US0075892.53B2

(12) United States Patent (10) Patent No.: US 7,589.253 B2 Green et al. (45) Date of Patent: Sep. 15, 2009

(54) FATTY ACID EPOXYGENASE GENES FROM Blee et al. (1993) “Regio-and stereoselectivity of cytochrome P-450 PLANTS AND USES THEREFOR IN and peroxygenase-dependent formation of CIS-12, 13-epoxy-9(Z)- MODIFYING FATTY ACID METABOLISM octadecenoic acid (vernolic acid) in Euphorbia lagascae'' Biochemi cal and Biophysical Research Communications 197(2):778-784. Blee et al. (1993) “Mechanism of reaction of fatty acid (75) Inventors: Allan Green, Bradoon (AU); Surinder hydroperoxides with soybean peroxygenase' The Journal of Biologi Singh, Downer (AU); Marit Lenman, cal Chemistry 268(3): 1708-1715. Lund (SE); Sten Stymine, Svalov (SE) Blee and Schuber (1990) “Efficient epoxidation of unsaturated fatty acids by a hydroperoxide-dependent oxygenase' The Journal of Bio (73) Assignee: Commonwealth Scientific and logical Chemistry 265(22): 12887-12894. Industrial Research Organisation, Bozak et al. (1990) “Sequence analysis of ripening-related Campbell (AU) cytochrome P-450 cDNAs from avocado fruit” Proc. Natl. Acad. Sci. USA 87:3904-3908. (*) Notice: Subject to any disclaimer, the term of this Dolferus et al. (1994) “Differential Interactions of promoter elements patent is extended or adjusted under 35 in stress responses of the Arabidopsis adh gene' Plant Physiol. U.S.C. 154(b) by 177 days. 105:1075-1087. Engeseth and Stymne (Feb. 1996) “Desaturation of oxygenated fatty (21) Appl. No.: 09/981,124 acids in Lesquerella and other oil seeds' Planta 198:238-245. Needleman and Wunsch (1970) “A General method applicable to the (22) Filed: Oct. 17, 2001 search for similarities in the amino acid sequence of two proteins' J. Mol. Biol. 48:443-453. (65) Prior Publication Data Shanklin et al. (1994) “Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, Stearoyl-coa US 2002/0166144 A1 Nov. 7, 2002 desaturase, and are conserved in alkane hydroxylase and Xylene monooxygenase” Biochemistry 33:12787-12794. Related U.S. Application Data Valvekens et al. (1988)"Agrobacterium tumefaciens-mediated trans (63) Continuation-in-part of application No. 09/059.769, formation of Arabidopsis thaliana root explants by using kanamycin filed on Apr. 14, 1998, now Pat. No. 6,329,518. Selection Proc. Natl. Acad. Sci. USA 85:5536-5540. Capdevila, J.H., et al., “Cytochrome P-450 arachidonate oxygenase” (60) Provisional application No. 60/050,403, filed on Jun. (1990) methods in enzymology 187:385-394. 20, 1997, provisional application No. 60/043,706, Christian , M.F. and Yu, S.J., “Cytochrome p-450-dependent filed on Apr. 16, 1997. monooxygenase activity in the velvetbean caterpillar, Anticarsia gemmatalis hubner'(1986) Comparative Biochemistry and Physiol (30) Foreign Application Priority Data ogy 83C(1):23-27. Romero, M.F. et al., “An epoxygenase metabolite of arachidonic acid Apr. 15, 1997 (AU) ...... PO6223.797 5.6 epoxy-eicosatrienoic acid mediates angiotensin-induced Apr. 15, 1997 (AU) PO6226797 natriuresis in proximal tubular epithelium”(1991) Advances in Prostaglandin, Thromboxane and Leukotriene Research 21:205-208. (51) Int. Cl. CI2N 5/82 (2006.01) (Continued) AOIH 5/00 (2006.01) Primary Examiner Elizabeth F McElwain (52) U.S. Cl...... 800/281; 435/419 (74) Attorney, Agent, or Firm John P. White; Cooper & (58) Field of Classification Search ...... 800/298, Dunham LLP 800/281,306, 317, 314, 322; 435/416, 468 See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention relates generally to novel genetic U.S. PATENT DOCUMENTS sequences that encode fatty acid epoxygenase enzymes, in 5,850,026 A * 12/1998 DeBonte et al...... 800/281 particular fatty acid A12-epoxygenase enzymes from plants 6,329,518 B1 12/2001 Green et al...... 536,236 that are mixed function monooxygenase enzymes. More par ticularly, the present invention exemplifies cDNA sequences FOREIGN PATENT DOCUMENTS from Crepis spp. and Vermonia galamensis that encode fatty WO WO 89,05852 6, 1989 acid A12-epoxygenases. The genetic sequences of the present WO WO96/10074 4f1996 invention provide the means by which fatty acid metabolism WO WO 97.37033 10, 1997 may be altered or manipulated in organisms, such as, for example, yeasts, moulds, bacteria, insects, birds, mammals OTHER PUBLICATIONS and plants, and more particularly in plants. The invention also Bafor et al. (1993) “Biosynthesis of vernoleate (cis-12 extends to genetically modified oil-accumulating organisms eposyoctadeca-cis-9-enoate) in microsomal preparations from devel transformed with the Subject genetic sequences and to the oils oping endosperm of Euphorbia lagascae''Archives of Biochemistry derived therefrom. The oils thus produced provide the means and Biophysics 303(1): 145-151. for the cost-effective raw materials for use in the efficient Banas et al. (Feb. 1997) In: Williams, J.P. Mobasher, K.U., Lem. production of coatings, resins, glues, plastics, Surfactants and N.W. (Eds) Physiol-ogy, biochemistry and molecular biology of lubricants. plant lipids. Kluwer Academic Publisher, Dordrecht. In press. “Biosynthesis of an Acetylenic Fatty Acid in Microsomal Prepara tions From Developing Seeds of Crepis alpina, pp. 57-59. 17 Claims, 19 Drawing Sheets US 7,589.253 B2 Page 2

OTHER PUBLICATIONS Hamberg and Fahlstadius (1992) On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.); Plant Physiol. 99.987 Laethem, R.M. et al., “Epoxidation of C1s unsaturated fatty acids by 995. cytochromes P4502C2 and P4502CAA” (Jun. 1996) Drug Metabo Heppard et al. (1996) “Developmental and Growth Temperature lism and Disposition 24(6):664-668. Regulation of Two Different Microsomal (O-6 Desaturase Genes in Soybeans”; Plant Physiol. 110:311-319. Lee, M. et al., Identification of non-heme dironproteins that catalyze Okuley et al. (1994) “Arabidopsis FAD2 Gene Encodes the Enzyme triple bond and epoxy group formation (May 8, 1998) Science That Is Essential for Polyunsaturated Lipid Synthesis”; Plant Cell 280:915-918. 6:147-158. Van de Loo, F.J. et al., “An oleate 12-hydroxylase from Ricinus U.S. Appl. No. 1 1/699.817, filed Jan. 30, 2007 and file history communis 1. is a fatty acyl desaturase homolog” (Jul. 1995) Proc. thereof, Allan Green et al. Natl. Acad. Sci. USA 92:6743-6747. * cited by examiner U.S. Patent US 7,589.253 B2

U.S. Patent Sep. 15, 2009 Sheet 10 of 19 US 7,589.253 B2

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FIGURE 10 US 7,589.253 B2 1. 2 FATTY ACD EPOXYGENASE GENES FROM BACKGROUND TO THE INVENTION PLANTS AND USES THEREFORN MODIFYING FATTY ACID METABOLISM There is considerable interest world-wide in producing chemical feedstock, Such as fatty acids, for industrial use RELATED APPLICATION DATA from renewable plant sources rather than from non-renewable petrochemicals. This concept has broad appeal to manufac This application is a continuation-in-part of U.S. Ser. No. turers and consumers on the basis of resource conservation 09/059,769, filed Apr. 14, 1998, now U.S. Pat. No. 6,329,518, and provides a significant opportunity to develop new indus issued Dec. 11, 2001, which claims the benefit of U.S. Pro trial crops for agriculture. visional Application No. 60/050,403, filed Jun. 20, 1997, and 10 There is a diverse array of unusual fatty acids in nature and U.S. Provisional Application No. 60/043,706, filed Apr. 16, these have been well characterized (Badam & Patil, 1981; 1997, and claims priority of Australian Patent Application Smith, 1970). Many of these unusual fatty acids have indus PO6223/97, filed Apr. 15, 1997, and Australian Patent Appli trial potential and this has led to interestin domesticating Such cation PO6226/97, filed Apr. 15, 1997, the contents of which species to enable agricultural production of particular fatty are hereby incorporated by reference. 15 acids. One class offatty acids of particular interest are the epoxy FIELD OF THE INVENTION fatty acids, consisting of an acyl chain in which two adjacent carbonbonds are linked by an epoxy bridge. Due to their high The present invention relates generally to novel genetic reactivity, they have considerable application in the produc sequences that encode fatty acid epoxygenase enzymes. In tion of coatings, resins, glues, plastics, Surfactants and lubri particular, the present invention relates to genetic sequences cants. These fatty acids are currently produced by chemical that encode fatty acid A12-epoxygenase enzymes as defined epoxidation of vegetable oils, mainly soybean oil and linseed herein. More particularly, the present invention provides oil, however this process produces mixtures of multiple and cDNA and genomic gene sequences that encode plant fatty isomeric forms and involves significant processing costs. acid epoxygenases, in particular from Crepis palaestina or 25 Attempts are being made by others to develop some wild Vermonia galamensis. The genetic sequences of the present plants that contain epoxy fatty acids (e.g. Euphorbia lagas invention provide the means by which fatty acid metabolism cae, or Vermonia galamensis) into commercial Sources of may be altered or manipulated in organisms such as yeasts, these oils. However, problems with agronomic suitability and moulds, bacteria, insects, birds, mammals and plants, in par low yield potential severely limit the commercial utility of ticular to convert unsaturated fatty acids to epoxy fatty acids 30 traditional plant breeding and cultivation approaches. therein. The invention extends to genetically modified oil The rapidly increasing sophistication of recombinant DNA accumulating organisms transformed with the subject genetic technology is greatly facilitating the efficiency of commer sequences and to the oils derived therefrom. The oils thus cially-important industrial processes, by the expression of produced provide the means for the cost-effective raw mate genes isolated from a first organism or species in a second rials for use in the efficient production of coatings, resins, 35 organism or species to confer novel phenotypes thereon. glues, plastics, Surfactants and lubricants, amongst others. More particularly, conventional industrial processes can be made more efficient or cost-effective, resulting in greater General yields per unit cost by the application of recombinant DNA Those skilled in the art will be aware that the present techniques. invention is Subject to variations and modifications other than 40 Moreover, the appropriate choice of host organism for the those specifically described herein. It is to be understood that expression of a genetic sequence of interest provides for the the invention includes all Such variations and modifications. production of compounds that are not normally produced or The invention also includes all such steps, features, compo synthesized by the host, at a high yield and purity. sitions and compounds referred to or indicated in this speci However, despite the general effectiveness of recombinant fication, individually or collectively, and any and all combi 45 DNA technology, the isolation of genetic sequences which nations of any two or more of said steps or features. encode important enzymes in fatty acid metabolism, in par Throughout this specification, unless the context requires ticular the genes which encode the fatty acid A12-epoxyge otherwise, the word “comprise', or variations such as “com nase enzymes responsible for producing 12,13-epoxy-9-oc prises' or “comprising, will be understood to imply the tadecenoic acid (vernolic acid) and 12,13-epoxy-9.15 inclusion of a stated integer or group of integers but not the 50 octadecadienoic acid, amongst others, remains a major exclusion of any other integer or group of integers. obstacle to the development of genetically-engineered organ Bibliographic details of the publications referred to by isms which produce these fatty acids. author in this specification are collected at the end of the Until the present invention, there were only limited bio description. chemical data indicating the nature offatty acid epoxygenase This specification contains nucleotide sequence informa 55 enzymes, in particular A12-epoxygenases. However, in tion prepared using the program PatentIn Version 3.1 pre Euphorbia lagascae, the formation of 12,13-epoxy-9-octade sented herein after the claims. Each nucleotide sequence is cenoic acid (vernolic acid) from linoleic acid appears to be identified in the sequence listing by the numeric indicator catalyzed by a cytochrome-P450-dependent A12 epoxyge <210> followed by the sequence identifier e.g. <210>1, nase enzyme (Bafor et al., 1993; Blee et al., 1994). Addition <210>2, etc. The length, type of sequence DNA, protein 60 ally, developing seed of linseed plants have the capability to (PRT), etc and source organism for each nucleotide sequence convert added vernolic acid to 12,13-epoxy-9.15-octadecadi are indicated by information provided in the numeric indica enoic acid by an endogenous A15 desaturase (Engeseth and tor fields <211 >, <212> and <213>, respectively. Nucleotide Stymine, 1996). Epoxy-fatty acids can also be produced by a sequences referred to in the specification are defined by the peroxide-dependent peroxygenase in plant tissues (Blee and term “SEQID NO:”, followed by the sequence identifier e.g. 65 Schuber, 1990). SEQID NO: 1 refers to the sequence in the sequence listing In work leading up to the present invention, the inventors designated as <400>1. sought to isolate genetic sequences which encode genes US 7,589.253 B2 3 4 which are important for the production of epoxy-fatty acids, (ii) transforming said gene construct into a cell or tissue of Such as 12,13-epoxy-9-octadecenoic acid (vernolic acid) or said plant; and 12,13-epoxy-9.15-octadecadienoic acid and to transfer these (iii) selecting transformants which express a functional genetic sequences into highly productive commercial oilseed epoxygenase encoded by the genetic sequence at a high plants and/or other oil accumulating organisms. 5 level in seeds. A further aspect of the invention provides a recombinant SUMMARY OF THE INVENTION epoxygenase polypeptide or functional enzyme molecule. A further aspect of the invention provides a recombinant One aspect of the invention provides an isolated nucleic epoxygenase which comprises a sequence of amino acids set acid which encodes or is complementary to an isolated 10 forth in any one of SEQID NOS: 2 or 4 or 6 or 20 or 20 or a nucleic acid which encodes a fatty acid epoxygenase. homologue, analogue or derivative thereof which is at least A second aspect of the invention provides an isolated about 50% identical thereto. More preferably, the percentage nucleic acid which hybridizes under at least low stringency identity to any one of SEQID NOS: 2 or 4 or 6 or 20 or 20 is conditions to at least 20 contiguous nucleotides of SEQ ID at least about 65%. NOs: 1 or 3 or 5 or 19 or 19, or a complementary sequence 15 A still further aspect of the invention provides a method of thereto. producing an epoxy fatty acid in a cell, tissue, organ or organ A further aspect of the invention provides isolated nucleic ism, said method comprising incubating a cell, tissue, organ acid comprising a sequence of nucleotides selected from the or organism which expresses an enzymatically active recom group consisting of binant epoxygenase with a fatty acid Substrate and preferably, (i) a nucleotide sequence that is at least 65% identical to a an unsaturated fatty acid Substrate, for a time and under con sequence selected from the group consisting of SEQ ID ditions sufficient for at least one carbon bond, preferably a NO: 1, SEQID NO:3, SEQID NO:5, and SEQID NO:19; carbon double bond, of said substrate to be converted to an (ii) a nucleotide sequence that encodes an amino acid epoxy group. sequence that is at least about 50% identical to a sequence A further aspect of the invention provides an immunologi selected from the group consisting of: SEQID NO: 2, SEQ 25 cally interactive molecule which binds to the recombinant ID NO: 4, SEQID NO: 6, and SEQID NO. 20; and epoxygenase polypeptide described herein or a homologue, (iii) a nucleotide sequence that is complementary to (i) or (ii). analogue or derivative thereof. A further aspect of the invention provides a gene construct that comprises the isolated nucleic acid Supra, in either the BRIEF DESCRIPTION OF THE DRAWINGS sense or antisense orientation, in operable connection with a 30 promoter sequence. FIG. 1 is a linear representation of an expression plasmid A further aspect of the invention provides a method of comprising an epoxygenase structural gene, placed operably altering the level of epoxy fatty acids in a cell, tissue, organ or under the control of the truncated napin promoter (FP1; right organism, said method comprising expressing a sense, anti hand hatched box) and placed upstream of the NOS termina sense, ribozyme or co-suppression molecule comprising the 35 tor sequence (right-hand stippled box). The epoxygenase isolated nucleic acid Supra in said cell, tissue, organ or organ genetic sequence is indicated by the right-hand open rectan ism for a time and under conditions sufficient for the level of gular box. The construct also comprises the NOS promoter epoxy fatty acids therein to be increased or reduced. (left-hand hatched box) driving expression of the NPTII gene A further aspect of the invention provides a method of (left-hand open box) and placed upstream of the NOS termi producing a recombinant enzymatically active epoxygenase 40 nator (left-hand stippled box). The left and right border polypeptide in a cell, said method comprising expressing the sequences of the Agrobacterium tumefaciens Tiplasmid are isolated nucleic acid Supra in said cell for a time and under also indicated. conditions sufficient for the epoxygenase encoded therefor to FIG. 2 is a schematic representation showing the alignment be produced. of the amino acid sequences of the epoxygenase polypeptide 45 of Crepis palaestina (Cpa12; SEQ ID NO: 2), a further A further aspect of the invention provides a method of epoxygenase derived from Crepis sp. other than C. palaestina producing a recombinant enzymatically active epoxygenase which produces high levels of vernolic acid (CrepX; SEQID polypeptide in a cell, said method comprising the steps of NO: 4), a partial amino acid sequence of an epoxygenase (i) producing a gene construct which comprises the iso polypeptide derived from Vernonia galamensis (Vgall; SEQ lated nucleic acid Supra placed operably under the con 50 ID NO: 6), a full-length amino acid sequence of an epoxyge trol of a promoter capable of conferring expression on nase polypeptide derived from Vermonia galamensis (SEQID said genetic sequence in said cell, and optionally an NO: 20), the amino acid sequence of the A12 acetylenase of expression enhancer element; Crepis alpina (Crep 1: SEQID NO: 8), the A12 desaturase of (ii) transforming said gene construct into said cell; and A. thaliana (L26296; SEQ ID NO: 9), Brassica iuncea (iii) selecting transformants which express a functional 55 (X91139; SEQ ID NO: 10), Glycine max (L43921; SEQ ID epoxygenase encoded by the genetic sequence at a high NO: 11), Solanum commersonii (X92847: SEQ ID NO: 12) level. and Glycine max (L43920; SEQ ID NO: 13), and the A12 A still further aspect of the invention provides a method of hydroxylase of Ricinus communis (U22378; SEQ ID NO: producing a recombinant and enzymatically active epoxyge 14). Underlined are three histidine-rich motifs that are con nase polypeptide in a transgenic plant comprising the steps 60 served in non-heme containing mixed-function monooxyge of: aSCS. (i) producing a gene construct which comprises the iso FIG. 3 is a copy of a photographic representation of a lated nucleic acid Supra placed operably under the con northern blot hybridization showing seed-specific expression trol of a seed-specific promoter and optionally an of the Crepis palaestina epoxygenase gene exemplified by expression enhancer element, wherein said genetic 65 SEQ ID NO: 1. Northern blot analysis of total RNA from sequences is also placed upstream of a transcription leaves (lane 1) and developing seeds (lane 2) of Crepis terminator sequence; palaestina. 15ug of total RNA was run on a Northern gel and US 7,589.253 B2 5 6 blotted onto Hybond N' membrane from Amersham accord arachidonic acid, it is particularly preferred that the subject ing to the manufacturer's instructions. The blot was hybrid nucleic acid is derived from a non-mammalian source. ized at 60° C. with a probe made from the 3' untranslated As used herein, the term "derived from shall be taken to region of SEQIDNO: 1. The blot was washed twice in 2xSSC indicate that a particular integer or group of integers has (NaCl Sodium Citrate buffer) at room temperature for 10 originated from the species specified, but has not necessarily minutes, then in 0.1XSSC at 60° C. for 20 min. been obtained directly from the specified source. FIG. 4 is a schematic representation of a binary plasmid The term “non-mammalian source' refers to any organism vector containing an expression cassette comprising the trun other than a mammal or a tissue or cell derived from same. In cated napin seed-specific promoter (Napin) and nopaline Syn the present context, the term "derived from a non-mammalian thase terminator (NT), with a BamHI cloning site there 10 Source' shall be taken to indicate that a particular integer or between, in addition to the kanamycin-resistance gene NPTII group of integers has been derived from bacteria, yeasts, operably connected to the nopaline synthase promoter (NP) birds, amphibians, reptiles, insects, plants, fungi, moulds and and nopaline synthase terminator (NT) sequences. The algae or other non-mammal. expression cassette is flanked by T-DNA left border (LB) and In a preferred embodiment of the present invention, the right-border (RB) sequences. 15 Source organism is any Such organism possessing the genetic FIG. 5 is a schematic representation of a binary plasmid capacity to synthesize epoxy fatty acids. More preferably, the vector containing an expression cassette which comprises Source organism is a plant Such as, but not limited to Chry SEQ ID NO: 1 placed operably under the control of a trun Santhemum spp., Crepis spp., Euphorbia spp. and Vermonia cated napin seed-specific promoter (Napin) and upstream of spp., amongst others. the nopaline synthase terminator (NT), in addition to the Even more preferably, the source organism is selected from kanamycin-resistance gene NPTII operably connected to the the group consisting of Crepis biennis, Crepis aurea, Crepis nopaline synthase promoter (NP) and nopaline synthase ter conyzaefolia, Crepis intermedia, Crepis Occidentalis, Crepis minator (NT) sequences. The expression cassette is flanked palaestina, Crepis vesicaria, Crepis xacintha, Euphorbia by T-DNA left border (LB) and right-border (RB) sequences. lagascae and Vermonia galamensis. Additional species are To produce this construct, SEQID NO: 1 is inserted into the 25 not excluded. BamHI site of the binary vector set forth in FIG. 4. In a particularly preferred embodiment of the present FIG. 6 is a graphical representation of gas-chromatography invention, the Source organism is a Crepis sp. comprising traces of fatty acid methyl esters prepared from oil seeds of high levels of Vernolic acid such as Crepis palaestina, untransformed Arabidopsis thaliana plants panel (a), or A. amongst others or alternatively, Vermonia galamensis. thaliana plants (transgenic line Cpal-17) which have been 30 Wherein the isolated nucleic acid of the invention encodes transformed with SEQID NO: 1 using the gene construct set a A6-epoxygenase or A9-epoxygenase enzyme or A12-ep forth in FIG. 5 panels (b) and (c). In panels (a) and (b), fatty oxygenase or A15-epoxygenase enzyme, or at least encodes acid methyl esters were separated using packed column sepa an enzyme which is not involved in the direct epoxidation of ration. In panel (c), the fatty acid methyl esters were separated arachidonic acid, the Subject nucleic acid may be derived using capillary column separation. The elution positions of 35 from any source producing said enzyme, including, but not Vernolic acid are indicated. limited to, yeasts, moulds, bacteria, insects, birds, mammals FIG. 7 is a graphical representation showing the joint dis and plants. tribution of epoxy fatty acids in selfed seed on T plants of The nucleic acid of the invention according to any of the Cpal2-transformed Arabidopsis thaliana plants as deter foregoing embodiments may be DNA, such as a gene, cDNA mined using gas chromatography. Levels of both Vernolic 40 molecule, RNA molecule or a synthetic oligonucleotide mol acid (X-axis) and 12,13-epoxy-9.15-octadecadienoic acid ecule, whether single-stranded or double-stranded and irre (y-axis) were determined and plotted relative to each other. spective of any secondary structure characteristics unless spe Data show a positive correlation between the levels of these cifically stated. fatty acids in transgenic plants. Reference herein to a “gene' is to be taken in its broadest FIG. 8 is a graphical representation showing the incorpo 45 context and includes: ration of ''C-label into the chloroform phase obtained from (i) a classical genomic gene consisting of transcriptional and/ lipid extraction of linseed cotyledons during labeled-sub or translational regulatory sequences and/or a coding strate feeding. Symbols used; 0, 'Coleic acid feeding: , region and/or non-translated sequences (i.e. introns, 5'- and '''C vernolic acid feeding. 3'-untranslated sequences); or FIG. 9 is a graphical representation showing the incorpo 50 (ii) mRNA or cDNA corresponding to the coding regions (i.e. ration of C-label into the phosphatidylcholine of linseed exons) and 5'- and 3'-untranslated sequences of the gene. cotyledons during labeled-substrate feeding. Symbols used; The term “gene' is also used to describe synthetic or fusion 0, ''Coleic acid feeding: , ''C vernolic acid feeding. molecules encoding all or part of a functional product. Pre FIG. 10 is a graphical representation showing the incorpo 55 ferred epoxygenase genes of the present invention may be ration of C-label into the triacylglycerols of linseed cotyle derived from a natural epoxygenase gene by standard recom dons during labeled-substrate feeding. Symbols used 0. binant techniques. Generally, an epoxygenase gene may be ''Coleic acid feeding: , ''C vernolic acid feeding. Subjected to mutagenesis to produce single or multiple nucle otide Substitutions, deletions and/or additions. DETAILED DESCRIPTION OF THE PREFERRED 60 Insertions are those variants in which one or more nucle EMBODIMENTS otides are introduced into a predetermined site in the nucle otide sequence, although random insertion is also possible One aspect of the present invention provides an isolated with suitable screening of the resulting product. Nucleotide nucleic acid which encodes or is complementary to an iso insertions include 5' and 3' terminal fusions as well as intra lated nucleic acid which encodes a fatty acid epoxygenase. 65 sequence insertions of single or multiple nucleotides. Wherein the isolated nucleic acid of the invention encodes Deletions are variants characterized by the removal of one an enzyme which is involved in the direct epoxidation of or more nucleotides from the sequence. US 7,589.253 B2 7 8 Substitutions are those variants in which at least one nucle Preferably, the substrate molecule for the epoxygenase of otide in the sequence has been removed and a different nucle the present invention is an unsaturated fatty acid comprising otide inserted in its place. Such a substitution may be “silent” at least one double bond. in that the Substitution does not change the amino acid defined Furthermore, epoxygenase enzymes may act upon any by the codon. Alternatively, a conservative Substitution may 5 number of carbon atoms in any one Substrate molecule. For alter one amino acid for another similar acting amino acid, or example, they may be characterized as A6-epoxygenase, an amino acid of like charge, polarity, or hydrophobicity. A9-epoxygenase, A12-epoxygenase or A15-epoxygenase enzymes amongst others. Accordingly, the present invention In the context of the present invention, the term “fatty acid is not limited by the position of the carbon atom in the sub epoxygenase' shall be taken to refer to any enzyme or func 10 strate upon which an epoxygenase enzyme may act. tional equivalent or enzymatically-active derivative thereof The term “A6-epoxygenase' as used herein shall be taken that catalyzes the biosynthesis of an epoxy fatty acid, by to refer to an epoxygenase enzyme which catalyzes the con converting a carbon bond of a fatty acid to an epoxy group and version of the A6 carbon bond of a fatty acid substrate to a A6 preferably, by converting a carbon double bond of an unsat epoxy group and preferably, catalyzes the conversion of the urated fatty acid to an epoxy group. Although not limiting the 15 A6 double bond of at least one unsaturated fatty acid to a A6 invention, a fatty acid epoxygenase may catalyze the biosyn epoxy group. thesis of an epoxy fatty acid selected from the group consist The term “A9-epoxygenase' as used herein shall be taken ing of: (i) 12,13-epoxy-9-octadecenoic acid (vernolic acid); to refer to an epoxygenase enzyme which catalyzes the con (ii) 12,13-epoxy-9.15-octadecadienoic acid; (iii) 15, 16-ep version of the A9 carbon bond of a fatty acid substrate to a A9 oxy-9,12-octadecadienoic acid; (iv) 9,10-epoxy-12-octade epoxy group and preferably, catalyzes the conversion of the cenoic acid; and (V) 9,10-epoxy-octadecanoic acid. A9 double bond of at least one unsaturated fatty acid to a A9 The term “epoxy’, or “epoxy group' or “epoxy residue' epoxy group. will be known by those skilled in the art to refer to a three As used herein, the term "A 12-epoxygenase' shall be taken member ring comprising two carbon atoms and an oxygen to refer to an epoxygenase enzyme which catalyzes the con 25 version of the A12 carbon bond of a fatty acid substrate to a atom linked by single bonds as follows: A12 epoxy group and preferably, catalyzes the conversion of the A12 double bond of at least one unsaturated fatty acid to a A12 epoxy group. As used herein, the term “A 15-epoxygenase' shall be taken 30 to refer to an epoxygenase enzyme which catalyzes the con version of the A15 carbon bond of a fatty acid substrate to a A 15 epoxy group and preferably, catalyzes the conversion of the A115 double bond of at least one unsaturated fatty acid to Accordingly, the term “epoxide” refers to a compound that a A15 epoxy group. comprise at least one epoxy group as herein before defined. 35 The present invention clearly extends to genetic sequences Those skilled in the art are aware that fatty acid nomencla which encode all of the epoxygenase enzymes listed Supra, ture is based upon the length of the carbon chain and the amongst others. position of unsaturated carbon atoms within that carbon In one preferred embodiment of the invention, the isolated chain. Thus, fatty acids are designated using the shorthand nucleic acid encodes a fatty acid epoxygenase enzyme which notation: 40 converts at least one carbonbond in palmitoleic acid (16:1^), (Carbon),(carbon double oleic acid (18:1°), linoleic acid (18:2^*), linolenic acid carbon double bond(A) position bonds), s (18:3^*'), or arachidonic acid (20:4::'''') to an epoxy bond. Preferably, the carbon bond is a carbon double bond. wherein the double bonds are cis unless otherwise indicated. More preferably, the isolated nucleic acid of the invention For example, palmitic acid (n-hexadecanoic acid) is a Satu 45 encodes a fatty acid epoxygenase enzyme that at least con rated 16-carbon fatty acid (i.e. 16:0), oleic acid (octadecenoic verts one or both double bonds in linoleic acid to an epoxy acid) is an unsaturated 18-carbon fatty acid with one double group. According to this embodiment, an epoxygenase which bond between C-9 and C-10 (i.e. 18:1°), and linoleic acid converts both the A9 and the A12 double bonds of linoleic acid (octadecadienoic acid) is an unsaturated 18-carbon fatty acid to an epoxy group may catalyze such conversions indepen with two double bonds between C-9 and C-10 and between 50 dently of each other Such that said epoxygenase is a A9-ep C-12 and C-13 (i.e. 18:2^*). oxygenase and/or a A12-epoxygenase enzyme as herein However, in the present context an epoxygenase enzyme before defined. may catalyze the conversion of any carbon bond to an epoxy In an alternative preferred embodiment, the fatty acid group or alternatively, the conversion of any double in an epoxygenase of the present invention is a A12-epoxygenase, unsaturated fatty acid Substrate to an epoxy group. In this 55 a A 15-epoxygenase or a A9-epoxygenase as herein before regard, it is well-known by those skilled in the art that most defined. mono-unsaturated fatty acids of higher organisms are 18-car More preferably, the fatty acid epoxygenase of the inven bonunsaturated fatty acids (i.e. 18:1°), while most polyun tion is a A12-epoxygenase as herein before defined. saturated fatty acids derived from higher organisms are In a particularly preferred embodiment of the invention, 18-carbon fatty acids with at least one of the double bonds 60 there is provided an isolated nucleic acid which encodes therein located between C-9 and C-10. Additionally, bacteria linoleate A12-epoxygenase, the enzyme which at least con also possess C16-mono-unsaturated fatty acids. Moreover, verts the A12 double bond of linoleic acid to a A12-epoxy the epoxygenase of the present invention may act on more group, thereby producing 12,13-epoxy-9-octadecenoic acid than a single fatty acid substrate molecule and, as a conse (vernolic acid). quence, the present invention is not to be limited by the nature 65 Although not limiting the present invention, the preferred of the Substrate molecule upon which the Subject epoxyge Source of the A12-epoxygenase of the invention is a plant, in nase enzyme acts. particular Crepis palaestina or a further Crepis sp. which is US 7,589.253 B2 10 distinct from C. palaestina but contains high levels of Ver Crepis sp (SEQID NO: 4), and Vermonia galamensis (SEQID nolic acid, or Vermonia galamensis. NO: 20), suggests functional similarity between these According to this embodiment, a A12-epoxygenase may polypeptides. In contrast, the amino acid sequences of these catalyze the conversion of palmitoleic acid to 9,10-epoxy epoxygenases have lower identity to the amino acid palmitic acid and/or the conversion of oleic acid to 9,10 sequences of a fatty acid desaturase or a fatty acid hydroxy epoxy-Stearic acid and/or the conversion of linoleic acid to lase. any one or more of 9,10-epoxy-12-octadecenoic acid or It is even more preferred that the epoxygenase of the 12,13-epoxy-9-octadecenoic acid or 9,10,12,13-diepoxy present invention at least comprises a sequence of amino Stearic acid and/or the conversion of linolenic acid to any one acids which comprises three histidine-rich regions as follows: or more of 9,10-epoxy-12, 15-octadecadienoic acid or 12,13 10 (i) His-Glu-Cys-Gly-His-His (SEQ ID NO: 15); epoxy-9.15-octadecadienoic acid or 15, 16-epoxy-octadeca dienoic acid or 9,10,12,13-diepoxy-15-octadecenoic acid or (ii) His-Arg-Asn-His-His (SEQID NO: 16); and 9,10,15, 16-diepoxy-12-octadecenoic acid or 12,13,15, 16 (iii) His-Val-Met-His-His (SEQ ID NO: 17) or His-Val diepoxy-9-octadecenoic acid or 9,10,12,13,15, 16-triepoxy Leu-His-His (SEQ ID NO: 18), Stearic acid and/or the conversion of arachidonic acid to any 15 wherein His designates histidine, Glu designates glutamate, one or more of 5,6-epoxy-8,11,14-tetracosatrienoic acid or Cys designates cysteine, Gly designates glycine, Arg desig 8.9-epoxy-5,11,14-tetracosatrienoic acid or 11,12-epoxy-5, nates arginine, ASn designates asparagine, Val designates 8,14-tetracosatrienoic acid or 14,15-epoxy-5,8,11-tetracosa Valine, Met designates methionine and Leu designates leu trienoic acid or 5.6.8,9-diepoxy-11,14-tetracosadienoic acid C1G. or 5,6,11,12-diepoxy-8,14-tetracosadienoic acid or 5,6,14. The present invention clearly extends to epoxygenase 15-diepoxy-8, 11-tetracosadienoic acid or 8,9,11,12-diep genes derived from other species, including the epoxygenase oxy-5,14-tetracosadienoic acid or 8.9.14,15-diepoxy-5, 11 genes derived from Chrysanthemum spp. and Euphorbia tetracosadienoic acid or 11,12,14,15-diepoxy-5.8- lagascae, amongst others. tetracosadienoic acid or 5.6,8,9,11,12-triepoxy-14 In a preferred embodiment, whilst not limiting the present tetracosenoic acid O 5,6,8,9,14, 15-triepoxy-11 25 invention, the epoxygenase genes of other species which are tetracosenoic acid O 5,6,11,12,14,15-triepoxy-8- encompassed by the present invention encode mixed-func tetracosenoic acid O 8,9,11,12,14,15-triepoxy-5- tion monooxygenase enzymes. The present invention further tetracosenoic acid, amongst others. extends to the isolated or recombinant polypeptides encoded Those skilled in the art may be aware that not all substrates by Such genes and uses of said genes and polypeptides. listed supra may be derivable from a natural source, but not 30 withstanding this, may be produced by chemical synthetic The invention described according to this embodiment means. The conversion of both natural and synthetic unsatur does not encompass nucleic acids which encode enzyme ated fatty acids to epoxy fatty acids is clearly within the scope activities other than epoxygenase activities as defined herein, of the present invention. in particular the A12-desaturase enzymes derived from Ara The present invention is particularly directed to those 35 bidopsis thaliana, Brassica iuncea, Brassica napus or Gly epoxygenase enzymes that are mixed-function monooxyge cine max, amongst others, which are known to contain similar nase enzymes, and nucleic acids encoding said enzymes, and histidine-rich motifs. uses of said enzymes and nucleic acids. Accordingly, it is In the present context, “homologues' of an amino acid particularly preferred that the nucleic acid of the invention sequence refer to those amino acid sequences or peptide encode a fatty acid epoxygenase which is a mixed-function 40 sequences which are derived from polypeptides, enzymes or monoOXygenase enzyme. proteins of the present invention or alternatively, correspond In the context of the present invention, the term “mixed Substantially to the amino acid sequences listed Supra, not function monooxygenase enzyme” shall be taken to refer to withstanding any naturally-occurring amino acid substitu any epoxygenase polypeptide that comprises an amino acid tions, additions or deletions thereto. sequence comprising three histidine-rich regions as follows: 45 For example, amino acids may be replaced by other amino (i) His-(Xaa)-His (SEQID NO:21 and SEQID NO: 22); acids having similar properties, for example hydrophobicity, (ii) His-(Xaa).--His-His (SEQ ID NO: 23 and SEQ ID hydrophilicity, hydrophobic moment, antigenicity, propen NO: 24); and sity to form or break C-helical structures or B-sheet structures, (iii) His-(Xaa)-His-His (SEQ ID NO: 23 and SEQ ID and so on. Alternatively, or in addition, the amino acids of a NO: 24), 50 homologous amino acid sequence may be replaced by other amino acids having similar properties, for example hydro wherein His designates histidine, Xaa designates any natu phobicity, hydrophilicity, hydrophobic moment, charge or rally-occurring amino acid residue as set forth in Table 1 antigenicity, and so on. herein, the integer (Xaa) refers to a sequence of amino Naturally-occurring amino acid residues contemplated acids comprising three or four repeats of Xaa, and the integer 55 (Xaa) refers to a sequence of amino acids comprising two herein are described in Table 1. or three repeats of Xaa. A homologue of an amino acid sequence may be a syn In the exemplification of the invention described herein, thetic peptide produced by any method knownto those skilled the inventors provide isolated cDNAs that comprise nucle in the art, Such as by using Fmoc chemistry. otide sequences encoding the A12-epoxygenase polypeptides 60 Alternatively, a homologue of anamino acid sequence may of Crepis palaestina and Vermonia galamensis. Each exem be derived from a natural source, such as the same or another plified full-length amino acid sequence encoded by said species as the polypeptides, enzymes or proteins of the cDNAs which includes the three characteristic amino acid present invention. Preferred sources of homologues of the sequence motifs of a mixed-function monooxygenase amino acid sequences listed Supra include any of the sources enzyme as herein before defined. Close sequence identity 65 contemplated herein. between the amino acid sequences of the A12-epoxygenase 'Analogues' of an amino acid sequence encompass those enzymes from C. palaestina (SEQID NO: 2), an unidentified amino acid sequences which are Substantially identical to the US 7,589.253 B2 11 12 amino acid sequences listed Supra notwithstanding the occur rence of any non-naturally occurring amino acid analogues TABLE 1-continued therein. Preferred non-naturally occurring amino acids contem Three-letter One-letter plated herein are listed below in Table 2. Amino Acid Abbreviation Symbol The term "derivative' in relation to anamino acid sequence Histidine His H Isoleucine Ile I shall be taken to refer hereinafter to mutants, parts, fragments Leucine Leu L or polypeptide fusions of the amino acid sequences listed Lysine Lys K Supra. Derivatives include modified amino acid sequences or Methionine Met M peptides in which ligands are attached to one or more of the 10 Phenylalanine Phe F amino acid residues contained therein, such as carbohydrates, Proline Pro P enzymes, proteins, polypeptides or reporter molecules Such Serine Ser S Threonine Thr T as radionuclides or fluorescent compounds. Glycosylated, Tryptophan Trp W fluorescent, acylated or alkylated forms of the subject pep Tyrosine Tyr Y tides are also contemplated by the present invention. Addi 15 Valine Wall V tionally, derivatives may comprise fragments or parts of an Any amino acid as above Xaa X amino acid sequence disclosed herein and are within the Scope of the invention, as are homopolymers or heteropoly mers comprising two or more copies of the Subject sequences. Procedures for derivatizing peptides are well-known in the TABLE 2 art. Non-conventional Substitutions encompass amino acid alterations in which amino acid Code an amino acid is replaced with a different naturally-occurring C-aminobutyric acid Abu or a non-conventional amino acid residue. Such substitutions C-amino-C.-methylbutyrate Mgabu may be classified as "conservative', in which case an amino 25 aminocyclopropane-carboxylate Cpro aminoisobutyric acid Aib acid residue is replaced with another naturally-occurring aminonorbornyl-carboxylate Norb amino acid of similar character, for example Gly (>Ala, cyclohexylalanine Chexa Valg-Ileg-Leu, Asp (Glu, Lysg)Arg, Asng-Gln or cyclopentylalanine Cpen Phe{Trp{Tyr. D-alanine Dal 30 D-arginine Darg Substitutions encompassed by the present invention may D-aspartic acid Dasp also be "non-conservative', in which an amino acid residue D-cysteine Dcys which is present in a repressor polypeptide is substituted with D-glutamine Dglin an amino acid having different properties, such as a naturally D-glutamic acid Dglu occurring amino acid from a different group (e.g. Substituted D-histidine Dhis 35 D-isoleucine Dile a charged or hydrophobic amino acid with alanine), or alter D-leucine Deu natively, in which a naturally-occurring amino acid is Substi D-lysine Dlys tuted with a non-conventional amino acid. D-methionine Dmet D-ornithine Dorn Amino acid Substitutions are typically of single residues, D-phenylalanine Dphe but may be of multiple residues, either clustered or dispersed. D-proline Dpro 40 Amino acid deletions will usually be of the order of about D-serine Dser D-threonine Dthr 1-10 amino acid residues, while insertions may be of any D-tryptophan Dtrp length. Deletions and insertions may be made to the N-termi D-tyrosine Dtyr nus, the C-terminus or be internal deletions or insertions. D-valine Dval Generally, insertions within the amino acid sequence will be D-O-methylalanine Dmala Smaller than amino- or carboxyl-terminal fusions and of the 45 D-O-methylarginine Dmarg L-N-methylalanine Nimala order of 1-4 amino acid residues. L-N-methylarginine Nmarg The present invention clearly extends to the subject iso L-N-methylasparagine NmaSn lated nucleic acid when integrated into the genome of a cell as L-N-methylaspartic acid Nmasp an addition to the endogenous cellular complement of epoxy L-N-methylcysteine Nmcys 50 L-N-methylglutamine Nmgln genase genes. Alternatively, wherein the host cell does not L-N-methylglutamic acid Nmglu normally encode enzymes required for epoxy fatty acid bio L-N-methylhistidine Nmhis synthesis, the present invention extends to the Subject isolated L-N-methylisoleucine Nmile nucleic acid when integrated into the genome of said cell as an L-N-methyleucine Nimleu addition to the endogenous cellular genome. L-N-methyllysine Nmlys 55 L-N-methylmethionine Nmmet L-N-methylnorleucine Nmnle TABLE 1. L-N-methylnorvaline Nminva L-N-methylornithine Nmorn Three-letter One-letter L-N-methylphenylalanine Nmphe Amino Acid Abbreviation Symbol L-N-methylproline Nmpro L-N-methylserine Nmser Alanine Ala A. 60 L-N-methylthreonine Nimthr Arginine Arg R L-N-methyltryptophan Nmtrp Asparagine ASn N L-N-methyltryosine Nmtyr Aspartic acid Asp D L-N-methylvaline Nmval Cysteine Cys C L-N-methylethylglycine Nmetg Glutamine Gln Q L-N-methyl-t-butylglycine Nmtbug Glutamic acid Glu E 65 L-norleucine Nle Glycine Gly G L-norvaline Nwa US 7,589.253 B2 13 14

TABLE 2-continued TABLE 2-continued

Non-conventional Non-conventional amino acid amino acid Code C.-methyl-aminoisobutyrate L-C.-methylaspartate Masp C-methyl-y-aminobutyrate L-C.-methylcysteine Mcys C.-methylcyclohexylalanine L-C.-methylglutamine Mglin C.-methylcyclopentylalanine L-C.-methylhistidine Mhis D-O-methylasparagine L-C.-methylisoleucine Mile D-O-methylaspartate Dmasp 10 L-C.-methyleucine Meu D-O-methylcysteine L-C.-methylmethionine Mnet D-O-methylglutamine L-C.-methylnorvaline Mnva D-O-methylhistidine L-C.-methylphenylalanine Mphe D-O-methylisoleucine L-C.-methylserine Miser D-O-methylleucine L-C.-methyltryptophan Mtrp D-O-methyllysine Dmlys 15 L-C.-methylvaline Mval D-O-methylmethionine N-(N-(2,2-diphenylethyl)carbamylmethylglycine Nmbc D-O-methylornithine -carboxy-1-(2,2-diphenyl-ethylamino)cyclopropane D-O-methylphenylalanine D-N-methylphenylalanine Dnmphe D-O-methylproline D-N-methylproline Dnimpro D-O-methylserine D-N-methylserine Dnmser D-O-methylthreonine D-N-methylthreonine Dnmthr D-O-methyltryptophan N-(1-methylethyl)glycine Nval D-O-methyltyrosine N-methyla-napthylalanine Nmanap D-O-methylvaline N-methylpenicillamine Nmpen D-N-methylalanine N-(p-hydroxyphenyl)glycine Nhtyr D-N-methylarginine N-thiomethyl)glycine Ncys D-N-methylasparagine penicillamine Pen D-N-methylaspartate Dnmasp 25 L-C.-methylalanine Mala D-N-methylcysteine L-C.-methylasparagine Masin D-N-methylglutamine L-C.-methyl-t-butylglycine Mtbug D-N-methylglutamate L-methylethylglycine Metg D-N-methylhistidine L-C.-methylglutamate Mglu D-N-methylisoleucine L-C.-methylhomo phenylalanine Mhphe D-N-methyleucine Dnmleu 30 N-(2-methylthioethyl)glycine Nmet D-N-methyllysine L-C.-methyllysine Mlys N-methylcyclohexylalanine L-C.-methylnorleucine Mille D-N-methylornithine L-C.-methylornithine Morn C.-methyl-C-napthylalanine L-C.-methylproline Mpro C.-methylpenicillamine L-C.-methylthreonine Mithr N-(4-aminobutyl)glycine Nglu 35 L-C.-methyltyrosine Mtyr N-(2-aminoethyl)glycine L-N-methylhomo phenylalanine Nmhphe N-(3-aminopropyl)glycine N-(N-(3,3-diphenylpropyl) Nnbhmcarbamylmethyl)glycine Nnbhe N-amino-C.-methylbutyrate C-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine A second aspect of the present invention provides an iso N-(carbamylmethyl)glycine 40 lated nucleic acid which comprises the sequence of nucle N-(2-carboxyethyl)glycine otides set forth in any one of SEQID NOS:1 or 3 or 5 or 19 or N-(carboxymethyl)glycine N-cyclobutylglycine a complementary sequence thereto, or a homologue, ana N-cycloheptylglycine logue or derivative thereof. N-cyclohexylglycine For the purposes of nomenclature, the nucleotide sequence N-cyclodecylglycine Nicdec 45 N-cylcododecylglycine set forth in SEQID NO: 1 is derived from Crepis palaestina N-cyclooctylglycine and encodes the mixed function monooxygenase sequence or N-cyclopropylglycine mixed function monooxygenase-like sequence set forth in N-cycloundecylglycine N-(2,2-diphenylethyl)glycine SEQ ID NO: 2. As exemplified herein, the amino acid N-(3,3-diphenylpropyl)glycine Nbhe 50 sequence set forth in SEQID NO: 2 has epoxygenase activity, N-(3-guanidinopropyl)glycine more particularly A12-epoxygenase activity. N-(1-hydroxyethyl)glycine N-(hydroxyethyl))glycine The nucleotide sequence set forth in SEQID NO:3 corre N-(imidazolylethyl)glycine sponds to a cDNA derived from a Crepis sp. other than C. N-(3-indolylethyl)glycine palaestina comprising high levels of vernolic acid. The amino N-methyl-y-aminobutyrate Nmgabu 55 acid sequence set forth in SEQID NO: 4 corresponds to the D-N-methylmethionine N-methylcyclopentylalanine derived amino acid sequence of the Crepis sp. epoxygenase N-methylglycine gene provided in SEQID NO:3. N-methylaminoisobutyrate The nucleotide sequence set forth in SEQID NO: 5 corre N-(1-methylpropyl)glycine N-(2-methylpropyl)glycine sponds to amplified DNA derived from Vernonia galamensis D-N-methyltryptophan 60 using amplification primers derived from a consensus D-N-methyltyrosine sequence of mixed function monooxygenases, including the D-N-methylvaline Crepis spp. epoxygenase gene sequences of the invention. Y-aminobutyric acid The amplified DNA comprises a partial epoxygenase gene L-t-butylglycine L-ethylglycine sequence, which includes nucleotide sequences capable of L-homophenylalanine Hphe 65 encoding the histidine-rich motif His-Arg-Asn-His-His L-C.-methylarginine which is characteristic of mixed function monooxygenase enzymes. The amino acid sequence set forth in SEQID NO: US 7,589.253 B2 15 16 6 corresponds to the derived amino acid sequence of the tide to elicit a sufficient immune response for the production Vermonia galamensis epoxygenase gene provided in SEQID of monoclonal antibodies, synthetic Fab fragments of an anti NO: 5. body molecule, single-chain antibody molecule or other The nucleotide sequence set forth in SEQ ID NO: 19 immunointeractive molecule. derived from Vernonia galamensis and encodes the full As used herein, the term “enzymatically-active' shall be length mixed function monooxygenase set forth in SEQ ID taken to refer to the ability of a polypeptide molecule to NO: 2O. catalyze an enzyme reaction, in particular an enzyme reaction The nucleotide sequence set forth in SEQID NO: 7 relates which comprises the epoxygenation of a carbon bond in a to the partial sequence of a Crepis alpina acetylenase gene fatty acid substrate molecule. More particularly, whilst not which was used as a probe to isolate the nucleic acid com 10 limiting the invention, the term “enzymatically-active' may prising the nucleotide sequence set forth in SEQID NO: 1. also refer to the ability of a polypeptide molecule to catalyze The amino acid sequence set forth in SEQ ID NO: 8 corre the epoxygenation of A-9 or A-12 in a fatty acid substrate sponds to the derived amino acid sequence of said partial molecule such as linoleic acid or Vernolic acid. sequence of the C. alpina acetylenase gene. In an alternative embodiment, a preferred homologue, ana As used herein, the term “acetylenase' shall be taken to 15 logue orderivative of the nucleotide sequence set forth in any refer to an enzyme which is capable of catalyzing the conver one of SEQID NOs: 1 or 3 or 5 or 19, or a complementary sion of a carbon double bondina fatty acid substrate molecule sequence thereto, comprises a sequence of nucleotides which to a carbon triple bond or alternatively, which is capable of is at least 65% identical to at least 20 contiguous nucleotides catalyzing the formation of a carbon triple bond in a fatty acid therein, other than a nucleotide sequence which encodes a molecule. Crepis sp. acetylenase enzyme. The present invention clearly extends to the genomic gene More preferably, the percentage identity to any one of SEQ equivalents of the cDNA molecules exemplified in any one of ID NOS: 1 or 3 or 5 or 19 is at least about 85%. Even more SEQID NOs: 1, 3, 5, or 19. preferably, a homologue, analogue or derivative of SEQ ID In a most particularly preferred embodiment, the present NOS: 1 or 3 or 5 or 19 is at least about 90% and even more invention provides an isolated nucleic acid which comprises 25 preferably at least about 95% identical to at least 100 or 250 the nucleotide sequence set forth in any one of SEQID NOs: or 500 or 1000 contiguous nucleotides therein. 1, 3, 5, or 19 or a genomic gene equivalent of said nucleotide Reference herein to a percentage identity or percentage sequence or a homologue, analogue or derivative thereof. similarity between two or more nucleotide or amino acid For the present purpose, “homologues' of a nucleotide sequences shall be taken to refer to the number of identical or sequence shall be taken to refer to an isolated nucleic acid 30 similar residues in a nucleotide oramino acid sequence align which is substantially the same as the nucleic acid of the ment, as determined using any standard algorithm known by present invention or its complementary nucleotide sequence, those skilled in the art. In particular, nucleotide and/or amino notwithstanding the occurrence within said sequence, of one acid sequence identities and similarities may be calculated or more nucleotide Substitutions, insertions, deletions, or using the Gap program, which utilizes the algorithm of rearrangements. 35 Needleman and Wunsch (1970) to maximize the number of Analogues' of a nucleotide sequence set forth herein shall residue matches and minimize the number of sequence gaps. be taken to refer to an isolated nucleic acid which is substan The Gap program is part of the Sequence and Analysis Soft tially the same as a nucleic acid of the present invention or its ware Package of the Computer Genetics Group Inc., Univer complementary nucleotide sequence, notwithstanding the sity Research Park, Madison, Wis., United States of America occurrence of any non-nucleotide constituents not normally 40 (Devereux et al., 1984). present in said isolated nucleic acid, for example carbohy In a further alternative embodiment, a preferred homo drates, radiochemicals including radionucleotides, reporter logue, analogue or derivative of the nucleotide sequence set molecules such as, but not limited to DIG, alkaline phos forth in any one of SEQID NOs: 1, 3, 5, or 19 or a comple phatase or horseradish peroxidase, amongst others. mentary sequence thereto, hybridizes under at least low strin “Derivatives” of a nucleotide sequence set forth herein 45 gency conditions to at least 20 contiguous nucleotides derived shall be taken to refer to any isolated nucleic acid comprising from said sequence. significant sequence similarity to said sequence or a part More preferably, the stringency of hybridization is at least thereof. moderate stringency, even more preferably at least high Strin Generally, homologues, analogues or derivatives of the gency. nucleic acid of the invention are produced by synthetic means 50 For the purposes of defining the level of stringency, those or alternatively, derived from naturally-occurring sources. skilled in the art will be aware that several different hybrid For example, the nucleotide sequence of the present invention ization conditions may be employed. For example, a low may be subjected to mutagenesis to produce single or mul stringency may comprise a hybridization and/or a wash car tiple nucleotide Substitutions, deletions and/or insertions as ried out in 6xSSC buffer, 0.1% (w/v) SDS at 28°C. A mod indicated Supra. 55 erate stringency may comprise a hybridization and/or wash In one embodiment of the invention, preferred homo carried out in 2XSSC buffer, 0.1% (w/v) SDS at a temperature logues, analogues or derivatives of the nucleotide sequences in the range 45° C. to 65°C. A high Stringency may comprise set forth in any one of SEQID NOs: 1, 3, 5, or 19 or comple a hybridization and/or wash carried out in 0.1 xSSC buffer, mentary sequences thereto, encode immunologically-active 0.1% (w/v) SDS at a temperature of at least 65° C. or enzymatically-active polypeptides. 60 Generally, the stringency is increased by reducing the con As used herein, the term “immunologically-active' shall centration of SSC buffer, and/or increasing the concentration be taken to refer to the ability of a polypeptide molecule to of SDS in the hybridization buffer or wash buffer and/or elicit an immune response in a mammal, in particular an increasing the temperature at which the hybridization and/or immune response Sufficient to produce an antibody molecule wash are performed. Conditions for a hybridization and/or such as, but not limited to, an IgM or IgG molecule or whole 65 wash are well understood by one normally skilled in the art. serum containing said antibody molecule. The term “immu For the purposes of clarification of parameters affecting nologically-active also extends to the ability of a polypep hybridization between nucleic acids, reference can conve US 7,589.253 B2 17 18 niently be made to pages 2.10.8 to 2.10.16. of Ausubel et al. Homologues, analogues or derivatives of any one of SEQ (1987), which is herein incorporated by reference. ID NOs: 2 or 4 or 6 or 20 may further comprise a histidine-rich The isolated nucleic acids disclosed herein may be used to region as defined Supra. Even more preferably, the Subject isolate or identify homologues, analogues or derivatives epoxygenase at least comprises a sequence of amino acids thereof from other cells, tissues, or organ types, or from the which comprises three histidine rich regions as follows: cells, tissues, or organs of another species using any one of a (i) His-Glu-Cys-Gly-His-His (SEQ ID NO: 15); number of means known to those skilled in the art. (ii) His-Arg-Asn-His-His (SEQID NO: 16); and For example, genomic DNA, or mRNA, or cDNA may be (iii) His-Val-Met-His-His (SEQ ID NO: 17) or His-Val contacted, under at least low stringency hybridization condi Leu-His-His (SEQ ID NO: 18), tions or equivalent, with a hybridization effective amount of 10 or a homologue, analogue or derivative thereof. an isolated nucleic acid which comprises the nucleotide The invention described according to this alternative sequence set forth in any one SEQID NOs: 1, 3, 5, or 19 or a embodiment does not encompass the A12-desaturase complementary sequence thereto, or a functional part thereof, enzymes derived from Arabidopsis thaliana, Brassica jun and the hybridization detected using a detection means. cea, Brassica napus or Glycine max, amongst others. The detection means may be a reporter molecule capable of 15 The isolated nucleic acid of the present invention is useful giving an identifiable signal(e.g. a radioisotope such as Por for developing gene constructs comprising a sense molecule S or a biotinylated molecule) covalently linked to the iso wherein said gene constructs are designed for the expression lated nucleic acid of the invention. in a cell which does not normally express said nucleic acid or In an alternative method, the detection means is any known over-expression of said nucleic acid in a cell which does format of the polymerase chain reaction (PCR). According to normally express the said nucleic acid. this method, degenerate pools of nucleic acid primer mol Accordingly, a further aspect of the invention provides a ecules” of about 15-50 nucleotides in length are designed gene construct which comprises a sense molecule which is based upon the nucleotide sequences disclosed in SEQ ID operably connected to a promoter sequence. NOs: 1, 3, 5, or 19 or a complementary sequence thereto. The 25 The term “sense molecule' as used herein shall be taken to homologues, analogues or derivatives (i.e. the “template mol refer to an isolated nucleic acid which encodes or is comple ecule') are hybridized to two of said primer molecules, such mentary to an isolated nucleic acid which encodes a fatty acid that a first primer hybridizes to a region on one strand of the epoxygenase wherein said nucleic acid is provided in a format template molecule and a second primer hybridizes to a Suitable for its expression to produce a recombinant polypep complementary sequence thereof, wherein the first and sec 30 tide when said sense molecule is introduced into a host cell by ond primers are not hybridized within the same or overlap transfection or transformation. ping regions of the template molecule and wherein each Those skilled in the art will be aware that a gene construct primer is positioned in a 5'- to 3'-orientation relative to the may be used to “transfect a cell, in which case it is introduced position at which the other primer is hybridized on the oppo into said cell without integration into the cell's genome. Alter site Strand. Specific nucleic acid copies of the template mol natively, a gene construct may be used to “transform a cell, ecule are amplified enzymatically in a polymerase chain reac in which case it is stably integrated into the genome of said tion, a technique that is well known to one skilled in the art. cell. The primer molecules may comprise any naturally-occur A sense molecule that comprises a fatty acid epoxygenase ring nucleotide residue (i.e. adenine, cytidine, guanine, thy gene sequence or homologue, analogue or derivative thereof, midine) and/or comprise inosine or functional analogues or 40 may be introduced into a cell using any known method for the derivatives thereof, capable of being incorporated into a poly transfection or transformation of said cell. Wherein a cell is nucleotide molecule. The nucleic acid primer molecules may transformed by the gene construct of the invention, a whole also be contained in an aqueous mixture of other nucleic acid organism may be regenerated from a single transformed cell, primer molecules or be in a Substantially pure form. using any method known to those skilled in the art. The detected sequence may be in a recombinant form, in a 45 Thus, the epoxygenase genes described herein may be used virus particle, bacteriophage particle, yeast cell, animal cell, to develop single cells or whole organisms which synthesize or a plant cell. Preferably, the related genetic sequence origi epoxy fatty acids not normally produced by wild or naturally nates from another plant species. occurring organisms belonging to the same genera or species A third aspect of the present invention provides an isolated as the genera or species from which the transfected or trans nucleic acid which encodes the amino acid sequence set forth 50 formed cell is derived, or to increase the levels of such fatty in any one of SEQID NOs: 2 or 4 or 6 or 20 or a homologue, acids above the levels normally found in such wild or natu analogue or derivative thereof. rally-occurring organisms. In one embodiment contemplated herein, preferred homo In an alternative preferred embodiment, the isolated logues, analogues or derivatives of the amino acid sequences nucleic acid of the invention is capable of reducing the level set forth in SEQID NOS: 2, 4, 6, or 20 are immunologically 55 of epoxy fatty acids in a cell, when expressed therein, in the active or enzymatically-active polypeptides as defined Supra. antisense orientation or as a ribozyme or co-suppression mol In an alternative embodiment of the invention, preferred ecule, under the control of a suitable promoter sequence. homologues, analogues or derivatives of the amino acid Co-suppression is the reduction in expression of an endog sequence set forth in any one of SEQID NOS: 2, 4, 6, or 20 enous gene that occurs when one or more copies of said gene, comprise a sequence of amino acids which is at least 65% 60 or one or more copies of a Substantially similar gene are identical thereto, other thana Crepis sp. acetylenase polypep introduced into the cell. The present invention also extends to tide. More preferably, homologues, analogues or derivatives the use of co-suppression to inhibit the expression of an of SEQID NOS: 2 or 4 or 6 or 20 which are encompassed by epoxygenase gene as described herein. the present invention are at least about 85% identical, even In the context of the present invention, an antisense mol more preferably at least about 90% identical and still even 65 ecule is an RNA molecule which is transcribed from the more preferably at least about 95% identical, and still more complementary Strand of a nuclear gene to that which is preferably at least about 99%-100% identical thereto. normally transcribed to produce a “sense' mRNA molecule US 7,589.253 B2 19 20 capable of being translated into a polypeptide. The antisense tissue-specificity or development-specificity of expression of molecule is therefore complementary to the sense mRNA, or the sense molecule which is required. a part thereof. Although not limiting the mode of action of the Reference herein to a “promoter' is to be taken in its antisense molecules of the present invention to any specific broadest context and includes the transcriptional regulatory mechanism, the antisense RNA molecule possesses the sequences of a classical eukaryotic genomic gene, including capacity to form a double-stranded mRNA by base pairing the TATA box which is required for accurate transcription with the sense mRNA, which may prevent translation of the initiation, with or without a CCAAT box sequence and addi sense mRNA and Subsequent synthesis of a polypeptide gene tional regulatory elements (i.e. upstream activating product. sequences, enhancers and silencers) which alter gene expres Ribozymes are synthetic RNA molecules which comprise 10 sion in response to developmental and/or external stimuli, or a hybridizing region complementary to two regions, each of at in a tissue-specific manner. In the context of the present least 5 contiguous nucleotide bases in the target sense mRNA. invention, the term “promoter also includes the transcrip In addition, ribozymes possess highly specific endoribonu tional regulatory sequences of a classical prokaryotic gene, in clease activity, which autocatalytically cleaves the target which case it may include a -35 box sequence and/or a -10 sense mRNA. A complete description of the function of 15 box transcriptional regulatory sequences. ribozymes is presented by Haseloff and Gerlach (1988) and In the present context, the term “promoter' is also used to contained in International Patent Application No. WO89/ describe a synthetic or fusion molecule, or derivative which 05852. The present invention extends to ribozymes which confers, activates or enhances expression of said sense mol target a sense mRNA encoding an epoxygenase polypeptide ecule in a cell. Preferred promoters may contain additional described herein, thereby hybridizing to said sense mRNA copies of one or more specific regulatory elements, to further and cleaving it, such that it is no longer capable of being enhance expression of the sense molecule and/or to alter the translated to synthesize a functional polypeptide product. spatial expression and/or temporal expression of said sense According to this embodiment, the present invention pro molecule. For example, copper-responsive regulatory ele vides a ribozyme or antisense molecule comprising a ments may be placed adjacent to a heterologous promoter sequence of contiguous nucleotide bases which are able to 25 sequence driving expression of a sense molecule to confer form a hydrogen-bonded complex with a sense mRNA copper inducible expression thereon. encoding an epoxygenase described herein, to reduce trans Placing a sense, antisense, ribozyme or co-suppression lation of said mRNA. Although the preferred antisense and/or molecule under the regulatory control of a promoter sequence ribozyme molecules hybridize to at least about 10 to 20 nucle means positioning said molecule Such that expression is con otides of the target molecule, the present invention extends to 30 trolled by the promoter sequence. A promoter is usually, but molecules capable of hybridizing to at least about 50-100 not necessarily, positioned upstream or 5' of a nucleic acid nucleotide bases in length, or a molecule capable of hybrid which it regulates. Furthermore, the regulatory elements izing to a full-length or Substantially full-length epoxygenase comprising a promoter are usually positioned within 2kb of mRNA. the start site of transcription of the sense, antisense, ribozyme It is understood in the art that certain modifications, includ 35 or co-suppression molecule or chimeric gene comprising ing nucleotide Substitutions amongst others, may be made to same. In the construction of heterologous promoter/structural the antisense and/or ribozyme molecules of the present inven gene combinations it is generally preferred to position the tion, without destroying the efficacy of said molecules in promoter at a distance from the gene transcription start site inhibiting the expression of the epoxygenase gene. It is there that is approximately the same as the distance between that fore within the scope of the present invention to include any 40 promoter and the gene it controls in its natural setting, i.e., the nucleotide sequence variants, homologues, analogues, or gene from which the promoter is derived. As is known in the fragments of the said gene encoding same, the only require art, Some variation in this distance can be accommodated ment being that said nucleotide sequence variant, when tran without loss of promoter function. Similarly, the preferred scribed, produces an antisense and/or ribozyme molecule positioning of a regulatory sequence element with respect to 45 a heterologous gene to be placed under its control is defined which is capable of hybridizing to the said sense mRNA by the positioning of the element in its natural setting, i.e., the molecule. genes from which it is derived. Again, as is known in the art, The present invention extends to gene constructs designed Some variation in this distance can also occur. to facilitate expression of a sense molecule, an antisense Examples of promoters Suitable for use in gene constructs molecule, ribozyme molecule, or co-suppression molecule 50 of the present invention include promoters derived from the which is capable of altering the level of epoxy fatty acids in a genes of viruses, yeasts, moulds, bacteria, insects, birds, cell. mammals and plants which are capable of functioning in In a particularly preferred embodiment, the sense mol isolated cells or whole organisms regenerated therefrom. The ecule, an antisense molecule, ribozyme molecule, co-Sup promoter may regulate the expression of the sense, antisense, pression molecule, or gene targeting molecule which is 55 ribozyme or co-suppression molecule constitutively, or dif capable of altering the epoxy fatty acid composition of a cell ferentially with respect to the tissue in which expression derived from plantor other organism comprises a sequence of occurs or, with respect to the developmental stage at which nucleotides set forth in any one of SEQID NOs: 1,3,5, or 19 expression occurs, or in response to external stimuli Such as or a complementary strand, homologue, analogue or deriva physiological stresses, pathogens, or metal ions, amongst tive thereof. 60 others. Those skilled in the art will also be aware that expression of Examples of promoters include the CaMV 35S promoter, a sense, antisense, ribozyme or co-suppression molecule may NOS promoter, octopine synthase (OCS) promoter, Arabi require the nucleic acid of the invention to be placed in oper dopsis thaliana SSU gene promoter, napin seed-specific pro able connection with a promoter sequence. The choice of moter, P. promoter, BK5-Timm promoter, lac promoter, tac promoter for the present purpose may vary depending upon 65 promoter, phage lambdaw or promoters, CMV promoter the level of expression of the sense molecule required and/or (U.S. Pat. No. 5,168,062), T7 promoter, lacUV5 promoter, the species from which the host cell is derived and/or the SV40 early promoter (U.S. Pat. No. 5,118,627), SV40 late US 7,589.253 B2 21 22 promoter (U.S. Pat. No. 5,118,627), adenovirus promoter, Suitable selectable marker genes contemplated herein baculovirus P10 or polyhedrin promoter (U.S. Pat. Nos. include the amplicillin resistance (Amp'), tetracycline resis 5,243,041, 5,242,687, 5,266,317, 4,745,051 and 5,169,784), tance gene (Tc), bacterial kanamycin resistance gene (Kan), and the like. In addition to the specific promoters identified phosphinothricin resistance gene, neomycin phosphotrans herein, cellular promoters for so-called housekeeping genes ferase gene (mptII), hygromycin resistance gene, B-glucu are useful. ronidase (GUS) gene, chloramphenicol acetyltransferase Preferred promoters according to this embodiment are (CAT) gene and luciferase gene, amongst others. those promoters which are capable of functioning in yeast, A further aspect of the present invention provides a trans mould or plant cells. More preferably, promoters suitable for fected or transformed cell, tissue, organ or whole organism use according to this embodiment are capable of functioning 10 which expresses a recombinant epoxygenase polypeptide ora in cells derived from oleaginous yeasts, oleaginous moulds or ribozyme, antisense or co-suppression molecule as described oilseed crop plants, such as flax sold under the trademark herein, or a homologue, analogue or derivative thereof. LinolaTM (hereinafter referred to as “LinolaTM flax”), sun Preferably, the isolated nucleic acid is contained within a flower, safflower, soybean, linseed, sesame, cottonseed, pea gene construct as described herein. The gene construct of the nut, olive or oil palm, amongst others. 15 present invention may be introduced into a cell by various In a more preferred embodiment, the promoter may be techniques known to those skilled in the art. The technique derived from a genomic clone encoding an epoxygenase used may vary depending on the known Successful techniques enzyme, preferably derived from the genomic gene equiva for that particular organism. lents of epoxygenase genes derived from Chrysanthemum Means for introducing recombinant DNA into bacterial spp., Crepis spp. including C. palaestina or other Crepis sp., cells, yeast cells, or plant, insect, fungal (including mould), Euphorbia lagascae or Vermonia galamensis, which are avian or mammalian tissue or cells include, but are not limited referred to herein. to, transformation using CaCl and variations thereof, in par In a more preferred embodiment, the promoter may be ticular the method described by Hanahan (1983), direct DNA derived from a highly-expressed seed gene, such as the napin uptake into protoplasts (Krens et al., 1982; Paszkowski et al. gene, amongst others. 25 1984), PEG-mediated uptake to protoplasts (Armstrong et al. The gene construct of the invention may further comprise a 1990) microparticle bombardment, electroporation (Fromm terminator sequence and be introduced into a Suitable host et al., 1985), microinjection of DNA (Crossway et al., 1986), cell where it is capable of being expressed to produce a microparticle bombardment of tissue explants or cells (Chris recombinant polypeptide gene product or alternatively, a tou et al., 1988; Sanford, 1988), vacuum-infiltration of tissue ribozyme or antisense molecule. 30 with nucleic acid, or in the case of plants, T-DNA-mediated The term “terminator” refers to a DNA sequence at the end transfer from Agrobacterium to the plant tissue as described of a transcriptional unit which signals termination of tran essentially by An et al. (1985), Herrera-Estrella et al. (1983a, Scription. Terminators are 3'-non-translated DNA sequences 1983b, 1985). containing a polyadenylation signal, which facilitates the For microparticle bombardment of cells, a microparticle is addition of polyadenylate sequences to the 3'-end of a pri 35 propelled into a cell to produce a transformed cell. Any Suit mary transcript. Terminators active in cells derived from able ballistic cell transformation methodology and apparatus viruses, yeasts, moulds, bacteria, insects, birds, mammals and can be used in performing the present invention. Exemplary plants are known and described in the literature. They may be apparatus and procedures are disclosed by Stomp et al. (U.S. isolated from bacteria, fungi, viruses, animals and/or plants. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat. No. Examples ofterminators particularly suitable for use in the 40 4.945.050). When using ballistic transformation procedures, gene constructs of the present invention include the nopaline the gene construct may incorporate a plasmid capable of synthase (NOS) gene terminator of Agrobacterium tumefa replicating in the cell to be transformed. ciens, the terminator of the Cauliflower mosaic virus (CaMV) Examples of microparticles Suitable for use in Such sys 35S gene, the Zein gene terminator from Zea mays, the tems include 1 to 5um gold spheres. The DNA construct may Rubisco Small subunit (SSU) gene terminator sequences, Sub 45 be deposited on the microparticle by any Suitable technique, clover stunt Virus (SCSV) gene sequence terminators, any Such as by precipitation. rho-independent E. coli terminator, amongst others. In a particularly preferred embodiment, wherein the gene Those skilled in the art will be aware of additional pro construct comprises a “sense' molecule, it is particularly moter sequences and terminator sequences which may be preferred that the recombinant epoxygenase polypeptide pro Suitable for use in performing the invention. Such sequences 50 may readily be used without any undue experimentation. duced therefrom is enzymatically active. The gene constructs of the invention may further includean Alternatively, wherein the cell is derived from a multicel origin of replication sequence which is required for replica lular organism and where relevant technology is available, a tion in a specific cell type, for example a bacterial cell, when whole organism may be regenerated from the transformed said gene construct is required to be maintained as an episo 55 cell, in accordance with procedures well known in the art. mal genetic element (e.g. plasmidor cosmid molecule) in said Those skilled in the art will also be aware of the methods cell. for transforming, regenerating and propagating other type of Preferred origins of replication include, but are not limited cells, such as those of fungi. to, the fl-ori and cofE1 origins of replication. In the case of plants, plant tissue capable of Subsequent The gene construct may further comprise a selectable 60 clonal propagation, whether by organogenesis or embryogen marker gene or genes that are functional in a cell into which esis, may be transformed with a gene construct of the present said gene construct is introduced. invention and a whole plant regenerated therefrom. The par As used herein, the term “selectable marker gene' includes ticular tissue chosen will vary depending on the clonal propa any gene which confers a phenotype on a cell in which it is gation systems available for, and best Suited to, the particular expressed to facilitate the identification and/or selection of 65 species being transformed. Exemplary tissue targets include cells which are transfected or transformed with a gene con leaf disks, pollen, embryos, cotyledons, hypocotyls, megaga struct of the invention or a derivative thereof. metophytes, callus tissue, existing meristematic tissue (e.g., US 7,589.253 B2 23 24 apical meristem, axillary buds, and root meristems), and (ii) transforming said gene construct into a cell or tissue of induced meristem tissue (e.g., cotyledon meristem and hypo said plant; and cotyl meristem). (iii) selecting transformants which express the epoxyge The term “organogenesis', as used herein, means a process nase encoded by the genetic sequence at a high level in by which shoots and roots are developed sequentially from 5 seeds. meristematic centers. In a more particularly preferred embodiment, the plant is The term "embryogenesis', as used herein, means a pro an oilseed species that normally produces significant levels of cess by which shoots and roots develop together in a con linoleic acid, for example LinolaTM flax, oilseed rape, Sun certed fashion (not sequentially), whether from Somatic cells flower, safflower, soybean, linseed, sesame, cottonseed, pea or gametes. 10 nut, olive or oil palm, amongst others. The regenerated transformed plants may be propagated by In an even more particularly preferred embodiment, the a variety of means, such as by clonal propagation or classical plant is an oilseed species that normally produces significant breeding techniques. For example, a first generation (or T1) levels of linoleic acid, for example Linola TM flax, Sunflower transformed plant may be selfed to give homozygous second or safflower, amongst others. generation (or T2) transformant, and the T2 plants further 15 Enzymatically active recombinant epoxygenases propagated through classical breeding techniques. described herein are particularly useful for the production of The regenerated transformed organisms contemplated epoxy fatty acids from unsaturated fatty acid Substrates. The herein may take a variety of forms. For example, they may be present invention especially contemplates the production of chimeras of transformed cells and non-transformed cells; specific epoxy fatty acids in cells or regenerated transformed clonal transformants (e.g., all cells transformed to contain the organisms which do not normally produce that specific epoxy expression cassette); grafts of transformed and untrans fatty acid. formed tissues (e.g., in plants, a transformed root stock Accordingly, a further aspect of the invention provides a grafted to an untransformed Scion). method of producing an epoxy fatty acid in a cell, tissue, A further aspect of the invention provides a method of organ or organism, said method comprising incubating a cell, altering the level of epoxy fatty acids in a cell, tissue, organ or 25 tissue, organ or organism which expresses an enzymatically organism, said method comprising expressing a sense, anti active recombinant epoxygenase of the present invention with sense, ribozyme or co-suppression molecule as described a fatty acid Substrate molecule, preferably an unsaturated herein in said cell for a time and under conditions sufficient fatty acid substrate molecule, for a time and under conditions for the level of epoxy fatty acids therein to be increased or sufficient for at least one carbon bond of said substrate to be reduced. 30 converted to an epoxy group. In a preferred embodiment, the subject method comprises In an alternative embodiment, the subject method further the additional first step of transforming the cell, tissue, organ comprises the additional first step of transforming or trans or organism with the sense, antisense, ribozyme or co-Sup fecting the cell, tissue, organ or organism with a nucleic acid pression molecule. which encodes said recombinant epoxygenase or a homo As discussed Supra the isolated nucleic acid may be con 35 logue, analogue or derivative thereof, as herein before tained within a gene construct. described. As discussed Supra the isolated nucleic acid may According to this embodiment, the cell, organ, tissue or be contained within a gene construct. organism in which the Subject sense, antisense, ribozyme or According to this embodiment, the cell, organ, tissue or co-Suppression molecule is expressed may be derived from a organism in which the Subject epoxygenase is expressed is bacteria, yeast, fungus (including a mould), insect, plant, bird 40 derived from a bacteria, yeast, fungus (including a mould), or mammal. insect, plant, bird or mammal. More preferably, the cell, Because a recombinant epoxygenase polypeptide may be organ, tissue or organism is derived from a yeast, plant or produced in the regenerated transformant as well as ex vivo, fungus, even more preferably from an oleaginous yeast or one alternative preferred embodiment of the present invention plant or fungus, or from an oilseed plant which does not provides a method of producing a recombinant enzymatically 45 normally express the recombinant epoxygenase of the inven active epoxygenase polypeptide in a cell, said method com tion. prising the steps of: Amongst the main economic oilseed plants contemplated (i) producing a gene construct which comprises the cDNA herein, high-linoleic genotypes of flax, Sunflower, corn and or genomic epoxygenase genetic sequence of the inven safflower are preferred targets. Soybean and rape seed are 50 alternative targets but are less Suitable for maximal epoxy tion placed operably under the control of a promoter fatty acid synthesis because of their lower levels of linoleic capable of conferring expression on said genetic acid Substrate and the presence of an active A15-desaturase sequence in said cell, and optionally an expression competing with the epoxygenase for the linoleic acid Sub enhancer element; Strate. (ii) transforming said gene construct into said cell; and 55 An alternative embodiment is the transformation of (iii) selecting transformants which express the epoxyge LinolaTM (low linolenic acid flax) with the epoxygenase of nase encoded by the genetic sequence at a high level. the invention. Linola TM flax normally contains around 70% A particularly preferred embodiment of the present inven linoleic acid with very little of this (<2%) being subsequently tion provides a method of producing a recombinant enzymati converted to linolenic acid by A15-desaturase (Green, 1986). cally active epoxygenase polypeptide in a transgenic plant 60 Preferred unsaturated fatty acid substrates contemplated comprising the steps of herein include, but are not limited to, palmitoleic acid, oleic (i) producing a gene construct which comprises the cDNA acid, linoleic acid, linolenic acid, and arachidonic acid, or genomic epoxygenase genetic sequence of the inven amongst others. tion placed operably under the control of a seed-specific In plant species that naturally contain high levels of Ver promoter and optionally an expression enhancer ele 65 nolic acid, the A12-epoxygenase therein may be very efficient ment, wherein said genetic sequences is also placed at carrying out the epoxidation of linoleic acid. As a conse upstream of a transcription terminator sequence; quence, the present invention particularly contemplates the US 7,589.253 B2 25 26 expression of recombinant A12-epoxygenase derived from lytic function into those encoding alternative catalytic func Euphorbia lagascae, Vernonia spp. and Crepis spp. at high tions. For example, the A12 epoxygenase of the instant inven levels in transgenic oilseeds during seed oil synthesis, to tion may be converted to a A12 acetylenase by replacing produce high levels of vernolic acid therein. portions of its C-terminal and N-terminal sequences with the Accordingly, linoleic acid is a particularly preferred Sub equivalent domains from the Crepis alpina A12 acetylenase. strate according to this embodiment of the invention. Addi Similarly, the reverse domain Swapping may also be per tional Substrates are not excluded. formed. The products of the substrate molecules listed supra will be As a further refinement, Such changes in catalytic function readily determined by those skilled in the art, without undue can similarly be effected by making specific changes (e.g. experimentation. Particularly preferred epoxy fatty acids pro 10 addition, Substitution or deletion) to only those amino-acids duced according to the present invention include 12,13-ep within each domain that are critical for determining the rel oxy-9-octadecenoic acid (vernolic acid) and 12,13-epoxy-9. evant catalytic function (such as by site-directed mutagen 15-octadecadienoic acid, amongst others. esis). Conditions for the incubation of cells, organs, tissues or Accordingly, a further aspect of the present invention con organisms expressing the recombinant epoxygenase in the 15 templates a synthetic fatty acid gene comprising a sequence presence of the substrate molecule will vary, at least depend of nucleotides derived from an epoxygenase gene as ing upon the uptake of the Substrate into the cell, tissue, organ described herein, wherein said synthetic fatty acid gene or organism, and the affinity of the epoxygenase for the Sub encodes a polypeptide with epoxygenase or acetylenase or strate molecule in the particular environment selected. Opti hydroxylase or desaturase activity, wherein said polypeptide mum conditions may be readily determined by those skilled either comprises an amino acid sequence which differs from in the relevant art. a naturally-occurring epoxygenase or acetylenase or The present invention clearly extends to the isolated oil hydroxylase ordesaturase enzyme, or said polypeptide exhib containing epoxy fatty acids, and/or the isolated epoxy fatty its catalytic properties which are different from a naturally acid itself produced as described herein and to any products occurring epoxygenase or acetylenase or hydroxylase or derived therefrom, for example coatings, resins, glues, plas 25 desaturase enzyme or said polypeptide comprises a sequence tics, Surfactants and lubricants, amongst others. of amino acids which are at least about 60% identical to a part The inventors have shown further that the mixed function of SEQID NO: 2 or 4 or 6 or 20 or homologue, analogue or monooxygenases (MMO) which perform catalytic functions derivative of said part. Such as desaturation, acetylenation, hydroxylation and/or Preferably, the synthetic fatty acid gene of the invention is epoxygenation, form a family of genes sharing considerable 30 derived from a A12 epoxygenase gene. nucleotide and amino acid sequence similarity. For example, In one embodiment, the synthetic fatty acid gene of the the desaturase, acetylenase, hydroxylase and/or epoxygenase invention encodes a fusion polypeptide in which the N-ter enzymes which act on Substrate molecules having a similar minal and/or C-terminal amino acids of any one of SEQ ID chain length and position of any carbon double bond(s) (if NOs: 2 or 4 or 6 or 20 are replaced, in-frame, by amino acid present) are more closely related to each other than to 35 sequences of a different member of the same family. enzymes acting upon other Substrates, and may be considered In a particularly preferred embodiment, the N-terminal to be a “family”. and/or C-terminal amino acids of SEQID NO: 2 or 4 or 6 or Without being bound by any theory or mode of action, the 20 are replaced by the corresponding regions of the acetyle sequence similarity between the members of any gene family nase, desaturase or hydroxylase polypeptides set forth in FIG. has its basis in the identity of the substrate involved and the 40 2.More preferably, at least about 30 amino acid residues from biochemical similarity of the reaction events occurring at the the N-terminal and/or C-terminal regions of any one of SEQ target carbon bond during the modification reaction, Suggest ID NOS: 2 or 4 or 6 or 20 are replaced, in-frame, by the ing that divergent sequences within a family may comprise corresponding regions of the acetylenase, desaturase or catalytic determinants or at least a functional part thereof hydroxylase polypeptides set forth in FIG. 2. which contributes to the specific catalytic properties of the 45 In an alternative embodiment, the synthetic fatty acid gene family members. of the invention encodes a fusion polypeptide in which the One example of a family is the desaturase, acetylenase, N-terminal and/or C-terminal amino acids of a fatty acid hydroxylase and/or epoxygenase enzymes which catalyze acetylenase or fatty acid hydroxylase or fatty acid desaturase desaturation, acetylenation, hydroxylation and/or epoxygen are replaced, in-frame, by the N-terminal and/or C-terminal ation respectively, of the A12 position of linoleic acid (here 50 region of any one of SEQID NOs: 2 or 4 or 6 or 20. inafter referred to as the “C18A12-MMO family”). The In a particularly preferred embodiment, the N-terminal present inventors have compared the nucleotide and amino and/or C-terminal amino acids of a fatty acid acetylenase or acid sequences of members of the C18A12-MMO family to fatty acid hydroxylase or fatty acid desaturase are replaced, determine the divergent regions thereof which potentially in-frame, by the N-terminal and/or C-terminal region of any comprise the determinants of alternative catalytic functions at 55 one of SEQID NOs: 2 or 4 or 6 or 20. Even more preferably, the A12 position (hereinafter referred to as “putative catalytic the fatty acid acetylenase or fatty acid hydroxylase or fatty determinants'). acid desaturase is selected from the list set forth in FIG. 2. Furthermore, the presence of such families of fatty acid Even still more preferably, at least about 30 amino acid modifying MMOs is contemplated with respect to other fatty residues from the N-terminal and/or C-terminal regions of a acid chain length and double bond positions. For example, the 60 fatty acid acetylenase or fatty acid hydroxylase or fatty acid C18A15-desaturase is contemplated to belong to a family of desaturase are replaced, in-frame, by the N-terminal and/or related enzymes capable of desaturation, acetylenation, C-terminal region of any one of SEQID NOS: 2 or 4 or 6 or 20. hydroxylation and/or epoxidation of the A15 position in C18 Accordingly, the present invention extends to any variants fatty acid substrates, the C18A15-MMO family. of the epoxygenase enzymes referred to herein, wherein said By producing synthetic genes in which these catalytic 65 variants are derived from an epoxygenase polypeptide as determinants have been interchanged (referred to as “domain described herein and exhibit demonstrable acetylenase or Swapping) it is possible to convert genes encoding one cata hydroxylase or desaturase activity, and either comprises an US 7,589.253 B2 27 28 amino acid sequence which differs from a naturally-occur high levels of the epoxy fatty acid vernolic acid in its seed oil. ring acetylenase or hydroxylase or desaturase enzyme, or Seeds from Crepis palaestina were shown to contain 61.4 exhibit catalytic properties which are different from a natu weight% of vernolic acid and 0.71 weight% of the acetylenic rally-occurring acetylenase or hydroxylase or desaturase fatty acid crepenynic acid of total fatty acids (Table 3). enzyme, or comprise a sequence of amino acids which are at least about 60% identical to any one of SEQID NOs: 2 or 4 or TABLE 3 6 or 20. As with other aspects of the invention, the variants Fatty acid composition of lipids derived from seeds of described herein may be produced as recombinant polypep Crepis alpina, Crepis palaestina and Euphorbia lagascae tides or in transgenic organisms, once the Subject synthetic 10 Relative distribution (weight%) genes are introduced into a Suitable host cell and expressed therein. Fatty acid Crepis alpina Crepis palaestina Euphorbia The recombinant polypeptides described herein or a homo Palmitic 3.9 S.1 4.3 logue, analogue or derivative thereof, may also be immuno Stearic 1.3 2.3 1.8 logically active molecules. 15 Oleic 1.8 6.3 22.0 Linoleic 14.0 23.0 1O.O A further aspect of the present invention provides an immu Crepyninic 75.0 0.7 O nologically-interactive molecule which is capable of binding Vernolic O 61.4 58.0 to a recombinant epoxygenase polypeptide of the invention. Other 4.0 1.2 3.9 Preferably, the recombinant epoxygenase polypeptide to which the immunologically-interactive molecule is capable Calculated from the area 96 of total integrated peak areas in gas liquid chro of binding comprises a sequence of amino acids set forth in matographic determination of methyl ester derivatives of the seed lipids any one of SEQ ID NOS: 2, 4, 6, or 20, or a homologue, analogue or derivative thereof. Example 2 In one embodiment, the immunologically interactive mol ecule is an antibody molecule. The antibody molecule may be 25 Biochemical Characterization of Linoleate monoclonal or polyclonal. Monoclonal or polyclonal anti A 12-Epoxygenases in Euphorbia lagascae and bodies may be selected from naturally occurring antibodies to Crepis palaestina an epitope, or peptide fragment, or synthetic epoxygenase peptide derived from a recombinant gene product or may be The enzyme, linoleate A12-epoxygenase synthesizes ver specifically raised against a recombinant epoxygenase or a 30 nolic acid from linoleic acid. Linoleate A12-epoxygenases homologue, analogue or derivative thereof. derived from Euphorbia lagascae and Crepis palaestina are Both polyclonal and monoclonal antibodies are obtainable localized in the microsome. The enzymes from these species by immunization with an appropriate gene product, or at least can remain active in membrane (microsome) fractions epitope, or peptide fragment of a gene product. Alternatively, prepared from developing seeds. fragments of antibodies may be used. Such as Fab fragments. 35 Preparations of membranes from Euphorbia lagascae and The present invention extends to recombinant and synthetic assays of their epoxygenase activities were performed as antibodies and to antibody hybrids. A “synthetic antibody' is described by Bafor et al. (1993) with incubations containing considered herein to include fragments and hybrids of anti NADPH, unless otherwise indicated in Table 4. Lipid extrac bodies tion, separation and methylation as well as GLC and radio The antibodies contemplated herein may be used for iden 40 GLC separations were performed essentially as described by tifying genetic sequences which express related epoxygenase Kohnet al. (1994) and Bafor et al. (1993). polypeptides encompassed by the embodiments described Preparations of membranes from Crepis alpina and Crepis herein. palaestina were obtained as follows. Crepis alpina and Cre The only requirement for successful detection of a related pis palaestina plants were grown in green houses and seeds epoxygenase genetic sequence is that said genetic sequence is 45 were harvested at the mid-stage of development (17-20 days expressed to produce at least one epitope recognized by the after flowering). Cotyledons were squeezed out from their antibody molecule. Preferably, for the purpose of obtaining seed coats and homogenized with mortar and pestle in 0.1M expression to facilitate detection, the related genetic sequence phosphate buffer, pH 7.2 containing 0.33M sucrose, 4 mM is placed operably behind a promoter sequence, for example NADH, 2 mM CoASH, 1 mg of bovine serum albumin/ml and the bacterial lac promoter. According to this preferred 50 4,000 units of catalase/ml. The homogenate was centrifuged embodiment, the antibodies are employed to detect the pres for 10 min at 18,000xg and the resulting supernatant centri ence of a plasmid or bacteriophage which expresses the fuged for 60 min at 150,000xg to obtain a microsome pellet. related epoxygenase. Accordingly, the antibody molecules Standard desaturase, acetylenase and epoxygenase assays are also useful in purifying the plasmid or bacteriophage with microsomal membranes from Crepis species were per which expresses the related epoxygenase. 55 formed at 25°C. with microsomal preparations equivalent to The subject antibody molecules may also be employed to 0.2 mg microsomal protein resuspended in fresh homogeni purify the recombinant epoxygenase of the invention or a zation buffer and 10 nmol of either 1-C 18:1-CoA or naturally-occurring equivalent or a homologue, analogue or 1-C18:2-CoA (specific activity 85,000 dp.m./nmol) in a derivative of same. total volume of 360 ul. When NADPH was used as coreduc 60 tant, the membranes were resuspended in homogenization Example 1 buffer comprising NADPH in place of NADH. Biochemical characterization of the microsomal linoleate Characterization of epoxy fatty acids in Euphorbia lagas A 12-epoxygenase derived from Euphorbia lagascae and Cre cae and Crepis spp. Seed from the wild species Euphorbia pis palaestina was carried out and data obtained were com lagascae and from various Crepis species were screened by 65 pared to the biochemical characteristics of oleate A12-desatu gas liquid chromatography for the presence of epoxy fatty rase and linoleate A12-acetylenase enzymes derived from acids. As shown in Table 3, Euphorbia lagascae contains very microsomal preparations of Crepis alpina (Table 4). US 7,589.253 B2 29 30 As shown in Table 4, the Crepis palaestina linoleate A12 cDNA using primers derived from the deduced amino acid epoxygenase exhibits similar biochemical features to the sequences of plant mixed-function monooxygenases. linoleate A12-acetylenase and oleate A12-desaturase from The D12V fragment was subsequently random-labeled and Crepis alpina, in so far as all three enzymes require O, work used to Screen the Crepis palaestina cDNA library Supra on equally well with either NADH or NADPH as the coreduc- 5 Hybond N' membrane filters from Amersham as prescribed tants, and are inhibited by cyanide but not by carbon monox by the manufacturer using standard hybridization conditions. ide. Additionally, none of these enzymes are inhibited by This approach resulted in the purification of a recombinant monoclonal antibodies against cytochrome P450 reductase. bacteriophage, designated Cpa12. The data in Table 4 Suggest that the Crepis palaestina The nucleotide sequence of the Cpa 12 cDNA was deter linoleate A12-epoxygenase belongs to the same class of mined and is set forth in SEQID NO: 1. enzyme as the Crepis alpina microsomal oleate A12-desatu The Cpa12 cDNA appeared to be full-length. A schematic rase and linoleate A12-acetylenase. representation of an expression vector comprising the Cpa12 In contrast, the Euphorbia lagascae linoleate A12-epoxy cDNA is presented in FIG. 1. The gene construct set forth genase requires NADPH as the coreductant, is not inhibited 15 therein is designed for introduction into plant material for the by cyanide, but is inhibited by carbon monoxide (Table 4). production of a transgenic plant which expresses the Subject Additionally, the inventors have discovered that the Euphor epoxygenase. Those skilled in the art will recognise that bia lagascae linoleate A12-epoxygenase is inhibited by similar expression vectors may be produced, without undue monoclonal antibodies raised against a cytochrome P450 experimentation, and used for the production of transgenic reductase enzyme. These data Suggest that the Euphorbia 2 plants which express any of the genetic sequences of the lagascae linoleate A12-epoxygenase belongs to the cyto 0 instant invention, by replacing the Cpal2 cDNA with another chrome P450 class of proteins and is therefore not related structural gene sequence. biochemically to the Crepis palaestina linoleate A12-epoxy As shown in FIG. 2, the nucleotide sequence of the Crep1 cDNA encoded a polypeptide which was closely related at the genase. amino acid level, at least, to an acetylenase enzyme of C. TABLE 4 25 alpina (Bafor et al. 1997: International Patent Application No. PCT/SE97/00247). Comparison of the biochemical characteristics of The 1.4 kb insert from pCpal2 was sequenced (SEQ ID epoxygenases, acetylenases and desaturases derived NO. 1) and shown to comprise an open reading frame which from Crepis spp. and Euphorbia lagascae encodes a polypeptide of 374 amino acids in length. The Enzyme Activity (% of control 30 deduced amino acid sequence of Cpal2 showed 81% identity and 92% similarity to the A12-acetylenase from Crepis alpina C. alpina C. palaestina E. lagascae and approximately 60% identity and 80% similarity with C. alpina linoleate linoleate linoleate oleate A12- A12- A12- A12 plant microsomal A12-desaturase proteins (FIG. 2). How Treatment desaturase acetylenase epoxygenase epoxygenase 35 ever, the polypeptide encoded by Cpal2 comprised significant differences in amino acid sequence compared to non-epoxy Carbon 85 84 88 3 monoxide genase enzymes. In particular, the Cpa12 has a deletion of six Anti-P450 96 91 94 33 contiguous amino acids in the 5-terminal region compared to reductase all the microsomal A12 desaturases, and a deletion of two antibodies contiguous amino acids in the 3'-terminal region compared to (CSAs) 40 KCN 16 O 35 92 the Crep 1 A12 acetylenase (FIG. 2). minus NADH 95 73 94 1OO Although membrane-bound fatty acid desaturase genes plus NADPH (control) show limited sequence homologies, they all contain three minus NADPH 100 1OO 100 11 regions of conserved histidine-rich motifs as follows: plus NADH (control) (control) (control) (i) His-(Xaa). His (SEQ ID NO: 21 and SEQ ID NO: 45 22); (ii) His-(Xaa)-His-His (SEQ ID NO: 23 and SEQ ID Example 3 NO: 24); and (iii) His-(Xaa)-His-His (SEQ ID NO: 23 and SEQ ID Strategy for Cloning Crepis palaestina Epoxygenase 50 NO: 24), Genes wherein His designates histidine, Xaa designates any natu rally-occurring amino acid residue as set forth in Table 1 Cloning of the Crepis palaestina epoxygenase genes relied herein, the integer (Xaa) refers to a sequence of amino on the characteristics of the C. palaestina and C. alpina acids comprising three or four repeats of Xaa, and the integer enzymes described in the preceding Examples. 55 (Xaa) refers to a sequence of amino acids comprising two In particular, poly(A)-- RNA was isolated from developing or three repeats of Xaa. These histidine-rich regions are Sug seeds of Crepis palaestina using a QuickPrep Micro mRNA gested to be apart of the active center of the enzyme (Shanklin purification kit (Pharmacia Biotechnology) and used to Syn et al., 1994). thesize an oligosaccharide d(T)-primed double stranded The amino acid sequence encoded by the Cpal2 cDNA cDNA. The double stranded cDNA was ligated to EcoRI/NotI 60 comprises three histidine-rich motifs similar, but not identi adaptors (Pharmacia Biotechnology) and a cDNA library was cal, to the histidine-rich motifs of the A12-desaturase constructed using the ZAP-cDNA Gigapack cloning kit enzymes. These data Suggest that the Cpa12 cDNA encodes (Stratagene). an enzyme which belongs to the mixed function monooxy Single-stranded cDNA was prepared from RNA derived genase class of enzymes. from the developing seeds of Crepis alpina, using standard 65 The analysis of fatty acids presented in Example 1 Supra procedures. A PCR fragment, designated as D12V (SEQ ID indicated that Vernolic acid was at least present in the seeds of NO: 7), was obtained by amplifying the single-stranded Crepis palaestina. This enzyme may in fact be present exclu US 7,589.253 B2 31 32 sively in the seeds of C. palaestina. The expression of the Cpal2 gene was examined using the 3' untranslated region of TABLE 5 the Cpal2 cDNA clone as a hybridization probe on northern Vernolic acid levels in transgenic A. thaliana blots of mRNA derived from developing seeds and leaves of lines expressing SEQID NO: 1 C. palaestina. As shown in FIG.3, the Cpal2 gene was highly 5 expressed in developing seeds but no expression could be Vernolic acid detected in leaves. These data are consistent with the enzyme (weight% of total seed fatty To Plant No. acids) activity profile of C. palaestina linoleate A12-epoxygenase in Cpal-4 1.4 these tissues. Cpal-5 1.1 10 Cpal-8 2.7 Example 4 Cpal-9 O.9 Cpal-13 O.9 Cpal-15 1.1 Demonstration of Epoxygenase Activity for the C. Cpal-17 15.8 palaestina Clones Cpal-21 1.3 15 Cpal-23 1.4 Cpal-24 1.O Confirmation that the cDNA clones of C. palaestina Cpal-25 1.2 encode an epoxygenase was obtained by transforming Ara Cpal-26 1.1 bidopsis thaliana, with each individual candidate clone. The untransformed control line O.O transformed tissue was examined for the presence of epoxy fatty acids that A. thaliana would not otherwise produce. Additionally, the level of hydroxy fatty acids was determined, Alternatively, or in addition, putative fatty acid epoxyge as such fatty acids can be formed from the metabolism of an nase sequences described herein are each transformed into epoxy fatty acid, by the action of endogenous A. thaliana Linum usitatissimum (flax) and Arabidopsis thaliana under epoxide hydrolases (Blee and Schuber, 1990). the control of the napin seed-specific promoter. Transgenic The epoxygenase cDNA comprising SEQ ID NO: 1 was 25 flax and Arabidopsis thaliana plants are examined for pres cloned into the Binary vector construct set forth in FIG. 4. ence of epoxy fatty acids in developing seed oils. Previous Briefly, the cDNA sequence was sub-cloned from the pCpal2 work has shown that if epoxy fatty acids are fed to developing plasmid (FIG. 1) into the binary plasmid, by digesting pCpal2 flax embryos they are incorporated into triglycerides (Ex with EcoRI and end-filling the restriction fragment using T4 ample 10). DNA polymerase enzyme. The Binary vector (FIG. 4) was 30 Alternatively, yeast are also transformed with the epoxy made linear using BamHI and also end-filled using T4 DNA genase clones of the invention and assayed for production of polymerase. For the end-filling reactions, 1 lug of cDNA insert epoxy fatty acids. or linearized Binary vector DNA was resuspended in 50 ul of T4 DNA polymerase buffer (33 mM Tris-acetate pH 7.9, 66 Example 6 mM potassium acetate, 10 mM magnesium acetate and 5 mM 35 DDT) supplemented with 100 mM of each dNTP and 0.1 mg/ml BSA and 3 units of T4 DNA polymerase, and incu Mass Spectroscopy Confirmation of Epoxy Fatty bated for 6 minincubation at 37°C. The reaction was stopped Acids in T. Arabidopsis Seed Borne on Primary To by heating at 75DC for 10 mins. The blunt-ended cDNA and Transgenic Plants Binary vector DNA were ligated using T4 DNA ligase and 40 standard ligation conditions as recommended by Promega. Gas chromatography of methyl esters prepared from seed Clones were selected in which the SEQID NO: 1 sequence lipids of T1 seed of Cpa12-transformed Arabidopsis thaliana was inserted behind the napin promoter, in the sense orienta plants (Example 5) revealed the presence of two additional tion, thereby allowing for expression of the epoxygenase fatty acids compared to the untransformed controls. The first polypeptide. The Binary plasmid harboring SEQID NO: 1, in 45 of these compounds had a retention time equivalent to that of the sense orientation, operably under control of the truncated a Vernolic acid standard. The second compound had a longer napin promoter, is represented schematically in FIG. 5. retention time and was putatively identified as 12,13-epoxy The Binary plasmid set forth in FIG. 5 was transformed 9, 15-octadecadienoic acid, an expected derivative of vernolic into Agrobacterium strain AGLI using electroporation and acid, resulting from desaturation at the A15 position by the used to transform Arabidopsis thaliana. Transgenic A. 50 endogenous Arabidopsis thaliana A15-desaturase. thaliana plants were obtained according to the method Confirmation of the exact identity of the two peaks was described by Valvekens et al. (1988) and Dolferus et al. obtained by mass spectroscopy of diols which were prepared (1994). from the epoxy fatty acid fraction derived from Cpal2-trans Transgenic plants and untransformed (i.e. control) plants formed plants. The diols were converted further to trimethyl were grown to maturity. Mature seed of each plant was ana 55 silyl ethers and analyzed by GC-MS DB23 on a fused silica lyzed for fatty acid composition by standard techniques. Pri capillary column (Hewlett-Packard 5890 II GC coupled to a mary transformant (T) plants were established and T1 seed Hewlett Packard 5989AMS working in electron impact at 70 was harvested from each plant and analyzed for fatty acid eV 15). The total ion chromatogram showed two peaks as composition by gas chromatography. Twelve To plants were follows: shown to contain vernolic acid in their T1 seed lipids at 60 (i) The first eluting peak had prominent ions of mass 73, concentrations ranging from 0.9% to 15.8% of total fatty 172, 275, and 299, indicating that the epoxy group was acids, while untransformed control plants contained no ver positioned at C-12 of a C18 fatty acid and that a double nolic acid (Table 5). The highest-expressing plant line was bond occurred between the epoxy group and the car Cpal-17, for which the GLC elution profiles (from packed boxyl terminus. This mass spectra was identical to the column and capillary column analysis) is presented in FIG. 6. 65 spectra of a trimethylsilyl ether derivative of diols pre The GLC elution profile from packed column for the untrans pared from pure vernolic acid (12,13-epoxy-9-octade formed control is also shown in FIG. 6. cenoic acid); and US 7,589.253 B2 33 34 (ii) the second eluting peak had prominentions of mass 73, trol of the napin promoter was germinated and T1 plants were 171,273, and 299, indicating the presence of two double established from five To lines (Nos. 4, 8, 13, 17& 21 in Table bonds and an epoxy group positioned at C-12 of a C18 5). The T2 seed was harvested from each T1 plant and ana fatty acid, consistent with the mass spectrum for 12,13 lyzed for fatty acid composition. The progeny of transformant epoxy-9.15-octadecadienoic acid. Nos. 4, 8, 13 and 21 (Table 5) segregated as expected for presence of Vernolic acid, with those plants containing ver Example 7 nolic acid ranging up to 3.1% (Table 6). All T1 plants that contained vernolic acid (i.e. epoxy 18:1 Fatty Acid Analysis of Cpal2 Transgenic Arabidopsis in Table 6) also contained 12,13-epoxy-9.15-octadecadienoic Plants 10 acid (i.e. epoxy 18:2 in Table 6: see also FIG. 7), indicating that some of the vernolic acid synthesized by the Cpal2 The T1 seed derived from transformed Arabidopsis epoxygenase was Subsequently desaturated by the endog thaliana plants expressing the Cpal2 cDNA clone under con enous A15-desaturase.

TABLE 6 Fatty acid composition of selfed seeds borne on T plants derived from five primary Cpal2 transformants of Arabidopsis thaliana Fatty Acid Plant Non-epoxy fatty acids Epoxy fatty acids

No. 16:O 18:0 18:1 8:2 8:3 20:O 20:1 22:O 22:1 18:1 18:2

4-1 8.3 3.9 15.S. 23.9 20.6 2.8 6.5 7 .6 4-2 7.6 4.1 20.3 7.8 8.0 3.4 9.7 8 2.0 O.82 O.63 4-3 8.4 4.3 26.O 3.5 6.1 2.8 9.0 8 .6 2.03 0.72 4-4 7.6 4.O 25.2 4.3 6.O 2.8 9.8 2.1 7 1.99 O.92 4-5 7.2 3.6 15.6 23.1 9.9 3.1 9.7 .6 2.1 4-6 7.0 3.7 19.2 7.8 8.4 3.2 20.3 9 2.1 O.87 O.33 4-8 7.4 3.9 16.O 23.6 201 3.1 8.7 .6 8 4-9 7.6 4.O 24.8 3.4 5.9 2.8 20.4 2.3 8 2.30 1.07 4-10 7.6 4.2 24.0 3.5 6.2 31, 20.4 9 8 97 O.83 4-11 7.4 3.9 15.O 23.2 20.4 3.3 8.8 7 2.0 4-12 8.7 4.O 20.7 7.0 7.5 2.6 7.2 7 .5 38 O.74 4-13 7.2 4.1. 21.9 6.4 7.7 3.2 21.0 7 9 .14 O45 8-1 8.1 3.9 26.1 S.O 6.O 2.6 9.S 2.0 .6 .79 O.82 8-3 8.7 4.2 31.6 1.5 4.O 2.2 8.5 9 .4 2.38 1.13 8-4 8.5 4.1 27.2 S.1 6.1 2.5 8.9 8 .4 70 O.84 8-5 9.1 4.2 27.7 4.7 6.2 2.4 8.3 7 .5 70 O.82 8-6 9.8 4.O 26.O 7.2 7.2 2.3 6.9 .6 .2 36 O.71 8-7 1O.O 3.S 15.2 25.3 22.3 2.3 4.4 7 7 8-8 8.4 4.3 32.2 0.7 3.3 2.S. 20.3 .6 .5 92 O.82 8-9 9.8 3.6 15.9 253 22.O 2.4 4.5 .6 3 8-10 7.5 3.9 24.4 5.9 5.8 2.8 20.2 2.2 8 70 O.82 8-11 7.6 3.8 15.4 23.6 9.8 2.9 9.4 .5 8 8-12 9.4 3.7 24.2 6.7 6.7 2.2 7.6 0.9 .2 .46 O.65 8-13 10.3 4.3 25.3 7.1 7.9 2.2 6.O 8 3 48 0.73 3-1 7.0 4.3 33.3 8.1 1.1 2.7 23.1 7 .6 2.42 1.26 3-2 7.2 4.3 30.4 9.6 2.7 2.8 22.0 8 .6 2.48 1.37 3-3 7.6 3.9 S. 6 23.6 9.7 3.0 9.1 7 8 3-4 7.7 4.0 S.2 22.5 9.3 3.1 8.0 .6 7 3-5 8.0 4.2 6.3 22.2 7.5 4.4 94 2.0 2.0 3-6 7.9 4.4 25.7 14.7 5.8 2.9 21.2 .6 7 1.56 O.63 3-7 7.9 4.0 6.O 23.3 9.6 3.0 9.1 .6 8 3-9 8.0 4.0 6.1 23.6 20.0 2.9 8.7 .6 .6 3-10 8.7 4.2 34.6 9.6 2.5 2.2 9.1 .5 .2 2.21 1.01 3-11 8.7 4.0 7.6 24.3 89 2.8 7.1 .6 .4 3-12 8.9 4.2 26.4 14.6 6.O 2.5 7.5 .6 .2 1.62 O.74 3-13 9.0 4.4 27.9 14.4 5.3 2.5 8.9 .5 .4 1.30 O.77 3-14 9.2 4.2 7.2 23.8 8.8 2.7 7.9 7 .5 3-15 8.4 4.2 9.7 20.9 8.6 2.7 7.7 .4 .5 O.40 O16 3-16 8.2 4.3 23.O. 17.1 7.3 2.8 9.3 .5 .5 0.97 O42 3-17 8.3 4.1 5.7 23.9 9.9 2.8 7.6 .6 9 7-1 7.6 4.1 5.8 23.7 9.6 2.6 20.3 7 7 7-2 8.3 4.1 6.4 24.4 20.1 2.3 6.8 .5 .4 7-3 8.1 4.1 6.4 24.3 20.O 2.5 7.6 .6 .4 21-1 8.1 4.3 26.9 14.5 S.O 2.9 9.9 .5 .5 1.64 O.63 21-2 8.2 4.O 27.9 11.8 3.2 2.5 9.8 7 .5 2.18 O.91 21-3 8.8 3.7 6.4 24.4 20.6 2.5 7.3 7 .4 21-4 7.9 3.9 9.6 19.8 7.8 2.7 8.7 7 7 O.66 O46 21-5 7.2 4.2 26.5 12.9 4.4 3.O 215 O.9 8 1.78 O.84 21-6 8.3 4.2 27.4 13.9 5.4 2.6 9.9 7 .5 1.66 O.65 21-7 7.2 4.2 26.8 13.5 34 30 21.9 7 8 1.74 O.8O 21-8 7.4 3.8 6.3 23.6 94 3.2 9.2 7 9 21-9 7.2 4.O 28.1 118 3.S 3.0 22.5 9 9 2.15 1.OS 21-10 7.2 4.2 26.1 13.8 4.6 3.O 22.3 7 8 1.64 O.82 US 7,589.253 B2 35 36

TABLE 6-continued Fatty acid composition of selfed seeds borne on T plants derived from five primary Cpal2 transformants of Arabidopsis thaliana Fatty Acid Plant Non-epoxy fatty acids Epoxy fatty acids

No. 16:0 18:0 18:1 18:2 18:3 20:O 20:1 22:O 22:1 18:1 18:2

21-11 7.1 4.2 29.2 115 12.7 3.0 22.5 18 1.8 2.2O 1.09 21-12 7.2 4.1 26.2 13.6 14.2 3.1 224 1.8 1.9 1.71 21-13 7.1 4.3 33.7 7.1 10.O 2.7 24.1 2.0 18 3. OS 1.47 21-14 7.4 3.7 16.9 21.9 19.6 3.1. 19.2 1.8 2.0 O.29 tr 21-15 7.7 3.6 15.6 24.3 20.2 2.9 18.1 1.8 1.8

Example 8 that these plants were kanamycin resistant. Ten T1 seeds from each plant were analyzed individually for fatty acid compo Fatty Acid Analysis of Cpal2 Transgenic Linola sition using standard techniques. Plants As shown in Table 7, seed from AP20 segregated into 3 The binary plasmid construct described above comprising classes, comprised of three seeds with no Vernolic acid, two the Cpal2 cDNA clone (FIG. 5) was transformed into Agro having greater than 0.7% vernolic acid, and five having inter bacterium tumefaciens Strain AGL1, using electroporation. mediate levels (0.13-0.47%) of vernolic acid. The transformed A. tumefaciens was used to infect Linum 25 Similarly, seeds from AP21 segregated into 3 classes com usitatissimum var. Eyre explants as described by Lawrence et prised of five seeds having no Vernolic acid, two having all (1989), except that MS media was used as the basal greater than 0.25% vernolic acid and three having an inter medium for the induction of roots on regenerated shoot mate mediate level (0.09-0.14%) of vernolic acid (Table 8). rial. Thus, a total of twelve seeds were obtained which con Two primary Linola transformants (To plants) designated 30 tained vernolic acid. Eight of the twelve AP20 and AP21 AP20 and AP21 were confirmed as being transgenic by PCR seeds containing Vernolic acid also contained 12,13-epoxy using primers directed against the Cpal2 gene and by showing 9, 15-octadecadienoic acid.

TABLE 7 Fatty acid composition of 10 individual T1 seeds from Linola Cpal2 primary transformant AP20 Non-epoxy fatty acids Epoxy fatty acids

seed 16: O 18:0 18:1 18:2 18:3 20:O 20:1 22:O 22:1 18:1 18:2

6.4 3.6 17.8 68.1 2.0 O.2 O6 6.O 3.5 25.4 60.8 1.4 O.2 O.2 O.70 O.23 6.O 3.9 20.4 64.6 2.1 O.3 O.6 6.3 3.S. 28.3 57.3 1.3 O.2 O.2 1.4 O.34 O.28 5.2 4.8 24.9 61.2 1.6 O.3 O.2 O.1 0.37 5.8 4.1 233 63.1 1.9 O.2 O.2 O.2 O.47 5.9 43 217 64.1 2.2 O.2 O.2 O.2 O.13 O.12 5.9 3.3 22.3 65.2 2.0 O.2 O.2 O.1 O.2 S.6 4.O 25.2 614 1.7 O.2 O.2 O.1 O.84 1 6.2 4.4 27.4 57.9 1.7 O.2 O.2 O.2 O.S4

TABLE 8 Fatty acid composition of 10 individual T1 seeds from Linola Cpal2 primary transformant AP21 Non-epoxy fatty acids Epoxy fatty acids

seed 16: O 18:0 18:1 18:2 18:3 20:O 20:1 22:O 22:1 18:1 18:2

6.1 4.2 35.2 SO.8 1.3 2.0 5.7 SO 32.9 53.3 1.4 O.2 O.2 O.2 O.14 O.21 5.9 4.0 35.1 SO.8 1.3 O.2 O.2 O.1 1.5 7.5 4.1 38.8 455 12 O.2 O.3 1.7 5.8 S.O 28.8 S7.3 1.3 O.2 O.2 O.1 0.37 O.O6 5.8 5.0 44.1 41.4 1.4 O.2 O.2 O.2 6.5 4S 27.9 58.6 1.3 O.2 O.1 O.1 6.9 4.6 37.6 48.1 1.2 O.10 O.19 US 7,589, 253 B2 37 38

TABLE 8-continued Fatty acid composition of 10 individual T1 seeds from Linola Cpal2 primary transformant AP21 T Non-epoxy fatty acids Epoxy fatty acids

Seed 16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 18:1 18:2

9 6.2 4.7 33.7 S2.1 1.3 O.2 O.2 O.2 O.09 10 6.1 4.8 29.7 56.6 1.3 O.2 O.2 O.1 O.2S

Four T1 plants were established from the kanamycin-re vernolic acid are produced by the transgenic LinolaTM flax sistant seedlings of AP20. All four plants were subsequently plants during seed oil synthesis, when the epoxygenase gene shown to produce vernolic acid in their T2 seed (Table 9). is expressed at high levels. Levels of 18:2 epoxy fatty acids were not analyzed in these T2 Additionally, the inventors have shown that labeled ver seed. nolic acid fed to developing flax seeds is not degraded but is

TABLE 9 Fatty acid composition of T2 seeds from Linola Cpal2T1 progeny of AP20 epoxy fatty T2 Non-epoxy fatty acids acid

seed 16:0 18:0 18:1 18:2 18:3 20:O 20:1 22:O 22:1 18:1

A. 3.4 3.O 27.4 6SS O-6 l8 l8 l l O.O6 B 3.5 31 30.2 626 O.6 l8 l8 l l O.O7 C 3.6 2.7 33.3 59.8 O.6 l8 l8 l l O.O7 D 3.4 31 28.2 64.6 O.6 l8 l8 l l O.11 na. = not analyzed

Example 9 incorporated into storage lipids at all three positions of the triglyceride molecule (see Example 10). Consistent with Producing Epoxy Fatty Acids in Transgenic 35 these data, high levels of Vernolic acid synthesized by the Organisms introduced epoxygenase are readily deposited into the seed oil triglycerides of this species. Production of an oil rich in vernolic acid was achieved by Example 10 transforming the epoxygenase gene described herein, in par- 40 ticular SEQIDNO: 1, into Arabidopsis thaliana, as described Incorporation of Oleic Acid and Vernolic Acid into in the preceding Examples. As shown in Table 5, transgenic A. the Lipids of Developing Linseed Cotyledons thaliana lines expressing SEQID NO: 1 produce high levels of vernolic acid in their seeds relative to other fatty acids. In Detached developing linseed cotyledons (six pairs in each particular, in one transgenic line (Cpal-17), the Vernolic acid 45 incubation, duplicate incubations) at mid stage of seed devel opment (20 days after flowering) were incubated with 10 produced is as much as 15.2% (w/w) of total seed fatty acid nmol of the ammonium salts of either 1-CIvernolic acid COntent. (specific activity 3000dp.m./nmol) or 1-''Coleic acid (spe Production of an oil rich invernolic acid is also achieved by cific activity 5000 d.p.m./nmol) in 0.2 ml phosphate buffer transforming the epoxygenase gene described herein, in any so pH 7.2 for 30 minat30DC. The cotyledons were then rinsed one of SEQID NOs: 1, 3, 5, or 19 and preferably any one of three times with 1 ml of distilled water and either extracted SEQ ID NOs: 1 or 3 or 5 or 19, into any oil accumulating immediately in an Ultra Turrax according to Bligh and Dyer organism that normally has very high levels of linoleic acid (1959) or incubated further in 0.5 m. 0.1 M phosphate buffer and minimal other competing enzyme activities capable of pH 7.2 for 90 or 270 min before extraction. An aliquot of the utilizing linoleic acid as a Substrate. The genetic sequences of 55 lipids in the chloroform phase was methylated and separated the invention are placed operably under the control of a pro on silica gel TLC plates in n-hexane/diethyletherfacetic acid moter which produces high-level expression in oilseed, for (85:15:1). The rest of the lipids in the chloroform phase of example the napin seed-specific promoter. each sample were applied on two separate silica gel TLC In one alternative approach to the transformation of A. plates and the plates were developed in chloroform/methanol/ 60 acetic acid/water (85:15:10:3.5 by Vol) for polar lipids sepa thaliana, high-linoleic genotypes of flax, Sunflower, corn or ration and in n-hexane/diethylether/acetic acid (60:40:1.5) safflower are transformed with the epoxygenase of the inven for neutral lipid separation. Lipid areas with migration cor tion. High levels of vernolic acid are produced by the trans responding to authentic standards were removed and radio genic plants during seed oil synthesis, when the epoxygenase activity in each lipid were quantified by liquid Scintillation gene is expressed at high levels. 65 counting. Alternatively, LinolaTM (low linolenic acid) flax is trans The recovery of ''C-label in the chloroform phase is formed with the epoxygenase of the invention. High levels of depicted in FIG.8. Somewhat more than half of added radio US 7,589.253 B2 39 40 activity from both|''Coleic acid and 'Clvernolic acid was thus obtained was then ligated to EcoRI/NotI adaptors (Phar taken up by the cotyledons and recovered as lipophilic Sub macia Biotechnology) and a cDNA library was constructed stances after the 30 min pulse labeling. This quantity using the ZAP-cDNA Gigapack cloning kit (Stratagene). The remained virtually unchanged during the further 270 min of cDNA library on Hybond N+ membrane filters (Amersham) incubation with both substrates. Separation of radioactive 5 was screened with the random-labeled D12V fragment (SEQ methyl esters of the lipids showed that most of the radioac ID NO: 7) derived from Crepis alpina as prescribed by the tivity (92%) from '''Clvernolic acid feeding experiments manufacturer, using standard hybridization conditions. This resided in compounds with the same migration as methyl resulted in the purification of a recombinant bacteriophage Vernoleate indicating that the epoxy group remained intact in designated CrepX. the linseed cotyledons throughout the 270 min incubation. 10 The nucleotide sequence of the CrepX cDNA was deter About 28% of the activity from '''Clvernolic acid feeding mined and is set forth in SEQID NO:3. The deduced amino which was present in the chloroform phase resided in phos acid sequence of CrepX (SEQ ID NO: 4) comprises a 374 phatidylcholine after 30 min and the radioactivity decreased amino acid protein having 97% identity to the Cpa 12 A12 to only 5% at 300 min of incubation (FIG.9). epoxygenase sequence, but only 57% identity to the Arabi About 22% of the activity from '''Coleic acid feeding 15 dopsis thaliana L26296 A12-desaturase sequence. This which was present in the chloroform phase resided in phos clearly demonstrates the presence of a gene in another Crepis phatidylcholine after 30 min and the radioactivity decreased sp. having high Vernolic acid content, which gene is highly to about 11% at 300 min of incubation (FIG.9). homologous to the Cpa12 A12-epoxygenase gene and is About 32% of the activity from '''Clvernolic acid feeding clearly not a desaturase gene. which was present in the chloroform phase resided in tria cylglycerols after 30 min and the radioactivity increased to Example 12 over 60% at 300 min of incubation (FIG. 10). The diacylg lycerols contained some 24% of the activity in the ''C Cloning of A12-Epoxygenase Genes from Vermonia Vernolic acid feeding experiments and this quantity remained galamensis rather constant over the incubation periods. 25 About 5% of the activity from '''Coleic acid feeding Following the general strategy outlined in the preceding which was present in the chloroform phase resided in tria example, a homologue of the Cpal2 A12-epoxygenase gene cylglycerols after 30 min and the radioactivity increased to was also obtained from Vermonia galamensis, containing high 18% at 300 min of incubation (FIG. 10). The diacylglycerols levels of Vernolic acid in its seeds. contained some 19% of the activity after 30 min in the ''C 30 A partial A12-epoxygenase-like sequence was obtained oleic acid feeding experiments and this quantity remained from V. galamensis, by preparing first strand cDNA templates rather constant over the incubation periods. using total RNA from developing seeds as a template. A PCR The above experiment shows that linseed cotyledons do fragment (550 nucleotides in length), designated as Vgall, not metabolize the epoxy group of Vernolic acid to any great was obtained by amplifying the single-stranded cDNA, using extent. Further it shows that linseed cotyledons possess 35 primers derived from the deduced amino acid sequence of mechanisms to efficiently remove vernolic acid from mem plant mixed function monooxygenases. The nucleotide brane lipids and incorporate them into triacylglycerols. sequence of the amplified DNA was determined using stan dard procedures and is set forth in SEQID NO: 5. Example 11 Alignment of the deduced amino acid sequence of the 40 Vgal1 PCR fragment (SEQID NO: 6) with the full sequence Cloning of A12-Epoxygenase Genes from an of Cpa12 A12-epoxygenase and the Arabidopsis thaliana Unidentified Crepis Species L26296 A12-desaturase (FIG. 2) demonstrates that the ampli fied Vgall sequence encodes an amino acid sequence corre Homologues of the Cpa12 A12-epoxygenase gene were sponding to the region spanning amino acid residues 103-285 obtained from species other than C. palaestina which are rich 45 of the Cpal2 polypeptide. Within this region, the Vgal1 in epoxy fatty acids, by cloning the members of the gene sequence showed greater amino acid identity with the Cpa12 family of A12 mixed function monooxygenases that are A 12-epoxygenase sequence (67%) than with the A. thaliana highly expressed in developing seeds and comparing their A 12-desaturase sequence (60%), Suggesting that the ampli amino acid sequence to those of known A12-desaturase and fied DNA corresponds to an epoxygenase rather than a A 12-epoxygenase sequences. 50 desaturase sequence. Such genes were cloned either by Screening developing The corresponding full-length. A 12-epoxygenase sequence seed cDNA libraries with genetic probes based on either the was obtained from V. galamensis, and the nucleotide Cpa12 gene (SEQID NO: 1) or the D12V fragment (SEQID sequence of the full-length clone determined (SEQ ID NO: NO: 7), or by amplifying PCR fragments using primers 19). The deduced amino acid sequence of the full-length Vgal designed against conserved sequences of the plant A12 mixed 55 A 12-epoxygenase polypeptide (SEQ ID NO: 20) comprises function monooxygenases, as described herein. Putative A12 384 amino acids comprising all three conserved mixed func epoxygenase sequences show greater overall sequence iden tion monooxygenase consensus sequences for epoxygenases tity to the A12-epoxygenase sequences disclosed herein, than as set forth in SEQID NOs: 15, 16, and 18 (see FIG. 2). to the known A12-desaturase sequences. In one example of this approach, a full-length A12-epoxy 60 Example 13 genase-like sequence was obtained from an unidentified Cre pis sp. containing high levels of Vernolic acid in its seed oils Demonstration of Epoxygenase Activity for the V. and known not to be Crepis palaestina. Poly(A)-- RNA was galamensis Clone isolated from developing seeds of this Crepis sp. using a QuickPrep Micro mRNA purification kit (Pharmacia Bio 65 Confirmation that the full-length cDNA clone of V. gala technology) and used to synthesize an oligosaccharide dCT)- mensis encodes an epoxygenase is obtained by transforming primed double-stranded cDNA. The double stranded cDNA Arabidopsis thaliana with a binary vector comprising the US 7,589.253 B2 41 42 isolated cDNA clone in the sense orientation and in operable 4. Bafor, M., Smith, M. A., Jonsson, L., Stobart, K. and connection with a promoter as described in the preceding Stymine, S. (1993) Arch. Biochem. Biophys. 303, 145-151. examples. Transformed tissue is examined for the presence of 5. Bafor, M., Banas, A., Wilberg, E., Lenman, M., Stahl, U. epoxy fatty acids that A. thaliana would not otherwise pro and Stymine, S. (1997) In: Williams, J. P., Mobasher, K.U., duce. Additionally, the level of hydroxy fatty acids is deter 5 Lem, N.W. (eds) Physiology, biochemistry and molecular mined, as Such fatty acids can beformed from the metabolism biology of plant lipids. Kluwer Academic Publisher, Dor of an epoxy fatty acid, by the action of endogenous A. drecht. In-press. thaliana epoxide hydrolases (Blee and Schuber, 1990). 6. Blee and Schuber (1990).J. Biol. Chem. 265, 12887-12894. The V. galamensis cDNA (SEQID NO:19) was cloned into 7. Blee, E., Wilcox, A. L., Marnett, J. M., Schuber, F. (1993) a binary vector construct, Such as that shown in FIG. 4. 10 J. Biol. Chem. 268, 1798-1715. essentially as described in the preceding examples. The 8. Blee, E., Stahl, S., Schuber, F. and Stymine, S. (1994) Binary plasmid harboring SEQID NO: 19 was transformed Biochem. Biophys. Res. Comm. 197,778-784 into Agrobacterium strain AGLI using electroporation and 9. Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. used to transform A. thaliana. Transgenic A. thaliana plants Physiol. 230, 379-288. were obtained according to the method described by Valvek 15 10. Bozak, K. R. Yu, H., Sirevag, R. and Christoffersen, R. E. ens et al. (1988) and Dolferus et al. (1994). (1990) Proc. Natl. Acad. Sci. USA 87,3904-3908. Transgenic plants and untransformed (i.e. control) plants 11. Christou, P., McCabe, D. E., Swain, W. F. (1988). Plant are grown to maturity. Mature seed of each plant are analyzed Physiol 87,671-674. for fatty acid composition by standard techniques. Primary 12. Crossway et al. (1986) Mol. Gen. Genet. 202, 179-185. transformant (To) plants are established and T1 seed are har 13. Devereux, J., Haeberli, P. and Smithies, O. (1984). Nucl. Vested from each plant and analyzed for their fatty acid com Acids Res. 12,387-395. position by gas chromatography. To plants are shown to con 14. Dolferus et al. Plant Physiol. (1994) 105, 1075-1087. tain higher levels of epoxy fatty acids in their T1 seed lipids 15. Engeseth, N. & Stymine, S. (1996) Planta 198, 238-245 than the seeds of untransformed control plants. 16. Fromm et al. (1985) Proc. Natl. Acad. Sci. (USA) 82, Gas chromatography of methyl esters prepared from seed 25 5824-5828. lipids of T1 seed of Vgal transformed Arabidopsis thaliana 17. Haseloff, J. and Gerlach, W. L. (1988). Nature 334, 586 plants is performed to show the presence of additional fatty 594. acids compared to the untransformed controls. The retention 18. Herrera-Estrella et al. (1983a) Nature 303, 209-213. time of these compounds permits their identification as epoxy 19. Herrera-Estrella et al. (1983b) EMBO J. 2,987-995. fatty acids, and/or derivatives of epoxy fatty acids that are 30 20. Herrera-Estrella et al. (1985) In: Plant Genetic Engineer produced by the action of endogenous desaturase enzymes on ing, Cambridge University Press, NY. pp 63-93. the epoxy fatty acids. 21. Kohn, G., Hartmann, E., Stymine, S. & Beutelmann, P. Confirmation of the exact identity of the epoxy fatty acid (1994).J. Plant Physiol. 144, 265-271 products and derivatives thereof is obtained by mass spec 22. Krens, F. A., Molendijk, L. Wullems, G.J. and Schilp troscopy of the diols from the epoxy fatty acid fraction of 35 eroort, R. A. (1982). Nature 296, 72-74. transformed plants. The diols are converted further to trim 23. Lawrence, G.J., Ellis, J. G. Finnegan, E.J., Dennis, E. S. ethylsilyl ethers and analyzed by GC-MS DB23 on a fused and Peacock, W.J. (1989) In: Breeding Research: The Key silica capillary column (Hewlett-Packard 5890 IIGC coupled to Survival of the Earth (Iyama, S. and Takeda, G. eds) 6th to a Hewlett Packard 5989AMS working in electron impact International Congress of SABRAO. pp 535-538. 40 24. Lazo, G. R., Stein, P. A. and Ludwig, R. A. (1991). at 70eV 15). Bio/technology 9,963-967. 25. Needleman and Wunsch (1970).J. Mol. Biol. 48, 443-453. REFERENCES 26. Pazkowski et al. (1984) EMBO.J. 3, 2717-2722. 27. Pietrzak, M., Shillito, R. D., Hohn, T. and Potrykus, I. 1. An et al. (1985) EMBO.J. 4:277-284. 45 (1986). Nucl. Acids Res. 14,5857-5868. 2. Ausubel, F. M., Brent, R., Kingston, RE, Moore, D. D., 28. Sanger. F., Nicklin, S. and Coulson, A. R. (1977) Proc. Seidman, J. G., Smith, J. A., and Struhl, K. (1987). In: Natl. Acad. Sci. (USA) 72, 5463-5467. Current Protocols in Molecular Biology. Wiley Inter 29. Shanklin, J., Whittle, E. and Fox, B.G. (1994) Biochem science (ISBN 047150338). istry 33, 12787-12794. 3. Badami, R. C., and Patil, K. B. (1981) Progress in Lipid 50 30. Valvekens et al. (1988) Proc. Natl. Acad. Sci. (USA) 85, Research, 19, 119-53. 5536-5540.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 24

SEO ID NO 1 LENGTH: 1358 TYPE: DNA ORGANISM: Crep is palaestina FEATURE; NAME/KEY: CDS LOCATION: (30) ... (1151) OTHER INFORMATION: US 7,589.253 B2 43 44

- Continued

<4 OO SEQUENCE: 1. gagaagttga ccataaat catttat caac atg ggt gcc ggc ggit cgt. ggt cgg 53 Met Gly Ala Gly Gly Arg Gly Arg a Ca tog gala a.a.a. tcg gtc atg gala cgt gt C toa gtt gat CC a gta acc Thir Ser Glu Lys Ser Wall Met Glu Arg Wall Ser Wall Asp Pro Wall Thir 1O 15 2O tto to a Ctg agt gaa ttg aag Cala gca at C cott c cc Cat tgc ttic cag 149 Phe Ser Luell Ser Glu Lell Glin Ala Ile Pro Pro His Cys Phe Glin 25 3 O 35 4 O aga tot gta at C cgc toa tot tac tat gtt gtt Cala gat citc. att att 197 Arg Ser Wall Ile Arg Ser Ser Wall Wall Glin Asp Luell Ile Ile 45 SO 55 gcc tac at C ttic tac tto citt gcc aac aca atc. cott act citt cott 245 Ala Ile Phe Tyr Phe Lell Ala Asn Thir Ile Pro Thir Luell Pro 60 65 70 act agt Cta gcc tac tta gct cc c gtt tgg tto tgt Cala gct 293 Thir Ser Luell Ala Tyr Lell Ala Pro Wall Trp Phe Cys Glin Ala 7s 85 agc gt C citc. act ggc tta tgg at C citc. ggc CaC gaa ggt CaC Cat 341 Ser Wall Luell Thir Gly Lell Trp Ile Luell Gly His Glu Cys Gly His His 90 95 1 OO gcc titt agc aac tac a Ca tgg titt gac gac act gtg ggc ttic at C citc. 389 Ala Phe Ser Asn Tyr Thir Trp Phe Asp Asp Thir Wall Gly Phe Ile Luell 105 11O 115 12O

CaC to a titt citc. citc. a CC cc.g tat ttic tot tgg a.a.a. tto agt CaC cgg 437 His Ser Phe Luell Lell Thir Pro Phe Ser Trp Phe Ser His Arg 125 13 O 135 aat CaC Cat to c aac a Ca agt tog att gat aac gat gaa gtt tac att 485 Asn His His Ser Asn Thir Ser Ser Ile Asp ASn Asp Glu Wall Ile 14 O 145 15 O cc.g a.a.a. agc aag t cc a.a.a. citc. gcg cgt at C tat a.a.a. citt citt aac aac 533 Pro Ser Lys Ser Lell Ala Arg Ile Lell Luell Asn Asn 155 16 O 1.65

C Ca cott ggt cgg Ctg ttg gtt ttg att at C atg tto a CC Cta gga titt 581 Pro Pro Gly Arg Lell Lell Wall Luell Ile Ile Met Phe Thir Luell Gly Phe 17 O 17s 18O cott tta tac citc. ttg a Ca aat att to c ggc aag tac gac agg titt 629 Pro Luell Tyr Luell Lell Thir Asn Ile Ser Gly Lys Asp Arg Phe 185 190 195 2OO gcc aac CaC ttic gac c cc atg agt CC a att titc gaa cgt. gag cgg 677 Ala Asn His Phe Asp Pro Met Ser Pro Ile Phe Glu Arg Glu Arg 2O5 21O 215 titt cag gt C ttic citt tcg gat citt ggt citt citt gcc gtg titt tat gga 72 Phe Glin Wall Phe Lell Ser Asp Luell Gly Luell Luell Ala Wall Phe Gly 22O 225 23 O att a.a.a. gtt gct gta gca aat a.a.a. gga gct gct tgg gta gcg tgc atg 773 Ile Wall Ala Wall Ala Asn Lys Gly Ala Ala Trp Wall Ala Cys Met 235 24 O 245 tat gga gtt cc.g gta tta ggc gta titt acc titt tto gat gtg at C acc 821 Gly Wall Pro Wall Lell Gly Wall Phe Thir Phe Phe Asp Wall Ile Thir 25 O 255 26 O tto ttg CaC CaC a CC Cat Cag tog tog cott Cat tat gat to a act gala 869 Phe Luell His His Thir His Glin Ser Ser Pro His Asp Ser Thir Glu 265 27 O 27s 28O tgg aac tgg at C aga 999 gcc ttg to a gca atc. gat agg gac titt gga 917 Trp Asn Trp Ile Arg Gly Ala Luell Ser Ala Ile Asp Arg Asp Phe Gly 285 290 295 tto Ctg aat gtt tto Cat gat gtt aca CaC act Cat gtC atg Cat 965 US 7,589.253 B2 45 46

- Continued

Phe Luell Asn Ser Wall Phe His Asp Wall Thir His Thir His Wal Met His 3OO 305 31 O

Cat ttg titt to a tac att C Ca CaC tat Cat gca aag gag gca agg gat O13 His Luell Phe Ser Tyr Ile Pro His His Ala Lys Glu Ala Arg Asp 315 32O 3.25 gca at C aag cca atc ttg ggc gac titt tat atg atc gac agg act coa Ala Ile Lys Pro Ile Leu Gly Asp Phe Tyr Met Ile Asp Arg Thr Pro 33 O 335 34 O att tta a.a.a. gca atg tdg aga gag ggc agg gag tgc atg tac at C gag 109 Ile Luell Ala Met Trp Arg Glu Gly Arg Glu Cys Met Tyr Ile Glu 345 350 355 360 cott gat agc aag ctic aaa ggit gtt tat tgg tat cat aaa ttg 151 Pro Asp Ser Llys Lieu Lys Gly Wall Tyr Trp Tyr His Llys Lieu. 365 37O tgat catatg caaaatgcac atgcatttitc aaaccct cta gttacgtttgttctatotat 211 aataaaccogc cggtcCtttg gttgactatg cctaa.gc.cag gccaaacagt taaataatat 271 cgg tatgatg tgtaatgaaa gtatgtggitt gtctggttitt gttgctatga aagaaagtat 331

gtcaaaaaaa aaaaaaa. 358

<210 SEQ ID NO 2 <211 LENGTH: 374 &212> TYPE : PRT <213> ORGANISM: Crepis palaestina

<4 OO SEQUENCE: 2

Met Gly Ala Gly Gly Arg Gly Arg Thir Ser Glu Lys Ser Val Met Glu 1. 5 1O 15

Arg Wall Ser Val Asp Pro Wall Thir Phe Ser Luell Ser Glu Lieu Lys Glin 2O 25 3O

Ala Ile Pro Pro His Cys Phe Glin Arg Ser Wall Ile Arg Ser Ser Tyr 35 4 O 45

Wall Wall Glin Asp Lieu. Ile Ile Ala Ile Phe Tyr Phe Leu Ala SO 55 6 O

Asn Thir Ile Pro Thir Lell Pro Thir Ser Luell Ala Tyr Lieu Ala Trp 65 70 8O

Pro Wall Trp Phe Cys Glin Ala Ser Wall Luell Thr Gly Lieu. Trp Ile 85 90 95

Lell Gly His Glu Cys Gly His His Ala Phe Ser Asn Tyr Thir Trp Phe 1OO 105 11 O

Asp Asp Thir Val Gly Phe Ile Luell His Ser Phe Lieu. Lieu. Thr Pro Tyr 115 12 O 125

Phe Ser Trp Llys Phe Ser His Arg Asn His His Ser Asn. Thir Ser Ser 13 O 135 14 O

Ile Asp Asn Asp Glu Val Ile Pro Ser Llys Ser Lys Lieu Ala 145 150 155 160

Arg Ile Llys Lieu. Lieu. Asn Asn Pro Pro Gly Arg Lieu. Lieu Val Lieu. 1.65 17O 17s

Ile Ile Met Phe Thir Lieu. Gly Phe Pro Luell Tyr Lieu. Lieu. Thir Asn. Ile 18O 185 19 O

Ser Gly Lys Arg Phe Ala Asn His Phe Asp Pro Met Ser 195 2O5

Pro Ile Phe Lys Glu Arg Glu Arg Phe Glin Wall Phe Lieu. Ser Asp Lieu. 21 O 215 22O

Gly Luell Luell Ala Wall Phe Gly Ile Wall Ala Wall Ala Asn Lys 225 23 O 235 24 O US 7,589.253 B2 47 48

- Continued

Gly Ala Ala Trp Val Ala Cys Met Tyr Gly Val Pro Val Lieu. Gly Val 245 250 255 Phe Thr Phe Phe Asp Val Ile Thr Phe Lieu. His His Thr His Glin Ser 26 O 265 27 O Ser Pro His Tyr Asp Ser Thr Glu Trp Asn Trp Ile Arg Gly Ala Leu 27s 28O 285 Ser Ala Ile Asp Arg Asp Phe Gly Phe Lieu. Asn. Ser Val Phe His Asp 29 O 295 3 OO Val Thr His Thr His Val Met His His Leu Phe Ser Tyr Ile Pro His 3. OS 310 315 32O Tyr His Ala Lys Glu Ala Arg Asp Ala Ile Llys Pro Ile Lieu. Gly Asp 3.25 330 335 Phe Tyr Met Ile Asp Arg Thr Pro Ile Leu Lys Ala Met Trp Arg Glu 34 O 345 35. O Gly Arg Glu. Cys Met Tyr Ile Glu Pro Asp Ser Llys Lieu Lys Gly Val 355 360 365 Tyr Trp Tyr His Lys Lieu. 37 O

<210 SEQ ID NO 3 <211 LENGTH: 1309 &212> TYPE: DNA <213> ORGANISM: Crepis sp. &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: (937) ... (937) <223> OTHER INFORMATION: N is any nucleotide residue <221 NAME/KEY: CDS <222> LOCATION: (26) ... (1147) &223> OTHER INFORMATION: <221 NAMEAKEY: misc feature <222> LOCATION: (901) ... (901) <223> OTHER INFORMATION: N is any nucleotide residue <4 OO SEQUENCE: 3 tgttgaccat aaatcatcta t caac atg ggit gcc ggc ggc cgt ggt cq9 tog 52 Met Gly Ala Gly Gly Arg Gly Arg Ser gala aag tog gt C atg gaa cit gtc. tca gtt gat cca gta acc titc. tca 1OO Glu Lys Ser Val Met Glu Arg Val Ser Val Asp Pro Val Thr Phe Ser 10 15 2O 25 citg agt gat ttg aag caa goa atc cct coa cat togc titc cag cqa tot 148 Lieu. Ser Asp Lieu Lys Glin Ala Ile Pro Pro His Cys Phe Glin Arg Ser 3 O 35 4 O gtc atc cqt to a tict tat tac gtt gtt cag gat citc ata att gcc tac 196 Val Ile Arg Ser Ser Tyr Tyr Val Val Glin Asp Lieu. Ile Ile Ala Tyr 45 SO 55 atc ttic tac ttic ctit gcc aac aca tat at c cct aat citc cct cat cot 244 Ile Phe Tyr Phe Leu Ala Asn Thr Tyr Ile Pro Asn Lieu Pro His Pro 60 65 70 cta goc tac tta gct togg ccg citt tac togg titc tdt caa got agc gtc 292 Lieu Ala Tyr Lieu Ala Trp Pro Leu Tyr Trp Phe Cys Glin Ala Ser Val 7s 8O 85

Ct c act ggg tta tig atc Ct c ggc cat gala tet ggt cac cat gcc tat 34 O Lieu. Thr Gly Lieu. Trp Ile Lieu. Gly His Glu. Cys Gly His His Ala Tyr 9 O 95 1 OO 105 agc aac tac aca togg gtt gac gac act gtg ggc titc atc at c cat to a 388 Ser Asn Tyr Thr Trp Val Asp Asp Thr Val Gly Phe Ile Ile His Ser 11O 115 12 O titt ct c ct c acc ccg tat titc. tct togg aaa tac agt cac cqg aat cac 436

US 7,589.253 B2 51

- Continued <221 NAMEAKEY: misc feature <222> LOCATION: (937) ... (937) <223> OTHER INFORMATION: N is any nucleotide residue <221 NAMEAKEY: misc feature <222> LOCATION: (901) ... (901) <223> OTHER INFORMATION: N is any nucleotide residue <4 OO SEQUENCE: 4 Met Gly Ala Gly Gly Arg Gly Arg Ser Glu Lys Ser Wal Met Glu Arg 1. 5 1O 15 Val Ser Val Asp Pro Val Thr Phe Ser Lieu. Ser Asp Lieu Lys Glin Ala 2O 25 3O Ile Pro Pro His Cys Phe Glin Arg Ser Val Ile Arg Ser Ser Tyr Tyr 35 4 O 45 Val Val Glin Asp Lieu. Ile Ile Ala Tyr Ile Phe Tyr Phe Lieu Ala Asn SO 55 6 O Thr Tyr Ile Pro Asn Lieu Pro His Pro Leu Ala Tyr Lieu Ala Trp Pro 65 70 7s 8O Lieu. Tyr Trp Phe Cys Glin Ala Ser Val Lieu. Thr Gly Lieu. Trp Ile Leu 85 90 95 Gly His Glu. Cys Gly His His Ala Tyr Ser Asn Tyr Thr Trp Val Asp 1OO 105 11 O Asp Thr Val Gly Phe Ile Ile His Ser Phe Leu Lleu. Thr Pro Tyr Phe 115 12 O 125 Ser Trp Llys Tyr Ser His Arg Asn His His Ser Asn Thr Ser Ser Ile 13 O 135 14 O Asp Asn Asp Glu Val Tyr Ile Pro Llys Ser Lys Ser Llys Lieu Lys Arg 145 150 155 160 Ile Tyr Lys Lieu. Lieu. Asn. Asn Pro Pro Gly Arg Lieu. Lieu Val Lieu Val 1.65 17O 17s Ile Met Phe Thr Lieu. Gly Phe Pro Leu Tyr Lieu Lleu. Thr Asn Ile Ser 18O 185 19 O Gly Lys Llys Tyr Asp Arg Phe Ala Asn His Phe Asp Pro Met Ser Pro 195 2OO 2O5 Ile Phe Lys Glu Arg Glu Arg Phe Glin Val Phe Lieu. Ser Asp Lieu. Gly 21 O 215 22O Lieu. Lieu Ala Val Phe Tyr Gly Ile Llys Val Ala Val Ala Asn Lys Gly 225 23 O 235 24 O Ala Ala Trp Val Ala Cys Met Tyr Gly Val Pro Val Lieu. Gly Val Phe 245 250 255 Thr Phe Phe Asp Val Ile Thr Phe Lieu. His His Thr His Glin Ser Ser 26 O 265 27 O Pro His Tyr Asp Ser Thr Glu Trp Asn Trp Ile Arg Gly Ala Leu Ser 27s 28O 285 Ala Ile Asp Xaa Asp Phe Gly Phe Lieu. Asn. Ser Val Phe His Asp Wall 29 O 295 3 OO Thr His Thr His Val Met His His Leu Phe Ser Tyr Ile Pro His Tyr 3. OS 310 315 32O His Ala Lys Glu Ala Arg Asp Ala Ile Llys Pro Ile Lieu. Gly Asp Phe 3.25 330 335 Tyr Met Ile Asp Arg Thr Pro Ile Lieu Lys Ala Met Trp Arg Glu Gly 34 O 345 35. O Arg Glu. Cys Met Tyr Ile Glu Pro Asp Ser Lys Lieu Lys Gly Val Tyr 355 360 365 Trp Tyr His Llys Lieu

US 7,589.253 B2 55

- Continued Tyr Ile Pro Llys Wall Lys Ser Llys Val Lys Ile Tyr Ser Lys Ile Lieu. SO 55 6 O Asn Asn Pro Pro Gly Arg Val Phe Thir Lieu Ala Phe Arg Lieu. Ile Val 65 70 7s 8O Gly Phe Pro Leu Tyr Lieu Phe Thr Asn Val Ser Gly Lys Llys Tyr Glu 85 90 95 Arg Phe Ala Asn His Phe Asp Pro Met Ser Pro Ile Phe Thr Glu Arg 1OO 105 11 O Glu. His Val Glin Val Lieu Lleu Ser Asp Phe Gly Lieu. Ile Ala Val Ala 115 12 O 125 Tyr Val Val Arg Glin Ala Val Lieu Ala Lys Gly Gly Ala Trp Val Met 13 O 135 14 O Cys Ile Tyr Gly Val Pro Val Lieu Ala Val Asn Ala Phe Phe Val Lieu. 145 150 155 160 Ile Thr Tyr Lieu. His His Thr His Leu Ser Leu Pro His Tyr Asp Ser 1.65 17O 17s Ser Glu Trp Asp Trp Lieu. Arg 18O

<210 SEQ ID NO 7 &2 11s LENGTH: 177 &212> TYPE: DNA <213> ORGANISM: Crepis alpina &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (1) ... (177) &223> OTHER INFORMATION:

<4 OO SEQUENCE: 7 gaa to ggt cac cat gcc titc agc gaC tac Cag tig gtt gaC gac aat 48 Glu Cys Gly His His Ala Phe Ser Asp Tyr Glin Trp Val Asp Asp Asn 1. 5 1O 15 gtg ggc titc atc ctic cac tog titt ct c atg acc ccg tat titc. tcc tigg 96 Val Gly Phe Ile Lieu. His Ser Phe Leu Met Thr Pro Tyr Phe Ser Trp 2O 25 3O aaa tac agc cac cqg aac cac cat gcc aac aca aat tcc citt gac aac 144 Llys Tyr Ser His Arg Asn His His Ala Asn. Thir Asn. Ser Lieu. Asp Asn 35 4 O 45 gat gala gtt tac at C C cc aaa agc aag gcc aaa. 177 Asp Glu Val Tyr Ile Pro Llys Ser Lys Ala Lys SO 55

<210 SEQ ID NO 8 <211 LENGTH: 59 &212> TYPE: PRT <213> ORGANISM: Crepis alpina

<4 OO SEQUENCE: 8 Glu Cys Gly His His Ala Phe Ser Asp Tyr Glin Trp Val Asp Asp Asn 1. 5 1O 15 Val Gly Phe Ile Lieu. His Ser Phe Leu Met Thr Pro Tyr Phe Ser Trp 2O 25 3O Llys Tyr Ser His Arg Asn His His Ala Asn. Thir Asn. Ser Lieu. Asp Asn 35 4 O 45 Asp Glu Val Tyr Ile Pro Llys Ser Lys Ala Lys SO 55

<210 SEQ ID NO 9 <211 LENGTH: 383 &212> TYPE: PRT US 7,589.253 B2 57

- Continued <213> ORGANISM: Arabidopsis thaliana <4 OO SEQUENCE: 9 Met Gly Ala Gly Gly Arg Met Pro Val Pro Thir Ser Ser Lys Llys Ser 1. 5 1O 15 Glu Thir Asp Thir Thr Lys Arg Val Pro Cys Glu Lys Pro Pro Phe Ser 2O 25 3O Val Gly Asp Lieu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 4 O 45 Ile Pro Arg Ser Phe Ser Tyr Lieu. Ile Ser Asp Ile Ile Ile Ala Ser SO 55 6 O Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Lieu Lleu Pro Gln Pro 65 70 7s 8O Lieu. Ser Tyr Lieu Ala Trp Pro Lieu. Tyr Trp Ala Cys Glin Gly Cys Val 85 90 95 Lieu. Thr Gly Ile Trp Val Ile Ala His Glu. Cys Gly His His Ala Phe 1OO O5 11 O Ser Asp Tyr Glin Trp Lieu. Asp Asp Thr Val Gly Lieu. Ile Phe His Ser 115 12 O 125 Phe Lieu. Leu Val Pro Tyr Phe Ser Trp Llys Tyr Ser His Arg Arg His 13 O 135 14 O His Ser Asn Thr Gly Ser Lieu. Glu Arg Asp Glu Val Phe Val Pro Llys 145 150 155 160 Glin Llys Ser Ala Ile Llys Trp Tyr Gly Lys Tyr Lieu. Asn. Asn Pro Lieu. 1.65 17O 17s Gly Arg Ile Met Met Lieu. Thr Val Glin Phe Val Lieu. Gly Trp Pro Leu 18O 185 19 O Tyr Lieu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala Cys 195 2OO 2O5 His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Lieu. Glin 21 O 215 22O Ile Tyr Lieu. Ser Asp Ala Gly Ile Lieu Ala Val Cys Phe Gly Lieu. Tyr 225 23 O 235 24 O Arg Tyr Ala Ala Ala Glin Gly Met Ala Ser Met Ile Cys Lieu. Tyr Gly 245 250 255 Val Pro Lieu. Lieu. Ile Val Asn Ala Phe Lieu Val Lieu. Ile Thr Tyr Lieu 26 O 265 27 O Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp Asp 27s 28O 285 Trp Lieu. Arg Gly Ala Lieu Ala Thr Val Asp Arg Asp Tyr Gly Ile Lieu. 29 O 295 3 OO Asn Llys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His Leu 3. OS 310 315 32O Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala Ile 3.25 330 335 Llys Pro Ile Leu Gly Asp Tyr Tyr Glin Phe Asp Gly Thr Pro Trp Tyr 34 O 345 35. O Val Ala Met Tyr Arg Glu Ala Lys Glu. Cys Ile Tyr Val Glu Pro Asp 355 360 365 Arg Glu Gly Asp Llys Lys Gly Val Tyr Trip Tyr Asn. Asn Llys Lieu. 37 O 375 38O

<210 SEQ ID NO 10 <211 LENGTH: 384 US 7,589.253 B2 59 60

- Continued

&212> TYPE : PRT <213> ORGANISM: Brassica juncea

<4 OO SEQUENCE: 10

Met Gly Ala Gly Gly Arg Met Glin Wall Ser Pro Ser Pro Lys Ser 1. 5 15

Glu Thir Asp Thir Lell Arg Wall Pro Glu Thir Pro Phe Thir 2O 25

Wall Gly Glu Luell Ala Ile Pro Pro His Phe Arg Ser 35 4 O 45

Ile Pro Arg Ser Phe Ser Tyr Luell le Trp Asp Ile Ile Wall Ala Ser SO 55 6 O

Cys Phe Wall Ala Thir Thir Phe Pro Lell Lell Pro His Pro 65 70

Lell Ser Wall Ala Trp Pro Luell Trp Ala Glin Gly Wall Wall 85 90 95

Lell Thir Gly Wall Trp Wall Ile Ala Glu Cys Gly His His Ala Phe 11 O

Ser Asp Tyr Glin Trp Lell Asp Asp Wall Gly Lell Ile Phe His Ser 115 12 O 125

Phe Luell Luell Wall Pro Phe Ser Ser His Arg Arg His 13 O 135 14 O

His Ser Asn Thir Gly Ser Lell Glu Arg Asp Glu Wall Phe Wall Pro Lys 145 150 155 160

Ser Asp Ile Trp Gly Lys Lell Asn Asn Pro Luell 1.65 17O 17s

Arg Thir Wall Met Lell Thir Wall Glin Phe Thir Lell Gly Trp Pro Luell 18O 185 19 O

Trp Ala Phe Asn Wall Ser Gly Arg Pro Pro Glu Gly Phe Ala 195

His Phe His Pro Asn Ala Pro Ile ASn Asp Arg Glu Arg Luell 21 O 215 22O

Glin Ile Wall Ser Asp Ala Gly Ile Luell Ala Wall Gly Luell 225 23 O 235 24 O

Arg Ala Ala Ala Glin Gly Wall Ala Ser Met Wall Luell 245 250 255

Gly Wall Pro Luell Lell Ile Wall Asn Ala Phe Luell Wall Lell Ile Thir 26 O 265 27 O

Lell Glin His Thir His Pro Ser Luell Pro His Asp Ser Ser Glu Trp 27s 285

Asp Trp Luell Arg Gly Ala Lell Ala Thir Wall Asp Arg Asp Gly Ile 29 O 295 3 OO

Lell Asn Wall Phe His Asn Ile Thir Asp Thir His Wall Ala His His 3. OS 310 315

Lell Phe Ser Thir Met Pro His His Ala Met Glu Wall Thir Lys Ala 3.25 330 335

Ile Pro Ile Lell Gly Asp Tyr Glin Phe Asp Gly Thir Pro Trp 34 O 345 35. O

Wall Ala Met Trp Arg Glu Ala Glu Cys Ile Tyr Wall Glu Pro 355 360 365

Asp Arg Glin Gly Glu Lys Gly Wall Phe Trp Tyr Asn Asn Luell 37 O 375 38O

<210 SEQ ID NO 11 US 7,589.253 B2 61

- Continued

<211 LENGTH: 367 &212> TYPE : PRT <213> ORGANISM: Glycine max

<4 OO SEQUENCE: 11

Met Gly Ala Gly Gly Arg Thir Asp Wall Pro Pro Ala Asn Arg Lys Ser 1. 5 15

Glu Wall Asp Pro Lell Arg Wall Pro Phe Glu Pro Glin Phe Ser 2O 25

Lell Ser Glin Ile Ala Ile Pro Pro His Phe Glin Arg Ser 35 4 O 45

Wall Luell Arg Ser Phe Ser Tyr Wall Wall Tyr Asp Lell Thir Ile Ala Phe SO 55 6 O

Cys Luell Tyr Wall Ala Thir His Phe His Lell Lell Pro Gly Pro 65 70

Lell Ser Phe Arg Gly Met Ala Ile Trp Ala Wall Glin Gly Cys Ile 85 90 95

Lell Thir Gly Wall Trp Wall Ile Ala His Glu Cys Gly His His Ala Phe 105 11 O

Ser Asp Tyr Glin Lell Lell Asp Asp Ile Wall Gly Lell Ile Luell His Ser 115 12 O 125

Ala Luell Luell Wall Pro Phe Ser Trp Ser His Arg Arg His 13 O 135 14 O

His Ser Asn Thir Gly Ser Lell Glu Arg Asp Glu Wall Phe Wall Pro Lys 145 150 155 160

Gly Arg Wall Luell Thir Lell Ala Wall Thir Luell Thir Lell Gly Trp Pro Luell 1.65 17O 17s

Luell Ala Luell Asn Wall Ser Gly Arg Pro Tyr Asp Arg Phe Ala 18O 185 19 O

His Asp Pro Tyr Gly Pro Ile Ser Asp Arg Glu Arg Luell Glin 195 2O5

Ile Tyr Ile Ser Asp Ala Gly Wall Luell Ala Wall Wall Gly Luell Phe 21 O 215 22O

Arg Luell Ala Met Ala Lys Gly Luell Ala Trp Wall Wall Wall Gly 225 23 O 235 24 O

Wall Pro Luell Luell Wall Wall Asn Gly Phe Luell Wall Lell Ile Thir Phe Luell 245 250 255

Glin His Thir His Pro Ala Lell Pro His Thir Ser Ser Glu Trp Asp 26 O 265 27 O

Trp Luell Arg Gly Ala Lell Ala Thir Wall Asp Arg Asp Tyr Gly Ile Luell 285

Asn Lys Wall Phe His Asn Ile Thir Asp Thir His Wall Ala His His Luell 29 O 295 3 OO

Phe Ser Thir Met Pro His His Ala Met Glu Ala Thir Ala Ile 3. OS 310 315

Pro Ile Luell Gly Glu Tyr Arg Phe Asp Glu Thir Pro Phe Wall 3.25 330 335

Ala Met Trp Arg Glu Ala Arg Glu Ile Wall Glu Pro Asp 34 O 345 35. O

Glin Ser Thir Glu Ser Gly Wall Phe Trp Tyr Asn Asn Luell 355 360 365

<210 SEQ ID NO 12 <211 LENGTH: 383 &212> TYPE : PRT US 7,589.253 B2 63 64

- Continued

<213> ORGANISM: Solanum commersonii

<4 OO SEQUENCE: 12

Met Gly Ala Gly Gly Arg Met Ser Ala Pro ASn Gly Glu Thir Glu Wall 1. 15

Lys Arg Asn Pro Lell Glin Wall Pro Thir Ser Pro Pro Phe Thir 2O 25

Wall Gly Asp Ile Ala Ile Pro Pro His Phe Glin Arg Ser 35 4 O 45

Lell Ile Arg Ser Phe Ser Tyr Wall Wall Tyr Asp Lell Ile Luell Wall Ser SO 55 6 O

Ile Met Wall Ala Asn Thir Phe His Lell Lell Pro Ser Pro 65 70

Ile Ala Trp Pro Ile Trp Ile Glin Gly Cys Wall 85 90 95

Thir Gly Ile Trp Wall Asn Ala His Glu Cys Gly His His Ala Phe 105 11 O

Ser Asp Tyr Glin Trp Wall Asp Asp Thir Wall Gly Lell Ile Luell His Ser 115 12 O 125

Ala Luell Luell Wall Pro Phe Ser Trp Ser His Arg Arg His 13 O 135 14 O

His Ser Asn Thir Gly Ser Lell Glu Arg Asp Glu Wall Phe Wall Pro Lys 145 150 155 160

Pro Ser Glin Lell Gly Trp Ser Lys Lell Asn Asn Pro Pro 1.65 17s

Gly Arg Wall Luell Ser Lell Thir Ile Thir Luell Thir Lell Gly Trp Pro Luell 18O 185 19 O

Luell Ala Phe Asn Wall Ser Gly Arg Pro Asp Arg Phe Ala 195

His Tyr Asp Pro Gly Pro Ile Asn ASn Arg Glu Arg Luell Glin 21 O 215

Ile Phe Ile Ser Asp Ala Gly Wall Luell Gly Wall Luell Luell Tyr 225 23 O 235 24 O

Arg Ile Ala Luell Wall Gly Luell Ala Trp Luell Wall Wall Tyr Gly 245 250 255

Wall Pro Luell Luell Wall Wall Asn Gly Phe Luell Wall Lell Ile Thir Luell 26 O 265 27 O

Glin His Thir His Pro Ser Lell Pro His Asp Ser Thir Glu Trp Asp 27s 28O 285

Trp Luell Arg Gly Ala Lell Ala Thir Asp Arg Asp Gly Wall Luell 29 O 295 3 OO

Asn Wall Phe His Asn Ile Thir Asp Thir His Wall Wall His His Luell 3. OS 310 315

Phe Ser Thir Met Pro His Asn Ala Met Glu Ala Thir Ala Wall 3.25 330 335

Pro Luell Luell Gly Asp Glin Phe Asp Gly Thir Pro Ile Tyr 34 O 345 35. O

Glu Met Trp Arg Glu Ala Lys Glu Luell Tyr Wall Glu Asp 355 360 365

Glu Ser Ser Glin Gly Gly Wall Phe Trp Tyr Lys Asn Luell 37 O 375 38O

<210 SEQ ID NO 13 <211 LENGTH: 387 US 7,589.253 B2 65 66

- Continued

&212> TYPE : PRT <213> ORGANISM: Glycine max

<4 OO SEQUENCE: 13

Met Gly Lieu Ala Lys Glu Thir Thir Met Gly Gly Arg Gly Arg Wall Ala 1. 5 15

Wall Glu Wall Glin Gly Pro Luell Ser Arg Wall Pro Asn Thir 25

Pro Pro Phe Thir Wall Gly Glin Luell Lys Ala Ile Pro Pro His 35 4 O 45

Phe Glin Arg Ser Lell Lell Thir Ser Phe Ser Tyr Wall Wall Asp SO 55 6 O

Lell Ser Phe Ala Phe Ile Phe Ile Ala Thir Thir Phe His Luell 65 70

Lell Pro Glin Pro Phe Ser Lell Ile Ala Trp Pro Ile Trp Wall Luell 85 90 95

Glin Gly Luell Lell Thir Gly Wall Trp Wall Ile Ala His Glu Gly 105 11 O

His His Ala Phe Ser Glin Trp Wall Asp Asp Wall Wall Gly Luell 115 12 O 125

Thir Luell His Ser Thir Lell Lell Wall Pro Phe Ser Trp Ile Ser 13 O 135 14 O

His Arg Arg His His Ser Asn Thir Gly Ser Luell Asp Arg Glu Wall 145 150 155 160

Phe Wall Pro Pro Ser Wall Ala Trp Phe Ser Tyr Luell 1.65 17s

Asn Asn Pro Luell Gly Arg Ala Wall Ser Luell Luell Wall Thir Luell Thir Ile 18O 185 19 O

Gly Trp Pro Met Tyr Lell Ala Phe Asn Wall Ser Gly Arg Pro Asp 195

Ser Phe Ala Ser His His Pro Ala Pro Ile Ser Asn Arg 21 O 215

Glu Arg Luell Luell Ile Tyr Wall Ser Asp Wall Ala Lell Phe Ser Wall Thir 225 23 O 235 24 O

Ser Luell Arg Wall Ala Thir Luell Lys Gly Lell Wall Trp Luell Luell 245 250 255

Wall Gly Wall Pro Lell Luell Ile Wall ASn Gly Phe Luell Wall Thir 26 O 265 27 O

Ile Thir Tyr Luell Glin His Thir His Phe Ala Luell Pro His Asp Ser 27s 28O 285

Ser Glu Trp Asp Trp Lell Lys Gly Ala Luell Ala Thir Met Asp Arg Asp 29 O 295 3 OO

Tyr Gly Ile Luell Asn Lys Wall Phe His His Ile Thir Asp Thir His Wall 3. OS 310 315

Ala His His Luell Phe Ser Thir Met Pro His His Ala Met Glu Ala 3.25 330 335

Thir Asn Ala Ile Lys Pro Ile Luell Gly Glu Glin Phe Asp Asp 34 O 345 35. O

Thir Pro Phe Ala Lell Trp Arg Glu Ala Arg Glu Luell Tyr 355 360 365

Wall Glu Pro Asp Glu Gly Thir Ser Glu Lys Gly Wall Trp 37 O 375 38O

Asn 385 US 7,589.253 B2 67

- Continued

<210 SEQ ID NO 14 <211 LENGTH: 387 &212> TYPE: PRT <213> ORGANISM: Ricinus communis

<4 OO SEQUENCE: 14 Met Gly Gly Gly Gly Arg Met Ser Thr Val Ile Thr Ser Asn Asn Ser 1. 5 1O 15 Glu Lys Lys Gly Gly Ser Ser His Lieu Lys Arg Ala Pro His Thir Lys 2O 25 3O Pro Pro Phe Thr Lieu. Gly Asp Leu Lys Arg Ala Ile Pro Pro His Cys 35 4 O 45 Phe Glu Arg Ser Phe Val Arg Ser Phe Ser Tyr Val Ala Tyr Asp Val SO 55 6 O Cys Lieu Ser Phe Lieu. Phe Tyr Ser Ile Ala Thr Asn Phe Phe Pro Tyr 65 70 7s 8O Ile Ser Ser Pro Leu Ser Tyr Val Ala Trp Leu Val Tyr Trp Leu Phe 85 90 95 Gln Gly Cys Ile Lieu. Thr Gly Leu Trp Val Ile Gly His Glu. Cys Gly 1OO 105 11 O His His Ala Phe Ser Glu Tyr Glin Lieu Ala Asp Asp Ile Val Gly Lieu. 115 12 O 125 Ile Val His Ser Ala Lieu. Leu Val Pro Tyr Phe Ser Trp Llys Tyr Ser 13 O 135 14 O His Arg Arg His His Ser Asn. Ile Gly Ser Lieu. Glu Arg Asp Glu Val 145 150 155 160 Phe Val Pro Llys Ser Lys Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Ser 1.65 17O 17s Asn Asn Pro Pro Gly Arg Val Lieu. Thir Lieu Ala Ala Thr Lieu. Lieu. Lieu 18O 185 19 O Gly Trp Pro Lieu. Tyr Lieu Ala Phe Asin Val Ser Gly Arg Pro Tyr Asp 195 2OO 2O5 Arg Phe Ala Cys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg 21 O 215 22O Glu Arg Lieu. Glin Ile Tyr Ile Ala Asp Lieu. Gly Ile Phe Ala Thir Thr 225 23 O 235 24 O Phe Val Lieu. Tyr Glin Ala Thr Met Ala Lys Gly Lieu Ala Trp Val Met 245 250 255 Arg Ile Tyr Gly Val Pro Leu Lieu. Ile Val Asn Cys Phe Leu Val Met 26 O 265 27 O Ile Thr Tyr Lieu Gln His Thr His Pro Ala Ile Pro Arg Tyr Gly Ser 27s 28O 285 Ser Glu Trp Asp Trp Lieu. Arg Gly Ala Met Val Thr Val Asp Arg Asp 29 O 295 3 OO Tyr Gly Val Lieu. Asn Llys Val Phe His Asn. Ile Ala Asp Thir His Val 3. OS 310 315 32O Ala His His Leu Phe Ala Thr Val Pro His Tyr His Ala Met Glu Ala 3.25 330 335 Thr Lys Ala Ile Llys Pro Ile Met Gly Glu Tyr Tyr Arg Tyr Asp Gly 34 O 345 35. O Thr Pro Phe Tyr Lys Ala Lieu. Trp Arg Glu Ala Lys Glu. Cys Lieu. Phe 355 360 365 Val Glu Pro Asp Glu Gly Ala Pro Thr Glin Gly Val Phe Trp Tyr Arg US 7,589.253 B2 69

- Continued

37 O 375 38O Asn Llys Tyr 385

<210 SEQ ID NO 15 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase peptide motif

<4 OO SEQUENCE: 15 His Glu. Cys Gly His His 1. 5

<210 SEQ ID NO 16 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase peptide motif

<4 OO SEQUENCE: 16 His Arg Asn His His 1. 5

<210 SEQ ID NO 17 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase peptide motif <4 OO SEQUENCE: 17

His Wal Met His His 1. 5

<210 SEQ ID NO 18 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase peptide motif <4 OO SEQUENCE: 18

His Wall Lieu. His His 1. 5

<210 SEQ ID NO 19 <211 LENGTH: 11.99 &212> TYPE: DNA <213> ORGANISM: Vernonia galamensis &220s FEATURE: <221 NAME/KEY: CDS <222> LOCATION: (44) . . (1195) &223> OTHER INFORMATION:

<4 OO SEQUENCE: 19 tattacacat ttacactgat ctdttaatca aatttcaaac aaa atg gga gct ggit 55 Met Gly Ala Gly 1. ggc ca atgaat acc acc gat gat gat cag aag aat Ctc ttic caa cc 103 Gly Arg Met Asn. Thir Thr Asp Asp Asp Gln Lys Asn Lieu. Phe Glin Arg 5 10 15 2O gta cca gcc toc aaa cca cca ttc. tcc titg got gat citt aag aaa goc 151 Val Pro Ala Ser Llys Pro Pro Phe Ser Lieu Ala Asp Lieu Lys Lys Ala 25 3O 35 ata cca ccc cac togt titc caa aga toc ct c ct c cq t t catct tac tat 199 Ile Pro Pro His Cys Phe Glin Arg Ser Leu Lieu. Arg Ser Ser Tyr Tyr 4 O 45 SO gtg gtt cat gat citc gtc gta goc tac git c titt tac tat ct c goc aac 247 Val Val His Asp Lieu Val Val Ala Tyr Val Phe Tyr Tyr Lieu Ala Asn US 7,589.253 B2 71 72

- Continued

55 60 65 a Ca tac at C cott citt citt c cc to c cott citt gcc tac tta tta gct tgg 295 Thir Tyr Ile Pro Lell Lell Pro Ser Pro Luell Ala Tyr Lell Luell Ala Trp 70 7s 8O c cc citt tac tgg tto tgt Cag ggt agc at C citc. a CC ggit gtC tgg gt C 343 Pro Luell Tyr Trp Phe Cys Glin Gly Ser Ile Luell Thir Gly Wall Trp Wall 85 9 O 95 1OO atc. ggt Cat gala tgt ggc CaC Cat gcc ttic gac tat Cala tgg at a 391 Ile Gly His Glu Cys Gly His His Ala Phe Ser Asp Glin Trp Ile 105 110 115 gac gac act gtg ggc tto atc. citt CaC tot gca citc. tto acc cott tat 439 Asp Asp Thir Wall Gly Phe Ile Luell His Ser Ala Lell Phe Thir Pro 12O 125 13 O tto tot tgg a.a.a. tac agt CaC cgt aat CaC Cat gcc aac aca aac tot 487 Phe Ser Trp Tyr Ser His Arg Asn His His Ala Asn Thir Asn Ser 135 14 O 145 citt gat aac gat gaa gta tac at C cott a.a.a. gtt a.a.a. t cc aag gt C aag 535 Lell Asp Asn Asp Glu Wall Tyr Ile Pro Wall Lys Ser Lys Wall Lys 15 O 155 16 O att tat to c a.a.a. atc. citt aac aac cott cott ggt cgc gtt ttic acc ttg 583 Ile Ser Ile Lell Asn Asn Pro Pro Gly Arg Wall Phe Thir Luell 1.65 17O 17s 18O gct ttic aga ttg atc. gtg ggit titt cott tta tac citt tto acc aat gtt 631 Ala Phe Arg Luell Ile Wall Gly Phe Pro Luell Tyr Lell Phe Thir Asn Wall 185 190 195 toa ggc aag a.a.a. tac gaa cgt titt gcc aac Cat titt gat cc c atg agt 6.79 Ser Gly Lys Lys Tyr Glu Arg Phe Ala Asn His Phe Asp Pro Met Ser 2OO 2O5 21 O c cc att ttic acc gag cgt gag Cat gta Cala gtc ttg citt tot gat titt 727 Pro Ile Phe Thir Glu Arg Glu His Wall Glin Wall Lell Lell Ser Asp Phe 215 22 O 225 ggit citc. at a gca gtt gct tac gtg gtt cgt Cala gct gta Ctg gct a.a.a. 775 Gly Luell Ile Ala Wall Ala Tyr Wall Wall Arg Glin Ala Wall Luell Ala 23 O 235 24 O gga ggt gct tgg gtg atg tgc att gga gtt cott gtg Ctg gcc gta 823 Gly Gly Ala Trp Wall Met Cys Ile Gly Wall Pro Wall Luell Ala Wall 245 250 255 26 O aac gca ttic titt gtt tta atc. act citt CaC CaC acg Cat citc. to a 871 Asn Ala Phe Phe Wall Lell Ile Thir Luell His His Thir His Luell Ser 265 27 O 27s

Ctg cott CaC tat gat tcg act gala tgg gac tgg atc. aag gga gct ttg 919 Lell Pro His Tyr Asp Ser Thir Glu Trp Asp Trp Ile Lys Gly Ala Luell 28O 285 29 O tgc acc at C gac aga gat tto gga ttic ttg aat agg gtt ttic CaC gac 96.7 Cys Thir Ile Asp Arg Asp Phe Gly Phe Luell ASn Arg Wall Phe His Asp 295 3OO 3. OS gtg aca CaC acc Cat gtg ttg Cat Cat ttg ata tcg tac att cott Cat O15 Wall Thir His Thir His Wall Lell His His Luell Ile Ser Ile Pro His 31 O 315 32O tat Cat gca aag gag gca aga gac gcc at C a.a.a. cc.g gtg ttg ggc gala Tyr His Ala Lys Glu Ala Arg Asp Ala Ile Lys Pro Wall Luell Gly Glu 3.25 330 335 34 O tac tat aag at C gac agg a Ca cc.g at C gtg aag gca atg tgg agg gala 111 Lys Ile Asp Arg Thir Pro Ile Wall Lys Ala Met Trp Arg Glu 345 350 355 gca aag aat gca tat a Ca ttg agg Ctg atg aag ata gcg agc acc aag 1.59 Ala Lys Asn Ala Tyr Thir Lell Arg Luell Met Lys Ile Ala Ser Thir Lys 360 365 37 O gca Cat act ggt a CC a Ca a.a.a. gcc aga t cc taag 199 US 7,589.253 B2 73 74

- Continued Ala His Thr Gly Thr Thr Ser Cys Lys Ala Arg Ser 375

<210 SEQ ID NO 2 O <211 LENGTH: 384 &212> TYPE : PRT <213> ORGANISM: Vernonia galamensis

<4 OO SEQUENCE:

Met Gly Ala Gly Gly Arg Met Asn Thir Thir Asp Asp Asp Glin Lys Asn 1. 5 15

Lell Phe Glin Arg Wall Pro Ala Ser Lys Pro Pro Phe Ser Luell Ala Asp 2O 25

Lell Lys Ala Ile Pro Pro His Phe Glin Arg Ser Luell Lieu. Arg 35 4 O 45

Ser Ser Wall Wall His Asp Luell Wall Wall Ala Wall Phe Tyr SO 55 6 O

Tyr Luell Ala Asn Thir Tyr Ile Pro Luell Luell Pro Ser Pro Luell Ala Tyr 65 70 8O

Lell Luell Ala Trp Pro Lell Trp Phe Cys Glin Gly Ser Ile Lieu. Thir 85 90 95

Gly Wall Trp Wall Ile Gly His Glu Cys Gly His His Ala Phe Ser Asp 105 11 O

Glin Trp Ile Asp Asp Thir Wall Gly Phe Ile Lell His Ser Ala Lieu 115 12 O 125

Phe Thir Pro Phe Ser Trp Ser His Arg Asn His His Ala 13 O 135 14 O

Asn Thir Asn Ser Lell Asp Asn Glu Wall Tyr Ile Pro Val Lys 145 150 155 160

Ser Wall Ile Ser Ile Luell ASn Asn Pro Pro Gly Arg 1.65 17O 17s

Wall Phe Thir Luell Ala Phe Arg Luell Ile Wall Gly Phe Pro Luell Tyr Lieu. 18O 185 19 O

Phe Thir Asn Wall Ser Gly Lys Glu Arg Phe Ala Asn His Phe 195

Asp Pro Met Ser Pro Ile Phe Thir Glu Arg Glu His Wall Glin Wall Lieu 21 O 215 22O

Lell Ser Asp Phe Gly Lell Ile Ala Wall Ala Tyr Wall Wall Arg Glin Ala 225 23 O 235 24 O

Wall Luell Ala Gly Gly Ala Trp Wall Met Ile Gly Wall Pro 245 250 255

Wall Luell Ala Wall Asn Ala Phe Phe Wall Luell Ile Thir Luell His His 26 O 265 27 O

Thir His Luell Ser Lell Pro His Tyr Asp Ser Thir Glu Trp Asp Trp Ile 285

Gly Ala Luell Thir Ile Asp Arg Asp Phe Gly Phe Luell Asn Arg 29 O 295 3 OO

Wall Phe His Asp Wall Thir His Thir His Wall Luell His His Luell Ile Ser 3. OS 310 315

Ile Pro His Tyr His Ala Glu Ala Arg Asp Ala Ile Llys Pro 3.25 330 335

Wall Luell Gly Glu Ile Asp Arg Thir Pro Ile Wall Lys Ala 34 O 345 35. O

Met Trp Arg Glu Ala Asn Ala Thir Luell Arg Lell Met Lys Ile 355 360 365 US 7,589.253 B2 75 76

- Continued

Ala Ser Thr Lys Ala His Thr Gly Thr Thr Ser Cys Lys Ala Arg Ser 37 O 375 38O

<210 SEQ ID NO 21 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase consensus motif &220s FEATURE: <221 NAME/KEY: MISC FEATURE <222> LOCATION: (2) ... (4) <223> OTHER INFORMATION: Xaa at position 2 is any amino acid; Xaa at position 3 is any amino acid; Xaa at position 4 is any amino acid;

<4 OO SEQUENCE: 21

His Xaa Xala Xala His 1. 5

<210 SEQ ID NO 22 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase consensus motif &220s FEATURE: <221 NAME/KEY: MISC FEATURE <222> LOCATION: (2) ... (5) <223> OTHER INFORMATION: Xaa at position 2 is any amino acid; Xaa at position 3 is any amino acid; Xaa at position 4 is any amino acid; Xaa at position 5 is any amino acid; <4 OO SEQUENCE: 22

His Xaa Xala Xala Xala His 1. 5

<210 SEQ ID NO 23 <211 LENGTH: 5 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase consensus motif &220s FEATURE: <221 NAME/KEY: MISC FEATURE <222> LOCATION: (2) ... (3) <223> OTHER INFORMATION: Xaa at position 2 is any amino acid; Xaa at position 3 is any amino acid;

<4 OO SEQUENCE: 23

His Xaa Xala His His 1. 5

<210 SEQ ID NO 24 <211 LENGTH: 6 &212> TYPE: PRT <213> ORGANISM: mixed function monooxygenase consensus motif &220s FEATURE: <221 NAME/KEY: MISC FEATURE <222> LOCATION: (2) ... (4) <223> OTHER INFORMATION: Xaa at position 2 is any amino acid; Xaa at position 3 is any amino acid; Xaa at position 4 is any amino acid; <4 OO SEQUENCE: 24

His Xaa Xala Xala His His 1. 5

We claim: 60 (ii) His-(Xaa)-His-His (SEQ ID NO; 23) or 1. A process for producing a transgenic plant comprising His-(Xaa)-His-His (SEQID NO: 24); and a) transforming a cell or tissue of a plant with a nucleic acid (iii) His-(Xaa)-His-His (SEQID NO. 23) or encoding a polypeptide having the following three his- His-(Xaa)-His-His (SEQID NO: 24), tidine-rich regions (i), (ii) and (iii): 65 wherein His designates histidine, Xaa designates any (i) His-(Xaa)-His (SEQID NO: 21) or naturally-occurring amino acid, (Xaa) refers to a His-(Xaa)-His (SEQ ID NO: 22); sequence of three amino acids, (Xaa) refers to a US 7,589.253 B2 77 78 sequence of four amino acids, and (Xaa) refers to a 11. A process for producing a transformed plant cell com sequence of two amino acids, prising introducing into the plant cell a nucleic acid encoding wherein the polypeptide comprises a sequence of amino a polypeptide having the following three histidine-rich acids at least 65% identical to the sequence of amino regions (i), (ii) and (iii): acids set forth in SEQID NO: 2, 5 (i) His-(Xaa). His (SEQID NO: 21) or wherein the nucleic acid encodes an expoxygenase, and His-(Xaa). His (SEQ ID NO: 22); wherein the nucleic acid is under the control of a pro (ii) His-(Xaa)-His-His (SEQID NO. 23) or moter conferring transcription of the nucleic acid in His-(Xaa)-His-His (SEQ ID NO:24); and the plant; (iii) His-(Xaa)-His-His (SEQID NO. 23) or 10 His-(Xaa)-His-His (SEQ ID NO:24), b) regenerating the transformed cellor tissue to produce the wherein His designates histidine, Xaa designates any transgenic plant; and naturally-occurring amino acid, (Xaa) refers to a c) examining the transgenic plant or part thereof for the sequence of three amino acids, (Xaa) refers to a presence of epoxy fatty acids to determine whether the sequence of four amino acids, and (Xaa) refers to a transgenic plant has epoxy fatty acids. 15 sequence of two amino acids, 2. The process of claim 1, wherein the plant is Arabidopsis wherein the polypeptide comprises a sequence of amino acids thaliana, flax, oilseed rape, Sunflower, safflower, soybean, at least 65% identical to the amino acid sequence set forth in sesame, cottonseed, peanut, olive or oil palm. SEQID NO: 2, 3. The process of claim 1, wherein the plant is flax, Sun wherein the nucleic acid encodes an expoxygenase, and flower, corn, or safflower. wherein the nucleic acid is under the control of a promoter 4. The process of claim 1, further comprising a step of conferring transcription of the nucleic acid in a plant cell Selecting a transgenic plant expressing an epoxygenase. and is stably integrated into the genome of the cell, 5. The process of claim 4, wherein the plant is Arabidopsis thereby producing the transformed plant cell. thaliana, flax, oilseed rape, Sunflower, safflower, soybean, 12. The process of claim 11, wherein the plant cell is from 25 Arabidopsis thaliana, flax, oilseed rape, Sunflower, safflower, sesame, cottonseed, peanut, olive or oil palm. Soybean, Sesame, cottonseed, peanut, olive or oil palm. 6. The process of claim 4, wherein the promoter is a seed 13. The process of claim 11, wherein the nucleic acid is specific promoter. from a plant that synthesizes epoxy fatty acids. 7. The process of claim 4, further comprising producing 14. The process of claim 13, wherein the plant is of Chry seed of the plant. 30 Santhemum Bpp., Crepis spp., Euphorbia spp., or Vermonia 8. The process of claim 7, further comprising selecting seed spp. having 12,13-epoxy-9-octadecenoic acid at a level of greater 15. The process of claim 11, wherein the promoter is a than 0.7%(w/w) of the total seed fatty acid content. seed-specific promoter. 9. The process of claim 4, wherein the nucleic acid is from 16. The process of claim 1, wherein the promoter is a a plant that synthesizes epoxy fatty acids. 35 seed-specific promoter. 10. The process of claim 9, wherein the plant is of Chry 17. The process of claim 16, further comprising producing Santhemum spp., Crepis spp., Euphorbia spp., or Vermonia seed of the transgenic plant. spp. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 7,589.253 B2 Page 1 of 1 APPLICATION NO. : 09/981124 DATED : September 15, 2009 INVENTOR(S) : Green et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the cover page, Item * Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 177 days Delete the phrase by 177 days and insert -- by 504 days --

Signed and Sealed this Fifteenth Day of June, 2010

David J. Kappos Director of the United States Patent and Trademark Office