A method for producing Euglena into which a target foreign gene has been introduced, and Euglena obtained by this method.

Abstract

The objective is to obtain Euglena that can express a target foreign gene, and in which the expression level of that gene is improved. [Solution] A method for producing Euglena that can express a target foreign gene, comprising the step of inserting double-stranded DNA with modified terminal regions that encodes at least the target foreign gene into the Euglena genome.

Description

[Technical Field]

[0001] This invention relates to a method for producing Euglena into which a target foreign gene has been introduced, and to Euglena obtained by this method. [Background technology]

[0002] Euglena is a protist that is classified in both the animal and plant kingdoms because it has the ability to grow autotrophically by performing photosynthesis in chloroplasts, and also possesses the ability to move using flagella. Euglena is characterized by the absence of a cell wall in its cellular structure, but is covered by a soft tissue called pectil, which is mainly composed of protein.

[0003] Euglena possesses a high carbon dioxide absorption capacity and exhibits good growth by performing photosynthesis even in the presence of extremely high carbon dioxide concentrations of 40%. Under anaerobic conditions, Euglena ferments and produces wax esters from paramylon, a storage polysaccharide β1,3-glucan. These wax esters can be easily converted into biodiesel. In other words, Euglena is an organism capable of fuel production in parallel with carbon dioxide reduction.

[0004] Industrial uses of Euglena have also been proposed in various other fields.

[0005] Various attempts have been made to introduce nucleic acids into Euglena. For example, there have been reports of successful cases in which double-stranded RNA was introduced into Euglena by electroporation and specific mRNA was eliminated by RNAi (Non-Patent Documents 1 and 2).

[0006] Furthermore, there have been reports of nuclear genome transformation being performed on Euglena using the particle gun method (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Iseki, M. et al., “A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis,” Nature, 2002, 415 [Non-Patent Document 2] Ishikawa, T. et al., "Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization," Biochemical Journal, 426, pp. 125-134 [Non-Patent Document 3] Ogawa, T. et al., “Enhancement of photosynthetic capacity in Euglena gracilis by expression of cyanobacterial fructose-1,6- / sedoheptulose-1,7-bisphosphatase leads to increases in biomass and wax ester production”, Biotechnology for Biofuels, volume 8,80 (2015) [Overview of the project] [Problems that the invention aims to solve]

[0008] The inventors of this invention have succeeded in obtaining a transformant Euglena in which double-stranded DNA containing a target foreign gene has been inserted into the genome of Euglena through their own research, but have found that the expression level of the target foreign gene in such transformants is insufficient. The object of this invention is to obtain a Euglena that retains the target foreign gene in an expressible manner and in which the expression level of the target foreign gene is improved. [Means for solving the problem]

[0009] The inventors of the present invention conducted diligent research to solve the above problems and found that the problems can be solved by inserting double-stranded DNA with modified terminal regions that encode at least the target foreign gene into the Euglena genome. The present invention was completed by further research based on this finding and includes the following embodiments.

[0010] Section 1. A method for producing Euglena capable of expressing a target foreign gene, comprising the step of inserting a double-stranded DNA with modified terminal regions that encodes at least the target foreign gene into the Euglena genome. Section 2. The method according to claim 1, wherein the terminal region is a region including at least one base from the terminal. Section 3. The method according to claim 1 or 2, wherein the modification is a change from a phosphodiester bond to a phosphorothioate bond. Section 4. The method according to any one of claims 1 to 3, wherein the double-stranded DNA is inserted so as to be placed under the control of an endogenous high-expression promoter in the Euglena genome. Section 5. The method according to item 4, wherein the double-stranded DNA is inserted by genome editing. Section 6. The method according to claim 4 or 5, wherein the endogenous high-expression promoter is the ALP1 promoter. Section 7. The method according to any one of claims 1 to 6, wherein the double-stranded DNA further includes a region encoding a drug resistance gene. Section 8. The method according to item 7, further comprising the step of culturing the Euglena obtained in the above step in the presence of the drug. Section 9. The method according to any one of items 1 to 8, wherein the double-stranded DNA is inserted into the Euglena genome by electroporation. Section 10. Euglena obtained by the method described in any one of items 1 to 8. [Effects of the Invention]

[0011] According to the present invention, Euglena that can retain an objective foreign gene in an expressible manner, and Euglena with improved expression level thereof can be obtained.

Brief Description of Drawings

[0012] [Figure 1] It is a schematic diagram of an example of transformation by introduction of phosphorothioate-modified (S-modified) DNA. [Figure 2] The results of verifying the effect of phosphorothioate modification in random gene introduction are shown. The vertical axis represents the luminescence intensity, which reflects the expression intensity of the introduced NanoLuc luciferase gene. The horizontal axis represents the number of subculture generations under drug selection (excluding the first generation). "Control" represents the control, "Nlucneo" represents a fusion gene of the PNO promoter, NanoLuc luciferase gene and the selection marker gene neor of G418, and double-stranded DNA containing the GAPDH terminator when expressed, and "S-Nlucneo" represents a double-stranded DNA containing a fusion gene of the PNO promoter, NanoLuc luciferase gene and neor, and GAPDH terminator, and both ends are phosphorothioated, when expressed, respectively. [Figure 3] The results of verifying that the integration of full-length DNA has been achieved in the transformant (6th generation isolated strain) by introduction of S-modified DNA are shown (the upper part is a schematic diagram of the introduced DNA, and the lower part shows the analysis results of the integrated DNA (size confirmation by electrophoresis and base sequence confirmation by sequencing), respectively). [Figure 4] The luciferase activity data in each strain isolated by the capillary method after random gene introduction of S-modified DNA containing the luciferase gene are shown. The vertical axis represents the luminescence intensity, and the horizontal axis represents the name of the isolated strain. The upper part shows a schematic diagram of the introduced DNA. [Figure 5]When the introduced DNA (expression cassette containing the PNO promoter) shown in Figure 4 and the expression cassette containing the CA (carbonic anhydrase) promoter (top panel) were randomly introduced and their respective expression intensities were examined, a higher expression intensity was observed when the DNA containing the CA promoter was introduced, as shown in the middle panel. This was thought to be a result of the high-expression promoter of the ALP1 gene at the insertion site functioning (bottom panel). [Figure 6] This is a schematic diagram of the gene targeting method in the example. [Figure 7] This is a schematic diagram of the state after gene targeting in the example. [Figure 8] This report shows the results of an investigation into the effect of phosphorothioate modification on gene targeting near the stop codon region of the ALP-1 gene. The vertical axis represents luminescence intensity, reflecting the expression intensity of the introduced NanoLuc luciferase gene. The horizontal axis, "RNP present," indicates the case where double-stranded DNA was cleaved during target integration, and "RNP absent," indicates the case where double-stranded DNA was not cleaved during target integration. "Control" represents the control group, "ALP1-Nlucneo" represents the case where double-stranded DNA containing a fusion gene of the NanoLuc luciferase gene and the G418 selection marker gene neor was expressed, and "S-modified ALP1-Nlucneo" represents the case where double-stranded DNA containing a fusion gene of the NanoLuc luciferase gene and neor, with both ends modified with phosphorothioate, was expressed. [Figure 9] This data shows luciferase activity in gene targeting of S-modified DNA containing the luciferase gene. The vertical axis represents luminescence intensity, and the horizontal axis represents the name of the isolated strain. [Modes for carrying out the invention]

[0013] 1. Manufacturing method of the present invention The present invention provides a method for producing Euglena that can express the target foreign gene, comprising the step of inserting double-stranded DNA with modified terminal regions that encodes at least the target foreign gene into the Euglena genome.

[0014] In this invention, Euglena can generally be any species belonging to the order Euglenales, and is not particularly limited. The species is not particularly limited, but examples include Euglena gracilis, Euglena gracilis var. bacillaris, Euglena viridis, Astasia longa, and Rapaza viridis.

[0015] In the present invention, Euglena gracilis is particularly preferred as the Euglena because (i) sterile strains can be easily obtained, and (ii) it can adapt to both heterotrophic and autotrophic growth environments.

[0016] The present invention is characterized by using double-stranded DNA with modified terminal regions that encode at least one target foreign gene. By using double-stranded DNA with modified terminal regions, deletion of the terminal region of the DNA during transformation can be suppressed, and as a result, the expression level of the target gene can be improved. The inventors' studies have shown that deletion during transformation is suppressed by performing terminal modifications known to confer nuclease resistance. Therefore, the mechanism of the above-mentioned deletion is assumed to be that the double-stranded DNA introduced during transformation is partially degraded by nucleases in the body. For this reason, the modification can be any modification that can confer nuclease resistance and is not particularly limited.

[0017] Examples of modifications include the conversion of phosphodiester bonds to phosphorothioate bonds, methylation modifications of nucleic acids (such as N6-methyladenine, O6-methylguanine, 5-methylcytosine, and O4-methylthymine), and Locked Nucleic Acid (trademark). The conversion of phosphodiester bonds to phosphorothioate bonds is preferred.

[0018] Double-stranded DNA in which phosphodiester bonds are replaced with phosphorothioate bonds in the terminal region can be prepared, for example, as follows: Determine the sequence of the double-stranded DNA to be introduced into the genome, and prepare a primer that is complementary to the terminal region of that sequence and in which the phosphodiester bonds are replaced with phosphorothioate bonds. By performing PCR using this primer and the above double-stranded DNA as a template, double-stranded DNA with modified terminal regions can be obtained.

[0019] The terminal region of the double-stranded DNA to be modified is not particularly limited, but it is preferably a region containing at least one base from the end, preferably a region containing at least three bases from the end, and more preferably a region containing at least five bases from the end. The purpose of the modification is solely to suppress deletion of the terminal region, and our studies have already shown that deletion is suppressed by modifying a region containing several bases from the end. Therefore, it is obvious that the same effect will be achieved even if a region containing several or more bases from the end is modified, and the extent to which the terminal region is extended can be set as appropriate.

[0020] In the manufacturing method of the present invention, it is preferable to insert the double-stranded DNA encoding the target gene so that it is positioned under the control of an endogenous high-expression promoter in the Euglena genome. Our studies have shown that this can further improve the expression level of the target gene.

[0021] By inserting the double-stranded DNA encoding the target gene under the control of an endogenous high-expression promoter in the Euglena genome, the target gene is thought to be highly expressed by the action of the endogenous high-expression promoter in the Euglena genome. Therefore, the endogenous high-expression promoter is not particularly limited and can be selected as appropriate. Examples of endogenous high-expression promoters include the ALP1 promoter (Acetylation lowers binding affinity-like protein-1 promoter), the actin promoter, and the GAPDH promoter. The ALP1 promoter is preferred as the endogenous high-expression promoter.

[0022] To place double-stranded DNA under the control of an endogenous high-expression promoter, the insertion site can be set as appropriate, but for example, the insertion site may be set near the stop codon region of the gene to be translated by the promoter.

[0023] Genome editing can be used as a means to insert double-stranded DNA encoding a target gene into the Euglena genome so that it is placed under the control of an endogenous high-expression promoter. For example, the CRISPR / Cas system can be used for genome editing. Specific examples of the CRISPR / Cas system include CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas type1-D. CRISPR-Cas9 is preferred as the CRISPR / Cas system.

[0024] In the manufacturing method of the present invention, it is preferable that the double-stranded DNA further includes a region encoding a drug resistance gene. By using this to select a drug, Euglena that can express the target gene can be efficiently selected. Specifically, the manufacturing method of the present invention in the above embodiment further includes a step of culturing the Euglena obtained in the above step in the presence of the drug.

[0025] The drug resistance gene is not particularly limited as long as it can serve as an effective drug selection marker for Euglena, but examples include resistance genes to G418, hygromycin, and zeosin. The drug resistance gene is preferably a resistance gene to G418.

[0026] Examples of G418 resistance genes include Klebsiella pneumoniae Neomycin-kanamycin phosphotransferase type II (nptII), but any gene with equivalent function is acceptable and is not limited to this. Examples of zeosin resistance genes include Streptoalloteichus hindustanus bleomycin resistance gene (Sh ble), but any gene with equivalent function is acceptable and is not limited to this.

[0027] While the conditions for this culture are not particularly limited, it is preferable to culture at around pH 5 when using G418 or hygromycin as the drug. Furthermore, when using zeosin as the drug, it is preferable to culture at pH 6-8. Although pH around 5.0 is generally considered advantageous for Euglena growth, culturing under these conditions is advantageous because it allows for more stable zeosin production and more efficiently obtains Euglena that can express drug resistance genes and target foreign genes.

[0028] When using drugs that are stable against acids and bases, the culture conditions can be set without being particularly affected by pH conditions.

[0029] The above culture conditions are not particularly limited, but examples include the following: Culture is performed on KH plate selective medium. The drug concentration in the medium is not particularly limited, but when introducing the G418 resistance gene, an example is 10-100 μg / ml of G418. Furthermore, other drugs may be added to the medium as needed. Although not particularly limited, when introducing the G418 resistance gene, for example, 50-500 μg / ml of cefotaxime may also be added to the medium. This is advantageous because it prevents bacterial contamination during the procedure.

[0030] The number of cells at the start of culture is not particularly limited, but for example, 1 × 10⁶ 4 ~1 × 10 8 For cells and other organisms that undergo an isolation process, examples include 1 to 5 cells.

[0031] The culture period is not particularly limited, but examples include 2 to 7 days.

[0032] The drug selection process may be performed only once, or it may be repeated two or more times as needed. If repeated two or more times, the number of cells at the start of culture may be gradually reduced as needed, although this is not particularly limited. For example, it may be reduced to about one-fifth to one-half of the number at the previous stage.

[0033] The target foreign gene is not particularly limited. A desired foreign gene can be selected according to each objective. For example, pyruvate:NADP+ oxidoreductase can be selected to enhance Euglena cell proliferation or wax ester fermentation. By increasing the expression level of pyruvate:NADP+ oxidoreductase, the amount of acetyl-CoA in mitochondria can be increased.

[0034] Furthermore, 3-ketoacyl-CoA thiolase can be selected to homogenize the wax esters produced by Euglena. By increasing the expression level of 3-ketoacyl-CoA thiolase, the maximum amount of acyl-CoA that can be accumulated for the longest period of reaction can be increased.

[0035] Furthermore, citrate synthase can be selected to control the amount of carbon influx into the TCA cycle in Euglena. By increasing the expression level of citrate synthase, the amount of storage polysaccharides that serve as raw materials for wax esters within cells can be increased.

[0036] Furthermore, by increasing the expression level of 2-oxoglutarate decarboxylase, the only irreversible reaction in the TCA cycle, which is aimed at facilitating carbon metabolism in Euglena, cell growth can be promoted.

[0037] In the present invention, "retaining the gene in an expressible state" means, without any particular limitations, that the drug resistance gene and the target foreign gene are retained in an expressible state for at least 10 generations in subculturing in the absence of the drug against which the introduced drug resistance gene is resistant. In the above, the number of generations in subculturing is more preferably 15 generations, and even more preferably 20 generations. Whether or not the gene is retained in an expressible state can be confirmed by performing RT-PCR on mRNA extracted from Euglena using a primer capable of amplifying the gene.

[0038] In the present invention, it is preferable that the drug resistance gene and the target foreign gene are under the control of at least the Euglena endogenous promoter. The Euglena endogenous promoter is not particularly limited, but examples include pyruvate:NADP+ oxidoreductase, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, bifunctional glyoxylate pathway enzyme, and α-tubulin. Pyruvate:NADP+ oxidoreductase is particularly preferred.

[0039] In the present invention, it is preferable that the drug resistance gene and the target foreign gene are incorporated into the genome. While not particularly limited, it is preferable that they are incorporated into the genome by homologous recombination. Whether or not they are incorporated into the genome can be confirmed by Southern blotting, but in some cases the copy number of the introduced gene is not sufficiently high and a clear signal cannot be obtained by Southern blotting. In such cases, RT-PCR can be performed on mRNA extracted from Euglena that has been continuously subcultured for 20 generations in the absence of the drug to which the introduced drug resistance gene is resistant, using primers capable of amplifying the introduced gene. If the target sequence is amplified, it can be determined that the gene has been incorporated into the genome. In other words, the detection of the introduced gene by RT-PCR from cells that have been continuously subcultured in the absence of selective pressure indicates a high probability that the introduced gene has been incorporated into the genome. [Examples]

[0040] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0041] 1. Biological samples We used Euglena gracilis strain Z (wild strain Euglena).

[0042] 2. Culture method Euglena were cultured in mixed nutrition using the heterotrophic medium Koren-Hutner medium (KH medium) (Koren and Hutner 1967) (Table 1). 3 mL of KH medium, adjusted to pH 5.0, was dispensed into test tubes and sterilized by autoclaving at 121°C for 15 min. Euglena (1.2-1.8 × 10¹⁶) that reached the stationary phase after approximately 7 days of culture were added to this medium. 7 60 μL of (cells / mL) was inoculated and cultured with shaking at 27°C under continuous light irradiation for 24 hours. Cells cultured aerobically on day 4, corresponding to the mid-to-late phase of logarithmic growth, were used for gene transfer experiments.

[0043] [Table 1]

[0044] 3. Measurement of cell count Cells were fixed by mixing Euglena culture medium and Lugol's solution in a 1:1 ratio. After this cell solution was diluted as needed, the number and volume of cells were measured using a particle analyzer CDA-1000 (Sysmex). The cell solution was diluted using Cellpack solution (Sysmex). 10x Lugol's solution: 1 g of iodine Potassium iodide 2 g Fill up with 300 mL of H2O.

[0045] 4-1. Preparation of DNA fragments used for random gene transfer The plasmid DNA used as the PCR template was the same as that used in Nakazawa et al. (2023) Algal Res. The promoter sequence and Nluc-neo were added to the multi-cloning site of the pCMV / Zeo plasmid using in-fusion cloning. r After inserting the sequence and terminator sequence, the DNA was amplified by PCR using 1594Fw primer (5′-ACACCGAACTGAGATACCTACAGCGTGAGC-3′ (SEQ ID NO: 1)) and 3348Rv primer (5′-GAGAAAATACCGCATCAGGCGCCATTCGC-3′ (SEQ ID NO: 2)) and FavorPrep TM The DNA was purified using a GEL / PCR Purification Kit (Favorgen). For the preparation of modified DNA, S-1594Fw primer (5′-A*C*A*C*C*GAACTGAGATACCTACAGCGTGAGC-3′ (SEQ ID NO: 1)) and S-3348Rv primer (5′-G*A*G*A*A*AATACCGCATCAGGCGCCATTCGC-3′ (SEQ ID NO: 2)) (asterisks indicate phosphorothioate modification) were used, with the rest of the procedure being the same as when using unmodified oligonucleotides.

[0046] 4-2. Preparation of DNA fragments for gene targeting The plasmid DNA used as the PCR template was the same as that used in Nakazawa et al. (2023) Algal Res. An oligo DNA with a homologous sequence (any number of bases from 5 to 30; in this example, a 20-base homologous sequence) near the genome break site at its 5′ end was used for Nluc-neo r The gene region was amplified by PCR (excluding the promoter and terminator regions). The primers used in the example are as follows: S-ALP1_nostopFw-NlucStartFw primer (5′-T*G*A*G*G*ATTTTTTTGCCGTCTCACAGCTATGGTGTTCACCTTGGAGGACT-3′(Sequence No. 3)), S-ALP1_stopRv-NeoStopRv primer (5′- A*T*C*A*G*GAGTATCAAGCTGTGTCAGAAGAACTCGTCAAGAAG-3′(Sequence No. 4))

[0047] 5. Measurement of DNA concentration The concentration of purified DNA was measured using a double-stranded DNA (dsDNA) specific fluorescent reagent. The reagent used was the QuantiFluor One dsDNA system (Promega), and a lambda DNA standard of 400 ng / μl was used as the standard DNA. Measurements were performed using a Quantus Fluorometer according to the protocol. If the sample DNA concentration was higher than that of the standard DNA, it was diluted with ultrapure water before measurement.

[0048] 6. GrRNA-mediated cleavage of genomic DNA In the example, a CRISPR RNA with a target sequence near the stop codon of the genomic DNA region encoding Alba-like protein 1 (ALP1), which the inventor identified as a candidate high-expression gene from the Euglena genome (Figure 5), was designed, and Alt-R modified crRNA was synthesized by Integrated DNA Technology (IDT). The target genome sequence used was 5′-AGGAGTATCAAGCTGTGAGA-3′ (Sequence ID 5).

[0049] 7. Preparation of ribonucleotide-Cas9 complexes Ribonucleotide-Cas9 complexes were prepared according to the STAR protocol of Nomura et al. (2020). 0.6 μl of Art-R modified crRNA and 0.6 μl of Art-R modified tracrRNA were mixed in a plastic tube, heat-treated at 95°C for 5 minutes, and then cooled to room temperature at a rate of 1°C / second. The cooled samples were treated with IDT's Alt-R TM 0.8 μl of Sp HiFi Cas9 Nuclease V3 was added, and the mixture was allowed to stand at 20°C for 15 minutes. After that, it was stored at -20°C until use.

[0050] 8-1. Introduction of dsDNA by electroporation 3-5 μg of dsDNA prepared according to method 4-1 was mixed with Euglena cells in an electroporation cuvette, and the DNA was introduced into the cells by electrical pulses using the method of Nakazawa et al. (2023) Algal Research. After more than 24 hours had elapsed since electroporation, a portion of the cells were subcultured in fresh Koren-Hutner medium and grown (first-generation cells). Furthermore, a portion of the cells were subcultured in Koren-Hutner medium containing 10 μg / ml of G418 sulfate as a selective agent, and the growth to the stationary phase was repeated. The analysis described below was performed using cells in the stationary phase.

[0051] 8-2. Simultaneous introduction of ribonucleotide Cas9 complex and dsDNA by electroporation 300-700 ng of dsDNA prepared according to method 4-2, along with ribonucleotide-Cas9 complexes and Euglena cells, were mixed in an electroporation cuvette, and electrical pulses were applied according to the STAR protocol described by Nomura et al. (2020). The electroporation apparatus and conditions used were those described by Nagamine et al. (2024) Bioresource Technology. After electroporation, the cells were cultured in the dark and rotated for at least two days, and then a portion was subcultured in Koren-Hutner medium and grown until the stationary phase (first-generation cells). Furthermore, a portion was subcultured in Koren-Hutner medium containing 10 μg / ml of G418 sulfate as a selective agent, and this process was repeated until the stationary phase was reached. The analyses described below were performed using cells in the stationary phase.

[0052] 9. Luminescence measurement using Nano-Glo assay Measurements were performed according to Nakazawa et al. (2023) Algal Research. Liquid cultures containing transformants were placed in 250 μl plastic tubes and sonicated on ice. The resulting cell lysates were centrifuged at 17500 g for 1 minute, and 25 μl of the supernatant was used for luciferase activity measurement using a Nano-Glo lucirefase assay system (Promega). Luminescence was measured using a Turner Biosystems 20 / 20n Luminometer. The obtained luminescence intensity data were standardized using separately measured cell concentration data.

[0053] 10. Isolation of transformed organisms and genomic DNA analysis Transformants were isolated using the limiting dilution method, which involves diluting cells 2-fold in each row of a 96-well plate, or by visual isolation under a microscope using glass capillaries. The isolated cells were grown by static culture in Koren-Hutner medium containing 10 μg / ml of G418 sulfate. Cells showing sufficient growth under drug selection were used as candidate transformants for subsequent analyses.

[0054] Nluc-neo is used as the foreign DNA for the candidate transformant.r Whether the gene was inserted was determined by genomic PCR (using Takara ExPremire DNA polymerase) with Euglena crude extract (e.g., Kaneka Simple DNA Extraction Kit Version 2) as a template to amplify the full length of the Nluc-neo gene. r Furthermore, to determine whether the DNA up to the terminal regions of the promoter and terminator regions of the introduced DNA was retained, the product obtained by PCR using 1594Fw primer (5′-ACACCGAACTGAGATACCTACAGCGTGAGC-3′ (SEQ ID NO: 1)) and 3348Rv primer (5′-GAGAAAATACCGCATCAGGCGCCATTCGC-3 (SEQ ID NO: 2)′) with the Euglena crude extract prepared in the same manner as a template was subjected to Sanger sequencing for confirmation.

[0055] Test results The effect of phosphorothioate modification (S modification) in random gene introduction was verified. A schematic diagram of the transformation is shown in Fig. 1. As shown in Fig. 2, higher luminescence intensity was confirmed in the group introduced with S-modified DNA. Specifically, under drug selection, it was about 10,000 times that of the unmodified in the third generation.

[0056] It was confirmed that the integration of the full-length DNA was achieved in the transformant (6th generation isolated strain) by the introduction of S-DNA (Fig. 3). It was confirmed that strains integrated in full length could be easily obtained by the introduction of S modification.

[0057] When the luciferase activity in the random gene introduction of S-DNA containing the luciferase gene was measured, the luminescence intensity of the isolated strain was at the same level as that of the cells in the third generation after DNA introduction grown under drug selection without isolation (Fig. 2), on the order of 10 8 orders (Fig. 4).

[0058] When the introduced DNA (expression cassette containing the PNO promoter) shown in Figure 4 and the expression cassette containing the CA (carbonic anhydrase) promoter (upper panel of Figure 5) were randomly introduced and their respective expression efficiencies were examined, a higher expression efficiency was observed when the DNA containing the CA promoter was introduced, as shown in the middle panel of Figure 5. This was thought to be a result of the high-expression promoter of the ALP1 gene at the insertion site functioning (lower panel of Figure 5).

[0059] Based on these results, gene targeting was performed on the region near the stop codon of the ALP-1 gene. A schematic diagram of the gene targeting method is shown in Figure 6, and a schematic diagram of the state after gene targeting is shown in Figure 7. The results of verifying the effect of phosphorothioate modification are shown in Figure 8. It was confirmed that integration into the high-expression region was successful, and the proportion of transformants increased by approximately 10 times due to S conversion (Figure 8). Strains showing higher luminescence intensity than before were easily isolated (Figure 9).

Claims

1. A method for producing Euglena capable of expressing a target foreign gene, comprising the step of inserting a double-stranded DNA with modified terminal regions that encodes at least the target foreign gene into the Euglena genome.

2. The method according to claim 1, wherein the terminal region is a region including at least one base from the terminal.

3. The method according to claim 1 or 2, wherein the modification is a change from a phosphodiester bond to a phosphorothioate bond.

4. The method according to claim 1 or 2, wherein the double-stranded DNA is inserted so as to be placed under the control of an endogenous high-expression promoter in the Euglena genome.

5. The method according to claim 4, wherein the double-stranded DNA is inserted by genome editing.

6. The method according to claim 4, wherein the endogenous high-expression promoter is the ALP1 promoter.

7. The method according to claim 1 or 2, wherein the double-stranded DNA further includes a region encoding a drug resistance gene.

8. The method according to claim 7, further comprising the step of culturing the Euglena obtained in the above step in the presence of the drug.

9. The method according to claim 1 or 2, wherein the double-stranded DNA is inserted into the Euglena genome by electroporation.

10. Euglena obtained by the method described in claim 1 or 2.

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