TW202313961A - Shewanella bacteria and use thereof - Google Patents

Abstract

Shewanella woodyi strain GS001 (accession number NITE BP-03460) or mutant strains thereof. A composition containing shewanella woodyi strain GS001 or mutant strains thereof. A dry powder or extract of shewanella woodyi strain GS001 or mutant strains thereof.

Description

Bacteria of the genus Shewanella, and uses thereof

The present invention relates to a bacterium of the genus Shewanella and its use. In particular it relates to a novel strain of Shewanella woodyi. In addition, it relates to a composition, a dry powder, and an extract comprising the aforementioned novel strain. This application claims priority based on Japanese Patent Application No. 2021-094382 filed in Japan on June 4, 2021, and the content thereof is hereby cited.

Eicosapentaenoic acid (EPA) is a type of ω3 fatty acid having an anti-obesity effect, a neutral lipid lowering effect, and the like. Fish are known to contain a lot of EPA. However, in the case of manufacturing EPA from fish, there is a possibility that heavy metal components that are bioconcentrated in fish may be contained, and there is concern about health damage caused by heavy metals. In addition, there is also the possibility of overfishing of fish that would involve limited marine resources.

Marine bacteria such as Shewanella are known to produce fatty acids such as EPA (for example, Patent Document 1, Non-Patent Documents 1 to 3). However, bacteria of the genus Shewanella often do not proliferate well unless a medium containing animal-derived components is used (Non-Patent Documents 3 and 4). Therefore, industrial production of EPA using bacteria of the genus Shewanella has not yet been realized. [Prior Art Literature] [Patent Document]

Patent Document 1: Japanese Patent Publication No. 7-61272 [Non-patent literature]

Non-Patent Document 1: Masataka Satomi, Hiroshi Oikawa, Yutaka Yano, Shewanella marinintestina sp. nov., Shewanella schlegeliana sp. nov., and Shewanella marinatus Shewanella sairae sp. nov., a novel eicosapentaenoic-acid-producing marine bacteria isolated from sea-animal intestines., International System International Journal of Systematic and Evolutionary Microbiology (2003), 53, 491-499. Non-Patent Document 2: Jinwei Zhang, J. Grant Burgess, Shewanella electrodiphila sp. nov., a psychrotolerant bacterium isolated from Mid-Atlantic Ridge deep-sea sediments -Atlantic Ridge deep-sea sediments.), International Journal of Phylogenetic Microbiology (2015), 65, 2882-2889. Non-Patent Document 3: Nobuo Suzuki, Kazuro Yazawa, Kazuro Watanabe, Yukari Akahori, Chinatsu Ishikawa, Sei Kondo, Kiyokatsu Takada, discussing the conditions for mass cultivation of eicosapentaenoic acid-producing bacteria SCRC-2738 , Journal of the Fisheries Society of Japan (Nippon Suisan Gakkaishi) 58(2), 323-328 (1992). Non-Patent Document 4: Esra Ersoy Omeroglu, Ismail Karaboz and Mert Sudagidan, Characteristic and genetic diversity of bioluminescent Shewanella woodyi strains isolated from Izmir Bay, Turkey the Gulf of Izmir, Turkey.), Folia Microbiol (2014) 59(1), 79-92.

[Problem to be solved by the invention]

In order to industrially produce EPA using bacteria of the genus Shewanella, it is preferable that bacteria of the genus Shewanella having a high EPA-producing ability proliferate well in a completely synthetic medium containing glucose or the like as the sole organic carbon source.

Therefore, the present invention provides a Shewanella bacterium, a composition comprising the aforementioned Shewanella bacterium, and a dry powder and extract of the aforementioned Shewanella bacterium; wherein, the Shewanella Bacteria of the genus Bacteria proliferate well on glucose as the sole organic carbon source, and have high EPA production capacity. [Means to solve the problem]

This disclosure includes the following aspects. [1] A strain of Shewanella xylinum GS001 (registration number NITE BP-03460) or a mutant thereof. [2] A composition comprising Shewanella xylinum GS001 strain or a mutant strain thereof. [3] The composition described in [2], which is food, feed, or bait, or cosmetics. [4] A dried powder of Shewanella xylinum strain GS001 or a mutant thereof. [5] An extract of Shewanella xylinum strain GS001 or a mutant thereof. [Effect of Invention]

According to the present disclosure, there can be provided a bacterium of the genus Shewanella, a composition comprising the bacterium of the genus Shewanella, and a dry powder and extract of the bacterium of the genus Shewanella; wherein, the bacterium of the genus Shewanella Bacteria of the genus Bacteria proliferate well with glucose as the sole organic carbon source, and have high EPA production capacity.

[Mode for Carrying Out the Invention]

The term "comprise" (comprise) means that constituent elements other than the intended constituent elements may be included. The term "consist of" (consist of) means not including constituent elements other than the intended constituent elements. The term "consist essentially of" (consist essentially of) means that an aspect that exerts a special function (a aspect that completely loses the effect of the invention, etc.) does not include constituent elements other than the target constituent elements. In this specification, when described as "comprising ~" (comprise), the aspect "consist of" (consist of) and the aspect "consist essentially of" (consist essentially of) are included.

Bacteria can be isolated. The so-called "isolated" means the state of being separated from the natural state or other components. Those who are "separated" can be those that do not substantially contain other ingredients. "Essentially not containing other components" means that the content of other components contained in the isolated components is negligible. The content of other components included in the isolated components can be, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, or 0.5% by mass or less , or 0.1% by mass or less. The bacteria described in this specification may be isolated bacteria.

The so-called "proliferation using glucose as the only organic carbon source" refers to the growth in a medium containing only glucose as an organic carbon source. The so-called "synthetic medium" refers to a medium with defined chemical components contained in the medium. Synthetic media are prepared by mixing chemical components with defined chemical substance names. The so-called "semi-synthetic medium" refers to a medium in which a mixture of substances with unclear chemical composition (ie, substances derived from organisms) is added to a synthetic medium. Examples of biologically derived substances include yeast extract, protein, casamino acid, meat extract, and the like.

In this specification, the term "eicosapentaenoic acid" or "EPA" includes eicosapentaenoic acid (free form) and its derivatives. Examples of derivatives of eicosapentaenoic acid include esterified type, phospholipid-bound type, phosphatidylglycerol-bound type, monoglyceride type, diglyceride type, and triglyceride type. type, salts of such, etc.

＜Single isolate of Shewanella xylinum＞ In one aspect, the present invention provides a Shewanella woodyi GS001 strain (registration number NITE BP-03460) or a mutant strain thereof.

Shewanella xylinum strain GS001 (hereinafter referred to as "GS001 strain") is a strain isolated from the intestinal tract of deep-sea fish Glossanodon semifasciata. The GS001 strain was identified as Shewanella xylinum by phylogenetic analysis based on the 16S rDNA sequence (see FIG. 2 ). The 16S rDNA sequence of the GS001 strain is shown in Sequence ID No. 1.

The GS001 strain has a characteristic of being able to grow well using glucose as the sole organic carbon source. For example, when the GS001 strain is cultured with shaking (150 rpm) at 15° C. in a 100 mL flask containing 50 mL of modified M9 liquid medium (modified M9+Glc liquid medium) containing 10% (w/v) glucose, it can Proliferate to a bacterial density of 5 or more in _OD600nm in 48 hours. The GS001 strain has a characteristic of being able to grow well on a semi-synthetic medium containing glucose and yeast extract. For example, the GS001 strain was aerated at 15° C. (1.0 vvm air, 150-750rpm), and when the glucose solution as the organic carbon source and the ammonium aqueous solution as the nitrogen source are respectively fed at an appropriate speed, it can proliferate to a dry cell weight of 35 g/L or more in 75 hours. The GS001 strain is characterized by a so-called high EPA content. For example, the GS001 strain was cultured with aeration at 15°C (1vvm air, 150-750rpm) in a 3L tank-type fermenter with 2L of modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract At this time, the EPA content per unit weight of dry cells can be maintained above 0.5 g/100 g dry cell weight. The GS001 strain has a characteristic of being able to proliferate at a sodium chloride concentration as low as 0.5% (w/v). For example, when the GS001 strain is cultured with shaking (150 rpm) at 15°C in a 100 mL flask containing 50 mL of the modified M9+Glc medium whose concentration of sodium chloride was changed to 0.5% (w/v), it can Proliferate to a bacterial density of 3 or more in _OD600nm in 48 hours. The GS001 strain is suitable for the industrial production of EPA because of its above-mentioned characteristics.

The GS001 strain was deposited with the deposit number NITE P-03460 on the date of April 15, 2021 at the National Institute of Technology and Evaluation Patent Microorganism Deposit Center (Kazusa Kazusa, Kisarazu City, Chiba Prefecture, Japan) 2-5-8 Room 122), transferred to International Depository on the date of April 4, 2022 under deposit number NITE BP-03460. Name of Depositor: Jiang Yuan Yue Address of depositor: 631 Sakado, Sakura City, Chiba Prefecture, Japan The depositor grants the applicant the authority mentioned in this application regarding the deposited organisms. The depositor gives the applicant the consent of the intention to deposit living things that can be used by the public.

The so-called "mutant strain" refers to a strain that produces a mutation in the genome of the original strain. Variations can be spontaneous or artificial. The method of artificially mutating the gene body is not particularly limited. The methods of artificially causing mutations include, for example, irradiation of high-energy electromagnetic waves such as ultraviolet irradiation and radiation irradiation; chemical treatment with nitrous acid, etc.; genetic engineering techniques such as gene introduction and genome editing, etc. . The mutant strain is preferably: the ratio of the variation of the whole genome of the original strain is, for example, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3% or less, or 0.1% the following. The 16S rDNA sequence of the mutant strain may also have the nucleotide sequence described in Sequence Identification No. 1.

The mutant strain of GS001 strain (hereinafter referred to as "GS001 mutant strain") is preferably: the relative growth rate (relative growth rate) when cultured with glucose as the only organic carbon source, and the GS001 strain cultured under the same conditions at the same level or above. For example, when cultured under the same culture conditions, the relative proliferation rate of the GS001 mutant strain is preferably: the relative proliferation rate of the GS001 strain multiplied by 0.7 (or 0.75, 0.8, 0.85, 0.9, 0.95, 0.97, 0.98, 0.99 or 1 ) above the value. For example, the GS001 mutant strain is preferably: use a 100mL flask containing 30mL of improved M9 liquid medium (improved M9+Glc medium) containing 10% glucose, and shake it at 15°C (150rpm) to 48 Hours to proliferate to a bacterial density with an _OD600nm of 5 or more.

Preferably, the GS001 mutant strain has a relative proliferation rate when cultured in a semi-synthetic medium containing glucose and yeast extract, which is at or above the same level as the GS001 strain cultured under the same conditions. For example, when cultured under the same culture conditions, the relative proliferation rate of the GS001 mutant strain is preferably: the relative proliferation rate of the GS001 strain multiplied by 0.7 (or 0.75, 0.8, 0.85, 0.9, 0.95, 0.97, 0.98, 0.99 or 1 ) above the value. For example, the GS001 mutant strain is preferably: use a 3L cylinder fermenter with 2L of improved M9+Glc liquid medium containing 0.5% (w/v) yeast extract, and carry out aerated culture at 15°C (1vvm air , 150 to 750 rpm), the cells proliferate to a dry cell weight of 30 g/L or more in 75 hours.

Preferably, the GS001 mutant strain has an EPA content per unit weight of dry cells when cultured with glucose as the sole organic carbon source, which is at the same level as that of the GS001 strain cultured under the same conditions. For example, preferably when cultured under the same culture conditions, the EPA content per unit weight of the dry cells of the GS001 mutant strain is the EPA content of the GS001 strain multiplied by 0.7 (or 0.75, 0.8, 0.85, 0.9, 0.95, 0.97, 0.98 , 0.99 or 1) around the value.

The GS001 mutant strain is preferably: when cultured in a semi-synthetic medium containing glucose and yeast extract, the EPA content per unit weight of dry cells is at the same level as that of the GS001 strain cultured under the same conditions. For example, preferably when cultured under the same culture conditions, the EPA content per unit weight of the dried cells of the GS001 mutant strain is the EPA content of the GS001 strain multiplied by 0.7 (or 0.75, 0.8, 0.85, 0.9, 0.95, 0.97, 0.98, 0.99 or 1) or so. For example, the GS001 mutant strain was cultured with aeration at 15°C (1vvm air, 150-750rpm) in a 3L tank-type fermenter with 2L of modified M9+Glc liquid medium containing 0.5% (w/v) yeast extract At this time, the EPA content per unit weight of dry cells is preferably maintained at about 0.5 g/100 g dry cell weight.

Preferably, the GS001 mutant strain is able to proliferate in a medium containing 0.5% (w/v) sodium chloride. For example, the relative growth rate when cultured in a medium containing glucose as the sole organic carbon source and containing 0.5% (w/v) sodium chloride is preferably equal to or higher than that of the GS001 strain cultured under the same conditions. For example, when cultured under the same culture conditions, the relative proliferation rate of the GS001 mutant strain is preferably: the relative proliferation rate of the GS001 strain multiplied by 0.7 (or 0.75, 0.8, 0.85, 0.9, 0.95, 0.97, 0.98, 0.99 or 1 ) above the value. For example, the GS001 mutant strain is preferably: use a 100mL flask with 50mL of the modified M9+Glc medium whose sodium chloride concentration has been changed to 0.5% (w/v), and culture it with shaking at 15°C (150rpm) At this time, it proliferated to a bacterial density of 3 or more in _OD600nm in 48 hours.

The GS001 strain and the GS001 mutant strain can be cultured using a medium generally used for culturing heterotrophic bacteria. Examples of the medium include those containing organic carbon sources, nitrogen sources, phosphorus sources, trace elements (zinc, boron, cobalt, copper, manganese, molybdenum, iron, nickel, etc.) and the like. Examples of organic carbon sources include glucose, dextran, starch, and yeast extract. Examples of nitrogen sources include ammonium salts, nitrates, nitrites, amino acids, and protein compounds. As a phosphorus source, a phosphate etc. are mentioned, for example. The medium may be, for example, a minimal medium such as an M9 medium or a modified medium of the M9 medium (improved M9 medium, etc.) with an organic carbon source added.

When using a synthetic medium, glucose is mentioned as a preferable organic carbon source. When using a semi-synthetic medium, yeast extract is mentioned as a preferable organic carbon source. Semi-synthetic media may contain glucose and yeast extract as organic carbon sources.

The medium preferably contains 0.5-5% (w/v) sodium chloride. The sodium chloride concentration of the medium is more preferably 0.5-3% (w/v).

The medium can be either a solid medium or a liquid medium. For maintenance, it is preferred to use a solid medium. When performing growth, it is preferable to use a liquid medium.

The pH of the medium includes, for example, pH6-9. The pH of the medium is, for example, preferably pH 7-8.5, more preferably pH 7.5-8.5.

Examples of the culture temperature include 0 to 30°C. The culture temperature is preferably from 10 to 30°C, more preferably from 12 to 20°C, further preferably from 15 to 18°C.

The culture may be static culture, aeration culture, or shaking culture. From the viewpoint of preventing hypoxia, it is preferable to perform aeration culture or shaking culture.

During the culture period, the GS001 strain or the GS001 mutant strain can also be suitably subcultured. When the maintenance culture is performed on a solid medium, the subculture interval is, for example, half a month to one month. When the growth is performed in a liquid medium, the subculture interval is, for example, 2 days to 10 days, or 2 to 5 days.

The GS001 strain and the GS001 mutant strain contained EPA at a high level in the cells. Therefore, the GS001 strain or the GS001 mutant strain can be used to produce EPA. Extraction of EPA from the GS001 strain or the GS001 mutant strain can be performed by a known method. For example, the cells are recovered from the culture fluid of the GS001 strain and the GS001 mutant strain by centrifugation or filtration, and the total lipids are extracted from the cells by organic solvents such as chloroform, methanol, and toluene. The amount of extraction solvent used for cells is not particularly limited. The amount of the extraction medium can be, for example, about 1 to 1000 times (preferably about 5 to 200 times) the amount of the cells. The extraction operation can generally be performed under normal pressure at a range from normal temperature to the boiling point of the solvent. The extraction operation may be performed only once or multiple times. For example, a fresh extraction medium can be added to the cell residue that has been subjected to the extraction operation once, and the extraction operation can be performed again. After the extraction operation, cell residues can also be removed by centrifugation, filtration, ultrafiltration, etc. as needed. In addition, the extraction solvent can also be removed by distillation under reduced pressure using heating, an evaporator, or the like. Furthermore, various refining treatments can also be performed to refine EPA. For the purification treatment, for example, salting out, dialysis, recrystallization, reprecipitation, solvent extraction, adsorption, concentration, filtration, gel filtration, ultrafiltration, various chromatography methods (thin layer chromatography, column chromatography, ion exchange chromatography, high-speed liquid chromatography, adsorption chromatography, etc.), but is not limited thereto. Specific examples of the method for extracting EPA from the GS001 strain or the GS001 mutant strain include the methods described in Examples described later.

EPA can be used as nutritional supplements, food additives, and the like.

＜Composition＞ In one aspect, the present invention provides a composition comprising Shewanella xylinum GS001 strain or a mutant strain thereof.

GS001 strain and GS001 mutant strain contain useful components such as EPA. Therefore, the GS001 strain or the GS001 mutant strain can be used in various compositions.

The type of composition comprising the GS001 strain or the GS001 mutant strain is not particularly limited. Examples of the composition include food, feed, bait, cosmetics and the like.

Foods include, for example, confectionery (caramel, candy, etc.), iced confectionery (ice cream, etc.), dairy products (yoghurt, etc.), various processed foods, and various seasonings. Foodstuffs, various beverages, functional foods, nutritional supplements, supplements, etc. As the feed, for example, various pet foods can be cited. As a bait, the bait for ornamental fish, the bait for cultured fish, etc. are mentioned, for example. Cosmetics include, for example, skin cosmetics (lotions, emulsions, beauty essences, creams, etc.), hair cosmetics (hair styling products, shampoos, conditioners, etc.) ), make-up cosmetics (foundation, blush, eye shadow, lipstick, etc.).

The GS001 strain or the GS001 mutant strain can be added as an additive to the above-mentioned various compositions. In addition, it can also be mixed with other ingredients and prepared as food, feed, bait, cosmetics and the like. Other components can be appropriately selected according to the use of the composition.

For example, when the composition is food, feed, or bait, components usable for food, feed, or bait can be used without particular limitation. Components that can be used in food, feed, or bait include, for example, fish, vegetables, grains, dairy products, fermented products, spices, proteins, amino acids, sugars, various seasonings, and sweeteners , flavoring agents, fragrances, fats and oils, vitamins, thickeners, gelling agents, antioxidants, preservatives, chelating agents, pH regulators, coloring agents, etc., but are not limited to these.

For example, when the composition is a cosmetic, components usable for cosmetics can be used without particular limitation. In terms of ingredients that can be used in cosmetics, for example, hydrocarbons, lipids (fats such as animal and vegetable oils and mineral oils, waxes, fatty acid esters, fatty acids, ceramides, etc.), alcohols (higher alcohols, etc.) , lower alcohols and polyols, etc.) alcohols, proteins (collagen, etc.), polysaccharides (hyaluronic acid, chitosan, cellulose, xanthan gum, etc.), various polymer compounds (poly Ethylene glycol, silicone, etc.), ingredients derived from animals, plants or microorganisms, amino acids, inorganic minerals, various surfactants, ultraviolet absorbers, whitening agents, anti-inflammatory agents, blood circulation promoters, anti-aging agents, moisturizing agents , vitamins, thickeners, gelling agents, antioxidants, preservatives, antibacterial agents, chelating agents, pH regulators, powders, fragrances, coloring agents, etc., but are not limited to these.

＜Dry powder＞ In one aspect, the present invention provides a dry powder of GS001 strain or GS001 mutant strain.

The term "dried powder of the GS001 strain" refers to the dried and powdered cells of the GS001 strain. The term "dried powder of the GS001 mutant strain" means that the cells of the GS001 mutant strain were dried and made into a powder form. The dry powder of the GS001 strain or the GS001 mutant strain can be produced by a known method. For example, the GS001 strain or the GS001 mutant strain is cultured, and the cells are recovered by centrifugation or the like. Then, a dry powder can be obtained by drying the cells and making a powder. The method of drying the cells is not particularly limited, and examples thereof include natural drying, heat drying, reduced-pressure drying, freeze-drying, and the like. Dried cells can also be physically crushed to make a powder. The cells may be washed, sterilized, etc. before being dried. For the washing treatment, for example, washing liquids such as water and buffer solutions can be used. As the method of sterilization treatment, for example, treatment by sterilization with hypochlorous acid, ultraviolet treatment, ozone treatment, heat treatment, etc. are mentioned.

Useful components such as EPA contained in the GS001 strain or GS001 mutant strain are concentrated by making dry powder. Therefore, the dry powder of GS001 strain or GS001 mutant strain can be used as food, feed, or bait. In addition, the dry powder of the GS001 strain or the GS001 mutant strain can be used as an additive in food, feed, bait, or cosmetics.

＜Extract＞ In one aspect, the present invention provides an extract of GS001 strain or GS001 mutant strain.

The so-called "extract of GS001 strain" refers to a cell component extracted from cells of GS001 strain. The so-called "extract of GS001 mutant strain" refers to a cell component extracted from cells of GS001 mutant strain. The extract of the GS001 strain or the GS001 mutant strain may also be a cell destruction product obtained by destroying the cells of the GS001 strain or the GS001 mutant strain. Examples of methods for disrupting cells include mechanical disruption using a homogenizer or the like, ultrasonic treatment, freeze-thaw treatment, and the like. In addition, the extract of the GS001 strain or the GS001 mutant strain may also be a cell lysate obtained by lysing the cells of the GS001 strain or the GS001 mutant strain. As a method for lysing cells, for example, enzyme treatment using enzymes such as protease and lysozyme, surfactant treatment, and the like can be mentioned. The extract of the GS001 strain or the GS001 mutant strain may be obtained by adding an extraction solvent to cells, cell disruptors, or cell lysates of the GS001 strain or the GS001 mutant strain and performing extraction treatment. As the extraction solvent, the same ones as above can be used.

By making the extract, useful components such as EPA contained in the GS001 strain or the GS001 mutant strain are concentrated. Therefore, the extract of GS001 strain or GS001 mutant strain can be used as food, feed, or bait. In addition, the extract of GS001 strain or GS001 mutant strain can be used as an additive in food, feed, bait, or cosmetics.

In one embodiment, the present disclosure provides a method for producing EPA, which comprises culturing GS001 strain or GS001 mutant strain. In one embodiment, the present disclosure provides a method for producing EPA, which comprises culturing GS001 strain or GS001 mutant strain using a medium containing glucose as an organic carbon source. In one embodiment, the present disclosure provides a method for producing EPA, which includes: cultivating GS001 strain or GS001 mutant strain using a medium containing glucose as an organic carbon source; recovering the cells of GS001 strain or GS001 mutant strain; Bacteria extract EPA. In one embodiment, the present disclosure provides a method for cultivating GS001 strain or GS001 mutant strain, which comprises cultivating GS001 strain or GS001 mutant strain using a medium containing glucose as an organic carbon source. In one embodiment, the present disclosure provides a method for producing a GS001 strain or a GS001 mutant strain, which comprises culturing the GS001 strain or the GS001 mutant strain using a medium containing glucose as an organic carbon source. [Example]

Hereinafter, although an Example demonstrates this invention, this invention is not limited to a following Example.

<Isolation of bacteria belonging to the genus Shewanella with high glucose assimilability> Using Marine Broth medium (manufactured by Difco), bacteria were isolated from the intestinal tract of a deep-sea fish, Aquamarina chinensis. The colonies that appeared in the Marine Broth cold weather medium were inoculated into 50 mL of the modified M9+Glc liquid medium that had been placed in a 100 mL Erlenmeyer flask for culture. The culture temperature was set at 15° C. and shaking culture was performed using a BioShaker (rotational speed: 150 rpm). When the OD ₆₀₀ of the medium reaches 4-5, take 500 μL of the culture solution and inoculate it into a new 50 mL modified M9+Glc liquid medium. The aforementioned subculture was carried out for about 6 months, and the strains with good proliferation in the improved M9+Glc liquid medium and high EPA content were selected. As a result, the GS001 strain was obtained as a strain that proliferates well in the modified M9+Glc liquid medium and has a high EPA content. The compositions of the improved M9+Glc liquid medium are shown in Tables 1-3. The improved M9+Glc medium is made by adding iron solution (Fe solution) (Table 2), trace metal solution (Trace Metal solution) (Table 3), and glucose to the M9 minimum nutrient medium.

[Table 1] Improved M9+Glc liquid medium Na ₂ HPO ₄ ･12H ₂ O 15 g/L KH ₂ PO ₄ 3 g/L (NH ₄ ) ₂ SO ₄ 2.65 g/L NaCl 20 g/L iron solution 10 mL/L trace metal solution 1 mL/L 1M MgSO ₄ 10 mL/L Vitamin B1 0.01 g/L 0.1M _CaCl2 10 mL 50% glucose solution 20 mL/L pH8.0

[Table 2] iron solution FeSO ₄ ･7H ₂ O 2.1 g/L EDTA 5.2 g/L

[table 3] trace metal solution H ₃ BO ₃ 10 mg/L _MnCl2 5 mg/L _CoCl2 190 mg/L NiCl ₂ ･6H ₂ O twenty four mg/L CuCl ₂ ･2H ₂ O 10 mg/L _ZnSO4 144 mg/L NaMo ₄ ･2H ₂ O 36 mg/L

Figure 1 shows the results of growing the GS001 strain under the same culture conditions as above using 50 mL of the modified M9+Glc liquid medium placed in a 100 mL Erlenmeyer flask. The GS001 strain reached an OD ₆₀₀ of about 5 to 6 in about 48 hours from the initiation of culture, showing good growth.

＜Identification of GS001 strain＞ Using the suspension of the GS001 strain as a template, PCR was performed using the following primer set to amplify 16S rDNA. Forward primer: GTTTGATCCTGGCTCA (SEQ ID NO. 2) Reverse primer: TACCAGGGTATCTAATCC (SEQ ID NO. 3)

Sequence analysis of the amplified fragment was carried out to determine the 16S rDNA sequence of the GS001 strain (SEQ ID NO: 1). A BLAST search was performed using the 16S rDNA sequence of the GS001 strain as a query sequence. From the results of the BLAST search, the GS001 strain was identified as Shewanella xylinum. Fig. 2 shows the results of the phylogenetic analysis of the GS001 strain based on the 16S rDNA sequence. The system analysis system is referenced in Satoi et al. (Masataka Satoi et al., International Journal of Phylogenetic Microbiology (2003), 53, 491-499.) and Zhang et al. (Jinwei Zhang, et al., International Journal of Phylogenetic Microbiology ( 2015), 65, 2882-2889.) method. The 16SrRNA sequences of 19 species of Shewanella bacteria including Shewanella xylinum and related species were collected from the DDBJ database, and their sequences and the 16srRNA sequence of GS001 strain were analyzed using software "CLC Genomics Workbench" (manufactured by CLC bio Co., Ltd.) to create a phylogenetic tree (cluster analysis (cluster analysis) is based on the neighbor joining method (Neighbor joining method, NJ method), genetic distance (Genetic distance) is based on the Kimura model (Kimura model) to carry out).

＜Cultivation of GS001 strain in semi-synthetic medium＞ A modified M9+Glc liquid medium (M9+Glc+Y.E. liquid medium) containing 0.5% (w/v) yeast extract was used as a semi-synthetic medium. The GS001 strain was inoculated into a 2L modified M9+Glc+Y.E. liquid medium that had been placed in a 3L tank-type fermenter for cultivation. The culture temperature was set at 15°C, and aeration culture was carried out (1vvm air, 150-750rpm). The culture system was carried out using two cylinder fermenters.

(Determination of dry cell weight) Centrifuge the culture solution of any volume at 10,000×g at 4°C for 5 to 10 minutes to obtain a precipitate. This was washed with a 1% (w/v) ammonium acetate aqueous solution, and the step of centrifuging again was performed twice. The pellet was resuspended in a small amount of 1% (w/v) aqueous ammonium acetate and transferred to a pre-weighed resin tube. After freezing at -80°C, freeze-drying was performed to obtain dry cells. The short resin tube containing the cells is weighed again to obtain the dry cell weight per unit volume.

(Measurement of EPA) Take about 30 mg of dried cells into a screw cap test tube (screw tube), and add 0.5 mL of extraction medium. The extraction solvent is a mixed solvent mixed in the ratio of toluene:methanol=52:48 (v/v). Add 1 mL of 0.5M sodium hydroxide-methanol solution to the solution mixed with extraction medium and dried cells. The cap was tightened, and the test tube was heated at 100° C. for 5 minutes using a heat block or the like. Thereafter, the test tube was left to cool sufficiently. Add 1 mL of boron trifluoride-methanol solution (12-15% (w/v) of boron trifluoride concentration) into the test tube. The cap is tightened, and the test tube is heated at 100° C. for 5 minutes using a heating block or the like. Thereafter, the test tube was left to cool sufficiently again. 1 mL of hexane and saturated saline were added to the test tube and mixed well. Centrifuge the mixed sample solution with the aforementioned saturated saline solution at 1450×g at room temperature for 5 minutes, take an appropriate amount of the resulting upper layer (organic layer) into a vial, and perform gas chromatography (GC) analysis .

The changes in _OD600 and dry cell weight are shown in Figure 3A. The EPA content per unit weight of dry cells is shown in Figure 3B. In Fig. 3A, OD J1 and DCW J1 respectively show the OD ₆₀₀ and dry cell weight in the first vat fermenter. In Fig. 3A, OD J2 and DCW J2 show the OD ₆₀₀ and dry cell weight in the second vat fermenter, respectively. The dry cell weight of GS001 strain reached above 35g/L in any cylinder fermenter, showing good proliferation. Furthermore, the reached dry cell weight of bacteria of the genus Shewanella reported so far is about 10.3 to 10.5 g/L. (Nobuo Suzuki et al. Journal of Fisheries Society of Japan 58(2), 323-328(1992).) In Fig. 3B, J1 shows the EPA content per unit weight of dry cells in the first vat fermenter. In FIG. 3B , J2 shows the EPA content per unit weight of dry cells in the second vat fermenter. Strain GS001 showed high EPA content in either tank fermenter.

<Effect of NaCl concentration on proliferation of GS001 strain> The modified M9+Glc liquid medium whose NaCl concentration was changed to 0.5-10% (w/v) was used to investigate the effect of NaCl concentration on the proliferation of GS001 strain. Shaking culture (150 rpm) was performed at 15° C. using 50 mL of the modified M9+Glc liquid medium already placed in a 100 mL Erlenmeyer flask. After 48 hours from the start of the culture, the culture solution was collected and the OD at a wavelength of 600 nm was measured.

The results are shown in Figure 4. GS001 strain can proliferate in the NaCl concentration range of 0.5-5% (w/v). The GS001 strain proliferates particularly well in the NaCl concentration range of 0.5 to 3% (w/v).

The previously reported effects of NaCl concentrations on the proliferation of Shewanella xylinum strains (Esra Ersoy Omeroglu et al., Leaf Microbiology (2014) 59:79-92) and GS001 strains are summarized in Table 4 and Table 5. In Table 4 and Table 5, data other than GS001 strain are quoted from Table 2 (Table 2) of Esra Ersoy Omeroglu et al., Leaf Microbiology (2014) 59:79-92. "+" means breedable, "-" means not breedable, "N.D." means not implemented.

[Table 4] Shewanella woodyi strains NaCl concentration [%(w/v)] Se2Lu26 Se3Lu42 U-8A U-8B U-1 X-4 X-7 0 - - - - - - - 0.5 - - - - - - - 1 + + + + + + + 3 + + + + + + + 5 + + + + + + + 7 + + - + + + + 9 - - - - - - - 10 - - - - - - - 11 - - - - - - -

[table 5] Shewanella woodyi strains NaCl concentration [%(w/v)] X-2 X-6 E-7 H-4 SW ^* GS001 0 - - - - ND - 0.5 - - - - ND + 1 + + + ND ND + 3 + + + ND ND + 5 + + + ND - + 7 + + + + + ND 9 - - - - ND - 10 - - - - ND - 11 - - - - ND - *Brenner et al. (2005) The Proteobacteria, 480-491 ; Zhao et al. (2005) Int J Syst Evol Microbiol 55:1511-1520; Makemson et al. (1997) Int J Syst Bacteriol 47:1034-1039; Ivanova et al. (2003) Int J Syst Bacteriol 53:1471-1477; Ivanova et al. (2003) Int J Syst Bacteriol Journal of Science 53:577-582 (Shewanella woodi (S. woodyi))

Strain GS001 can also reproduce at 0.5% (w/v) NaCl concentration. On the other hand, no other Shewanella xylinum strains have been reported to be able to reproduce at a NaCl concentration of 0.5% (w/v). From this result, it was confirmed that the GS001 strain can reproduce at a lower salt concentration than Shewanella xylinum strains reported so far.

＜Proliferation test using a synthetic medium using glucose as the sole organic carbon source＞ GS001 strain or Shewanella electrodiphila (Shewanella electrodiphila) ATCC BAA-2408 (hereinafter "ATCC BAA-2408") was precultured (preculture) for 48 hours using the improved M9+Glc liquid medium (cultivation temperature 15°C, shaking Cultivation (rotational speed: 150rpm). Inoculate the pre-cultured GS001 strain or ATCC BAA-2408 into 50 mL of the modified M9+Glc liquid medium that has been placed in a 100 mL flask for cultivation. The culture temperature was set at 15° C., and shaking culture was performed using a BioShaker (rotational speed: 150 rpm). The electrode-loving Shewanella lineage has been reported to reproduce well with glucose as the sole organic carbon source (Jinwei Zhang, and J. Grant Burgess. PLoS One. 2017 Nov 27;12(11) :). The growth rate of Shewanella electrodeophilia when glucose is used as the sole organic carbon source is an index of glucose assimilation energy.

The results are shown in FIG. 5 . Compared with ATCC BAA-2408, the GS001 strain has a shorter induction period (lag phase) and a faster proliferation rate. From these results, it was confirmed that the glucose assimilation ability of the GS001 strain was higher than that of ATCC BAA-2408.

As mentioned above, although the preferred embodiment of this invention was described and illustrated, it should be understood that these are only illustrations of this invention, and should not be considered as limiting. Additions, omissions, substitutions, and other changes may be made without departing from the spirit or scope of the present invention. Therefore, the present invention is not considered to be limited by the foregoing description, but is only limited by the scope of the appended patent application.

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FIG. 1 shows the results of culturing a single isolate (GS001 strain) of Shewanella woodyi using 50 mL of the modified M9+glucose (Glc) liquid medium that had been placed in a 100 mL Erlenmeyer flask. Figure 2 shows the results of phylogenetic analysis of Shewanella xylinum strain GS001 based on the 16S rDNA sequence. Fig. 3A shows the results of a culture test of Shewanella xylinum GS001 strain using a jar fermenter. Fig. 3B shows the results of measuring the EPA content per unit weight of dry cells in a culture test of Shewanella xylinum GS001 strain using a vat fermenter. Fig. 4 shows the results of investigation of the effect of NaCl concentration on the growth of the GS001 strain. Fig. 5 shows the results of a proliferation test using a modified M9+Glc liquid medium using Shewanella xylinum GS001 strain or Shewanella electrodiphila ATCC BAA-2408.

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Claims (5)

A Shewanella woodyi GS001 strain (registration number NITE BP-03460) or a mutant thereof. A composition comprising Shewanella xylinum GS001 strain or its mutant strain. Such as the composition of claim 2, which is food, feed, bait, or cosmetics. A dry powder of Shewanella xylinum GS001 strain or its mutant strain. An extract of Shewanella xylinum GS001 strain or a mutant strain thereof.

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