MXPA02001086A - Nucleic acid sequences encoding polyenoic fatty acid isomerase and uses thereof. - Google Patents

Abstract

Nucleic acid sequences and methods are provided for producing conjugated fatty acids in host cells. Nucleic acid sequences are obtained from an algal sources which encode polyenoic fatty acid isomerase. The nucleic acid sequences can be used in expression constructs to direct the expression of the PFI sequences in host cells. Expression of PFI in transgenic host cells provides for the production of conjugated fatty acids in the host cell.

Description

NUCLEIC ACID SEQUENCES THAT CODIFY FOR POLYENOUS FATTY ACID ISOMERASE AND USES THEREOF CROSS REFERENCE TO RELATED REQUESTS This application claims priority of the provisional patent application of E.U.A. with serial number 60 / 146,458 filed July 30, 1999, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD The present invention relates to sequences and constructions of nucleic acid and amino acids, and methods related thereto.

BACKGROUND OF THE INVENTION Novel improved means and / or vegetable oil compositions are required to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources. Depending on the use of the intended oil, different oil compositions are desired. For example, sources of edible oil that contain the minimum possible amounts of saturated fatty acids are suitable for dietary reasons SSBßSSS? - * Íía_ £ ___. AJí-- > t ~ i--? f - ___, "" iWitoiiíii and alternatives are also needed for current sources of highly saturated oil products, such as tropical oils. In addition, oil compositions containing rare or exotic fatty acid species that have dietary benefits are also necessary in the art. Conjugated fatty acids, such as conjugated linoleic acid (CLA), are gaining recognition for their health benefits in animal nutrition and human nutrition. Conjugated fatty acid is a general term for fatty acids that contain double bonds alternating with simple bonds. For example, conjugated linoleic acid refers to a series of geometric and positional isomers of linoleic acid (a 18-carbon molecule containing double bonds at the cis-9 and cis-12 positions). One of the different isomers of CLA, the cis-9, trans-10, cis-12 isomers, have received the most attention. Recent data suggest (Parks, ef al. (1999), Lipids, 34: 235-243) that trans-10, cis-12 is the biologically active form. However, it is recognized that other isomers of CLA, and / or other conjugated fatty acids, may also show that they have biological activities. CLA is now recognized as a dietary supplement and an effective inhibitor of epidermal carcinogenesis and neoplasia of the anterior stomach in mice, and of rat and mammary tumors in rats induced by carcinogen. In addition, CLA has been shown to reduce LDL and atherosclerosis in hamsters and rabbits, reduce body fat and increase body mass ^ • - > - ^ iii sparse in chickens, pigs, rats and mice, increase feed efficiency in chickens and pigs, reduce PGE2 serum in rats, increase bone mass in mice and chickens, as well as reduce weight loss during immune challenge in mice, chickens and rats. In this way, the identification of objective enzymes and sources for nucleic acid sequences of said target enzymes capable of producing conjugated fatty acids in host cells is necessary in the art. Finally, useful constructions of nucleic acid having the necessary elements to provide a phenotypic modification and host cells containing said constructions are needed.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to polyenoic fatty acid isomerases (PF1), and in particular to PF1 polypeptides and polynucleotides. The polypeptides and polynucleotides of the present invention include those derived from plant and fungal sources. In another aspect of the invention, polynucleotides encoding novel novel polypeptides, particularly polynucleotides encoding PF1, are provided. In a further aspect, the invention relates to oligonucleotides derived from PF1 proteins and oligonucleotides which include partial or complete PF1 coding sequences.

In addition, it is an aspect of the present invention, provide recombinant DNA constructs which can be used either for transcription and / or expression of PF1. In particular, constructs are provided which are capable of transcription and / or expression in host cells. Particularly preferred constructs are those capable of transcription and / or expression in plant cells. Even in another aspect of the present invention, methods for producing PF1 in a host cell or progeny thereof are provided. In particular, the host cells are transformed or transfected with a DNA construct which can be used for transcription and / or expression of PF1. Recombinant cells containing PF1 are also part of the present invention. In a further aspect, the present invention relates to methods for using polynucleotide and polypeptide sequences to modify the fatty acid composition in a host cell, particularly in seed oil from oilseed crops. In particular, the modified fatty acid composition comprises an altered amount of conjugated fatty acids. Vegetable cells containing said modified fatty acids are also contemplated herein. Modified plants, seeds and oils obtained by the expression of the vegetable proteins of PF1 are also considered part of the invention. the e. l, ._. m_ < _í ... ._ ii, .. »> .__2..

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, nucleotide sequences encoding a protein, polypeptide or peptide are provided, which are active in the formation of conjugated fatty acids from polyenoic fatty acid substrates. Said sequences are referred to herein as polyeneoic fatty acid isomerases (also referred to as PF1). The novel nucleic acid sequences find use in the preparation of constructs to direct their expression in a host cell. In addition, novel nucleic acid sequences find use in the preparation of plant expression constructs to modify the fatty acid composition of a plant cell. A polyphenolic fatty acid isomerase nucleic acid sequence of this invention includes a nucleic acid sequence encoding a protein, polypeptide, or peptide fragment, which can be obtained from a source which is active in the formation of conjugated fatty acids from a polyunsaturated fatty acid substrate in a plant host cell, i.e., in vivo, or in an environment similar to plant cell, i.e., in vitro. As used herein, "conjugate" refers to the interaction of electron systems pi where the carbon chain contains alternating double and single bonds (C = CC = C) so that the electrons of the double bonds are Close enough to interact with each other. This unlike "isolated" double links, where < * * Í? Rk .i I the pi electron systems are separated by a saturated carbon (C = C-C-C = C) or "accumulated" where the double bonds share a central carbon (C = C = C). "An environment similar to plant cell" means that any necessary condition is available in that environment (ie, factors such as temperatures, pH, lack of inhibitory substances) which will allow the enzyme to function. The fatty acids used as substrates by the protein encoded by the polynucleotide sequence of the present invention include any polyunsaturated fatty acid substrate. Such fatty acid substrates include, but are not limited to dienes, trienes, tetraenes, pentanes and hexanes. The fatty acid substrates of particular interest in the present invention include but are not limited to linoleic acid, linolenic acid, stearidonic, eicosapentanoic acid, dihomo-α-linolenic acid, adrenal acid, eicostrienonic acid, α-linolenic acid, docosahexaenoic acid and arachidonic acid.

Isolated proteins, polypeptides and polynucleotides A first aspect of the present invention relates to isolated PF1 polypeptides. Such polypeptides include isolated polypeptides listed in the sequence lig, as well as polypeptides and fragments thereof, particularly those polypeptides that exhibit PF1 activity and also those polypeptides having at least about 50-79% identity, preferably about at least 80% identity, and even preferably near at least 90% identity, with a polypeptide sequence selected from the group of sequences set forth in the sequence lig, and also includes portions of said polypeptides, wherein said portion of the polypeptide, preferably includes at least 30 amino acids and preferably includes at least 50 amino acids. "Identity", as understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing these sequences. In the art, "identity" also means the degree of sequence relationship between polypeptide or polynucleotide sequences, as determined by the matching between rows of said sequences. "Identity" can be easily calculated by known methods including but not limited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey (1 | 994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J Applied Math, 48: 1073 (1988). The methods for determining identity are designed to give the greatest agreement among the sequences under test. In addition, the methods to determine the i_j_¡ ___, __.; _?. +.-a »» *. »..-... M_t_auam * idefltt? lad are encoded in publicly available programs. Computational programs that can be used to determine identity between two sequences include, but are not limited to GCG (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984); pack of five BLAST programs , three designed for investigations of nucleotide sequences (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence investigations (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al. al., Genome Analysis, 1: 543-559 (1997).) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul S., et al., NCBI NLM NIH, Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol., 215: 403-410 (1990).) The known algorithm of Smith Waterman can also be used to determine identity.Parameters for comparing polypeptide sequences typically include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 by Hentikoff and Hentikoff, Proc. Nati Acad. Sci USA 89: 10915-10919 (1992) Penalty for space: 12 Penalty for space length: 4 A program that can be used with these parameters is publicly available as the "space" program of Genetics Computer Group, Madison Wisconsin. The above parameters together with fc ^ il fe ^^ Ka- ial ^ fe - ^^^ - ^ fe-a. ^^ «** fr. * ¡T, ^. ^ - ^, s ^^ *. ^ *. ^ * & > * & & ^ ". ^ *, _ *, * - »k t no final space sanctions are the default parameters for peptide comparisons. The parameters for comparing polynucleotide sequences include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: equalities = + 10; inequalities = 0 Penalty for space: 50 Penalty for length of space: 3 A program which can be used with these parameters is publicly available as the "space" program of Genetics Computer Group, Madison Wisconsin. The above parameters are the predefined parameters for nucleic acid comparisons. The invention also includes polypeptides of the formula: wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Ri and R3 are any amino acid residue, n is an integer between 1 and 1000, and R2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from the group set forth in the sequence listing and preferably SEQ ID Nos: 2 and 4. In the formula, R2 is oriented such that its amino terminal residue is on the left, bound to R1 f and its carboxy terminal residue is on the right, attached to R3. Any stretch of amino acid residues denoted either by • binds the group R, where R is greater than 1, can be a heteropolymer or a homopolymer, preferably a heteropolymer. The polypeptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising a sequence selected from the group of a sequence contained in SEQ ID Nos: 1 and 3. The polypeptides of the present invention have been shown to have PF1 activity and are of interest because to which PF1 is involved in the production of conjugated fatty acids from polyacetic fatty acyl substrate molecules. The polypeptides of the present invention can be a mature protein or can be part of a fusion protein. Fragments and variants of the polypeptides are also considered part of the invention. A fragment is a polypeptide variant which has an amino acid sequence that is completely the same as part but not completely of the amino acid sequence of the previously described polypeptides. The fragment may be "independent" or comprised within a larger polypeptide of which the fragment forms a part or a region, preferably as a single continuous region. Preferred fragments are biologically active fragments which are those fragments that mediate activities of the polypeptides of the invention, including those with similar activity or improved activity or with decreased activity.

Also included are those fragments that are antigenic or immunogenic in an animal, particularly a human. Variants of the polypeptide also include polypeptides that vary from the sequences set forth in the sequence listing by conservative amino acid substitutions, which are substitutions of a residue by another residue with similar characteristics and / or properties. In general, such substitutions are between Ala, Val, Leu e lie; between Ser and Thr; between Asp and Glu; between Asn and Gln; between Lys and Arg; or between Phe and Tyr.

Particularly preferred are variants in which 5 to 10; 1 to 5; 1 to 3 or an amino acid (s) are substituted, deleted, or added in any combination. Variants that are fragments of the polyethers of the invention can be used to produce the corresponding total length polypeptide by peptide synthesis. Therefore, these variants can be used as intermediates to produce the full-length polypeptides of the invention. Another aspect of the present invention relates to isolated PF1 polynucleotides. The polynucleotide sequences of the present invention include isolated polynucleotides encoding the polypeptides of the invention having a deduced amino acid sequence selected from the group of sequences set forth in the sequence listing and to other polynucleotide sequences closely related to said sequences and variants of the same.

The invention also provides an identical polynucleotide sequence throughout its length to each coding sequence as set forth in the sequence listing. The invention also provides the coding sequence for the mature polypeptide or a fragment thereof, as well as the coding sequence for the mature polypeptide or a fragment thereof in a reading frame with other coding sequences, such as those coding for a leader sequence. or secretory, a sequence of pre-, pro- or prepro-protein. The polynucleotide can also include non-coding sequences, including, but not limited to, 5 'and 3' non-coding sequences, such as transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence that codes for additional amino acids. For example, a marker sequence can be included to facilitate purification of the fused polypeptide. The polynucleotides of the present invention also include polynucleotides that comprise a structural gene and naturally associated sequences that control gene expression. The invention also includes polynucleotides of the formula: X- (R?) N- (R2) - (R3) nY wherein, at the 5 'end, X is hydrogen and at the 3' end Y is hydrogen or a metal, Ri and R3 are any nucleic acid residue, n is an integer between 1 and 3000, preferably between 1 and 1000 and R2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence _V? ¡£ -. TO ? . ??? is selected from the group set forth in the sequence listing and preferably SEQ ID Nos: 1 and 3. In the formula, R2 is oriented such that its 5 'end residue is on the left, joined to Ri, and its 3 'end residue is on the right, joined to R3. Any stretch of nucleic acid residue denoted by either group R, wherein R is greater than 1, can be a heteropolymer or a homopolymer, preferably a heteropolymer. The invention also relates to variants of the polynucleotides described herein that encode variants of the polypeptides of the invention. Variants that are fragments of the polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention. Preferred embodiments are polynucleotides that encode for polypeptide variants wherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residue of a polypeptide sequence of the invention is substituted, added or deleted, in any combination. Particularly preferred are substitutions, additions and deletions that are silent so as not to alter the properties or activities of the polynucleotide or polypeptide. The nucleotide sequences coding for polyenoic fatty acid isomerases can be obtained from natural sources or can be partially or completely synthesized artificially. They may correspond directly to an endogenous polyeneoic fatty acid isomerase to a natural source or contain modified amino acid sequences, such as sequences that have been mutated, truncated, or ¿ItÁ. ?.? t. * ,. ?AHA . aadfeajj ^ similar. The polyeneoic fatty acid isomerases can be obtained through a variety of methods, including but not limited to partial or homogeneous purification of protein extracts, protein model formation, nucleic acid probes, antibody preparations and comparisons of sequences. Normally, a polyenoic fatty acid isomerase will be derived completely or in part from a natural source. A natural source includes but is not limited to prokaryotic and eukaryotic sources, which include bacteria, yeast, plants, including algae and the like. Of particular interest are polyeneoic fatty acid isomerases which are obtainable from algae sources, including those obtained from Ptilota, Bossiella, Lithotham, for example P.filicina, or polyeneoic fatty acid isomerases which are obtainable through the use of these sequences. "Obtainable" refers to those polyenoic fatty acid isomerases having sequences sufficiently similar to those of the sequences provided herein to provide a biologically active polyeneoic fatty acid isomerase. Further preferred embodiments of the invention are those at least about 50-79% identical over their entire length to a polynucleotide that encodes a polypeptide of the invention, and polynucleotides that are complementary to said polynucleotides. Preferred are polynucleotides comprising a region that is at least about 80% identical over the entire length to a polynucleotide that encodes a polypeptide of the invention and polynucleotides that are complementary to it. Particularly preferred are at least about 90% identical polynucleotides over their entire length, and those of at least about 95% identical are especially preferred. In addition, those with at least about 97% identity are highly preferred and those with at least about 98% and 99% identity, are particularly preferred, with those of at least about 99% being most preferred. Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same function or biological activity as the mature polypeptides encoded by the polynucleotides set forth in the sequence listing. The invention also relates to polynucleotides that hybridize the sequences described above. In particular, the invention relates to polynucleotides that hybridize under severe conditions to the polynucleotides described above. As used herein, the terms "severe conditions" and "severe hybridization conditions" mean that hybridization will generally occur if there is at least about 95% and preferably, at least about 97% identity between the sequences. An example of severe hybridization conditions is incubation overnight at 42 ° C in a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6) , 5x Denhardt solution, 10% dextran sulfate, and 20 The micrograms / milliliters of denatured salmon sperm DNA, fragmented, followed by washing the hybridization support in 0.1x SSC at approximately 65 ° C. Other hybridization and washing conditions are also known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989), particularly Chapter 11. The invention also provides a polynucleotide comprising essentially a polynucleotide sequence obtainable by selection of a suitable library containing the complete gene for a polynucleotide sequence exposed in the sequence listing under severe hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment of the same; and isolating said polynucleotide sequence. Fragments useful for obtaining said polynucleotide include, for example, probes and primers as described herein. As discussed herein with respect to polynucleotide analyzes of the invention, for example, polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA or genomic DNA to isolate full-length cDNA or genomic clones coding for a polypeptide and for isolating cDNA or genomic clones from other genes having high sequence similarity to a polynucleotide set forth in the sequence listing. Said probes will generally comprise at least 15 bases. Preferably, said probes will generally comprise at least 15 bases. Preferably, said probes will have at least 30 bases and can have at least 50 bases. Particularly preferred probes will have inclusive, between 30 bases and 50 bases. The coding region of each gene comprising or consisting of a polynucleotide sequence set forth in the sequence listing can be isolated by selection using a DNA sequence provided in the sequence listing to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to select a library of cDNA, genomic DNA or mRNA to identify elements of the library that hybridize the probe. For example, synthetic oligonucleotides corresponding to the N-terminal sequence of the PF1 peptide are prepared. The partial sequences thus prepared are then used as probes to obtain clones of PF1 from a library prepared from Ptilota phylline, a red seaweed. Alternatively, when low degeneracy oligonucleotides are prepared from particular PF1 peptides, such probes can be used directly to select libraries for PF1 gene sequences. In particular, the selection of cDNA libraries in phage vectors is useful in such methods due to lower levels of background hybridization. Normally, a PF1 sequence obtained from the use of nucleic acid probes will show approximately 60-70% sequence identity between the target PF1 sequence and the coding sequence eleven. . ? . < 1f.?_. .A _ & -. * _ .. .F S such as a probe. However, extensive sequences can also be obtained with only about 50-60% sequence identity. The nucleic acid probes can be a large fragment of the nucleic acid sequence, or it can be a shorter oligonucleotide probe. When larger nucleic acid fragments (greater than about 100 bp) are used as probes, it can be selected at lower severities in order to obtain sequences from the target sample which has 20-50% deviation (i.e. -80% sequence homology) of the sequences used as a probe. The oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding a PF1 enzyme, but must have at least about 10, preferably at least about 15 and preferably at least about 20 nucleotides. A greater degree of sequence identity is advisable when using shorter regions as opposed to larger regions. In this way, it may be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes to detect and recover other related PF1 genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al., PNAS E.U.A (1989) 86: 1934-1938). jlfainfeá m, jt-teá áajtj, ^ The person skilled in the art will appreciate that in many cases, an isolated cDNA sequence will be incomplete, because the region encoding the polypeptide is truncated with respect to the 5 'terminus of the cDNA. This is a consequence of the reverse transcriptase, an enzyme with low "processing capacity" (a measure of the ability of the enzyme to remain bound to the template during the polymerization reaction) employed during the synthesis of the first strand of cDNA. There are different methods available and which are known to the person skilled in the art to obtain full-length cDNA, or to extend short cDNAs, for example, those based on the Rapid Amplification of End of cDNA (RACE) method (see for example, Frohman et al. (1988) Proc. Nati, Acad. Sci. USA 85: 8998-9002). Recent modifications of the technique, exemplified by the Marathoná technology (Clonetech Laboratories, Inc.) for example, have been significantly simplified by obtaining full-length cDNA sequences. The polynucleotides and polypeptides of the invention can be used, for example, in the transformation of different host cells, as discussed hereinafter. The invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl terminal amino acids, or amino acids within the mature polypeptide (eg, when the mature form of the protein has more than one polypeptide chain). Such sequences, for example, can play a role in the processing of a protein from a precursor to a mature form, allow protein transport, shorten or lengthen the protein half-life, or facilitate manipulation of the protein in analysis or production. . It is contemplated that cellular enzymes can be used to remove any additional amino acid from the mature protein. A precursor protein, having the mature form of the polypeptide fused to one or more prosequences, may be an inactive form of the polypeptide. Inactive precursors are usually activated when prosequences are removed. Some or all of the prosequences can be removed before activation. Said precursor protein is generally called proprotein. The polynucleotide and polypeptide sequences can also be used to identify additional sequences which are homologous to the sequences of the present invention. The most preferred and convenient method is to store the sequence in a computer-readable medium, for example, floppy disk, CD ROM, hard disk drives, external disk drives and DVDs, and then use the stored sequence to search a sequence database with known search tools. Examples of public databases include the DNA Database of Japan (DDBJ) (http: //www.ddbj.nig.ac.jp/); Genebank (http://www.ncbi.nlm.nih.gov/web/Genbank/lndex, htlm); and the Nucleic Acid Sequence Database of the European Molecular Biology Laboratory (EMBL) (http://www.ebi.ac.uk/ebi docs / embl db.html). A number of different search algorithms are available to the person skilled in the art, an example of which is the set of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence investigations (BLASTN, BLASTX and TBLASTX) and two designed for protein sequence investigations (BLASTP and TBLASTIN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994)).; Birren, et al., Genome Analysis, 1: 543-559 (1997)). Additional programs are available in the art for the analysis of identified sequences, such as sequence alignment programs, programs for the identification of related sequences more distantly, and the like, and are known to the person skilled in the art.

Plant constructions and methods of use It is of particular interest to use the nucleotide sequences, or polynucleotides, in recombinant DNA constructs, to direct the transcription and / or expression of the PF1 sequences of the present invention in a host plant cell. Expression constructs generally comprise a functional promoter in a plant cell operably linked to a nucleic acid sequence encoding a polyenoic fatty acid isomerase of the present invention and a functional transcription termination region in a plant cell. Those skilled in the art will recognize that there are a number of promoters that are functional in plant cells that have been described in the literature. In addition, specific promoters of organelles and plastids are also contemplated such as functional promoters of chloroplast or plastid and promoters operable by chloroplast or plastid. A set of promoters are constitutive promoters such as the CaMV35S or FMV35S promoters that produce high levels of expression in most plant organs. Enhanced or duplicated versions of the CaMV35S or FMV35S promoters are useful in the practice of this invention (Odell, et al (1985) Nature 313: 810-812; Roger, U.S. Patent No. 5,378,619). In addition, it may also be preferred to cause expression of the PF1 gene in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc., and the selected promoter must have the desired tissue and development specificity. Of particular interest is the expression of the nucleic acid sequences of the present invention from regions of transcription initiation, which are preferably expressed in a plant-seed tissue. Examples of said preferential seed transcription initiation sequences include those sequences derived from sequences encoding plant storage protein genes or genes involved in fatty acid biosynthesis in oilseeds. Examples of such promoters include the 5 'regulatory regions of said genes such as napin (Kridl et al., Seed Sci. Res. 1: 209: 219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP. , estearoil-ACP * * * ^ ^ ^ ^? ^ ^ - - * & _ -, * ^ ___ kám *? ^ Á desaturase, subunit a 'of b-conglycinin soybean (soybeans). , (Chen et al., Proc. Nati, Acad. Sci. 83: 8560-8564 (1986))) and oleosin. It would be convenient to direct the localization of proteins that confer PF1 to a particular subcellular compartment, for example, to the mitochondrion, endoplasmic reticulum, vacuoles, chloroplast or other plastid compartment. For example, when the genes of interest of the present invention are oriented to plastids, such as chloroplasts for expression, the constructs will also employ the use of sequences to direct the gene towards the plastid. Said sequences are referred to herein as chloroplast transit peptides (CTP) or plastid transit peptides (PTP). In this way, when the gene of interest is not directly inserted into the plastid, the expression construct will additionally contain a gene encoding a transit peptide to direct the gene of interest towards the plastid. Chloroplast transit peptides can be derived from the gene of interest, or they can be derived from a heterologous sequence having a CTP. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Che ,. 264: 17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer ef al. (1993) Biochem. Biophys. Res Commun. 196: 1414-1421; and, Shah et al. (1986) Science 233: 478-481. Additional transit peptides for the translocation of the PF1 protein to the endoplasmic reticulum (ER), or vacuole may also find use in the constructions of the present invention.

Depending on the intended use, the constructs may contain the nucleic acid sequence encoding the entire PF1 protein or a portion thereof. For example, when antisense inhibition of a given PF1 protein is desired, the entire PF1 sequence is not necessary. In addition, when the PF1 sequences used in constructions are intended for use as probes, it may be convenient to prepare constructs containing only a particular portion of a PF1 coding sequence, for example, a sequence that is found to code for a PF1 region. highly conserved The person skilled in the art will recognize that there are different methods for the inhibition of expression of endogenous sequences in a host cell. Such methods include but are not limited to antisense suppression (Smith, et al (1988) Nature 334: 724-726), cosuppression (Napoli, et al. (1989) Plant Cell 2: 279-289), ribozymes (publication of PCT WO 97/10328), and sense and antisense combinations Waterhouse, ef al. (1998) Proc. Nati Acad. Sci. USA 95: 13959-13964. Methods for the deletion of endogenous sequences in a host cell, usually employ the transcription or transcription and translation of at least a portion of the sequence to be deleted. Said sequences may be homologous to coding regions, as well as non-coding for the endogenous sequence. The regulatory regions of transcription termination may also be provided in plant expression constructs of this invention. The transcription termination regions can be Í ~ * k.? Á., .. *, *, .. £.-__-_.- .. provided by the DNA sequence coding for the polyenoic fatty acid isomerase or a convenient transcription termination region derived from a different gene source, eg, the transcription termination region which is naturally associated with the region of initiation of transcription. The person skilled in the art will recognize that any convenient transcription termination region that is capable of terminating transcription in a plant cell can be employed in the constructions of the present invention. Alternatively, constructs can be prepared to drive expression of the PF1 sequences directly from the host plant cell plastid. Such constructions and methods are known in the art and are described generally, for example in Svab, ef al. (1990) Proc. Nati Acad. Sci. USA 87: 8526-8530 and Svab and Maliga (1993) Proc. Nati Acad. Sci. USA 90: 913-917 and in the patent of E.U.A. No. 5,693,507. A plant cell, tissue, organ or plant in which recombinant DNA constructs containing the expression constructs have been introduced is considered transformed, transfected or transgenic. A transgenic or transformed plant or cell also includes progeny of the cell or plant and progeny produced from a cross program that employs said transgenic plant as a parent in a cross and which has an altered phenotype resulting from the presence of a sequence of nucleic acid of PFl.

Transcription constructs or plant expression having a plant PFl as the DNA sequence of interest for increased or decreased expression thereof, can be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Especially preferred are hardened oilseed crops. Plants of interest include, but are not limited to, rapeseed (Cañóla and tall erucic acid varieties), sunflower, safflower, cotton, soybean, peanut, palm and coconut oil, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention is equally applicable to dicotyledonous and monocotyledonous species and will be readily applicable to new and / or improved transformation and regulation techniques. Of particular interest, is the use of vegetable PFl constructs in plants which have been genetically engineered to produce a particular fatty acid in the vegetable seed oil, where TAG in the seeds of unmanipulated plants of the manipulated species, do not naturally contain that acid particular fat. In this way, the expression of novel PFl in plants may be advisable for the incorporation of unique fatty acyl groups in the sn-3 position. Additional plant genetic manipulation applications for PF1 proteins of this invention include their use in preparing structured plant lipids containing TAG molecules having desirable fatty acyl groups incorporated at particular positions in the TAG molecules. It is contemplated that gene sequences can be synthesized, either completely or in part, especially when it is advisable to provide preferred plant sequences. Thus, all or a portion of the desired structural gene (that portion of the gene encoding the PF1 protein) can be synthesized using codons preferred by a selected host. The preferred host codons can be determined, for example, from the codons used most frequently in the proteins expressed in a desired host species. One skilled in the art will readily recognize that preparations of antibodies, nucleic acid probes (DNA and RNA) and the like can be prepared and used to select and recover "homologous" or "related" PF1 from a variety of plant sources. Homologous sequences are found when there is a sequence identity, which can be determined by comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions between a known PF1 and a candidate source. Conservative changes, such as Glu / Asp, Val / lie, Ser / Thr, Arg / Lys and Gln / Asn can also be considered in the determination of sequence homology. The amino acid sequences are considered homologous with only 25% sequence identity between the two complete mature proteins. (See in a way l »tí? ' .-. i.kÁi i '1 «* ari i. , ._,. , general, Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986). Thus, other PFl can be obtained from the specific exemplified PFl sequences exemplified herein. Furthermore, it will be evident that natural and synthetic PF1 can be obtained, including modified amino acid sequences and starting materials for synthetic protein model formation from the exemplified PF1 and PF1 which are obtained through the use of said sequences exemplified Modified amino acid sequences include sequences that have been mutated, truncated, increased and the like, whether said sequences are partially or completely synthesized. The sequences that are actually purified from plant preparations or that are identical or code for identical proteins thereof, without considering the method used to obtain the protein or sequence, are also considered as naturally derived. For immunological analysis, antibodies to the PF1 protein can be prepared by injecting rabbits or mice with the purified protein or portion thereof, said methods of antibody preparation being known to those skilled in the art. Monoclonal or polyclonal anticues can be produced, although polyclonal anticuefos are usually more useful for gene isolation. Western analysis can be performed to determine that a related protein is present in a crude extract of the desired plant species, as determined by the * i _á¡máu mH ^^ * faith & .-, fei ._. -... - .. - *, __. .- > . . ,. ^ __ > * > . n? * Í \ \ cross-reaction with anti-convolutions for P filicin PFl protein. When cross-reactivity is observed, the genes that code for the related proteins are isolated by selection of expression libraries that represent the desired plant species. Expression libraries can be constructed in a variety of commercially available vectors, including Lambda gt11, as described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, second edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The nucleic acid sequences associated with plant PF1 proteins can be used for a variety of uses. For example, recombinant constructs can be prepared which can be used as probes, or which will direct expression of the PF1 protein in host cells to produce a rapid source of the enzyme and / or to modify the triglyceride composition found therein. . Other useful applications can be found when the host cell is a plant host cell, either in vitro or in vivo. For example, by expressing a PF1 protein in a host plant cell, different conjugated fatty acids can be produced in a given plant tissue. Similarly, for some applications it may be desirable to decrease the amount of PF1 endogenously expressed in a plant cell through different gene suppression technologies discussed supra. It is appreciated that expression constructs containing the polynucleotide sequences of the present invention find use with additional expression constructs having sequences responsible for the alteration of fatty acids in a host cell. Examples of such sequences include, but are not limited to, thioesterases, desaturases, elongases, KASas, and the like. The modification of fatty acid compositions can also affect the fluidity of plant membranes. Different concentrations of lipids have been observed in cold-hardened plants, for example. Through this invention, features can be introduced which will provide cooling tolerance. Regulatory regulatory regions of constitutive or inducible transcription initiation by temperature may have special applications for such uses. As discussed above, the nucleic acid sequence encoding a plant PFl of this invention may include genomic sequence, cDNA or mRNA. By "coding" means that the sequence corresponds to a particular amino acid sequence either in a sense or antisense orientation. By "extrachromosomal" it is meant that the sequence is outside the plant genome of which it is naturally associated. By "recombinant" is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes and the like. Once the desired plant PFl nucleic acid sequence is obtained, it can be manipulated in a variety of ways. When the sequence involves non-coding flanking regions, the t-faj *. * ^ ». flanking regions can be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions and insertions can be made in the natural sequence. In addition, all or part of the sequence can be synthesized. In the structural gene, one or more codons may be modified to provide a modified amino acid sequence, or one or more codon mutations may be introduced to provide a convenient restriction site or other purpose involved with construction or expression. The structural gene can be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites or the like. The nucleic acid or amino acid sequences encoding a plant PFl of this invention may be combined with other non-native or heterologous sequences in a variety of ways. By heterologous sequences, any sequence that is not naturally bound to the plant PFl is referred to, including, for example, combinations of nucleic acid sequences from the same plant, which are not naturally bound. The DNA sequence encoding a plant PFl of this invention can be used together with all or part of the gene sequences normally associated with the PF1. In its components, a DNA sequence encoding PF1 is combined into a DNA construct having, in the 5 'to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation l * j > i.a-? .. M3.I > < fc & a »in a host cell, the DNA sequence coding for plant PFl and a region determining transcription and translation. Potential host cells include prokaryotic and eukaryotic cells. A host cell can be unicellular or it can be found in a differentiated or undifferentiated multicellular organism depending on the intended use. The cells of this invention can be distinguished by having a plant PFl external to the wild type cell present therein, for example, by having a recombinant nucleic acid construct encoding a plant PFl therein. The methods used for the transformation of the host plant cell are not critical to the present invention. The transformation of the plant preferably is permanent, that is, by integration of expression constructs induced in the genome of the host plant, so that the induced constructions pass to successive generations of the plants. A person skilled in the art will recognize that there is a great variety of transformation techniques in the art, and more and more new techniques are available. Any technique that is suitable for the target host plant may be employed within the scope of the present invention. For example, constructs can be introduced in a variety of ways including, but not limited to, a DNA standard, a plasmid or an artificial chromosome. The introduction of the constructs into the target plant cells can be achieved by a variety of techniques, including but not being . j ".A i ÍA __: A. * .A. to . v Jl. limited to, co-precipitation of calcium-phosphate-DNA, electroporation, microinjection, infection by Agrobacterium, transformation of liposomes or microprojectiles. The person skilled in the art can refer to the literature for details, and select suitable techniques for use in the methods of the present invention. Normally, there will be a structural gene included with the DNA construct, which has the regulatory regions necessary for expression in a host and aids in the selection of transforming cells. The gene can help in the resistance to a cytotoxic agent, for example, antibiotic, heavy metal, toxin, etc., complementation provides prototrophy to an auxotrophic host, viral immunity or similar. Depending on the number of different host species, the expression construct or its components are introduced, one or more markers can be used, where different conditions are used for the selection, for the different hosts. When the Agrobacterium is used for a plant cell transformation, a vector that can be introduced into the Agrobacterium host for the T-DNA homologous recombination or with the Ti- or Ri- plasmid plasmid is present in the Agrobacterium host. The Ti- or Ri plasmid that contains the T-DNA for recombination, can be assembled (capable of causing the formation of roughness) or disarmed (unable to cause the formation of roughness), the latter being permissible, as long as the genes vir are present in the transformed Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and roughness. In some examples where Agrobacterium is used as the vehicle to transform host plant cells, the expression or transcription construct bordered by the border region (s) of T-DNA will be inserted into a large scale vector host capable of replicating in E. Coli and in Agrobacterium, the wide-scale host vectors are described in the literature. The one that is commonly used is pRK2 or derivatives thereof. For examples refer to Ditta, ef al., (Roe Nat. Acad. Sci., U.S.A. (1980) 77: 7347-7351) and to the document EPA 0 120 515, which are incorporated herein by way of reference. Alternatively, the sequences to be expressed in plant cells can be inserted into a vector containing separate replication sequences, one of which stabilizes the vector in E. Coli, and the other in Agrobacterium. See, for example, McBride and Summerfeit (Plant Mol. Biol (1990) 14: 269-276), where the origin of replication of pRiHRI is used (Jouanin, ef al.Mol.Genet.Genet. (1985) 201: 370-374) and provides an aggregate stability of the plant expression vectors in Agrobacterium host cells. Included with the expression construct and the T-DNA, there will be one or more markers, which allows the selection of the transformed Agrobacterium and the transformed plant cells. Several markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, aminoglycoside G418, hygromycin, or the like. The particular label used is not essential for this invention. The preferred marker will depend on the host in particular and the specific construction that is used for the transformation of said host. For the transformation of plant cells using Agrobacterium, the plants can be combined and incubated with the modified Agrobacterium for a sufficient time to make it possible for said plant cell to be transformed by the Agrobacterium. The bacteria are then killed, and the plant cells are cultured in an appropriate selective medium. Once a callus is formed, the formation of the rod can be stimulated by using the appropriate plant hormones according to known methods, and the rods are transferred to a root medium for the regeneration of the plants. Then the plants can be grown to produce seeds and the seeds can be used to establish repetitive generations and for the isolation of vegetable oils. There are many possible ways to obtain the plant cells of this invention that contain multiple expression constructs. Any means for producing a plant comprising a construct having a DNA sequence encoding the polyenoic fatty acid isomerase of the present invention, and at least one other construct having another DNA sequence encoding an enzyme, are encompassed by the present invention. For example, the expression construction can be used to transform a plant at the same time as the second construction, either by the inclusion of both expression constructs in a single transformation vector, or by using separate vectors, each of which expresses the desired genes. The second construction can be introduced in a plant that has already been transformed with the PFl expression construction, or alternatively, transformed plants, one that expresses the construction PFl and one that expresses the second construction, can be crossed to join the constructions on the same floor.

Other constructions and methods of use The invention also relates to vectors that include a polynucleotide or polynucleotides of the invention, host cells that have been genetically transformed with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can be employed to produce the protein using RNAs derived from the DNA constructs of the invention. For recombinant production, the host cells can be genetically modified to incorporate systems or expression portions thereof, or polynucleotides of the present invention. The introduction of a polynucleotide into a host cell can be effected by the methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989). Such methods include, but are not limited to, calcium phosphate transfection, dextran-mediated transfection of DEAE, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, ballistic loading by scraping and infection. Representative examples of suitable hosts include bacterial cells, such as streptococcal cells, staphylococci, enterococci, E. Coli, streptomyces, and Bacillus subtilis, fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes; and plant cells as described above. A variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, but are not limited to, chromosomal, episomal vectors, and vectors derived from viruses, eg, vectors of bacterial plasmids, bacteriophages, transpositors, yeast epidomes, insects, yeast chromosomal elements., viruses such as vaculovirus, turkey virus, such as SB40, vaccinia virus, adenovirus, bird pustular virus, seudorabies virus and retroviruses, and vectors derived from combinations of such viruses, such as those derived from plasmid and genetic elements. bacteriophages, such as cosmids and phagemids. The constructions of expression systems may contain control regions that regulate, as well as engender expression. Generally, any system or vector that is suitable for maintaining, propagating or expressing polynucleotides and / or expressing a polypeptide in a host can be used. The appropriate DNA sequence can be inserted into an expression chosen by any of a variety of routine and well known techniques, such as, for example, those described in Sambrook et al, Molecular Cloning, A Laboratory Manual, (supra). Appropriate secretion signals, either homologous or heterologous, can be incorporated into the expressed polypeptide to allow secretion of the protein in the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. The polypeptides of the present invention can be recovered and purified from recombinant cell cultures, by a variety of well-known methods, including, but not limited to, the precipitation of ammonium sulfate or ethanol, extraction of acids, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl appetite chromatography, and lecithin chromatography. It is more preferable to use high performance liquid chromatography (HPLC) for purification. Any of the well-known techniques for redoubling the proteins can be used to regenerate an active confirmation if the polypeptide is denatured during isolation and / or purification.

The oil combinations obtained from the host cells expressing the PF1 sequences of the present invention can be used in a variety of industrial, animal feeding and human nutrition applications. The oil that is produced by the methods of the present invention, which contains the conjugated fatty acids, has a variety of uses. Methods for the use of various conjugated fatty acids, for example conjugated linoleic acid (CLA), are known in the art, for example. A variety of methods for the use of CLA are described in the patents of E.U.A. 5,428,072, 5,430,066, 5,504,114, 5,554,646, 5,585,400, 5,674,901, 5,760,082,5,760,083, 5,770,247, 5,804,210, 5,814,663, 5827,885, 5,851, 572, 5,855,917. In this manner, the oil compositions of the present invention having an altered content of conjugated fatty acid, have a use in the preparation of foods, food products, processed foods, food ingredients, food additive compositions, or dietary supplements that contain oils and / or fats. Examples of such uses include, but are not limited to, margarines, butters, shortenings, cooking oils, frying oils, spreads, spreads, mayonnaises, and vitamin / mineral supplements. Additional examples include, but are not limited to, covers, dairy products such as cheese and processed cheese, processed meat and imitations of meat, pasta, cereals, sauces, desserts including frozen and freestanding desserts, dipping sauces. French fries, baked goods, dried pasta, cookies, snack bars, confections, chocolates, beverages, non-extracted seeds, and non-extracted seeds that have been cultivated, crushed, ground, rolled, extruded, laminated, degraded, dehydrated or processed. some other way, but still contain the oils, etc. Which are described here. The oil compositions of the present invention having altered and conjugated fatty acid also have a use in pharmaceutical compositions comprising an effective amount of the conjugated fatty acid composition, together with a pharmaceutically acceptable carrier, excipient or diluent. These pharmaceutical compositions can be found in the form of a solid or a liquid. The solids may be in the form of powder, granule, tablet, tablet, gel or extrudate; Liquids can be solutions or suspensions. Now that the invention has been described in a general manner, it will be easier to understand by referring to the following examples which are included solely for the purpose of illustration, and which are not intended to limit the present invention. * l -4 ^ «t.» < *. * & EXAMPLES EXAMPLE 1 Identification of a polyeneic fatty acid isomerase nucleic acid sequence A. Preparation of the complementary DNA library The total RNA of the red seaweed Ptilota filicin was isolated for use in the construction of complementary DNA libraries (cDNA). The fresh material was cultivated in liquid nitrogen with a mortar / grinder. Approximately 5 g of P. fílicina powder was mixed with 5 ml of an extraction buffer (1% SDS, 10 mM EDTA, 0.2 M NaAC, pH 4.8) and 3 ml of acid phenol (pH 4.3). The mixture was incubated at 60 ° C for 30 minutes, stirring it every 5 minutes. The mixture was cooled to room temperature and 3 ml of chloroform was added. After stirring at room temperature for 10 minutes, the mixture was centrifuged at 5000 x g for 30 minutes. The aqueous phase was extracted once with 6 ml of phenol / chloroform (1: 1 v / v) for 10 minutes and then centrifuged at 5000 x g for 5 minutes. The upper aqueous layer was recovered, extracted once with 6 ml of chloroform. After the last extraction, the RNA was precipitated from the aqueous layer with an equal volume of 4M LiCl on ice overnight, and centrifuged at 10000 x g for 30 minutes. The precipitated RNA was washed with 70% ethanol, dried in vacuo and dissolved in water.

The resulting total RNA was used to prepare cDNA libraries using the SuperScript plasmid system for the synthesis of cDNA and the plasmid cloning kit (BRL Life-Technologies, Gaithersburg, MD). To identify the candidate nucleic acid sequences, a pair of synthetic oligonucleotides was prepared (5'-GAYYYNGAYGAYACNATHGC-3", 5'-TGYTGNBWRTADATYTCNAC-3 ' (Y = CT, N = ATGC, H = ACT, B = GCT, W = AT, R = AG, D = AGT) (SEQ ID NO: 5 and 6)) corresponding to the 38 N-terminal amino acids y (DDFDDTIAWGAGYSGLSAAFTLVKKGYTNVEIYSQQY , SEQ ID NO: 7) (Wise (1995) Biosynthesis and enzymology of conjugated polyenoic fatty acid production in macrophytic marine algae, Ph.D. Thesis State University, Corvalis, OR, UMI Dissertation Services) for use in PCR reactions to amplify the probes for use in the hybridization screen of the cDNA library of P. filicina. The PCR amplification included an initial denaturation step of 95 ° C for 5 minutes followed by 5 cycles of 94 ° C (30 sec.) -45 ° C (30 sec.) -72 ° C (30 sec.) And 30 cycles (94 ° C (30 sec.) -52 ° C (30 sec.) -72 ° C (30 sec.). PCR products between 70 and 150 bp were gel purified and used as molds for a second PCR reaction After amplification, the PCR products were cloned into pCR2.1 using the TA cloning system from Invitrogen Co. Genomic library screens were run using standard colony staining protocols (Sambrook, et al. Molecular Cloning, A Laboratory Manual, (supra)). i > . * k *. afc-á m ljsfi & * .a »» - i- > _ * & .a * - * - v - s * * * -. ^ "* ** -» .- »«. * _ «& ,, - ^ - * ^ - $ ¿, s«, a ^ * fe Two cDNA sequences were identified that had been hybridized in the oligonucleotide probe . These two DNA sequences, referred to as PFI-B2 (SEQ ID NO: 1) and PFI-F3 (SEQ ID NO: 3), contained the pSPORTI cloning vector (BRL Life-Technologies, Gaithersburg, MD ), pCGN10100 and pCGN10101, respectively. The deduced amino acid sequence for PFI-B2 (SEQ ID NO: 2) and PFI-F3 (SEQ ID NO: 4) were also determined.

EXAMPLE 2 Preparation of the construction 2A. Bacterial expression constructions A series of constructs were prepared to express the PF1 sequences in host cells. For expression in E. coli, constructs containing labels or those lacking such label sequences were prepared. The pCGN10102 vector was designed to express the protein encoded by the PFI-B2 sequence with the native leader peptide with a His-tag sequence with 6 C-terminal residues in the PQE-60 vector (Qiagen). The pCGN10103 vector is similar to pCGN10102, except that it contains the sequence encoding the PFI-F3 protein with its native leader peptide. A set of constructs were also prepared to express the PF1 sequences in E. coli without the leader sequences. The vector Má * »i iluAi * .. •» .teta -. pCGN10104 contains the sequence PFl-B2 of pCGN10100 without the leader peptide cloned in the vector pQE-60 with a His tag of 6 residues in the C-terminus of PF1. The vector pCGN10105 is similar to pCGN10104, except that it contains the sequence coding for pFI-F3 of pCGN10101. Finally, for expression in E. coli, a set of constructs were prepared which lacked His tag of 6 C-terminal residues. The pCGN10106 construct contains the coding sequence PFI-B2 of pCGN10100 which includes the native leader peptide in the pQE-60 vector. The vector pCGN10107 is similar to pCGN10106, except that it contains the coding sequence PFI-F3, including the native leader peptide. For the expression of PF1 in the cytoplasm of E. coli, the pQE60 system from Qiagen Inc. was used. The PF1 coding region with and without the putative leader peptide was amplified with PCR using the forward primers: 5'-CGCCATGGCTTTGAATAGAGTTCTTCAC- 3 'or 5'-CGCCATGGACGATTTTGATGACACGATTGC-3' (SEQ ID NO: 8 and 9), the reverse primer: 5'-CGAGATCTGAAGAAATCCTTGATCAAATTATCCG-3 '(SEQ ID NO: 10). The Ncol sites (underlined) were introduced in the forward primers while the BglU site (underlined) was introduced in the reverse primer. The PCR products were subcloned in pCR2.1 using the TA cloning system (Invitrogen Co.). Then the resulting product was sent for sequencing. After the sequence confirmation, the inserts were cut using the complete digestion of BglU and the partial digestion of? / cab followed by the gel purification using the i_ * i gel purification system from Qiagen Inc. Subcloning of the inserts into the pQE60 vector and the expression of the recombinant proteins were made as recommended by the manufacturer. The E. coli transformants were cultured at different temperatures at an OD600 of 0.7 to 0.8 before being induced by 1 mM 1-mo-propyl-β-D-thiogalactopyranoside for 1 to 5 hours. The induced cells were then harvested and subjected to lysis by sonication, followed by enzymatic tests. The plasmid expression of the PF1 was carried out by fusing the alkaline phosphotase leader peptide (PhoA) of E. coli PF1 without its native leader peptide. The coding region of the fusion protein was designed to be driven by the native promoter of the alkaline phosphothase gene (phoA) of E. coli. Three primers were designated: PPF, 5 'AAGCTTTGGAGATTATCGTC-3' (SEQ ID NO: 11), was derived from the sequence upstream of the phoA promoter; PPM, 5'-TCGTGTCATC AA TCATGGGCTTTTGTCACAGGGGTAA-3 '(SEQ ID NO: 12), contained the partial coding sequence of the PhoA leader peptide (underlined) and the partial coding region of PF1 without the leader peptide; and EPR, 5'-GCAGGATCCGTATCGAGCTC T GATT CG-3 '(SEQ ID NO: 13), was derived from the current stream below the PF1 coding region of the cDNA clone. To make the fusion construct, two PCR reactions were carried out. The first PCR reaction was done using PPF primers paired with PPM and E. coli K-12 genomic DNA, as a template. The second PCR reaction was carried out using primers PPF and EPR.

Molds for the second PCR reaction were generated by mixing 1 ml of the first PCR product, and 1 ml of plasmid DNA diluted 1: 50 of the pFl cDNA clone. The final PCR product was cloned in pCR2.1 using the Topo-TA cloning system (Invitrogen Co.) and the insert was verified by sequencing both sepals. The E. coli transformants were cultured in an ECLB medium at 37 ° C to an early stationary phase and subsequently harvested for protein analysis by centrifugation. Plant Expression Constructions A series of constructs were prepared for the expression of the PF1 coding sequences in host plant cells. The constructs are prepared to direct the expression of the PF1 coding sequences constitutively, as well as preferentially, in particular, plant tissues. A plasmid containing the napin cassette derived from pCGN3223 (described in USPN5, 639,790, which is incorporated herein by reference in its entirety) was modified to make it more useful for cloning large fragments of DNA containing multiple restriction sites , and to allow the cloning of multiple napin fusion genes into binary transformation plant vectors. An adapter comprised the autoagnelated oligonucleotide of the sequence CGCGATTTAAATGGCGCGCCCTGACAGGCCGCCTGCAGGGCGCGCCATT TAAAT (SEQ ID NO: 14), was ligated into the cloning vector pBCSK + (estratagen) after digestion with restriction endonuclease BssHIl to the construction vector pCGN7765. Plasmids pCGN3223 and pCGN7765 were digested with Notl and ligated together. The resulting vector, pCGN7770, contains the main structure with the napin specific expression cassette of pCGN3223. The cloning cassette, pCGN7787, had essentially the same regulatory elements as pCGN7770, with the exception of the napin regulatory regions of pCGN7770 that have been replaced with the CAMV 35S double promoter and the tml polyadenylation region and transcriptional determination. A binary vector for plant transformation, pCGN5139, was constructed from pCGN1558 (McBride and Summmerfeit, (1990) Plant Molecular Biology, 14: 269-276). The multiple licker of pCGN1558 was replaced as a Hindlll / Asp718 fragment with a multiple linker containing unique restriction endonuclease sites, Ascl, Pací, Xbal, SwaI, BamHl, and Notl. The restriction endonuclease sites Asp 718 and Hindlll are retained in pCGN5139. A series of binary turbo vectors were constructed to allow rapid cloning of DNA sequences into binary vectors containing transcriptional initiation regions (promoters) and transcriptional termination regions. The plasmid pCGN8618 was constructed by ligating the oligonucleotides 5'-TCGAGGATCCGACGGCCGCAAGCTTCCTGACAGG-3 '(SEQ ID NO: 15) and 5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO: 16) in the dually digested pCGN7770 Sall / Xhol. A fragment containing the napin promoter, the multiple linker and the 3 'napin region was separated from pCGN8618 by digestion with Asp7181; To the fragment the ends were flattened by filling it in the 5 'overhangs with the Klenow fragment, then it was ligated into pCGN5139 which had been digested with Asp7181 and Hindlll and the ends were flattened by filling it in the 5' overhangs with the Klenow fragment. A plasmid containing the insert oriented in such a way that the napin promoter was closer to the flattened Asp7181 site of pCGN5139 and the 3 'napin was closer to the flattened Hindlll site. Subsequently, these regions were subjected to sequence analysis to confirm the orientation of the insert and the integrity of the cloning junctions. The resulting plasmid was designated pCGN8622. The plasmid pCGN8619 was constructed by ligating the oligonucleotides 5 'TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO: 17) and 5 'and 5' TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3 '(SEQ ID NO: 18) in the dually digested pCGN7770 with Sall / Xhol a fragment which contained the napin promoter, the multiple linker and the 3 'napin region was removed from pCGN8619 by digestion, with Asp7181; the fragment was flattened by filling it into the 5 'overhangs with the Klenow fragment, then it was ligated into pCGN5139 which had been digested with Asp7181 and Hindlll, the ends were flattened by filling it in the 5' overhang with the Klenow fragment. A plasmid containing the insert oriented for the promoter ? 3. . ^ __. »- - the napin was closer to the flattened Asp7181 site of pCGN5139 and the 3 'napin was closer to the flattened Hindlll site, subjected to sequence analysis to confirm the orientation of the insert and the integrity of the joints of cloning. The resulting plasmid was designated 5 pCGN8623. The plasmid pCGN8620 was constructed by ligating the oligonucleotides 5'-TCGAGGATCCGCGGCCGCAAGCTTCCTCCTGCAGGAGCT-3 '(SEQ ID NO: 19) and 5'- CCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO: 20) 10 in pCGN7787 doubly digested with Sal I / Sacl. A fragment containing the d35S promoter, the multiple linker and the 3 'tml region was removed from pCGN8620 by complete digestion with Asp7181 and partial digestion with Notl. To the fragment the ends were flattened filling it in the 5 'overhangs with the Klenow fragment, then it was ligated in pCGN5139 which 15 had been digested with Asp718l and Hindlll, and the ends were flattened by filling it into the 5 'overhangs with the Klenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closer to the flattened Asp7181 site of pCGN5139 and the 3 'end tml was closer to the flattened Hindlll site, was subjected to sequence analysis for 20 confirm the orientation of the insert and the integrity of the cloning junctions. The resulting plasmid was designated pCGN8624. The plasmid pCGN8621 was constructed by ligating the oligonucleotides 5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT- 3 '(SEQ ID NO: 21) and 5'- GGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO: 22) in pCGN7787 doubly digested with Sal I / Sacl. A fragment containing the d35S promoter, the multiple linker and the 3 'tml region was removed from pCGN8621 by complete digestion with Asp7181 and partial digestion with Notl. The fragment was flattened by filling the ends in the 5 'overhangs with the Klenow fragment then ligated into pCGN5139, which had been digested with Asp718l and Hindlll and the ends were flattened by filling it in the 5' overhangs with the Klenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closer to the flattened Asp718l site of pCGN5139 and the 3 'tml terminus was closer to the flattened Hindlll site, was subjected to sequence analysis to confirm the orientation of the insert and the integrity of cloning unions. The resulting plasmid was designated pCGN8625. To clone in plant expression constructions, regions coding for PF1 with or without the putative leader peptide were amplified by PCR using forward primers, PLF1 5'-GGATCCGCGGCCGCATGTCTTTGAATAGAGTTCTTC-3 '(with the leader peptide) (SEQ ID NO: 23) and PLF2 5'- GGATCCGCGGCCGCATGGATTTTGATGACACGATTGC -3 '(without the leader peptide) (SEQ ID NO: 24), and the reverse primer PLR 5'-CCTGCAGGAAGCTTCTAGAAGA AATCCT TGATC-3' (SEQ ID NO: 25). The? / Ofl sites (underlined) were placed upstream of the initiator codons (in bold) on the primers PLF1 and PLF2, whereas the site v Psfl (underlined) was placed downstream of the stop codon (in bold) in PLR. The PCR products were first cloned into pCR2.1 (Invitrogen) and the presence of the inserts that possessed the correct sequence was verified by the sequencing of both strains. Two constructions that used the coding sequence PFI-B2 were prepared in the pCGN8622 vector for expression from the napin promoter. The vector pCGN10108 contains the PFI-B2 coding sequence containing the native leader sequence. The vector pCGN10109 contains the PFI-B2 coding sequence that does not have the native leader sequence. Two constructs employing the PFI-B2 coding sequence were prepared in the vector pCGN8624 for expression from the 35S promoter. The vector pCGN10110 contains the PFI-B2 coding sequence, which contains the native leader sequence. The vector pCGN10111 contains the coding sequence PFI-B2 which lacks the native leader sequence. Two constructions that use the coding sequence PFI-F3 were prepared in the pCGN8622 vector for expression from the napin promoter. The vector pCGN10112 contains the coding sequence PFI-F3 which contains the native leader sequence. The vector pCGN10113 contains the PFI-F3 coding sequence that does not have the native leader sequence. A single construct employing the PFI-F3 coding sequence was prepared in the vector pCGN8624 for expression from the promoter 1 * 1 J. _ &_ _b r *. . I will go._ _? 35S. The vector pCGN10114 contains the PFI-B2 coding sequence containing the native leader sequence.

EXAMPLE 3 Transformation and analysis of the host cell To express the PF1 protein in E. coli, constructions were made using the QIAexpressionist system (Qiagen). The transformation and induction of M15 cells were carried out according to the manufacturers' protocol. The preparation and transformation of a competent yeast cell was performed, using the Frozen-EZ yeast transformation kit (Zymo Research). The expression vector that was used was pYES2 (Invitrogen) and the selected yeast strain was INVd (Invitrogen). A variety of methods have been developed to insert a DNA sequence of interest into the genome of a host plant to obtain transcription and translation of the sequence to effect phenotypic changes. Transgenic Brassica plants obtained by transformation mediated by Agrobacterium were obtained, as described by Radke et al. (Theor. Appl. Genet. (1988) 75: 685-694; Plant Cell Reports (1992) 11: 499-505). Transgenic Arabidopsis thaliana plants can be obtained by transformation mediated by Agrobacterium as described by Valverkens et al. as; • * - -j al., (Proc. Nat. Acad. Sci. (1988) 85: 5536-5540), or as described by Bent et al. ((1994), Science 265: 1856-1860), or Bechtold et al. ((1993), C.R.Acad.Sci, Life Sciences 316: 1194-1199). Other plant species can be transformed in a similar way using related techniques. Alternatively, microprojectile bombardment methods, such as those described by et al. (Bio / Technology 10: 286-291) to obtain nuclearly transformed plants. The expressed PF1 protein, as well as the PF1 protein of P. filicina, wild-type, was evaluated as described here. Enzyme activity was evaluated as described by Wise ((1995) PhD Thesis, supra) with some modifications. Preparation of the crude extract of Ptilota filicina protein: frozen tissue was cultivated in a mortar / grinder with liquid nitrogen. Approximately 200 mg of tissue powder was mixed with 1 ml of extraction buffer (100 mM NaH2PO4, 5 mM EGTA, 5 mM DTT, and 5 mM MgCl2, pH6.5) and homogenized with a homogenizer. The homogenate was centrifuged in microfuge at 14 k rpm for 5 minutes and the supernatant was collected for enzymatic tests. Similar methods were used for the preparation of the crude protein extract from the transgenic material (E. coli, yeast, Schizochitrium, and Arabidopsis): the test materials were decomposed and homogenized in the extraction buffer and centrifuged in microfuge. The supernatant was used for the enzyme test.

The collected supernatant was analyzed for the PF1 activity in the following manner. Approximately 20 ml of crude protein extract was mixed with 300 ml of the reaction buffer (100 mM NaH2PO4, pH7.2 with 0.02% Tween 20) and the spectrophotometer (DU650, Beckman) was drained. The reaction was started by adding 2 ml of the stored substrate solution and the formation of conjugated fatty acid was measured by sling length analysis between 200 nm and 300 nm. Specifically, the formation of conjugated diene and triene was measured by absorbance monitoring at 278nm and 234nm, respectively. The stored substrate solution was prepared by adding free polyunsaturated fatty acids in 95% ethanol to a final concentration of 25 mg / ml. All publications and patent applications mentioned in this specification indicate the level of experience of those skilled in the art to which this invention is directed. All publications and patent applications will be listed here for reference, to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated as a reference. Although the above invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

.. ?? LIST OF SEQUENCES < 110 > Zheng, Wei Yuan, Ling Metz, James G. < 120 > SEQUENCES OF NUCLEIC ACIDS CODING POLYENOIC FATTY ACID ISOMERASE AND USES THEREOF < 130 > MTC6711 < 140 > < 141 > < 150 > 60 / 146,458 < 151 > 1999-07-30 < 160 > 27 < 170 > PatentIn Ver. 2.1 < 210 > 1 211 > 1507 < 212 > DNA < 213 > Ptilota filicina < 400 > 1 cgcaaaatgt ctttgaatag agttcttcac attttcctta tcgcatatct cgcatgcact 60 gccctaaccc atgattttga tgacacgatt gccgttgtgg gagctggcta ctctggactg 120 agcgctgctt ttactctcgt caagaaaggg tacaccaacg ttgagattta cgaatcccag 180 ggcgaagttg gggggtatgt ctactctgtt gactataaca acgtcgcgca tgacctggcc 240 acgtacgctc tgactcctgc atactggaaa ttccaggagg ccatgaaaag tatcggcgtt 300 gggttttgtg agctcgatgt tgcaattgtg caaacgaatt ctacgcctgt ctcagtcccg 360 ttcgagaaat ggatggccgc ctactgggct gcgaaagtcc caaacccact caacctcgtg 420 aggaaggtct cgactcaagt ttcgacgtac gttgaagttt ggaagaagct cttcaatatg 480 gacttcattg acacgagcac gaagcgcact aatcgcctct ttccgttgaa gaccaacgac 540 gtcgacgtcc ttgcccaatt ttcaatgccc atgaaagatt ttgttgcatt gcataagctg 600 gacttgctcg agcctctttt tatccaggca accgactccc aggcgtacgg tccgtatgac 660 cactctacta acgacaccgg catggtgtgg ttccctccga accttttcaa cggtgaggaa 720 aataccgttc catgtggtac gtataactcg atgcagtcca tggccgagca catggccgaa 780 gcaaaggagt tggttgaaga cacgttccac atgaatacga aggtgacgaa aatctctcgc 840 gccaccgatg gatctagtcc atccctcttg gaagaaggtg tagctacgcc gaagctcttc 900 tcagtacgaa gacaccataa caagctgccg tctgcgaacc gtgccgaagt tgtgacacct 960 aggagcggga ctgcttccga ggccgccgat acgtacgagg agctacaaat gttctctgct 1020 cttctcgaga cgaatcgcag cgatgccatt ccgacgacag gcttcttgat ggtggatgcg 1080 tagctcacga gacgcaatta ccctaacacc gggttttggg gttgtttgaa tgctgagcgt 1140 attcggatga cgcggaggct gaatgctatt ctaagctcgg atactgtgac gcgcgtcagc 1200 actatacaga gccatctact gcgtgcaaac aacgaacgca tcgacttttc tctcgacgag 1260 aagattcagc aggtgaagac caatcttgcg acgtgggact cggctacctg gaccaatcta 1320 acctcccgta cgttcggtgg atatttccag aggtggagga cgccggatgt tatgggtcaa 1380 atctggctga aagccgtgga cattcaagga gaaggagatg tgtactacgt caactcggct 1440 gcatgcgggt t cgagtccgt cggccacgtt ttcgattgcg cggataattt gatcaaggat 1500 tttttct 1507 < 210 > 2 < 211 > 500 < 212 > PRT < 213 > Ptilota filicina < 400 > 2 Met Ser Leu Asn Arg Val Leu His He Phe Leu He Ala Tyr Leu Ala 1 5 10 15- Cys Thr Ala Leu Thr His Asp Phe Asp Asp Thr He Ala Val Val Gly 20 25 30 Ala Gly Tyr Ser Gly Leu Ser Ala Ala Phe Thr Leu Val Lys Lys Gly 35 40 45 Tyr Thr Asn Val Glu He Tyr Glu Ser Gln Gly Glu Val Gly Gly Tyr 50 55 60 Íti. _.:A > i..J_.

Val Tyr Ser Val Asp Tyr Asn Asn Val Wing His Asp Leu Wing Thr Tyr 65 70 75 80 Wing Leu Thr Pro Wing Tyr Trp Lys Phe Gln Glu Wing Met Lys Ser He 85 90 95 Gly Val Gly Phe Cys Glu Leu Asp Val Ala He Val Gln Thr Asn Ser 100 105 110 Thr Pro Val Ser Val Pro Phe Glu Lys Trp Met Wing Ala Tyr Trp Wing 1 15 120 125 Wing Lys Val Pro Asn Pro Leu Asn Leu Val Axg Lys Val Ser Thr Gln 130 135 140 Val Ser Thr Tyr Val Glu Val Trp Lys Lys Leu Phe Asn Met Asp Phe 145 150 155 160 He Asp Thr Ser Thr Lys Arg Thr Asn Arg Leu Phe Pro Leu Lys Thr 165 170 175 Asn Asp Val Asp Val Leu Wing Gln Phe Ser Met Pro Met Lys Asp Phe 180 185 190 Val Ala Leu His Lys Leu Asp Leu Leu Glu Pro Leu Phe He Gln Wing 195 200 205 Thr Asp Ser Gln Wing Tyr Gly Pro Tyr Asp Thr Thr Pro Wing Leu Tyr 210 215 220 Tyr Met Val Trp Phe Pro Pro Asn Leu Phe Asn Gly Glu Glu Asn Thr 225 230 235 240 Val Pro Cys Gly Thr Tyr Asn Ser Met Gln Ser Met Ala Glu His Met 245 250 255 i i??:: t A. -H. ** Í *? t t w - ^^^ »t a '£ - - ^ * > -neR-aa sjffrá »é I & j Ala Glu Trp Leu Lys Ser Lys Gly Val Thr Phe His Met Asn Thr Lys 260 265 270 Val Thr Lys He Ser Arg Ala Thr Asp Gly Ser Ser Pro Ser Leu Leu 275 280 285 Glu Glu Gly Val Wing Thr Pro Lys Leu Phe Asp Thr He He Ser Thr 290 295 300 Asn Lys Leu Pro Be Ala Asn Arg Ala Glu Val Val Thr Pro Leu Leu 305 310 315 320 Pro Lys Glu Arg Glu Wing Wing Asp Thr Tyr Glu Glu Leu Gln Met Phe 325 330 335 10 Be Ala Leu Leu Glu Thr Asn Arg Be Asp Ala He Pro Thr Thr Gly 340 345 350 Phe Leu Met Val Asp Wing Asp Wing He He Wing His Asp Pro Asn Thr 355 360 365 Gly Phe Tf Gly Cys Leu Asn Wing Glu Arg Arg Gly Gly Tyr Ser Asp 15 370 375 380 Glu Asn Ala He Leu Ser Ser Asp Thr Val Thr Arg Val Ser Ala He 385 390 395 400 Tyr Tyr Tyr Thr Glu Arg Wing Asn Asn Glu Arg He Asp Phe Ser Leu 405 410 415? 0 Asp Glu Lys He Gln Gln Val Lys Thr Asn Leu Wing Thr Tf Asp Ser 420 425 430 Wing Thr Tf Thr Asn Leu Thr Ser Arg T r Phe Gly Gly Tyr Phe Gln 435 440 445 á riau *? ?? > _ &-t_b.t í, Arg Tf Arg Thr Pro Asp Val Met Gly Gln Lys Pro Tf Asn Leu Ala 450 455 460 Asp He Gln Gly Glu Gly Asp Val Tyr Tyr Val A = n Ser Ala Ala Cys 465 470 475 480 Gly Phe Glu Ser Val Gly His Val Phe Asp Cys Wing Asp Asn Leu He 485 490 495 Lys Asp Phe Phe 500 < 210 > 3 10 < 211 > 1539 < 212 > DNA < 213 > Ptilota filicina < 400 > 3 atgtctttga atagagttct tcacattttc cttatcgcat atctcgcatg cactgcccta 60 acccatgatt ttgatgacac gattgccgtt gtgggagctg gctactctgg actgagcgct 120 gcttttactc tcgtcaagaa agggtacacc aacgttgaga tttacgaatc ccagggcgaa 180 15 gttggggggt atgtctactc tgttgactat aacaacgtcg cgcatgacct ggccacgtac 240 gctctgactc ctgcatactg gaaattccag gaggccatga aaagtatcgg cgttgggttt 300 tgtgagctcg atgttgcaat tgtgcaaacg aattctacgc ctgtctcagt cccgttcgag 360 aaatggatgg ccgcctactg ggctgcgaaa gtcccaaacc cactcaacct cgtgaggaag 420 gtctcgactc aagtttcgac gtácgttgaa gtttggaaga agctcttcaa tatggacttc 480 attgacacga gcacgaagcg cactaatcgc ctctttccgt tgaagaccaa cgacgtcgac 540 gtccttgccc aattttcaat gcccatgaaa gattttgttg cattgcataa gctggacttg 600 tttttatcca ctcgagcctc tcccaggcgt ggcaaccgac tgacacgaca acggtccgta 660 20 ccggcactct actacatggt gtggttccct ccgaaccttt tcaacggtga ggaaaatacc 720 gttccatgtg gtacgtataa ctcgatgcag tccatggccg agcacatggc cgaatggttg 780 aagagcaaag gagtcacgtt ccacatgaat acgaaggtga cgaaaatctc tcgcgccacc 84 0 gatggatcta gtccatccct cttggaagaa ggtgtagcta cgccgaagct cttcgacacc 900 ataatcagta cgaacaagct gccgtctgcg aaccgtgccg aagttgtgac acctctgctt 960 ccgaaggagc gggaggccac caatacgtac gaggagctac aaatgttctc tgctcttctc 1020 gagacgaatc gcagcgatgc cattccgacg acaggcttct tgcggacgca tgatggtgga 1080 ¡____ ¿? _li ___ ^^ ... ...., -. ^. ._, a ^, ^, ^ .-. ^ ^ - ^^. -. ^^ acgabcctga attatagctc caccgggrtt tggggttgtt gcgtcgcgga tgaatgctga 1,140 ggctattcgg atgagaatgc tattctaagc tcggatactg tgacgcgcgt cagcgccatc 1200 tactactata cagagcgtgc aaacaacgaa cgcatcgact tttctctcga cgagaagatt 1260 cagcaggtga agaccaatct tgcgacgtgg gactcggcta cctggaccaa tctaacttcc 1320 cgtacgttcg gtggatattt ccagaggtgg aggacgccgg atgttatggg tcaaaagccg 1380 ctgacattca tggaatctgg aggagaagga gatgtgtact acgtcaacgc ggctgcatgc 1440 ccgtcggcca gggttcgagt cgttttcgat tgcgcggata arttgatcaa ggatttcttc 1500 tagataaaca caacagaagt agactgccgc caaagtctg 1539 < 210 > 4 < 211 > 500 < 212 > PRT < 213 > Ptilota phylline < 400 > 4 Met Ser Leu Asn Arg Val Leu His He Phe Leu He Wing Tyr Leu Wing 1 5 10 15 Cys Thr Ala Leu Thr His Asp Phe Asp Asp Thr He Ala Val Val Gly 20 25 30 Ala Gly Tyr Ser Gly Leu Ser Ala Ala Phe Thr Leu Val Lys Lys Gly 35 40 45 Tyr Thr Asn Val Glu He Tyr Glu Ser Gln Gly Glu Val Gly Gly Tyr 50 55 60 Val Tyr Ser Val Asp Tyr Asn Asn Val Wing His Asp Leu Wing Thr Tyr 65 70 75 80 Ala Leu Thr Pro Ala Tyr Tf Lys Phe Gln Glu Ala Met Lys Ser He 85 90 95 Gly Val Gly Phe Cys Glu Leu Asp Val Ala He Val Gln Thr Asn Ser 100 105 110 ^ - ~ * * ü Thr Pro Val Ser Val Pro Phe Glu Lys Tf Met Wing Wing Tyr Tf Wing 115 120 125 Wing Lys Val Pro Asn Pro Leu Asn Leu Val Arg Lys Val Ser Thr Gln 130 135 140 Val Ser Thr Tyr Val Glu Val Tf Lys Lys Leu Phe Asn Met Asp Phe 145 150 155 160 He Asp Thr Ser Thr Lys Arg Thr Asn Arg Leu Phe Pro Leu Lys Thr 165 170 175 Asn Asp Val Asp Val Leu Wing Gln Phe Ser Met Pro Met Lys Asp Phe 180 185 190 Val Ala Leu His Lys Leu Asp Leu Leu Glu Pro Leu Phe He Gln Wing 195 200 205 Thr Asp Ser Gln Wing Tyr Gly Pro Tyr Asp Thr Thr Pro Wing Leu Tyr 210 215 220 Tyr Met Val Tf Phe Pro Pro Asn Leu Phe Asn Gly Glu Glu Asn Thr 225 230 235 240 Val Pro Cys Gly Thr Tyr Asn Ser Met Gln Ser Met Ala Glu His Met 245 250 255 Wing Glu Tf Leu Lys Ser Lys Gly Val Thr Phe His Met Asn Thr Lys 260 265 270 Val Thr Lys He Ser Arg Ala Thr Asp Gly Ser Ser Pro Ser Leu Leu 275 280 285 Glu Glu Gly Val Wing Thr Pro Lys Leu Phe Asp Thr He He Ser Thr 290 295 300 , - ^ s _U Asn Lys Leu Pro Be Ala Asn Arg Ala Glu Val Val Thr Pro Leu Leu 305 310 315 320 Pro Lys Glu Arg Glu Wing Thr Asn Thr Tyr Glu Glu Leu Gln Met Phe 325 330 335 Be Ala Leu Leu Glu Thr Asn Arg Be Asp Ala He Pro Thr Thr Gly 340 345 350 Phe Leu Met Val Asp Wing Asp Wing He He Wing His Asp Pro Asp Thr 355 360 365 Gly Phe Tf Gly Cys Leu Asn Wing Glu Arg Arg Gly Gly Tyr Ser Asp 370 375 380 Glu Asn Ala He Leu Ser Ser Asp Thr Val Thr Arg Val Ser Ala He 385 390 395 400 Tyr Tyr Tyr Thr Glu Arg Wing Asn Asn Glu Arg He Asp Phe Ser Leu 405 410 415 Asp Glu Lys He Gln Gln Val Lys Thr Asn Leu Wing Thr Tf Asp Ser 420 425 430 Wing Thr Tf Thr Asn Leu Thr Ser Arg Thr Phe Gly Gly Tyr Phe Gln 435 440 445 Arg Tf Arg Thr Pro Asp Val Met Gly Gln Lys Pro Tf Asn Leu Ala 450 455 460 Asp He Gln Gly Glu Gly Asp Val Tyr Tyr Val Asn Ala Wing Cys Wing 465 470 475 480 Gly Phe Glu Ser Val Gly His Val Phe Asp Cys Wing Asp Asn Leu He 485 490 495 Lys Asp Phe Phe 500 < 210 > 5 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide < 220 > < 221 > difference mise. < 222 > (3) .. (6) < 223 > y = c or t, n = a or t or g or c < 220 > < 221 > difference mise. < 222 > (9) < 223 > y = c or t < 220 > - Í difference mise. < 222 > (12) < 223 > y = c ort < 220 > < 221 > difference mise. < 222 > (15) < 223 > n = a or t or g or c .j__, < 220 > < 221 > difference mise. < 222 > (18) < 223 > h = a or c or t < 400 > 5 gayyyngayg ayacnathgc 20 < 210 > 6 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucledtido < 220 > < 221 > difference mise. < 222 > (3) < 223 > y = c or t < 220 > < 221 > difference mise. < 222 > (6) .. (9) < 223 > n = a or t or g or c, b = g or c or t, w = a or t, r = a org < 220 > < 22 ^ > difference mise. < 222 > (12) < 223 > d = a or g or t < 220 > < 221 > difference mise. < 222 > (15) < 223 > y = c or t < 220 > < 221 > difference mise. < 222 > (18) < 223 > n = a or t or g or c < 400 > 6 tgytgnbwrt adatytcnac < 210 > 7 < 211 > 38 < 2 2 > PRT < 213 > Ptilota filicina < 400 > 7 Asp Asp Phe Asp Asp Thr He Wing Val Val Gly Wing Gly Tyr Ser Gly 1 5 10 15 Leu Ser Ala Ala Phe Thr Leu Val Lys Lys Gly Tyr Thr Asn Val Glu 20 25 30 He Tyr Ser Gln Gln Tyr 35 < 210 > 8 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Initiator forward < 400 > 8 cgccatggct ttgaatagag ttcttcac 28 < 210 > 9 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > 10 < 223 > Description of the Artificial Sequence: Initiator forward < 400 > 9 cgccatggac gattttgatg acacgattgc 30 < 210 > 10 < 211 > 34 15 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Reverse Initiator < 400 > 10 cgagatctga agaaatcctt gatcaaatta tccg 34 20 < 210 > 11 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence ¿Í ___________ ii ________ Q_J ___ ^ ¡_ .......... , _ ..Í.-A.Á _ ^ .., ..__ ^ A ..._. Í ...... ... ... .. .. ......... .. ^,.:. ^. '__, ".., ... ás > ^ _. ._-._.__.: - j? t..j__l .--.?.? < 220 > < 223 > Description of the Artificial Sequence: PPF Initiator < 400 > 11 aagctttgga gattatcgtc 20 < 210 > 12 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > 10 < 223 > Description of the Artificial Sequence: PPM Initiator < 400 > 12 tcgtgtcatc aaaatcatgg gcttttgtca caggggtaa 39 < 210 > 13 < 211 > 27 15 < 212 > DNA ^^ Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: EPR Initiator < 400 > 13 gcaggatccg tatcgagctc tgattcg 27 20 < 210 > 14 < 211 > 56 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Adapter < 400 > 14 cgcgatttaa atggcgcgcc ctgcaggcgg ccgcctgcag ggcgcgccat ttaaat 56 < 210 > 15 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > _23 > Description of the Artificial Sequence: Ligadoc Oligonucleotide < 400 > 15 tcgaggatcc gcggccgcaa gcttcctgca gg 32 < 210 > 16 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide linker < 400 > 16 tcgacctgca ggaagcttgc ggccgcggat ce 32 < 210 > 17 < 211 > 32 < 212 > DNA Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide linker < 400 > 17 tcgacctgca ggaagcttgc ggccgcggat ce 32 < 210 > 18 < 211 > 32 < 212 > DNA < 213 > Artificial Sequence < 220 > 10 < 223 > Description of the Artificial Sequence: Oligonucleotide linker < 400 > 18 tcgaggatcc gcggccgcaa gcttcctgca gg 32 < 210 > 19 15 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide linker 0 < 400 > 19 tcgaggatcc gcggccgcaa gcttcctgca ggaget 36 < 210 > 20 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Ligador < 400 > 20 cctgcaggaa gcttgcggcc gcggatcc 28 < 210 > 21 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide Ligador < 400 > 21 tcgacctgca ggaagcttgc ggccgcggat ccagct 36 < 210 > 22 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Oligonucleotide linker < 400 > 22 ggatccgcgg ccgcaagctt cctgcagg 28 < 210 > 23 < 211 > 36 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Initiator forward PLF1 < 400 > 23 ggatccgcgg ccgcatgtct ttgaatagag ttcttc 36 < 210 > 24 < 211 > 37 < 212 > DNA < 213 > Artificial Secuance < 220 > < 223 > Desire of the Artificial Sequence: Initiator forward PLF2 < 400 > 24 ggatccgcgg ccgcatggat tttgatgaca cgattgc 37 < 210 > 25 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Reverse Initiator PLR < 400 > 25 cctgcaggaa gcttctagaa gaaatccttg ate 33 < 210 > 26 < 211 > 1920 < 212 > DNA < 213 > Ptyas mucosus < 400 > 26 gggcaatcag ctgttgccgt ctcactggtg aaaagaaaac ccaccctggc ggcccaatac 60 gcaaccgcct ctccccggcg cgttggccga ttcattaatg cagctggcac gacaggtttc 120 ccgactgaaa agcgggcagt agcgcaacgc aattaatgtg agttagetca ctcattaggc 180 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 240 acaatttcac acagaaacag ctatgaccat agetetaata gattacgcca CGACTCACTA 300 tagggaaagc tggtacgcct gcaggtaccg gtccggaatt cccgggtcga cccacgcgtc 360 cgcaaaatgt ctttgaatag agttcttcac attttcctta tegeatatet cgcatgcact 420 gccctaaccc atgattttga tgacaegatt gccgttgtgg gagctggcta ctctggactg 480 agcgctgctt ttactctcgt caag'aaaggg tacaccaacg ttgagattta cgaatcccag 540 ggcgaagttg gggggtatgt ctactctgtt gactataaca acgtcgcgca tgacctggcc 600 acgtacgctc tgactcctgc atactggaaa ttccaggagg ccatgaaaag tatcggcgtt 660 gggttttgtg agetegatgt tgcaattgtg caaaegaatt ctacgcctgt ctcagtcccg 720 ttcgagaaat ggatggccgc ctactgggct gcgaaagtcc caaacccact caacctcgtg 780 aggaaggtct cgactcaagt ttcgacgtac gttgaagttt ggaagaaget cttcaatatg 840 gacttcattg acacga GCAC gaagcgcact aatcgcctct ttccgttgaa gaccaacgac 900 gtcgacgtcc ttgeccaatt ttcaatgccc atgaaagatt ttgttgcatt gcataagctg 960 gacttgctcg agcctctttt tatccaggca accgactccc aggcgtacgg tccgtatgac 1020 cactctacta acgacaccgg catggtgtgg ttccctccga accttttcaa cggtgaggaa 1080 aataccgttc catgtggtac gtataactcg atgcagtcca tggccgagca catggccgaa 1,140 gcaaaggagt tggttgaaga cacgttccac atgaatacga aggtgacgaa aatctctcgc 1200 gccaccgatg gatetagtec atccctcttg gaagaaggtg tagctacgcc gaagetette 1260 gacaccataa tcagtacgaa caagctgccg tctgcgaacc gtgccgaagt tgtgacacct 1320 aggagcggga ctgcttccga ggccgccgat acgtacgagg agctacaaat gttctctgct 1380 ettetegaga cgaatcgcag egatgecatt ccgacgacag gcttcttgat ggtggatgcg 1440 tagetcaega gacgcaatta ccctaacacc gggttttggg gttgtttgaa tgctgagcgt 1500 attcggatga cgcggaggct gaatgetatt etaagetegg atactgtgac gcgcgtcagc 1560 actatacaga gccatctact gcgtgcaaac aacgaacgca tcgacttttc tctcgacgag 1620 aagattcagc aggtgaagac caatcttgcg aacgtgggac tcggctacct gaccaatcta 1680 acctcccgta cgttcggtgg to tatttccag aggtggagga cgccggatgt tatgggtcaa 1740 aagccgtgga atctggctga cattcaagga gaaggagatg tgtactacgt caactcggct 1800 gcatgcgggt tcgagtccgt cggccacgtt ttcgattgcg cggataattt gatcaaggat 1860 tttttctaga taaacacaac agaagtagac tgccgtcaaa gtctggttac ctcatggaaa 1920 < 210 > 27 < 211 > 1860 < 212 > DNA < 213 > Ptyas mucosus < 400 > 27 gcgcgttggc cgattcatta atcagctggc acgacaggtt tcccgcgaaa acggccatgg 60 agcgcaacgc aattaatgta agttagctca ctcattaggc accccaggct tttacacttt 120 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 180 agctatgacc atgattacgc caagctctaa tacgactcac tatagggaaa gctggtacgc 240 ctgcaggtac cggtccggaa ttcccgggtc gacccacgcg tccgctctcg cagaaaagct 300 gcatttccct cttctctcaa aatgtctttg aatagagttc ttcacatttc ccttatcgca 360 tatctcgcat gcactgccct aacccatgat tttgatgaca cgattgccgt tgtgggagct 420 ggctactctg gactgagcgc tgcttttact ctcgtcaaga aagggtacac caacgttgag 480 atttacgaat cccagggcga agttgggggg tatgtctact ctgttgacta taacaacgtc 540 gcgcatgacc tggccacgta cgctctgact cctgcatact ggaaattcca ggaggccatg 600 aaaagtatcg gcgttgggtt ttgtgagctc gatgttgcaa ttgtgcaaac gaattctacg 660 cctgtctcag tcccgttcga gaaatggatg gccgcctact gggctgcgaa agtcccaaac 720 ccactcaacc tcgtgaggaa ggtctcgact cgtacgttga caagtttcga agtttggaag 780 aagctcttca atatggactt cattgacacg agcacgaagc gcactaatcg cctctttccg 840 ttgaag acca acgacgtcga cgtccttgcc tgcccatgaa caattttcaa agattttgtt 900 gcattgcata agctggactt gctcgagcct ctttttatcc aggcaaccga ctcccaggcg 960 tacggtccgt atgacacgac accggcactc tactacatgg tgtggttccc tccgaacctt 1020 ttcaacggtg aggaaaatac cgttccatgt ggtacgtata actcgatgca gtccatggcc 1080 gagcacatgg ccgaatggtt gaagagcaag gagtcacgtt ccacatgaat tacgaaggtg 1140 acgaaaatct ctcgcgccac cgatggatct agtccatccc tcttggaaga aggtgtagct 1200 acgccgaagc tcttcgacac cataatcagt acgaacaagc tgccgtctgc gaaccgtgcc 1260 gaagttgtga cacctctgct tccgaaggag cgggaggcca ccaatacgta cgaggagcta 1320 caaatgttct ctgctcttct cgagacgaat cgcagcgatg ccattccgac gacaggcttc 1380 ttgatggtgg atgcggacgc aattatagct cacgaccctg acaccgggtt ttggggttgt 1440 ttgaatgctg agcgtcgcgg aggctattcg gatgagaatg ctattctaag ctcggatact 1500 gtgacgcgcg tcagcgccat ctactactat acagagcgtg caaacaacga acgcatcgac 1560 ttttctctcg acgagaagat tcagcaggtg aagaccaatc ttgcgacgtg ggactcggct 1620 acctggacca atctaacttc ccgtacgttc ggtggatatt tccagaggtg gaggacgccg 1680 gatgttatgg gtcaaaagcc gtggaatctg gctgacattc aaggagaagg agatgtgtac 1740 tacgtcaacg cggctgcatg cgggttcgag tccgtcggcc acgtttcgat ttgcgcggat 1800 aatttgatca aggatttctt ctagataaac acaacagaag tagactgcgc ccaaagtctg 1860 . Á & i

Claims (24)

NOVELTY OF THE INVENTION CLAIMS

1. An isolated DNA sequence that codes for an active enzyme in the formation of conjugated fatty acids from polyacetic fatty acyl substrates.

2. The isolated DNA sequence according to claim 1, further characterized in that said nucleic acid acid sequence encodes polyeneoic fatty acid isomerase.

3. The isolated DNA sequence according to claim 1, further characterized in that said nucleic acid sequence is isolated from a source of eukaryotic cells.

4. The isolated DNA sequence according to claim 3, further characterized in that said source of eukaryotic cells is selected from the group consisting of fungal cells and plant cells.

5. The isolated DNA sequence according to claim 4, further characterized in that said DNA sequence is Ptilota filicina.

6. The isolated DNA sequence according to claim 4, further characterized in that said polyeneoic fatty acid isomerase protein is encoded by a sequence that includes a l '- ^ _ t_ÉÍ__ÍÍ_ll_tt Íl¡MÍ_ÉÍÍ ^ í ?? t -_. ¡.. ..., _ > á «i,. < _a »« - «f ', --- .I. _. . ,. - »» .._.... »-» _, "» ".,.. .. _. _. 8... _" .... .... AMt. Faith ??.?. nucleic acid sequence selected from the group consisting of SEQ ID Nos: 1 and 3.

7. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 2.

8. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

9. An isolated polynucleotide selected from the group consisting of: a) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, b) an isolated polynucleotide comprising SEQ ID NO: 1; c) an isolated polynucleotide comprising a nucleotide sequence having at least about 70% identity with that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; d) an isolated polynucleotide; comprising a nucleotide sequence having at least about 80% identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; e) a polynucleotide isolated ottid comprising a nucleotide sequence having at least about 90% identity with that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; f) an isolated polynucleotide comprising a nucleotide sequence having at least about 95% identity with that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; g) an isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO: 1 or a fragment thereof; and h) an isolated polynucleotide «" «*» - * - complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), (f), or (g)

10. An isolated polynucleotide selected from the group consisting of: a) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 4; b) an isolated polynucleotide comprising SEQ ID NO: 3; c) an isolated polynucleotide comprising a nucleotide sequence having at least about 70% identity to that of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; d) an isolated polynucleotide comprising a nucleotide sequence having at least about 80% identity with that of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; e) an isolated polynucleotide comprising a nucleotide sequence having at least about 90% identity with that of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; f) an isolated polynucleotide comprising a nucleotide sequence having at least about 95% identity with that of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; g) an isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO: 3 or a fragment thereof; and h) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), (f), or (g).

11. A nucleic acid construct comprising, when operatively linked to the components in the 5 'to 3' direction of transcription: a transcriptional initiation region; and a sequence of polynucleotides encoding an active enzyme in the formation of conjugated fatty acids from polyaneic fatty acyl substrates.

12. The nucleic acid construct according to claim 11, further characterized in that said enzyme is fatty acid polyenoic acid.

13. A host cell comprising a DNA construct according to claim 11.

14. The host cell according to claim 13, further characterized in that said host cell is selected from the group consisting of bacterial, insect cells , fungal, mammal and vegetable.

15. A non-human organism transformed with the construction of claim 11 or 12.

16. The organism according to claim 15, further characterized in that said organism is selected from the group consisting of bacterial, insect, fungal organism, of mammal and vegetable.

17. The organism according to claim 16, further characterized in that said organism is a vegetable.

18. A method for producing a recombinant host cell, characterized in that it comprises: transforming or transfecting a cell with a nucleic acid construct, comprising a transcriptional initiation region and a polynucleotide sequence that encodes an active enzyme in the formation of conjugated fatty acids from - «S 'y5'" *; polyenoic grade acyl substrates, such that said host cell, under appropriate culture conditions, produces a polyeneoic fatty acid isomerase protein.

19. The method according to claim 18, further characterized in that said host cell is selected from the group consisting of plant cells, bacterial cells, yeast cells, and algae cells.

20. A non-human organism transformed by the method of claim 18 or 19.

21. The organism according to claim 20, further characterized in that said organism is selected from the group consisting of bacteria, yeast, algae and vegetables.

22. The organism according to claim 21, further characterized in that said organism is a vegetable.

23. A method for producing a recombinant host cell, characterized in that it comprises: transforming or transfecting a cell with a nucleic acid construct, comprising a transcriptional initiation region and a polynucleotide sequence selected from the group consisting of a polynucleotide according to with claim 9 and a polynucleotide according to claim 10, so that said host cell, under appropriate culture conditions, produces a polyeneoic fatty acid isomerase protein.

24. - The method according to claim 23, further characterized in that said polynucleotide sequence comprises the nucleotide sequence described in SEQ ID NO: 1. The method according to claim 23, further characterized in that said host cell is a plant cell. 26. A non-human organism transformed according to the method of claim 23 or 24. 27. The organism according to claim 26, further characterized in that the organism is a vegetable. 28.- A method for modifying the fatty acid composition in a host cell, said method comprises: transforming a host cell with a nucleic acid construct, comprising a transcriptional initiation region and a polynucleotide sequence that encodes an active enzyme in the formation of conjugated fatty acids from polyenoic fatty acyl substrates, such that said host cell, under appropriate culture conditions, produces a polyeneoic fatty acid isomerase protein. 29. The method according to claim 28, further characterized in that said production of a polyenoic fatty acid surfactant produces an increase of conjugated fatty acids in said host cell. 30. The method according to claim 28, further characterized in that said polynucleotide sequence is in an orientation selected from the group consisting of sense orientation and antisense orientation. 31. The method according to claim 28, further characterized in that said host cell is selected from the group consisting of a plant cell, a bacterial cell, and a fungal cell. 32. A non-human organism transformed by the method of any of claims 28 to 30. 33.- The organism according to claim 32, further characterized in that said organism is selected from the group consisting of plant, bacteria and fungi. 34. The organism according to claim 33, further characterized in that said organism is a vegetable.