KR20220072912A - Novel strain of Bacillus subtilis producing menaquinone-7 and method for producing menaquinone-7 using the same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis strain producing menaquinone-7 and a method for producing menaquinone-7 using the same, wherein the novel Bacillus subtilis strain is 1-hydroxy-2-naph due to UV irradiation As a strain mutated to have resistance to local acid, it is possible to produce menaquinone-7 at a high concentration.

Description

Novel strain of Bacillus subtilis producing menaquinone-7 and method for producing menaquinone-7 using the same}

The present invention relates to a novel Bacillus subtilis strain for producing menaquinone-7 and a method for producing menaquinone-7 using the same.

Vitamin K is produced in the body, but its amount is small, and it is a nutrient that is attracting attention as it is known to have various effects such as blood clotting control, cardiovascular disease prevention, osteoporosis prevention, anticancer, and female hormone regulation in the body. Vitamin K is a generic term for fat-soluble vitamins having a naphthoquinone ring structure, and vitamin K1 (phylloquinone) derived from plants and vitamin K2 (menaquinone) derived from microorganisms are representative. Vitamin K2 can be divided into menaquinone-1 ~ menaquinone-14 according to the length of the isoprenyl side chain attached to naphthoquinone, and menaquinone-4 and menaquinone-7 are mainly used commercially.

Menaquinone-7 is produced not only by microbial production but also by chemical synthesis. Menaquinone-7 produced by chemical synthesis is trans-form and isomer in the side chain isoprenyl unit. While all cis-forms (isomers) exist, menaquinone-7 produced in microorganisms does not have a cis-form in the isoprenyl unit and all trans-forms (all transform) structure. The structural difference of these side chains affects the absorption rate and bioactivity of menaquinone-7, and the absorption rate and bioactivity of menaquinone-7 produced by microorganisms compared to menaquinone-7 produced by chemical synthesis This is known to be high.

Therefore, recently, research to develop a method for mass production of menaquinone-7 from microorganisms is being actively conducted. Korean Patent No. 10-1196338 and Korean Patent Publication No. 10-2016-0045661 disclose strains producing menaquinone-7, but the production of menaquinone-7 of these strains is insignificant and suitable for commercial use. There is a problem that it doesn't. Therefore, many studies are still needed to discover new microorganisms capable of producing menaquinone-7 at high concentrations.

Korean Patent Registration No. 10-1196338 Korean Patent Publication No. 10-2016-0045661

An object of the present invention is to provide a Bacillus subtilis DS-VK4 strain (Accession No.: KCCM12812P) that produces menaquinone-7 as a novel Bacillus subtilis strain.

Another object of the present invention is to provide a method for producing menaquinone-7 using the strain.

The present inventors have studied to develop a new strain or mutant capable of industrially mass-producing menaquinone-7 among vitamin K2. As a result, menaquinone-7 production is increased by inducing mutations in the gene involved in the production of menaquinone-7. By confirming the increase, the present invention was completed.

One aspect of the present invention provides a Bacillus subtilis DS-VK4 strain producing menaquinone-7.

The Bacillus subtilis DS-VK4 strain is a mutant strain having resistance to 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid) due to mutagenesis by ultraviolet (ultraviolet, UV), Mena It is a strain with improved menaquinone-7 production by weakening, inactivating, or enhancing the activity of factors involved in quinone-7 production.

As used in the present invention, “weakened activity” means that the expression of genes encoding proteins, such as enzymes, transcription factors, and transport proteins, is reduced compared to the native strain, the wild-type strain, or the strain before modification. Weakening of this activity is when the activity of the protein itself is reduced compared to the activity of the protein possessed by the original microorganism through nucleotide substitution, insertion, deletion, or a combination thereof, and inhibition of the expression or translation of the encoding gene When the overall degree of enzymatic activity in the cell is low compared to the native strain, the wild type strain or the strain before modification, combinations thereof are also included.

"Inactivation" as used in the present invention refers to a case in which the expression of a gene encoding a protein such as an enzyme, a transcription factor, a transport protein, etc. is not expressed at all compared to the native strain, the wild type strain, or the strain before modification, or even if it is expressed, its activity This means that there is no

As used in the present invention, “enhanced activity” means that the expression of a gene encoding a protein such as an enzyme, a transcription factor, or a transport protein is newly introduced or increased, so that the expression level is increased compared to the native strain, the wild type strain, or the strain before modification. do. The enhancement of this activity is when the activity of the protein itself is increased compared to the activity of the protein possessed by the original microorganism through nucleotide substitution, insertion, deletion, or a combination thereof, and the expression or translation increase of the gene encoding the gene is increased. For example, when the overall degree of enzymatic activity in the cell is higher than that of the native strain, the wild type strain, or the strain before modification, a combination thereof is also included.

This Bacillus subtilis DS-VK4 strain was deposited with the Korea Microorganism Conservation Center on October 22, 2020 and was given an accession number KCCM12812P.

According to one embodiment of the present invention, the Bacillus subtilis DS-VK4 strain may have resistance to 1-hydroxy-2-naphthoic acid.

More specifically, the Bacillus subtilis DS-VK4 strain is 100 ppm or more of 1-hydroxy-2-naphthoic acid, specifically 100 to 200 ppm, 100 to 150 ppm, or 100 ppm tolerant to a concentration of it could be

According to an embodiment of the present invention, the present inventors apply ultraviolet rays to the Bacillus subtilis KCCM11796P strain isolated from nature at a wavelength of 200 to 300 nm (preferably, 240 to 270 nm), 50 to 500 seconds (preferably, 70 to 200 seconds) to induce mutations by irradiation 1 to 4 times (preferably, 4 times), and menaquinone-7 production is the highest among mutations with resistance to 100 ppm of 1-hydroxy-2-naphthoic acid. By selecting an excellent DS-VK4 strain, a Bacillus subtilis mutant (DS-VK4 strain) with improved menaquinone-7 production ability compared to the Bacillus subtilis KCCM11796P strain was obtained and deposited at the Korea Microorganism Conservation Center on October 22, 2020 (Accession No. KCCM12812P).

Here, 'improved productivity' means that the productivity of menaquinone-7 is increased compared to the parent strain. The parent strain means a wild-type or mutant strain to be mutated, and includes a target to be directly mutated or transformed with a recombinant vector. In the present invention, the parent strain may be a Bacillus subtilis KCCM11796P strain.

According to one embodiment of the present invention, the Bacillus subtilis DS-VK4 strain exhibits increased menaquinone-7 production ability compared to the parent strain, and in particular, the menaquinone-7 production is more than doubled compared to the parent strain, specifically As such, it may be increased by 2 to 20 times, 2 to 10 times, 4 to 20 times, or 4 to 10 times.

According to an embodiment of the present invention, it was confirmed that the Bacillus subtilis DS-VK4 strain increased menaquinone-7 production by about 4.5 times compared to the parent strain.

In addition, another aspect of the present invention comprises the steps of: a) culturing the Bacillus subtilis DS-VK4 strain in a medium; And b) provides a method for producing menaquinone-7 comprising the step of recovering menaquinone-7 from the strain or the culture medium.

The culture may be made according to an appropriate medium and culture conditions known in the art, and those skilled in the art can easily adjust the medium and culture conditions for use. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, for example, batch culture, continuous culture, fed-batch culture, or a combination culture thereof, but is not limited thereto.

According to one embodiment of the present invention, the medium should meet the requirements of a specific strain in an appropriate manner, and may be appropriately modified by a person skilled in the art.

According to one embodiment of the present invention, the medium may include various carbon sources, nitrogen sources and trace element components. Carbon sources that can be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto. Nitrogen sources that can be used include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture, but is not limited thereto. Sources of phosphorus that may be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. In addition, precursors suitable for the culture medium may be used. The medium or individual components may be added batchwise or continuously by an appropriate method to the culture medium during the culturing process, but is not limited thereto.

According to one embodiment of the present invention, it is possible to adjust the pH of the culture medium by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microorganism culture medium in an appropriate manner during culture. In addition, it is possible to suppress the formation of bubbles by using an antifoaming agent such as fatty acid polyglycol ester during culture. Additionally, in order to maintain the aerobic state of the culture medium, oxygen or oxygen-containing gas (eg, air) may be injected into the culture medium, for example, the stirring speed may be set to 100 to 300 rpm, 100 to 250 rpm, 150 to 300 rpm , or 150 to 250 rpm. The temperature of the culture medium may be usually 20 °C to 45 °C, for example, 25 °C to 40 °C. The incubation period may be continued until a useful substance is obtained in a desired production amount, for example, 6 to 24 hours, 6 to 48 hours, 12 to 24 hours, 12 to 48 hours, 10 to 240 hours, 10 to 180 hours. , 60 to 240 hours, or 60 to 180 hours.

According to one embodiment of the present invention, the medium is glycerol (glycerol) 1 to 5% by weight, sucrose (sucrose) 0.1 to 2% by weight, soy peptone (soy peptone) 4 to 8% by weight based on the total weight, yeast 0.1 to 1% by weight of yeast extract, 0.1 to 1% by weight of K ₂ HPO ₄ and 0.05 to 0.5% by weight of MgSO ₄ may be included.

This medium may be an optimized medium to increase the productivity of menaquinone-7 from the Bacillus subtilis DS-VK4 strain.

According to an embodiment of the present invention, the Bacillus subtilis DS-VK4 strain is glycerol 3%, sucrose 1%, soy peptone 6%, yeast extract 0.6%, K ₂ HPO ₄ 0.5% and MgSO ₄ 0.15% It was cultured for 16 hours or 120 hours at a temperature of 37° C. and a speed of 200 rpm in the containing GSSY medium.

According to one embodiment of the present invention, the step of recovering the cultured strain and menaquinone-7 from the culture medium is menaquinone produced from the medium using a suitable method known in the art according to the culture method- 7 can be collected or retrieved. e.g. centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion) may be used, but the present invention is not limited thereto.

According to an embodiment of the present invention, the culture medium of Bacillus subtilis DS-VK4 strain and 2-propanol:n-hexane (1:2) are mixed in a volume ratio of 2:1, and the layers are separated by centrifugation at low speed. After induction, the solvent layer was separated, and menaquinone-7 was separated from the separated solvent layer through HPLC analysis.

According to one embodiment of the present invention, the step of recovering menaquinone-7 may include a step of purifying menaquinone-7.

The novel Bacillus subtilis strain according to the present invention is a strain mutated to have resistance to 1-hydroxy-2-naphthoic acid due to ultraviolet irradiation, and can produce menaquinone-7 at a high concentration.

Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Preparation of menaquinone-7 producing mutants

1-1. Determination of 1-hydroxy-2-naphthoic acid treatment concentration

Bacillus subtilis KCCM11796P strain was provided from Daesang Food Research Institute and was used as the parent strain for this experiment.

The parent strain was inoculated into 3ml of LB broth and incubated at 37°C and 200rpm for 16 hours. The culture solution was diluted 100 times with physiological saline (0.85% NaCl) and spread on LB solid medium containing 1-hydroxy-2-naphthoic acid concentration (0, 50, 100, 150 ppm), followed by incubation at 37°C for 48 hours. .

As a result, as shown in Table 1 below, the parent strain was grown under conditions in which 1-hydroxy-2-naphthoic acid was absent or the concentration was 50 ppm, whereas 1-hydroxy-2-naphthoic acid was 100 ppm or more. It was confirmed that it did not grow in Through these results, 1-hydroxy-2-naphthoic acid treatment concentration for screening 1-hydroxy-2-naphthoic acid resistant strain (resistant strain) was set to 100 ppm.

1-hydroxy-2-naphthoic acid concentration Viability of the parent strain ^* 0 ppm +++++ 50 ppm ++ 100 ppm - 150 ppm - *(-): poor growth, (+): excellent growth

1-2. 1-hydroxy-2-naphthoic acid resistant strain selection

The parent strain was inoculated into 2ml of LB broth and cultured for 16 hours at 37°C and 200rpm. 250 μl of the seed culture was inoculated into 25 ml of LB broth and cultured at 37° C. and 200 rpm until OD _{600 nm} = 0.5. Cells were recovered by centrifugation at 12,000 rpm, 4° C., for 10 minutes, and then washed twice with physiological saline. Then, the cells were suspended in physiological saline so that OD _600nm = 1.0. 3ml of the suspension was evenly spread on a culture dish (60X15mm), placed in a multipurpose sterilizer (RD-920H/D, Rios Co., Ltd.), and UV irradiated with a wavelength of 254nm for 100 seconds. After resting the suspension for 2 hours in dark conditions, it was smeared on LB agar medium with a concentration of 100 ppm of 1-hydroxy-2-naphthoic acid and cultured at 37° C. for 48 hours. After that, the cells were recovered and the UV irradiation process was repeated 4 times.

The culture solution was diluted 100-fold with physiological saline, spread on LB solid medium containing 1-hydroxy-2-naphthoic acid for each concentration (0, 50, 100, 150 ppm), and then cultured at 37° C. for 48 hours.

As a result, as shown in Table 2 below, the strain irradiated with UV as a mutant showed increased resistance to 1-hydroxy-2-naphthoic acid compared to the parent strain.

1-hydroxy-2-naphthoic acid concentration viability ^* parent strain UV-irradiated strains 0 ppm +++++ +++++ 50 ppm ++ ++++ 100 ppm - ++ 150 ppm - + *(-): poor growth, (+): excellent growth

1-3. 1-hydroxy-2-naphthoic acid resistant strain selection

Four colonies of 1-hydroxy-2-naphthoic acid-resistant strains (DS-VK1, DS-VK2, DS-VK3 and DS-VK4) selected under the condition of 1-hydroxy-2-naphthoic acid concentration of 100 ppm were tested, respectively. It was inoculated into 3 ml of GSSY medium (see Table 3 below) in a test tube and cultured at 37° C., 200 rpm, and 120 hours. In 2 ml of the culture medium, 1 ml of 2-propanol:n-hexane (1:2) was added as a solvent, vortexed for 10 minutes, and centrifuged at 12,000 rpm for 5 minutes to separate the solvent layer. After filtering the separated solvent layer with a 0.45 μm PTFE filter, menaquinone-7 was analyzed using an HPLC analyzer (Agilent technologies 1260 infinity LC system). Analysis conditions were analyzed under the conditions shown in Table 4 below, and a menaquinone-7 peak was confirmed in 10.3 min.

GSSY medium composition medium ingredients density Glycerol 3% Sucrose One% Soy peptone 6% Yeast Extract 0.6% K ₂ HPO ₄ 0.5% MgSO ₄ 0.15%

Buffer Methanol 60%, Ethanol 40% Column Apollo C18 column
(5㎛, 150mm×4.6mm) Detector UV at 248nm Temperature 40℃ Run time 15 min flow rate 1ml/min

As a result, as shown in Table 5 below, the 1-hydroxy-2-naphthoic acid resistant strain DS-VK4 is the parent strain or other 1-hydroxy-2-naphthoic acid resistant strains (DS-VK1, DS-VK2 and DS-VK3) was found to have superior menaquinone-7 production ability.

strain Menaquinone-7 production parent strain 3.4 mg/L DS-VK1 7.2 mg/L DS-VK2 8.1 mg/L DS-VK3 10.7 mg/L DS-VK4 15.6 mg/L

The present inventors deposited the Bacillus subtilis DS-VK4 strain at the Korea Microorganism Conservation Center on October 22, 2020 and were given an accession number KCCM12812P, and then used it to produce menaquinone-7.

Example 2. Confirmation of viability and menaquinone-7 production ability of DS-VK4 strain

The DS-VK4 strain was inoculated into 3ml of LB broth and cultured at 37°C, 200rpm, for 16 hours. 50ml of GSSY medium (see Table 3 above) was put into a 500ml baffled flask, 500 μl of the seed culture solution was inoculated, and cultured at 37° C., 200rpm, 120 hours.

In order to confirm the viability of the DS-VK4 strain, the culture medium was diluted 100-fold with physiological saline after the end of the culture, and the OD was measured at a wavelength of 600 nm using a spectrophotometer.

In order to confirm the menaquinone-7 production ability of the DS-VK4 strain, 1 ml of the solvent 2-propanol:n-hexane (1:2) was added to 2 ml of the culture medium, vortexed for 10 minutes, and centrifuged at 12,000 rpm for 5 minutes to remove the solvent layer. separated. After filtering the separated solvent layer with a 0.45 μm PTFE filter, menaquinone-7 was analyzed under the conditions of Table 4 using an HPLC analyzer (Agilent technologies 1260 infinity LC system).

As a result, as shown in Table 6 below, the DS-VK4 strain showed a 4.5-fold increase in menaquinone-7 production compared to the parent strain. Through these results, it could be confirmed that the parent strain was mutated to have resistance to 1-hydroxy-2-naphthoic acid due to UV irradiation, thereby producing menaquinone-7 at a high concentration.

strain viability Menaquinone-7 production parent strain ++ 7.2 mg/L DS-VK4 ++++ 32.6 mg/L

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Korea Microorganism Conservation Center (Overseas) KCCM12812P 20201022

Claims (3)

Bacillus subtilis DS-VK4 strain producing menaquinone-7 (accession number: KCCM12812P).
The method according to claim 1,
The strain is a strain having resistance to 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid).
a) culturing the strain of claim 1 in a medium; and
b) a method for producing menaquinone-7 comprising the step of recovering menaquinone-7 from the strain or the culture medium in which the strain is cultured.