Fatty acid epoxygenase genes from plants and uses therefor in modifying fatty acid metabolism

Abstract

The present invention relates generally to novel genetic sequences that encode fatty acid epoxygenase enzymes, in particular fatty acid Δ12-epoxygenase enzymes from plants that are mixed function monooxygenase enzymes. More particularly, the present invention exemplifies cDNA sequences from Crepis spp. and Vernonia galamensis that encode fatty acid Δ12-epoxygenases. The genetic sequences of the present invention provide the means by which fatty acid metabolism may be altered or manipulated in organisms, such as, for example, yeasts, moulds, bacteria, insects, birds, mammals and plants, and more particularly in plants. The invention also extends to genetically modified oil-accumulating organisms transformed with the subject genetic sequences and to the oils derived therefrom. The oils thus produced provide the means for the cost-effective raw materials for use in the efficient production of coatings, resins, glues, plastics, surfactants and lubricants.

Description

FIELD OF THE INVENTION

[0002] The present invention relates generally to novel genetic sequences that encode fatty acid epoxygenase enzymes. In particular, the present invention relates to genetic sequences that encode fatty acid Δ12-epoxygenase enzymes as defined herein. More particularly, the present invention provides cDNA and genomic gene sequences that encode plant fatty acid epoxygenases, in particular from Crepis palaestina or Vernonia galamensis. The genetic sequences of the present invention provide the means by which fatty acid metabolism may be altered or manipulated in organisms such as yeasts, moulds, bacteria, insects, birds, mammals and plants, in particular to convert unsaturated fatty acids to epoxy fatty acids therein. The invention extends to genetically modified oil-accumulating organisms transformed with the subject genetic sequences and to the oils derived therefrom. The oils thus produced provide the means for the cost-effective raw materials for use in the efficient production of coatings, resins, glues, plastics, surfactants and lubricants, amongst others.

GENERAL

[0003] Those skilled in the art will be aware that the present invention is subject to variations and modifications other than those specifically described herein. It is to be understood that the invention includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0004] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0005] Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

[0006] This specification contains nucleotide sequence information prepared using the program Patentin Version 3.1 presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier [e.g. <210>1, <210>2, etc]. The length, type of sequence [DNA, protein (PRT), etc] and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields <211>,<212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term “SEQ ID NO:”, followed by the sequence identifier [e.g. SEQ ID NO:1 refers to the sequence in the sequence listing designated as <400>1].

BACKGROUND TO THE INVENTION

[0007] There is considerable interest world-wide in producing chemical feedstock, such as fatty acids, for industrial use from renewable plant sources rather than from non-renewable petrochemicals. This concept has broad appeal to manufacturers and consumers on the basis of resource conservation and provides a significant opportunity to develop new industrial crops for agriculture.

[0008] There is a diverse array of unusual fatty acids in nature and these have been well characterized (Badam & Patil, 1981; Smith, 1970). Many of these unusual fatty acids have industrial potential and this has led to interest in domesticating such species to enable agricultural production of particular fatty acids.

[0009] One class of fatty acids of particular interest are the epoxy-fatty acids, consisting of an acyl chain in which two adjacent carbon bonds are linked by an epoxy bridge. Due to their high reactivity, they have considerable application in the production of coatings, resins, glues, plastics, surfactants and lubricants. These fatty acids are currently produced by chemical epoxidation of vegetable oils, mainly soybean oil and linseed oil, however this process produces mixtures of multiple and isomeric forms and involves significant processing costs.

[0010] Attempts are being made by others to develop some wild plants that contain epoxy fatty acids (e.g. Euphorbia lagascae, or Vernonia galamensis) into commercial sources of these oils. However, problems with agronomic suitability and low yield potential severely limit the commercial utility of traditional plant breeding and cultivation approaches.

[0011] The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating the efficiency of commercially-important industrial processes, by the expression of genes isolated from a first organism or species in a second organism or species to confer novel phenotypes thereon. More particularly, conventional industrial processes can be made more efficient or cost-effective, resulting in greater yields per unit cost by the application of recombinant DNA techniques.

[0012] Moreover, the appropriate choice of host organism for the expression of a genetic sequence of interest provides for the production of compounds that are not normally produced or synthesized by the host, at a high yield and purity.

[0013] However, despite the general effectiveness of recombinant DNA technology, the isolation of genetic sequences which encode important enzymes in fatty acid metabolism, in particular the genes which encode the fatty acid Δ12-epoxygenase enzymes responsible for producing 12,13-epoxy-9-octadecenoic acid (vernolic acid) and 12,13-epoxy-9,15-octadecadienoic acid, amongst others, remains a major obstacle to the development of genetically-engineered organisms which produce these fatty acids.

[0014] Until the present invention, there were only limited biochemical data indicating the nature of fatty acid epoxygenase enzymes, in particular Δ12-epoxygenases. However, in Euphorbia lagascae, the formation of 12,13-epoxy-9-octadecenoic acid (vernolic acid) from linoleic acid appears to be catalyzed by a cytochrome-P450-dependent Δ12 epoxygenase enzyme (Bafor et al., 1993; Blee et al., 1994). Additionally, developing seed of linseed plants have the capability to convert added vernolic acid to 12,13-epoxy-9,15-octadecadienoic acid by an endogenous Δ15 desaturase (Engeseth and Stymne, 1996). Epoxy-fatty acids can also be produced by a peroxide-dependent peroxygenase in plant tissues (Blee and Schuber, 1990).

[0015] In work leading up to the present invention, the inventors sought to isolate genetic sequences which encode genes which are important for the production of epoxy-fatty acids, such as 12,13-epoxy-9-octadecenoic acid (vernolic acid) or 12,13-epoxy-9,15-octadecadienoic acid and to transfer these genetic sequences into highly productive commercial oilseed plants and/or other oil accumulating organisms.

SUMMARY OF THE INVENTION

[0016] One aspect of the invention provides an isolated nucleic acid which encodes or is complementary to an isolated nucleic acid which encodes a fatty acid epoxygenase.

[0017] A second aspect of the invention provides an isolated nucleic acid which hybridizes under at least low stringency conditions to at least 20 contiguous nucleotides of SEQ ID NOS:1 or 3 or 5 or 19 or 19, or a complementary sequence thereto.

[0018] A further aspect of the invention provides isolated nucleic acid comprising a sequence of nucleotides selected from the group consisting of:

[0019] (i) a nucleotide sequence that is at least 65% identical to a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 19;

[0020] (ii) a nucleotide sequence that encodes an amino acid sequence that is at least about 50% identical to a sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 20; and

[0021] (iii) a nucleotide sequence that is complementary to (i) or (ii).

[0022] A further aspect of the invention provides a gene construct that comprises the isolated nucleic acid supra, in either the sense or antisense orientation, in operable connection with a promoter sequence.

[0023] A further aspect of the invention provides a method of altering the level of epoxy fatty acids in a cell, tissue, organ or organism, said method comprising expressing a sense, antisense, ribozyme or co-suppression molecule comprising the isolated nucleic acid supra in said cell, tissue, organ or organism for a time and under conditions sufficient for the level of epoxy fatty acids therein to be increased or reduced.

[0024] A further aspect of the invention provides a method of producing a recombinant enzymatically active epoxygenase polypeptide in a cell, said method comprising expressing the isolated nucleic acid supra in said cell for a time and under conditions sufficient for the epoxygenase encoded therefor to be produced.

[0025] A further aspect of the invention provides a method of producing a recombinant enzymatically active epoxygenase polypeptide in a cell, said method comprising the steps of:

[0026] (i) producing a gene construct which comprises the isolated nucleic acid supra placed operably under the control of a promoter capable of conferring expression on said genetic sequence in said cell, and optionally an expression enhancer element;

[0027] (ii) transforming said gene construct into said cell; and

[0028] (iii) selecting transformants which express a functional epoxygenase encoded by the genetic sequence at a high level.

[0029] A still further aspect of the invention provides a method of producing a recombinant and enzymatically active epoxygenase polypeptide in a transgenic plant comprising the steps of:

[0030] (i) producing a gene construct which comprises the isolated nucleic acid supra placed operably under the control of a seed-specific promoter and optionally an expression enhancer element, wherein said genetic sequences is also placed upstream of a transcription terminator sequence;

[0031] (ii) transforming said gene construct into a cell or tissue of said plant; and

[0032] (iii) selecting transformants which express a functional epoxygenase encoded by the genetic sequence at a high level in seeds.

[0033] A further aspect of the invention provides a recombinant epoxygenase polypeptide or functional enzyme molecule.

[0034] A further aspect of the invention provides a recombinant epoxygenase which comprises a sequence of amino acids set forth in any one of SEQ ID NOS: 2 or 4 or 6 or 20 or 20 or a homologue, analogue or derivative thereof which is at least about 50% identical thereto. More preferably, the percentage identity to any one of SEQ ID NOS: 2 or 4 or 6 or 20 or 20 is at least about 65%.

[0035] A still further aspect of the invention provides a method of producing an epoxy fatty acid in a cell, tissue, organ or organism, said method comprising incubating a cell, tissue, organ or organism which expresses an enzymatically active recombinant epoxygenase with a fatty acid substrate and preferably, an unsaturated fatty acid substrate, for a time and under conditions sufficient for at least one carbon bond, preferably a carbon double bond, of said substrate to be converted to an epoxy group.

[0036] A further aspect of the invention provides an immunologically interactive molecule which binds to the recombinant epoxygenase polypeptide described herein or a homologue, analogue or derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 is a linear representation of an expression plasmid comprising an epoxygenase structural gene, placed operably under the control of the truncated napin promoter (FP1; right-hand hatched box) and placed upstream of the NOS terminator sequence (right-hand stippled box). The epoxygenase genetic sequence is indicated by the right-hand open rectangular box. The construct also comprises the NOS promoter (left-hand hatched box) driving expression of the NPTII 71 gene (left-hand open box) and placed upstream of the NOS terminator (left-hand stippled box). The left and right border sequences of the Agrobacterium tumefaciens Ti plasmid are also indicated.

[0038] FIG. 2 is a schematic representation showing the alignment of the amino acid sequences of the epoxygenase polypeptide of Crepis palaestina (Cpa12; SEQ ID NO: 2), a further epoxygenase derived from Crepis sp. other than C. palaestina which produces high levels of vernolic acid (CrepX; SEQ ID NO: 4), a partial amino acid sequence of an epoxygenase polypeptide derived from Vernonia galamensis (Vgal1; SEQ ID NO: 6), a full-length amino acid sequence of an epoxygenase polypeptide derived from Vernonia galamensis (SEQ ID NO: 20), the amino acid sequence of the Δ12 acetylenase of Crepis alpina (Crep1; SEQ ID NO: 8), the Δ12 desaturase of A. thaliana (L26296; SEQ ID NO: 9), Brassica juncea (X91139; SEQ ID NO: 10), Glycine max (L43921; SEQ ID NO: 11), Solanum commersonii (X92847; SEQ ID NO: 12) and Glycine max (L43920; SEQ ID NO: 13), and the Δ12 hydroxylase of Ricinus communis (U22378; SEQ ID NO: 14). Underlined are three histidine-rich motifs that are conserved in non-heme containing mixed-function monooxygenases.

[0039] FIG. 3 is a copy of a photographic representation of a northern blot hybridization showing seed-specific expression of the Crepis palaestina epoxygenase gene exemplified by SEQ ID NO: 1. Northern blot analysis of total RNA from leaves (lane 1) and developing seeds (lane 2) of Crepis palaestina. 15 μg of total RNA was run on a Northern gel and blotted onto Hybond N+ membrane from Amersham according to the manufacturer's instructions. The blot was hybridized at 60° C. with a probe made from the 3′ untranslated region of SEQ ID NO: 1. The blot was washed twice in 2×SSC (NaCl-Sodium Citrate buffer) at room temperature for 10 minutes, then in 0.1×SSC at 60° C. for 20 min.

[0040] FIG. 4 is a schematic representation of a binary plasmid vector containing an expression cassette comprising the truncated napin seed-specific promoter (Napin) and nopaline synthase terminator (NT), with a BamHI cloning site there between, in addition to the kanamycin-resistance gene NPII operably connected to the nopaline synthase promoter (NP) and nopaline synthase terminator (NT) sequences. The expression cassette is flanked by T-DNA left border (LB) and right-border (RB) sequences.

[0041] FIG. 5 is a schematic representation of a binary plasmid vector containing an expression cassette which comprises SEQ ID NO: 1 placed operably under the control of a truncated napin seed-specific promoter (Napin) and upstream of the nopaline synthase terminator (NT), in addition to the kanamycin-resistance gene NPTII operably connected to the nopaline synthase promoter (NP) and nopaline synthase terminator (NT) sequences. The expression cassette is flanked by T-DNA left border (LB) and right-border (RB) sequences. To produce this construct, SEQ ID NO: 1 is inserted into the BamHI site of the binary vector set forth in FIG. 4.

[0042] FIG. 6 is a graphical representation of gas-chromatography traces of fatty acid methyl esters prepared from oil seeds of untransformed Arabidopsis thaliana plants [panel (a)], or A. thaliana plants (transgenic line Cpal-17) which have been transformed with SEQ ID NO: 1 using the gene construct set forth in FIG. 5 [panels (b) and (c)]. In panels (a) and (b), fatty acid methyl esters were separated using packed column separation. In panel (c), the fatty acid methyl esters were separated using capillary column separation. The elution positions of vernolic acid are indicated.

[0043] FIG. 7 is a graphical representation showing the joint distribution of epoxy fatty acids in selfed seed on T1 plants of Cpal2-transformed Arabidopsis thaliana plants as determined using gas chromatography. Levels of both vernolic acid (x-axis) and 12,13-epoxy-9,15-octadecadienoic acid (y-axis) were determined and plotted relative to each other. Data show a positive correlation between the levels of these fatty acids in transgenic plants.

[0044] FIG. 8 is a graphical representation showing the incorporation of 14C-label into the chloroform phase obtained from lipid extraction of linseed cotyledons during labeled-substrate feeding. Symbols used; ♦, [14C] oleic acid feeding; ▪, [14C] vernolic acid feeding.

[0045] FIG. 9 is a graphical representation showing the incorporation of 14C-label into the phosphatidyl choline of linseed cotyledons during labeled-substrate feeding. Symbols used; ♦, [14C] oleic acid feeding; ▪, [14C] vernolic acid feeding.

[0046] FIG. 10 is a graphical representation showing the incorporation of 14C-label into the triacylglycerols of linseed cotyledons during labeled-substrate feeding. Symbols used♦, [14C]oleic acid feeding; ▪, [14C] vernolic acid feeding.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] One aspect of the present invention provides an isolated nucleic acid which encodes or is complementary to an isolated nucleic acid which encodes a fatty acid epoxygenase.

[0048] Wherein the isolated nucleic acid of the invention encodes an enzyme which is involved in the direct epoxidation of arachidonic acid, it is particularly preferred that the subject nucleic acid is derived from a non-mammalian source.

[0049] As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.

[0050] The term “non-mammalian source” refers to any organism other than a mammal or a tissue or cell derived from same. In the present context, the term “derived from a non-mammalian source” shall be taken to indicate that a particular integer or group of integers has been derived from bacteria, yeasts, birds, amphibians, reptiles, insects, plants, fungi, moulds and algae or other non-mammal.

[0051] In a preferred embodiment of the present invention, the source organism is any such organism possessing the genetic capacity to synthesize epoxy fatty acids. More preferably, the source organism is a plant such as, but not limited to Chrysanthemum spp., Crepis spp., Euphorbia spp. and Vernonia spp., amongst others.

[0052] Even more preferably, the source organism is selected from the group consisting of: Crepis biennis, Crepis aurea, Crepis conyzaefolia, Crepis intermedia, Crepis occidentalis, Crepis palaestina, Crepis vesicaria, Crepis xacintha, Euphorbia lagascae and Vernonia galamensis. Additional species are not excluded.

[0053] In a particularly preferred embodiment of the present invention, the source organism is a Crepis sp. comprising high levels of vernolic acid such as Crepis palaestina, amongst others or alternatively, Vernonia galamensis.

[0054] Wherein the isolated nucleic acid of the invention encodes a Δ6-epoxygenase or Δ9-epoxygenase enzyme or Δ12-epoxygenase or Δ15-epoxygenase enzyme, or at least encodes an enzyme which is not involved in the direct epoxidation of arachidonic acid, the subject nucleic acid may be derived from any source producing said enzyme, including, but not limited to, yeasts, moulds, bacteria, insects, birds, mammals and plants.

[0055] The nucleic acid of the invention according to any of the foregoing embodiments may be DNA, such as a gene, cDNA molecule, RNA molecule or a synthetic oligonucleotide molecule, whether single-stranded or double-stranded and irrespective of any secondary structure characteristics unless specifically stated.

[0056] Reference herein to a “gene” is to be taken in its broadest context and includes:

[0057] (i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5′- and 3′-untranslated sequences);or

[0058] (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5′- and 3′- untranslated sequences of the gene.

[0059] The term “gene” is also used to describe synthetic or fusion molecules encoding all or part of a functional product. Preferred epoxygenase genes of the present invention may be derived from a natural epoxygenase gene by standard recombinant techniques. Generally, an epoxygenase gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions.

[0060] Insertions are those variants in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence, although random insertion is also possible with suitable screening of the resulting product. Nucleotide insertions include 5′ and 3′ terminal fusions as well as intra-sequence insertions of single or multiple nucleotides.

[0061] Deletions are variants characterized by the removal of one or more nucleotides from the sequence.

[0062] Substitutions are those variants in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be “silent” in that the substitution does not change the amino acid defined by the codon. Alternatively, a conservative substitution may alter one amino acid for another similar acting amino acid, or an amino acid of like charge, polarity, or hydrophobicity.

[0063] In the context of the present invention, the term “fatty acid epoxygenase” shall be taken to refer to any enzyme or functional equivalent or enzymatically-active derivative thereof that catalyzes the biosynthesis of an epoxy fatty acid, by converting a carbon bond of a fatty acid to an epoxy group and preferably, by converting a carbon double bond of an unsaturated fatty acid to an epoxy group. Although not limiting the invention, a fatty acid epoxygenase may catalyze the biosynthesis of an epoxy fatty acid selected from the group consisting of: (i) 12,13-epoxy-9-octadecenoic acid (vernolic acid); (ii) 12,13-epoxy-9,15-octadecadienoic acid; (iii) 15,16-epoxy-9,12-octadecadienoic acid; (iv) 9,10-epoxy-12-octadecenoic acid; and (v) 9,10-epoxy-octadecanoic acid.

[0064] The term “epoxy”, or “epoxy group” or “epoxy residue” will be known by those skilled in the art to refer to a three member ring comprising two carbon atoms and an oxygen atom linked by single bonds as follows: 1

[0065] Accordingly, the term “epoxide” refers to a compound that comprise at least one epoxy group as herein before defined.

[0066] Those skilled in the art are aware that fatty acid nomenclature is based upon the length of the carbon chain and the position of unsaturated carbon atoms within that carbon chain. Thus, fatty acids are designated using the shorthand notation:

(Carbon)total(carbon double bonds)totalcarbon double bond(Δ) position

[0067] wherein the double bonds are cis unless otherwise indicated. For example, palmitic acid (n-hexadecanoic acid) is a saturated 16-carbon fatty acid (i.e. 16:0), oleic acid (octadecenoic acid) is an unsaturated 18-carbon fatty acid with one double bond between C-9 and C-10 (i.e. 18:1Δ9), and linoleic acid (octadecadienoic acid) is an unsaturated 18-carbon fatty acid with two double bonds between C-9 and C-10 and between C-12 and C-13 (i.e. 18:2Δ9,12).

[0068] However, in the present context an epoxygenase enzyme may catalyze the conversion of any carbon bond to an epoxy group or alternatively, the conversion of any double in an unsaturated fatty acid substrate to an epoxy group. In this regard, it is well-known by those skilled in the art that most mono-unsaturated fatty acids of higher organisms are 18-carbon unsaturated fatty acids (i.e. 18:1Δ9), while most polyunsaturated fatty acids derived from higher organisms are 18-carbon fatty acids with at least one of the double bonds therein located between C-9 and C-10. Additionally, bacteria also possess C16-mono-unsaturated fatty acids. Moreover, the epoxygenase of the present invention may act on more than a single fatty acid substrate molecule and, as a consequence, the present invention is not to be limited by the nature of the substrate molecule upon which the subject epoxygenase enzyme acts.

[0069] Preferably, the substrate molecule for the epoxygenase of the present invention is an unsaturated fatty acid comprising at least one double bond.

[0070] Furthermore, epoxygenase enzymes may act upon any number of carbon atoms in any one substrate molecule. For example, they may be characterized as Δ6-epoxygenase, Δ9-epoxygenase, Δ12-epoxygenase or Δ15-epoxygenase enzymes amongst others. Accordingly, the present invention is not limited by the position of the carbon atom in the substrate upon which an epoxygenase enzyme may act.

[0071] The term “Δ6-epoxygenase” as used herein shall be taken to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ6 carbon bond of a fatty acid substrate to a Δ6 epoxy group and preferably, catalyzes the conversion of the Δ6 double bond of at least one unsaturated fatty acid to a Δ6 epoxy group.

[0072] The term “Δ9-epoxygenase” as used herein shall be taken to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ9 carbon bond of a fatty acid substrate to a Δ9 epoxy group and preferably, catalyzes the conversion of the Δ9 double bond of at least one unsaturated fatty acid to a Δ9 epoxy group.

[0073] As used herein, the term “Δ12-epoxygenase” shall be taken to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ12 carbon bond of a fatty acid substrate to a Δ12 epoxy group and preferably, catalyzes the conversion of the Δ12 double bond of at least one unsaturated fatty acid to a Δ12 epoxy group.

[0074] As used herein, the term “Δ15-epoxygenase” shall be taken to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ15 carbon bond of a fatty acid substrate to a Δ15 epoxy group and preferably, catalyzes the conversion of the Δ15 double bond of at least one unsaturated fatty acid to a Δ15 epoxy group.

[0075] The present invention clearly extends to genetic sequences which encode all of the epoxygenase enzymes listed supra, amongst others.

[0076] In one preferred embodiment of the invention, the isolated nucleic acid encodes a fatty acid epoxygenase enzyme which converts at least one carbon bond in palmitoleic acid (16:1Δ9), oleic acid (18:1Δ9), linoleic acid (18:2Δ9,12), linolenic acid (18:3Δ9,12,15), or arachidoniic acid (20:4Δ5,8,11,14) to an epoxy bond. Preferably, the carbon bond is a carbon double bond.

[0077] More preferably, the isolated nucleic acid of the invention encodes a fatty acid epoxygenase enzyme that at least converts one or both double bonds in linoleic acid to an epoxy group. According to this embodiment, an epoxygenase which converts both the Δ9 and the Δ12 double bonds of linoleic acid to an epoxy group may catalyze such conversions independently of each other such that said epoxygenase is a Δ9-epoxygenase and/or a Δ12-epoxygenase enzyme as herein before defined.

[0078] In an alternative preferred embodiment, the fatty acid epoxygenase of the present invention is a Δ12-epoxygenase, a Δ15- epoxygenase or a Δ9-epoxygenase as herein before defined.

[0079] More preferably, the fatty acid epoxygenase of the invention is a Δ12- epoxygenase as herein before defined.

[0080] In a particularly preferred embodiment of the invention, there is provided an isolated nucleic acid which encodes linoleate Δ12-epoxygenase, the enzyme which at least converts the Δ12 double bond of linoleic acid to a Δ12-epoxy group, thereby producing 12,13-epoxy-9-octadecenoic acid (vernolic acid).

[0081] Although not limiting the present invention, the preferred source of the Δ12-epoxygenase of the invention is a plant, in particular Crepis palaestina or a further Crepis sp. which is distinct from C. palaestina but contains high levels of vernolic acid, or Vernonia galamensis.

[0082] According to this embodiment, a Δ12-epoxygenase may catalyze the conversion of palmitoleic acid to 9,10-epoxy-palmitic acid and/or the conversion of oleic acid to 9,10-epoxy-stearic acid and/or the conversion of linoleic acid to any one or more of 9,10-epoxy-12-octadecenoic acid or 12,13-epoxy-9-octadecenoic acid or 9,10,12,13-diepoxy-stearic acid and/or the conversion of linolenic acid to any one or more of 9,10-epoxy-12,15-octadecadienoic acid or 12,13-epoxy-9,15-octadecadienoic acid or 15,16-epoxy-octadecadienoic acid or 9,10,12,13-diepoxy-15-octadecenoic acid or 9,10,15,16-diepoxy-12-octadecenoic acid or 12,13,15,16-diepoxy-9-octadecenoic acid or 9,10,12,13,15,16-triepoxy-stearic acid and/or the conversion of arachidonic acid to any one or more of 5,6-epoxy-8,11,14-tetracosatrienoic acid or 8,9-epoxy-5,11,14-tetracosatrienoic acid or 11,12-epoxy-5,8,14-tetracosatrienoic acid or 14,15-epoxy-5,8,11 -tetracosatrienoic acid or 5,6,8,9-diepoxy-11,14-tetracosadienoic acid or 5,6,11,12-diepoxy-8,14-tetracosadienoic acid or 5,6,14,15-diepoxy-8,11-tetracosadienoic acid or 8,9,11,12-diepoxy-5,14-tetracosadienoic acid or 8,9,14,15-diepoxy-5,11-tetracosadienoic acid or 11,12,14,15-diepoxy-5,8-tetracosadienoic acid or 5,6,8,9,11,12-triepoxy-14-tetracosenoic acid or 5,6,8,9,14,15-triepoxy-11-tetracosenoic acid or 5,6,11,12,14,15-triepoxy-8-tetracosenoic acid or 8,9,11,12,14,15-triepoxy-5-tetracosenoic acid, amongst others.

[0083] Those skilled in the art may be aware that not all substrates listed supra may be derivable from a natural source, but notwithstanding this, may be produced by chemical synthetic means. The conversion of both natural and synthetic unsaturated fatty acids to epoxy fatty acids is clearly within the scope of the present invention.

[0084] The present invention is particularly directed to those epoxygenase enzymes that are mixed-function monooxygenase enzymes, and nucleic acids encoding said enzymes, and uses of said enzymes and nucleic acids. Accordingly, it is particularly preferred that the nucleic acid of the invention encode a fatty acid epoxygenase which is a mixed-function monooxygenase enzyme.

[0085] In the context of the present invention, the term “mixed-function monooxygenase enzyme” shall be taken to refer to any epoxygenase polypeptide that comprises an amino acid sequence comprising three histidine-rich regions as follows:

[0086]

(i)   His-(Xaa)3-4-His (SEQ ID NO: 21 and SEQ ID NO: 22);
(ii)  His-(Xaa)2-3-His-His (SEQ ID NO: 23 and SEQ ID NO: 24);
(iii) His-(Xaa)2-3-His-His (SEQ ID NO: 23 and SEQ ID NO: 24),

[0087] (i)

[0088] (ii)

[0089] (iii)

[0090] and

[0091] wherein His designates histidine, Xaa designates any naturally-occurring amino acid residue as set forth in Table 1 herein, the integer (Xaa)3-4 refers to a sequence of amino acids comprising three or four repeats of Xaa, and the integer (Xaa)2-3 refers to a sequence of amino acids comprising two or three repeats of Xaa.

[0092] In the exemplification of the invention described herein, the inventors provide isolated cDNAs that comprise nucleotide sequences encoding the Δ12-epoxygenase polypeptides of Crepis palaestina and Vernonia galamensis. Each exemplified full-length amino acid sequence encoded by said cDNAs which includes the three characteristic amino acid sequence motifs of a mixed-function monooxygenase enzyme as herein before defined. Close sequence identity between the amino acid sequences of the Δ12-epoxygenase enzymes from C. palaestina (SEQ ID NO: 2), an unidentified Crepis sp (SEQ ID NO: 4), and Vernonia galamensis (SEQ ID NO: 20), suggests functional similarity between these polypeptides. In contrast, the amino acid sequences of these epoxygenases have lower identity to the amino acid sequences of a fatty acid desaturase or a fatty acid hydroxylase.

[0093] It is even more preferred that the epoxygenase of the present invention at least comprises a sequence of amino acids which comprises three histidine-rich regions as follows:

(i)	His-Glu-Cys-Gly-His-His	(SEQ ID NO: 15);
(ii)	His-Arg-Asn-His-His	(SEQ ID NO: 16);
		and
(iii)	His-Val-Met-His-His	(SEQ ID NO: 17) or
	His-Val-Leu-His-His	(SEQ ID NO: 18),

[0094] (i)

[0095] (ii)

[0096] (iii)

[0097] wherein His designates histidine, Glu designates glutamate, Cys designates cysteine, Gly designates glycine, Arg designates arginine, Asn designates asparagine, Val designates valine, Met designates methionine and Leu designates leucine.

[0098] The present invention clearly extends to epoxygenase genes derived from other species, including the epoxygenase genes derived from Chrysanthemum spp. and Euphorbia lagascae, amongst others.

[0099] In a preferred embodiment, whilst not limiting the present invention, the epoxygenase genes of other species which are encompassed by the present invention encode mixed-function monooxygenase enzymes. The present invention further extends to the isolated or recombinant polypeptides encoded by such genes and uses of said genes and polypeptides.

[0100] The invention described according to this embodiment does not encompass nucleic acids which encode enzyme activities other than epoxygenase activities as defined herein, in particular the Δ12-desaturase enzymes derived from Arabidopsis thaliana, Brassica juncea, Brassica napus or Glycine max, amongst others, which are known to contain similar histidine-rich motifs.

[0101] In the present context, “homologues” of an amino acid sequence refer to those amino acid sequences or peptide sequences which are derived from polypeptides, enzymes or proteins of the present invention or alternatively, correspond substantially to the amino acid sequences listed supra, notwithstanding any naturally-occurring amino acid substitutions, additions or deletions thereto.

[0102] For example, amino acids may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break α-helical structures or β-sheet structures, and so on. Alternatively, or in addition, the amino acids of a homologous amino acid sequence may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

[0103] Naturally-occurring amino acid residues contemplated herein are described in Table 1.

[0104] A homologue of an amino acid sequence may be a synthetic peptide produced by any method known to those skilled in the art, such as by using Fmoc chemistry.

[0105] Alternatively, a homologue of an amino acid sequence may be derived from a natural source, such as the same or another species as the polypeptides, enzymes or proteins of the present invention. Preferred sources of homologues of the amino acid sequences listed supra include any of the sources contemplated herein.

[0106] “Analogues” of an amino acid sequence encompass those amino acid sequences which are substantially identical to the amino acid sequences listed supra notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.

[0107] Preferred non-naturally occurring amino acids contemplated herein are listed below in Table 2.

[0108] The term “derivative” in relation to an amino acid sequence shall be taken to refer hereinafter to mutants, parts, fragments or polypeptide fusions of the amino acid sequences listed supra. Derivatives include modified amino acid sequences or peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are also contemplated by the present invention. Additionally, derivatives may comprise fragments or parts of an amino acid sequence disclosed herein and are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject sequences.

[0109] Procedures for derivatizing peptides are well-known in the art.

[0110] Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as “conservative”, in which case an amino acid residue is replaced with another naturally-occurring amino acid of similar character, for example Gly⇄Ala, Val⇄Ile⇄Leu, Asp⇄Glu, Lys⇄Arg, Asn⇄Gln or Phe⇄Trp⇄Tyr.

[0111] Substitutions encompassed by the present invention may also be “non-conservative”, in which an amino acid residue which is present in a repressor polypeptide is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (e.g. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

[0112] Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

[0113] Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxyl-terminal fusions and of the order of 1-4 amino acid residues.

[0114] The present invention clearly extends to the subject isolated nucleic acid when integrated into the genome of a cell as an addition to the endogenous cellular complement of epoxygenase genes. Alternatively, wherein the host cell does not normally encode enzymes required for epoxy fatty acid biosynthesis, the present invention extends to the subject isolated nucleic acid when integrated into the genome of said cell as an addition to the endogenous cellular genome.

	TABLE 1
		Three-letter	One-letter
	Amino Acid	Abbreviation	Symbol
	Alanine	Ala	A
	Arginine	Arg	R
	Asparagine	Asn	N
	Aspartic acid	Asp	D
	Cysteine	Cys	C
	Glutamine	Gln	Q
	Glutamic acid	Glu	E
	Glycine	Gly	G
	Histidine	His	H
	Isoleucine	Ile	I
	Leucine	Leu	L
	Lysine	Lys	K
	Methionine	Met	M
	Phenylalanine	Phe	F
	Proline	Pro	P
	Serine	Ser	S
	Threonine	Thr	T
	Tryptophan	Trp	W
	Tyrosine	Tyr	Y
	Valine	Val	V
	Any amino acid as above	Xaa	X

[0115]

TABLE 2
Non-conventional
amino acid                 Code
α-aminobutyric acid        Abu
α-amino-α-methylbutyrate   Mgabu
aminocyclopropane-         Cpro
carboxylate
aminoisobutyric acid       Aib
aminonorbornyl-            Norb
carboxylate
cyclohexylalanine          Chexa
cyclopentylalanine         Cpen
D-alanine                  Dal
D-arginine                 Darg
D-aspartic acid            Dasp
D-cysteine                 Dcys
D-glutamine                Dgln
D-glutamic acid            Dglu
D-histidine                Dhis
D-isoleucine               Dile
D-leucine                  Dleu
D-lysine                   Dlys
D-methionine               Dmet
D-ornithine                Dorn
D-phenylalanine            Dphe
D-proline                  Dpro
D-serine                   Dser
D-threonine                Dthr
D-tryptophan               Dtrp
D-tyrosine                 Dtyr
D-valine                   Dval
D-α-methylalanine          Dmala
D-α-methylarginine         Dmarg
D-α-methylasparagine       Dmasn
D-α-methylaspartate        Dmasp
D-α-methylcysteine         Dmcys
D-α-methylglutamine        Dmgln
D-α-methylhistidine        Dmhis
D-α-methylisoleucine       Dmile
D-α-methylleucine          Dmleu
D-α-methyllysine           Dmlys
D-α-methylmethionine       Dmmet
D-α-methylornithine        Dmorn
D-α-methylphenylalanine    Dmphe
D-α-methylproline          Dmpro
D-α-methylserine           Dmser
D-α-methylthreonine        Dmthr
D-α-methyltryptophan       Dmtrp
D-α-methyltyrosine         Dmty
D-α-methylvaline           Dmval
D-N-methylalanine          Dnmala
D-N-methylarginine         Dnmarg
D-N-methylasparagine       Dnmasn
D-N-methylaspartate        Dnmasp
D-N-methylcysteine         Dnmcys
D-N-methylglutamine        Dnmgln
D-N-methylglutamate        Dnmglu
D-N-methylhistidine        Dnmhis
D-N-methylisoleucine       Dnmile
D-N-methylleucine          Dnmleu
D-N-methyllysine           Dnmlys
N-methylcyclohexylalanine  Nmchexa
D-N-methylornithine        Dnmorn
L-N-methylalanine          Nmala
L-N-methylarginine         Nmarg
L-N-methylasparagine       Nmasn
L-N-methylaspartic acid    Nmasp
L-N-methylcysteine         Nmcys
L-N-methylglutamine        Nmgln
L-N-methylglutamic acid    Nmglu
L-N-methylhistidine        Nmhis
L-N-methylisolleucine      Nmile
L-N-methylleucine          Nmleu
L-N-methyllysine           Nmlys
L-N-methylmethionine       Nmmet
L-N-methylnorleucine       Nmnle
L-N-methylnorvaline        Nmnva
L-N-methylornithine        Nmorn
L-N-methylphenylalanine    Nmphe
L-N-methylproline          Nmpro
L-N-methylserine           Nmser
L-N-methylthreonine        Nmthr
L-N-methyltryptophan       Nmtrp
L-N-methyltyrosine         Nmtyr
L-N-methylvaline           Nmval
L-N-methylethylglycine     Nmetg
L-N-methyl-t-butylglycine  Nmtbug
L-norleucine               Nle
L-norvaline                Nva
α-methyl-aminoisobutyrate  Maib
α-methyl-γ-aminobutyrate   Mgabu
α-methylcyclohexylalanine  Mchexa
α-methylcylcopentylalanine Mcpen
α-methyl-α-napthylalanine  Manap
α-methylpenicillamine      Mpen
N-(4-aminobutyl)glycine    Nglu
N-(2-aminoethyl)glycine    Naeg
N-(3-aminopropyl)glycine   Norn
N-amino-α-methylbutyrate   Nmaabu
α-napthylalanine           Anap
N-benzylglycine            Nphe
N-(2-carbamylethyl)glycine Ngln
N-(carbamylmethyl)glycine  Nasn
N-(2-carboxyethyl)glycine  Nglu
N-(carboxymethyl)glycine   Nasp
N-cyclobutylglycine        Ncbut
N-cycloheptylglycine       Nchep
N-cyclohexylglycine        Nchex
N-cyclodecylglycine        Ncdec
N-cylcododecylglycine      Ncdod
N-cyclooctylglycine        Ncoct
N-cyclopropylglycine       Ncpro
N-cycloundecylglycine      Ncund
N-(2,2-diphenylethyl)      Nbhm
glycine
N-(3,3-diphenylpropyl)     Nbhe
glycine
N-(3-guanidinopropyl)      Narg
glycine
N-(1-hydroxyethyl)glycine  Nthr
N-(hydroxyethyl))glycine   Nser
N-(imidazolylethyl))       Nhis
glycine
N-(3-indolylyethyl)        Nhtrp
glycine
N-methyl-γ-aminobutyrate   Nmgabu
D-N-methylmethionine       Dnmmet
N-methylcyclopentylalanine Nmcpen

[0116]

Claims

1. An isolated nucleic acid that encodes a plant fatty acid epoxygenase polypeptide.

2. The isolated nucleic acid according to claim 1 wherein the epoxygenase is a mixed-function monooxygenase that catalyzes the epoxygenation of a carbon bond in a fatty acid molecule.

3. The isolated nucleic acid according to claim 2, wherein the carbon bond is a double bond in an unsaturated fatty acid molecule.

4. The isolated nucleic acid according to claim 1 wherein the epoxygenase is a Δ12-epoxygenase.

5. The isolated nucleic acid according to claim 1 wherein the plant is Crepis spp. or Vernonia spp.

6. The isolated nucleic acid according to claim 5 wherein the plant is Crepis palaestina.

7. The isolated nucleic acid according to claim 5 wherein the plant is Vernonia galamensis.

8. An isolated nucleic acid encoding a plant fatty acid epoxygenase polypeptide comprising a nucleotide sequence selected from the group consisting of: (i) a sequence having at least about 65% identity to a sequence selected from the group consisting of SEQ ID No: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 19 as determined using the default parameters of the Sequence and Analysis Software Package of the Computer Genetic Group (GCG) at the University of Wisconsin; (ii) a sequence encoding an amino acid sequence that is at least about 65% identical to a sequence selected from the group consisting of SEQ ID No: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 20 as determined using the default parameters of the Sequence and Analysis Software Package of the GCG at the University of Wisconsin; and (iii) a sequence that is complementary to (i) or (ii).

9. The isolated nucleic acid according to claim 8 comprising a nucleotide sequence that is at least about 65% identical a sequence selected from the group consisting of SEQ ID No: 1, SEQ ID NO: 3, SEQ ID NO: 5and SEQ ID NO: 19.

10. An isolated nucleic acid from the plant Vernonia that encodes a fatty acid epoxygenase polypeptide wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of: (i) the sequence set forth in SEQ ID NO: 19; (ii) a sequence encoding the amino acid sequence set forth in SEQ ID NO: 20; and (iii) a sequence that is complementary to (i) or (ii).

11. An isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 19 or a complementary nucleotide sequence thereto.

12. The isolated nucleic acid according to claim 1 wherein said nucleic acid comprises a nucleotide sequence that hybridizes under at least low stringency conditions to at least 20 contiguous nucleotides complementary to a sequence selected from the group consisting of SEQ ID No: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 19.

13. A gene construct that comprises the isolated nucleic acid according to claim 1 operably connected to a promoter sequence, wherein said nucleic acid is capable of being transcribed in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring epoxygenase gene.

14. A gene construct that comprises the isolated nucleic acid according to claim 8 operably connected to a promoter sequence, wherein said nucleic acid is capable of being transcribed in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring epoxygenase gene.

15. A gene construct that comprises the isolated nucleic acid according to claim 10 operably connected to a promoter sequence, wherein said nucleic acid is capable of being transcribed in the sense or antisense orientation relative to the direction of in vivo transcription of a naturally-occurring epoxygenase gene.

16. A method of altering the level of epoxy fatty acids in a plant or a cell, tissue, or organ of said plant, said method comprising introducing a gene construct comprising the isolated nucleic acid of claim 1 to a plant cell, tissue, or organ, regenerating said cell, tissue or organ into a whole plant and growing said plant under conditions sufficient to express said isolated nucleic acid in a cell, tissue or organ of said plant.

17. The method according to claim 16, wherein the isolated nucleic acid is expressed to produce a functional epoxygenase polypeptide in said cell, tissue, or organ.

18. The method of claim 16 wherein the plant organ is a seed.

19. The method of claim 16 wherein the plant cell, tissue or organ produces high levels of linoleic acid in its seed in its native state.

20. A method of producing a recombinant enzymatically active epoxygenase polypeptide in a plant cell, said method comprising: (i) producing a gene construct that comprises the isolated nucleic acid of claim 1 operably under the control of a promoter that is operable in said plant cell; (ii) transforming said gene construct into said cell; and (iii) selecting transformants which express a functional epoxygenase encoded by the genetic sequence at a high level.

21. A plant transformed with the isolated nucleic acid according to claim 1.

22. A progeny of the plant according to claim 21 wherein said progeny also comprises said nucleic acid.

23. The plant of claim 21 selected from the group consisting of: Arabidopsis thaliana, Linola™ flax, oilseed rape, sunflower, safflower, soybean, linseed, sesame, cottonseed, peanut, olive and oil palm.

24. The plant of claim 23 consisting of an A. thaliana plant.

25. The plant of claim 23 consisting of a Linola™ flax plant.