New regulations for microorganisms

Abstract

An object of the present invention is to provide a novel microorganism belonging to the order Limnochordales. 【Means for solving the problem】 The novel microorganism of the present invention is the H3_T3d strain (accession number: NITE P-04317) belonging to the order Limnochordales. This H3_T3d strain is a thermophilic bacterium. Furthermore, this H3_T3d strain has the following scientific properties. · It is a Gram-negative bacillus. · It grows by static culture in SYP_KW medium at 65°C, but the growth amount increases by shaking culture. · The growth temperature in SYP_KW medium is 47°C to 68°C, and the optimum growth temperature is 65°C. · The growth pH of SYP_KW medium at 65°C is 7 to 9. · Even when sodium chloride in SYP_KW medium at 65°C is added to a final concentration of 4%, growth is confirmed. · The GC content of genomic DNA is 62.3%. · The most major cellular fatty acid is anteiso-C17:0. · It has β-1,4-glucosidase.

Description

Technical Field

[0001] The present invention relates to novel microorganisms, and particularly to novel microorganisms belonging to the order Limnochordales.

Background Art

[0002] As microorganisms belonging to or close to the order Limnochordales, there is the article in the International Journal of Systematic and Evolutionary Microbiology, Volume: Vol. 65, Issue 8, Pages: 2378 - 2384, Publication Year: 2015, DOI: 10.1099 / ijs.0.000267, Authors: Miho Watanabe, Hisaya Kojima, Manabu Fukui (Institute of Low Temperature Science, Hokkaido University). In Title Limnochorda pilosa gen. nov., sp. nov., a moderately thermophilic, facultatively anaerobic, pleomorphic bacterium and proposal of Limnochordaceae fam. nov., Limnochordales ord. nov. and Limnochordia classis nov. in the phylum Firmicutes which is described as follows. This paper describes a new strain HC45 isolated from the sediment of Lake Harutori in Kushiro City, Hokkaido, names it Limnochorda pilosa, and further proposes a new family (Limnochordaceae), order (Limnochordales), and class (Limnochordia).

Prior Art Documents

Patent Documents

[0003] [Non-Patent Document 1] International Journal of Systematic and Evolutionary Microbiology, Vol. 65, Issue 8, pp. 2378-2384, Publication Year: 2015, DOI: 10.1099 / ijs.0.000267, Authors: Miho Watanabe, Hisaya Kojima, Manabu Fukui (Institute of Low Temperature Science, Hokkaido University) [Overview of the project] [Problems that the invention aims to solve]

[0004] The present invention aims to provide a novel microorganism belonging to the order Limnocordales. [Means for solving the problem]

[0005] The present inventors discovered novel microorganisms from high-temperature compost made from sewage sludge collected at a sewage treatment plant in Osaka Prefecture, and completed the present invention by isolating and identifying these microorganisms. The aforementioned problems are solved by novel microorganisms and thermophilic bacteria having the following configurations (1) to (7). (1) This is a novel microorganism belonging to the order Limnocordales, specifically strain H3_T3d (accession number: NITE P-04317). (2) A novel microorganism described in (1) above, which is a thermophilic bacterium. (3) A novel microorganism described in (1) above, having the following scientific properties. • It is a Gram-negative bacillus. At 65°C, the organism grows under static culture conditions in SYP_KW medium, but growth increases with shaking culture. The optimal growth temperature in SYP_KW medium is 47°C to 68°C, with an optimal growth temperature of 65°C. The optimal pH for growth in SYP_KW medium at 65°C is 7-9. Growth was observed even when sodium chloride was added to SYP_KW medium at 65°C up to a final concentration of 4%. The GC content of genomic DNA is 62.3%. • The most prominent bacterial fatty acid is anteiso-C17:0. (4) A novel microorganism according to (1) above, having the base sequence shown in Sequence ID No. 1. (5) A novel microorganism according to (1) above, possessing β-1,4-glucosidase. (6) A thermophilic bacterium having a 16S rRNA gene consisting of a nucleotide sequence having 98.7% or more homology to the nucleotide sequence shown in Sequence ID No. 1 of the novel microorganism described in (1) above. (7) A thermophilic bacterium having a DNA-DNA hybridization homology of 70% or more with the novel microorganism described in (1) above. [Effects of the Invention]

[0006] The novel microorganism obtained by this invention is a new species of the order Limnocordales. This novel microorganism possesses heat-resistant enzymes that decompose organic substances such as proteins, cellulose, and fatty acids, and is useful for the decomposition of organic matter at high temperatures. [Brief explanation of the drawing]

[0007] [Figure 1] This shows a molecular phylogenetic tree based on the 16S rRNA gene of the novel microorganism of the present invention. [Figure 2] This shows a scanning electron microscope image of the novel microorganism of the present invention. [Figure 3] This shows a transmission microscope image of the novel microorganism of the present invention. [Figure 4] The image shows zymographic images of β-1,4-glucosidase activity confirmed in the culture supernatant concentrate and cell-free extract of the novel microorganism of the present invention. [Modes for carrying out the invention]

[0008] The microorganism of the present invention is a novel microorganism belonging to the order Limnocordales. [Background to the invention] This invention was completed by discovering novel microorganisms from high-temperature compost made from sewage sludge collected at a sewage treatment plant in Osaka Prefecture, and then isolating and identifying them.

[0009] (Isolation and identification of bacteria) The H3_T3d strain was isolated from compost produced by aerobic fermentation at high temperature using sewage sludge as a raw material. Specifically, 3 g of the compost was mixed with 1 mL of distilled water and cultured at 65°C for 3 days to obtain a culture medium. The compost was extracted using three times the volume of boiling water, and the resulting extract was filtered through a 0.1 μm PVDF membrane filter (Durapore(R) membrane filter 0.1 μm) to obtain the extract medium. The cured compost was added to the autoclaved extract medium, and static culture was repeatedly performed at 60-65°C to obtain a compost culture solution. After removing the supernatant from this compost culture solution by centrifugation (SAKUMA SS-1500, 15000 × g, 5 min), the grown bacteria were added to a 30% glycerol solution and stored at -80°C to obtain the stored bacteria. The aforementioned stored bacterial cells were added to SYP_KW medium (details of this medium are described below) and subjected to static culture at 60°C, followed by repeated smear culture at 60°C in SYP_KW solid medium with 0.8% Gelan Gum added, to isolate the H3_T3d strain. This strain was deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE) at the applicant's request, and accession number NITE P-04317 was obtained. A BLAST homology search of the 16S rRNA gene sequence of this strain was performed using the National Center for Biotechnology Information. The result showed that it was most similar to Limnochorda pilosa (HC45), with a homology of 88.7%, indicating that it is an independent bacterium, at least at the genus level. In other words, it was determined to be a novel microorganism. This H3_T3d strain has the nucleotide sequence shown in Sequence ID No. 1. Subsequently, 16S rRNA gene sequences of various microorganisms including related species of H3_T3d were collected from the National Center for Biotechnology Information, a gene database, and a phylogenetic tree was constructed by the neighbor-joining method using the ClustalX 2.1 program. The obtained phylogenetic tree is shown in FIG. 1. The numbers in parentheses indicate GenBank accession numbers, and H3_T3d indicates the strain isolated according to the present invention.

[0010] [Growth Medium] The growth medium was prepared with the composition shown in Table 1.a, adjusted to pH 8.5 with sodium hydroxide, and then 10 mL each of the solutions shown in Table 1.b, Table 1.c, and Table 1.d was filtered through a 0.2 μm membrane filter (ADVANTEC(R) DISMIC 13CP020AS) and added. This medium will be hereinafter referred to as the SYP_KW medium. In addition, during the test for examining the growth pH range, in addition to sodium hydroxide, sodium carbonate or sodium bicarbonate solution was filter-sterilized through a 0.2 μm filter and added to the medium after autoclaving. Table 1.a JPEG0007848971000002.jpg4681Table 1.b JPEG0007848971000003.jpg2881Table 1.c JPEG0007848971000004.jpg1882Table 1.d JPEG0007848971000005.jpg17,81

[0011] [Chemical Characteristics] 1) It is a Gram-negative bacillus. 2) It grows by static culture in the SYP_KW medium at 65°C. The growth amount increases by shaking culture. 3) The growth temperature in the SYP_KW medium is 47°C to 68°C, and the optimum growth temperature is �5°C. 4) The growth pH of the SYP_KW medium at 65°C is 7 to 9. 5) Growth is observed even when sodium chloride is added to SYP_KW medium at 65°C up to a final concentration of 4%. 6) The GC content of genomic DNA is 62.3%. 7) The most prominent bacterial fatty acid is anteiso-C17:0.

[0012] [form] As is clear from the scanning electron microscope image in Figure 2 (JSM-7500F; JEOL Ltd.) and the transmission microscope image in Figure 3 (JEM-1400Plus; JEOL Ltd., EM-14830RUBY2; JEOL Ltd.), the H3_T3d strain of the present invention is a rod-shaped bacterium.

[0013] [Genome analysis] Genome analysis and GC content measurement were performed using the following methods. After inoculation into SYP_KW medium, 290 mg of wet bacterial cells were collected from 200 mL of culture medium of the H3_T3d strain, which had been sufficiently grown at 60°C and 150 rpm (TAITEC Bioshaker BR-23FH), using a centrifuge (SAKUMA SS-1500, 4800×g, 10 minutes). After freezing at -80°C, genome extraction and sequencing analysis were performed at Biotechnical Research Institute Co., Ltd.

[0014] The genome extraction method was performed using the following procedure: DNA was extracted using a Genomic-tip 20G (QIAGEN). DNA concentration was measured using a QuantiFluor dsDNA System and a Quantus Fluorometer (Promega), confirming that the extracted genome concentration was 440 ng / μL. Electrophoresis was performed using a 5200 Fragment Analyzer System and an Agilent HS Genomic DNA 50kb Kit (Agilent Technologies) to confirm that the extracted genome quality was suitable for library preparation and sequencing analysis.

[0015] Library preparation and sequencing analysis were performed using the following procedure. Specifically, genomes were generated using 1.8 × solution volume of DNA Clean Beads (MGL Tech Co., Ltd) and Short Read Eliminator XL (PacBio), and the library was prepared using SMRTbell Prep Kit 3.0 (PacBio). Subsequently, polymerase complexes of the library were formed using Revio Polymerase Kit (Pac Bio), and sequencing was performed using Revio (Pac Bio).

[0016] Data analysis was performed using the following procedure: Overhang adapter sequences were removed from the sequences obtained by sequencing using SMRT Link (ver. 13.1.0.221970) to form subreads. After creating a consensus sequence (CCS) by aligning these subreads, CCS reads with an average quality of less than 20 per read were removed to obtain HiFi reads, and reads shorter than 1000 base pairs were removed using Filtlong. Subsequently, HiFi reads longer than 1000 base pairs were assembled using Flye (ver. 2.9.3-b1797) under default conditions to obtain the sequence of a 3,241,083 bp circular genome. The completeness of the obtained genome was checked using CheckM2 (ver. 1.0.1), and the completeness was 97.6% and contamination was 0.50%, indicating a high-quality genome.

[0017] [Analysis of fatty acids] Fatty acid methyl ester analysis was performed using the following procedure: After inoculating SYP_KW medium with the H3_T3d strain, 62.7 mg of wet bacterial cells were collected from 60 mL of culture solution grown sufficiently at 65°C and 150 rpm (TAITEC Bioshaker BR-23FH) using a centrifuge (SAKUMA SS-1500, 4800 × g, 10 min), frozen at -80°C, and then analyzed by Biochromat Co., Ltd. The analysis at Biochromatomato Co., Ltd. was performed using the following procedure. Specifically, 19.352 mg of wet bacterial cells were saponified (hydrolyzed), methylated, and the resulting solution extracted with an organic solvent was analyzed using GC / MS (Gas chromatograph (7890 GC System), Agilent Technologies, Inc., Mass spectrometer (5977B MSD), Agilent Technologies, Inc.). The analytical conditions are shown in the table. The obtained peaks were identified using search software (MassHunter unknown analysis: Biochromatomato's original database), and the component with the highest hit was selected based on the Retention Index (RI) using n-Alkane and mass spectral information. Among the detected fatty acid species, those with an abundance of more than 10% are shown as major fatty acids in Table 3.

[0018] Table 2 GC / MS measurement conditions JPEG0007848971000006.jpg118159Table 3 JPEG0007848971000007.jpg3093 [Industrial applicability]

[0019] The novel microorganism obtained by this invention is a new genus and species of the Limnocordales order. This novel microorganism possesses β-1,4-glucosidase activity that cleaves the β-1,4-glucosidic bonds found in cellulose and other materials. Cellulose is a major component of plant cell walls and plant fibers, and is found in large quantities not only in woody waste but also in kitchen waste and sludge. However, because the β-1,4-glucosidic bond is a very stable structure, it is difficult to decompose. The β-1,4-glucosidase possessed by this novel microorganism has been shown to be active at high temperatures, making it beneficial for efficient decomposition of organic matter under high-temperature conditions. [Confirmation of β-1,4-glucosidase activity] β-1,4-glucosidase activity was confirmed by zymography in the culture supernatant concentrate and cell-free extract of H3_T3d. Specifically, H3_T3d cells were cultured in SYP_KW medium at 60°C at 150 rpm (TAITEC Bioshaker BR-23FH) for 40 hours with shaking, and the cells were harvested at 5,800 × g to obtain wet cells and culture supernatant. The weight of the wet cells was measured, and 100 mg of wet cells was dissolved in 1 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 20% ​​glycerol to obtain a cell suspension. Subsequently, the cell suspension was subjected to sonication on ice. This was repeated for 1 minute of disruption followed by 1 minute of standing, for a total of 15 minutes of disruption. The obtained lysates were centrifuged at 19,600 × g, and the supernatant was used as the cell-free extract. For the culture supernatant, ammonium sulfate was added to the volume of the culture supernatant to a 70% saturation, and dissolved in a glass beaker using a stirrer and stirring bar. Sodium hydroxide was added as needed to ensure that the pH was kept below 7.5 and above 8.5. The culture supernatant, in which the ammonium sulfate had completely dissolved, was stored at 4°C for one to three nights. It was centrifuged at 5,800 × g, and the precipitate containing extracellular proteins was collected. The collected precipitate was washed by suspending it in a 70% saturated ammonium sulfate aqueous solution, and then washed again by centrifugation at 5,800 × g. This washing procedure was repeated once more. The obtained precipitate was dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 20% ​​glycerol. The volume of this solution was equivalent to that of the cell-free extract, and the resulting sample was used as the cell supernatant concentrate. Samples for electrophoresis were prepared from the obtained cell supernatant concentrate and cell-free extract. Specifically, the cell supernatant concentrate and cell-free extract were mixed with 4× sample buffer in a 3:1 ratio. The 4× sample buffer consisted of 200 mM Tris-HCl buffer (pH 6.8), 20% glycerol, 8% sodium dodecyl sulfate, and 0.01% bromophenol blue. This sample was subjected to electrophoresis on a polyacrylamide gel containing carboxymethylcellulose. Specifically, a mixture of 1500 μL of 30% acrylamide-0.8% N,N'-methylenebisacrylamide solution, 1500 μL of 1.5 M Tris-HCl (pH 8.8), 2300 μL of MilliQ, 60 μL of 10% sodium dodecyl sulfate, and 600 μL of 1% carboxymethylcellulose solution was prepared by adding 50 μL of 10% ammonium persulfate and 5 μL of N,N,N',N'-tetramethylethylenediamine to form a gel, which was then separated to obtain the gel. Furthermore, a mixture of 450 μL of 30% acrylamide-0.8% N,N'-methylenebisacrylamide solution, 750 μL of 0.5 M Tris-HCl (pH 6.8), 1700 μL of MilliQ, and 30 μL of 10% sodium dodecyl sulfate was prepared by adding 50 μL of 10% ammonium persulfate and 5 μL of N,N,N',N'-tetramethylethylenediamine to a concentrated gel. The concentrated gel was then placed on top of the separation gel to prepare the electrophoresis gel. The electrophoresis buffer was prepared as follows: 20 mL of 25 mM Tris-192 mM glycine buffer (pH 8.3) and 20 μL of 10% SDS were mixed to 200 mL using MilliQ. The electrophoretic gels were filled with electrophoretic buffer, and electrophoresis was performed at a fixed current of 20 mA per gel at 4°C for approximately 1.5 to 3 hours, until the bromophenol blue reached the bottom of the gel. The electrophoretic gel was immersed in 100 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 7.5) and 0.2% Triton-X, and shaken for 10 minutes. Then, it was immersed in 100 mM dipotassium hydrogen phosphate / potassium dihydrogen phosphate buffer (pH 7.5) and allowed to stand overnight at a specified temperature to allow the enzyme reaction to occur. After that, the solution was discarded and the gel was washed with RO water, and the electrophoretic gel was immersed in Congo Red solution and shaken until it was sufficiently stained red. The sufficiently stained electrophoretic gel was destained with 1 M sodium chloride aqueous solution, and β-1,4-glucosidase activity was detected by observing the halo. As a result, as shown in the photograph in Figure 4, β-1,4-glucosidase activity was detected both inside and outside the H3_T3d bacterial cell over a wide temperature range from 65°C to 90°C.

Claims

1. A novel microorganism belonging to the order Limnochordales, strain H3_T3d (accession number: NITE P-04317).

2. A novel microorganism according to claim 1, which is a thermophilic bacterium.

3. A novel microorganism according to claim 1, having the following scientific properties. 1) It is a bacillus that is Gram-negative. 2) At 65°C, it grows under static culture in SYP_KW medium, but the growth rate increases with shaking culture. 3) The growth temperature in SYP_KW medium is 47°C to 68°C, with the optimal growth temperature being 65°C. 4) The growth pH of SYP_KW medium at 65°C is 7-9. 5) Growth was observed even when sodium chloride was added to SYP_KW medium at 65°C up to a final concentration of 4%. 6) The GC content of genomic DNA is 62.3%. 7) The most prominent bacterial fatty acid is anteiso-C17:

0.

4. A novel microorganism according to claim 1, having the base sequence shown in Sequence ID No.

1.

5. A novel microorganism according to claim 1, possessing β-1,4-glucosidase.

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