Cyanobacterial delta 6-desaturase, nucleic acid encoding the same and process for producing delta 6- desaturase inducing the gama-linolenic acid production

The present invention relates to the enzyme A6-cyanobacterial desaturase, the nucleic acid encoding it and a process for inducing the production of gamma-linolenic acid (GLA), essential fatty acid required in mammalian food and the use of the enzyme A6-desaturase in the production of plants with content. raised by GLA, with frost resistance properties.

It is known that linoleic acid (18: 2) (LA) is converted to gamma-linolenic acid (18: 3) (GLA) by the enzyme A6-desaturase. When this enzyme or the nucleic acid encoding it is transferred into LA-producing cells, GLA is produced.

Unsaturated fatty acids, such as linoleic acids (C₁₈ Δ^9,12 ] and α-linolenic (C18 Δ^{9 12 15} ) are essential dietary constituents that cannot be synthesized by vertebrates, as vertebrate cells can introduce double bonds at the Δ position⁹ of fatty acids, but cannot introduce additional double bonds between the double bond Δ⁹ and the methyl terminal of the fatty acid chain. Since they are precursors 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 γ-linolenic acid (GLA C18 Δ⁶·⁹·¹² ] which, in turn, can be converted to arachidonic acid (20: 4), an important fatty acid in a critical manner as it is an essential precursor to most prostaglandins.

The dietary supply of linoleic acid, due to its resulting conversion to GLA and arachidonic acid, satisfies the dietary requirement for GLA and arachidonic acid. However, a correlation between consumption of saturated fatty acids and health risks, such as hypercholesterolemia, atherosclerosis and other chemical disorders that correlate with susceptibility to coronary heart disease, has been demonstrated, as unsaturated fatty acids have been associated with decreased blood cholesterol concentration and reduced risk of atherosclerosis. The therapeutic benefits of dietary GLA may be due to the fact that GLA is a precursor of arachidonic acid and thus contributes subsequently to prostaglandin synthesis. Accordingly, consumption of GLA, which is more unsaturated, rather than linoleic acid, has potential health benefits. However, the GLA is not actually present in. any commercial crop plant.

Linoleic acid is converted to GLA by the enzyme Δε-desaturase. Δδ-desaturase, an enzyme of approximately 359 amino acids, has the membrane binding domain and the active part for desaturation of fatty acids. When this enzyme is transferred to cells that endogenously produce linoleic acid (LA) and not GLA, GLA is produced.

The problem solved by the invention is the presentation of nucleic acid encoding cyanobacterial A6-desaturase, the amino acid sequence of this enzyme and a method of inducing the production of gamma-linolenic acid in a deficient organism or in which this acid is lacking, by obtaining a recombinant organism contained in the nucleus or nucleic acid encoding the enzyme A6-desaturase.

The nucleic acid encoding the cyanobacterial ΔΒ-desaturase enzyme, according to the invention removes the above disadvantages, by having the nucleotide sequence represented by SEQ. ID N0: 3.

The cyanobacterial Δβ-desaturase according to the invention removes the disadvantages above, by having the amino acid sequence represented by SEQ. ID N0: 2.

The process of producing the enzyme Δ-6 cyanobacterial desaturase induces the production of gamma-linolenic acid (GLA) in a deficient plant or where gamma-linolenic acid (GLA) and linoleic acid (LA) are missing, eliminating the above disadvantages, in that it comprises introducing isolated DNA sequences encoding cyanobacterial Δβ-desaturase and DNA sequences encoding Δ12-desaturase into a bacterial vectorRO 113256 Bl riofag or cosmid containing sequences required for integration of foreign DNA, transferring the vector containing sequences DNA was said in an organism by known techniques.

The present invention provides a nucleic acid comprising Δβ-desaturase genes. More specifically, the nucleic acid comprises the promoter, the coding region and the end regions of the Δ6 desaturase gene. The present invention is further directed to recombinant constructs comprising a coding region of ΔΒ-desaturase in functional combination with heterologous regulatory sequences. The nucleic acids and recombinant constructs of the present invention are usable in the production of GLA in transgenic organisms.

The present invention, by providing the gene encoding Δβ-desaturase, allows the production of transgenic organisms containing functional ΔΒ-desaturase and which produces GLA. In addition to allowing large quantities of GLA to be produced, the present invention provides for new sources of GLA diet.

The present invention is directed to an isolated gene of Δβ-desaturase. Specifically the isolated gene comprises the Δθ-desaturase promoter, the coding region and the end regions.

The present invention is further directed to expression vectors comprising the Δ6 desaturase promoter, the coding region and the end regions.

The present invention is also directed to expression vectors comprising Δβ-desaturase, the coding region of Δθ-desaturase in functional combination with heterologous regulatory sequences, respectively elements that are not derived from ΔΒ-desaturase genes.

The cells and organisms comprising the vectors of the present invention and the progeny of such organisms are also provided for in the present invention.

The present invention further provides isolated bacterial ΔΒ-desaturase and is further directed to an isolated nucleic acid encoding bacterial Δθ-desaturase.

The present invention further provides a process for producing plants with high gamma-linolenic acid (GLA) content, which comprises transforming the plant cell with nucleic acid isolated from the present invention and regenerating the plant with increased GLA content from the plant cell.

A process for producing the tolerance to cooling is also provided by the present invention.

The present invention provides an isolated nucleic acid encoding ΔΒ-desaturase. To identify the nucleic acid encoding ΔΒ-desaturase, the DNA is isolated from an organism that produces GLA. The so-called organism can be, for example, an animal cell, certain fungi (eg Mortierella), certain bacteria (eg Synechocystis) or certain plants (lamb tongue, Oenothera, currant). Genomic DNA isolation can be accomplished by a variety of methods well known to one skilled in the art, as exemplified by Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY Isolated DNA is fragmented by physical methods or enzymatic digestion and cloned into a suitable vector, for example a bacteriophage or cosmid vector, by any of a variety of well-known methods that can be found in references such as Sambrook et al. (1989) . Expression vectors containing the DNA of the present invention are specifically examined here. DNA encoding Δβ-desaturase can be identified by taking advantage of functional analysis. The vector containing fragmented DNA is transferred, for example by infection, transconjugation, transfection into a host organism that produces linoleic acid, but not GLA. As used herein, the term "transformation" generally refers to the incorporation of foreign DNA into the host cell. Methods for introducing the combined DNA reRO 113256 Bl into a host organism are known to one skilled in the art and can be found, for example, in Sambrook et al. (1989). The production of GLA by these organisms (respectively gain of function) is tested for example by gas, chromatography or other methods known to those skilled in the art. Organisms that are induced to produce GLA, respectively have gained function by introducing the vector, are identified as expressing DNA encoding A6-desaturase, and the so-called DNA is recovered from organisms. The recovered DNA can again be fragmented, cloned with expression vectors and functionally tested through the above procedures to define with more particularity the DNA encoding A6-desaturase.

A random DNA is isolated from the cyanobacteria Synechocystis Pasteur Culture Collection (POC] 6803, American Type Culture Collection (ATCC) 27184, cloned into a cosmid vector and introduced by cross-conjugation into a GLA-deficient Anabaena cyanobacteria species, PCC 7120, ATCC 27893. The production of GLA from Anabaena linoleic acid is tracked by gas chromatography, and the corresponding DNA fragment is isolated.

Isolated DNA is sequenced by methods well known to one skilled in the art, as found for example in Sambrook et al. (1989).

In accordance with the present invention, a DNA containing A6-desaturase genes was isolated. More specifically, a 3,588-kilobase (kb) DNA containing A6-desaturase genes was isolated from the cyanobacterium Synechocystis. The 3,588-kb nucleotide sequence of DNA was determined and shown in SEQ ID NO: 1. define potential coding regions are present from nucleotide 317 to 1507 and from nucleotide 2002 to 3081. To define the nucleotides responsible for encoding A6-desaturase, the 3,588-kb fragment conferring A6-desaturase activity is split into two sub-fragments, each containing only an open interpreted structure. The ORF fragment 1 contains nucleotides 1 to 1704, while the CRF fragment 2 contains nucleotides 1705 to 3588. Each fragment is subcloned in both directions, front and reverse, into a conjugated expression vector (AM542, Wolk and others, 1984, Proc. Natl. - Acad. Sci. USA 81, 1561], which contains the cyanobacterial promoter of carboxylase. The resulting constructs - respectively ORF 1 (F], ORF 1 [R], ORF 2 (F] and ORF 2 (R], are conjugated to the wild type of Anabaena PCC 7120 by standard methods (see, for example, Wolk and others, 1984, Proc. Natl. - Acad. Sci. USA 81, 1561], cells conjugated by Anabaena are identified as Neo green colonies^R on a dark brown background of non-conjugated cells, dead after two weeks of growth in a selective medium (standard mineral medium BG11N + containing 30 µg / ml of neomycin according to Rippka et al., 1979, J. Gen. Microbial 111, 1). The green colonies are selected and grown in a selective liquid medium (BG111N + with 15 µg / ml neomycin). Lipids are extracted by standard methods (e.g. Dahmer et al., 1989, Joural of American Oii Chemical Society 66, 543] from the resulting transconjugates containing the ORF 1 and ORF 2 constructs facing forward or reverse. For comparison lipids are also extracted from cultures wild type of Anabaena and Synechocystis. Fatty acid methyl esters are examined by liquid gas chromatography (GLC), for example with a Tracor 560 gas-liquid chromatograph equipped with a hydrogen flame ionization detector and capillary column. The results of the GLC analysis are shown in Table 1.

RO 113256 Bl

8

Table 1

Manifestation of fatty acids C_1P In wild-type or transgenic cyanobacteria

<td>SOURCE</td><td> 18 ; 0</td><td> 18 : 1</td><td> 18 : 2</td><td>γ-1 8: 3</td><td>α-1 8: 3</td><td> 18 ; 4</td>

<td>Anabaena (the wild type)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td>Anabaena + ORF 1 (F)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td>Anabaena + ORF 1 (R)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td>Anabaena + ORF 2 (F)</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>Anabaena + ORF 2 (R)</td><td> +</td><td> +</td><td> +</td><td> -</td><td> +</td><td> -</td>

<td>Synechocystis (wild type)</td><td></td><td> +</td><td> +</td><td> +</td><td> -</td><td> -</td>

As estimated by the analysis of 20 GLC, Anabaena with deficient GLA acquires the production function of GLA when the construction contains ORF 2 in face orientation and is introduced by transconjugation. Transconjugants containing 25 trophies with ORF 2 in reverse orientation to the carboxylase promoter or ORF 1 in either orientation do not indicate GLA production. This analysis demonstrates that the simple interpreted open structure (ORF 2) in the 1884 bp fragment encodes A6-desaturase. The fragment 1884 bp is shown as SEQ. ID NO: 3. This is substantiated by the general similarity of the hydropathic profiles 35 between A6-desaturase and Δ12-desaturase (Wada et al., 1990, Nature 347), as shown in Fig. 1, as (A) and respectively ( B).

Isolated nucleic acids encoding 40 Δδ-desaturase can be identified from other organisms that produce GI_A by the gain-of-function analysis described above or by nucleic acid hybridization techniques using isolated nucleic acid encoding Anabaena A6-desaturase as a sample. hybridization. Both methods, genomics and DNA donation, are known to those skilled in the art and are examined in the present invention. The hybridization sample may comprise the entire DNA sequence disclosed as SEQ. ID NO: 1 or a restrictive fragment or other DNA fragment thereof, an oligonucleotide sample. Methods for cloning homologous genes by cross-hybridization are known to those skilled in the art and can be found, for example, in Sambrook et al. (1989) and Beltz et al. (1983) Methods in Enzymology 100, 266.

Transgenic organisms that gain the function of GLA production by introducing DNA encoding Δθ-desaturase also gain the function of producing octadecatetraenoic acid (18: Δ^{6, 9 12 15} ). Octadecatetraenoic acid is normally present in fish oils and in some plant species in the Boraginaceae family (Craig et al., 1964, J. Amer. Oii Chem. 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 subsequent desaturation of α-linolenic acid by the aid of A6-desaturase or as a result of GLA desaturation in the presence of Δ15 desaturase.

The 359 amino acids encoded by ORF 2, respectively the open interpreted structures encoding A6-desaturase, are shown as SEQ sequences. ID NO: 2. The present invention further examines other nucleotide sequences encoding amino acids of the SEQ. ID NO: 2. It is in the field of knowledge of the specialists in the field of a

RO 113256 Bl identifies such sequences that result, for example, as a result of genetic code degeneration. Moreover, one who is skilled in the art can determine, by analyzing the gain of function described above, the smaller subfragments of the 1884 bp fragment containing ORF 2 encoding A6-desaturase.

The present invention examines any such fragment of polypeptides of A6-desaturase and nucleic acids as a result that retain the activity of converting LA into GLA.

With respect to a further aspect of the present invention, a vector containing the 1884 bp fragment or a smaller fragment containing the promoter, the coding sequence and the terminal region of the Δ6 desaturase is transferred to an organism, for example, cyanobacteria, wherein the A6-desaturase promoter. and the terminal regions are functional. Accordingly, organisms producing recombinant A6-desaturase are provided in this invention. Still another aspect of the present invention provides isolated A6-desaturase, which can be purified from recombinant organisms by standard methods of protein purification (for example, see Ausubel et al., 1987, Current Protocols in Molecular Biology, Green Publishing Associates, New York ).

The vectors containing DNA encoding A6-desaturase are also provided in the present invention. It is obvious to those skilled in the art that the appropriate vectors can be constructed to guide the expression of the A6-desaturase coding sequence in a variety of organisms. Replicable expression vectors are especially preferred. The replicable expression vectors as described herein are DNA or RNA molecules designed for controlled expression of the desired gene, respectively of the A6-desaturase gene. It is preferable that the vectors be plasmids, bacteriophages, cosmids or viruses. Shuttle vectors, such as described by Wolk et al., 1984, Proc. Natl.

Acad. Sci. US, 1561-1565 and Bustos et al., 1991, J. Bacteriol., 174, 75257533 are also examined in accordance with the present invention. Sambrok et al., 1989, Goeddel ed., 1990, Methods in Enzimology 185 Academic Press and Perbal, 1988, A Practical Guide to Molecular Cloning, John Wiley and sons, Inc., provide a detailed overview of vectors in which nucleic acid is present. which encodes this A6-desaturase can be introduced and expressed. Such vectors also contain nucleic acid sequences that can effect the expression of nucleic acids encoding A6-desaturase. Sequence elements capable of expressing a gene product include promoters, acceleration elements, anti-activation sequences, transcription termination signals, and polyadenylation moieties. Both specific, constitutive and tissue promoters are examined. For the transformation of plant cells, the mosaic virus of the flowering sow (Cauliflower -Ca MV) promoter 35S and the promoters that are adapted during the maturation of the planting seed are of particular interest. All of these promoters and transcriptional adaptation elements, alone or in combination, are examined for use in the present replicable expression of vectors and are known to those skilled in the art. The promoter Ca MV 35S is described, for example, by Restrepo et al., 1990, Plant Cell 2, 987. Genetically engineered and mutated, adaptation sequences are also examined.

Specialists in this field can determine the vectors and regulatory elements corresponding to expression in a particular host cell. For example, the promoter-containing vector in the carboxylase-encoding gene of Anabaena is operably linked to the coding region of A6-desaturase and subsequently operably linked to the terminal signal in Synechocystis is suitable for the expression of A6-desaturase in cyanobacteria. . "Operably linked" in this context means that the promoter and

The terminal sequences function effectively to regulate transcription. As a further example, the vector suitable for expression of A6-desaturase in transgenic plants may comprise a seed-specific promoter sequence derived from heliantinine, napine, or glycine operably linked to the coding region of A6-desaturase and further operably linked. a signal for termination of seeding or of the end signal of nopaline synthetase.

In particular, the regulatory elements of heliantinin disclosed in U.S. Patent No. 682354, filed April 8, 1991 and incorporated herein by reference, are examined as promoter elements to guide the expression of A6-desaturase of the present invention.

Modifications of the nucleotide sequences or regulatory elements disclosed herein, which maintain the functions examined herein, are within the scope of this invention. Such changes include insertions, substitutions and deletions, but specifically substitutions that reflect the degeneracy of the genetic code.

Standard techniques for constructing such hybrid vectors are well known to those skilled in the art and can be found in the incorporated references, such as Sambrok et al. (1989), or in any of the large number of recombinant DNA technology laboratory manuals that is available in a wide field. A variety of strategies are available for ligated DNA fragments, the choice of which depends on the terminal nature of the DNA fragments. They are further examined in accordance with the present invention for including in the hybrid vectors other elements of the nucleotide sequence that facilitate donation, expression or processing, for example of the sequences encoding peptide signal, the sequence encoding KDEL, which is required for protein retention in the lattice. endoplasmic or sequences encoding transit peptides that target A6-desaturase to chloroplast.

Such sequences are known to those skilled in the art. A peptide-optimized transit is described, for example, by Van den Broeck et al. (1985) Nature 313, 558. The prokaryotic and eukaryotic signal sequences are disclosed, for example, by Michaelis et al. (1982) Ann. Rev. Microbiol. 36, 425.

Another aspect of the present invention provides organisms, other than cyanobacteria, that contain DNA encoding A6-desaturase of the present invention. The transgenic organisms examined according to the present invention include bacteria, cyanobacteria, fungi and plants and animals. DNA isolated from the present invention may be introduced into a host by methods known to those skilled in the art, for example, infection, transfection, transformation, or conjugation. Techniques for transferring DNA from the present invention to such organisms are widely known and are provided in references such as Sambrok et al. (1989).

A variety of plant transformation methods are known. The A6 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 transformation methods such as protoplast culture (Horsch) et al., 1984, Science 223, 496; DeBlock et al., 1984, EMBOJ. 2, 2143; Barton et al., 1983, Cell 32, 1033) may also be used and are within the scope of this invention. In a preferred embodiment, the plants are transformed with vectors derived from Agrobacterium. However, other methods are available to introduce the A6-desaturase gene of the present invention into plant cells. Such alternative methods include biolistic interpretation (Klein et al., 1987, Nature 327, 70), electroporation, chemically induced DNA uptake, and the use of viruses or pollen as vectors.

When required for the transformation method, the A6 gene desaturates

RO 113256 Bl of the present invention may be introduced into a plant transformation vector, for example, the binary vector described by Bevan (1984) Nucleic Acids Res. 12, 8111. Plant transformation vectors can be derived by modifying the natural gene as a transfer system of Agrobacterium tumefaciens. The natural system comprises large Ti (tumor-inducing) plasmids containing a large segment, known as T-DNA, which is transferred to transformed plants. Another segment of the plasmid Ti, the vir region, is responsible for T-DNA transfer. The TADN region is bounded by terminal replication, in the modified binary vectors the tumor induction genes have been eliminated, and the functions of the vir region are used to transfer foreign DNA bounded by the T-DNA border sequences. The T regions also contain a selectable indicator on antibiotic resistance and a multiple donation position for insertion of transfer sequences. Such projected species are known as "disarmed" A. tumefaciens species and allow an efficient transformation of the T-region sequences into nuclear genomes of plants.

Surface sterilized leaf disks are inoculated with foreign "disarmed" DNA containing A. tumefaciens, grown in culture medium for two days and then transferred to the antibiotic-containing environment. The transformed shoots are selected after rooting in the environment containing the appropriate antibiotic transferred to the soil and regenerated.

Another aspect of the present invention provides transgenic plants or offspring of these plants containing DNA isolated from the present invention. Both monocotyledonous and dicotyledonous plants are examined. Plant cells are transformed with isolated DNA encoding A6-desaturase, by any of the plant transformation methods described above. The transformed plant cell, usually in a leaf node or leaf culture, is regenerated into a completely transgenic plant by methods well known to those skilled in the art (e.g. Horsch et al., 1985, Science 227, 1129 ). In a preferred embodiment, the transgenic plant is sunflower, rapeseed for oil seed, corn, tobacco, peanuts or soy. As the offspring of transformed plants inherit DNA encoding A6-desaturase, seeds or cuttings of transformed plants are used to maintain the transgenic line of the plant.

The present invention further provides a method for the production of transgenic plants with a high GLA content. This method includes introducing DNA encoding A6-desaturase into plant cells where GLA is lacking or only low, but containing LA, as well as regenerating plants with increased GLA content in transgenic cells. In particular, plants that are grown for harvest as a transgenic organism are examined, including, but not limited to, sunflower, soybean, oil seed rape, corn, peanuts and tobacco.

The present invention further provides a process for providing transgenic organisms containing GLA. This method comprises introducing DNA encoding A6-desaturase into an organism that is lacking or has low GLA levels but contains LA.

In another embodiment of the invention, the method comprises introducing one or more expression vectors containing DNA encoding A6-desaturase in organisms that are deficient in both GLA and LA. Correspondingly, organisms that are deficient in both GLA and LA are induced to produce LA by the expression of Al2-desaturase, and GLA is then generated as a result of the expression of A6-desaturase. Expression vectors comprising DNA encoding Δ12-desaturase and Δ6 desaturase can be constructed by methods of known recombinant technology.

RO 113256 Bl to those working in the art (Sambrok et al., 1989) and published sequence of Δ12-desaturase (Wada et al., 1990, Nature [London] 347, 2DO-2D3). Additionally, it has been found in accordance with the present invention that nucleotides 2003-3081 of SEQ. ID N0: 1 encodes cyanobacterial Al2-desaturase. Suitably, this sequence can be used to construct the expression subject vector. In particular, plants cultivated commercially for harvesting as transgenic organisms are examined, including but not limited to sunflower, soybean, oilseed rape, corn, peanuts and tobacco.

The present invention is further directed to a method of inducing frost tolerance of plants. Frost sensitivity may be due to the lipid transition phase in cell membranes. The temperature of the transition phase depends on the degree of unsaturation of the fatty acids in the cell membrane and in this way by increasing the degree of unsaturation, for example by introducing Δ6desaturase to convert LA to GLA, it can induce or improve the frost resistance. Suitably, the present method comprises introducing DNA encoding Δθ-desaturase into a plant cell, as well as regenerating the plant with improved frost resistance from said transformed plant cell. In a preferred embodiment, the plant is sunflower, soybean, beans, oilseed rape, corn, peanuts or tobacco.

The present invention has the following advantages:

- Providing a diet with high gamma-linolenic acid (GLA) content, more unsaturated than linoleic acid (LA). GLA consumption, rather than LA, has potential health benefits, being associated with lower cholesterol levels, reduced risk of atherosclerosis and other chemical disorders, leading to decreased susceptibility to coronary heart disease. In addition, the therapeutic benefits of dietary GLA may result from the fact that GLA is a precursor of arachidonic acid and thus subsequently contributes to prostaglandin synthesis;

- the production of recombinant plants with high GLA content for commercial culture;

- the production of recombinant plants with induced or improved frost resistance by increasing the GLA content in the membranes of the plant cells.

The following are 6 examples of embodiments of the invention, according to FIG. 1-? 4, which represents:

FIG. 1, the hydropathic profiles of the deduced amino acid sequences of Synechocystis A6-desaturase (Diagram A) and Δ12-desaturase (Diagram B). The opening regions of the putative membrane are indicated by compact lines. The hydrophobic index was calculated for a window size of 19 amino acid residues (Kyte et al., 1982, J. Molec. Biol. 157];

FIG. 2, the profiles of gas-liquid chromatography of the calbatic (Diagram A) and transgenic (Diagram B) type of Anabaena;

FIG. 3, the map of the cosmid maps cSy75, cSy13 and cSy7 with overlapping and subclonal regions. The origin of the subclonal regions of cSy75 cSy75-3.5 and cSy7 are indicated by dashed diagonal lines. The restrictive parts that have been inactivated are in brackets;

FIG. 4 gas chromatography profiles of wild type (Diagram A) and transgenic (Diagram B) tobacco.

The following examples further illustrate aspects of the present invention.

Example 1. Species and culture conditions

Synechocystis (PCC 6803, ATCC 27184), Anabaena (PCC 7120, ATCC 27893), and 5ynechococus (PCC 7942, ATCC 33912) are grown photoautotrophic at 30 ° C in BG11N + medium (Rippka et al., 1979, J. Gen. Microbiol. 111, 1-61) under the illumination of incandescent lamps (60 pE. M '². s'¹). Cosmids and plasmids are selected and propagated in Escherichia coli species

RO 113256 Bl □ H5a on LB medium supplemented with antibiotics at standard concentrations as described by Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, New York, USA.

Example 2. Synechocystis Cosmid Genomic library construction

Total genomic DNA from Synechocystis (PCC 6803) is partially digested with Sau3A and fractionated on a sucrose gradient (Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York). Fractions containing 30 to 40 kb DNA fragments are selected and ligated into a dephosphorylated BamHI portion of the cosmid vector, pDUCA7 [Buikema et al., 1991, J. Bacteriol. 173, 1879-1885). Binding DNA is packaged in vitro as described by Ausubel et al. (1987), and the packaged phage is propagated in E. coli DH5a containing the methylase helper plasmid Aval and Eco 4711, pRL528 as described by Buikema et al. (1991). A total of 1152 colonies are isolated at random and individually maintained in 12 microtiter plates with 96 network.

Example 3. The expression of gain of function of the GLA In Anabaena

Anabaena [PCC 7120) is a filamentous cyanobacterium deficient in GLA, but contains significant amounts of linoleic acid (LA), a precursor for GLA (Fig. 2; Table 2). The library of Synechocystis cosmid described in example 2 is conjugated in Anabaena (CCP 7120) to identify the transconjugants that produce GLA. Anabaena cells are grown in the mid-log phase in BG11N + liquid medium and resuspended in the same medium to a final concentration of approximately 2 x 10⁸ cells per ml. A culture in the "mid-log" phase of E.coli RP4 (Burkardt et al., 1979, J. Gen. Microbiol. 114, 341-348) grown in LB containing ampicillin is washed and resuspended in fresh LB medium. Anabaena and RP4 are then mixed and spread evenly on BG11N + plates containing 5% LB. The genomic cosmid library is replicated on LB plates containing 50 pg / ml kanamycin and 17.5 pg / ml chloramphenicol and is further applied on BG11N + plates containing Anabaena and RP4. After 24 hours incubation at 30 ° C, 30 µg / ml neomycin is placed below. Incubation at 30 ° C is continued until the transconjugants appear.

The individual transconjugants are isolated after conjugation and grown in 2 ml BG11N + liquid medium with 15 µg / ml neomycin. Fatty acid methyl esters are prepared from wild type cultures and cultures containing ten transconjugant pools, as follows. The wild type and transgenic cyanobacterial cultures are harvested by centrifugation and washed twice with distilled water. Fatty acid methyl esters are extracted from these cultures, as described by Dahmer et al. (1989) J. Amer. Sheep Chem. Shock. 66, 543-548 and are analyzed by gas-liquid chromatography (GLC) using a Tracor-560 equipped with a hydrogen flame ionization detector as well as the capillary column (30 mx 0.25 mm linked to FSOT Superox II, Altech Associates Inc., IL). Retention times and standard co-chromatography (obtained from Sigma Chemical Co.) are used to identify fatty acids. The average fatty acid composition is determined as the ratio of the surface area of ​​each C 18 fatty acid normalized to an internal standard.

Representative GLC profiles are shown in FIG. 2. C 18 fatty acid methyl esters are shown. The peaks are identified by comparing elution times with known standards of fatty acid methyl esters and are confirmed by gas chromatography-mass spectrometry. Diagram A depicts the analyzes of gas-liquid chromatography of wild-type Anabaena fatty acids. The arrow indicates the migration time of gamma-linolenic acid. Diagram B is a gas-liquid profile chromatography of fatty acids from Anabaena transconjugants with pAM542 + 1.8F. Two pools producing acid

RO 113256 Gamma-linolenic acid (25 basins representing 250 transconjugants) are identified as producing gamma-linolenic acid. The individual transconjugants of each positive gamma-linolenic acid basin are analyzed in 5 regarding the production of gamma-linolenic acid; two independent transconjugants; , AS13 and AS75, one from each basin, are identified as expressing significant levels of gamma-linolenic acid and they contained cosmids, cSy13 and cSy75 respectively (Fig. 3). The cosmids overlap in a region of approx

7.5 kb in length. A 3.5 kb Nhel fragment of cSy75 is cloned into vector 15 pDUCA7 and transferred to Anabaena resulting in a gain-of-function expression of the gamma-linolenic wall (Table 2).

Two NhelcHind III subfragments (1.8 and 1.7 kb) of the 3.5 kb fragment 20 Nhel of cSy75 - 3.5 are subcloned into "pBLUSCRIPT" (Stratagene] (Fig. 3) for sequencing. Standard molecular biology techniques are used as described by Maniatis et al. 25 (1982) and Ausubel et al. (1987). Dideoxy sequencing is performed (Sânger et al., 1977, Proc. Natl. Acad. Sci. USA 74, 5464-5467) of pBS1.8 with "SEQUENASE" (United States Bioche- mical) on both sides using specific oligonucleotide primers synthesized by Advanced DNA Technologies Laboratory (Biology Department, Texas A and M University). DNA sequence analysis is performed with gas-liquid chromatography (Madison, WI) software, as often read by Devereux et al. (1984) Nucleic Acids Pes. 12, 387-396.

Both NhelcHind III subfragments are transferred into a conjugal expression vector, AM542, in both forward and reverse directions relative to a cyanobacterial carboxylase promoter and introduced into Anabaena by conjugation. Transconjugants containing 1.8 kb in the face orientation (AM5421,8F) produce significant amounts of gamma-linolenic acid and octadecatetraenoic acid (Fig. 2; Table 2). Transconjugates containing other constructs, either 1.8-kb reverse or front-facing and 1.7-kb reverse, do not produce detectable levels of gamma-linolenic acid (Table 2).

Fig. 2 compares the C 18 fatty acid profile of an extract of the wild type Anabaena (Figure 2A) with that of the transgenic Anabaena containing 1.8 kb fragment of cSy75-3.5 in face orientation (Fig. 2B). Gas-liquid chromatographic analysis of AM542-1.8F fatty acid methyl esters indicates a peak with a retention time identical to that of standard gamma-linolenic acid. Analysis of this peak by gas chromatography-mass spectrometry (GC-MS) confirms that it has the same type of mass fragmentation as the gamma-linolenic acid reference sample. Transgenic duckweed with modified levels of polyunsaturated fatty acids is similar to the wild type in terms of growth rate and morphology.

Table 2

Composition of fatty acids C 18 In wild-type and transgenic cyanobacteria

<td>SPECIES</td><td> 18 : 0</td><td> 18 : 1</td><td>ACID 18: 2</td><td>FAT% a-18: 3</td><td>γ-18: 3 18: 4</td>

<td colspan="6">THE WILD TYPE</td>

<td>Synechocystis (PCC sp. 6803)</td><td> 13,6</td><td> 4,5</td><td> 54,5</td><td> -</td><td> 27,3</td>

<td>Anabaena (PCC sp. 7120)</td><td> 2,9</td><td> 24,8</td><td> 37,1</td><td> 35,2</td><td> -</td>

<td>Synechococcus (PCC sp. 7942)</td><td> 20,6</td><td> 79,4</td><td> -</td><td> -</td><td> -</td>

<td colspan="6">TRANSCONJUGANȚI Anabaena</td>

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Table 2 [continued]

<td>cSy 75</td><td> 3,8</td><td> 24,4</td><td> 22,3</td><td> 9,1</td><td> 27,9</td><td> 12,5</td>

<td>cSy 75-3.5</td><td> 4,3</td><td> 27,6</td><td> 18,1</td><td> 3,2</td><td> 40,4</td><td> 6,4</td>

<td>pAM 542-1,8F</td><td> 4,2</td><td> 13,9</td><td> 12,1</td><td> 19,1</td><td> 25,4</td><td> 25,4</td>

<td>pAM 542-1,8R</td><td> 7,7</td><td> 23,1</td><td> 38,4</td><td> 30,8</td><td> -</td><td> -</td>

<td>pAM 542-1,7F</td><td> 2,8</td><td> 27,8</td><td> 36,1</td><td> 33,3</td><td> -</td><td> -</td>

<td>pAM 542-1,7R</td><td> 2,8</td><td> 25,4</td><td> 42,3</td><td> 29,6</td><td> -</td><td> -</td>

<td colspan="7">TRANSFORM Synechococcusâ</td>

<td>pAM854</td><td> 27,8</td><td> 72,2</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>pAM854 -Δ¹²</td><td> 4,0</td><td> 43,2</td><td> 46,0</td><td> -</td><td> -</td><td> -</td>

<td>pAM854 -Δ⁶</td><td> 18,2</td><td> 81,8</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td>pAM854 -Δ⁶ and Δ¹²</td><td> 42,7</td><td> 25,3</td><td> 19,5</td><td> -</td><td> 16,5</td><td> -</td>

Note: 18: 0, stearic acid; 18: 1, oleic acid; 18: 2, linoleic acid; 18: 3 (a), «-linolenic acid; 18: 3 (γ), γ-linolenic acid; 18: 4, octadecatetraenoic acid.

Example 4. Transformation of Sy- 20 nechococcus with Δ6 and Δ12 desaturase

A third cosmid, cSy7, containing a Δ12-desaturase gene is isolated by screening the Synechococcus 25 genomic library with an oligonucleotide synthesized from the published Al2-desaturase gene sequence Synechocystis (Wada et al., 1990, Nature [London] 347, 200203 ). A 1.7 kb downstream fragment of this cosmid containing Al2-desaturase genes is identified and used as a sample to demonstrate that cSy13 contains not only A6-desaturase genes but also Δ12-desaturase genes (Fig. 3). Genomic Southern blot analysis further shows that both Δθ-desaturase and Δ12-desaturase genes are unique in the Synechocystis genome, so that both functional genes that are involved in C 18 fatty acid desaturation are closely linked in the Synechocystis genome.

Synechococcus unicellular cyanobacteria (PCC 7942) is deficient in both linoleic acid and 45 in gamma-linolenic acid (3). The Δ12-desaturase and Δθ-desaturase genes are cloned individually and together in pAM854 (Bustos et al., 1991, J. Bacteriol. 174, 7525-7533), 50 an alternative vector containing the sequences required for foreign DNA integration into the Synechococcus genome (Golden et al., 1987, Methods in Enzymol.

153, 215-231). Synechococcus is transformed with these gene constructs and colonies are selected. Fatty acid methyl esters are extracted from transgenic Synechococcus and analyzed by gas-liquid chromatography.

Table 2 shows that the main fatty acids of the wild type Synechococcus are stearic acid (18: O) and oleic acid (18: 1). Synechococcus transformed with ΡΑΜ854-Δ12 express linoleic acid (18: 2) in addition to major fatty acids. Transformers with ρΑΜ854-Δ6 and Δ12 both produce linoleate and gamma-linolenic acid (Table 2). These results indicate that Synechococcus containing both Δ12-desaturase and A6-desaturase genes gained the ability to introduce a second double bond at the Δ12 position and a third double bond at the Δ6 position of the C 18. fatty acids. observed changes in the fatty acid composition in the transformant containing ρΑΜ854-Δ6, indicating that in the absence of the substrate synthesized by Δ12-desaturase, A6-desaturase is inactive. This experiment further confirms that the 1.8 kb NhekHind III fragment (Fig. 3) contains both coding and promoter regions of the A6-desaturase gene in Synechocystis. Transgenic synechococcus with modified levels of polyunsaturated fatty acids is similar to the wild type in terms of growth rate and morphology.

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Example 5. Nucleotide sequence of A6-desaturase

The nucleotide sequence of the 1.8 kb fragment of cSy75-3.5 including the functional gene of A6-desaturase is determined. An open structure of amino acid polypeptide 359 is identified (Fig. 4). Hydropathic KyteDoolittle analysis (Kyte et al., 1982, J. Mol. Biol. 157, 105-132) identified two regions of hydrophobic amino acids that may represent transmembrane domains (fig. 1 A); moreover, the hydropathic profile of Δθ-desaturase is similar to that of the gene of Δ12-desaturase (Fig. 1B) (Wada et al.) and A9-desaturase (Thiede et al., 1986, J. Biol. Chem. 261, 13230 -13 235). However, the sequence similarity between Synechocystis Δ6 desaturase and Δ12-desaturase is less than 40% at the nucleotide level and about 18% at the amino acid level.

Example 6. Transfer of A6-cyanobacterial desaturase into tobacco

The cyanobacterial A6-desaturase gene is mobilized into a plant expression vector and transferred to tobacco using Agrobacterium-mediated gene transfer techniques. To ensure that the transferred desaturase is properly expressed in the developing leaves and seeds and that the desaturase gene product is targeted for the endoplasmic reticulum or chloroplast, different expression cassettes with Synechocystis Δδ-desaturase open-interpreting structures (ORF) are constructed. The components of these boxes include; (i) a 35S promoter or seed-specific promoter derived from the sunflower gene heliantinin to drive the expression of the A6-desaturase gene in all plant tissues or only in seeds that develop; (ii) a putative peptide signal from either the carrot extension gene or the sunflower heliantinin gene targeting a novel Δθ-desaturase synthesized in ER; (iii) a lumen ER (KDEL) retention signal sequence at the COOH- terminus of A6-ORF desaturase; and (iv) a transit peptide optimized to introduce Δδ-desaturase into chloroplast. The 35S promoter is a derivative of pRTL2 described by Restrepo et al. (1990). The optimized transit peptide sequence is described by Van den Broeck et al. (1985). The carrot peptide extension signal is described by Chen and others (1985) EMBOJ. 9.2145.

Transgenic tobacco plants are produced containing a chimeric cyanobacterial desaturase gene encompassed by the Synechocystis Δθ-desaturase gene fused to an endoplasmic reticulum retention sequence (KDEL) and an extensive CaMV 35S promoter-driven peptide signal. PCR amplifications of transgenic tobacco genomic DNA show that Δ6-deΞaturase genes are incorporated into the tobacco genome. The fatty acid methyl esters of the leaves of these transgenic tobacco plants are extracted and analyzed by gas-liquid chromatography (GLC). This transgenic tobacco accumulates significant amounts of gamma-linolenic acid (Fig. 4). Fig. 4 depicts the fatty acid methyl esters as determined by gas-liquid chromatography. The peaks are identified by comparing the elution times with known standards of fatty acid methyl esters. Correspondingly, the cyanobacterial genes involved in fatty acid metabolism can be used to generate transgenic plants with modified fatty acid compositions.

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