JP7269580B2 - Mutant of Green Algae with Decreased Fat Degradation and Increased Fat Productivity, and Use Thereof - Google Patents

Description

IPOD FERM BP-10484 IPOD FERM BP-22179 IPOD FERM BP-22254

TECHNICAL FIELD The present invention relates to mutant green algae in which lipolysis is reduced and lipid accumulation and lipid productivity are increased due to mutations in lipid droplet protein genes, and uses thereof.

Products such as biodiesel and bio jet fuel are produced using triacylglycerols (hereinafter referred to as "TAG") produced by unicellular eukaryotic photosynthetic organisms (hereinafter referred to as "eukaryotic microalgae") as raw materials. Although research is being conducted widely worldwide, production costs are currently high, and production on a commercial basis is difficult (Non-Patent Document 1). Therefore, further technical development is ongoing, one of which is the improvement of algal oil productivity.

Eukaryotic microalgae are known to accumulate carbohydrates and lipids in their cells under stress conditions such as nitrogen, phosphorus, or sulfur deficiency. Accumulated lipids (mainly TAG) are accumulated in intracellular organelles called lipid droplets, which are surrounded by a single phospholipid membrane. A large number of proteins called lipid droplet proteins (hereinafter referred to as 'LDP') are present in the phospholipid monolayer of intracellular oil droplets. Various types of LDP exist depending on species such as animals, fungi, and plants, and it is becoming clear that they have functions related to the formation of oil droplets and lipid metabolism. There are many (Non-Patent Document 2).

Among algae, Chlamydomonas, Dunaliella, Haematococcus belonging to Viridiplantae, Chlorophyta, and Chlorophyceae belong to Major lipid droplet protein (MLDP) and LDPs called oil globule protein (OGP) have been found (Non-Patent Documents 3, 4, 5). These green algae LDPs have sequence homology, and it has been shown that gene expression increases at the same time as oil accumulation increases during nitrogen starvation, and that they are localized in the membrane of oil droplets (Non-Patent Documents 3 and 4). , 5). Suppression of the gene expression of MLDP (CHLREDRAFT_192823; NCBI accession number XP_001697668) identified in Chlamydomonas was shown to increase the oil droplet size, but did not change the amount of fat accumulated (Non-Patent Document 3).

Coccomyxa sp. Obi strain and Coccomyxa sp. strains) are known. Also in the algae Lobosphaera belonging to the same Trebouxiophyceae, a protein having sequence similarity to MLDP (Oil globule protein) has been found as an oil droplet protein (Non-Patent Document 6).

The Obi strain is the same strain as the unicellular green alga Pseudochoricystis ellipsoidea MBIC11204 strain described in Patent Document 1, and has been deposited under Accession No. FERM BP-10484. The KJ strain is closely related to the Obi strain, but has approximately twice the oil productivity (oil production rate per volume of culture medium) of the Obi strain. Are listed. This strain has been deposited under accession number FERM BP-22254. The Obi strain and the KJ strain grow well even in a medium with a pH of 3.5 or less, can be cultured in the open culture system shown in Patent Document 3, and can be continuously produced outdoors by the method shown in Patent Document 4. It can be carried out.

The present inventors have deciphered the genome sequences of the Obi strain and the KJ strain, and have worked to improve their breeding and culture techniques. In order to breed strains with improved oil productivity, for example, a method of improving the light utilization efficiency of photosynthesis (Patent Document 5), a method of promoting the activity of enzymes involved in oil production (Patent Document 6), or oil A method for suppressing decomposition (Patent Document 7) and the like are conceivable. So far, several strains with mutations in the TAG lipase gene with sequence similarity to sugar-dependent 1 (SDP1) of higher plants have been isolated from Obi and KJ strains by treatment with mutagen agents ( Patent document 7). The SDP1 mutant in the KJ strain inhibited lipid degradation, but did not increase lipid productivity under nitrogen deficiency.

Patent No. 4748154 Patent No. 6088375 Patent No. 6235210 Patent No. 5810831 JP 2013-102715 A JP 2017-046643 A JP 2017-046645 A

Chisti Y. (2013) Constraints to commercialization of algal fuels. J. Biotechnol. 167: 201-214. Huang AHC. (2018) Plant lipid droplets and their associated proteins: potential for rapid advances. Plant Physiol. 176: 1894-1918. Moellering ER, Benning C. (2010) RNA Interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell. 9: 97-106. Davidi L, Katz A, Pick U. (2012) Characterization of major lipid droplet proteins from Dunaliella. Planta. 236: 19-33. Peled E, Leu S, Zarka A, Weiss M, Pick U, Khozin-Goldberg I, Boussiba S. (2011) Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (Chlorophyceae). Lipids. 46: 851-861 . Siegler H, Valerius O, Ischebeck T, Popko J, Tourasse NJ, Vallon O, Khozin-Goldberg I, Braus GH, Feussner I. (2017) Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa. BMC Plant Biol. 17 : 98.

Increasing the fat and oil productivity of eukaryotic microalgae can be considered as a powerful means of cost reduction required for commercialization of biofuel production. Degradation of lipids occurs simultaneously with the synthesis of lipids in cells, but in eukaryotic microalgae mutants with reduced ability to decompose lipids (lipolysis), the amount of lipids accumulated (weight of lipids per dry weight of algae) increases, Fats and oils productivity may increase as a result. In addition, by culturing such eukaryotic microalgae mutants, it is possible to reduce the production cost of fats and oils used for biofuels and the like.

Accordingly, an object of the present invention is to provide eukaryotic microalgae with reduced fat-degrading capacity and improved fat-accumulation and fat-productivity.

As a result of intensive research to solve the above problems, eukaryotic microalgae in which the gene encoding a specific LDP (LDP1) is mutated has a reduced ability to decompose oils and fats, and an increase in fat accumulation and fat productivity. The discovery led to the completion of the present invention.

That is, the present invention includes the following.
(1) A eukaryote having an amino acid sequence having at least 50% sequence identity with the conserved region of LDP shown in SEQ ID NO: 7 or SEQ ID NO: 8 and having reduced function of a protein localized on the membrane surface of oil droplets A eukaryotic microalgae mutant, which has increased intracellular lipid accumulation and lipid productivity and reduced lipolysis compared to the parent strain.
(2) The eukaryotic microalgae mutant according to (1), wherein the gene encoding the protein has been disrupted.
(3) The eukaryotic microalgae mutant according to (1), which has reduced expression of the gene encoding the protein.
(4) The eukaryotic microalgae mutant according to (1), wherein the translation efficiency of the gene encoding the protein is reduced.
(5) The eukaryotic microalgae mutant according to any one of (1) to (4), which belongs to the phylum Chlorophyta.
(6) The eukaryotic microalgae mutant according to (5), belonging to Trebouxiophyceae.
(7) The eukaryotic microalgae mutant according to (6), which belongs to the genus Coccomyxa.
(8) A method for producing oils and fats, comprising a step of culturing the eukaryotic microalgae mutant according to any one of (1) to (7).

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to create eukaryotic microalgae mutants with increased intracellular fat accumulation and fat productivity and decreased fat degradation. In addition, by culturing the mutant eukaryotic microalgae according to the present invention, it is possible to reduce the production cost of fats and oils to be used as biofuels.

Antenna chlorophyll-depleted mutant (5P strain) derived from Obi strain and LDP1 gene mutant (ldp1-1) derived from 5P strain were cultured in test tubes under nitrogen-deficient (1/2A7) medium under continuous light conditions for 14 days. After that, it was transferred to a nitrogen-sufficient (MA5) medium and cultured in the dark for 3 days, and the amount of oil accumulated was measured every 24 hours. In the graph, the vertical axis indicates the accumulated amount of fat and oil, and the horizontal axis indicates the time after transferring to a nitrogen-sufficient medium and transferring in the dark. Shows a list of arrays. This is a continuation of Figure 2-1. This is a continuation of Figure 2-2. This is a continuation of Figure 2-3. This is a continuation of Figure 2-4. This is a continuation of Figure 2-5.

The present invention will be described in detail below.

By reducing the function of the LDP protein named LDP1, the present invention is a eukaryotic microalgae mutation that has increased intracellular lipid accumulation and lipid productivity and decreased lipolysis compared to the parent strain. Regarding the body.

A mutant derived from the Obi strain obtained by treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was mutated to the LDP1 gene (genomic sequence) shown in SEQ ID NO: 2. had

Major lipid droplet protein (MLDP) (Non-Patent Document 3), which has been shown to be related to the size of oil droplets in LDP of Chlamydomonas, Donaliella (Non-Patent Document 4) of the same Chlorophyceae, Haematococcus (Non-Patent Document Two proteins having about 20 to 30% amino acid sequence identity with LDP of 5) were found in Obi strain and KJ strain, respectively, and they were named LDP1 protein and LDP2 protein. The amino acid sequences of the KJ strain LDP1 protein and LDP2 protein are shown in SEQ ID NO: 5 and SEQ ID NO: 9, respectively. In addition, the amino acid sequences of LDP1 protein and LDP2 protein of Obi strain are shown in SEQ ID NO: 6 and SEQ ID NO: 10, respectively.

On the other hand, the LDP1 gene (genomic sequence) of the KJ strain and the nucleotide sequence of its CDS are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. In addition, the LDP1 gene (genomic sequence) of the Obi strain and the nucleotide sequence of its CDS are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

The amino acid sequences of LDP1 from the Obi and KJ strains show about 98% sequence identity with each other. The N-terminal 9 amino acid residues and the C-terminal 24 amino acid residues of the LDP1 proteins of the KJ and Obi strains show no similarity with the amino acid sequences of other LDPs. Therefore, the central portion excluding these N-terminal and C-terminal portions is defined as the conserved region of the LDP1 protein. The amino acid sequences of the conserved regions of the LDP1 proteins of the KJ and Obi strains are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 and the amino acid sequence of LDP2 of Obi strain or KJ strain show about 44% (119/268) identity. Compared to the oil globule protein (Non-Patent Document 6), which is the LDP of the algae Lobosphaera belonging to the Treboxiaphyceae, the conserved region of the LDP2 protein is about 52% (139/266), and the conserved region of the LDP1 protein is about 45% ( 121/268).

The LDP genes of Chlamydomonas, Donaliella, and Lobosphaera have been shown to increase in expression during oil accumulation (Non-Patent Documents 3 to 6). Similar to these known LDP genes, the LDP2 gene was upregulated during nitrogen starvation, but the LDP1 gene was not upregulated during nitrogen starvation.

The mutant strain derived from the Obi strain and the gene-disrupted strain derived from the KJ strain in which the LDP1 gene was disrupted showed an increase in the amount of intracellular lipid accumulation and an increase in lipid productivity compared to the parent strain. Furthermore, the present inventors have found that these LDP1 gene-disrupted strains are inhibited from decomposing fats and oils, and have completed the present invention.

In the present invention, examples of eukaryotic microalgae include eukaryotic microalgae belonging to green algae, diatoms or Bacillariophyceae, Eustigmatophyceae, and the like.

Green algae include, for example, green algae belonging to the Treboxia algae. Green algae belonging to the Treboxia algae include, for example, the genus Trebouxia, the genus Chlorella, the genus Botryococcus, the genus Choricystis, the genus Coccomyxa, the genus Pseudococcomyxa. Green algae belonging to Specific strains belonging to Treboxia algae include the Obi strain (accession number FERM BP-10484) and its mutant strain P. ellipsoidea 5P strain (accession number FERM BP-22179; hereinafter sometimes referred to as "5P strain". ) and KJ strain (accession number FERM BP-22254). On February 15, 2005, the Obi strain was entrusted to the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (1-1-1-Chuo-6, Higashi, Tsukuba, Ibaraki, Japan 305-8566). It was deposited under the number FERM P-20401 and has been transferred to the international deposit under the Budapest Treaty under the accession number FERM BP-10484. The Obi strain is available from the National Institute of Technology and Evaluation, Patent Organism Depository (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818, Japan). The 5P strain was entrusted to the Independent Administrative Institution National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (1-1-1 Central 6, Higashi Tsukuba, Ibaraki 305-8566 Japan) on October 21, 2011. Deposited as No. FERM P-22179, and further to the National Institute of Technology and Evaluation Patent Organism Depositary Center (NITE-IPOD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818 Room 120) It has been transferred to an international deposit under the Budapest Treaty under accession number FERM BP-22179. As of June 4, 2013, the KJ strain was registered with the National Institute of Technology and Evaluation, Patent Organism Depositary Center (NITE-IPOD) (2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan). -8 Room 120) under the accession number FERM P-22254, and has been transferred to the international deposit under the Budapest Treaty under the accession number FERM BP-22254. The Obi strain and the 5P strain have the same gene encoding the LDP1 protein (genomic DNA nucleotide sequence: SEQ ID NO: 2, CDS nucleotide sequence: SEQ ID NO: 4, full-length amino acid sequence: SEQ ID NO: 6, conserved region amino acid sequence: SEQ ID NO: 8).

Green algae other than those belonging to Treboxia algae include, for example, the genus Tetraselmis, the genus Ankistrodesmus, the genus Dunalliella, the genus Neochloris, the genus Chlamydomonas, and the squid (= Scenedesmus) Examples include green algae belonging to the genus and the like.

Furthermore, diatoms include eukaryotic microalgae belonging to the genus Fistulifera, the genus Phaeodactylum, the genus Thalassiosira, the genus Cyclotella, the genus Cylindrotheca, the genus Skeletonema, and the like. be able to. Moreover, the genus Nannochloropsis is mentioned as an eutectic algae.

The eukaryotic microalgae mutant according to the present invention is a eukaryotic microalgae mutant obtained by subjecting the aforementioned eukaryotic microalgae as a parent strain to a method for reducing the function of the LDP1 protein. Here, the function of the LDP1 protein is to localize on the membrane surface of oil droplets, for example, to prevent binding between oil droplets, and / or to prevent lipolytic enzymes in oil droplets such as SDP1 lipase (Patent Document 7). It means promoting TAG decomposition.

In the present invention, the LDP1 protein is at least 50%, preferably at least 65%, particularly preferably at least 80% the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 (that is, the amino acid sequence of the conserved region of the LDP1 protein). %, most preferably at least 85%, at least 90%, at least 95%, 100% sequence identity and proteins localized to the membrane surface of oil droplets.

In addition, the LDP1 protein is at least 50%, preferably at least 65%, particularly preferably at least 80%, most preferably at least 85%, at least 90%, the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, Proteins consisting of amino acid sequences with at least 95%, 100% sequence identity and localized to the membrane surface of oil droplets are included.

Examples of the LDP1 gene include genes encoding the LDP1 protein described above. In addition, the LDP1 gene is at least 50%, preferably at least 58%, particularly preferably at least 65%, at least 80%, most preferably at least 85% of the coding region of the mRNA shown in SEQ ID NO: 3 or SEQ ID NO: 4. %, at least 90%, at least 95%, or 100% sequence identity, and encoding a protein localized on the membrane surface of oil droplets.

In many eukaryotic microalgae, multiple LDP1 genes such as alleles, synonymous genes, etc. may exist, and in the present invention, at least one or more of these LDP1 genes are meant.

In the present invention, eukaryotic microalgae mutants according to the present invention can be obtained by subjecting eukaryotic microalgae having the LDP1 gene described above to a method for reducing the function of the LDP1 protein.

Specifically, the method of reducing the function of the LDP1 protein according to the present invention includes a method of randomly introducing mutations with drugs, radiation, ultraviolet rays, etc., and phenotypically selecting strains having mutations in the LDP1 gene. be done. Methods for selectively disrupting the LDP1 gene include gene disruption by genome editing.

Furthermore, as a method of reducing the function of LDP1 protein, for example
(1) introduce a mutation targeting the LDP1 gene to disrupt the gene;
(2) suppressing the transcription of the LDP1 gene and reducing the expression of the gene;
(3) suppressing the translation of the LDP1 gene and reducing the translation efficiency of the gene;
method.

(1) Method of introducing mutations targeting the LDP1 gene
As a method for introducing mutations targeting the LDP1 gene, gene knockout methods called ZFN, TALEN, or CRISPR/Cas (Gaj T, Gersbach CA, Barbas CF 3rd. (2013) ZFN, TALEN, and CRISPR/Cas-based methods (for genome engineering. Trends Biotechnol. 31:397-405.) can be used to create mutants lacking the gene.

(2) A method for suppressing transcription of the LDP1 gene and reducing expression of the gene
Methods for suppressing the transcription of the LDP1 gene include methods for introducing mutations into the promoter region of the gene in eukaryotic microalgae of interest.

Another example is a method of introducing mutations into genes involved in the positive expression control of the genes to reduce their functions.

Alternatively, there is a method of introducing a mutation into a gene involved in negative expression control of the gene so that the negative expression control always works.

(3) A method for suppressing translation of the LDP1 gene and reducing the translation efficiency of the gene
Methods for suppressing translation of the LDP1 gene include the so-called RNA interference method (Cerutti H et al., 2011, Eukaryot Cell, 10, 1164) and the antisense method.

Furthermore, the present invention includes a method for mass-culturing the eukaryotic microalgae mutant according to the present invention described above to produce oils and fats containing TAG. Mass culture methods include an open culture system disclosed in Patent Document 3, a continuous culture method disclosed in Patent Document 4, and the like. After culturing, fats and oils containing TAG can be obtained by, for example, hexane extraction from the culture.

A list of the sequences is shown in FIG. 2 together with the sequence table.

EXAMPLES The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] Isolation of LDP1 gene mutant strain and evaluation of oil productivity
Using the 5P strain (Patent Document 5, accession number FERM BP-22179), which is a mutant with reduced antenna chlorophyll content derived from the Obi strain, as the parent strain, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) After mutagenesis with , we obtained several mutants with increased lipid accumulation by screening using the amount of lipid accumulated under nitrogen deficiency as an index. In this screening, oils and fats accumulated in cells were fluorescently stained using Nile Red or BODIPY, and cells with high fluorescence intensity were selected using a cell sorter. As a result of comparative analysis of the genome sequence of the strain with high oil productivity and the parent strain 5P strain, the LDP1 gene (genomic sequence: SEQ ID NO: 2, CDS sequence: SEQ ID NO: 4, full-length amino acid sequence: SEQ ID NO: 6, conserved region Amino acid sequence: SEQ ID NO: 8) There is a single base deletion in the first half, and a frameshift mutation occurs after the 15th phenylalanine residue, so this mutation was named ldp1-1. In addition, the strain having this mutation was named ldp1-1 mutant.

The 5P strain and the ldp1-1 mutant were incubated in a test tube under continuous light conditions in an acidic (pH 3.5) nitrogen-deficient medium (1/2A7: Takahashi et al., 2017, Algal Res, 32, 300) for 13 days. When culturing and comparing the fat productivity, as shown in Table 1, the fat accumulation amount in the ldp1-1 mutant increased to about 1.3 times that of the parent strain, and the fat productivity after 13 days of culture was about 1.6 times that of the parent strain. doubled.

To further explore the reason for the increased lipid productivity in the ldp1-1 mutant, we investigated the lipolytic activity of both strains. First, oil was accumulated by growing in a nitrogen-deficient medium. When this strain is transferred to a nitrogen-sufficient medium and placed in the dark, the wild strain decomposes the accumulated lipids because it cannot acquire energy through photosynthesis. Therefore, when the 5P strain and the ldp1-1 mutant were transferred to a nitrogen-sufficient medium (MA5: Imamura et al., 2012, J Gen Appl Microbiol, 58, 1) and continuously cultured in the dark for 3 days, the 5P strain However, lipolysis was inhibited in the ldp1-1 mutant (Fig. 1). From these results, it was inferred that the LDP1 protein is necessary for the degradation of fats and oils in the dark, and that the lack of this protein suppresses the degradation of fats and oils, thereby improving fats and oils productivity.

[Example 2] Isolation of LDP1 gene disrupted strain and evaluation of oil productivity The ldp1-1 mutant shown in Example 1 had mutations in about 35 genes other than the ldp1-1 mutation, so the mutant ldp1-1 could not be determined to be the causative gene mutation of the phenotype shown by . Therefore, we attempted to disrupt the LDP1 gene using the CRISPR/Cas9 system, one of the genome editing technologies.

First, the LDP1 gene (genome sequence: SEQ ID NO: 1, CDS sequence: SEQ ID NO: 3, full-length amino acid sequence: SEQ ID NO: 5, conserved region amino acid sequence: SEQ ID NO: 7) of the KJ strain (accession number FERM BP-22254) was identified. A gRNA target sequence for targeted cleavage was designed and gRNA was synthesized. After forming a complex between this gRNA and purified Cas9 protein, this complex was introduced into the parental cell line by electroporation. The electroporated cell population was then grown in a nitrogen-deficient medium to allow intracellular lipid accumulation. After that, the cell clusters that accumulated oil were transferred to a nitrogen-sufficient medium and cultured in the dark. From the results of Example 1, it was predicted that lipolysis of the LDP1 gene-disrupted strain would not occur, although lipolysis of the parent strain would occur rapidly by culturing in the dark. For this reason, cells with a high accumulation of oil and fat even after dark culture were selected from the above cell group cultured in the dark using a cell sorter, and the selected cells were seeded on a plate to obtain a large number of colonies.

DNA extraction was performed on each of the strains derived from these colonies, and the gene region containing the gRNA target sequence was PCR amplified. As shown in Table 2, within the gRNA target sequence, there is a recognition sequence for the restriction enzyme HinfI at a position one base away from the PAM sequence. Therefore, the obtained PCR fragment was treated with the restriction enzyme HinfI and electrophoresed, and colonies having mutant DNA in which the PCR fragment was not cleaved were selected. As a result, multiple strains (kjldp1-1 to 1-4) with mutations in the target sequence of the LDP1 gene were obtained, with four types of mutations (Table 2). All these strains had a frameshift mutation in the LDP1 gene.

These strains were cultured in test tubes for 14 days in an acidic, nitrogen-deficient medium under continuous light conditions, and the lipid productivity was compared. Productivity increased over the parent strain. Furthermore, when it was transferred to a nitrogen-sufficient medium and continuously cultured in the dark for 4 days, the amount of oil accumulated in the KJ strain (KJ-WT) decreased from 52.6% to 28.4%, but in the LDP1 gene disruption strain, after 4 days The fat and oil accumulation amount was about 60%, and fat and oil decomposition was suppressed (Table 3).

FERM BP-10484
FERM BP-22179
FERM BP-22254

Claims (8)

A protein having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8, wherein the protein is localized on the membrane surface of oil droplets and has reduced function in eukaryotes. A eukaryotic microalgae mutant, which has increased intracellular lipid accumulation and lipid productivity and reduced lipolysis compared to the parent strain. 2. The eukaryotic microalgae mutant of claim 1, wherein the gene encoding said protein has been disrupted. 2. The eukaryotic microalgae mutant of claim 1, which has reduced expression of the gene encoding said protein. 2. The eukaryotic microalgae mutant of claim 1, wherein the gene encoding said protein has reduced translation efficiency. A eukaryotic microalgae mutant according to any one of claims 1 to 4, belonging to the phylum Chlorophyta. 6. A eukaryotic microalgae mutant according to claim 5, belonging to the Trebouxiophyceae. 7. A eukaryotic microalgae mutant according to claim 6, belonging to the genus Coccomyxa. A method for producing oils and fats, comprising the step of culturing the eukaryotic microalgae mutant according to any one of claims 1 to 7.

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