molecules or herbicides. This new fatty acid hydroxylase is typical of higher plants and differs from fatty acid hydroxylases already isolated from other living microorganisms.

[Table 1]

Since 1974, there have been numerous reports showing that plant P450 is triggered by a vast number of physical, physiological and chemical factors. However, in many cases, it was not known what isomers were induced by what induction mechanism. Below are three different cloned P450 species, CYP73A1, CYP76B1
The triggering mechanism and the functional characteristics of CYP94A1 and CYP94A1 are shown below.

CYP73A1 is a cinnamic acid 4-hydroxylase, which first catalyzes the oxidation reactions involved in the common phenylpropanoid enzyme catalysis to produce lignin, flavonoids,
Produces protective molecules, anti-UV protective agents. Add this enzyme to Triton X114 until homogeneous
Purified by phase fractionation and cloned using native antibodies raised against the pure protein (Teutsch et al., 1993). The data suggest that the induction of cinnamic acid 4-hydroxylase activity is mainly due to gene activation. After incision treatment and aminopyrine treatment, experiments were performed on reaction time and reaction route. The timing of activity-induced changes, protein and transcript status confirmed that C4H was mainly induced by increased CYP73A1 mRNA in both cut and aminopyrine treated tissues. However, the post-transcriptional reaction mechanism may also contribute to the control of C4H activity.

CYP76B1 is an alkoxycoumarin O-dealkylase (Batard et al., 1995), the true physiological function of which is not yet well known. This protein was purified in the same manner as used for CYP73A1 and the microsequenced peptide (B
The gene was cloned using cytochrome P-450 primers derived from atard et al., 1998). After slicing the tuber tissue and aging these in water,
The steady state concentration of CYP76B1 alone or in the presence of various drugs was determined. As a result, it was found that the expression of P450 does not occur in response to mechanical stress but strongly depends on chemical treatment. Therefore, CYP76B
1 seems to be a good marker for chemical stress and environmental pollution.

From these data, it is understood that the possibility that the “plant P450 induction mechanism” exists is extremely high. To date, more than 60 physiologically active effects of P450 have been identified, and further characterization of 500-600 physiologically active effects is expected to be completed in the near future. The secondary plant metabolic pathway of these physiologically active effects of plant P450 is versatile. Thus, induction of plant P450 enzymes is likely to be comparable in the pathway to induction of other enzymes. This means
This is especially true for all enzymes involved in the synthesis of protective compounds, which are induced by mechanical cuts, infections and stress conditions. The situation induced by chemical inducers is even more intriguing and very speculative. The method of treating plants with chemicals is a type of stress that is "painful" for plants, which will derive signals from one of the various signaling chains. On the other hand, some chemicals that induce P450 in the body of an animal also induce plant P450 on the same substrate at the same time, and some of these plant P450s even have site selectivity. In animals, plants and B. megaterium, fatty acid intrachain hydroxylases are selectively induced by phenobarbital. In contrast, in animals and plants, clofibrate selectively induces ω-hydroxylase. These data suggest that during evolution, regulatory mechanisms as well as catalytic function may be preserved.
Finally, some variants (from biochemical, phylogenetic, and possibly structural aspects) catalyze the major synthetic steps of jasmonic acid and salicylic acid, respectively, to oxidize allene synthase (Song et al., 1993) or Benzoic acid 2-hydroxylase (Leon et al., 1995
It should be emphasized here that some of them form P450s.

Among the seedlings of Vicia sativa, clofibrate is highly efficient (20 times more efficient) and stimulates the catalytic activity of cytochrome P450 in omega hydroxylation of lauric and oleic acids. DEHP and 2,4-D have a similar stimulating effect on lauric omega hydroxylase in the same substance. Recently, based on an internal peptide sequence, we found that cD encoding plant fatty acid omega hydroxylase
NA was isolated. After expression in yeast, these omega-hydroxylase substrates have different chain lengths (C10-C18) and different degrees of unsaturation (C18: 1, C18: 2, C18: 3). Characterization of the specificity of CYP94A1 omega hydroxylated fatty acids was performed. Northern blot analysis of RNA collected from clofibrate-treated Vicia sativa seedlings revealed that very rapid (30 minutes later) and large accumulation of CYP94A1 transcripts occurred. This suggests that the clofibrate receptor is involved in signal transduction. To elucidate the mechanism by which clofibrate regulates CYP94A1 and the possible involvement of PPAR in this regulatory mechanism, we isolated the promoter sequence of CYP94A1. We are currently searching for major coordinator factors. In addition, studies of peroxisome proliferation with clofibrate at the concentration of acyl-CoA oxidase in Vicia sativa have already begun.

In addition, the present inventors have recently noticed that treatment of dark yellowed Vicia sativa seedlings with the plant hormone methyl jasmonate (MetJA) increases cytochrome P450 content. . This suggests that this plant defense molecule may act via the CYP94A1 pathway (Pinot et al., 1998). Seedlings MetJA 1
Treatment with a mM solution for 48 hours stimulated ω-hydroxylation of lauric acid 14-fold compared to control samples (153 pmol / min / mg protein: 11 pmo
l / min / mg protein). This evoked response was dose dependent. Increased activity (2
.7 times), the detection was already possible 3 hours after the treatment. Activity increased as a function of time, reaching a steady level after 24 hours. Analysis by Northern blot showed that a transcript encoding fatty acid ω-hydroxylase CYP94A1
It began to accumulate 1 hour after exposure to tJA and reached the highest concentration after 3-6 hours. Under the same conditions, enzymatic hydrolysis of 9,10-epoxystearic acid did not affect the microsomal hydroxylase activity of the water-soluble epoxidized product by MetJA treatment. Therefore, in the general reaction mechanism of plant defense molecules, it is considered that the regulation function of fatty acid microsomal ω-hydroxylation by methyl jasmonate plays a major role.

[0057] Over the past few years, we have determined that at least 3 cells present in Vicia sativa
We have cloned and characterized various fatty acid ω-hydroxylases. These P450 enzymes have the ability to introduce alcohol groups at the terminal carbons of fatty acids having various chain lengths and various degrees of unsaturation. These new enzymes are the first members of the CYP94 family. Extensive work has been done on mammalian ω-hydroxylating enzymes (Simpson, 1997) and these enzymes have
It is classified in the YP4A clan. CYP4A is known to be involved in the metabolism of arachidonic acid and to produce physiologically important metabolites. These substances are also involved in fatty acid catabolism (Gibson, 1989). In plants, fatty acid ω-hydroxylase is involved in epidermal biosynthesis (Kolattukudy, 1981). Furthermore, ω-hydroxy fatty acids have recently been reported to play an important role in plant defense mechanisms (Schweizer, 1996). It may also be involved in the cork-forming reaction of green beans treated with inducers (Bolwell, 1997).

To characterize the physiological role of fatty acid ω-hydroxylase, we have developed a method that uses transgenic tobacco lines. To this day,
Three new clones belonging to the CYP94 family (CYP94A4, CYP94A5 and CYP94A6) have been isolated from tobacco cDNA libraries. To modulate the activity of these P450s in vivo, the two coding sequences were incorporated into a T-DNA vector in the sense and antisense orientation to transform tobacco leaf discoid tissue. Although expression of a morphological phenotype in these transgenic lines is expected, epidermal quality and quantity, as well as its resistance to pathogens, may be affected at the same time.

Aryl phenoxy compounds, such as the lipid-lowering drug clofibrate and the herbicide 2,4-D, are chemicals known as inducers of fatty acid hydroxylase activity and peroxisomes. In plants, long-chain fatty acid omega hydroxylase is believed to play a very important role in the epidermis synthesis that protects plants from the external environment. First cloned P450 from plant
The dependent fatty acid omega hydroxylase, CYP94A1, was isolated by using a mechanistic inhibitor of P450 apoprotein labeled with a radioisotope.
By functionally expressing this novel P450 in S. cerevisiae, CYP9
It was found that the methyl end groups of saturated (C10-C16) and unsaturated (C18: 1, C18: 2 and C18: 3) fatty acids were predominantly oxidized at 4A1. This plant enzyme, like animal omega hydroxylase, was strongly induced by clofibrate treatment. The rapid accumulation of CYP94A1 detected its transcript less than 20 minutes after exposing Vicia sativa seedlings to clofibrate. The rapid induction of CYP94A1 effectively transformed fatty acids (FA) into the keratinocytes required for repair and protection. The possible role of hydroxylated fatty acids as a natural inducer of the plant defense mechanism has hitherto been unexpected prospects for naturally occurring plants to study new regulatory pathways for protective compounds against chemical or pathogen stress Was to open.

With respect to the books, articles and patents cited herein, all of them have been set forth in their entirety for reference.

The following examples are provided to illustrate the present invention and should not be construed as limiting the practice of the present invention in any way.

Example I-Clone A [CYP94A1] Saturated and unsaturated fatty acid (FA) omega hydroxylases, ie microsomal cytochrome P450 dependent hydroxylases, are capric acid (C10: 0), lauric acid
(C12: 0), myristic acid (C14: 0), palmitic acid (C16: 0), oleic acid (C18: 1), linoleic acid (C18: 2 and its enantiomers [9E, 12Z]; [9Z, 12E ]) And linolenic acid (C
18: 3) are catalyzed by reactions for transforming them into the corresponding omega hydroxy acids, respectively, and clone A has a function of encoding the omega hydroxylase. In addition, two sulfur-containing lauric acid analogs, 10-methylsulfinyldecanoic acid (10S-LAU) and (8-propylsulfinyloctanoic acid (8S-LAU), actively correspond to their 10- and Respectively converted to 8-sulfoxide (
(Fig. 3).

As previously indicated for incubating microsomes from V. sativa, a series of unsaturated lauric acid analogs having double or triple bonds at carbon positions 8, 9 and 10 were identified as omega May be hydroxylated. further,
11-Dodecenoic acid, a lauric acid analog with an ethylene group at the end, is also
Clone A) may be converted to 11-epoxylauric acid (Weissba
rt et al., 1992; Pinot et al., 1992, 1993; Helvig et al., 1997).

[0064] (CYP94A1 [Clone A] Isolation) radiolabeled P450 apoproteins ^(1-14 C) and 11-dodecyne acid (11-DDYA) (S
alaun et al., 1988; Helvig et al., 1997), and a newly developed method for lauric omega hydroxylase-specific peptide sequences from clofibrate-treated V. sativa microsomes (Salaun et al., 1986). Determined by Chemically labeled proteins (approximately 53 kDa) were isolated by first performing a continuous SDS-PAGE analysis and then subjecting to "in gel" V8 proteolysis. The peptide obtained by this method was transferred to a nylon membrane (IMMOBILON (registered trademark)) and sequenced by the Edman degradation method. The sequences of the four peptides were determined. Only two of them showed similarity to P450. Later, these two peptides were found in the deduced amino acid sequence of clone A (SEQ ID NO: 4).

The first peptide contained an 18-20 amino acid hydrophobic domain typical of the membrane anchor found in all microsomal P450s. After isolation of the clone, the peptide is the peptide deduced from the N-terminal amino acid sequence of the enzyme, ie, MFQFLLEVLLPYLLPLLLYILPF peptide microsequence It was confirmed to correspond to.

[0066] The second peptide had the sequence LMNLYPPVPMMNAKEVVVXVLLXQ. Computer search of the peptide for all known cytochrome P450 enzymes resulted in the beginning of the domain (
The CYP4 family contains mammalian fatty acid omega hydroxylases, ie, peptide p3, murine CYP4A1, murine CYP4B1, and murine CYP4A3, and about 130 residues from the C-terminal group in some P450s belonging to this family). Similarity was found to exist. After cloning and sequencing, of the first 10 amino acids, only 8 shown in bold were CYP94A1 (SEQ ID NO: 4, residues 370-394; SMRLYPPVPMDSKEAVND
DVLPDGW) in the corresponding domain. This implies that the peptide is contaminated with other proteins. Examination of the sequence of 428 cytochrome P450s showed that two methiones were never present consecutively. The following PCR primer sequences (peptide and nucleic acid sequences contained in SEQ ID NOs: 4 and 3, respectively) were estimated.

Embedded image

[0067] V. sativa seedlings were clofibrate-treated by RT-PCR, and this primer was used with an oligo (dT) primer to generate a 661 bp probe on all RNA collected therefrom. The conditions in this case were as follows. That is, denaturation at 93 ° C. for 5 minutes, a denaturation cycle of 93 ° C. for 1 minute was repeated 30 times, hybridization at 48 ° C. for 2 minutes, stretching at 72 ° C. for 3 minutes, and stretching at 72 ° C. for 10 minutes to carry out the reaction. Stopped.

Poly (A) RNA was collected from a 48-hour clofibrate-treated V. sativa seedling and a λZAP cDNA library was prepared therefrom according to the manufacturer's instructions (Stratagene).
The library was screened rigorously using a 650 bp probe.
(Α- ³² dCTP) to label the probe randomly, 5 x SSC, 0.5% SDC
, 5 x Denhardt's solution, 100 μg / ml salmon semen DNA, 2-mM EDTA, and 50 at pH 6.0.
Hybridization was performed in -mM sodium phosphate at 65 ° C for 24 hours. After hybridization, the soil was washed twice with 2 × SSC and 0.1% SDS at room temperature for 15 minutes, and twice with 0.2 × SSC and 0.1% SDS at 55 ° C. for 30 minutes. . One clone (1862 bp) VAGHI11 was isolated and sequenced. The clone was found to encode a new cytochrome P450, CYP94A1 (FIG. 1).

(Heterologous Expression in Yeast) As a result of performing complete sequencing of the Saccharomyces cerevisiae genome, this yeast has only four types of P450s, none of which has a catalytic effect on the hydroxylation reaction of fatty acids. It turned out to be just things. In addition, the yeast can be grown with minimal expression of endogenous P450 (ie, no P450 is detected by spectroscopy). By expressing the function of CYP94A1 (clone A) in a genetically designed yeast, its catalytic activity was evaluated. Urban et al. (199
0) using a system developed by Saccharomyces cerevis
expressed in iae. All of these methods (eg, P450 cDNA, pYeDP60 shuttle
Pompon et al. (1996) discuss the sub-cloning of vectors, transformation of yeast, and growth conditions that allow expression of cloned P450 genes. Clone A
(SEQ ID NO: 3) was obtained by cloning PCR into the expression vector pYeDP60 using BamH1 and EcoR1 restriction sites as described below. Sense primer

Embedded image Antisense primer

Embedded image

The amplification sequence was verified using Stratagene Pfu polymerase according to the manufacturer's instructions to avoid errors caused by the polymerase. Yeast strain WAT1 (Urban et al., 1990) was used in an expression vector by the method of Schiestl and Gietz (1989).
Was transformed.

Characterization of CYP94A1 [clone A] Preparation of microsomes: CYP94A1 (plone) in pYeDP60 according to the method of Pompon et al. (1996).
A yeast strain WAT11 transformed with clone A) was grown and CPY94A1 was induced.
The culture was started by isolating the cultivation from the isolated colony. After growth, cells
Centrifugation was performed at 4500C at 7500g for 10 minutes. Wash the pellet with TEK (2ml TEK / g cells)
Centrifugation was performed at 4 ° C at 7,500 g for 10 minutes. The pellet was resuspended in 1 ml of TES and glass beads were added to the level of the liquid surface. In a cold room, cells were broken by hand shaking well for 5 minutes using a plastic conical 30-ml Falcon tube containing 0.5 mm diameter glass beads. More than 90% of the cells were lysed. This homogenate is combined with a washing solution in which glass beads are washed twice with 5 ml of TES, and mixed at 4 ° C.
Centrifugation was performed at 7,500 g × 10 minutes. Further, the obtained supernatant was centrifuged at 4 ° C. and 100,000 g × 45 minutes. The resulting pellet was resuspended in 2 ml of TEG using a coarse-grained ceramic homogenizer to obtain the following microsome design fractions. Microsomes did not lose activity when stored at -20 ° C for several weeks. WAT11 cells transformed only with the pYeDP60 expression vector were subjected to the same procedure as a control. TEK: Tris-HCl 50 mM, pH 7.5; EDTA 1 mM; KCl 100 mM TES: Tris-HCl 50 mM, pH 7.5; EDTA 1 mM; Sorbitol 600 mM TEG: Tris-HCl 50 mM, pH 7.5; EDTA 1 mM ; Glycerin 20%

Measurement of P450 content: The microsomes were diluted 5-fold with TEG and the P450 content was measured by the method of Omura and Sato (1964) using an extinction coefficient of 91 / cm / mM (FIG. 2).

Measurement of activity: The method described above (Weissbart et al., 1992; Pinot et al., 1992, 1993; Bouche
According to r et al., 1996), the metabolite production rate of the transformed yeast microsome containing the radiolabeled substrate during the culture was measured, and the enzyme activity was determined from this. Final volume 0.2 ml
Among the standard solutions, 0.19-0.43 mg of microsomal protein, phosphate buffer (p
H: 7.4) contained 20 mM and 100 μM of radiolabeled substrate. Omega hydroxylase activity was measured in the presence of a 0.6-mM NADPH + regeneration system and 375-μM β-mercaptomethanol. The ethanol solution containing the radiolabeled substrate was evaporated in a stream of argon, after which other fractions required for the culture were added. 27
The reaction was started by adding NADPH at ° C., and after culturing for 10 minutes, the reaction was stopped by adding 0.2-ml of acetonitrile-acetic acid (99.8 / 0.2 v / v). After extraction with 2 x 600-μl of diethyl ether, the organic phase is added dropwise onto a thin layer of silica plate,
Diethyl ether-light oil (boiling point: 40-60 ° C) -formic acid mixture (7 for C16 substrate)
0/30/1 v / v / v, 50/50/1 v / v / v for C18 substrate). The plate was scanned with a belt hold thin layer scanner. For accurate kinetic measurements, the radioactive spot was scraped into a counting bottle and the product was quantified by the liquid scintillation method. In this experiment, all reaction products were identified by GC / MS spectroscopy. Table 2 shows the activity of CYP94A1 (clone A) containing various fatty acid substrates.

[Table 2]

FIG. 4 (capric acid, lauric acid, myristic acid and palmitic acid), FIG. 5 (oleic acid, linoleic acid and linolenic acid), and FIG. 6 (C18: 2-9E, 12Z acid; C18: 2-
9Z, 12E acid; 8S-LAU acid and 10S-LAU acid). For each substrate, two chromatograms are shown (in the case where NADPH is present [A: hydroxylase is activated] and when it is not present [hydroxylase is not activated]).

Example II Clone B [CYP94A2] Clone B encodes ω-MAH (omega myristate hydroxylase), a microsomal cytochrome P-450 dependent hydroxylase. The hydroxylase catalyzes a reaction that transforms myristic acid (C14: 0) to produce 14-hydroxytetradecanoic acid (ie, the terminal methyl group is hydroxylated). Low levels of transformation of lauric acid (C12: 0) and palmitic acid (C16: 0) to produce the corresponding omega hydroxy fatty acids were also observed.

(Isolation of VAGH811) The λZAP cDNA library prepared from clofibrate-treated Vicia sativa seedlings was screened with clone A as described below to obtain the clone. V. sativa seedlings were treated with clofibrate for 48 hours, poly (A) RNA was collected therefrom, and a λZP cDNA library was prepared from the poly (A) RNA according to the manufacturer's instructions (Stratagene). Screened with low rigor. During the isolation of clone A, a 661 bp DNA fragment was prepared by the RT-PCR method and used for screening. The screening was started from the position 1201 in FIG. The fragment was radiolabeled with ³² P by random priming. Hybridization overnight at 55 ° C, 5 x SSC, 0.5% SDS
, 5x Denhardt's solution, 100 μg / ml salmon semen DNA, 2 mM EDTA, and 100 mM
In sodium phosphate at pH 6.0. After hybridizing, remove dirt 2
xSSC, 0.1% SDS, wash twice at room temperature for 15 minutes each, further 0.2 x SSC, 0.1%
The plate was washed twice with SDS at 45 ° C. for 30 minutes each. One of the isolated clones (1437 bp), VAGH811, was sequenced and found to encode a new cytochrome P450 (FIG. 7). The 5 'end of this clone was incomplete.
However, the sequence of this incomplete clone was used to encode the sequence of VAG811 from Vicia sativa seedlings treated with clofibrate for 96 hours, containing 5'-RAC containing poly (A) RNA.
E primers were synthesized. This clone is hereinafter referred to as clone B (FIG. 7).

(Heterologous Expression in Yeast) The catalytic activity of CYP94A2 (clone B) was evaluated by expressing its function in genetically designed yeast by the method described above for clone A. The coding sequence of CYP94A2 (clone B, SEQ ID NO: 5) was PCR cloned into the expression vector pYeDP60 using the SmaI and SacI restriction sites (peptide contained in SEQ ID NO: 6) as described below. Array). Sense primer:

Embedded image Antisense primer:

Embedded image

The Boehringer HiFi® polymerase was used according to the manufacturer's instructions. This was verified on the amplified sequences to avoid errors caused by the polymerase. Yeast strain WAT11 (Urban et al., 1990) was transformed according to the method of Schiestl and Gietz (1989).

(Characterization of CYP94A2 [clone B]) Preparation of microsomes: CYP94A2 (clone B) was incorporated into a pYeDP60 expression vector, yeast strain WAT11 was transformed with the pYeDP60 expression vector, and the method described for clone A was followed. The yeast strain WAT11 was grown to induce microsomes.

Evaluation of P450: Microsomes were diluted 5-fold with TEG and the amount of P450 was measured by the method of Omura and Sato (1964) using an extinction coefficient of 91 / cm / mM (FIG. 8).

Activity measurement: Enzyme activity was measured by the method described above for clone B.
All reaction products identified in these experiments were identified using plant microsomes.
For the identification, a rechromatography method using a genuine compound as a control and a GC / MS method were used. Table 3 shows the activity of ω-MAH using various fatty acids as substrates.

[Table 3]

The actual TLC radiochromatograms are shown in FIG. 9 (capric acid), FIG. 10 (lauric acid), FIG. 11 (myristic acid), FIG. 12 (palmitic acid), FIG. 13 (stearic acid), and FIG. Oleic acid). Two chromatograms are shown for each substrate. One case includes NADPH (A: hydroxylase has activity), and the other case does not include NADPH (B: hydroxylase has no activity).

(Kinetic Variables and Specific Activity) For CYP94A2 (clone B), apparent Km and Vmax values for myristic acid were measured in separate experiments. These values are 3.8 μM and 80-mol 1
It was 4-hydroxymyristic acid / min / mol-ω-MAH.

Example III-Clone C [CYP94A3] CYP94A3 encodes a cytochrome P450-dependent hydroxylase, wherein the cytochrome P450-dependent hydroxylases are capric acid (C10: 0), lauric acid (C12: 0),
And catalyzes the oxidation reaction of the methyl terminal of myristic acid (C14: 0). It also catalyzes, to a lesser extent, the omega hydroxylation of palmitic acid (C16: 0), oleic acid (C18: 1), and linoleic acid (C18: 2).

(Isolation of CYP94A3 [clone C]) A λZap cDNA library was prepared from clofibrate-treated Vicia sativa seedlings, and this library was
By strict screening at bp), clone C was obtained (from the sequence encoding the heme binding domain at the poly A tail). Hybridization in 5 x SSC, 0.5% SDS, 5 x Denhardt's solution, 100 μg / ml salmon semen DNA, 2 mM EDTA, and 50 mM sodium phosphate, pH 6.0, 65 ° C, 24 hours went. After the hybridization was completed, the resultant was washed twice with 2 × SSC and 0.1% SDS at room temperature for 15 minutes to remove dirt. Further, the plate was washed twice with 0.2 × SSC and 0.1% SDS at 55 ° C. for 30 minutes. Fragments were radiolabeled with ³² P by the random priming method. This clone (1600 bp) was sequenced and found to encode a new cytochrome P450 (Figure 15). By comparing the sequence to CYP94A2, CYP94
A3 was found to be missing nine nucleotides. The first nine nucleotides of CYP94A2 were added in front of the incomplete CYP94A3 (clone C) sequence for heterologous expression. The full-length cDNA was then isolated,
It was found to be identical to the cDNA used in these activity evaluation experiments.

(Heterologous Expression in Yeast) The catalytic activity of CYP94A3 (clone C) was evaluated by expressing the function of a preceding clone in a genetically engineered yeast by the method described above. CYP94A3 (clone C; SEQ ID NO
: 7) was cloned by PCR into the expression vector pYeDP60 as described below using the SmaI and SacI restriction sites (peptide sequence contained in SEQ ID NO: 8). Sense primer:

Embedded image Antisense primer:

Embedded image

The sequence was amplified using Boehringer HiFi® polymerase according to the manufacturer's instructions and the resulting amplified sequence was verified for polymerase-free errors. The yeast strain WAT11 (Urban et al., 1990) was transformed with Schiestl and
Transformation was performed according to the method of Gietz (1989).

(Characterization of CYP94A3 [clone C]) Preparation of Microsomes: Yeast Strain with pYeDP60 Incorporating CYP94A3 (Clone C)
WAT11 was transformed, the yeast strain was grown by the method described above, and clone C was induced with respect to the preceding clone. The microsomes do not lose their activity when stored at -20 ° C for 5-6 weeks. WAT transformed with pYeDP60 expression vector alone
The same operation was performed on 11 cells, and this was used as a comparative control sample.

Evaluation of P450: Microsomes were diluted 5-fold with TEG, and the amount of P450 was measured by the method of Omura and Sato (1964) using the extinction coefficient at 91 / cm / mM.

Measurement of activity: The enzyme activity on the preceding clone was measured by the method described above. Table 4 shows the activity values of CYP94A3 (clone C) containing various fatty acid substrates.

[Table 4]

FIG. 17 (capric acid and lauric acid), FIG. 18 (myristic acid and palmitic acid)
, And FIG. 19 (oleic acid and linoleic acid) show TLC radiochromatograms. Two chromatograms are shown for each substrate, one in the presence of NADPH (A: hydroxylase has activity) and the other in the absence of NADPH (B: Hydroxylase has no activity).

Example IV-Clone D [CYP81B1]; Intramolecular hydroxylase [IC-LAH] of capric acid, lauric acid, and myristic acid Clone D contains capric acid (C10: 0), lauric acid (C12 : 0) and ω-2, ω-3 and ω-4 hydroxylation reactions of myristic acid (C14: 0) and Helianthus tu
It has the function of encoding microsomal P450 collected from berosis. The major metabolite is the omega-3 hydroxylate.

(Isolation of CYP81B1 [clone D]) The xenobiotic-inducing 7-ethoxycoumarin o-deethylase collected from H. tuberosus was purified to obtain a P450-enriched fraction containing a mixture of 5-6 P450 proteins. Fractions were isolated (Batard et al., 1995). A polyclonal antibody was raised against this P450-enriched fraction. On the other hand, the keratinocytes of H. tuberosus were sliced and aged for 24 hours in the presence of 20 mM aminopyrine. Using the polyclonal antibody, a λZAPII cDNA library prepared from the matured product was screened, and clone D was grown therefrom. Positive clones (56) were isolated and examined for the presence of the P450 consensus sequence using the PCR technique described by Meijer et al. (1993). From the 15 clones, PCR fragments of the expected size were obtained. These were labeled and hybridized with resting keratinocytes or complete RNA prepared from cut or aminopyrine-treated keratinocytes. One of the 15 clones corresponded to a 2.2 kb transcript. The transcript is hardly detectable in dormant keratinocytes and cut keratinocytes, but is induced by aminopyrine treatment. Sequencing of the insert revealed that the sequence encoded about 150 nucleotides lacking P450 at the N-terminal group. The library was rescreened to isolate longer cDNAs, lacking only 5 nucleotides. Poly (A) RNA was collected from keratinocyte tissue treated with aminopyrine for 24 hours, and this poly (A) RNA could be used to obtain missing coding sequences by 5'-RACE. Was. In this way, the complete sequence was reconstructed and named clone C (FIG. 20).

(Heterologous Expression in Yeast) The catalytic activity of CYP81B1 (clone D) was evaluated by expressing its function in yeast. A yeast strain providing an environment suitable for plant P450 expression (membrane structure and the presence of plant P450 reductase) was genetically engineered and used for heterologous expression. The strain WAT11, the expression vector, subcloning of the coding sequence, yeast growth, transformation, and preparation of yeast microsomes are described in Pompon et al. (1996). CYP81B1 (clone D) cDN from which sequences other than the coding sequence have been removed
A was transformed into pYe under the control of a galactose-inducible promoter (GAL10-CYC1).
It was expressed in a DP60 expression vector. CYP81B1 (clone D) was subcloned and inserted into this vector using Pfu DNA polymerase (Stratagene), and the modified cDNA was checked to ensure that there were no errors due to PCR. I bitten.

The P450 content and the catalytic activity were measured in microsomes prepared from the transformed yeast and the control yeast (control yeast = yeast transformed with an empty plasmid). No metabolism of P450s or fatty acids was detected in the control microsomes. CY
The transformed yeast was grown for 16 hours in the microsomal membrane prepared from P81B1 (clone D) in the presence of galactose, and the P450 content was measured by the method of Omura and Sato (1964) (FIG. 21). The P450 content was about 202 pmol / mg protein (ie, about 1% of the microsomal protein). Aromatic compounds, sterols, herbicides,
The catalytic activity of 20 or more types of potentially radioactively labeled substrates containing glycerol and fatty acids was examined. Fatty acid metabolism was assessed by the method of Salaun et al. (1981).

The molecules metabolized by CYP81B1 (clone D) are C10: 0, C12: 0 and C14: 0
(Table 5). Metabolism was dependent on the presence of NADPH.

(Table 5: Substrate specificity of CYP81B1) The activity was measured using a radiolabeled substrate. The amount of metabolites (the sum of the three hydroxylated products) was quantified by radio TLC. No activity was detected in the absence of control yeast (transformed with an empty expression vector) or NADPH.

[Table 5]

(Kinetic Variables and Specific Activities) For capric acid and lauric acid, the apparent Vmax and Km of the reaction were measured. In microsomes collected from yeast overexpressing Arabiodopsis reductase (ie, WAT11 strain), in the case of capric acid, the enzyme turnover rate was 41 ± 0.8.
/ Min, Km value 903 ± 168 nM, in the case of lauric acid, enzyme turnover rate 30.7 ± 1.4 / min, Km
The reaction progressed at a value of 788 ± 400 nM.

Characterization of metabolites The results of previous work by the present inventors (Salaun et al., 1981) show that lauric acid is 8-, 9- and It is shown to be converted to 10-hydroxylated metabolites (25:60:15 respectively). The product obtained in plant microsomes was characterized by GC-MS. The activity of lauric acid intrachain hydroxylase was also detected in corn and tulip microsomes and was induced by aminopyrine, phenobarbital and other xenobiotics (Adele et al., 1981; Salaun et al., 1982; Salaun et al.). ,
1986; Fonne-Pfister et al., 1988).

The TLC profile in FIG. 22 shows that CYP81B1 (clone D) shows that
Figure 2 shows that it encodes P450 catalyst formation. ω-2, ω-3, ω-4 hydroxylated metabolites were produced from three fatty acid substrates (capric acid, lauric acid, and myristic acid) at the same rate as in plant microsomes. Other minor products have been detected after culturing lauric acid with yeast-expressed enzymes. The structure of this metabolite is currently under investigation. In the case of lauric and myristic acids, the presence of three metabolites and their proportions were confirmed by HPLC (FIG. 23).

The results of previous studies we have performed show that the same enzyme performs allyl hydroxylation or epoxidation of the unsaturated acid lauric acid (Salaun et al., 1989, 1992, 1993) and 9- And the likelihood of catalyzing the sulfoxidation of 11-thiadodecanoic acid (Bosch, 1992) is very high. Unsaturated analogues (of the Z sequence) have also been found to be metabolized with a high degree of stereoselectivity (Salaun et al., 1992).

Example V Clone E (CYP94A4) Cytochrome P450-dependent hydroxylases include capric acid (C10: 0), lauric acid (C12: 0), myristic acid (C14: 0), and palmitic acid (C16: 0). ), Oleic acid (C
18: 0), catalyzes the methyl end group oxidation of linoleic acid (C18: 2) and linolenic acid (C18: 3), and CYP94A4 encodes a cytochrome P450-dependent hydroxylase. The highest activity is obtained at C14: 0 and C12: 0.

(Isolation of CYP94A4 [clone E]) Lambda-Zap cDNA library prepared from TMV-infected tobacco leaves was
By screening with 4A1, A2 and A3, clone E was obtained as follows (Dr. M. Legrand, IBMP [Strasbourg]).

Tobacco (Nicotiana tabacum var. Samsun NN) leaves were infected with TMV for 48 hours, poly (A ⁺ ) RNA was collected from the leaves, and a λZAP cDNA library was prepared from the poly (A ⁺ ) RNA. did. This library was screened with low stringency using a mixture of CYP94A1, A2 and A3 coding sequences as a probe. This probe was radioactively labeled with ³² P by a random priming method. Fifteen clones larger than 1500 pb were isolated and sequenced. Ten of these were full length, indicating that these clones encode a new cytochrome P450 of the CYP94 family. This clone was named CYP94A4 (FIG. 24).

Heterogeneous Expression in Yeast The catalytic activity of CYP94A4 (clone E) was demonstrated in yeast specifically designed by the method described previously for the clones previously described herein. The function was developed and evaluated. The coding sequence of clone E (SEQ ID NO: 9) was cloned by PCR into the expression vector pYeDP60 using the BamHI restriction site as follows. Sense primer (BamHI)

Embedded image Antisense primer (KpnI)

Embedded image

Boehringer HIFI® polymerase was used according to the manufacturer's instructions to verify that the amplified sequences did not contain polymerase-induced errors. Yeast strain WAT11 (Urban et al., 1990) was transformed according to the method of Schiestl and Gietz (1989).

(Preparation of Microsome) Yeast (strain WAT11) was transformed with pYeDP60 incorporating clone E, and the yeast was grown and microsomes were induced by the method described above. WA that does not transform
The same operation was performed on T11 cells, which was used as a comparative control sample.

(Measurement of the amount of P450) The microsome was diluted 5-fold with TEG, and was diluted by the method of Omura and Sato (1964).
The amount of P450 was measured using the extinction coefficient of / cm / mM (FIG. 2).

(Measurement of Activity) The enzyme activity was measured by the method described above. All reaction products identified in these experiments have previously been identified on plant microsomes by rechromatography using pure compounds and by GC / MS spectroscopy. Complete kinetic studies were performed for each substrate.

[Table 6]

Example VI-Clone F [CYP94A5] CYP94A5 encodes a cytochrome P450-dependent hydroxylase, wherein cytochrome P450-dependent hydroxylase is lauric acid (C12: 0), myristic acid (C14: 0).
), Catalyzes the oxidation of the methyl end groups of palmitic acid (C16: 0), oleic acid (C18: 1), linoleic acid (C18: 2), and linolenic acid (C18: 3). However, the efficiency varies greatly depending on the type of the lipid. The highest activity is obtained at C14: 0 and C18: 2.

(Isolation of CYP94A5 [clone F]) A lambda-Zap cDNA library was prepared from tobacco leaves infected with TMV, and this lambda-Zap cDNA library was screened with CYP94A1, A2 and A3, and Clone F was obtained (Dr. M. Legrand, IBMP [Strasbourg]).

The λZAP cDNA library was transferred to tobacco (Nicotiana t.) Infected with TMV for 48 hours.
abacum var. Samsun NN) was prepared from poly (A ⁺ ) RNA collected from leaves.
The cDNA library was screened with low stringency using a mixture of CYP94A1, A2 and A3 coding sequences as a probe. This probe was radiolabeled with ³² P by random priming. Fifteen clones larger than 1500 pb were isolated and sequenced. Two full-length clones were found to encode a new cytochrome P450 of the CYP94 family. This new cytochrome
P450 was named CYP94A5 (FIG. 25).

(Heterologous Expression in Yeast) The catalytic activity of CYP94A5 (clone F) was evaluated by expressing the function in yeast designed for ad hoc purposes by the method described for the preceding clone. .

Reformatting of Clone F The coding sequence of clone F (SEQ ID NO: 11) was cloned by PCR into the vector pYeDP60 using the BamHI restriction site as described below. The peptide sequence contained in SEQ ID NO: 12 is as follows. Sense primer (BamHI)

Embedded image Antisense primer (KpnI)

Embedded image

[0115] Boehringer HIFI® polymerase was used according to the manufacturer's instructions to verify that there were no polymerase-induced errors in the amplified sequences. Yeast strain WAT11 (Urban et al., 1990) was transformed according to the method of Schiestl and Gietz (1989).

(Preparation of Microsome) Yeast (strain WAT11) was transformed with pYeDP60 incorporating clone F, and the yeast was grown and clone F was induced by the method described above for the preceding clone. Untransformed WAT11 cells were subjected to the same procedure as a control sample.

(Measurement of the amount of P450) The microsome was diluted 5-fold with TEG, and the ratio of 91 / cm / was determined by the method of Omura and Sato (1964).
P450 was quantified using the extinction coefficient in mM (FIG. 2).

(Measurement of activity) The enzyme activity was measured by the method described above. All of the products identified in these experiments have already been identified by rechromatography and GC / MS using pure compounds in experiments previously performed on plant microsomes. Complete kinetic studies were performed for each substrate.

[Table 7]

[Table 8]

Example VII Clone G [CYP94A6] CYP94A6 encodes cytochrome P450. Currently, its catalytic activity is under evaluation. CYP94A6 is expected to have a fatty acid hydroxylase activity because it exhibits a signature sequence characteristic of enzymes of this class.

(Isolation of CYP94A6 [clone G]) Lambda-Zap cDNA libraries prepared from TMV-infected tobacco leaves (by Dr. M. Legrand of IBMP [Strasbourg] M. Legrand) were used for CYP94A1, A2 as follows. And A3 to obtain clone G.

[0121] Tobacco (Nicotiana tabacum var. Samsun NN) leaves were infected with TMV for 48 hours, poly (A ⁺ ) RNA was collected from the tobacco leaves, and λZAP cD was obtained from the poly (A ⁺ ) RNA.
An NA library was prepared and this λZAP cDNA library was screened with low stringency using a mixture of the CYP94A1, A2 and A3 coding sequences as a probe. This probe was radiolabeled with ³² P by random priming. Fifteen clones larger than 1500 pb were isolated and sequenced. One complete clone was found to encode a new cytochrome P450 of the CYP94 clan. This clone was named CYP94A6. Ndel restriction site ACATAT at position 594 (Figure
The complete sequence of CYP94A6 and sequence-specific primers were obtained by performing reverse PCR on genomic tobacco (Nicotiana tabacum var. Samsun NN) DNA using 26).

(Heterogeneous Expression in Yeast) CYP94A6 was expressed in yeast by the method described above, and the produced protein was detected by Western blotting. The catalytic activity of clone G is expressed in a specially designed enzyme whose function is currently being evaluated.

(Reform of Clone G) Using the BamHI restriction site, the coding sequence of clone G (SEQ
ID NO: 13) was cloned by PCR into the vector pYeDP60. (Peptide sequence contained in SEQ ID NO: 14) Sense primer (BamHI)

Embedded image Antisense primer (KpnI)

Embedded image The amplified sequences were verified for polymerase errors using Boehringer HIFI® polymerase as per the manufacturer's instructions. Schiestl and Gietz (19
The yeast strain WAT11 (Urban et al., 1990) was transformed by the method of 89).

(Preparation of microsome) [0124] Clone G was incorporated into pYeDP60, yeast (strain WAT11) was transformed with this pYeDP60, and the yeast was grown by the method described above to induce microsomes. The same operation was performed on untransformed WAT11 cells, and this was used as a comparative control sample.

(Measurement of the amount of P450) The microsome was diluted 5-fold with TEG, and was subjected to the method of Omura and Sato (1964) at 91 / cm /
The amount of P450 was measured using the extinction coefficient of mM.

(Preparation of transgenic tobacco expressing CYP94A4, CYPA5, CYPA6) Tobacco (Nicotiana tabacum L. var Xanthi) was cloned into clones E (CYP94A4), F
(CYPA5) and G (CYPA6) were transformed in the "open-repeat-open" reading frame in the sense and antisense orientation. The coding sequence was cloned into the vector pFB8 (Atanassova et al., Plant J. 1995, 8, pp 465-477) which was specifically constructed in our laboratory. This transformation was performed using the disc-shaped tissue of tobacco leaves and the method of Horsch (Scie
1985, 227, pp 1227-1237) via the tumor bacterium (strain LBA 4404).

Reformatting of CYP94A4, CYPA5, CYPA6 coding sequence The coding sequence was cloned by PCR into the vector pFB8 using the BamHI and KpnI restriction sites shown in bold below. (SEQ ID NOs: 4 and 3 for 94A4, SE for 94A5 and 94A6, respectively)
Peptide and diffusion sequences contained in Q ID NOs: 13 and 12) a) Sequence in sense direction in pFB8 Sense primer (KpnI)

Embedded image Antisense primer (BamHI)

Embedded image b) Sequence in pFB8 in antisense direction Sense primer (BamHI)

Embedded image Antisense primer (KpnI)

Embedded image At present, plants transformed in both sense and antisense directions are growing on seeds of T1, and the results will be forthcoming soon.

(Signature of fatty acid omega hydroxylase) The present inventors identified the peptide sequence (SEQ ID NO: 2). This is shown in FIGS.
, 15, 24, 25 and 26 are indicated by double underlining. This sequence is a unique signature found in all plant fatty acid omega hydroxylases that have been characterized to date. S (AVS) AL (TVS) WFFWL (LIV) where (AVS) is one of A, V or S, (TVS) is one of T, V or S,
(LIV) means one of L, I or V. This signature sequence is C
YP86A1 (SEQ ID NO: 1), CYP86A5, CYP94A1, CYP94A2, CYP94A3, CYP94A4, CYP
Present in 94A5, and CYP94A6. All signature sequences except for CYP94A6 (now under characterization) have omega hydroxylase activity. This signature sequence has been found to be absent in CYP81B1, an intramolecular hydroxylase, as a result of sequence adjustment.

When this signature sequence was searched from among all the fatty acid genes stored in GeneBank, it was found that 12 sequences existed. All of these were isomeric forms of cytochrome P450. Some sequences were duplicated due to recloning of the same gene in different laboratories.

As a result of scanning all the sequences included in the database (the number of non-overlapping sequences was 364,804 in the total number of sequences in GenBank, EMBL, DDBI, and PDB), this signature was found to be According to the application of the present invention, it was confirmed that there was no gene other than the target gene of the present invention, even if collected from all plants, animals or microorganisms showing the same type of catalytic activity. . Therefore, the isolated genes representing the signature are all related to the gene of the present invention, and are considered to exhibit the same type of catalytic activity as the gene of the present invention. (References)

With respect to the present invention, what has been described herein is what is believed to be practical at present. Accordingly, the present invention is not limited or limited to the embodiments disclosed herein. The invention applies to all modifications and equivalents falling within the spirit and scope of the claims appended hereto.

Accordingly, it is easy for those skilled in the art to make changes to the description of the invention without departing from the novel aspects of the invention. Accordingly, all such modifications are intended to be included in the scope of the present invention.

[Sequence list]

[Brief description of the drawings]

FIG. 1. Nucleotide sequence of CYP94A1 (clone A) and protein translation derived therefrom (SEQ ID NOs: 3 and 4, respectively). Open reading frame nucleotides are shown in capital letters. Representative heme binding domains that make up the signature of P450 are underlined.

FIG. 2 is a carbon monoxide difference spectrum of yeast microsomes expressing CYP94A1 (clone A). Microsomes prepared by the method of Pompon et al. (1996) (10 mg protein / ml)
Was diluted 5-fold, reduced with a few drops of sodium hyposulfite and divided into two cuvettes. Use a Shimadzu MP2000 dual beam spectrometer to increase the baseline to 40
Recorded between 0 and 500 nm. Carbon monoxide was blown into the cuvette containing the sample and the spectrum of the resulting P450-CO complex was recorded. As a result of measuring absorbance at 91 / cm / mM, the amount of CYP94A1 was 176 pmol / mg protein.

FIG. 3. Chemical structures of sulfur-containing lauric acid analogs and sulfoxylated metabolites.

FIG. 4 shows the reaction product of capric acid (C10: 0), lauric acid (C12: 0), myristic acid (C14: 0), and palmitic acid (C16: 0) produced by CYP94A1 (clone A). Radio chromatogram. After collecting microsomes from the yeast transformed with the fatty acid and culturing them, the reaction mixture was extracted by the method described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH, and S: residual substrate.

FIG. 5 is a radiochromatogram of a reaction product generated from oleic acid (C18: 1), linoleic acid (C18: 2), and linolenic acid (C18: 3) by CYP94A1 (clone A). Microsomes were collected from yeast transformed with unsaturated fatty acids, and after culturing, the reaction mixture was extracted by the method described above and analyzed by TLC. A: In the presence of NADPH,
B: in the absence of NADPH (comparison control) and S: residual substrate.

FIG. 6 shows that 9Z, 12E-octadecadienoic acid (C18: 2-9E, 12Z), CYP94A1 (clone A)
Radio of reaction products formed from 9E, 12Z-octadecadienoic acid (C18: 2-9Z, 12E), 8-propylsulfinyloctanoic acid (8S-LAU), and 10-methylsulfinyldecanoic acid (10S-LAU) Chromatogram. After collecting microsomes from the yeast transformed with unsaturated fatty acids and culturing them, the reaction mixture was extracted by the method described above, and TLC (C
18: 2-9E, 12Z and C18: 2-9Z, 12E) or HPLC (8S-LAU and 10S-LAU). A
: In the presence of NADPH, B: in the absence of NADPH (comparison control), and S: residual substrate.

FIG. 7. Nucleotide sequence of VAGH811 (incomplete at 5 ′ end) [also called clone B (complete cDNA encoding CYP94A2)] and putative protein translation (ωMAH or CYP94A2)
(SEQ ID NOs: 5 and 6, respectively). The consensus heme binding domain that constitutes the P450 signature is underlined.

FIG. 8 is a carbon monoxide difference spectrum of yeast microsome (clone B) expressing ω-MAH. Microsomes (10 mg-protein) prepared by the method described by Pompon et al. (1996)
/ ml) was diluted 5-fold, reduced with a few drops of sodium hyposulfite and split into two cuvettes. 400 and 50 using Shimadzu MP2000 dual beam spectrometer
A baseline was recorded during 0 nm. Carbon monoxide was blown into the sample cuvette and the spectrum of the P450-CO complex was recorded. As a result of measuring the amount of ω-MAH based on the absorption coefficient at 91 / cm / mM, it was 80 pmol / ml microsome.

FIG. 9 is a radiochromatogram of a reaction product generated from capric acid by ω-MAH. ¹⁴ After culturing with C-capric acid, the reaction mixture was extracted by the method described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH.

FIG. 10 is a radiochromatogram of a reaction product generated from lauric acid by ω-MAH. ¹⁴ After incubation with C-lauric acid, the reaction products were extracted as described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH.

FIG. 11 is a radiochromatogram of a reaction product generated from myristate by ω-MAH. After culturing with ¹⁴ C-myristic acid, the reaction product was extracted as described above,
Analyzed in C. A: in the presence of NADPH, B: in the absence of NADPH.

FIG. 12 is a radiochromatogram of a reaction product generated from palmitic acid by ω-MAH. After culturing with ¹⁴ C-palmitic acid, the reaction product was extracted as described above and TL
Analyzed in C. A: in the presence of NADPH, B: in the absence of NADPH.

FIG. 13 is a radiochromatogram of a reaction product generated from stearic acid by ω-MAH. After incubation with ¹⁴ C-stearic acid, the reaction products were extracted as described above and
Analyzed in C. A: in the presence of NADPH, B: in the absence of NADPH.

FIG. 14 is a radiochromatogram of a reaction product generated from oleic acid by ω-MAH. ¹⁴ After incubation with C-oleic acid, the reaction products were extracted as described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH.

FIG. 15: Nucleotide sequence and predicted protein translation of CYP94A3 (clone C). SEQ ID
NO is 7 and 8, respectively. Compared to clone B, 9 nucleotides are missing at the 5 'end.

FIG. 16 is a carbon monoxide difference spectrum of yeast microsomes expressing CYP94A3 (clone C). Microsomes (10 mg protein / ml) prepared by the method described by Pompon et al. (1996) were diluted 5-fold, reduced with a few drops of sodium bisulfite, and split into two cuvettes. . Using a Shimadzu MP2000 dual beam spectrometer, 400 and
A baseline was recorded during 500 nm. Carbon monoxide was blown into the sample cuvette and the spectrum of the P450-CO complex was recorded. As a result of measuring the amount of CYP94A3 based on the extinction coefficient at 91 / cm / mM, it was 550 pmol / ml microsome.

FIG. 17 is a radiochromatogram of a reaction product generated from capric acid (C10: 0) and lauric acid (C12: 0) by CYP94A3 (clone C). Microsomes were collected from the yeast transformed with the fatty acid and cultured, and then the reaction mixture was extracted by the method described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH (comparative control), and S: residual substrate.

FIG. 18. Myristic acid (C14: 0) and palmitic acid (C1) were expressed by CYP94A3 (clone C).
6: 0) Radiochromatogram of the reaction product generated from Microsomes were collected from the yeast transformed with the fatty acid and cultured, and then the reaction mixture was extracted by the method described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH (comparative control), and S: residual substrate.

FIG. 19 is a radiochromatogram of a reaction product generated from oleic acid (C18: 1) and linoleic acid (C18: 2) by CYP94A3 (clone C). Microsomes were collected from the yeast transformed with the fatty acid and cultured, and then the reaction mixture was extracted by the method described above and analyzed by TLC. A: in the presence of NADPH, B: in the absence of NADPH (comparative control), and S: residual substrate.

FIG. 20. Nucleotide sequence and predicted protein translation of CYP81B1 (clone D) (SEQ ID NOs: 15 and 16, respectively). The consensus heme binding domain that constitutes the P450 signature is underlined.

FIG. 21 is a carbon monoxide difference spectrum of yeast microsomes expressing CYP81B1 (clone D). Microsomes (10 mg protein / ml) prepared by the method described by Pompon et al. (1996) were diluted 5-fold, reduced with a few drops of sodium bisulfite, and split into two cuvettes. . Using a Shimadzu MP2000 dual beam spectrometer, 400 and
A baseline was recorded during 500 nm. Carbon monoxide was blown into the sample cuvette and the spectrum of the P450-CO complex was recorded. As a result of measuring the amount of CYP81B1 based on the extinction coefficient at 91 / cm / mM, it was 202 pmol / mg protein.

FIG. 22: 100 μM of C10: 0 (a), C12: 0 (b), C14: 0 (c) radioactively labeled with ¹⁴ C was added to Hel
ianthus tuberosus (H.tub., 1.2 mg protein) or transgenic yeast (CYP81B1
, 0.1 mg protein) with 600 μM NADPH
After culturing at 27 ° C. for 45 minutes, the obtained metabolite was analyzed by TLC. After stopping the reaction with one volume of acetonitrile-acetic acid (99.8 / 0.2), the culture medium was changed to a TLC-silica plate (Me
The solution was dropped directly on 60 F254 manufactured by rck and developed with a mixture of ether-petroleum benzene-formic acid (70/30 / 0.2). Radiometer thin layer scanner (Berthold LB 2723)
) Was used to confirm the location of the reaction product.

FIG. 23. Obtained by culturing 100 μM C12: 0 (a) and C14: 0 (b) together with microsomes of transgenic yeast (0.1 mg protein) and 600 mM NADPH at 27 ° C. for 45 minutes. Of the metabolites obtained by radio HPLC analysis. 1 volume of acetonitrile-acetic acid (99.8 / 0.2 v / v
), The culture medium was extracted twice with ether. The ether was evaporated in an argon atmosphere and 1.6 mm x 15 cm using a Beckman HPLC ODS 5 μm.
On the column, as eluent H ₂ O-acetonitrile - acetic acid (C12: 0 for the volume ratio 75/25 / 0.2, C14: 0 68/32 / 0.2 by volume with respect to) use To separate the metabolites. The flow rate was 2 ml / min. The radioactivity of the HPLC eluate was monitored on a computerized online solid scintillation counter (Ramona D, Isomess).

FIG. 24. Nucleotide sequence and predicted protein translation of CYP94A4 (clone E) prepared from tobacco leaves infected with tobacco mosaic virus (SEQ ID NOs: 9 and 10, respectively).

FIG. 25. Nucleotide sequence and predicted protein translation of CYP94A5 (clone F) prepared from tobacco leaves infected with tobacco mosaic virus (SEQ ID NOs: 11 and 12, respectively).

FIG. 26. Nucleotide sequence and putative protein translation of CYP94A6 (clone G) prepared from tobacco leaves infected with tobacco mosaic virus (SEQ ID NOs: 13 and 14, respectively).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12Q 1/68 A 9/02 C12N 15/00 ZNAA C12Q 1/68 5/00 C (72 Inventor Bambunist, Irene France Straubourg, Rue Goethe, 21 (72) Inventor Le Boucan, Renault France Strasbourg, Rue des Frons, 16 Ah (72) Inventor Elbi, Christiane Sarburbourg, France, Le Molière , 19 (72) Inventor Batal, Yannick Strasbourg, Rüdreman, France, 5 (72) Inventor Cavelo-Yurtado, Francisco Spain Cordoba, Bajo 2, Pinto Velazquez 5 (72) Inventor Werk-Reichhardt Daniel France Funding shy-time, Ryu de Baghdad, 3 (72) inventor salon, Jean-Pierre, France Strasbourg, Ryu de Kyapyusan, 68 (72) inventor Deyurusu, Francis France Berunorushaimu, Ryu de Ranshan'nu Nekoru, 7

Claims (27)

[Claims] 1. An isolated nucleic acid encoding a plant fatty acid hydroxylase selected from the group consisting of omega hydroxylase, in-chain hydroxylase, and functional derivatives thereof. 2. An isolated nucleic acid encoding a plant fatty acid hydroxylase,
An isolated nucleic acid wherein the plant fatty acid hydroxylase is an omega hydroxylase having a peptide sequence of SEQ ID NO: 2 or a functional derivative thereof, and hydroxylates a fatty acid substrate at a terminal. 3. The isolated nucleic acid of claim 2, wherein said omega hydroxylase is selected from the group consisting of CYP94A1, CYP94A2, CYP94A3, CYP94A4, CYP94A5 and CYP94A6. 4. The isolated nucleic acid according to claim 1, wherein the plant fatty acid hydroxylase is an intramolecular hydroxylase (CYP81) or a functional derivative thereof, and hydroxylates a fatty acid substrate near the terminal. 5. The isolated nucleic acid according to claim 4, wherein the intramolecular hydroxylase is CYP81B1. 6. A recombinant nucleic acid comprising the isolated nucleic acid according to any one of claims 1 to 5. 7. The recombinant nucleic acid according to claim 6, further comprising a regulatory region suitable for expression of a plant fatty acid hydroxylase in a host cell. 8. A host cell comprising the recombinant nucleic acid of claim 6. 9. The host cell of claim 8, wherein said host cell is selected from the group consisting of a bacterial cell, a fungal cell, and a plant cell. 10. A transgenic plant comprising the transgenic nucleic acid according to claim 6. 11. A host cell comprising the recombinant nucleic acid according to claim 7. 12. The host cell of claim 11, wherein said host cell is selected from the group consisting of a bacterial cell, a fungal cell, and a plant cell. 13. A transgenic plant comprising the transgenic nucleic acid according to claim 7. A plant fatty acid hydroxylase encoded by the isolated nucleic acid according to any one of claims 1 to 5. 15. A composition comprising the plant fatty acid hydroxylase according to claim 14 as an essential component. 16. A polypeptide produced by the recombinant nucleic acid according to claim 7. 17. A composition comprising the polypeptide of claim 16 as an essential component. 18. A method for isolating an additional fatty acid hydroxylase gene from a plant using the isolated nucleic acid according to any one of claims 1 to 5. 19. The method of claim 18, wherein said isolated nucleic acid is used as a labeled probe hybridized to a series of nucleic acids collected from a plant to select additional nucleic acid encoding a fatty acid hydroxylase gene. . 20. The method of claim 18, wherein the primers synthesized according to the conserved nucleotide sequence of the isolated nucleic acid amplify nucleic acids from a series of plants to select for nucleic acids encoding additional fatty acid hydroxylase genes. Method. 21. An isolated nucleic acid selected by the method of claim 18. 22. A method for changing a fatty acid composition in a plant, wherein the isolated nucleic acid according to any one of claims 1 to 5 is introduced into a plant to produce a transgenic plant. The above method, wherein the fatty acid hydroxylase is expressed in a transgenic plant, and the fatty acid substrate in the transgenic plant is hydroxylated or epoxidized. 23. The method of claim 22, wherein said fatty acid substrate is a medium-chain fatty acid. 24. The method of claim 23, wherein said medium chain fatty acid is selected from the group consisting of caprylic fatty acids, lauric fatty acids, and myristic fatty acids. 25. The method of claim 22, wherein said fatty acid substrate is a long chain fatty acid. 26. The long chain fatty acid is selected from the group consisting of palmitic fatty acids, oleic fatty acids, linoleic fatty acids, and linolenic fatty acids.
the method of. 27. The fatty acid substrate has a fatty acid having an odd number of a single prime, a fatty acid having an in-chain hydroxy group, a fatty acid having an intramolecular epoxy group, a thia fatty acid, an ether fatty acid, and an ester bond. 26. The method of claim 25, wherein the method is selected from the group consisting of modified fatty acids and modified fatty acids having an amide bond.