JP7852909B2 - Proteins, genes, vectors, fish, methods for producing unsaturated fatty acids, and methods for producing fish - Google Patents

Description

This invention relates to proteins and their uses. More specifically, this invention relates to proteins, genes, vectors, fish, methods for producing unsaturated fatty acids, and methods for producing fish.

Docosahexaenoic acid (DHA) has important physiological activity for the development and maintenance of animal nerve tissue, and is known as an essential fatty acid, especially in marine fish. Many marine fish cannot synthesize DHA themselves and therefore depend on obtaining it from their feed.
When farming these marine fish, it is necessary to add fish oil rich in DHA as a source of lipids to their feed. However, problems have been pointed out regarding this fish oil, including shortages of the main raw material, anchovies, soaring prices due to increased demand, and the risk of bioaccumulation of marine pollutants.

To address these issues, vegetable oils are attracting attention as an alternative source of fats and oils. They have the advantage of being able to be supplied stably and in large quantities, as well as eliminating the possibility of bioaccumulation of pollutants.
However, it has a major drawback: it contains absolutely no DHA, an essential fatty acid for marine fish. If vegetable oil is used as the soybean source, survival and growth will be significantly impaired.

DHA is synthesized stepwise, starting with alpha-linolenic acid, which is abundant in vegetable oils. This process involves desaturating enzymes that introduce double bonds into the carbon chains of fatty acids, and chain elongating enzymes that extend the carbon chains.

However, the desaturating enzymes in many marine fish possess only Δ6 desaturation activity (hereinafter also referred to as Δ6 activity), and since the reactions beyond Δ5 desaturation do not proceed, they cannot synthesize DHA.

Therefore, the inventor has been conducting research to create a new type of marine fish that can be raised using only vegetable oil as a source of fat by improving the DHA synthesis pathway of marine fish so that they can synthesize DHA themselves (see Patent Document 1).

Japanese Patent Publication No. 2015-181477

Patent Document 1 describes the construction of a chimeric enzyme combining the Δ4 fatty acid desaturates and Δ5/Δ6 fatty acid desaturates of the rabbitfish. Furthermore, there is a need for technology to efficiently produce fish that possess all Δ4/Δ5/Δ6 desaturates activity by utilizing genome editing or the like.
The present invention has been made in view of the above circumstances, and aims to provide a protein, gene, vector, fish, a method for producing unsaturated fatty acids, and a method for producing fish for producing fish possessing Δ4/Δ5/Δ6 unsaturated enzyme activity.

In other words, the present invention includes the following embodiments.
[1] A protein comprising a sequence containing any one of the following amino acid sequences (a) to (c), and having Δ4, Δ5 and Δ6 fatty acid unsaturation activity.
(a) In the amino acid sequence represented by Sequence ID No. 1,
(b) an amino acid sequence comprising substitutions of asparagine at amino acid position 281, methionine at amino acid position 288, serine at amino acid position 298, and threonine at amino acid position 325 with hydrophobic residues; (a) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the portion of the amino acid sequence represented in (a) other than positions 281, 288, 298, and 325; (c) an amino acid sequence having 70% or more identity in the portion of the amino acid sequence represented in (a) other than positions 281, 288, 298, and 325; [2] the protein according to [1], wherein the amino acid at position 281 of the amino acid sequence represented by Sequence ID No. 1 is isoleucine.
[3] The protein according to [1] or [2], wherein the amino acid at position 288 of the amino acid sequence represented by Sequence ID No. 1 is leucine.
[4] The protein described in any one of [1] to [3], wherein the amino acid at position 298 of the amino acid sequence represented by Sequence ID No. 1 is alanine.
[5] The protein described in any one of [1] to [4], wherein the amino acid at position 325 of the amino acid sequence represented by Sequence ID No. 1 is isoleucine.
[6] A protein comprising a sequence containing any one of the following amino acid sequences (e) to (g), and having Δ4, Δ5 and Δ6 fatty acid unsaturation activity.
(e) an amino acid sequence represented by Sequence ID No. 3; (f) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in a portion of the amino acid sequence represented by (e) other than amino acid positions 281, 288, 298, and 325; (g) an amino acid sequence that has 70% or more identity in a portion of the amino acid sequence represented by (e) other than amino acid positions 281, 288, 298, and 325; a gene encoding the protein described in any one of the items [7][1] to [6].
A vector containing the genes described in [8] and [7].
Fish comprising any one of the proteins described in [9], [1] to [6], the gene or its transcript described in [7], or the vector described in [8].
A method for producing unsaturated fatty acids, comprising reacting the fish described in [10] and [9] with a fatty acid substrate to produce fatty acids in which the Δ4, Δ5, and Δ6 sites are unsaturated.
[11] The method for producing an unsaturated fatty acid according to [10], wherein the fatty acid substrate is α-linolenic acid, docosapentaenoic acid, eicosatetraenoic acid, or eicosatrienoic acid.
[12] A method for producing fish, comprising injecting a donor DNA having homology arms at both ends into a fertilized egg of a fish, the donor DNA having at least 70% identity with an amino acid sequence having at least the sequence from position 281 to 325 of the amino acid sequence represented by Sequence ID No. 2, and an amino acid sequence other than amino acid positions 281, 288, 298, and 325, and performing homologous recombination by genome editing to introduce a predetermined mutation into the target DNA of the fish.
[13] A method for producing fish, comprising comparing the amino acid sequences of the protein responsible for the function in a marine species lacking a specific function, and in a migratory or river species belonging to the same order that has evolved from the marine species and acquired the function, identifying the amino acid or responsible amino acid region responsible for the function, and conferring the function to the marine species by substituting the responsible amino acid or responsible amino acid region of the marine species with that of the migratory or river species.

According to the present invention, it is possible to provide a technique for efficiently producing fish that possess all Δ4/Δ5/Δ6 desaturates activity.

(A) This figure shows the structure of the chimeric enzyme. The amino acid region of wild-type freshwater flounder is shown in the upper panel, the amino acid region of wild-type senegalsol is shown in the lower panel, and the chimeric enzyme is schematically represented in between. (B) This is a graph showing the conversion efficiency of the protein shown in Figure 1(A). (A) This figure shows the mutation sites of each enzyme. (B) This graph shows the conversion efficiency of the protein shown in Figure 2(A).

Protein
The Fads2 (fatty acid desaturate 2) protein of Senegalensis, a member of the family Soleaceae, is a desaturase that introduces double bonds into the carbon chains of fatty acids. The full-length amino acid sequence of the wild-type Fads2 protein of Senegalensis is shown in Sequence ID No. 1.

It is known that the position of double bond introduction in desaturates varies depending on the species of organism and enzyme. Desaturation activity that introduces a double bond to the nth carbon from the carboxyl terminus of a fatty acid is denoted as Δn activity, and enzymes that retain this activity are denoted as Δn desaturates.
In the DHA synthesis pathway, DHA is synthesized when α-linolenic acid is sequentially acted upon by Δ6-unsaturates, Δ5-unsaturates, and Δ4-unsaturates.

Senegalsoor is a marine fish. While the desaturates of many marine fish possess only Δ6 activity, the desaturates of Senegalsoor primarily catalyzes Δ4 desaturation but does not catalyze Δ6 desaturation.
Meanwhile, the inventor discovered that the desaturase enzyme of freshwater flounder belonging to the family Soleidae, which originally originated in the sea but have moved into freshwater, may be a triplicate enzyme capable of catalyzing all desaturation reactions.

Therefore, the inventors created a protein with a mutation in the transmembrane region of senegalsol desaturates that resembles that of freshwater flounder, and by evaluating the conversion efficiency of substrate fatty acids, they discovered a protein with Δ4/Δ5/Δ6 activity.

<An unsaturated enzyme protein with mutations in asparagine at amino acid position 281, methionine at amino acid position 288, serine at amino acid position 298, and threonine at amino acid position 325>
In one embodiment, the present invention provides a protein comprising a sequence containing any one of the following amino acid sequences (a) to (c), and having Δ4, Δ5, and Δ6 fatty acid unsaturation activity.
(a) In the amino acid sequence represented by Sequence ID No. 1,
(b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the portion of the amino acid sequence other than positions 281, 288, 298, and 325 of the amino acid sequence represented in (a) above. (c) an amino acid sequence in which 70% or more of the portion of the amino acid sequence other than positions 281, 288, 298, and 325 of the amino acid sequence represented in (a) above has been replaced by a hydrophobic residue.

The amino acid sequence represented by Sequence ID No. 1 is the full-length amino acid sequence of the wild-type Fads2 protein of senegalsol.
In (a), the substitution of asparagine at amino acid position 281 is preferably alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, or tyrosine, with isoleucine being more preferred.
In (a), the substitution of methionine at amino acid position 288 is preferably alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, or tyrosine, with leucine being more preferred.
In (a), the substitution of serine at amino acid position 298 is preferably alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, or tyrosine, with alanine being more preferred.
In (a), the substitution of threonine at amino acid position 325 is preferably alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, or tyrosine, with isoleucine being more preferred.
In (a), the amino acid sequence represented by Sequence ID No. 2, which has all of N281I, T325I, S298A, and M288L, is preferred.

In (b), the number of deleted, inserted, substituted, or added amino acids is 1 to 130, preferably 1 to 110, more preferably 1 to 90, more preferably 1 to 65, more preferably 1 to 45, even more preferably 1 to 20, and most preferably 1 to 10.

In (c), identity is preferably 75% or more, more preferably 80% or more, particularly preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more.
The present invention, which imparts Δ4, Δ5, and Δ6 fatty acid unsaturation activity to desaturates, is not limited to senegalsol, but is also applicable to proteins of identity or greater than those defined in (b) and (c), for example, desaturates of the superfamily Soleidae in the order Pleuronectiformes.
Here, the sole of the Japanese flounder exhibits 86.39% identity with the wild-type Fads2 protein of Senegal sole, 77.51% identity with the black sole, 78.91% identity with the Japanese flounder, and 81.86% identity with the Japanese yellowtail.

In this invention, "Δ4 fatty acid unsaturation activity" refers to the desaturation activity that introduces a double bond to the fourth carbon from the carboxyl terminus of a fatty acid. "Δ5 fatty acid unsaturation activity" refers to the desaturation activity that introduces a double bond to the fifth carbon from the carboxyl terminus of a fatty acid. "Δ6 fatty acid unsaturation activity" refers to the desaturation activity that introduces a double bond to the sixth carbon from the carboxyl terminus of a fatty acid.

The protein of this embodiment further has the following characteristics: (d) In the amino acid sequence represented by SEQ ID NO: 1, the substitution of phenylalanine at amino acid position 282 is preferably alanine, valine, glycine, isoleucine, leucine, proline, tryptophan, or tyrosine, with isoleucine being more preferred.

<A chimeric desaturase protein in which amino acids 266-337 are substituted with the corresponding amino acid sequence of freshwater flounder.>
In one embodiment, the present invention relates to a protein comprising a sequence containing any one of the following amino acid sequences (e) to (g), and having Δ4, Δ5, and Δ6 fatty acid unsaturation activity.
(e) The amino acid sequence represented by Sequence ID No. 3 (f) The amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the portion of the amino acid sequence represented by (e) other than amino acid positions 281, 288, 298, and 325 (g) The amino acid sequence in which 70% or more of the amino acid sequence represented by (e) is identical in the portion of the amino acid sequence other than amino acid positions 281, 288, 298, and 325

In (e), the amino acid sequence represented by SEQ ID NO: 3 is a sequence in which the amino acid sequence of positions 266-337 of the senegal sole wild type represented by SEQ ID NO: 1 is replaced with the amino acid sequence of positions 266-337 of the freshwater flounder wild type represented by SEQ ID NO: 4.

In (f), the number of deleted, inserted, substituted, or added amino acids is 1 to 130, preferably 1 to 110, more preferably 1 to 90, more preferably 1 to 65, more preferably 1 to 45, even more preferably 1 to 20, and most preferably 1 to 10.

In (g), the degree of identity is preferably 75% or more, more preferably 80% or more, particularly preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more.

≪Genes that code for proteins≫
In one embodiment, the present invention provides a gene encoding the above-described desaturating enzyme protein variant.

Examples of such genes include those encoding proteins that consist of a sequence containing one of the following base sequences (h) to (q) and possess Δ4, Δ5, and Δ6 fatty acid unsaturation activity.

(h) The base sequence represented by Sequence ID No. 5 (the base sequence of the amino acid sequence represented by Sequence ID No. 2)
(i) A nucleotide sequence in which one to several nucleotides are deleted, inserted, substituted, or added at positions other than positions 841-843, 862-864, 892-894, and 973-975 of the nucleotide sequence represented by SEQ ID NO:
(j) A base sequence in which the base sequence represented by Sequence ID No. 5 has a identity of 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more.
(k) A base sequence that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by Sequence ID No. 5. (l) Degenerate isomers of the base sequences of (h) to (k).

(m) The base sequence represented by Sequence ID No. 6 (the base sequence of the amino acid sequence represented by Sequence ID No. 3)
(n) A nucleotide sequence in which one to several nucleotides are deleted, inserted, substituted, or added at positions other than positions 841-843, 862-864, 892-894, and 973-975 of the nucleotide sequence represented by SEQ ID NO: 6
(o) A base sequence in which the base sequence represented by Sequence ID No. 6 has a identity of 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more.
(p) A base sequence that can hybridize under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by Sequence ID No. 6. (q) Degenerate isomers of the base sequences (m) to (p) above.

In (i) and (n), the number of bases that may be deleted, inserted, substituted, or added is 1 to 400, preferably 1 to 330, more preferably 1 to 260, even more preferably 1 to 200, particularly preferably 1 to 130, particularly preferably 1 to 65, and most preferably 1 to 30.

In (k) and (p), "stringent conditions" can refer to conditions in which hybridization is performed by incubation at 55-70°C for several hours to overnight in a hybridization buffer consisting of 5×SSC (composition of 20×SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1% by weight N-lauroyl sarcosine, 0.02% by weight SDS, 2% by weight nucleic acid hybridization blocking reagent, and 50% formamide. The washing buffer used for washing after incubation is preferably a 1×SSC solution containing 0.1% by weight SDS, more preferably a 0.1×SSC solution containing 0.1% by weight SDS.

For amino acids other than methionine and tryptophan, multiple codons correspond to a single amino acid. This is called genetic code degeneracy. In (l) and (q), a degenerate isomer of a base sequence refers to another base sequence that corresponds to the amino acid encoded by a given base sequence.

≪Vector≫
In one embodiment, the present invention provides a vector containing the gene of the present invention described above.
The vector is not particularly limited, and conventionally known vectors such as plasmid vectors and viral vectors can be used. Examples of plasmid vectors include vectors having promoters for expression in animal cells, such as the CAG promoter, EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, and HSV-tk promoter.
Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated (AAV) vectors, vaccinia virus vectors, lentivirus vectors, herpesvirus vectors, alphavirus vectors, EB virus vectors, papillomavirus vectors, Formy virus vectors, and Sindbisvirus vectors.

＜＜Fish＞＞
In one embodiment, the present invention provides fish containing the protein of the present invention, or the gene or its transcript, or the vector of the present invention.
The introduction of proteins, genes, etc., into fish according to the present invention follows standard methods such as transfection, injection, and electroporation.
The proteins, genes, etc., introduced into fish may coexist with the endogenous wild type, or they may replace the endogenous wild type as described in the <<Methods for Manufacturing Fish>>.

<<Method for producing unsaturated fatty acids>>
In one embodiment, the present invention provides a method for producing unsaturated fatty acids by reacting the fish of the present invention with a fatty acid substrate to produce fatty acids in which the Δ4, Δ5, and Δ6 sites are unsaturated.
By adding fatty acid substrates to the culture tank as feed, the fatty acid substrates can be applied to the aforementioned fish. Examples of fatty acid substrates include alpha-linolenic acid, docosapentaenoic acid, eicosatetraenoic acid, or eicosatrienoic acid. These fatty acids are desaturated within the fish body, and eicosapentaenoic acid and docosahexaenoic acid are synthesized.

<<Methods for producing fish>>
In one embodiment, the present invention provides a method for producing fish, which involves comparing the amino acid sequences of the protein responsible for a particular function in a marine species lacking a specific function, and in a migratory or river species belonging to the same order that has evolved from the marine species and acquired the function, identifying the amino acid or responsible amino acid region responsible for the function, and then substituting the responsible amino acid or responsible amino acid region of the marine species with that of the migratory or river species to confer the function to the marine species.

Several species of flatfish inhabit rivers around the world. Generally, flatfish require docosahexaenoic acid (DHA) as an essential fatty acid and cannot survive without consuming food containing it. However, freshwater prey organisms are poor in highly unsaturated fatty acids, including DHA, and it was unclear how flatfish that have migrated from the sea into rivers meet their fatty acid requirements.
In this invention, we conducted a detailed analysis of the fatty acid metabolic pathways of three types of flounder: marine species that spend their entire lives in the sea, migratory species that are born in the sea and spend most of their lives in rivers, and riverine species that complete their entire lives in freshwater. We found that while the ancestral marine species cannot synthesize DHA, both the migratory and riverine species have acquired the ability to synthesize DHA.
This strongly suggests that the ability to synthesize DHA is a key factor in the success or failure of marine fish species when they venture into freshwater environments.

As an example, by comparing the amino acid sequences of the Fads2 protein in senegalsol, which has an enzyme that mainly catalyzes Δ4 desaturation, and freshwater flounder, which has a trifunctional enzyme that can catalyze all desaturation reactions, it is possible to confer DHA synthesis ability to senegalsol by introducing mutations in the responsible amino acids N281I, T325I, S298A, and M288L into the Fads2 gene of senegalsol, or by substituting them into TM2 (transmembrane region 2) of freshwater flounder, which is the amino acid region responsible for DHA synthesis.
This invention is not limited to DHA synthesis ability, but can be applied to any function that is lacking in marine species but acquired in migratory or riverine species.

The acquisition of DHA synthesis ability in the Fads2 protein of senegalsol, that is, the acquisition of Δ5 and Δ6 fatty acid unsaturation activity of the Fads2 protein, can be induced, for example, by introducing mutations into the Fads2 gene in the genome of the target senegalsol using conventional methods.
The method for introducing the mutation can be carried out, for example, by genome editing technology using homologous recombination, ZFN, TALEN, CRISPR-CAS9, etc. The method for introducing the mutation may also be carried out by a mutagenesis method such as site-directed mutagenesis. Alternatively, the method for introducing the mutation may also be carried out by a random mutagenesis method. Examples of random mutagenesis methods include irradiation with alpha rays, beta rays, gamma rays, X-rays, etc., treatment with mutagenic agents such as ethyl methanesulfonate (EMS) and ethynylnitrosourea (ENU), and heavy ion beams. The above-mentioned methods for introducing mutations may also be carried out using commercially available kits, for example.

In one embodiment, the present invention provides a method for producing fish, which involves injecting a donor DNA having homology arms at both ends into a fish fertilized egg, and performing homologous recombination by genome editing to introduce a predetermined mutation into the target DNA of the fish. This DNA encodes an amino acid sequence having at least the sequence from positions 281 to 325 of the amino acid sequence represented by Sequence ID No. 2, with at least 80% identity in the portion other than amino acid positions 281, 288, 298, and 325.

As an example, a modified partial sequence of the Senegalsol Fads2 gene containing mutations N281I, T325I, S298A, and M288L, approximately 300 bp in length, is connected to approximately 1 kb upstream and downstream of its homologous sequence on the genome as homologous arms. This constructs a donor DNA template for knock-in via homologous recombination repair.
Next, donor DNA, Cas9 protein, and guide RNA are injected into the cytoplasm of senegalsol fertilized eggs. The genome-edited fertilized eggs are cultured and hatched, and then reared using conventional aquaculture methods. Senegalsol homozygous for the N281I, T325I, S298A, and M288L gene mutation alleles can be obtained by breeding and mating senegalsol heterozygous for the gene mutation alleles.

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[Example 1]
Many marine fish have desaturase enzymes that only possess Δ6 activity, and the reaction beyond Δ5 desaturation does not proceed, making it impossible to synthesize DHA.
The inventor discovered that the desaturase enzyme of flounder (also known as freshwater flounder), a species of flounder originally from the sea that inhabits freshwater areas, is a triplicate enzyme capable of catalyzing all desaturation reactions.
The gene encoding this enzyme is orthologized to those of other marine fish, and this special function is thought to have been acquired by freshwater flounder as an adaptation to the freshwater environment, where DHA is much scarcer than in the ocean.

The sole species *Senegalensis*, which is farmed in Europe, also belongs to this group. Unlike many marine fish, the desaturase enzyme in *Senegalensis* primarily catalyzes Δ4 desaturation, but it has been shown to be largely incapable of catalyzing Δ6 desaturation.

The enzymatic activity of senegal sole and freshwater flounder desaturates was measured. Specifically, budding yeast into which the genes for each desaturate were introduced was cultured in the presence of substrate fatty acids. The fatty acid composition of the cultured yeast was then analyzed by gas chromatography, and the conversion efficiency of each enzyme, or the efficiency of how much of the reaction it catalyzed, was calculated using the following formula from the peak areas of the substrate taken up by the yeast and the products metabolized within the yeast.
Conversion efficiency (%) = 100 × product area / (substrate area + product area)

The results of the enzyme activity test are shown in Table 1.

As shown in Table 1, the desaturation activities of Δ4, Δ6, and Δ5 in senegal sole and freshwater flounder were compared. The amino acid sequences of these enzymes were nearly 90% identical, suggesting that the Δ6 responsible site was located in the 10% of amino acid residues that differed between the enzymes.

Therefore, in order to identify the Δ6 responsible site, we began creating chimeric enzymes. By exchanging parts of the amino acid sequences of freshwater flounder and senegalsol (hereinafter also referred to as SEN), if the activity is converted, that region can be considered the Δ6 responsible site.
First, we created chimeric enzymes as shown in Figure 1(A), focusing on a region with a concentration of specific amino acids found only in freshwater flounder and transmembrane region 2 (hereinafter also referred to as TM2, corresponding to amino acid sequences 266-337 in freshwater flounder), which previous studies had suggested controls the function of desaturates. The freshwater flounder-type amino acid region is shown in the upper panel, the senegalsol wild-type amino acid region is shown in the lower panel, and the chimeric enzyme is schematically represented between them. These were introduced into yeast, and functional analysis was performed using Δ4 and Δ6 substrates. The results are shown in Figure 1(B). A change in activity due to the exchange of TM2 was confirmed. Chimeras 4 and 6, in which freshwater flounder TM2 was introduced into the senegalsol enzyme, showed a significant increase in Δ6 activity. From this, it became clear that TM2 has a site responsible for Δ6.

[Example 2]
To identify which amino acids in TM2 are important for Δ6 activity, the amino acids in the TM2 of the senegalsol enzyme were replaced with those of the freshwater flounder. The mutation sites and conversion efficiencies of each enzyme are shown in Figure 2. SEN-mt9, SEN-mt11, and SEN-mt17 were able to achieve Δ6 conversion efficiencies of over 10%, comparable to SEN-freshwater flounder TM2 (chimera 4 in Figure 1), in which the entire TM2 molecule was replaced. In particular, mt17 showed high activity, with both Δ6 and Δ4 conversion efficiencies exceeding 10%.
Furthermore, the Δ5 enzyme activity was compared. The results are shown in Table 2.

As shown in Table 2, mt17 exhibited a conversion efficiency of over 10% in the total desaturation reaction.

According to the present invention, it is possible to provide a technique for efficiently producing fish that possess all Δ4/Δ5/Δ6 desaturates activity.

Claims (8)

A protein comprising a sequence containing any one of the following amino acid sequences (a) to (c), and having Δ4, Δ5, and Δ6 fatty acid unsaturation activity.
(a) Amino acid sequence represented by Sequence ID No. 2
(b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the portion of the amino acid sequence represented in (a) other than amino acid positions 281, 288, 298, and 325, and which has amino acid positions 281, 288, 298, and 325 of the amino acid sequence represented in (a).
(c) An amino acid sequence having 90 % or more identity in the portion of the amino acid sequence represented in ( a) other than amino acid positions 281, 288, 298, and 325, and having amino acid positions 281, 288, 298, and 325 of the amino acid sequence represented in (a). A protein comprising a sequence containing one of the following amino acid sequences (e) to (g), and having Δ4, Δ5, and Δ6 fatty acid unsaturation activity.
(e) The amino acid sequence represented by Sequence ID No. 3 (f) An amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added in the portion of the amino acid sequence represented by (e) other than amino acid positions 281, 288, 298, and 325, and which has amino acid positions 281, 288, 298, and 325 of the amino acid sequence represented by (e)
(g) an amino acid sequence having 90 % or more identity in the portion of the amino acid sequence represented in ( e) other than amino acid positions 281, 288, 298, and 325, and which has amino acid positions 281, 288, 298, and 325 of the amino acid sequence represented in (e) A gene encoding the protein described in claim 1 or 2 . A vector comprising the gene described in claim 3 . A fish comprising the protein according to claim 1 or 2 , the gene or transcript according to claim 3 , or the vector according to claim 4 . A method for producing unsaturated fatty acids, comprising reacting the fish described in claim 5 with a fatty acid substrate to produce fatty acids in which the Δ4, Δ5, and Δ6 sites are unsaturated. The method for producing an unsaturated fatty acid according to claim 6 , wherein the fatty acid substrate is α-linolenic acid, docosapentaenoic acid, eicosatetraenoic acid, or eicosatrienoic acid. A method for producing fish, comprising injecting DNA encoding an amino acid sequence having at least the sequence from position 281 to 325 of the amino acid sequence represented by Sequence ID No. 2, and an amino acid sequence having 90 % or more identity in the portion other than amino acid positions 281, 288, 298, and 325 of the amino acid sequence represented by Sequence ID No. 2, and donor DNA having homology arms at both ends thereof, into a fertilized egg of a fish, performing homologous recombination by genome editing, and introducing a predetermined mutation into the target DNA of the fish.