KR102557415B1 - Microorganism strain having improved growth characteristics under photolithoautotrophic condition and use thereof - Google Patents

Abstract

It relates to a mutant strain of Rhodobacter spheroides in which growth rate is increased and carotenoid production ability is increased under photoinorganic autotrophic growth conditions, and a method for producing carotenoids by culturing the same.

Description

Microorganism strain having improved growth characteristics under photolithoautotrophic condition and use thereof}

It relates to a mutant strain of Rhodobacter spheroides in which growth rate is increased and carotenoid production ability is increased under photoinorganic autotrophic growth conditions, and a method for producing carotenoids by culturing the same.

Rhodobacter sphaeroides is a photosynthetic bacterium belonging to purple non-sulfur bacteria, and is a rod-shaped or spherical gram-negative bacterium with a width of 0.5 to 1.2 μm and a length of about 2 to 2.5 μm, It can grow in a variety of environmental conditions. For example, in an anaerobic state, photoorganic autotrophic growth using light, carbon dioxide (CO ₂ ) as a carbon source and inorganic hydrogen as an electron donor, or photoorganic heterotrophic growth using organic matter as a carbon source and electron donor or both, and chemoheterotrophic growth is possible under aerobic conditions.

Rhodobacter spheroides has various metabolic pathways and is used to produce useful substances such as polyhydroxybutyrate (PHB), hydrogen (H ₂ ) and carotenoids. In addition, through these characteristics, Rhodobacter spheroides is used for soil and water purification or carbon dioxide reduction in the environmental field, or in various industries such as agriculture, food, and medicine, for the production of bioactive substances or high value-added biomaterials such as bioplastics. It has excellent economic efficiency.

Therefore, it is required to develop a technology that maximizes industrial usefulness by promoting the growth of Rhodobacter spheroides.

Domestic Patent Publication No. 10-2020-0022748 (2020.03.04)

Under this background, the present invention provides a mutant strain with increased growth rate and enhanced carotenoid production ability compared to non-mutant control under photoinorganic autotrophic growth conditions.

Accordingly, as an example, a Rhodobacter sphaeroides mutant strain KCTC 14365BP with enhanced growth rate under light inorganic autotrophic growth conditions is provided.

As another example, it provides a carotenoid production method comprising culturing the mutant strain of Rhodobacter spheroides.

As another example, a composition for producing carotenoids comprising the mutant strain of Rhodobacter spheroides or a culture of the strain is provided.

According to one aspect of the present invention, a mutant strain of Rhodobacter spheroides with increased growth rate under photo-organismal autotrophic growth conditions is provided. In one embodiment, the mutant strain has an enhanced growth rate and enhanced carotenoid production ability under photoinorganic autotrophic growth conditions.

According to another aspect of the present invention, there is provided a method for producing carotenoids comprising culturing the mutant strain of Rhodobacter spheroides.

According to another aspect of the present invention, there is provided a composition for producing carotenoids comprising the mutant strain of Rhodobacter spheroides or a culture of the strain.

Specifically, in the present invention, in order to increase the growth rate under autotrophic growth conditions of Rhodobacter spheroides, random mutations are applied to select species whose growth rate is significantly improved compared to parent or wild strains under photoinorganic autotrophic growth conditions And, as of November 12, 2020, the strain was deposited at KCTC (Korean Colletion for Type Cultures, Korea Research Institute of Bioscience and Biotechnology Biological Resources Center) and was given accession number KCTC 14365BP. It was confirmed that the strain not only increased the growth rate under photoautotrophic conditions, but also had excellent total carotenoid production ability.

As used herein, "mutant strain" or "mutant strain" refers to a strain that has a change in DNA sequence compared to the non-mutated parent strain. Such a mutation may be a change in DNA sequence by insertion, addition, substitution, deletion or inversion of bases in the DNA sequence. Also, the mutation may be a missense, nonsense or frameshift mutation. In one embodiment, the mutant strain of the present application is a strain induced to mutate by treating N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen using Rhodobacter spheroid (KCTC1434) as a parent strain.

In this application, "photoautotrophic growth" and "photoautotrophic growth" are used interchangeably, using light as an energy source, carbon dioxide (CO ₂ ) as a carbon source, and inorganic molecules such as hydrogen as an electron donor. refers to the type of growth used.

The strain of the present application is a strain whose growth rate is dramatically improved compared to the parent strain or wild strain under photoinorganic autotrophic growth conditions, and can fix carbon dioxide, which is a problem as a greenhouse gas, as a carbon source in microorganisms by performing photoinorganic autotrophic growth. It has a function as a biological capture method that has high concentrations of carbon dioxide, so that yield and productivity can be improved in the production process of high value-added materials using carbon dioxide because high concentrations of carbon dioxide can be used quickly, and a method of culturing Rhodobacter using organic acids or sugars. Unlike, there is an advantageous effect in that there is little reported about the conversion and production of carbon dioxide into high value-added materials through the cultivation of Rhodobacter using carbon dioxide as a carbon source.

In addition, the strain of the present application is characterized by an increased production ability of useful substances such as carotenoids compared to parent strains or wild strains.

As used herein, "carotenoid" is a compound widely distributed in nature as a useful pigment group of the C30-C50 series among terpene-based compounds widely known as isoprenoid compounds. Carotenoids show various colors such as brown (fucoxanthin), red (lycopene), and orange (β-carotene) depending on their molecular structure. there is. In addition, carotenoids can be used as natural pigments in the food industry due to their unique pigments, and have various biological functions such as precursors of vitamin A, scavenging active oxygen, antioxidant, and inhibiting cancer cell proliferation in the human body. It is used as a functional food and pharmaceutical material because of its preventive effect. In addition, it is known as a cosmetic material such as prevention of skin damage from excessive UV exposure and inhibition of melanin biosynthesis through improvement of immune function. Until now, about 750 carotenoids have been isolated from nature and most of them have been produced through plant extraction or chemical synthesis, but carotenoid production through these processes has problems such as by-product generation, side effects, and reduced production yield.

Rhodobacter spheroides has a complete pathway for producing carotenoids, but the yield is low. The mutant strain of Rhodobacter spheroides provided herein has an enhanced ability to produce carotenoids compared to its parent strain or wild strain, and thus can be widely used in various fields such as food, medicine, and cosmetics.

The present invention also provides a method for producing carotenoids, comprising the step of culturing the mutant strain of Rhodobacter spheroides defined above.

A medium used for culturing a strain must suitably satisfy the requirements of a particular strain. The medium may contain various carbon sources, nitrogen sources, phosphorus and trace element components. The specific type of medium may be a medium such as LB (Luria Bertani) broth, SD (Sabouraud Dextrose) broth or YM (Yeast medium) broth, Sistrom medium, but is not limited thereto.

The culture medium uses carbon dioxide as a carbon source and is preferably cultured under photoanaerobic conditions. In addition, other carbon sources may be included as needed, and monosaccharides, oligosaccharides, polysaccharides, single carbon substrates, or mixtures thereof may be exemplified. monosaccharides such as, for example, glucose and fructose; oligosaccharides such as sucrose, maltose or lactose; polysaccharides such as starch or cellulose; Single carbon substrates such as methanol, formaldehyde or formate can be exemplified. In addition, lower alcohols such as ethanol, propanol, and butanol; polyhydric alcohols such as glycerol; fatty acids such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid; Organic acids such as acetic acid, succinic acid, malic acid, and lactic acid may be exemplified, but are not limited thereto. These materials may be used individually or as a mixture.

Examples of the nitrogen source in the medium include ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates such as potassium nitrate, ammonia, and urea as inorganic salts, but are not limited thereto. Nitrogen sources can also be used individually or as a mixture, and the amount added varies depending on the type of nitrogen source and can be appropriately adjusted.

As the organic nitrogen source, for example, contifrica (including filtered products), pharmamedia, soybean meal, soybean powder, peanuts wheat, peptone, distillers soluble, dry yeast, yeast extract, casamino acid, glutamic acid, aspartate Acid, malt extract, corn steep liquor, etc. may be exemplified, but are not limited thereto. The inorganic nitrogen source and the organic nitrogen source are usually added to the starting medium, but may be additionally supplied sequentially or continuously.

Persons in the medium include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or a corresponding sodium-containing salt. In addition, the culture medium may include metal salts such as magnesium sulfate or iron sulfate required for growth, or may include essential growth substances such as amino acids and vitamins, but is not limited thereto.

Examples of inorganic salts include phosphates such as potassium dihydrogenphosphate, dipotassium hydrogenphosphate, disodium hydrogenphosphate, magnesium salts such as magnesium sulfate and magnesium chloride, iron salts such as iron sulfate and iron chloride, calcium chloride and calcium carbonate. Calcium salts such as sodium carbonate, sodium salts such as sodium chloride, manganese salts such as manganese sulfate, copper salts such as copper sulfate, zinc salts such as zinc sulfate, molybdenum salts such as sodium molybdate, nickel salts such as nickel sulfate, selenic acid One or two or more of selenium salts such as sodium, tungsten salts such as sodium tungstate, aluminum salts such as aluminum chloride, chromium salts such as chromium chloride, boric acid, and potassium iodide are used. The addition amount differs depending on the type of inorganic salt and can be appropriately adjusted.

As vitamins, for example, cyanocobamin, riboflavin, pantothenic acid, pyridoxine, thiamine, ascorbic acid, folic acid, niacin, p-aminobenzoic acid, biotin, inositol, choline and the like can be used. The addition ratio is different depending on the type of vitamins and can be appropriately adjusted.

In addition, an antifoaming agent can be used to suppress foaming of the culture medium. Any type of antifoaming agent may be used as long as it has an effect of suppressing the generation of bubbles or removing the generated bubbles and has a small inhibitory effect on the target strain. For example, an alcohol-based antifoaming agent, a polyether-based antifoaming agent, an ester-based antifoaming agent, a fatty acid-based antifoaming agent, a silicone-based antifoaming agent, a sulfonic acid-based antifoaming agent, and the like can be exemplified. The antifoaming agent may be usually added to the starting medium before sterilization, or additionally added continuously or intermittently during culture.

The above raw materials may be added in a batchwise or continuous manner by a method suitable for the culture during the cultivation process.

Cultivation (fermentation) may be carried out in a batch, fed-batch, or continuous manner depending on the recombinant microorganism. The initial pH of the medium can be adjusted to 2 to 12, preferably 6 to 9, more preferably 6.5 to 8.0, and as the pH adjusting agent, aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous sodium carbonate solution, ammonia water, ammonia gas, sulfuric acid aqueous solution Or mixtures thereof can be exemplified. The culture temperature may be usually 20°C to 45°C, preferably 25°C to 40°C. Cultivation may be continued until the maximum production of carotenoids is obtained.

Cultivation can be carried out in an appropriate culture vessel. The culture vessel can be appropriately selected according to the culture capacity, and examples thereof include test tubes, flasks, and fermenters. As an example, the culture can be carried out by appropriately increasing the strain by seed culture using a test tube, flask, or fermenter, and adding the resulting culture medium to a medium for producing carotenoids. The medium used for seed culture is not particularly limited as long as it is a medium in which the carotenoid-producing strain of the present application grows satisfactorily.

Methods for recovering carotenoids from fermentation media are known in the art. For example, carotenoids can be obtained from the medium by centrifugation, chromatography, extraction, filtration, precipitation, or a combination thereof. The production of carotenoids by microorganisms can be identified and quantified by suitable techniques well known in the art. As an example, HPLC techniques can be used to measure the amount of carotenoids produced. The HPLC technique also helps determine the purity of carotenoids produced by microorganisms.

In addition, the present invention provides a composition for producing carotenoids, comprising the aforementioned Rhodobacter spheroides mutant strain or a culture of the strain.

The culture refers to, for example, a culture broth, culture supernatant, cell concentrate, wet cell, dry cell, cell lysate, strain lysate, and the like. A culture supernatant can be prepared by subjecting the culture solution to centrifugation or filtration to remove cells from the culture solution. The microbial cell concentrate can be obtained by centrifugation or membrane filtration concentration of the culture solution. Wet cells can be obtained by centrifuging or filtering the culture solution. Dry cells can be obtained by drying wet cells or cell concentrates by a general drying method. Strain lysate can be obtained by subjecting the culture to chemical treatment using alkaline reagents or surfactants, biochemical treatment using lytic enzymes, lipolytic enzymes, and proteolytic enzymes, or physical treatment such as ultrasonication or grinding. there is.

Since the strain or culture of the strain contains carotenoids in high concentration, it can be used as a food, pharmaceutical or cosmetic composition. When used as a food composition, various flavors, natural carbohydrates, nutrients, vitamins, electrolytes, flavors such as synthetic flavors and natural flavors, colorants and enhancers, pectic acids and their Salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be included, but are not limited thereto.

Cosmetic compositions can be prepared in any form such as liquid, cream, paste, solid, etc. by conventional manufacturing methods according to each need. For example, astringent lotion, softening lotion, nutrient lotion, massage cream, essence, pack , It can be prepared in formulations such as lotion, cream, etc., but is not limited thereto. When the cosmetic composition is in the form of an emulsion, purified water, monohydric or polyhydric alcohol, fatty acid, oil, surfactant, etc. may be included in addition to the 'strain' of the present invention or its culture, and other flavoring agents, coloring agents, preservatives, etc. may be used. When the cosmetic composition of the present invention is in a solubilized state, the strain of the present invention or its culture and other ingredients may include purified water, a surfactant, monohydric or polyhydric alcohol, and the like. In addition, if the cosmetic composition of the present invention is in the form of an emulsion, flavoring agents, coloring agents, preservatives, etc. may be used as other ingredients. While fragrances, chelating agents, pigments, antioxidants, and preservatives are used, synthetic or natural materials such as proteins, minerals, and vitamins may be used for the purpose of improving physical properties, but are not limited thereto.

It can be used in various fields of food, medicine, and cosmetics by using a mutant strain of Rhodobacter spheroides with enhanced growth ability and increased carotenoid production ability under light inorganic autotrophic conditions.

Figure 1 shows the results of forming a dominant species through three rounds of continuous subculture of the NTG-induced Rhodobacter spheroides mutant population according to an embodiment of the present application.
Figure 2 shows the results of evaluating the growth rate of Rhodobacter spheroides mutant strains under light inorganic autotrophic growth conditions according to an embodiment of the present application.
Figure 3 shows the results of reproduction experiments evaluating the growth rate of Rhodobacter spheroides mutant strain YR-1 under light inorganic autotrophic growth conditions according to an embodiment of the present application.
Figure 4 shows the results of evaluating the total carotenoid production ability of Rhodobacter spheroides mutant strain YR-1 according to an embodiment of the present application.

Hereinafter, the present invention will be described in more detail by the following examples. However, these are only for exemplifying the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Rhodobacter spheroides mutagenesis

After treatment with NTG ( N -methyl- N' -nitro- N -nitrosoguanidine) to Rhodobacter spheroides wild-type strain (KCTC 1434) to induce mutation, a dominant species was formed through three consecutive subcultures of the mutant population. . Specifically, the wild-type Rhodobacter spheroid strain (KCTC 1434) was inoculated in Sistrom medium containing succinic acid at 1% concentration and cultured for 48 hours at 30°C and 150 rpm. The obtained culture medium was passaged, and 20 ml of cells with an OD ₆₆₀ of between 0.5 and 1 were taken and centrifuged at 4° C. and 10000 rpm for 20 minutes. Then, wash twice with 4ml of Tris-maleate buffer (0.05M, pH 6), dissolve again with 1ml of the same buffer, and dissolve NTG ( N -methyl- N' -nitro- N -nitrosoguanidine) at a concentration of 0.4 mg/ml. Cells were treated with 50 ml of Tris-maleate buffer at 30°C for 1 hour and then sufficiently cooled on ice. After washing once with Tris-maleate buffer, washing once more with Sistrom medium without organic acid, the cells were released with 2 ml of Sistrom medium without organic acid. Then, the NTG-treated cells were inoculated into Sistrom medium without organic acid, and then supplied with a gas having a ratio of 10% carbon dioxide, 60% hydrogen, and 30% argon. It was cultured under conditions of 30 ° C. and 150 rpm using light, and subcultures were performed at 2-week intervals under the same culture conditions.

As a result, as shown in FIG. 1, through 3 rounds of continuous subculture for about 40 days, compared to the parental wild-type strain, about 3 times in the photoautotrophic growth conditions of 10% carbon dioxide, 60% hydrogen, and 30% argon gas composition. Mutant populations with increased growth rates were obtained.

Example 2. Selection of Rhodobacter spheroides mutant strains

After three rounds of continuous passage of the NTG mutant population of Example 1, single colony isolation was performed to select mutant strains with enhanced growth rate under photoinorganic autotrophic growth conditions compared to the parental wild-type strain. Specifically, after diluting an appropriate amount of the mutant population of 3 rounds on a Sistrom solid plate containing succinic acid, it was smeared and cultured at 30 ° C for 2 days to secure colonies. A total of 30 colonies were selected and pre-cultured in Sistrom medium containing organic acid at 30°C and 150 rpm using light for 2 days, and then in Sistrom medium without organic acid (medium composition: KH ₂ PO ₄ 2.72 g /L, NH ₄ Cl 0.195 g/L, L-Glutamic acid 0.1 g/L, L-Aspartic acid 0.04 g/L, NaCl 0.5 g/L, Nitrilotriacetic acid 0.2 g/L, MgSO ₄ 7H ₂ O 0.3 g /L, CaCl ₂ 2H ₂ O 0.0334 g/L, FeSO ₄ 7H ₂ O 0.002 g/L, (NH ₄ ) ₆ Mo ₇ O ₂₄ (1% solution) 0.02 ml, Trace Elements Solution 0.1 ml (EDTA 1.765 mg/L, ZnSO ₄ 7H ₂ O 10.95 mg/L, FeSO ₄ 7H ₂ O 5 mg/L, CuSO ₄ 5H ₂ O 0.392 mg/L, Co(NO ₃ ) ₂ 6H ₂ O 0.248 mg/L L, H ₃ BO ₃ 0.114 mg/L, Vitamin Solution 0.1 ml (Nicotinic acid 1 mg/L, Thiamine HCl 0.5 mg/L, Biotin 0.01 mg/L)) was used to inoculate cells with an OD ₆₆₀ value of 0.1. Then, a gas of 10% carbon dioxide, 60% hydrogen, and 30% argon was supplied, and shaking culture was performed for 2 weeks under the condition of 30 ° C and 150 rpm in the presence of light. It was measured and confirmed.

As a result, as shown in FIG. 2, among the selected colonies, Rhodobacter sphaeroides YR-1, Rhodobacter spheroides colony-2, Rhodobacter spheroides colony-19, Rhodobacter spheroides colony 27, etc. It was confirmed that the growth rate was increased compared to this parent strain. Among them, the Rhodobacter spheroides YR-1 strain, which showed excellent growth, was deposited at KCTC (Korean Colletion for Type Cultures, Korea Research Institute of Bioscience and Biotechnology) on November 12, 2020, and was given accession number KCTC 14365BP .

In addition, as shown in FIG. 3, the Rhodobacter spheroides YR-1 strain was reproduced under the same culture conditions as the photoautotrophic conditions of 10% carbon dioxide, 60% hydrogen, and 30% argon for single colony isolation. In the experiment, the final value of OD ₆₆₀ was 1.665, indicating a growth enhancement of about 70.77% compared to the parent strain.

Example 3. Confirmation of carotenoid production ability of Rhodobacter spheroides mutant strain

In order to confirm the carotenoid production ability of the Rhodobacter spheroides YR-1 strain, the amounts of carotenoids of the wild-type and YR-1 strains were measured using HCl-assisted extraction. Specifically, after washing the cultured cells twice with sterile water, freeze-dried cells were obtained by removing moisture using a lyophilizer. Then, the ratio of 3M HCl and freeze-dried cells was 30:1, and then incubated for 30 minutes at 30°C and 100 rpm. Thereafter, centrifugation was performed at 10000 rpm for 20 minutes to remove the supernatant and collect only the cells. The recovered cells were released again using 1 ml of acetone, and then incubated for 30 minutes at 30°C and 100 rpm. And centrifugation was performed to remove the cells that had settled down, and only the supernatant was taken, and the absorbance was measured at 480 nm using a microplate reader to confirm the amount of total carotenoids.

As a result, as shown in FIG. 4, as a result of measuring the amount of total carotenoids, the Rhodobacter spheroids YR-1 strain produced 0.703 g/L and the parent strain 0.407 g/L of carotenoids, and the Rhodobacter spheroides YR -1 strain was found to be about 72.73% increased total carotenoid production ability compared to the parent strain.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be embodied in other specific forms without changing its technical spirit or essential features. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Korea Research Institute of Bioscience and Biotechnology KCTC14365BP 20201112

Claims (4)

Rhodobacter sphaeroides mutant strain KCTC 14365BP with increased growth rate under photoinorganic autotrophic growth conditions. According to claim 1,
The mutant strain is characterized in that the carotenoid production ability is enhanced, Rhodobacter spheroides mutant strain KCTC 14365BP. A method for producing carotenoids comprising culturing the mutant strain KCTC 14365BP of claim 1 or claim 2. A composition for producing carotenoids comprising the mutant strain KCTC 14365BP of claim 1 or a culture of the strain.