CA2120629C - Production of gamma linolenic acid by a .delta.6-desaturase - Google Patents

Abstract

Linoieic acid is converted into .gamma.-linolenic acid by the enzyme .DELTA.6-desaturase. The present invention is directed to an isolated nucleic acid comprising the .DELTA.6-desaturase gene. More particularly, the isolated nucleic acid comprises the promoter, coding region and termination regions of the .DELTA.6-desaturase gene. The present invention provides recombinant constructions comprising the .DELTA.6-desaturase coding region in functional combination with heterologous regulatory sequences. The nucleic acids and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms.

Description

PRODUCTION OF GANiMA LINOLENIC ACID

Linoleic acid (18:2) (LA) is tr_ansformed into gamma linolenic acid (18:3) (GLA) by the enzyme 66-desaturase. When this enzyme, or the nucleic acid encoding it, is transferred into LA-producing cells, GLA
is produced. The present inventiori provides a nucleic acid comprising the A6-desaturase qene. More specifically, the nucleic acid comprises the promoter, coding region and termination regions of the A6-desaturase gene. The present invention is further directed to recombinant constructions cornpris.ing aA6-desaturase coding region in functional combination with heterologous regulatory sequences. The nucleic acids lr' and recombinant constructions of the i_nstant invention are usef.ul in the production of. GLA in transaenic organisms.
Unsaturated fatty acids such as linoleic (C1P6~ "') and a-linolenic (C1pAQ 1'=ts) acids are essential dietary constituents that cannot be synthesized by vertebrates since vertebrate cells cari introduce double bonds at the A position of fatty acids but cannot introduce additional double bonds between the A" double bond and the methyl-terminus of the fatty acid chai.n. Because they are precursor.s of other products, linoleic and a-linolenic acids are essential fatty acids, and are usually obtained from plant sources.
Linoleic acid can be converted by mammals into Y-linol.enic acid (GLA, which can in turn be converted to atachidonic acid (20:4), a critically important fatty acid since it is an essential precursor of most prostagl.andins.

A t Z The dietary provision of linoleic acid, by virtue of its resulting conversion to GLA and arachidonic acid, satisfies the dietary need for GLA and arachidonic acid.
However, a relationship has been demonstrated between consumption of saturated fats and health risks such as hypercholesterolemia, atherosclerosis and other chemical disorders which correlate with susceptibility to coronary disease, while the consumption of unsaturated fats has been associated with decreased blood cholesterol concentration and reduced risk of atherosclerosis. The therapeutic benefits of dietary GLA may result fromGLA being a precursor to arachidonic acid and thus subsequently contributing to prostaglandin synthesis. Accordingly, consumption of the more unsaturated GLA, rather than linoleic acid, has potential health benefits. However, GLA is not present in virtually any commercially grown crop plant.
Linoleic acid is converted into GLA by the enzyme A6-desaturase. A6-desaturase, an enzyme of about 359 amino acids, has a membrane-bound domain and an active site.for desaturation offatty acids. When this enzyme is tzansferred into cells which endogenously produce linoleic acid but not GLA, GLA is produced. The present.
invention, by providing the gene encoding Afr-desaturase,.
allows the production of transgenic organisms which contain functional A6-desaturase and which produce GLA.
In addition to allowing production of large amounts of GLA, the present invention provides new dietary sources of GLA.
The present invention is directed to an isolated a6-desaturase gene. Specifically, the isolated gene .r ...i. .r- .. .
~. n ....... . .:.J.:.~ v. r'- ...i.. . . -" ... _ comprises the A6-desaturase promoter, coding region, and termination region.
The present invention is further directed to expression vectors comprising the A6-desaturase promoter, coding region and termination region.
The present invention is also directed to expression vectors comprising a A6-desaturase coding region in functional combination with heterologous regulatory regions, i.e. elements not derived from the A6-desaturase gene.
Cells and organisms comprising the vectors of the present invention, and progeny of such organisms, are also provided by the present invention.
The present invention further provides isolated bacterial A6-desaturase and is still further directed to an isolated nucleic acid encoding bacterial n6-desaturase.
The present invention further provides a method for producing plants with increased gamma linolenic acid (GLA) content which comprises transforming a plant cell with an isolated nucleic acid of the present invention and regenerating a plant with increased GLA content from said plant cell.
A method for producing chilling tolerant plants is also provided by the present invention.
In accordance with one embodiment of the present invention there is provided an isolated nucleic acid encoding bacterial e6-desaturase.
In accordance with another embodiment of the present invention there is provided a method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in. GLA and lino.leic acid (LA) which comprises transforming the organism with an isolated nucleic acid encoding bacterial A6-desaturase and an isolated nucleic acid encoding e12-desaturase.

-3a-In accordance with yet another embodiment of the present invention there is,provided a method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA and linoleic acid (LA) which comprises transforming the organism with at least one expression vector comprising the isolated nucleic acid as set out above and an isolated nucleic acid encoding A12-desaturase, wherein the organism is a bacterium or a plant.
In accordance with a futher embodiment of the present invention there is provided isolated bacterial L6-desaturase.
In accordance with a still futher embodiment of the present invention there is provided an isolated nucleic acid encoding cyano-bacterial Z~6-desaturase, wherein the isolated nucleic acid is isolatable from cyanobacteria that produces gamma linolenic acid.
In accordance with one embodiment of the present invention there is provided an isolated nucleic acid having the sequence of SEQ ID NO: 3.
In accordance with another embodiment of the present invention there is provided an isolated nucleic acid encoding a Synechocystis 06-desaturase.
In accordance with yet another embodiment of the present invention there is provided an isolated plant cell or bacterium transformed with at least the isolated nucleic acid described above.
In accordance with one embodiment of the present invention there is provided a method of producing a plant with increased gamma linolenic acid (GLA) content which comprises: transforming a plant cell with at least one isolated nucleic acid set out above and regenerating a plant with increased GLA content from the plant cell.
In accordance with another embodiment of the present invention there is provided a method of producing a plant -3b-with increased gamma linolenic acid (GLA) content which comprises transforming a plant cell with at least a vector comprising the isolated nucleic acid described above.
In accordance with yet another embodiment of the present invention there is'provided a method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA which comprises transforming the plant with at least the nucleic acid described above.
In accordance with a futher embodiment of the present ivention there is provided a method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA which comprises transforming the plant with at least the isolated nucleic acid described above.
In accordance with a still further embodiment of the present invention there is provided a method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA and linoleic acid (LA) which comprises transforming the plant with the isolated nucleic acid described above.
In accordance with one embodiment of the present invention there is provided a method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA and linoleic acid (LA) which comprises transforming the plant with at least one expression vector comprising an isolated DNA molecule encoding a Synechocystis Z~6-desaturase and the isolated nucleic acid described above.
In accordance with another embodiment of the present invention there is provided a method of inducing production of octadeca-tetraeonic acid in a plant deficient or lacking in gamma linolenic acid which comprises transforming the plant with at least the nucleic acid described above.
In accordance with yet another embodiment of the present invention there is provided a method of inducing -3c-production of the octadeca-tetraeonic acid in a plant deficient or lacking in gamma linolenic acid which comprises transforming the plant with a` least the isolated nucleic acid described above.
In accordance with a still further embodiment of the present invention there is provided a cyanobacterial A6-desaturase encoded by a nucleic acid from a cyanobacteria that produces gamma linolenic acid.
In accordance with a further aspect of the present invention, there is provided an isolated nucleic acid encoding a bacterial A6-desaturase, wherein the nucleic acid is selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino' acid sequence of SEQ ID NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID
NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).
In accordance with a further aspect of the present invention, there is provided an isolated bacterial A6-desaturase encoded by a nucleic acid selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).
In accordance with a further an isolated nucleic acid encoding cyanobacterial A6-desaturase, wherein the isolated nucleic acid is isolatable from a cyanobacteria that produces gamma linolenic acid, and is selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing -3d-under stringency conditions to t:ze complement of nucleic acid of (b).
In accordance with a further aspect of the present invention, there is provided an isolated nucleic acid encoding a Synechocystis 06-desaturase, wherein the nucleic acid is selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID
NO: 3, (c) a nucleic acid hybriciizing under stringency conditions to the complement of nucleic acid of (b).
In accordance with a further aspect of the present invention, there is provided a c:yanobacterial A6-desaturase encoded by a nucleic acid from a cvanobacteria that produces gamma linolenic acid, wherein the nucleic acid is selected from (a) a nucleic acid comp:-ising a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing inder stringency conditions to the complement of nucleic acid of (b).
Fig. 1 depicts the hydropathy profiles of the deduced amino acid sequences of Synechocystis A6-desaturase (Panel A) and A12-desaturase (Panel B).
Putative membrane spanning regions are indicated by solid bars. Hydrophobic index was calculated for a window size of 19 amino acid residues [Kyte, et al. (1982) J. Molec.
Biol. 157].

WO 97 'q67iz 212 0 6 2 9 PCT/US92/08746 1 Fig. 2 provides gas liquid chromatography profiles of wild type (Panel A) and transgenic (Panel B) Anabaena.
Fig. 3 is a diagram of maps of cosmid cSy75, cSyl3 and cSy7 with overlapping regions and subclones.
The origins of subclones of cSy75, oSy75-3.5 and cSy7 are.indicated by the dashed diagonal lines. Restriction sites that have been inactivated are in parentheses.
Fig. 4 provides gas liquid chromatography profiles of wild type (Panel A) and transgenic (Panel B) tobacco.
The present invention provides an isolated nucleic acid encoding A6-desaturase. To identify a nucleic acid encoding a6-desaturase, DNA is isolated from an organism which produces GLA. Said organism can be, for example, an animal cell, certain fungi (e.g.
Mortierella), certain bacteria (e.g. Synechocystis) or certain plants (borage, oenothera, currants). The isolation of genomic DNA can be accomplished by a variety of inethods well-known to one of ordinary skill in the art, as exemplifiedby Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY. The isolated DNA is fragmented by physical methods or enzymatic digestion and cloned into an appropriate vector, e.g. a bacteriophage or cosmid vector, by any of a variety of well-known methods which can be found in references such as Sambrook et al.
(1989). Expression vectors containing the DNA of the present invention are specifically contemplated herein.
DNA encoding A6-desaturase can be identified by gain of function analysis. The vector containing fragmented DNA
is transferred, for example by infection, 1 transconjugation, transfection, into a host organism that produces linoleic acid but not GLA.. As used herein, "transformation" refers generally to the incorporation of foreign DNA into a host cell. Methods for introducing recombinant DNA into a host organism are known to one of ordinary skill in the art and can be found, for example, in Sambrook et al. (1989).
Production of GLA by these organisms (i.e.', gain of function) is assayed, for example by gas chromatography or.other methods known to the ordinarily skilled artisan. Organismswhich are induced to produce GLA, i.e. have gained function by the introduction of the vector, are identified as expressing DNA encoding A6-desaturase, and said DNA is recovered from the organisms. The recovered DNA can again be fragmented, clonedwith expressionvectors, and functionally assessed by the above procedures to define with more particularity the DNA encoding e6-desaturase..
As an example of thepresent invention, random DNA is isolated from the cyanobacteria Synechocystis Pasteur Culture Collection (PCC) 6803, American Type Culture Collection (ATCC) 27184, cloned into a cosmid vector, and introduced by transconjugation into the GLA-deficient cyanobacterium AnaYSaena strain PCC 7120, ATCC
27893. Production of GLA from Anabaena linoleic acid is monitored by gas chromatography and the corresponding DNA fragment is isolated.
The isolated DNA is sequenced by methods well-known to one of ordinary skill in the art as found, for example, in Sambrook et a1. (1989).
In accordance with the present invention, a DNA
comprising a A6-desaturase gene has been isolated. More .1 ' 2120629.
WQ 9`" "n6712 PC'T/US92/08746 1 particularly, a 3.588 kilobase (kb) DNA comprising a 46-desaturase gene has been isolated from the cyanobacteria Synechocystis. The nucleotide sequence of the 3.588 kb DNA was determined and is shown in SEQ ID N0:1. Open reading frames defining potential coding regions are present from nucleotide 317 to 1507 and from nucleotide 2002 to 3081. To define the nucleotides responsible for encoding e6-desaturase, the 3.588 kb fragment that confers A6-desaturase activity is cleaved into two subfragments, each of 'which contains only one open reading frame. Fragment ORF1 contains nucleotides 1 through 1704, while fragment ORF2 contains nucleotides 1705 through 3588. Each fragment is subcloned in both forward and reverse orientations into a conjugal expression vector (AM542, Wolk et al. [1984] Proc. Natl.
Acad. Sci. USA 81, 1561) that contains a cyanobacterial carboxylase promoter. The resulting constructs (i.e.
0RF1(F), ORF1(R), ORF2(F) and ORF2(R)] are conjugated to wild-type Anabaena PCC 7120 by standard methods (see, for example, Wolk et al. (1984) Proc. Natl. Acad. Sci.
USA 81, 1561). Conjugated cells of Anabaena are identified as NeoR green colonies.on a brown background of dying non-conjugated cells after two weeks of growth on selective media (standard mineral media BG11N +
containing 30u.g/ml of neomycin according to Rippka et al., (1979) J. Gen Microbiol. 111, 1). The green colonies are selected and grown in selective liquid media (BG11N + with 15ug/mi neomycin). Lipids are extracted by standard methods (e.g. Dahmer et al., (1989) Journal of American Oil Chemical Society 66, 543) from the resulting transconjugants containing the forward and reverse oriented ORF1 and ORF2 constructs.

1 For comparison, lipids are also extracted from wild-type cultures of Anabaena and Synechocystis. The fatty acid methyl esters are analyzed by gas liquid chromatography (GLC), for example with a Tracor-560 gas liquid chromatograph equipped with a hydrogen flame ionization detector and a capillary column. The results of GLC
analysis are shown in Table 1.
Table 1: Occurrence of C18 fatty acids in wild-type and transgenic cyanobacteria -SO'URCE 18:0'.18:1 18:Z Y18:~' a18t:3 28:4 Anabaena + + + + -(wild type) Anabaena + ORF]. ( F ) + + + - +

Anabaena + ORF1(R) + + + - + -Anabaena + ORF2(F)' + + + + + +
Anabaena + ORF2(R) + + + - + -Synechocysti.s + + + + -(wild type) As assessed by GLC analysis, GLA deficient Anabaena gain the function of GLA production when the construct containing ORF2 in forward orientation is introduced by transconjugation. Transconjugants containing constructs with,ORF2 in reverse orientation to the carboxylasepromoter, or ORF1 in either orientation, show no GLA production. This analysis demonstrates that the singYeopen reading frame (ORF2) within the 1884 bp fragment encodes e6-desaturase. The 1884 bp fragment is shown as SEQ ID NO:3. This is substantiatedby the overall similarity of the hydropathy profiles between e6-desaturase and e12-WO 93/A6712 212 062 9 PCr/US92/08746 1 desaturase [Wada et al. (1990) Nature 3471 as shown in Fig. 1 as (A) and (B}, respectively.
Isolated nucleic acids encoding e6-desaturase can be identified from other GLA-producing organisms by the gain of function analysis described above, or by nucleic acid hybridization techniques using the isolated nucleic acid which encodes Anabaena e6-desaturase as a hybridization probe. Both genomic and cDNA cloning methods are known to the skilled artisan and are contemplated by the present invention. The hybridization probe can comprise the entire DNA sequence disclosed as SEQ. ID NOc1, or a restriction fragment or other DNA fragment thereof, including an oligonucleotide probe. Methods for cloning homologous genes by cross-hybridization are known to the ordinarilyskilled artisan and can be found, for example, in Sambrook (1989) and Beltz et al. (1983) Methods in Enzymology 100, 266.
Transgenic organisms which gain the function of GLA production by introduction of DNA encoding desaturase also gain the function of octadecatetraeonic acid (18s4A6,9-'-Z='-5) production. octadecatetraeonic acidis present normally in fish oils and in some plant species of the Boraginaceae family (Crai.g et al. [19641 J. Amer. Oi1Chem. Soc. 41, 209-211; Gross et al. [1976]
Can. J. Plant Sci. 56, 659-664). In the transgenic organisms of the present invention, octadecatetraenoic acid results from further desaturation of cc-linolenic acid by A6-desaturase or desaturation of GLA by a15-desaturase.
The 359 amino acids encoded by ORF2, i.e. the open reading frame encoding n6-desaturase, are shown as WO 93/06712 2120629 PCI'/US92/08746 SEQ. ID NOz2. The present invention further contemplates other nucleotide sequences which encode the amino acids of SEQ ID No:2. It is within the ken of the ordinarily skilled artisan to identify such sequences which result, for example, from the degeneracy of the genetic code. Furthermore, one of ordinary ski],1 in the art can determine, by the gain of function analysis described hereinabove, smaller subfragments of the 1884 bp fragment containing ORF2 which encode e6-desaturase.
The present invention contemplates any such polypeptide fragment of e6-desaturase and the nucleic acids therefor which retain activity for converting LA
to GLA.
In another aspect of the present invention, a vector containing the 1884 bp fragment or a smaller fragment containing the promoter, coding sequence and termination region of the t16-desaturase gene is transferred into an organism,for example, cyanobacteria, in which the A6-desaturase promoter and termination regions are functional. Accordingly, organisms producing recombinant A6-desaturase are provided by this invention: Yet another aspect of this invention provides isolated A6-desaturase, which can be purifiedfrom the recombinant organisms by standard methods of proteinpurification. (For example, see Ausubel t al. L1987] Current Protocolsin Molecular Biology, Green Publishing Associates, New York).
Vectors containing DNA encoding 46-desaturase are also provided by the present invention. It will be apparent to one of ordinary skill in the art that appropriate vectors can be constructed to direct the expression of the A6-desaturase coding sequence in a !~ 1 1 n n-r=,-. .--..-. _ .

WO 93 f 16'712 2120 629 PC17/US92/08746 _Z0_ l variety of organisms. Replicable expression vectors are particularly preferred. Replicable expression vectors as described herein are DNA or RNA molecules engineered for controlled expression of a desired gene, i.e. the e6-desaturase gene. Preferably the vectors are plasmids, bacteriophages, cosmids or viruses. Shuttle vectors, e.g. as described by Wolk et al. (1984) Proc.
Nat1. Acad. Sci. USA, 1561-1565 and Bustos et al. (1991) J. Bacteriol. 174, 7525-7533, are also contemplated in accordance with the present invention. Sambrook et al.
(1989). Goeddel, ed. (1990) Methods in Enzymology 185 Academic Press, and Perbal (1988) A Practical Guide to Molecular Cloning, John Wiley and Sons, Inc., provide detailed reviews of vectors into which a nucleic acid encoding the present e6-desaturase can be inserted and expressed. Such vectors also contain nucleic ucid sequences which can effect expression of nucleic acids encoding o6-desaturase. Sequence elements capable of effecting expression of a gene product include promoters, enhancer elements, upstream activating sequences, transcription termination signals and polyadenylation sites. Both constitutive and tissue specific promoters are contemplated. For transformation of plant cells, the cauliflower mosaic virus (CaMV) 35S
promoter and promoters which are regulated during plant seed maturation are of particular interest. All such promoter and transcriptional regulatory elements, singly or in combination, are contemplated for use in the present replicable expression vectors and are known to one of ordinary skill inthe art. The CaMV 355 promoter is described, for example, by Restrepo et al. (1990) Plant Cell 2, 987. Genetically engineered and mutated regulatory sequences are also contemplated.
The ordinary skil:Led artisan can determine vectors and regulatory elements suitable for expression in a particular host cell. For example, a vector comprising the promoter from the gene encoding the carboxylase of Anabaena operably linked to the coding region of A6-desaturase and further operably linked to a termination signal from Synechocystis is appropriate for expression of A6-desaturase in cyano-bacteria. "Operably linked" in this context means that the promoter and terminator sequences effectively function to regulate transcription. As a further example, a vector appropriate for expression of A6-desaturase in transgenic plants can comprise a,seed-specific promoter sequence derived from helianthinin, napin, or glycin operably linked to the n6-desaturase coding region and further operably linked to a seed termination signal or the nopaline synthase termination signal.
In particular, the helianthinin regulatory elements disclosed in, for example, Bogue et al: (1990) Mol. Gen.
Genet. 222:49; and T.L. Thomas et al.. (1991) "ABA regulation of gene expression in embryos and mature plants" in Environmental Plant Biology Series: Abscisic Acid:
Physiology and Biochemistry, Davies, W.J. and Jones, H.G.
(ed.), Bio Scientific Publishers LTD., Oxford, England, UK., are contemplated as promoter elements to direct the expression of the A6-desaturase of the present invention.
Modifications of the nucleotide sequences or regulatory elements disclosed herein which maintains the functions contemplated herein are within the scope of this invention.
Such modifications include insertions, substitutions and deletions, and specifically substitutions which reflect the degeneracy of the genetic code.

WO 93/06712 2120629 PCr/US92/08746 3. Standard techniques for the construction of such hybrid vectors are well-known to those of ordinary skill in the art and can be found in references such as =
Sambrook et al. (1989), or any of the myriad of laboratory manuals on recombinant DNA technology that are widely available. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA
fragments. It is further contemplated in accordance with the present invention to include in the hybrid vectors other nucleotide sequence elements which facilitate cloning,expression or processing, for example sequences encoding signal peptides, a sequence encoding KDEL, which is required for retention of proteins in the endoplasmic reticulum or sequences encoding transit peptideswhich direct e6-desaturase to the ahioroplast. Such sequences are known to one of ordinary skill in theart. An optimized transit peptide is described, for example, by Van den Broeck et al.
(1985) Nature 313, 358. Prokaryotic and eukaryotic signal sequences are disclosed, for example, by Michaelis et al. (1982)Ann. Rev. Microbiol. 36, 425.
A further aspect of the instant invention provides organisms other than,cyanobacteria which contain the DNA encoding the e6-desaturase of the present invention. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA

1 of the present invention into such organisms are widely known and.provided in references such as Sambrook et el.
(1989').
A variety of plant transformation methods are known. The e6-desaturase gene can be- introduced into plants by a leaf disk transformation-regeneration procedure as described by Horsch et al. (1985) Science 227, 1229. Other methods of transformation, such as protoplast culture (Horsch et al. (1984) Science 223, 496; DeHlock et al. (1984) EMBO J. 2, 2143; Barton et al. (1983) Cell 32, 1033) can also be used and are within the scope of this invention. In a preferred embodiment plants are transformed with Agrobacterium-dezived vectors. However, other methods are available to insert the ef-desaturase gene of the present invention into plant cells. Such alternative methods include biolistic approaches (Klein et al. (1987) Nature 327,.70)-,. electroporation, chemically-induced DNA, uptake, and use of viruses or pollen as vectors.
When necessary for the transformation method, the e6-desaturase gene of the presentinvention can be inserted into a plant transformation vector, e.g. the binary vector described by Bevan (1984) Nucleic Acids Res. 12, 8111. Plant transformation vectors can be derived by modifying the natural genetransfer system of Agrobaeterium tumefaciens. The natural system comprises large Ti (tumor-inducing)-plasmids containing a large segment, known as T-DNA, which is transferred to transformed plan,ts. Another segment of the Ti plasmid, the vir region, is responsible for T-DNA transfer. The T-DNA region is bordered by terminal repeats. In the modified binary vectors the tumor-inducing genes have r,r...,..~._..

WO 97M6712 2120629 PC.'rlUS92f08746 _14-1 been deleted and the functions of the vir region are =
utilized to transfer foreign DNA bordered by the T-DNA
border sequences. The T-region also contains a =
selectable marker for antibiotic resistance, and a multiple cloning site for inserting sequences for transfer. Such engineered strains are known as "disarmed" A. tumefaciens strains, and allow the efficient transformation of sequences bordered by the T-region into the nuclear genomes of plants.
Surface-sterilized leaf disks are inoculated with the "disarmed" foreign DNA-containing A. tumefaciens, cultured for.two days, and then transferred to antibiotic-containing medium. Transformed shoots are selected after rooting in medium containing the appropriate antibiotic, transferred to soil and regenerated.
Another aspect of the present invention provides transgenic plants or progeny of these plants containing the isolated DNA of the invention. Both monocotyledenous and dicotyledenousplants are contemplated. Plant cells are transformed with the isolated DNA encoding o6-desaturase by any of the plant transformation methods described above. The transformed.
plant cell, usually in a callus culture or leaf disk, is.-regenerated into a complete transgenic plant by methods well-known to one of ordinary skill in the art (e.g.
Horsch etal. (1985) Science 227, 1129). In a preferred embodiment, the transgenic plant is sunflower, oil seed rape, maize, tobacco, peanut or soybean. Since progeny of transformed plants inherit the DNA encoding a6-desaturase, seeds or cuttings from transformed plants are used to maintain the transgenic plant line.

~~ ~, J

1 The present invention further provides a method for providing transgenic plants with an increased content of GLA. This nethod includes introducing DNA
encoding e-6-desaturase into plant cells which lack or have low levels of GLA but contain LA, and regenerating plants with increased GLA content from the transgenic cells. In particular, commercially grown crop plants are contemplated as the transgenic organism, including, but not limited to, sunflower, soybean, oil seed rape, maize, peanut and tobacco.
The present invention further provides a method for providing transc3enicorganisms which contain GLA.
This method comprises introducing DNA encoding e6-desaturase into an organism which lacks or has low levels of GLA, but contains LA. In another embodiment, the method comprises introducing one or more expression vectors which comprise DNA encoding a12-desaturase and n6-desaturase. into organisms which are deficient in both GLA and LA. Accordingly, organisms deficient in both LA
and GLA are induced topXoduce LA by the expression of n12-desaturase, and GLA is then generated due to the expressiQn of e6-desaturase. Expression vectors comprising DNA encoding e12-desaturase, or a12-desaturase and ab-desaturase, can be constructed by methods of recombinant technology known to one of ordinary skill in the art (Sambrook et al., 1989) and the published sequence of e12-desaturase(Wada et al C19901 Nature (London) 347; 200-203. In addition, it has been discovered in accordance with the present invention that nucleotides 2002-3081 of SEQ. ID NO:1' encode cyanobacteriala12-desaturase. AccordingZy, this sequence can be used to construct the subject expression /11 ~P~ I1T'fT. r=o=r- .-, . ,......~~

' ' 2120629 WO 92 ~06712 Pcrrus92/08746 1 vectors. In particular, commercially grown crop plants are contemplated as the transgenic organism, including, but not limited to, sunflower, soybean, oil seed rape, maize, peanut and tobacco.
The present invention is further directed to a method of inducing chilling tolerance in plants.
Chilling sensitivity may be due to phase transition of lipids in cell membranes. Phase transition temperature depends upon the degree of unsaturation of fatty acids in membrane lipids, andthus increasing thedegree of unsaturation, for example by introducing e6-desaturase to convert LA to GLA, can induce or improve chilling resistance. Accordingly, the present method comprises introducing DNA encoding A6-desaturase into a plant cell, and regenerating a plant with improved chilling resistance from said transformedplant cell. In a preferred embodiment, the plant is a sunflower, soybean, oil seed rape, maize, peanut or tobacco plant.
The following examples further illustrate the present invention.-. 2124629-1 E7tAMPLE 1 Strains and Culture Conditions Synechocystis (PCC 6803, ATCC 27184), Anabaena (PCC
7120, ATCC 27893.) and S nechococcus (PCC 7942, ATCC
33912) were grown photoautotrophically at 30 C in BG11N+
medium (Rippka et al. [1979) J. Gen. Microbiol. iil, 1-61) under illuminaotion of incandescent lamps.
(604B.m-a.S'1). Cosmids and plasmids were selected and propagated in Escherichia co}.i strain DI15a on LB medium supplemented with antibiotics at standard concentrations as described by Maniatis et a1. (1982) Molecular Cloninc,z A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, New York.

., . .,.. ,. _ _ . _.

WO 9?/Q6712 2120629 PG7'/US92/08746 Construction of Synechocystis Cosmid Genomic Library Total genomic DNA from Synechocystis (PCC 6803) was partially digested with Sau3A and fractionated on a sucrose gradient (Ausubel et al. [19871 Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York). Fractions containing 30 to 40 kb DNA fragments were selected and ligated into the dephosphorylated BamHI site of the cosmid vector, pDUCA7 (Buikema et al. [19911 J.
Bacteriol. 173, 1879-1885). The ligated DNA was packaged in vitro as described by Ausubel et al. (1987), and packaged phage were propagated in E. coli DH5a containing the Aval and Eco4711 methylase helper plasmid, pRL528 as describedby Buikema et al. (1991).
A total of 1152 colonies were isolated randomly and maintained individually in twelve 96-well microtiter plates.

= 1 w0 93/06712 2120629 PcT/us92f08746 EX,AMPLE 3 Gain-of-Function Expression of GLA in Anabaena Anabaena (PCC 7120), a filamentous cyanobacterium, is deficient in GLA but contains significant amounts of linoleic acid, the precursor for GLA (Figure 2; Table 2). The Synechocystis cosmid library described in Example 2 was conjugated into Anabaena (PCC 7120) to identify transconjugants that produce GLA. Anabaena cellswere grown to mid-log phase in BG11N+ liquid medium and resuspended in the same medium to a final concentration of approximately 2x10e cells per ml. A mid-log phase culture of E. coli RP4 (Burkardt et al. [1979) J. Gen. Microbiol. 114, 341-348) grown in LB containing ampicillin was washed and resuspended in fresh LB medium. Anabaena and RP4 were then mixed and spread evenly on BG11N+ plates containing 5% LB. The cosmid genomiclibrary was replica plated onto LB plates containing 50 g/ml kanamycin and 17.5 ugjml chloramphenicol and wassubsequently patched onto BG11N+ plates containing Anabaena and RP4. After 24 hours of incubation at 30 C, 30 glml of neomycin was underlaid;-and incubation at 30 C was continued until transconjugants appeared.
Individual transconjdgants were isolated after.
conjugation and grown in 2 ml BG11N+ liquid medium with 15 g/mi neomycin. Fatty acid methyl esters were prepared from wild type cultures and cultures containing poolsof ten transconjugants as follows. Wild type and transgenic cyanobacterial cultures were harvested by centrifugation and.washed twice withdistilled water.
Fatty acidmethyl esters were extracted from these cultures as described by Dahmer et al. (1989) J. Amer.

rti - ( n n-r-r-r-. .~. .... .. . .

WO 9:2`96712 PC.'T/US92/08746 1 Oil. Chem. Soc. 66, 543-548 and were analyzed by Gas Liquid Chromatography (GLC) using a Tracor-560 equipped with a hydrogen flame ionization detector and capillary column (30 m x 0.25 mm bonded FSOT Superox II, Alitech Associates Inc., IL). Retention times and co-chromatography of standards (obtained from Sigma Chemical Co.) were used for identification of fatty acids. The average fatty acid composition was determined as the ratio of peak area of each C18 fatty acid normalized to an internal standard.
Representative GLC profiles are 'shown in Fig. 2.
C18 fatty acid methyl esters are shown. Peaks were identified by comparing the elution times with known standards of fatty acid methyl esters and were confirmed by gas chromatography-mass spectrometry. Panel A
depicts GLC analysis of fatty acids of wild type Anabaena. The arrow indicates the migration time of GLA. Panel B is a GLCprofile of fatty acids of transconjugants of Anabaena with pAM542+1.8F. Two GLA
producing pools (of25 pools representing 250 transconjugants) were identified that produced GLA.
Individual transconjugants of each GLA positive pool were analyzed for GLA production; two independent transconjugants, AS13 and AS75, one from each pool, were identified which expressed significant levels of GLA and which contained cosmids, cSy13 and cSy75, respectively (Figure 3). The cosmids overlap in a region approximately 7.5kb in length. A 3.5 kb NheI fragment of cSy75 was recloned in the vector pDUCA7 and transferred to Anabaena resulting in gain-of-function expression of GLA (Table 2).

c~ c~
WO 93/06712 2 j~+ 06 f~ 9 PCI'/US92/08746 1 Two Nhel/Hind III subfragments (1.8 and 1.7 kb) of the 3.5 kb Nhe I fragment of cSy75-3.5 were subcloned into "pBLUESCRIPT" (Stratagene) (Figure 3) for sequencing. Sta.ndard molecular biology techniques'were performed as described by Maniatis et al. (198*2) and Ausubel et al. (1987). Dideoxy sequencing (Sanger et al.
[19771 Proc. Natl. Acad. Sci. USA 74, 5463-5467) of pBS1.8 was performed with "SEQUENASE" (United States Biochemical) on both strands by using specific oligonucleotide primers synthesized by the Advanced DNA
Technologies Laboratory (Biology Department, Texas A & M
University). DNA sequence analysis was done with the GCG (Madison, WI). software as described by Devereux et al. (1984) Nucleic Acids Res. 12, 387-395.
Both Nhel/HindIII subfragments were transferred into.a conjugal expression vector, AM542, in both forward and reverse orientations with respect to a cyanobacterial carboxylase promoter and were introduced into. Anabaena by conjugation. Transconjugants .20 containing the 1.8 kb fragment in the forward orxentation (AM542-1.8F) produced significant quantities of GLA and octadecatetraenoic acid (Figure 2; Table 2).
Transconjugants containingothEr constructs, either reverse oriented 1.8 kb fragment or forward and reverse oriented 1.7 kb fragment, did not produce detectable levels of GLA (Table 2).
Figure 2 compares the C18 fatty acid profile of an extract from wild type Anabaena (Figure 2A) with that of transgenic Anabaena containing the 1.8 kb fragment of cSy75-3.5 in the forward orientation (Figure 2B). GLC
analysis of fatty acid methyl esters from AM542-1.8F
revealed a peak with a retention time identical to that WO 9" 16712 2120629 PCT/US92/08746 3- of authentic GLA standard. Analysis of this peak by gas chromatography-mass spectrometry (GC-MS) confirmed that it had the same mass fragmentation pattern as a GLA
reference sample. Transgenic Anabaena with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.

1 1 =

1 Table ?
Composition of C18 Fatty Acids in Wild Type and Trasgenic Cyanobacteri.a Strain Fatty acid M

18:0 18:1 18:2 18:3 18:3 .18:4 (a) (Y) Wi.ld type SynecT~ocystis. 13.6 4.5 54.5 - 27.3 -(sp.PCC6803) ' Anabaena 2.9 24.8 37.1 35.2 -(sp.PCC7120) Synechococcus 20.6 79.4 - - - -(Sp.PCC7942) 'Anabaena Transconjugants cSy75 3:8 24.4 22.3 9.1. 27.9 12.5 cSy75-3.5 4.3 27.6 18.1 3.2 40.4 6.4 pAM542-1.8F 4.2 13.9 12.1 19.1 25.4 25.4 gAM542-1.8R 7.7 23.1 38.4 30,8 - -pAM542-1.7F 2.8 27.8 36.1 33.3 - -pAM542-1.7R 2.8 25.4 42.3 29.6 - -Synechococcus Transformants pAlvf854 27.8 72.2 - - - -pAM854-tl12 4.0 43.2 46.0 pAM854-AO 18.2 81.8 - - -pAM854-A6 & A12 42.7 25.3 19.5 - 16.5 -18:0, stearic acid; 18.1, oleic acid; 18:2, linoleic acid; 18 : 3( a), a-linolenic acid; 18 : 3( Y), Y-linolenic acid; 18:4, octadecatetraenoic acid NVO 93l06712 11CUl!S92/0R746 1 F,XAMFLE 4 Transformation of Synechococcus with A6 and n12 Desaturase Genes A third cosmid, cSy7, which contains a a12-desaturase gene, was isolated by screening the S nechocystis genomic library with a oligonucleotide synthesized from the published Synechocystis e12-desaturase gene sequence (Wada et al. 119901 Nature (Loridon) 347, 200-203). A 1.7 kh Aval fra_qment frorn this cosmid cotitainirig the e12-desaturase gene was identified and used as a probe to demonstrate that eSy13 not only contains a n6-desaturase gene but also a n12-desaturase gerie (Figure 3). Getiomic Southern blot analysis further showed that both the e6-and e12-desaturase genes are unique in the Synechocystis genome so that both functional genes involved in C18 fatty acid desaturation are linked closely in the Synechocystis genome.
The unicellular cyanobacterium Synechococcus (PCC
7942) is deficient in both linoleic acid and GLA.).
The 612 and e6-desat.urase genes were cloned individually and together into pAM85+1 (Bustos et al. t1991) J.
Bacteriol. 174, 7525-7533), a shuttle vector that contains sequences necessary for the integration of foreign Dl3A into the getiome of Synechococcus (Golden et ~ al. 11987) Methods in ErtzyTnal. 153, 215-231).
Synechococcus was transformed with these gene constructs and colonies were selected. Fatty acid methyl esters were extracted from transgenic Synechococcus and analyzed by GLC.
Table 2 shows tha t: the principal fatty acids of wild type Synechococcus are stearic acid (1II:0) and 1 oleic acid (18:1). Synechococcus transformed with pAM854-ni2 expressed linoleic acid (18:2) in addition to the principal fatty acids. Transformants with pAM854-a6 and e12 produced both linoleate and GLA (Table 1).
These results indicated that Synechococcus containing both e12- and e6--desaturase genes has gained the .
capability of introducing a second double bond at the e12 position and a third double bond at the n6 position of C18 fatty acids. However, no changes i,n fatty acid composition was observed in.the transformant containing pAM854-e6, indicating that in the absence of substrate synthesized by the o12 desaturase, the a6-desaturase is inactive. This experiment further confirms that the 1.8 kb NheI/HindIII fragment (Figure 3) contains both coding 1,5 and promoter regions of the Synechocystis e6-des.aturase gene. Transgenic Synechococcus with altered levels of polyunsaturated fatty acids were similar to wild type in growth rate and morphology.

r WO 93 -"5712 212 062 9 PGTrvs92/08746 Nucl.eotide Sequence of e6-Aesaturase The nucleotide sequence of the 1.8 kb fragment of cSy75-3.5 including the functional a6-desaturase gene was determined. An open reading frame encoding a polypeptide of 359 amino acids was identified (Figure 4). A Kyte-Doolittle hydropathy-analysis (Kyte et al.
[1982] J. Mol. Biol. 157, 105-132) identified two regions of hydrophobic amino acids that could represent transmembrane domains (Figure 1A); furthermore, the hydropathic profile of the e6-desaturase is similar to that of the a12-desaturase gene (Figure 1B; Wada et al.) ande9-desaturases (Thiede et al. [1986] J. Biol. Chem.
261, 13230-R13235). However, the sequence similarity between the Synechocystis e6- and a12-desaturases is less than 40$ at the nucleotide level and approximately 18% at the amino acid level.

~ . ..

Transfer of Cyanobacterial A6-Desaturase into Tobacco The cyanobacterial o6-desaturase gene was mobilized into a plant expression vector and transferred to tobacco using Actrobacterium mediated gene transfer techniques. To ensure that the transferred desaturase is appropriately expressed in leaves and developing seeds and that the desaturase gene product is targeted to the endoplasmic reticulum or the chloroplast, various expression cassettes with Synechocystis e-desaturase open reading frame (ORF) were constructed.
Components of these cassettes include: (i) a 35S promoter or seed specific promoter derived from the sunflower helianthinin gene to drive n"-desaturase gene expression in all plant tissues or only in developing seeds respectively, (ii) a putative signal peptide either from carrot extensin gene or sunflower helianthinin gene to target newly synthesized A6-desatura.se into the ER, (iii) an ER lumen retention signal sequence (KDEL) at the COOH-terminal of the e6-desaturase ORF, and (iv) an optimized transit peptide to target n6-desaturase into the chloroplast. The 35S promoter is a derivative of pRTL2 described by Restrepo gt al (1990) Plant Cell, 2, 987, optimized transit peptide sequence is described by Van de Broeck gt al (1985) Nature 313, 358.
The carrot extensin signal peptide is described by Chen et al (1985) EMBO J. 9. 2:145.
Transgenic tobacco plants were produced containing a chimeric cyanobacterial desaturase gene, comprised of the Synechocystis e6-desaturase gene fused to an endoplasmic reticulum retention sequence (KDEL) and extensin signal peptide driven by the CaMV 35S promoter. PCR amplifications of transgenic tobacco genomic DNA indicate that the e6-desaturase gene was incorporated int.o the tobacco genome.
Fatty acid methyl esters of leaves of these transgenic tobacco plants were + 1 WO 93/06712 2120629 PCr/vs92i08746 _28_ l extracted and analyzed by Gas Liquid Chromatography (GLC). These transgenic tobacco accumulated significant amounts of GLA (Figure 4). Figure 4 shows fatty acid methyl esters as determined by GLC. Peaks were identified by comparing the elution times wxth known standards of fatty acid methyl ester. Accordingly, cyanobacterial genes involved in fatty acid metabolism can be used to generate transgenic plants with altered fatty acid compositions.

WO 93/06712 2 120629 PC!'/US92/08746 SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: Thomas, Terry L.
Reddy, Avutu S..
Nuccio, Michael Freyssinet, Georges L.

(ii) TITLE OF INVENTION: PRODUCTION OF GAMMA LINOLENIC

(iii) NUMBER OF SEQUENCES : 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Scully, Scott, Murphy & Presser (B) STREET: 400 Garden City Plaza (C) CITY: Garden City ( D ) STATE : New York (E) COUNTRY: United States (F) ZIP: 11530 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER : IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To be assigned (B) FILING DATE: 08-JAN-1992 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME; McNulty, William E.
(B) REGgSTRATION NUMBER: 22,.606 (C) REFERENCE/DOCKET NUMBER: $383Z
( ix ) TELECOMMUNICATION INFORMATION :
(A) TELEPHONE: (516) 742-4343 (B) TELEFAX: (516) 742-4366 (C) TELEX: 230 901 SANS UR

a WO 93/n~712 2120629 PGT/US92/08746 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3588 base pairs =
(S) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genamic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2002..3081 (xi) SEQUENCE DESCRIPTIONe SEQ.ID N0:1:

CACCTTGCCA GACCACGTTA GTTTGAGTGT TTCCGCCCTG GCGGCCCCGA TZ'TTZTCCTT 180 TGCGGCTTTG GGCAATCAGG CGATCGGGCA ATTGCGTT'.CG TTTGACCAGA CTTGGCCCAT 240 GATGAT't'T1T CTGGCCACCT TCATCTACGT TTCCATTGAT CAACATATTG CCCCAGTGGA= 600 CGCGTrGTAT TTZTCCGTGG GCATGATTAC CGGGGCCGGT GGCAAGGAAG AGGTGGCCGA 660 GGA'1.`ACAGAT AATCGT3TCT TGCATACGGC CCGCTCCCTG GGGGTGCCCG TAATTGTGGA 960 ' ' 2120629 GGAATTATTG GGTACCCATC TCGACTCTGG hGACGTGTTG TATZTAACCA TGCCCGCCAC 1440 lO TGCCCTAGAG CAACxTTGGC GATCGCCCCGTGCCACTGCT GATCCTCTGG ACTCTI'ITiT 1500 GG.TTTAGCAT GGGGGGATGG AACTCTTGAC TCGGCCCAAT GGTGATCAAG AAAGAACGCT 1560 TTGTCTATGT TTAGTATT'IT TAAGTTAACC AACAGCAGAG GATAACTTCC AAAAGAAATT 1620 ..TGCAAAAAAG TCAGATAAAA TAAAAGCTTC ACTTCGGTTT TATATTGTGA CCATGGTTCC 1740 CAGGCATCTG CTCTAGGGAG TT2'ZTCCGCT GCCTTTAGAG AGTATTTTCT CCAAGTCGGC 1800 TAACTCCCCC ATTTTTAGGC AAAATCATAT ACAGACTATC CCAATATTGC CAGAGCTTTG ' 1860 TZ'TATCTATT'TAAATTTATA A ATG CTA ACA GCG GAA AGA ATT AAA TTT ACC 2031 MetLeu Thr Ala Glu Arg Ile Lys Phe Thr Gln Lys Arg Gly Phe Arg Arg- Va1 Leu Asn Gln Arg Val Asp Ala Tyr TZ,'1' GCC GAGCAT GGC CTG ACC CAA AGG GAT AAT CCC TCC ATG TAT CTG 2127 Phe Ala Glu"His Gly Leu Thr Gln Arg Asp Asn Pro Ser Met Tyr Leu =

_32_ Lys Thr Leu Ile Ile Val Leu Trp Leu Phe Ser Ala Trp Ala Phe Val Leu Phe Ala Pro Val Ile Phe Pro Val Arg Leu Leu Gly Cys Met Val Leu Ala Ile Ala Leu Ala AlaPhe Ser Phe Asn Val Gly His Asp Ala Asn His Asn Ala Tyr Ser Ser Asn Pro His Ile Asn Arg Val Leu Gly 1.0 Met Thr Tyr Asp Phe Val Gly Leu Ser Ser Phe Leu Trp Arg Tyr Arg His Asn Tyr Leu His His Thr Tyr Thr Asn Ile Leu Gly His Asp Val GAA ATC CAT GGA GAT.GGC GCA GTA CGT ATG AGT CCT GAA CAA GAA CAT 2463 Glu Ile His Gly Asp Gly Ala Val Arq Met Ser Pro Glu Gln Glu His Val Gly Ile Tyr Arg Phe G1n Gin Phe Tyr Ile Trp Gly Leu Tyr Leu Phe Ile Pro Phe Tyr Trp Phe Leu Tyr:Asp Val Tyr Leu Val Leu Asn LysGly Lys Tyr His Asp His Lys Ile Pro Pro Phe G1n Pro Leu Glu Leu Ala Ser Leu Leu Gly Ile Lys Leu Leu Trp Leu Gly Tyr Val Phe GGC TTA CCT CTG GCT CTG GGC TTT TCC ATT CCT GAA GTA TTA ATl GGT 2703'*
GlX Leu Pro Leu Ala Leu Gly Phe Ser Ile Pro Glu Val Leu Ile Gly 3a WO 93/06712 MOM PCI'/US92/08746 GCT TCG GTA ACC TAT ATG ACC TAT GGC ATC GTG GTT TGC ACC AZ`C TTT 2751 Ala Ser Val Thr Tyr Met Thr Tyr Gly Ile Val Val Cys Thr lle Phe 235 240 .245 250 Met Leu Ala Hi,s Val Leu Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly Glu' Sez Gly Ala Ile Asp Asp Glu Trp Ala Ile Cys Gln Ile Arg Thr 270 . 275 280 Thr Ala Asn-Phe Ala Thr Asn Asn Pro Phe Trp Asn Trp Phe Cys Gly lo GGT TTA AAT CAC CAA GTT ACC CAC CAT CTT TTC CCC AAT ATT TGT CAT 2943 G1y Le.u Asn His Gin Val Thr His His Leu Phe Pro Asn Ile Cys His ATT CAC TAT CCC CAA TTG GAA AAT ATT ATT AAG GAT GTT TG'C CAA GAG 2991 Ile His Tyr Pro G1n Leu Glu Asn Ile Ile Lys Asp Val Cys Gln= Glu Phe Gly Val Glu Tyr Lys Val Tyr Pro Thr Phe Lys Ala Ala Ile Ala Ser Asn Tyr Arg Trp Leu GluAla Met Gly Lys Ala Ser TTGGGATTGA AGCAAAATGG CAAAATCCCT CGTAAATCTA TGATCGAAGC CTTTCTGTTG 3.148 CCCGCCGACC'AAATCCCCGA TGCTGACCAA AGGTTGATGT TGGCATTGCT CCAAACCCAC 3208 TTGCTCAAAT CCGCTGGGAT ATTGAAAGGC TTCACCACCT TTGGTTTCTA CCCTG.CTCAA 3328 TGGGAAGGAC AAACCGTCAG AATTGTTTAT TC'rGGTGACA CCATCACCGA CCCATCCATG 3388 TGGTCTAACC CAGCCCTGGC CAAGGCTTGG AgCAAGGCCA TGCAAATTCT=CCACGAGGCT 3448 AGGCCAGAAA AATTATATTG GCTCCTGATT TCTTCCGGCT ATCGCACCTA CCGA7TT'1TG 3508 a e PCi'/US92/08'746 ~ t 9~ ~j ~ 9 I AATT7.".,CATCC ATCAGCTAGC 3588 (2) INFORNMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 amino acids 5(8) TYPE: amino acid (D) TOPOLOGY: linear (ii.} MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Leu Thr Ala Glu Arg Ile Lys Phe Thr G1n Lys Arg Gly Phe Arg !. 5 10 15 Arg Val Leu Asn Gln Arg Vai Asp Ala Tyr Phe Ala Glu His G1y Leu .25 30 Thr Gln Arg Asp Asn Pro Ser Met Tyr Leu Lys Thr Leu Ile Ile Val Leu Trp Leu Phe Ser Ala Trp Ala Phe Val Leu Phe Ala Pro Val Ile Z~ Phe Pro Val Arg Leu Leu Gly Cys Met Val Leu Ala Ile Ala Leu Ala Ala Phe Ser Phe Asn Val Gly His Asp Ala Asn His Asn Ala Tyr Ser Ser Asn Pro His Iie Asn Arg Val Leu Gly Met Thr Tyr Asp Phe Val Gly Leu Ser Ser Phe Leu Trp Arg Tyr Arg His Asn Tyr Leu His His Thr Tyr Thr Asn Ile LeuGly His Asp Val Glu Ile His Gly Asp Gly Ala Val Arg Met Ser Pro Glu Gin Glu His Val Gly Ile Tyr Arg Phe Gin Gln Phe Tyr Ile Trp Giy Leu Tyr Leu. Phe Ile Pro Phe Tyr Trp / \ =
WO 93/06712 2120629 PC'T/US92/08746 1 Phe Leu Tyr Asp Val Tyr Leu Val Leu Asn Lys Gly Lys xyr His Asp 180 185 $90 His Lys Ile Pro Pro Phe Gln Pro Leu Glu Leu Ala Ser Leu Leu Gly Ile Lys Leu Leu Trp Leu Gly Tyr Val Phe Gly Leu Pro Leu Ala Leu Gly Phe Ser Ile Pro Glu Val Leu Ile Gly Ala Ser Val Thr Tyr Met Thr Tyr Gly Ile Val Val Cys Thr Ile Phe Met Leu Ala His Val Leu Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly Glu Ser Gly Ala ]Cle Asp Asp Glu Trp Ala Ile Cys Gln Ile Arg Thr Thr Ala Asn Phe Ala Thr Asn Asn Pro Phe Trp Asn Trp Phe Cys Gly Gly Leu Asn His Gln Val Thr His His Leu Phe Pro Asn Ile Cys His Ile His Tyr Pro Gln Leu Glu Asn Ile Ile Lys Asp ValCys Gin Glu Phe Giy Val Glu Tyr Lys Val Tyr Fro Thr Phe Lys Ala Ala I1e Ala Ser Asn Tyr Arg Trp Leu Glu Ala Met Gly Lys Ala Ser (2) INFOFtMATION FOR SEQ ID NO: 3, (i) SEQUENCE CHARACTERISTICSr (A) LENGTH. 1884 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)SEQUENCE DESCRIPTIaN: SEQ ID NO:3:

W0 93/06712 "g PG'r/L'S92/08746 AGCTTCACTT CGGT1.x'.CATA TTGTGACCAT GGTTCCCAGG CATCTGCTCT AGGGAGTTTT 60 AAAATTCTAG CAATGGACTC CCAGTTGGAA TAAATTrTTA GTCTCCCCCG GCGCTGGAGT 240 2T1'1'TTGTAG TTAATGGCGG TATAATGTGA AAGT'P1"ITTA TCTATTTAAA TTTATAAATG 300 CGGGTGGATG CCTACT'1TGC CGAGCATGGC CTGACCCAAA GGGATAATCC CTCCATGTAT 420 CTGAAAACCC TGATTATTGT GCTCTGGTTG TTTTCCGCTT GGGCCTTTGT GC2T'iTTGCT 480 lo CCAGTTATTT TTCCGGTGCG CCTACTGGGT TGTATGGTTT TGGCGATCGC CTTGGCGGCC 540 1.~'j CAATTITATA TTTGGGGTIT ATATCTTTTC ATTCCCTTTT ATTG'TTTCT CTACGATGTC 840 CTGGCTCTGG GCTTTTCCAT TCCTGGAAGTA TTAATrGGTG CTTCGGTAAC CTATATGACC 1020 20 CTCACCCCCG ATGGTGAATC'CGGTGCCATT GATGACGAGT GGGCTATTTG CCAAATTCGT 1140 ACCACGGCCA A2Ti"rGCCAC CAATAATCCC TTTTGGAACT GGZT'tL'6TGG CGGTTTAAAT 1200 AATATTATTA AGGATGTTTG CCAAGAGTTT GGTGTGGAAT ATAAAGTTTA TCCCACCTTC 1=320 w 93e06712 PGTrYJS32/09746 ~. GT'I'GCCCGCC GACCAAATCC CCGATGCTGA CCAAAGGTTG ATGTTGGCAT TGCTCCAAAC 1500 CATGTGGTCT AACCCAGCCC'TGGCCAAGGC TTGGACCAAG GCCATGCAAA TTCTCCACGA 1740

Claims (47)

WHAT IS CLAIMED IS:

1. An isolated nucleic acid encoding a bacterial .DELTA.6-desaturase, wherein said nucleic acid is selected from:
(a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID
NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).

2. The isolated nucleic acid of Claim 1 comprising the nucleotides of SEQ ID NO: 3.

3. The isolated nucleic acid of Claim 1 or 2, wherein said nucleic acid is contained in a vector.

4. The isolated nucleic acid of Claim 3 operably linked to a promoter and/or a termination signal capable of effecting expression of said isolated nucleic acid.

5. The isolated nucleic acid of Claim 4, wherein said promoter is a .DELTA.6-desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycin promoter, a napin promoter, or a helianthinin tissue-specific promoter.

6. The isolated nucleic acid of Claim 4, wherein said termination signal is a Synechocystis termination signal, a nopaline synthase termination signal, or a seed termination signal.

7. An isolated cell transformed with the nucleic acid of any one of Claims 1-6.

8. The isolated cell of Claim -7, wherein said cell is selected from a bacterial cell, a fungal cell, a plant cell or an animal cell.

9. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises:
(a) transforming a plant cell with the isolated nucleic acid of any one of Claims 1-6; and (b) regenerating a plant with increased GLA
content from said plant cell.

10. The method of Claim 9, wherein said plant is a sunflower, soybean, maize, tobacco, peanut or oil seed rape plant.

11. A method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA which comprises transforming said organism with the isolated nucleic acid of any one of Claims 1-6, wherein said organism is a bacterium or a plant.

12. A method of inducing production of gamma linoleic acid (GLA) in an organism deficient or lacking in GLA and linoleic acid (LA) which comprises transforming said organism with the isolated nucleic acid of any one of Claims 1-6 and an isolated nucleic acid encoding .DELTA.12-desaturase, wherein said organism is a bacterium or a plant.

13. A method of inducing production of gamma linolenic acid (GLA) in an organism deficient or lacking in GLA and linoleic acid (LA) which comprises transforming said organism with at least one expression vector comprising the isolated nucleic acid of any one of Claims 1-6 and an isolated nucleic acid encoding .DELTA.12-desaturase, wherein said organism is a bacterium or a plant.

14. The method of any one of Claims 12 or 13, wherein said isolated nucleic acid encoding .DELTA.6-desaturase comprises nucleotides 316 to 1507 of SEQ ID NO: 1.

15. A method of inducing production of octadeca-tetraeonic acid in an organism deficient or lacking in gamma linolenic acid which comprises transforming said organism with isolated nucleic acid of any one of Claims 1-6, wherein said organism is a bacterium or a plant.

16. An isolated bacterial .DELTA.6-desaturase encoded by a nucleic acid selected from:
(a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID
NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).

17. The isolated bacterial .DELTA.6-desaturase of Claim 16 which has an amino acid sequence of SEQ ID NO:
2.

18. An isolated nucleic acid encoding cyano-bacterial .DELTA.6-desaturase, wherein said isolated nucleic acid is isolatable from a cyanobacteria that produces gamma linolenic acid, and is selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID
NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).

19. An isolated nucleic acid having the sequence of SED ID NO: 3.

20. An isolated nucleic acid encoding a Synechocystis .DELTA.6-desaturase, wherein said nucleic acid is selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID
NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).

21. A vector comprising the nucleic acid of any one of Claims 18 to 20.

22. An expression vector comprising the isolated nucleic acid of any one of Claims 18 to 20 operably linked to a promoter capable of effecting expression of said isolated nucleic acid.

23. An expression vector comprising the isolated nucleic acid of any one Claims 18 to 20 operably linked to a promoter and a termination signal capable of effecting expression of said isolated nucleic acid.

24. The expression vector of claim 22 or 23, wherein said promoter is a .DELTA.6-desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycin promoter, a napin promoter, or a helianthinin tissue-specific promoter.

25. The expression vector of claim 23, wherein said termination signal is a Synechocystis termination signal, a nopaline synthase termination signal, or a seed termination signal.

26. An isolated bacterial or plant cell comprising the vector of any one of Claims 21 to 23.

27. A method of inducing production of gamma linolenic acid (GLA) in a bacteria deficient or lacking in GLA, wherein said bacteria produces linoleic acid which comprises transforming said bacteria with the vector of any one of Claims 21-23.

28. The method of any one of Claims 12 or 13, wherein said isolated nucleic acid encoding .DELTA.6-desaturase comprises nucleotides 2002 to 3081 of SEQ ID NO: 1.

29. A method of inducing production of octadecatetraeonic acid in a bacteria deficient or lacking in gamma linolenic acid, wherein said bacteria produces linoleic acid, which comprises transforming said bacteria with isolated nucleic acid of any one of Claims 18 to 20.

30. A method of inducing production of octadecatetraeonic acid in a bacteria deficient or lacking in gamma linolenic acid, wherein said bacteria produces linolenic acid which comprises transforming said bacteria with vector of any one of Claim 21 to 23.

31. An isolated plant cell or bacterium transformed with at least the isolated nucleic acid of any one of Claims 18 to 20.

32. An isolated plant cell or bacterium transformed with at least the vector of any one of Claims 21 to 25.

33. An isolated plant cell or bacterium transformed with the vector comprising the isolated nucleic acid of Claims 18 to 20, wherein said nucleic acid is operably linked to a producer capable of effecting the desired expression.

34. A plant cell or bacterium transformed with the vector comprising the isolated nucleic acid of Claims 18 to 20, wherein said DNA molecule is operably linked to a promoter and termination signal capable of effecting the desired expression.

35. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises: transforming a plant cell with at least one of the isolated nucleic acid of any one of Claims 18 to 20 and regenerating a plant with increased GLA content from said plant cell.

36. A method of producing a plant with increased gamma linolenic acid (GLA) content which comprises transforming a plant cell with at least a vector comprising the isolated nucleic acid of any one of Claims 18 to 20.

37. The method of Claim 35 or 36, wherein said plant is a sunflower, soybean, maize, tobacco, peanut or oil seed rape plant.

38. A method of inducing producyion of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA which comprises transforming said plant with at least the isolated nucleic acid of any one of Claims 18 to 20.

39. A method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA and linoleic acid (LA) which comprises transforming said plant with the isolated nucleic acid of Claim 18 or 20.

40. A method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA and linoleic acid (LA) which comprises transforming said plant with at least one expression vector comprising the isolated nucleic acid of Claim 18 or 20 and an isolated DNA molecule encoding .DELTA.12-desaturase.

41. The method of any one of Claims 39 or 40 wherein said isolated nucleic acid encoding .DELTA.6-desaturase comprises nucleotides 2002-3081 of SEQ ID NO: 1.

42. A method of inducing production of octadecatetraeonic acid in a plant deficient or lacking in gamma linolenic acid which comprises transforming said plant with at least the isolated nucleic acid of any one Claims 18 to 20.

43. A method of inducing production of octadecatetraeonic acid in a plant deficient or lacking in gamma linolenic acid which comprises transforming said plant with at least a vector comprising the isolated nucleic acid of any one of Claims 18 to 20.

44. A cyanobacterial .DELTA.6-desaturase encoded by a nucleic acid from a cyanobacteria that produces gamma linolenic acid, wherein said nucleic acid is selected from (a) a nucleic acid comprising a nucleotide sequence which encodes the amino acid sequence of SEQ ID
NO: 2, (b) a nucleic acid comprising the nucleotide sequence as set forth in SEQ ID NO: 3, (c) a nucleic acid hybridizing under stringency conditions to the complement of nucleic acid of (b).

45. The cyanobacterial .DELTA.6-desaturase of Claim 44, which has an amino acid sequence of SEQ ID NO: 2.

46. A method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA and linoleic acid (LA) which comprises transforming said plant with the isolated nucleic acid of Claim 19.

47. A method of inducing production of gamma linolenic acid (GLA) in a plant deficient or lacking in GLA and linoleic acid (LA) which comprises transforming said plant with at least one expression vector comprising the isolated nucleic acid of Claim 19 and an isolated DNA
molecule encoding .DELTA.12-desaturase.