WO2001009296A1 - 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 ENCODING POLYENOIC FATTY ACID ISOMERASE AND USES THEREOF

Cross-Reference to Related Applications

This application claims priority from U.S. provisional patent application Ser. No. 60/146,458 filed July 30, 1999, which is hereby incorporated by reference in its entirety.

Technical Field

The present invention is directed to nucleic acid and amino acid sequences and constructs, and methods related thereto.

Background Novel vegetable oils compositions and/or improved means to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources, are needed. Depending upon the intended oil use, various different oil compositions are desired. For example, edible oil sources containing the minimum possible amounts of saturated fatty acids are desired for dietary reasons and alternatives to current sources of highly saturated oil products, such as tropical oils, are also needed. Furthermore, oils compositions containing rare or exotic fatty acid species having nutritional benefits are also needed in the art.

Conjugated fatty acids, such as conjugated linoleic acid (CLA), are gaining recognition for their health benefits in animal feed and in human nutrition. Conjugated fatty acid is a general term for fatty acids containing double bonds alternating with single bonds. For example, conjugated linoleic acid refers to a series of positional and geometric isomers of linoleic acid (an 18 carbon molecule that contains double bonds in the cis-9 and cis-12 positions).

Of the various isomers of CLA, the cis-9, trans- 11 and trans- 10, cis-12 isomers have received the most attention. Recent data suggests (Parks, et al. (1999), Lipids, 34: 235-243) that the trans- 10, cis-12 is the biologically active form. However, it is recognized that other CLA isomers, and/or other conjugated fatty acids, may also be shown to have biological activities.

CLA is now recognized as a nutritional supplement and an effective inhibitor of epidermal carcinogenesis and forestomach neoplasia in mice, and of carcinogen-induced rat mammary and colon tumors. Furthermore, CLA has been shown to reduce LDL and atherosclerosis in hamsters and rabbits, reduce body fat and increase lean body mass in chickens, swine, rats and mice, increase feed efficiency in chickens and swine, reduce serum PGE2 in rats, increase bone mass in mice and chickens, as well as reducing weight loss during immune challenge in mice, chickens and rats. Thus, the identification of enzyme targets and sources for nucleic acid sequences of such enzyme targets capable of producing conjugated fatty acids in host cells is needed in the art. Ultimately, useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and host cells containing such constructs are needed.

Summary of the Invention

The present invention is directed to polyenoic fatty acid isomerases (PFI), and in particular to PFI 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 polypeptides, particularly, polynucleotides that encode PFI, are provided.

In a further aspect, the invention relates to oligonucleotides derived from the PFI proteins and oligonucleotides which include partial or complete PFI encoding sequences.

It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for either transcription and/or expression of PFI. In particular, constructs are provided which are capable of both transcription or and/or in host cells.

Particularly preferred constructs are those capable of both transcription and/or expression in plant cells.

In yet another aspect of the present invention, methods are provided for production of PFI in a host cell or progeny thereof. In particular, host cells are transformed or transfected with a DNA construct which can be used for transcription and/or expression of PFI. The recombinant cells which contain PFI are also part of the present invention.

In a further aspect, the present invention relates to methods of using polynucleotide and polypeptide sequences to modify the fatty acid composition in a host cell, particularly in seed oil of oilseed crops. In particular, the modified fatty acid composition comprises an altered amount of conjugated fatty acids. Plant cells having such a modified fatty acids are also contemplated herein.

The modified plants, seeds and oils obtained by the expression of the plant PFI proteins are also considered part of the invention.

Detailed description of the Invention In accordance with the subject invention, nucleotide sequences are provided that code for a protein, polypeptide or peptide, which are active in the formation of conjugated fatty acids from polyenoic fatty acid substrates. Such sequences are referred to herein as polyenoic fatty acid isomerases (also referred to as PFI). The novel nucleic acid sequences find use in the preparation of constructs to direct their expression in a host cell. Furthermore, the novel nucleic acid sequences find use in the preparation of plant expression constructs to modify the fatty acid composition of a plant cell.

A polyenoic fatty acid isomerase nucleic acid sequence of this invention includes any nucleic acid sequence that codes for a protein, polypeptide, or peptide fragment, obtainable 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 a plant cell-like environment, i.e. in vitro. As used herein, "conjugated" refers to the interaction of the pi electron systems when the carbon chain contains alternating double and single bonds (C=C-C=C) such that the electrons of the double bonds are close enough to interact with each other. This is in contrast to "isolated" double bonds where the pi electron systems are separated by a saturated carbon (C=C-C-C=C) or "cumulated" where the double bonds share a central carbon (C=C=C). "A plant cell-like environment" means that any necessary conditions are available in said environment (i.e., such factors as temperatures, pH, lack of inhibiting substances) which will permit 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, pentaenes and hexaenes. Fatty acid substrates of particular interest in the present invention include, but are not limited to, linoleic acid, linolenic acid, stearidonic, eicosapentaenoic acid, dihomo- γ-linolenic acid, adrenic acid, eicosatrienonic acid, α-linolenic acid, docosahexaenoic acid and arachidonic acid.

Isolated proteins, Polypeptides and Polynucleotides

A first aspect of the present invention relates to isolated PFI polypeptides. Such polypeptides include isolated polypeptides set forth in the Sequence Listing, as well as polypeptides and fragments thereof, particularly those polypeptides which exhibit PFI activity and also those polypeptides which have approximately at least 50-79% identity, more preferably approximately at least 80% identity, even more preferably approximately at least 90% identity, and most preferably approximately at least 95% identity to a polypeptide sequence selected from the group of sequences set forth in the Sequence Listing, and also includes portions of such polypeptides, wherein such portion of the polypeptide preferably include at least 30 amino acids and more preferably include at least 50 amino acids.

"Identity", as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. "Identity" can be readily 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 (1994); 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). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs which 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); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et 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 NTH, Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol, 215:403-410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.

Parameters for polypeptide sequence comparison typically include the following: Algorithm: Needleman and Wunsch, J Mol. Biol. 48:443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci USA 89:10915-10919 (1992) Gap Penalty: 12

Gap Length Penalty: 4

A program which can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wisconsin. The above parameters along with no penalty for end gap are the default parameters for peptide comparisons.

Parameters for polynucleotide sequence comparison include the following: Algorithm: Needleman and Wunsch, j. Mol. Biol. 48:443-453 (1970) Comparison matrix: matches = +10; mismatches = 0 Gap Penalty: 50 Gap Length Penalty: 3 A program which can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wisconsin. The above parameters are the default parameters for nucleic acid comparisons.

The invention also includes polypeptides of the formula: X-(R,)_n-(R₂)-(R₃)_nY wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R, 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 so that its amino terminal residue is at the left, bound to R_l5 and its carboxy terminal residue is at the right, bound to R₃ Any stretch of amino acid residues denoted by either R group, where R is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

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 .

Polypeptides of the present invention have been shown to have PFI activity and are of interest because PFI is involved in the production of conjugated fatty acids from polyenoic 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 to be a part of the invention. A fragment is a variant polypeptide which has an amino acid sequence that is entirely the same as part but not all of the amino acid sequence of the previously described polypeptides. The fragments can be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or a region, most 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 a 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 substitution of a residue by another residue with like characteristics and/or properties. In general, such substitutions are between Ala, Val, Leu and He; between Ser and Thr; between Asp and Glu; between Asn and Gin; 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 one amino acid(s) are substituted, deleted, or added, in any combination. Variants that are fragments of the polypeptides of the invention can be used to produce the corresponding full length polypeptide by peptide synthesis. Therefore, these variants can be used as intermediates for producing the full-length polypeptides of the invention. Another aspect of the present invention relates to isolated PFI polynucleotides.

The polynucleotide sequences of the present invention include isolated polynucleotides that encode 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 such sequences and variants thereof. The invention also provides a polynucleotide sequence identical over its entire 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 encoding a leader or secretory sequence, a pre-, pro-, or prepro- protein sequence. The polynucleotide can also include non-coding sequences, including for example, but not limited to, non-coding 5' and 3' sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence that encodes additional amino acids. For example, a marker sequence can be included to facilitate the purification of the fused polypeptide. Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression.

The invention also includes polynucleotides of the formula:

X-(R,)_n-(R₂-(R₃)_n-Y wherein, at the 5' end, X is hydrogen, and at the 3' end, Y is hydrogen or a metal, R, 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 selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: 1 and 3. In the formula, R₂ is oriented so that its 5' end residue is at the left, bound to R„ and its 3' end residue is at the right, bound to R₃. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.

The invention also relates to variants of the polynucleotides described herein that encode for 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 encoding polypeptide variants wherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues of a polypeptide sequence of the invention are substituted, added or deleted, in any combination. Particularly preferred are substitutions, additions, and deletions that are silent such that they do not alter the properties or activities of the polynucleotide or polypeptide.

Nucleotide sequences encoding polyenoic fatty acid isomerases may be obtained from natural sources or be partially or wholly artificially synthesized. They may directly correspond to a polyenoic fatty acid isomerase endogenous to a natural source or contain modified amino acid sequences, such as sequences which have been mutated, truncated, increased or the like. Polyenoic fatty acid isomerases may be obtained by a variety of methods, including but not limited to, partial or homogenous purification of protein extracts, protein modeling, nucleic acid probes, antibody preparations and sequence comparisons. Typically a polyenoic fatty acid isomerase will be derived in whole or in part from a natural source. A natural source includes, but is not limited to, prokaryotic and eukaryotic sources, including, bacteria, yeasts, plants, including algae, and the like.

Of special interest are polyenoic fatty acid isomerases which are obtainable from algae sources, including those which are obtained, from Ptilota, Bossiella, Lithotham, for example P. filicina, or from polyenoic fatty acid isomerases which are obtainable through the use of these sequences. "Obtainable" refers to those polyenoic fatty acid isomerases which have sufficiently similar sequences to that of the sequences provided herein to provide a biologically active polyenoic fatty acid isomerase. Further preferred embodiments of the invention that are approximately at least

50-79%) identical over their entire length to a polynucleotide encoding a polypeptide of the invention, and polynucleotides that are complementary to such polynucleotides. More preferable are polynucleotides that comprise a region that is approximately at least 80%) identical over its entire length to a polynucleotide encoding a polypeptide of the invention and polynucleotides that are complementary thereto. Polynucleotides approximately at least 90%) identical over their entire length are particularly preferred, those approximately at least 95%> identical are especially preferred. Further, those with approximately at least 97%o identity are highly preferred and those with approximately at least 98%> and 99% identity are particularly highly preferred, with those approximately at least 99% being the most highly preferred.

Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptides encoded by the polynucleotides set forth in the Sequence Listing.

The invention further relates to polynucleotides that hybridize to the above- described sequences. In particular, the invention relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" mean that hybridization will generally occur if there is approximately at least 95%> and preferably approximately at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation 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's solution, 10%> dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0. lx SSC at approximately 65°C. Other hybridization and wash conditions are well known and are 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 consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set for in the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers as described herein.

As discussed herein regarding polynucleotide assays 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 cDNAs or genomic clones encoding a polypeptide and to isolate cDNA or genomic clones of other genes that have a high sequence similarity to a polynucleotide set forth in the Sequence Listing. Such probes will generally comprise at least 15 bases. Preferably such probes will have at least 30 bases and can have at least 50 bases. Particularly preferred probes will have between 30 bases and 50 bases, inclusive. The coding region of each gene that comprises or is comprised by a polynucleotide sequence set forth in the Sequence Listing may be isolated by screening 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 screen a library of cDNA, genomic DNA or mRNA to identify members of the library which hybridize to the probe. For example, synthetic oligonucleotides are prepared which correspond to the N-terminal sequence of the PFI peptide. The partial sequences so prepared are then used as probes to obtain PFI clones from a gene library prepared from Ptilota filicina, a red marine algae. Alternatively, where oligonucleotides of low degeneracy can be prepared from particular PFI peptides, such probes may be used directly to screen gene libraries for PFI gene sequences. In particular, screening of cDNA libraries in phage vectors is useful in such methods due to lower levels of background hybridization. Typically, a PFI sequence obtainable from the use of nucleic acid probes will show approximately 60-70% sequence identity between the target PFI sequence and the encoding sequence used as a probe. However, lengthy sequences with as little as approximately 50-60%> sequence identity may also be obtained. The nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe. When longer nucleic acid fragments are employed as probes (greater than about 100 bp), one may screen at lower stringencies in order to obtain sequences from the target sample which have 20-50%> deviation (i.e., 50-80% sequence homology) from the sequences used as probe. Oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding an PFI enzyme, but should be at least about 10, preferably at least about 15, and more preferably at least about 20 nucleotides. A higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions. It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related PFI 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 USA (1989) 55:1934-1938).

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for 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 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction) employed during the first strand cDNA synthesis.

There are several methods available and are well know to the skilled artisan to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA Ends (RACE) (see, for example, Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002). Recent modifications of the technique, exemplified by the Marathona technology (Clonetech Laboratories, Inc.) for example, have significantly simplified obtaining full-length cDNA sequences. The polynucleotides and polypeptides of the invention can be used, for example, in the transformation of various host cells, as further discussed herein.

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 (for example, when the mature form of the protein has more than one polypeptide chain). Such sequences can, for example, play a role in the processing of a protein from a precursor to a mature form, allow protein transport, shorten or lengthen protein half-life, or facilitate manipulation of the protein in assays or production. It is contemplated that cellular enzymes can be used to remove any additional amino acids 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. The inactive precursors generally are activated when the prosequences are removed. Some or all of the prosequences may be removed prior to activation. Such precursor protein are generally called proproteins.

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 preferable 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 DVD, and then to use the stored sequence to search a sequence database with well known searching 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/Index.htlm); and the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL)

(http://www.ebi.ac.uk/ebi_docs/embl db.html). A number of different search algorithms are available to the skilled artisan, one example of which are the suite of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (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 more distantly related sequences, and the like, and are well known to the skilled artisan.

Plant Constructs and Methods of Use

Of particular interest is the use of the nucleotide sequences, or polynucleotides, in recombinant DNA constructs to direct the transcription and/or expression of the PFI sequences of the present invention in a host plant cell. The expression constructs generally comprise a promoter functional in a plant cell operably linked to a nucleic acid sequence encoding a polyenoic fatty acid isomerase of the present invention and a transcriptional termination region functional in a plant cell. Those skilled in the art will recognize that there are a number of promoters which are functional in plant cells that have been described in the literature. In addition, organelle and plastid specific promoters such as chloroplast or plastid functional promoters, and chloroplast or plastid operable promoters are also envisioned. One set of promoters are constitutive promoters such as the CaMV35S or FMV35S promoters that yield high levels of expression in most plant organs. Enhanced or duplicated versions of the CaMV35S and FMV35S promoters are useful in the practice of this invention (Odell, et al. (1985) Nature 313:810-812; Rogers, U.S. Patent Number 5,378, 619). In addition, it may also be preferred to bring about expression of the PFI gene in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc., and the promoter chosen should have the desired tissue and developmental specificity.

Of particular interest is the expression of the nucleic acid sequences of the present invention from transcription initiation regions which are preferentially expressed in a plant seed tissue. Examples of such seed preferential transcription initiation sequences include those sequences derived from sequences encoding plant storage protein genes or from genes involved in fatty acid biosynthesis in oilseeds. Examples of such promoters include the 5' regulatory regions from such genes as napin (Kridl et al, Seed Sci. Res. 7:209:219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean ά subunit of b-conglycinin (soy 7s, (Chen et al, Proc. Natl. Acad. Sci., 83:8560- 8564 (1986))) and oleosin.

It may be advantageous to direct the localization of proteins conferring PFI to a particular subcellular compartment, for example, to the mitochondrion, endoplasmic reticulum, vacuoles, chloroplast or other plastidic compartment. For example, where the genes of interest of the present invention will be targeted to plastids, such as chloroplasts for expression, the constructs will also employ the use of sequences to direct the gene to the plastid. Such sequences are referred to herein as chloroplast transit peptides (CTP) or plastid transit peptides (PTP). In this manner, where 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 to the plastid. The chloroplast transit peptides may be derived from the gene of interest, or may 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 Chem. 26V:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 8 .965-96S; Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and, Shah et al. (1986) Science 233:478-481. Additional transit peptides for the franslocation of the PFI protein to the endoplasmic reticulum (ER), or vacuole may also find use in the constructs of the present invention. Depending upon the intended use, the constructs may contain the nucleic acid sequence which encodes the entire PFI protein, or a portion thereof. For example, where antisense inhibition of a given PFI protein is desired, the entire PFI sequence is not required. Furthermore, where PFI sequences used in constructs are intended for use as probes, it may be advantageous to prepare constructs containing only a particular portion of a PFI encoding sequence, for example a sequence which is discovered to encode a highly conserved PFI region.

The skilled artisan will recognize that there are various 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) , co- suppression (Napoli, et al. (1989) Plant Cell 2:279-289), ribozymes (PCT Publication WO 97/10328), and combinations of sense and antisense Waterhouse, et al. (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964. Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence.

Regulatory transcript termination regions may be provided in plant expression constructs of this invention as well. Transcript termination regions may be provided by the DNA sequence encoding the polyenoic fatty acid isomerase or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. The skilled artisan will recognize that any convenient transcript termination region which is capable of terminating transcription in a plant cell may be employed in the constructs of the present invention. Alternatively, constructs may be prepared to direct the expression of the PFI sequences directly from the host plant cell plastid. Such constructs and methods are known in the art and are generally described, for example, in Svab, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917 and in U.S. Patent Number 5,693,507. A plant cell, tissue, organ, or plant into which the recombinant DNA constructs containing the expression constructs have been introduced is considered transformed, transfected, or transgenic. A transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a PFI nucleic acid sequence.

Plant expression or transcription constructs having a plant PFI as the DNA sequence of interest for increased or decreased expression thereof may be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Most especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, 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 applicable to dicotyledyons and monocotyledons species alike and will be readily applicable to new and/or improved transformation and regulation techniques.

Of particular interest, is the use of plant PFI constructs in plants which have been genetically engineered to produce a particular fatty acid in the plant seed oil, where TAG in the seeds of nonengineered plants of the engineered species, do not naturally contain that particular fatty acid. Thus, the expression of novel PFI in plants may be desirable for the incorporation of unique fatty acyl groups into the sn-3 position.

Further plant genetic engineering applications for PFI proteins of this invention include their use in preparation of structured plant lipids which contain TAG molecules having desirable fatty acyl groups incorporated into particular positions on the TAG molecules.

It is contemplated that the gene sequences may be synthesized, either completely or in part, especially where it is desirable to provide plant-preferred sequences. Thus, all or a portion of the desired structural gene (that portion of the gene which encodes the PFI protein) may be synthesized using codons preferred by a selected host. Host-preferred codons may 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 antibody preparations, nucleic acid probes (DNA and RNA) and the like may be prepared and used to screen and recover

"homologous" or "related" PFIs from a variety of plant sources. Homologous sequences are found when there is an identity of sequence, which may be determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions between a known PFI and a candidate source. Conservative changes, such as Glu/ Asp, Val/Ile, Ser/Thr, Arg/Lys and Gin/ Asn may also be considered in determining sequence homology. Amino acid sequences are considered homologous by as little as 25% sequence identity between the two complete mature proteins. (See generally, Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.)

Thus, other PFIs may be obtained from the specific exemplified Ptilota PFI sequences provided herein. Furthermore, it will be apparent that one can obtain natural and synthetic PFIs, including modified amino acid sequences and starting materials for synthetic-protein modeling from the exemplified PFIs and from PFIs which are obtained through the use of such exemplified sequences. Modified amino acid sequences include sequences which have been mutated, truncated, increased and the like, whether such sequences were partially or wholly synthesized. Sequences which are actually purified from plant preparations or are identical or encode identical proteins thereto, regardless of the method used to obtain the protein or sequence, are equally considered naturally derived.

For immuno logical screening, antibodies to the PFI protein can be prepared by injecting rabbits or mice with the purified protein or portion thereof, such methods of preparing antibodies being well known to those in the art. Either monoclonal or polyclonal antibodies can be produced, although typically polyclonal antibodies are more useful for gene isolation. Western analysis may be conducted to determine that a related protein is present in a crude extract of the desired plant species, as determined by cross- reaction with the antibodies to the P. filicina PFI protein. When cross-reactivity is observed, genes encoding the related proteins are isolated by screening expression libraries representing the desired plant species. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtl 1, 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 PFI proteins may be utilized for a variety of uses. For example, recombinant constructs can be prepared which may be employed as probes, or which will direct expression of the PFI protein in host cells to produce a ready source of the enzyme and/or to modify the composition of triglycerides found therein. Other useful applications may be found when the host cell is a plant host cell, either in vitro or in vivo. For example, by expressing a PFI protein in a host plant cell, various conjugate fatty acids may be produced in a given plant tissue. In a like manner, for some applications it may be desired to decrease the amount of PFI endogenously expressed in a plant cell by various gene suppression technologies discussed supra.

It is appreciated that the 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, KASes, and the like.

The modification of fatty acid compositions may also affect the fluidity of plant membranes. Different lipid concentrations have been observed in cold-hardened plants, for example. By this invention, one may be capable of introducing traits which will lend to chill tolerance. Constitutive or temperature inducible transcription initiation regulatory control regions may have special applications for such uses.

As discussed above, nucleic acid sequence encoding a plant PFI of this invention may include genomic, cDNA or mRNA sequence. By "encoding" is meant that the sequence corresponds to a particular amino acid sequence either in a sense or anti-sense orientation. By "extrachromosomal" is meant that the sequence is outside of 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 PFI nucleic acid sequence is obtained, it may be manipulated in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, transversions, deletions, and insertions may be performed on the naturally occurring sequence. In addition, all or part of the sequence may be synthesized. In the structural gene, one or more codons may be modified to provide for a modified amino acid sequence, or one or more codon mutations may be introduced to provide for a convenient restriction site or other purpose involved with construction or expression. The structural gene may 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 PFI of this invention may be combined with other non-native or heterologous sequences in a variety of ways. By heterologous sequences is meant any sequence which is not naturally found joined to the plant PFI, including, for example, combinations of nucleic acid sequences from the same plant which are not naturally found joined together. The DNA sequence encoding a plant PFI of this invention may be employed in conjunction with all or part of the gene sequences normally associated with the PFI. In its component parts, a DNA sequence encoding PFI is combined in a DNA construct having, in the 5' to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding plant PFI and a transcription and translation termination region.

Potential host cells include both prokaryotic and eukaryotic cells. A host cell may be unicellular or found in a multicellular differentiated or undifferentiated organism depending upon the intended use. Cells of this invention may be distinguished by having a plant PFI foreign to the wild-type cell present therein, for example, by having a recombinant nucleic acid construct encoding a plant PFI therein.

The methods used for the transformation of the host plant cell are not critical to the present invention. The transformation of the plant is preferably permanent, i.e. by integration of the introduced expression constructs into the host plant genome, so that the introduced constructs are passed onto successive plant generations. The skilled artisan will recognize that a wide variety of transformation techniques exist in the art, and new techniques are continually becoming available. Any technique that is suitable for the target host plant can be employed within the scope of the present invention. For example, the constructs can be introduced in a variety of forms including, but not limited to, as a strand of DNA, in a plasmid, or in an artificial chromosome. The introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to calcium-phosphate-DNA co-precipitation, electroporation, microinjection, Agrobacterium infection, liposomes or microprojectile transformation. The skilled artisan can refer to the literature for details and select suitable techniques for use in the methods of the present invention.

Normally, included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells. The gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the number of different host species, the expression construct or components thereof are introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts.

Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host. The armed plasmid can give a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle for transforming host plant cells, the expression or transcription construct bordered by the T-DNA border region(s) will be inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, there being broad host range vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al, (roc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0 120 515, which are incoφorated herein by reference. Alternatively, one may insert the sequences to be expressed in plant cells 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 Summerfelt (Plant Mol. Biol (1990) 14:269-216), wherein the pRiHRI (Jouanin, et al, Mol. Gen. Genet. (1985) 201 :370-374) origin of replication is utilized and provides for added stability of the plant expression vectors in host Agrobacterium cells.

Included with the expression construct and the T-DNA will be one or more markers, which allow for selection of transformed Agrobacterium and transformed plant cells. A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like. The particular marker employed is not essential to this invention. The preferred marker will depend upon the particular host and specific construct employed for transformation of such host. For transformation of plant cells using Agrobacterium, plants may be combined and incubated with the modified Agrobacterium for sufficient time to enable said plant cell to be transformed by the Agrobacterium. The bacteria are then killed, and the plant cells cultured in an appropriate selective medium. Once a callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.

There are several possible ways to obtain the plant cells of this invention which 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 construct can be used to transform a plant at the same time as the second construct either by inclusion of both expression constructs in a single transformation vector or by using separate vectors, each of which express desired genes. The second construct can be introduced into a plant which has already been transformed with the PFI expression construct, or alternatively, transformed plants, one expressing the PFI construct and one expressing the second construct, can be crossed to bring the constructs together in the same plant.

Other Constructs and Methods of Use

The invention also relates to vectors that include a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell free translation systems can be employed to produce such protein using RNAs derived from the DNA constructs of the invention. For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the present invention. Introduction of a polynucleotide into a host cell can be effected by 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, 2^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989). Such methods include, but are not limited to, calcium phosphate transfection, DEAE dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading ballistic introduction and infection. Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, enterococci, E. coli, streptomyces, and Bacillus subtilis cells; 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 melanoma cells; 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, and virus derived vectors, for example vectors from bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, such as SB40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations of such viruses, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector which is suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host can be used for expression. The appropriate DNA sequence can be inserted into the chosen expression by any of a variety of well-known and routine techniques, such as, for example, those set forth 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 the secretion of the protein into 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 any of a number of well known methods, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. It is most preferable to use high performance liquid chromatography (HPLC) for purification. Any of the well known techniques for protein refolding can be used to regenerate an active confirmation if the polypeptide is denatured during isolation and/or purification. The oil compositions obtained from host cells expressing the PFI sequences of the present invention find use in a variety of industrial, animal feed and human nutritional applications.

The oil produced by the methods of the present invention containing the conjugated fatty acids find a number of uses. For example methods for the use of various conjugated fatty acids, for example, conjugated linolenic acid (CLA) are known in the art. A number of methods for the use of CLA are described in US Patents 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, 5,827,885, 5,851,572, 5,855,917.

Thus, the oil compositions of the present invention having an altered conjugated fatty acid content find 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, dressings, spreads, mayonnaises, and vitamin mineral supplements. Additional examples include, but are not limited to toppings, dairy products such as cheese and processed cheese, processed meat and meat mimetics, pastas, cereals, sauces, desserts including frozen and shelf stable desserts, dips, chips, baked goods, pastries, cookies, snack bars, confections, chocolates, beverages, unextracted seed, and unextracted seed that has been ground, cracked, milled, rolled, extruded, pelleted, defatted, dehydrated, or otherwise processed, but which still contains the oils, etc., disclosed herein.

The oil compositions of the present invention having an altered conjugated fatty acid also find use in pharmaceutical compositions comprising an effective amount of the conjugated fatty acid composition, along with a pharmaceutically acceptable carrier, excipient, or diluent. These pharmaceutical compositions can be in the form of a solid or liquid. Solids can be in the form of a powder, a granule, a pill, a tablet, a gel, or an extrudate; liquids can be solutions or suspensions.

The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention. EXAMPLES

Example 1 Identification of a Polyenoic Fatty Acid Isomerase Nucleic Acid Sequence

A. Complementary DNA Library Preparation

Total RNA from the red marine algae Ptilota filicina was isolated for use in construction of complementary (cDNA) libraries. Fresh material was ground in liquid nitrogen with a mortar/pestle. Approximately 5 g P. filicina powder was mixed with 5 ml extraction buffer (1% SDS, 10 mM EDTA, 0.2 M NaAC, pH 4.8) and 3 ml acid phenol (pH 4.3). The mixture was incubated at 60°C for 30 min with shaking every 5 min. The mixture was cooled to room temperature and 3 ml chlorophorm was added. After shaking at room temperature for 10 min, the mixture was centrifuged at 5000 * g for 30 min. The aqueous phase was extracted once with 6 ml phenol chlorophorm (1:1 v/v) for 10 min and then centrifuged at 5000 x g for 5 min. The upper aqueous layer was recovered , extracted once with 6 ml chloroform. After the last extraction, RNA was precipitated from the aqueous layer with equal volume of 4M LiCl on ice overnight, and spun down at 10000 x g for 30 min. Precipitated RNA was washed with 70% ethanol, vacuum-dried and dissolved in water.

The resulting total RNA was used to prepare cDNA libraries using the Superscript plasmid system for cDNA synthesis and plasmid cloning kit (BRL Life-Technologies, Gaithersburg, MD). In order to identify candidate nucleic acid sequences, a pair of synthetic oligonucleotides (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)) were prepared corresponding to the 38 N-terminal amino acids (DDFDDTIAVVGAGYSGLSAAFTLVKKGYTNVEIYSQQY, SEQ ID NO:7)(Wise (1995) Biosynthesis and enzymology of conjugated polyenoic fatty acid production in macrophytic marine algae, Ph.D. Thesis, Oregon State University, Corvalis, OR, UMI Dissertation Services) for use in PCR reactions to amplify probes for use in hybridization screening of the P. filicina cDNA library. PCR amplification included an initial denaturation step of 95 °C for 5 min followed by a 5 cycles of 94°C (30 sec) - 45°C (30 sec) - 72°C (30 sec) and a 30 cycles of 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 templates for a second PCR reaction. Following amplification, PCR products were cloned into pCR2.1 using TA cloning system from Invitrogen Co. Library screenings were carried out using standard colony blot protocols (Sambrook, et al. Molecular Cloning, A Laboratory Manual, (supra)). Two cDNA sequences were identified as having hybridized with the oligonucleotide probe. These two DNA sequences, referred to as PFI-B2 (SEQ ID NO:l) and PFI-F3 (SEQ ID NO:3), contained in the pSPORTl cloning vector (BRL Life- Technologies, Gaithersburg, MD), pCGNlOlOO and pCGNlOlOl, respectively. The deduced amino acid sequence for both PFI-B2 (SEQ ID NO:2) and PFI-F3 (SEQ ID NO:4) are also determined.

Example 2 Construct Preparation

2A. Bacterial Expression Constructs

A series of constructs were prepared to express the PFI sequences in host cells. For expression in E. coli, constructs were prepared that either contained tags or lacked such tag sequences.

The vector pCGN10102 was designed to express the protein encoded by the PFI- B2 sequence with the native leader peptide with a C-terminal 6 residue His-tag sequence in the pQE-60 vector (Qiagen). The vector pCGN10103 is similar to pCGN10102, except it contains the sequence encoding the PFI-F3 protein with its native leader peptide. A set of constructs were also prepared to express the PFI sequences in E. coli without the leader sequences. The vector pCGN10104 contains the PFI-B2 sequence from pCGNlOlOO without the leader peptide cloned into the pQE-60 vector with a 6 residue His tag on the PFI C-terminus. The vector pCGN10105 is similar to ρCGN10104, except it contains the PFI-F3 encoding sequence from pCGNl 0101.

Finally, for expression in E. coli a set of constructs lacking the C-terminal 6 residue His tag were prepared. The construct pCGN10106 contains the PFI-B2 encoding sequence from pCGNlOlOO including the native leader peptide in the pQE-60 vector. The vector pCGN10107 is similar to pCGN10106 except containing the PFI-F3 encoding sequence, including the native leader peptide.

For expression of PFI in the cytoplasm of E. coli, the pQE60 system from Qiagen Inc. was used. The PFI coding region with and without the putative leader peptide was PCR amplified using forward primers: 5'-CGCCATGGCTTTGAATAGAGTTCTTCAC- 3' or 5^CGCCATGGACGATTTTGATGACACGATTGC-3'(SEQ ID NO:8 and 9), reverse primer: 5 '- CGAGATCTGAAGAAATCCTTGATCAAATTATCCG-3 '(SEQ ID NO: 10). The Ncol sites (underlined) were introduced in the forward primers while the Bglil site (underlined) was introduced in the reverse primer. The PCR products were subcloned in pCR2.1 using TA cloning system (Invitrogen Co.). The resulting product was then sent for sequencing. After sequence confirmation, the inserts were excised using complete digestion of BgHl and partial digestion of Ncol followed by gel purification using the gel purification system from Qiagen Inc. Subcloning of the inserts into pQE60 vector and expression of the recombinant proteins were done as recommended by the manufacturer. The E. coli transformants were grown at different temperatures to an OD600 of 0.7- 0.8 before being induced by 1 mM isopropyl-b-D-thiogalactopyranoside for 1-5 h. The induced cells were then harvested and lysed by sonication followed by enzyme assays.

Periplasmic expression of PFI was carried out by fusing E. coli alkaline phosphotase (PhoA) leader peptide and PFI without its native leader peptide. The fusion protein coding region was designed to be driven by E. coli alkaline phosphotase gene (phoA) native promoter. Three primers were designed: PPF, 5'-AAGCTTTGGAGATTA TCGTC-3'(SEQ ID NO:l 1), was derived from the sequence upstream of phoA promoter; PPM, 5 '-TCGTGTCATC AAAATCATGGGCTTTTGTCACAGGGGTAA-3 SEQ ID NO: 12), contained partial coding sequence of PhoA leader peptide (underlined) and partial coding region of PFI without leader peptide; and EPR, 5'- GCAGGATCCGTATCGAGCTC T GATT CG-3' (SEQ ID NO: 13) was derived from down stream sequence of PFI coding region of the cDNA clone. To make the fusion construct, two PCR reactions were conducted. The first PCR reaction was done using primers PPF paired with PPM and E. coli K-12 genomic DNA as template. The second PCR reaction was carried out using primers PPF and EPR. The templates for the second PCR reaction were generated by mixing 1 ml of the first PCR product and 1 ml of 1 :50 diluted plasmid DNA of the PFI cDNA clone. The final PCR product was cloned into pCR2.1 using the Topo-TA cloning system (Invitrogen Co. ) and the insert was verified by sequencing of both strands. The E. coli transformants were grown in ECLB media at 37°C to early stationary phase and subsequently harvested for protein analysis by centrifugation.

2B. Plant Expression Constructs A series of constructs are prepared for the expression of the PFI encoding sequences in host plant cells. Constructs are prepared to direct the expression of the PFI encoding sequences constitutively as well as preferentially in particular plant tissues.

A plasmid containing the napin cassette derived from pCGN3223 (described in USPN 5,639,790, the entirety of which is incorporated herein by reference) was modified to make it more useful for cloning large DNA fragments containing multiple restriction sites, and to allow the cloning of multiple napin fusion genes into plant binary transformation vectors. An adapter comprised of the self annealed oligonucleotide of sequence CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTG CAGGGCGCGCCATTTAAAT (SEQ ID NO: 14) was ligated into the cloning vector pBC SK+ (Stratagene) after digestion with the restriction endonuclease BssHII to construct vector pCGN7765. Plasmids pCGN3223 and pCGN7765 were digested with Notl and ligated together. The resultant vector, pCGN7770, contains the pCGN7765 backbone with the napin seed specific expression cassette from pCGN3223.

The cloning cassette, pCGN7787, has essentially the same regulatory elements as pCGN7770, with the exception of the napin regulatory regions of pCGN7770 have been replaced with the double CAMV 35S promoter and the tml polyadenylation and transcriptional termination region.

A binary vector for plant transformation, pCGN5139, was constructed from pCGN1558 (McBride and Summerfelt, (1990) Plant Molecular Biology, 14:269-276). The polylinker of pCGN1558 was replaced as a HindIII/Asp718 fragment with a polylinker containing unique restriction endonuclease sites, Ascl, Pad, Xbal, Swal, BamHI, and Notl. The Asp718 and Hindlll restriction endonuclease sites are retained in pCGN5139. A series of turbo binary vectors were constructed to allow for the rapid cloning of DNA sequences into binary vectors containing transcriptional initiation regions (promoters) and transcriptional termination regions. The plasmid pCGN8618 was constructed by ligating oligonucleotides 5 '-

TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO: 15) and 5'- TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO: 16) into Sall/Xhol double-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3' region was excised from pCGN8618 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt-ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the napin 3' was closest to the blunted Hindlll site. Subsequently, these regions were subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8622.

The plasmid pCGN8619 was constructed by ligating oligonucleotides 5'- TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC -3' (SEQ ID NO: 17) and 5'- TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO: 18) into Sall/Xhol double-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3' region was removed from pCGN8619 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt-ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the napin 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8623.

The plasmid pCGN8620 was constructed by ligating oligonucleotides 5'- TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT -3' (SEQ ID NO: 19) and 5'- CCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO:20) into Sall/Sacl double-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3' region was removed from pCGN8620 by complete digestion with Asp718I and partial digestion with Notl. The fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt-ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8624. The plasmid pCGN8621 was constructed by ligating oligonucleotides 5'-

TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT -3' (SEQ ID NO:21) and 5'- GGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO:22) into Sall/Sacl double-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3' region was removed from pCGN8621 by complete digestion with Asp718I and partial digestion with Notl. The fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hindlll and blunt-ended by filling in the 5' overhangs with Klenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to the blunted Hindlll site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8625.

For cloning in plant expression constructs, the PFI coding regions with or without the putative leader peptide were PCR-amplified using forward primers, PLF1 5'- GGATCCGCGGCCGCATGTCTTTGAATAGAGTTCTTC-3'(with the leader peptide) (SEQ ID NO:23) and PLF2 5 '-GGATCCGCGGCCGCATGGATTTT

GATGACACGATTGC-3' (without the leader peptide) (SEQ ID NO:24), and the reverse primer PLR 5'-CCTGCAGGAAGCTTCTAGAAGA AATCCT TGATC-3'(SEQ ID NO:25). The Notl sites (underlined) were placed upstream of the start codons (in boldface) in primers PLF1 and PLF2 while the Pstl site (underlined) was placed downstream of the stop codon (in boldface) in PLR. The PCR products were first cloned into pCR2.1

(Invitrogen) and the presence of inserts possessing the correct sequence was verified by sequencing of both strands. Two constructs employing the PFI-B2 encoding sequence were prepared in the vector pCGN8622 for expression from the napin promoter. The vector pCGN10108 contains the PFI-B2 encoding sequence containing the native leader sequence. The vector pCGN10109 contains the PFI-B2 encoding sequence lacking the native leader sequence. Two constructs employing the PFI-B2 encoding sequence were prepared in the vector pCGN8624 for expression from the 35S promoter. The vector pCGNlOl 10 contains the PFI-B2 encoding sequence containing the native leader sequence. The vector pCGNlOl 11 contains the PFI-B2 encoding sequence lacking the native leader sequence.

Two constructs employing the PFI-F3 encoding sequence were prepared in the vector pCGN8622 for expression from the napin promoter. The vector pCGNlOl 12 contains the PFI-F3 encoding sequence containing the native leader sequence. The vector pCGNlOl 13 contains the PFI-F3 encoding sequence lacking the native leader sequence.

A single construct employing the PFI-F3 encoding sequence was prepared in the vector pCGN8624 for expression from the 35S promoter. The vector pCGNlOl 14 contains the PFI-B2 encoding sequence containing the native leader sequence.

Example 3 Host Cell Transformation and Analysis

To express the PFI protein in E.coli, constructs were made using the QIAexpressionist system (Qiagen). Transformation and induction of the Ml 5 cells were performed according to the manufacturers protocol. Yeast competent cell preparation and transformation were performed using the

Frozen-EZ yeast transformation kit (Zymo Research). The expression vector employed was pYES2 (Invitrogen) and the yeast strain selected was INVcl (Invitrogen).

A variety of methods have been developed to insert a DNA sequence of interest into the genome of a plant host to obtain the transcription or transcription and translation of the sequence to effect phenotypic changes.

Transgenic Brassica plants were obtained by Agrobacterium-mediated transformation as described by Radke et al. (Theor. Appl. Genet. (1988) 75:685-694; Plant Cell Reports (1992) 11 :499-505). Transgenic Arabidopsis thaliana plants may be obtained by Agrobacterium-mediated transformation as described by Valverkens et 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 may be similarly transformed using related techniques.

Alternatively, microprojectile bombardment methods, such as described by Klein et al. (Bio/Technology 10:286-291) may also be used to obtain nuclear transformed plants. The expressed PFI protein, as well as the PFI protein from the wild-type P. filicina, was assayed as described herein. The enzyme activity was assayed as described by Wise ((1995) PhD Thesis, supra) with some modifications. Preparation of protein crude extract from Ptilota filicina: frozen tissue was ground in a mortar/pestle with liquid nitrogen. Approximately 200 mg of the tissue powder was mixed with 1 ml of the extraction buffer (100 mM NaH2PO4, 5 mM EGTA, 5 mM DTT, and 5 mM MgC12, pH6.5) and homogenized with a glass homogenizer. The homogenate was micro fuged at 14 K rpm for 5 min and the supernatant was collected for the enzyme assays. Similar methods were used for preparation of protein crude extract from transgenic material (E. coli, yeast, Schizochitrium, and Arabidopsis): The sample materials were broken and homogenized in the extraction buffer and microcentrifuged. The supernatant was used for the enzyme assay.

The collected supernatant was analyzed for PFI activity as follows. About 20 ml of the protein crude 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 blanked. The reaction was initiated by adding 2 ml of the stock substrate solution and the conjugated fatty acid formation was measured by wave length scanning between

200nm and 300nm. Specifically, the conjugated triene and diene formation was measured by monitoring absorbance at 278nm and 234nm, respectively. The stock 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 are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claim.

Claims

CLAIMSWhat is claimed is:

1. An isolated DNA sequence encoding an enzyme active in the formation of conjugated fatty acids from polyenoic fatty acyl substrates.

2. The isolated DNA sequence according to Claim 1 wherein said nucleic acid sequence encodes polyenoic fatty acid isomerase.

3. The isolated DNA sequence according to Claim 1 wherein said nucleic acid sequence is isolated from an eukaryotic cell source.

4. The isolated DNA sequence according to Claim 3 wherein said eukaryotic cell source is selected from the group consisting of fungal, and plant cells.

5. The DNA encoding sequence of Claim 4 wherein said DNA sequence is from Ptilota filicina.

6. The DNA encoding sequence of Claim 4 wherein said polyenoic fatty acid isomerase protein is encoded by a sequence which includes a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1 and 3.

7. An isolated polypeptide comprising the 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 which has at least approximately 70% identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; d) an isolated polynucleotide comprising a nucleotide sequence which has at least approximately 80%ι identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; e) an isolated polynucleotide comprising a nucleotide sequence which has at least approximately 90%> identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; f) an isolated polynucleotide comprising a nucleotide sequence which has at least approximately 95%> identity to 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 which has at least approximately 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 which has at least approximately 80%> identity to that of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; e) an isolated polynucleotide comprising a nucleotide sequence which has at least approximately 90%> identity to that of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; f) an isolated polynucleotide comprising a nucleotide sequence which has at least approximately 95%> identity to 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 as operably linked components in the 5' to 3' direction of transcription: a transcriptional initiation region; and a polynucleotide sequence encoding an enzyme active in the formation of conjugated fatty acids from polyenoic fatty acyl substrates.

12. The nucleic acid construct according to Claim 11, wherein said enzyme is polyenoic fatty acid isomerase.

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

14. The host cell according to Claim 13, wherein said host cell is selected from the group consisting of bacterial, insect, fungal, mammalian, and plant.

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

16. The organism according to claim 15 wherein such organism is selected from the group consisting of bacterial, insect, fungal, mammalian and plant.

17. The organism of claim 16 wherein such organism is a plant.

18. A method for producing a recombinant host cell, comprising: transforming or transfecting a cell with a nucleic acid construct comprising a transcriptional initiation region and a polynucleotide sequence encoding an enzyme active in the formation of conjugated fatty acids from polyenoic fatty acyl substrates, such that said host cell, under appropriate culture conditions, produces a polyenoic fatty acid isomerase protein.

19. The method according to claim 18 wherein said host cell is selected from the group consisting of plant cells, bacterial cells, yeast cells, and algal cells.

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

21. The organism according to claim 20 wherein said organism is selected from the group consisting of bacteria, yeast, algal and plant.

22. The organism of claim 21 wherein said organism is a plant.

23. A method for producing a recombinant host cell, comprising: 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 claim 9 and a polynucleotide according to claim 10, such that said host cell, under appropriate culture conditions, produces a polyenoic fatty acid isomerase protein.

24. The method according to claim 23 wherein said polynucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1.

25. The method according to claim 23 wherein 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 wherein the organism is a plant.

28. A method of modifying the fatty acid composition in a host cell, said method comprising: transforming a host cell with a nucleic acid construct comprising a transcriptional initiation region and a polynucleotide sequence encoding an enzyme active in the formation of conjugated fatty acids from polyenoic fatty acyl substrates, such that said host cell, under appropriate culture conditions, produces a polyenoic fatty acid isomerase protein.

29. The method according to Claim 28 wherein said production of a polyenoic fatty acid isomerase produces an increase of conjugated fatty acids in said host cell.

30. The method according to Claim 28 wherein 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 wherein 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 28-30.

33. The organism according to claim 32 wherein said organism is selected from the group consisting of plant, bacteria and fungi.

34. The organism of claim 33 wherein said organism is a plant.