KR20210115744A - Novel Strain of Streptomyces sp. SBC-4 Having Anti-bacterial Activity, and Uses thereof - Google Patents

Abstract

The present invention relates to an antibacterial composition containing a Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) strain or a culture solution thereof as an active ingredient. According to the present invention, the composition containing the Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) strain or the culture solution thereof exhibits antibacterial activity against various bacteria and can be applied to various bacterial infections caused by pathogenic harmful bacteria. In addition, the composition can be usefully used for antibacterial and antiseptic purposes in various fields such as cosmetic compositions, additive compositions for food or feed preservation, etc.

Description

A novel strain Streptomyces SBC-4 having antibacterial activity and uses thereof {Novel Strain of Streptomyces sp. SBC-4 Having Anti-bacterial Activity, and Uses thereof}

The present invention relates to a new strain SBC-4 of the genus Streptomyces having antibacterial activity and a use thereof, and more particularly, to a new strain Streptomyces spelluncae SBC-4 isolated from Moonmilk of Baekryong Cave located in Pyeongchang-gun, Gangwon-do. And it relates to an antibacterial composition comprising the same as an active ingredient.

Some of the substances that play an important role in the process of maintaining homeostasis of an organism are physiologically active substances derived from various living organisms. Until now, many studies have been conducted on numerous physiologically active substances, and among them, research on antibacterial substances is a very important area in life science and medicine.

It is known that all living things produce antibacterial substances for survival. A lot of research is being done on microorganisms that produce antibacterial substances. have been tried for a long time.

On the other hand, studying the role of microorganisms in a specific ecological environment is very important for understanding the mechanisms by which that ecological environment is maintained. For example, caverns are natural underground openings that do not receive sunlight, have limited nutrient supply, and where there are various interactions between biological activity and mineral supply (Akob and Kusel, 2011). According to the amount of light, the cave can be divided into four zones: entrance, twilight, transition, and deep dark zone (Barton, 2006). Also, compared to the external environment, the cave has high humidity (80-100%) and the partial pressure of carbon dioxide is often high. Lime caves contain various types of cavernous sediments such as soda straw, stalactite, stalagmite, and moonmilk composed mainly of calcium carbonate (CaCO _{3 ).} Moonmilk is a white sediment commonly observed on the walls and ceilings of limestone caves around the world, including in various countries and climates, and is a soft powdery sediment depending on the moisture content (Canaveras et al., 2006; Curry et al.) al., 2009; Cacchio et al., 2012).

In addition, moon milk is characterized in that it is composed of various forms having crystals or filaments of micrometer or nanometer length similar to microbial filaments (Canaveras et al., 2006; Curry et al., 2009; Cacchio et al. al., 2012; Canaberas et al., 1999; Hii and Forti, 2007).

Although it is not clear whether menstrual milk formation occurs through abiotic or biological processes, many studies have recently been reported on the discovery or involvement of various eukaryotic and prokaryotic activities in menstrual milk formation (Canaveras et al. , 2006; Cacchio et al., 2012; Canaberas et al., 1999; Northup and Lavioe, 2001; Barton and Northup, 2007; Portillo and Gonzalez, 2007; Rooney et al., 2010; Portillo and Gonzalez, 2011; Sanchez-Moral et al., 2012). Actively formed moon milk generally has wet traits, which are known to change into dry traits over time (Cacchio et al., 2012). Therefore, various microbial communities exist according to the active state of moon oil in the cave ecosystem, and rare microorganisms that cannot be seen in the general ecosystem can be observed depending on the specific moon oil activity period.

However, research on microorganisms present in the cave ecosystem is still insignificant, and achievements such as excavation of rare cave microorganisms have been reported. It is necessary to discover microorganisms with high commercialization potential.

Under this background, the inventors of the present inventors made diligent efforts to find new strains with antibacterial activity among rare cave microorganisms. As a result, a new strain of Streptomyces genus isolated from Wolyu of Baekryong Cave in Pyeongchang, Gangwon-do has excellent antibacterial activity and is useful as an antibacterial composition. After confirming that it can be used effectively, this invention was completed.

Accordingly, the main object of the present invention is to provide a novel strain of the genus Streptomyces having antibacterial activity, and a regulatory gene derived from the strain and specifically promoting the production of antibiotics at low temperature.

In addition, another object of the present invention is to provide an antibacterial composition using the novel strain of the genus Streptomyces.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides Streptomyces speluncae KNU-JSL-004 (accession number: KCTC 18803P) having antibacterial activity.

The term "Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 strain" is a novel Streptomyces spelluncae strain newly isolated and identified by the present inventors in Wolyu of Baekryong Cave, "SBC 4 strain", which is an abbreviation of "Streptomyces 4 from Baeg-nyong Cave" Alternatively, it may be used in combination with "SBC 4".

The present inventors investigated and analyzed the ecology of several rare microorganisms that grow wild in domestic caves and bioactive substances isolated from microorganisms for many years, and as a result, the antibacterial effect of Streptomyces spp. was studied intensively. As a result, it was confirmed that the Streptomyces speluncae KNU-JSL-004 strain of the present invention has excellent antibacterial activity against pathogenic microorganisms, unlike other isolated strains, and additionally, the new strain of Streptomyces genus It was confirmed that it is possible to control the production of antibacterial substances through a regulator gene that specifically promotes the production of antibiotics at low temperatures.

In addition, the present inventors compared and analyzed the MLST analysis of the new strains of the genus Streptomyces and the conventionally known strains and the fatty acid component constituting the cell membrane, confirming that the strain of the present invention is a new strain belonging to the genus Streptomyces by Streptomyces spell unkae (Streptomyces speluncae ) was named KNU-JSL-004, and it was deposited at the Korea Institute of Biotechnology and Biotechnology Biological Resources Center on January 7, 2020, and was given an accession number KCTC 18803P.

In the present invention, the Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) is characterized in that it has a regulator gene sequence of SEQ ID NO: 1 that specifically promotes the production of antibiotics at a low temperature of 14 ° C to 16 ° C.

According to another aspect of the present invention, the present invention provides an antibacterial composition containing the Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) strain, or a culture medium thereof as an active ingredient.

In the present invention, the Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture is characterized in that it exhibits antibacterial activity itself.

rin) 등의 그람 음성균에 항균 활성이 있으며, 진균류인 칸디다 속 균주, 구체적으로 칸디다 알비칸스(Candida albicans)에 항균 활성을 나타내나, 이에 한정되는 것은 아니다.The Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture medium has antibacterial activity against various pathogenic microorganisms. The strain or its culture medium of the present invention has antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, and specifically, antibacterial activity against Gram-positive bacteria such as Bacillus sp. strain, Staphylococcus sp. strain, Listeria sp. strain, Staphylococcus sp. And, more specifically Bacillus subtilis ( B. subtilis ), Staphylococcus aureus ( Staphylococcus aureus ), Listeria monocytogenes ( Listeria monocytogenes ), methicillin resistant Staphylococcus aureus (MRSA, It has antibacterial activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus ). In addition, the strain of the present invention or its culture medium has antibacterial activity against Gram-negative bacteria such as Escherichia sp. strain, Pseudomonas sp. strain, Salmonella strain, Klebsiella sp. strain, Mycobacterium tuberculosis, etc., more specifically Escherichia coli ), Pseudomonas aeruginosa aeruginosa ), Salmonella Typhimurium, Klebsiella Pneumonia pneumoniae ), Mycobacterium bovis bacille Calmette-Gu

rin) has antibacterial activity against Gram-negative bacteria, and exhibits antibacterial activity against Candida sp. strains, specifically Candida albicans , which are fungi, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the culture medium of the strain according to the present invention has a growth inhibitory effect against various pathogenic bacteria (see FIG. 9 and Table 1).

According to another aspect of the present invention, the present invention provides an antibacterial pharmaceutical comprising the Streptomyces speluncae KNU-JSL-004 (Accession Number: KCTC 18803P) strain having the antibacterial activity or a culture solution thereof as an active ingredient. It provides an enemy composition. The Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture solution having antibacterial activity of the present invention exhibits excellent antibacterial activity through direct influence on harmful microorganisms. It can be usefully used as an antifungal pharmaceutical composition.

On the other hand, Streptomyces spellunkae having the antibacterial activity of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) culture solution or its extract may be prepared in the form of a pharmaceutically acceptable salt. Specifically, salts can be formed by addition of acids, for example, inorganic acids (e.g. hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (e.g. acetic acid, trifluoroacetic acid, etc.) acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides such as hyaluronic acid, chondroitin sulfate, arginic acid), organic sulfonic acids including sulfonic acid sugar esters such as chondroitin sulfate (eg, methane, sulfonic acid, p-toluene sulfonic acid), etc. may be added to form salts.

Streptomyces spellunkae having the antibacterial activity of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture medium can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, Streptomyces spellunkae of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture solution can actually be administered in various oral or parenteral formulations. , prepared by using a diluent or excipient such as a surfactant. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, liulinji, glycerogelatin, and the like can be used.

In addition, Streptomyces spellunkae of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture solution can be used by mixing it with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and to increase stability or absorbency, glucose , carbohydrates such as sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

Streptomyces spellunkae of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) The effective dose of the strain or its culture is 0.01 to 10 mg/kg, preferably 0.1 to 1 mg/kg, and can be administered 1 to 3 times a day have.

The total effective amount of the pharmaceutical composition or the culture medium of the present invention may be administered to the patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, and multiple doses (multiple does) can be administered by a fractionated treatment protocol that is administered over a long period of time. Since the concentration is determined by considering various factors such as the age and health status of the patient as well as the administration route and number of treatments, the effective dosage of the patient is determined. It will be possible to determine an appropriate effective dosage according to the specific use of the novel strain JS-169 or its culture medium as a pharmaceutical composition.

According to another aspect of the present invention, the present invention provides an antibacterial cosmetic composition containing Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) or a culture solution thereof as an active ingredient having antibacterial activity to provide.

Streptomyces spellunkae of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) or its culture medium can be usefully used as an antibacterial cosmetic composition because it exhibits excellent antibacterial activity by directly affecting harmful microorganisms.

The cosmetic composition of the present invention contains the Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) strain as an active ingredient or ingredients commonly used in the cosmetic composition in addition to the culture solution thereof, for example, antioxidant conventional adjuvants such as agents, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), a nourishing lotion (milk lotion), a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder. .

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. are used as carrier components. can be

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.

Specific examples of the cosmetic of the present invention include face wash cream, face wash foam, cleansing cream, cleansing milk, cleansing lotion, massage cream, cold cream, moisture cream, emulsion, lotion, pack, after-saving cream, anti-tan cream, sun-tan oil, soap , body shampoo, hair shampoo, hair conditioner, hair treatment, hair care, hair growth, hair cream, hair liquid, set lotion, hair spray, hair bridge, color rinse, color spray, permanent wave solution, press powder, loose powder , eye shadow, hand cream, lipstick, and the like.

According to another aspect of the present invention, the present invention is Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) It provides a composition for preserving food or feed containing the strain or its culture medium as an active ingredient.

Food or feed preservatives are additives used to prevent deterioration, decay, discoloration and chemical changes of food or feed. These include disinfectants and antioxidants. , functional antibacterial agents such as inhibiting the growth of decaying microorganisms or sterilizing in cosmetics, pharmaceuticals, etc. Ideal conditions for these preservatives in food or feed should be non-toxic and effective even in a small amount. Streptomyces spellunkae of the present invention ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or its culture solution shows excellent antibacterial activity through direct effect on harmful microorganisms, so it can be widely used as a preservative for food or feed, cosmetic preservatives and pharmaceutical preservatives. can

In addition, according to another aspect of the present invention, the present invention is the Streptomyces speluncae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or a composition containing the culture medium, which requires treatment It provides a method for treating bacterial infections in animals other than humans, comprising the step of killing pathogenic bacteria by administering to a subject.

Although the treatment method according to the present invention is a method of treating animals other than humans, it does not mean that the treatment method is ineffective in humans. In addition, in consideration of having a disease caused by a bacterial infection in which symptoms can be improved by administration of the therapeutic composition according to the present invention in the case of a human, it can be sufficiently used for human treatment.

In the present invention, the term "animals other than humans" refers to horses, sheep, pigs, goats, camels, antelopes, except for humans, who have diseases caused by bacterial infection, whose symptoms can be improved by administration of the therapeutic composition according to the present invention. , animals such as dogs. By administering the therapeutic composition according to the present invention to animals other than humans, it is possible to effectively prevent and treat diseases caused by bacterial infection.

In the present invention, the term "administration" means introducing a predetermined substance to an animal by any suitable method, and the administration route of the therapeutic composition according to the present invention is oral or through any general route as long as it can reach the target tissue. It may be administered parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient to the target cell.

The preferred dosage of the therapeutic composition according to the present invention varies depending on the condition and weight of the animal to be treated, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition of the present invention is 0.01 to 10 mg/kg, preferably 0.1 to 1 mg/kg, and may be administered once to 3 times a day.

According to another aspect of the present invention, the present invention is isolated from Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) having antibacterial activity, 14 ℃ to 16 represented by SEQ ID NO: 1 It provides a regulator gene that specifically promotes the production of antibiotics at a low temperature of ℃.

The present inventors Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) as a result of whole genome sequencing analysis of strain A factor A factor through transcriptome analysis related to gene expression according to temperature conditions of 15°C and 30°C It was confirmed that the synthase A gene increased gene expression at low temperature (see FIG. 15).

As described above, the A factor synthase A gene, whose gene expression is increased at low temperature, is regulated by the γ-butyrolactone hormone, and can control the production of antibiotics in various microbial species. In fact, "factor A" synthesized by the A factor synthase A gene is well known to promote the activity of a master regulator of antibiotic production in Streptomyces griseus (see FIG. 16). . Therefore, the regulator gene of the present invention is a regulator gene that specifically promotes the production of antibiotics at low temperatures, and can be recombinated in genes of various strains and used to control the production of antibacterial substances. .

The features and advantages of the present invention are summarized as follows:

(a) the present invention is Streptomyces spellunkae ( Streptomyces speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) It provides an antibacterial composition comprising a strain or a culture solution thereof as an active ingredient.

(b) Streptomyces spellunkae of the present invention speluncae ) KNU-JSL-004 (Accession No.: KCTC 18803P) strain or a composition containing its culture medium exhibits antibacterial activity against various bacteria, and the composition of the present invention is applicable to various bacterial infections caused by pathogenic harmful bacteria. .

(c) In addition, the composition of the present invention can be usefully used for antibacterial and antiseptic purposes in various fields such as cosmetic compositions, additive compositions for food or feed preservation.

1 is a schematic diagram showing the location and appearance (A, B) of Baekryong Cave, and a photograph of the collected Dry Wolyu (C) and Wet Wolyu (D) colonies (the sample is collected in red (moonmilk) in (B). ) and blue dots (soil), the abbreviation DM stands for dry moonmilk, the abbreviation WM stands for wet moonmilk, and CS stands for calcite soil).
2 is an x-ray diffraction analysis result of dry and wet moon milk collected from Baekryong Cave.
3 is an SEM analysis result of dry moon milk and wet moon milk collected from Baekryong Cave.
4 is a metagenome analysis result for microorganisms present in dry moon milk, wet moon milk, and soil samples.
5 is a diagram showing microorganisms present in a significant proportion in Dry Monthly Milk and Wet Monthly Oil.
Figure 6 is a photograph of 9 strains (SBC 1 to 9) having the form of a Streptomyces strain isolated from Dry and Wet monthly milk.
7 is a systematic diagram prepared based on the results of 16S rRNA analysis for 9 isolates.
8 is a schematic diagram illustrating a cross streak assay.
9 is a photograph taken of the cross streak assay results for pathogenic microorganisms of 9 isolates.
10 is a table summarizing the results of MLST analysis of SBC 4 strains.
11 is a table summarizing the results of analyzing the entire genome sequence of the SBC 4 strain.
12 is a diagram showing a comparative analysis of the fatty acid component constituting the cell membrane of the SBC 4 strain and the Streptomyces Gardneri strain.
13 is a photograph showing the comparative analysis of the antibacterial activity of the SBC 4 strain according to the temperature conditions of 15°C and 30°C.
14 is a diagram showing comparative analysis by measuring MIC values for various pathogenic microorganisms in order to comparatively analyze the antibacterial activity of SBC 4 strains according to temperature conditions of 15°C and 30°C.
15 is a diagram illustrating a summary of 28 biosynthetic gene clusters based on the results of whole genome sequencing of SBC 4 strains.
16 is a graph showing changes in the expression of biosynthetic gene clusters measured at 15° C. and 30° C. temperature conditions.
17 is a schematic diagram illustrating a low-temperature-specific A factor synthase A gene expression regulation mechanism in SBC 4 strain.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Example 1. Collection of Moonmilk from Baekryong Cave

1-1. collection site

The cave sampled for this study is Baekryong Cave (37°16' 19.65"N, 128°34' 46.03"E, 1,875m) located in Pyeongchang-gun, Gangwon-do, located at the foot of Mt. , is the first cave in Korea with a Critical Zone Observatory (CZO) research site.

Baekryong Cave consists of a total of 4 caves, the main cave and 3 eggplant caves extending from east to west. The representative feature of Baekryong Cave is that it has a terrain that bends at 90° after going straight 15m from the entrance, so changes in external environmental factors such as sunlight appear, and the microenvironment inside the cave is very stable. The internal temperature inside Baengnyeong Cave is always maintained at 11.0°C to 13.5°C regardless of the season, and the relative humidity is 70% to 100%. Some sections allow people to enter for tourism purposes, but the number of people entering and leaving per day is limited, and access to other sections is strictly restricted.

1-2. monthly milk collection

Baekryong Cave has various cave products such as stalactites, stalagmites, stalactites, petrification, and moon oil.

Wolyu is a white precipitate often found on the ceilings, walls, and surfaces of rocks in limestone caves. exist in the form Traces of various types of microorganisms have been found in moonmilk in various caves around the world, and it is presumed that there are some physical factors as well as the action of microorganisms in the formation of moonmilk. In fact, it is well known that _{microorganisms can form CaCO 3} crystals outside the microorganisms while the _{CO 2} component created by oxidizing the material on the surface of the cave through various processes ^{combines with Ca 2+ ions on the wall.}

Baekryong Cave is 1.8 km long and consists of the main cave in area A and the remaining 4 caves, B, C, and D. The researchers were able to sample moon milk in dry form (see Fig. 1 C) and wet form (see Fig. 1 D) in Cave B, where human access is restricted, and in order to compare the microbial structure in these caves with the external environment, Soil samples were taken at the inlet.

Example 2. Metagenomic analysis of moon milk and microorganisms

2-1. moon milk analysis

First, it was confirmed through x-ray diffraction analysis that the two types of moon milk consist of calcite component (FIG. 2), and through SEM analysis, it was confirmed that the two types of moon oil had traces of microorganisms with different structures. could be (Fig. 3).

Specifically, it was confirmed that round-shaped microorganisms formed a colony in dry moon milk, and it was confirmed that filamentous microorganisms formed a colony in wet moon milk. And, it was confirmed that these unusual structures are actually _{composed of CaCO 3 component.}

Therefore, based on the above results, it was possible to hypothesize that different types of microbial communities exist in dry and wet moon milk, and metagenomic analysis was performed on microorganisms present in dry and wet moon milk.

2-2. Microbial metagenomic analysis

After diluting the harvested moon milk in sterile physiological saline (0.85% NaCl) to isolate the strain, 100 μl of the supernatant was spread on ISP4 (International Streptomyces Project 4) medium, and cultured for about 35 days in an incubator at 30 ° C. . A single colony of the strain was selected, plated on ISP4 medium, and cultured again for about 35 days in an incubator at 30°C.

Dry and wet moon milk samples were analyzed by 3 and soil samples by 4, and it was confirmed that the community structure of prokaryotic microorganisms in each sample was well grouped. It could be seen that it was markedly different from the In addition, it was confirmed that the predominant types of microorganisms were proteobacteria, actinobacteria, and acidobacteria in the order (see FIG. 4).

In addition, if we looked at the microorganisms analyzed in dry and wet moon milk in more detail, it was confirmed that different types of microorganisms existed predominantly in the two types of moon milk. It is speculated that these microorganisms may play an important role in the formation of moon milk and calcite. In particular, the chromatiales genus strain found in Dry moon milk is a species found in many other limestone caves, and the Streptomyces genus strain is known to actually act as a mineral deposit (FIG. 5) .

Therefore, although the two types of dry and wet moon milk were in the same oligotrophic environment, it could be inferred that certain detailed factors such as moisture content had a great influence on the formation of different microbial communities.

Next, in order to select a model microorganism that responds specifically to the cave environment, microorganisms that specifically dominate the moon milk were identified. To be used as a model microorganism, it must be able to be well cultured in a laboratory environment, be capable of experimental manipulation, and must have striking characteristics. Therefore, we selected a Streptomyces genus strain that is relatively easy to cultivate in a laboratory environment and secretes various secondary metabolites as a model strain.

Example 3. Isolation of Streptomyces sp.

Monthly milk was diluted in sterile normal saline, the supernatant was spread on an oligotrophic medium with insufficient nutrients, and cultured for 3 to 5 days to obtain various types of microorganisms. . Next, when visually observed, colonies having a streptomyces strain form were selected, isolated and cultured (see FIG. 6 ).

In addition, through 16S rRNA analysis of a total of 9 isolates, it was confirmed that these were microorganisms of the genus Streptomyces (see FIG. 7 ).

Example 4. Analysis of antibacterial activity of Streptomyces sp.

4-1. cross streak assay

Streptomyces (streptomyces) genus strains are well known to have characteristics of producing various secondary metabolites such as antibiotics or pigments.

Therefore, a cross streak assay experiment was performed to confirm whether the isolated 9 strains had antimicrobial activity.

Cross streak assay was performed by streaking 9 isolated strains in the center of the medium, culturing at 30° C. for 5 days, streaking the pathogenic microorganisms in the horizontal direction of the 9 isolated strains, and culturing at 37° C. for one day ( see Fig. 8).

As a result, it was confirmed that only the SBC4 strain among the 9 isolated strains inhibited the growth of pathogenic microorganisms, gram positive B. subtilis strains and gram negative E. coli and Salmonella strains ( see Fig. 9).

4-2. Minimum inhibitory concentration test

After culturing the SBC4 strain exhibiting antibacterial activity in a large culture environment as a result of cross streak assay, the microbial growth minimum inhibitory concentration (MIC) of the supernatant was evaluated.

Harmful bacteria are Bacillus subtilis ( B. subtilis ), Staphylococcus aureus ( Staphylococcus aureus ), Listeria monocytogenes ( Listeria ) monocytogenes ), MRSA, BCG, E. coli, Salmonella Typhimurium, Klebsiella pneumoniae pneumoniae ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Candida albicans ( Candida albicans ) strain was used. Each bacteria was inoculated by obtaining 2-3 colonies (5×10 ⁷ CFU/mL). For MIC measurement, after culturing each bacteria and fungus on a solid agar medium, ^{5 colonies were obtained according to} 1×10 5-6 CFU/mL, diluted in the medium and inoculated, and maintained at 37° C. for 2-3 hours. The culture supernatant of the SBC 4 strain used in this experiment was titrated to a concentration of 2 mg/mL and each diluted two-fold. The medium containing each harmful bacteria and sample was transferred to a 96 well plate and incubated at 37°C for 16 to 24 hours, and then measured with a Multi label Counter (Bio-rad Microplate reader) at 600 nm to determine the SBC for each harmful bacteria. The minimum inhibitory concentration value of the culture supernatant of 4 strains was obtained, and it is shown in [Table 1] below.

[Table 1]

As a result, the culture supernatant of the SBC 4 strain of the present invention is Bacillus subtilis ( B. subtilis ), Staphylococcus aureus ( Staphylococcus aureus ), Listeria monocytogenes ( Listeria ) monocytogenes ), Gram-positive bacteria such as MRSA, Escherichia coli, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Salmonella Typhimurium, Klebsiella Pneumonia pneumoniae ) and Mycobacterium tuberculosis (BCG), and has antibacterial activity against Gram-negative bacteria, and it was confirmed that it also exhibits antibacterial activity against the fungus Candida albicans.

Example 5. Identification of SBC 4 strain of the genus Streptomyces

Next, in order to confirm the genetic information of the SBC 4 strain, 16S rRNA analysis of the SBC 4 strain was performed.

Usually, if the similarity of the 16S rRNA gene is 97% to 98%, it can be determined that they are the same species. In the case of the SBC 4 strain, more than 10 similar species showing 16S rRNA sequence similarity of 99% or more were identified, so species classification and identification could not be performed only by 16S rRNA sequence analysis.

[Table 2]

Accordingly, MLST analysis, which is a more advanced species classification method, was performed. MLST analysis is an abbreviation of “Multilocus sequence typing” and aligns the sequences of several housekeeping genes (atpD, gyrB, recA, rpoB, trpB, and 16S rRNA genes) to determine the distance on the gene sequence of microorganisms. way to calculate. Usually, if the distance on the analyzed sequence is 0.007 or less, it is judged as the same species.

Referring to FIG. 10 , the SBC 4 strain was analyzed to have a distance of 0.018 from the S. gardneri strain, which is the closest type strain, as a result of MLST analysis.

Through the MLST analysis, it was confirmed that the SBC 4 strain was a new strain of the genus Streptomyces.

In addition, as a result of ANI (Average Nucleotide Identity) analysis, which analyzes genomic similarity at the nucleotide level between two genomes, it was confirmed that the SBC 4 strain was a new strain.

Finally, the fatty acid component constituting the cell membrane of the SBC 4 strain was investigated. As a result, it was confirmed that there is a clear difference in the fatty acid component constituting the cell membrane of the Streptomyces gardneri strain and the SBC4 strain of the present invention, which are close to each other in the gene sequence. It could be reconfirmed that it was a strain (see FIG. 12).

Therefore, the present inventors named the strain of the present invention having excellent antibacterial activity as Streptomyces speluncae KNU-JSL-004, and deposited it with the deposit number (KCTC 18803P) at the Korea Research Institute of Bioscience and Biotechnology. .

Example 6. Analysis of gene expression regulation of SBC 4 strain of the genus Streptomyces

The identification of SBC 4 strains of the genus Streptomyces that live in a cave environment and have antibacterial activity was completed, and the cave environment-specific epigenetic gene expression regulation of this microorganism was additionally analyzed. There are various environmental factors in the cave environment where the strain lives, but we paid attention to the relatively low temperature. Although the inside of Baekryong Cave is maintained at 10 ° C to 12 ° C, the growth activity was low when the SBC 4 strain was actually cultured at a temperature of 10 ° C to 12 ° C, so the temperature was set at 15 ° C. 30 ℃, which is known as the optimal culture temperature for Streptomyces genus strains, was set. Then, antibacterial substances were extracted from their cultures and their effects were checked, and at the same time, their transcriptome analysis was performed under the same conditions to find out which antibiotics they were.

As a result, referring to Table 3 and FIG. 13, when the antibiotics were extracted at two temperatures and cultured at 15° C., the pathogenic strains E. coli and Salmonella Typhimurium, and Bacillus subtilis and Stapylococcus aureus showed more effective antibacterial activity. I was able to confirm what I had.

[Table 3]

In addition, referring to FIG. 14 , it was confirmed that antibiotics were extracted at a temperature of 15° C. for all the tested pathogenic bacteria to have more effective antibacterial activity.

Additionally, through the result of whole genome sequencing analysis of SBC 4 strain, 28 biosynthetic gene clusters were analyzed (see FIG. 15 ) and related to gene expression according to temperature conditions of 15°C and 30°C. Through transcriptome analysis, it was confirmed that only one biosynthetic gene increased gene expression at 15°C (see FIG. 16 ).

As described above, the gene whose gene expression increases at 15° C. is the A factor synthase A gene, which is regulated by the γ-butyrolactone hormone, and is a gene known to control the production of antibiotics in various microbial species. In fact, “factor A” synthesized by the A factor synthase A gene is well known to promote the activity of a master regulator of antibiotic production in Streptomyces griseus (see FIG. 17). .

Therefore, through the above results, the new strain of Streptomyces genus SBC 4 of the present invention epigenetically produces antibacterial substances through a regulator gene that promotes the production of antibiotics specifically at low temperature at 15°C. was found to control the

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Institute of Bioscience and Biotechnology KCTC18803 202000107

<110> KNU-Industry Cooperation Foundation <120> Novel Strain of Streptomyces sp. SBC-4 Having Anti-bacterial Activity, and Uses thereof <130> PN200015AN <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 951 <212> DNA <213> Unknown <220> <223> Novel Strain of Streptomyces sp. : Streptomyces speluncae KNU-JSL-004 <400> 1 atggaccagg agtggcgagg agtgacggcg cgcgtccccg gagagttcgt tcaccgagcc 60 gaccccgcag acatcatccc cacgggctgg acacagttga cggagaaccg gttctccata 120 tccgcccgct ggcctgccgc gcatcccttc ttctctcccg tcgcagggga ccggcacgac 180 cccgtgctgg tggccgagac gatacgccag gccaccatgc tcgtcgctca tgccgagctc 240 ggggtacccg tggacgatca gttcgtcatg tggggcctcc attacgccgc cgaccccgag 300 gcgctcgccg tggacggcct ctcctcggac gtcaccgtcg acctcgtctg ctccgacctg 360 acccgtcgcg ccggcagcat ccgcaacctg cgtgccaccc tggtgctcac gcgcgacggc 420 cggcacctcg ccaccggcag tgccctggcc cgctgcacgt cggccctggc gtaccgccgg 480 atccgcggcg accgcatggc cgccctgggc aggccggtcc cgctcatacc gggcctggca 540 ccgcacctgg tcggccggga gaacgccaag gacgtcgtcc tggctccggg caccagaccg 600 gaccagtggc agctgcggat caacacccag cacaccaccc tcttccgccg cccgaacgac 660 cacgtaccgg gcatggtgct ccttgaggcc gcccgtcagg cggcgaccgt gacgaccggg 720 agccgcgcct tcctgcccac cgtcatggac gcctccttct cgcgctacgc cgagctcgac 780 agcccgtgct ggatcgagac cgaggtcgtc cccagctccg cctcctcggc caccaccgtc 840 cgcgtcctcg gccggcagga cggcaacgag gtgttccagt gcgcgctcac ctccccgtca 900 cgcgacctgg cggtcgccgg aggcggcctc ggcagccgcc tggccggttg a 951

Claims (8)

Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) with antibacterial activity. According to claim 1,
The Streptomyces speluncae (Streptomyces speluncae) KNU-JSL-004 (Accession No.: KCTC 18803P) is a regulator gene of SEQ ID NO: 1 that specifically promotes the production of antibiotics at a low temperature of 14 ° C to 16 ° C. A strain characterized in that it has a sequence. An antibacterial composition comprising the strain of claim 1 or 2, or a culture solution thereof as an active ingredient. 4. The method of claim 3,
The antibacterial composition is an antibacterial composition, characterized in that it has antibacterial activity against pathogenic microorganisms Bacillus subtilis, Staphylococcus aureus, and E. coli. A pharmaceutical composition comprising the composition of claim 3 as an active ingredient. A cosmetic composition comprising the composition of claim 3 as an active ingredient. A preservative composition for feed or food containing the composition of claim 3 as an active ingredient. Isolated from Streptomyces speluncae KNU-JSL-004 (Accession No.: KCTC 18803P) with antibacterial activity,
A regulator gene that specifically promotes the production of antibiotics at a low temperature of 14° C. to 16° C. represented by SEQ ID NO: 1.

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