STRAIN CAPABLE OF PRODUCING BUTYRIC ACID

Abstract

A strain that efficiently produces butyric acid and a method for producing butyric acid using the strain. Clostridium beijerinckii SIID27451-B11 strain (Accession No. NITE BP-02951), produces more butyric acid and less lactic acid and acetic acid in a culture solution after anaerobically culturing the strain at 37° C. for 72 hours, compared with Clostridium beijerinckii NBRC 109359T, which is a type strain of Clostridium beijerinckii.

Claims

1. A Clostridium beijerinckii SIID27451-B 11 strain (accession number NITE BP-02951).

2. The Clostridium beijerinckii SIID27451-B11 strain according to claim 1, having genome sequences shown in sequence numbers 1 to 5.

3. The Clostridium beijerinckii SIID27451-B11 strain according to claim 1, having a 16S rDNA sequence shown in sequence number 6.

4. The Clostridium beijerinckii SIID27451-B11 strain according to claim 1, which produces more butyric acid and less lactic acid and acetic acid in a culture solution after anaerobically culturing the strain at 37 ° C. for 72 hours, compared with Clostridium beijerinckii NBRC 109359T, which is a type strain of Clostridium beijerinckii.

5. A method for producing butyric acid using SIID27451-B11 strain according to claim 1.

6. The method for producing butyric acid using SIID27451-B11 strain according to claim 2.

7. The method for producing butyric acid using SIID27451-B11 strain according to claim 3.

8. The method for producing butyric acid using SIID27451-B11 strain according to claim 4.

Description

BRIEF DESCRIPTION OF DRAWING

[0012] FIG. 1 is a photograph showing a colony image of SIID27451-B11 strain.

[0013] FIG. 2 is a photograph showing a Gram staining image of SIID27451-B11 strain

[0014] FIG. 3 shows a simple molecular phylogenetic tree based on a 16S rDNA partial sequence of SIID27451-B11 strain.

DESCRIPTION OF EMBODIMENTS

[0015] The taxonomic characteristics of SIID27451-B11 strain were examined. Hereafter, SIID27451-B11 strain will be also referred to as B11 strain. All the following tests were performed at TechnoSuruga Laboratory Co, Ltd. unless otherwise noted.

[0016] (1) Morphological Observation

[0017] Anaerobic culture of B11 strain was performed at 30° C. for 48 hours using GAM Broth “Nissui” (Nissui Pharmaceutical, Japan)+agar as a culture medium. Stereomicroscopic colony observation and Gram staining were performed to observe colonies and cell morphology of specimens. The colony and Gram staining images are shown in FIG. 1 and FIG. 2, respectively. The cell morphology was Bacillus to oval Bacillus (1.0-1.5×3.0-5.0 μm), Gram staining was positive, and spore formation was observed. Colonies were cream in color.

[0018] (2) Physiological and Biochemical Properties

[0019] Anaerobic culture of B11 strain was performed at 30° C. for 48 hours using GAM Broth “Nissui” (Nissui Pharmaceutical, Japan)+agar as a culture medium. An anaerobic biochemical identification kit, API20A (bioMerieux, FRA) was used to perform tests. Test items and results are shown in Table 1.

TABLE-US-00001 TABLE 1 Test Items and results Test Items Reaction/Enzyme Results IND Indole Production* − URE Urease* − GLU Glucose** + MAN D-Mannitol** + LAC Lactose** + SAC Saccharose** + MAL Maltose** + SAL Salicin** + XYL D-Xylose** + ARA L-Arabinose** + GEL Gelatin hydrolysis* − ESC Esculin hydrolysis* + GLY Glycerin** + CEL D-Cellobiose** + MNE D-Mannose** + MLZ D-Melezitose** − RAF D-Raffinose** − SOR D-Sorbitol** + RHA L-Rhamnose** − TRE D-Trehalose** + CAT Catalase* + SPOR Spore + GRAM Gram staining + COCC Coccus − *Biochemical test, **Oxidation test +: Positive, −: Negative

[0020] In addition, additional tests were conducted on the following items. The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Additional Tests Test Items Results Starch hydrolysis + Growth in the presence of 5% NaQ + Growth in the presence of 6.5% NaQ + Growth in the presence of 7% NaQ + Milk coagulation test Coagulation +: Positive, −: Negative

[0021] (3) 16S rDNA Partial Sequence Analysis

[0022] The attribution of the specimen was inferred from the results of the analysis of 16S rDNA (16S rRNA gene) partial sequence (approximately 1500 bp) (Sequence No. 6). Anaerobic culture of B11 strain was performed at 30° C. for 7 days using GAM Broth “Nissui” (Nissui Pharmaceutical, Japan)+agar as a culture medium. The 16S rDNA partial sequence analysis was performed using the following conditions. [0023] DNA Extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan) [0024] PCR amplification: TKs Gflex DNA Polymerase (Takara Bio, Japan) [0025] Cycle Sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) [0026] Primer used

[0027] PCR amplification: 9F, 1510 R

[0028] Sequencing: 9F, 515F, 1099F, 536R, 926R, 1510R [0029] Sequencing: ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems) [0030] Determination of base sequence: ChromasPro 2.1 (Technelysium, AUS) [0031] BLAST homology search:

[0032] Analysis Software: ENKI (TechnoSuruga Laboratory, Japan)

[0033] Database: [0034] DB-BA14.1 (TechnoSuruga Laboratory) [0035] International Nucleotide Sequence Database (DDBJ/ENA (EMBL)/GenBank) [0036] Search date: Feb. 22, 2019 [0037] Simple molecular phylogenetic tree analysis:

[0038] Phylogenetic tree estimation: neighbor-joining method

[0039] Base substitution model: Kimura-2-parameter

[0040] Reliability evaluation of tree form: Bootstrap method (1,000 iterations)

[0041] The results are shown in Tables 3 and 4.

TABLE-US-00003 TABLE 3 BLAST search result of B11 strain on DB-BA: Homology to top 30 sequence Registered Name Strain name Accession No. Homology BSL Clostridium beijerinckii JCM1390 AB643462 1433/1437 (99.7%) Clostridium diolis DSM5431 AJ458418 1432/1437 (99.7%) Clostridium chromiireducens GCAF-1 AY22834 1418/1437 (98.7%) Clostridium saccharoperbutylacetonicum N1-4 U16122 1403/1417 (99.0%) Clostridium puniceum DSM2619 X71857 1417/1438 (98.5%) Clostridium saccharobutylicum NCP262 U16147 1394/1417 (98.4%) Clostridium butyricum JCM1391 AB595129 1405/1440 (97.6%) Clostridium paraputrificum JCM1293 AB536771 1385/1440 (96.2%) 1* Clostridium chatatabidum DSM5482 X71850 1378/1437 (95.9%) Clostridium uliginosum CK55 AJ276992 1368/1421 (96.3%) Clostridium sardiniense DSM2632 AB161367 1381/1441 (95.8%) Clostridium vincentii lac-1 X97432 1374/1439 (95.5%) Clostridium saudiense JCC HG726039 1375/1441 (95.4%) Clostridium monoliforme JCM9990 AB540985 1375/1441 (95.4%) 1* Clostridium baratii ATCC27638 X68174 1375/1441 (95.4%) 1* Clostridium tertium JCM6289 AB618789 1374/1441 (95.4%) 1* Clostridium sartagoforme DSM1292 Y18175 1372/1441 (95.2%) Clostridium disporicum DSM5521 Y18176 1372/1443 (95.1%) 1* Clostridium colicanis DSM13634 AJ420008 1365/1437 (95.0%) Clostridium gasigenes DSM12272 AF092548 1365/1441 (94.7%) Eubacterium tarantellae DSM3997 FR733677 1362/1439 (94.6%) 1* Clostridium quinii DSM6736 X76745 1365/1441 (94.7%) Eubacterium budayi JCM9989 AB018183 1364/1441 (94.7%) Clostridium chauvoei ATCC10092 U51843 1361/1442 (94.4%) 2  Clostridium septicum ATCC12464 U59278 1360/1442 (94.3%) 2  Eubacterium multiforme JCM6484 AB018184 1359/1440 (94.4%) Clostridium algidicarnis NCFB2931 AF127023 1349/1438 (93.8%) Eubacterium nitritogenes JCM6485 AB018185 1354/1437 (94.2%) 1* Clostridium putrefaciens DSM1291 AF127024 1348/1438 (93.7%) Clostridium carnis ATCC25777 M59091 1339/1431 (93.6%) 1* Note 1: BSL (Biosafety Level) shows Level 1* (opportunistic pathogens) or higher; blank cell means Level 1. Note 2: Shading indicates sequence data used for simple molecular phylogenetic analysis.

TABLE-US-00004 TABLE 4 BLAST search result of B11 strain on International Nucleotide Sequence Database: Homology to top 30 sequence Registered Name Strain Name Accession No. Homology Clostridium beijerinckii — LN908213 1435/1437 (99.9%) Clostridium beijerinckii AAU1 KC433940 1435/1437 (99.9%) Clostridium beijerinckii JCM 8031 AB678394 1435/1437 (99.9%) Clostridium beijerinckii JCM 7990 AB678386 1435/1437 (99.9%) Clostridium beijerinckii JCM 6287 AB640693 1435/1437 (99.9%) Clostridium beijerinckii JCM 7829 LC071788 1435/1437 (99.9%) uncultured Clostridium sp. — MF360200 1434/1437 (99.8%) Clostridium beijerinckii JCM 7837 LC258130 1434/1437 (99.8%) Clostridium beijerinckii B17 KC915012 1433/1435 (99.9%) Clostridium beijerinckii NRRL B-598 CP011966 1434/1437 (99.8%) Clostridium sp. MF28 CP014331 1434/1437 (99.8%) Clostridium beijerinckii BAS/B3/I/124 CP016090 1434/1437 (99.8%) Clostridium beijerinckii B12-KKU KT799795 1434/1437 (99.8%) Clostridium beijerinckii NCIMB 14988 CP010086 1434/1437 (99.8%) Clostridium beijerinckii JCM 7847 AB971810 1434/1437 (99.8%) Clostridium sp. G117 JX091678 1434/1437 (99.8%) Clostridium beijerinckii JCM 8028 AB678392 1434/1437 (99.8%) Clostridium beijerinckii JCM 8026 AB647333 1434/1437 (99.8%) Clostridium butyricum JCM 7840 AB647330 1434/1437 (99.8%) subsp. convexa Clostridium beijerinckii RZF1108 GQ375085 1434/1437 (99.8%) Clostridium beijerinckii SA-1 CP006777 1433/1437 (99.7%) Clostridium beijerinckii E102 JX267117 1433/1437 (99.7%) Clostridium beijerinckii E080 JX267098 1433/1437 (99.7%) Clostridium beijerinckii JCM 8025 AB647332 1433/1437 (99.7%) Clostridium beijerinckii NCIMB 8052 CP000721 1433/1437 (99.7%) Clostridium diolis S33-KKU KT799798 1431/1434 (99.8%) Clostridium beijerinckii JCM 1390 NR_113388 1433/1437 (99.7%) Clostridium beijerinckii CP23-KKU KT799797 1430/1433 (99.8%) Clostridium beijerinckii E11-KKU KT799794 1430/1433 (99.8%) Clostridium beijerinckii E092 JX267108 1432/1437 (99.7%)

[0042] A simple molecular phylogenetic tree based on the 16S partial rDNA sequence of B 11 strain is shown in FIG. 3. In the figure, the upper left line indicates a scale bar, and 0.01 on the scale bar means that the length of the scale bar indicates 1% base difference. The numbers on the branches indicate bootstrap values, which indicates the reliability of the tree, the T at the end of the strain name indicates the Type strain of the species, and BSL indicates the biosafety level (BSL1* (opportunistic pathogen) or higher is indicated). As can be seen in FIG. 3, B11 strain is estimated to be closely related to Clostridium beijerinckii and Clostridium diolis.

[0043] (4) Deposition of Strains

[0044] Based on the above results, SIID 27451-B11 strain was found to be a strain belonging to Clostridium. SIID 27451-B11 strain was domestically deposited to National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (#122, 2-5-8, KazusaKamatari, Kisarazu-shi, Chiba 292-0818, Japan), which is an International Depositary Authority (IDA) under the terms of the Budapest Treaty, under Accession Number NITE P-02951 on May 16, 2019 and have been transferred to the international deposit on Apr. 22, 2020 in the same institute under International Accession Numbers NITE BP-02951.

Type of microorganism: bacteria
Cell form: Bacillus
Characteristics: spore-forming positive (spore-forming)
Taxonomical position: Clostridium sp.
Culture condition: GAM Broth “Nissui”+agar
Culture temperature: 30° C.
Culture period: 48 hours
Culture method: anaerobic

[0045] (5) ANI (Average Nucleotide Identity) Analysis

[0046] Furthermore, to examine whether B11 strain is a new species, Average Nucleotide Identity (ANI) value is calculated for the top three species with the highest homology to B11 strain, namely Clostridium beijerinckii (homology 99.7%), Clostridium diolis (homology 99.7%), and Clostridium saccharoperbutylacetonicum (homology 99.0%) to determine whether the species are same or different. In ANI analysis a similarity (ANI: homology value) between a full-length genome sequence or draft genome sequence of a control strain (specimen) and a comparison strain is calculated on a computer to determine whether the species are same or different. The ANI value is calculated by using the publicly available program ANI Calculator (http://enve-omics.ce.gatech.edu/ani/index), and if the ANI value is 95% or greater, the species are determined to be the same, and if the ANI value is less than 95%, the species are determined to be different (new species).

[0047] The comparative species used in the ANI analysis are listed in Table 5 below.

TABLE-US-00005 TABLE 5 Species used in ANI analysis GenBank SIID Species Name Strain Name* Accession No. 27451-02 Clostridium beijerinckii DSM 791.sup.T GCA_002006445 27451-03 Clostridium diolis DSM 15410.sup.T GCA_008705175 27451-04 Clostridium N1-4.sup.T GCA_000340885 saccharoperbutylacetonicum *.sup.T at the end of strain name: Type strain

[0048] The results of the ANI analysis are shown in Table 6 below.

TABLE-US-00006 TABLE 6 ANI values (%) between specimens SIID27451-B11 Clostridium beijerinckii 95.81 (SIID27451-02) Clostridium diolis 95.76 (SIID27451-03) Clostridium 82.59 saccharoperbutylacetonicum (SIID27451-04) Clostridium beijerinckii (SIID27451-02) Clostridium diolis 98.47 (SIID27451-03)

[0049] Since the ANI value for SIID27451-B11 strain to Clostridium saccharoperbutylacetonicum is less than 95%, SIID27451-B11 strain and Clostridium saccharoperbutylacetonicum are determined to be different species. SIID27451-B11 strain provides the ANI value of 95% or greater to Clostridium beijerinckii and Clostridium diolis, respectively. The combination of Clostridium beijerinckii and Clostridium diolis also provides the ANI value of 95% or greater. Therefore, the above three specimens are determined to be the same species. It should be noted that, according to a report of Kobayashi et al. at 2020 (Kobayashi H, et al. Int J Syst Evol Microbiol 2020, 70: 2463-2466), Clostridium diolis was shown to be the same species as Clostridium beijerinckii, and the scientific names have been integrated into Clostridium beijerinckii. Based on the above, SIID27451-B11 strain was identified as Clostridium beijerinckii.

[0050] (6) Genome Analysis

[0051] DNA of SIID27451-B11 strain was extracted and subjected to next-generation sequencing and its data analysis.

<Extraction and Purification of DNA>

[0052] DNA of B11 strain was extracted and purified using Nucleobond AXG20 column (MACHEREY-NAGEL, DE), and a purity of the extracted DNA was measured using ultra micro-volume spectrophotometer NanoDrop One (Thermo Fisher Scientific, USA). The quality inspection acceptance criteria were as follows: purity: A260 (absorbance at 260 nm, the same hereinafter)/A280≥1.8 and A260/A230≥1.6, and 20 μg (concentration of 150 ng/μL) or more.

[0053] Next, fragmentation of the extracted DNA was confirmed by agarose electrophoresis. The setting conditions were 0.5% agarose, 30 V, and 1 hour. The electrophoresis results showed that, compared with the DNA molecular weight marker (k-Hind III digest), a band was observed around 23130 bp for the extracted DNA, and thus the extracted DNA was determined to be of good quality for use in next-generation sequencing because of its low fragmentation.

[0054] <Next Generation Sequencing>

[0055] Next-generation sequencing and its data analysis were commissioned to Hokkaido System Science Co., Ltd.

[0056] Extracted and purified DNA samples of SIID27451-B11 strain were subjected to next-generation sequencing using next-generation sequencer PacBio RS II to obtain genome sequence information.

[0057] The genome sequence information of Clostridium beijerinckii (C. beijerinckii) type strain (GCA_002006445.1) was obtained from NCBI (National Center for Biotechnology Information, U.S.A.) web site (https ://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/002/006/445/GCA_002006445.1_ASM200644v1).

[0058] The genome sequences information of SIID27451-B11 and Clostridium beijerinckii type strains were auto-annotated with “DFAST”, an automated annotation program for prokaryotes provided by National Institute of Genetics.

[0059] Based on the annotation information of the analyzed SIID27451-B11 and Clostridium beijerinckii type strains, draft genome sequences are compared between these two strains utilizing COG (Clusters of Orthologous Groups of protein) functional classification information. COG is a genetic function classification database for microorganisms provided by NCBI.

[0060] Table 7 shows a summary of the genomic information of the Clostridium beijerinckii type strain used, and Table 8 shows a summary of the genomic information of SIID27451-B11 strain.

TABLE-US-00007 TABLE 7 Sample Name: Clostridium beijerinckii Strain DSM 791 (Type Strain) Sample ID: GCA_002006445.1 Species Name: Clostridium beijerinckii Data Acquired from: NCBI (※1) GCA_002006445.1 Seq Name Type Length GC (%) Number of Contigs 264 sequence001 linear 148,805 30.0 Total Size (bp) 5,781,472 sequence002 linear 121,086 29.6 GC (%) 29.7 sequence003 linear 118,978 29.4 N50 Contig Size (bp) 43,059 sequence004 linear 113,134 30.0 Max Contig Size (bp) 148,805 sequence005 linear 107,222 30.3 Min Contig Size (bp) 512 ※ Only Top 5 in Config Size are selected ※

[0061] The 264 contig sequences of the Clostridium beijerinckii type strain shown in Table 7 are referred as sequence Nos. 7 to 270, respectively.

TABLE-US-00008 TABLE 8 Sample Name: 27451-B11 Sample ID: Ig18351 Species Name: Clostridium beijerinckii 27451-811 (Ig18351) Seq Name Type Length GG (%) Number of Contigs 5 sequence1 Circular 6,239,980 29.9 Total Size (bp) 6,372,138 sequence2 linear 45,420 29.7 GC (%) 29.9 sequence3 linear 34,111 29.3 N50 Contig Size (bp) 6,239,980 sequence4 linear 29,427 29.3 Max Contig Size (bp) 6,239,980 sequence5 linear 23,200 29.9 Min Contig Size (bp) 23,200 Total 6,372,138 29.9

[0062] Five contig sequences 1 to 5 of SIID27451-B11 strain shown in Table 8 are referred as Sequence Nos. 1, 2, 3, 4, and 5, respectively.

[0063] Table 9 shows the predicted number of genes (ORF/rRNA/t RNA) for each strain detected by the annotation processing using DFAST.

TABLE-US-00009 TABLE 9 Predicted number of genes for each strain Sample Name Sample ID ORF rRNA tRNA Clostridium beijerinckii GCA_002006445.1 5,132 15 86 strain DSM 791 (Type Strain) 27451-B11 Ig18351 5,722 46 94

[0064] Next, among all the ORF information detected by DFAST, we focused on the COG annotated ORF information, compared the information of the genes possessed by the type strain and B11 strain respectively, wherein the COG functional classifications of the genes have become apparent, and extracted the COG annotation information specifically detected only one of these two strains. Table 10 collectively shows the COG annotation information specifically detected for each of the Clostridium beijerinckii type strain and SIID27451-B11 strain.

TABLE-US-00010 TABLE 10 Comparison of annotation information of type strain and B11 strain indicates data missing or illegible when filed

[0065] From the above results, it can be seen that the Clostridium beijerinckii type strain and SIID27451-B11 strain have sequences with different annotations. This indicates that SIID27451-B11 strain is presumed to be a strain having different characteristics from the type strain of Clostridium beijerinckii species.

[0066] As mentioned above, as a result of considering the taxonomic characterization of B11 strain, B11 strain is identified as a Clostridium beijerinckii species, while genome analysis revealed that B11 strain has sequences with different annotation from that of the type strain. Therefore, to further confirm that B11 strain is a strain with different characteristics from the type strain of Clostridium beijerinckii, we compared B11 strain, a type strain of Clostridium butyricum, which is a representative butyric acid-producing bacterium, and the type strain of Clostridium beijerinckii for their ability to produce organic acids. Details are shown in Example 1 below.

EXAMPLES

Example 1: Test for Organic Acid-producing Ability

[0067] Each strain was anaerobically cultured at 37° C. for 72 hours using GAM Broth (Nissui Pharmaceutical, Japan) as a culture medium. The culture solution was filtered through a membrane filter with a pore size of 0.20 μm and used as a sample solution. The concentration of organic acids in the sample solution was determined by high-performance liquid chromatography. This test was conducted on February 13, 2020, at T TechnoSuruga Laboratory Co, Ltd. The measurement conditions were as follows: [0068] System: Shimadzu Organic Acid Analysis System (Shimadzu, Japan) [0069] Columns: Shim-pack SCR-102(H) 300 mm×8 mm ID, 2 columns in series [0070] Guard columns: Shim-pack SCR-102(H) 50 mm×6 mm ID [0071] Eluent: 5 mmol/L of p-toluene sulfonic acid [0072] Reaction solution: 5 mmol/L of p-toluene sulfonic acid, 100 μmol/L of EDTA, and 20 mmol/L of Bis-Tris [0073] Flow rate: 0.8 mL/min [0074] Oven temperature: 45° C. [0075] Detector: Conductivity Detector CDD-10A

[0076] The nine organic acids measured were succinic acid, lactic acid, formic acid, acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid, and n-valeric acid. The results are shown in Table 11 below.

TABLE-US-00011 TABLE 11 Test for organic acid-producing ability (μg/mL) succinic acetic formic acetic propionic iso-butyric n-butyric iso-valeric n-valeric Sample name acid acid acid acid acid acid acid acid acid Control (medium only) 163 131 30 164 63 Clostridium butyricum 158 1107 804 1147 62 1030 NBRC 13949T Clostridium beijerinckii 161 1038 26 1189 63 1530 NBRC 109359T 27451-B11 161 49 240 69 2828
Blank cells in the table are below the lower limit of quantitation. The lower limit of quantification is 5μg/mL for succinic acid, lactic acid, acetic acid, and propionic acid, and 10 μg/mL for formic acid, iso-butyric acid, n-butyric acid, iso-valeric acid, and n-valeric acid. The values for propionic acid include foreign substances.

[0077] It can be seen that B11 strain produced 2.74 times more n-butyric acid than Clostridium butyricum, which is a well-known butyric acid bacterium. Furthermore, it can be seen that B11 produced almost exclusively butyric acid when the amounts of organic acids contained in the medium (Control) are subtracted. In contrast, Clostridium butyricum produced lactic acid, formic acid, and acetic acid in addition to butyric acid. This means that B11 strain may be a very efficient and selective butyric acid-producing bacterium. In particular, B11 produced very little formic acid. Formic acid may have a deleterious effect on humans. Since B11 produces very little the formic acid, it is expected to be used for biological applications.

[0078] In addition, it can be seen that B11 strain produced different types and amounts of organic acids compared with Clostridium beijerinckii NBRC 109359T, the type strain of Clostridium beijerinckii. B11 strain produced lactic acid below the limit of quantification, less acetic acid, and more butyric acid compared with the type strain, Clostridium beijerinckii NBRC 109359T. Therefore, it has been determined that B11 strain is a strain having different characteristics from the type strain of Clostridium beijerinckii species.

[0079] [References to Deposited Biological Material]

(1) Name of depositary: National Institute of Technology and Evaluation, Patent Microorganisms Depositary

(2) Contact: #122, 2-5-8, KazusaKamatari, Kisarazu-shi, Chiba 292-0818, Japan, Telephone No. 0438-20-5580

[0080] (3) Accession number: NITE BP-02951
(4) Indication reference: SIID27451-B11
(5) Date of original deposit date: May 16, 2019

[Sequence Listing]

HM06P-1_ST25.txt

[0081]