KR102513450B1 - Novel ectoine synthase and uses thereof - Google Patents

Abstract

An object of the present invention is to provide a composition or method for producing ectoin using a novel ectoin synthetase isolated from a strain producing ectoin.

Description

Novel ectoine synthase and uses thereof {Novel ectoine synthase and uses thereof}

The present invention relates to a method for producing ectoin using a novel ectoin synthetase isolated from a strain producing ectoin.

Ectoin (1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a natural amino acid extract and heterocyclic compound component discovered in 1985 from microorganisms living in salt lakes. It is also a heterocyclic amino acid naturally produced by halophilic organisms in nature, and is produced as one of the osmotic counterbalance substances required by microorganisms to survive in a salty environment.

Ectoin, which is mainly used by being added to skin care cosmetics, maintains the skin's immune system to protect the skin from external stressors such as ultraviolet rays, cold air, heat, and dryness, and has the effect of preventing wrinkled skin texture due to aging. . Ectoin, with its excellent antioxidant effect, can prevent pigmentation caused by all levels of air pollution, including fine dust and ultrafine dust, and is known to be effective in preventing pollution-induced damage and skin aging. In addition, ectoin combines with surrounding water molecules to form a moisture film to moisturize dry and sensitive skin, softens skin texture softly and supplely, and protects various components including skin cells, enzymes and proteins. action can be provided.

In particular, ectoin is recognized as a stable ingredient that can be used on all skin types as it has been proven to have no side effects as an alternative to cortisone, a steroid component used for atopy, psoriasis, and eczema, due to its excellent anti-inflammatory effect. Thanks to these beneficial properties, ectoin can be usefully used for various purposes, such as protein stabilizers, cosmetic additives, PCR enhancers, and drying protectants for microorganisms.

Until now, ectoin has been mainly produced through a bacterial production route using Halomonas elongata, a type of halophilic bacterium, but it requires a high salt concentration in the production stage, complicated manufacturing steps and corrosion of production equipment. ectoin production rate is required to be improved.

Korean Patent Publication No. 2020-0026261 relates to ectoin-producing yeast, and discloses genetically modified yeast and a method for producing ectoin using the same, and Korean Patent No. 1707292 discloses ectoin as a compatible solute for enzyme stabilization and Regarding its derivatives, a method for producing them from various bacteria of the genus Halomonadaceae or Firmicutes is disclosed.

However, the ectoin synthase isolated from the Aestuariispira sp. SWCN16 strain of the present invention and its use for ectoin production have not been disclosed yet.

1. Korean Patent Publication No. 2020-0026261 2. Korea Patent No. 1707292

The present invention has been derived from the above needs, and the present inventors have found that the ectoin producing enzyme obtained from Aestuariispira sp. SWCN16 strain isolated from non-halophilic bacterium And it was discovered that ectoin could be produced with high efficiency when expressed in an ectoin-producing strain, thereby completing the present invention.

In order to solve the above problems, the present invention provides at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Provided is an ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain including.

In addition, the present invention provides nucleotide sequences encoding at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3. A vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain is provided.

In another embodiment of the present invention, at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 is provided. A composition for producing ectoin comprising an ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain is provided.

In another embodiment of the present invention, the present invention synthesizes at least one ectoin selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Provided is a composition for producing ectoin comprising a vector overexpressing ectoin synthase isolated from Aestuariispira sp. SWCN16 strain containing a nucleotide sequence encoding the enzyme.

The composition for producing ectoin further comprises at least one selected from the group consisting of sodium aspartate and KCl; pH is selected from 6 to 8 conditions; And the temperature is made in one or more conditions selected from the group consisting of; conditions selected from 25 to 40 ℃.

In another embodiment of the present invention, at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Preparing a vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain containing the encoding nucleotide sequence; transduction into an ectoin-producing strain; and culturing.

The ectoin-producing strain is E. coli, and the method for producing ectoin further comprises at least one selected from the group consisting of sodium aspartate and KCl; pH is selected from 6 to 8; And a step in which the temperature is selected from 25 to 40 ° C.; further comprising one or more steps selected from the group consisting of.

The ectoin synthetase isolated from the Aestuariispira sp. SWCN16 strain isolated from non-halophilic bacteria of the present invention can be usefully used for ectoin production.

Figure 1 shows the growth of the SWCN16 strain by NaCl concentration according to an embodiment of the present invention.
2 shows ectoin produced from the SWCN16 strain according to one embodiment of the present invention. ■, ectoin secreted into the medium from strain SWCN16; □, intracellular ectoin of SWCN16 strain; ●, ectoin secreted into the medium from H. elongata strain; ○, Intracellular ectoin of H. elongata strain.
3 shows the results of phylogeny analysis based on 16s rRNA according to an embodiment of the present invention.
Figure 4 shows the structure of the gene ectABC related to ectoin synthesis of the new strain SWCN16 according to an embodiment of the present invention.
5A to 5C show a comparison of amino acid sequences of ectoin synthase of SWCN16 according to an embodiment of the present invention.
6A shows the structure of an ectoin synthase gene according to an embodiment of the present invention.
Figure 6B shows the expressed ectoin synthase protein according to an embodiment of the present invention.
Figure 7 shows the results of searching for optimal conditions for bioconversion according to an embodiment of the present invention.
8 shows the amount of ectoin biosynthesis over time under the optimal bioconversion conditions for ectoin biosynthesis according to an embodiment of the present invention. ●, intracellular ectoin; ○, extracellular ectoin

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

In order to achieve the object of the present invention, the present invention provides at least one ectoin selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Provided is an ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain containing the synthetase.

The Aestuariispira sp. (Aestuariispira sp.) SWCN16 strain was isolated from a non-halophilic strain, and accession number is KCTC82028.

The ectoin synthetase gene of the Aestuariispira sp. SWCN16 strain has about 40 to 60% homology compared to the individual enzymes of phylogenetically adjacent strains, T. mesophila JCM18969 and T. marina KCTC62333 It represents only a novelty.

The ectoin synthesis is performed by ectoin synthetase A (L-2,4-diaminobutyric acid acetyltransferase, ectA), ectoin synthetase B (diaminobutyrate-2-oxoglutarate transaminase, ectB), and ectoin synthase C (L-ectoine synthase, ectC) gene. The ectoin synthetase of the present invention may be combined with ectoin-producing enzymes of other bacteria or only the ectoin synthetase of the strain of the present invention may be used. Preferably, ectoin synthetase A of SEQ ID NO: 1 and SEQ ID NO: 2 It may be an ectoin synthetase including ectoin synthetase B and ectoin synthetase C of SEQ ID NO: 3, but is not limited thereto.

In addition, the present invention provides nucleotide sequences encoding at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3. A vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain is provided.

In the present invention, the term "vector" can be used to remove or introduce a gene, and the vector is not limited thereto, but is not particularly limited as long as it can introduce an ectoin-producing enzyme into an ectoin-producing strain. It can be made using any known vector. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors , pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. Vectors usable in the present invention are not particularly limited, and known expression vectors can be used.

In the present invention, the term "overexpression" means that the expression of a gene or enzyme is increased compared to unmodified microorganisms. Increased expression of an enzyme is obtained by increasing the expression of a gene encoding the enzyme. Increasing the expression of a gene can be performed by any technique known to those skilled in the art. In this regard, particular reference may be made to the provision of a strong promoter upstream of the nucleic acid to be overexpressed or the introduction of multiple copies of said nucleic acid between a promoter, in particular a strong promoter and a terminator.

In another embodiment of the present invention, at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 is provided. A composition for producing ectoin comprising an ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain is provided.

In another embodiment of the present invention, the present invention synthesizes at least one ectoin selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Provided is a composition for producing ectoin comprising a vector overexpressing ectoin synthase isolated from Aestuariispira sp. SWCN16 strain containing a nucleotide sequence encoding the enzyme.

The composition encompasses all types of compositions including strains or all types of components produced by strains, preferably in the form of pharmaceutical compositions, food compositions, cosmetic compositions, feed compositions, etc., but is not limited thereto. .

The composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of compositions.

The pharmaceutical composition of the present invention may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, and powders , tablets, capsules, injections and solutions are more preferred. Such formulation may be performed by a method commonly performed in the pharmaceutical field, and may be preferably formulated according to each disease or component using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

When formulated, it may be prepared by additionally using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to water and liquid paraffin, which are commonly used simple diluents. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous agents and suspending agents. As the base of the suppository, witepsol, macrogol, tween, cacao butter, laurin paper, glycerogeratin and the like may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art.

When the composition of the present invention is included in a health functional food and used, the composition may be added as it is or used together with other health functional food or health functional food ingredients. Extracts, food additives, or natural carbohydrates may be included as additional components, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use. In general, the composition of the present invention can be added in an amount of preferably 15 parts by weight or less, more preferably 10 parts by weight or less, based on the raw material during production of food or beverage. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

There is no particular limitation on the type of health functional food that can contain the composition of the present invention, and specific examples include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, and ice cream. There are dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in a conventional sense, and may include foods used as feed for animals.

Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above, natural flavors (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can advantageously be used as flavoring agents.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its 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. In addition, it may include fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination.

In the present invention, health functional food (functional food) is the same term as food for special health use (FoSHU), medicine that is processed to efficiently display bioregulatory functions in addition to nutritional supply, medical care Means highly effective food. Here, "function (sex)" means to obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the preparation. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a drug for a long time using food as a raw material, and has excellent portability.

In another embodiment of the present invention, at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Preparing a vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain containing the encoding nucleotide sequence; transduction into an ectoin-producing strain; and culturing.

In the present invention, the ectoin-producing strain is not particularly limited as long as it is a strain capable of producing ectoin, and may preferably be E. coli, but is not limited thereto.

In the composition or production method for producing ectoin of the present invention, various conditions can be used if the composition or method can produce ectoin, and preferably, it further contains at least one selected from the group consisting of sodium aspartate and KCl, or the pH is 6 to 6. 8 or the temperature is 25 to 40 ° C., or a combination of the above conditions may be used, but it is not limited thereto as long as it is an additional method capable of increasing ectoin production.

The term "cultivation" of the present invention refers to a method of growing microorganisms in appropriately artificially controlled environmental conditions, and artificially controlled environmental conditions typically include nutrients, temperature, osmotic pressure, pH gas composition, light, etc. , What has a direct effect is the medium, which can be largely divided into liquid medium and solid medium, and all kinds of medium that can induce optimal growth can be used depending on the type of strain used.

In the present invention, the method of culturing the transformant, which is the transduced strain, can be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as ectoin can be produced from the transformant, but can be cultured continuously in a batch process or fed batch or repeated fed batch process. .

The medium used for culturing the strain must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include sugars and carbohydrates such as glucose, xylose, sucrose, lactose, fructose, maltose, starch, and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil, and palmitate. Acids, fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol and ethanol, organic acids such as acetic acid, and the like may be additionally used, and these materials may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or decomposition products thereof, defatted soybean cake or decomposition products thereof, may be used. can These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts as phosphorus. Persons that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above substances.

Regarding the incubation time of the strain, the strain encompasses all culture conditions for a certain period of time or more capable of producing ectoin, and is characterized in that it is cultured for preferably 24 hours, 48 hours or 72 hours or more, more preferably 24 hours. It is characterized in that it is cultured for more than an hour, but is not limited thereto.

The strain transduced in the present invention is not particularly limited thereto as long as it can be used to produce ectoin in high yield, but as an example, it may be a culture of the strain, a culture supernatant, a lysate, a fraction thereof, etc. , As another example, the culture supernatant obtained by centrifuging the culture of the strain, the lysate obtained by physically or ultrasonically treating the strain, and applying the culture, culture supernatant, lysate, etc. to methods such as centrifugation and chromatography It may be a fraction obtained by doing.

In addition, the method for producing ectoin may additionally include a step of recovering ectoin from a strain lysate or culture solution, and the recovery may be performed by dialysis, centrifugation, filtration, solvent extraction, spraying, drying, evaporation, precipitation, fractional dissolution, chromatography It may be performed by a method known in the art, such as chromatography or crystallization. For example, a method of centrifuging the strain lysate or culture medium to remove the transformed strain and applying the obtained supernatant to a solvent extraction method to recover the desired ectoin may be used. In addition, the characteristics of the desired ectoin Any method capable of recovering the ectoin by combining known experimental methods according to the method may be used without particular limitation.

The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.

<Example 1> Confirmation of physiological characteristics and ectoin synthesis of novel strains

To confirm the synthesis of ectoin from non-halophilic bacterium, seawater samples were diluted in MA and smeared and cultured. The grown colonies were inoculated into MB supplemented with 9% NaCl to select salt-tolerant strains (SWCN16; KCTC82028), and the selected strains were inoculated into MB to confirm ectoin synthesis. In order to confirm the optimal growth salinity of SWCN16, 0-15% NaCl was added to MB medium without salt, respectively, and the growth rate was confirmed. As a result, SWCN16 showed the highest growth rate at 5-8% NaCl (FIG. 1).

In order to produce ectoine under relatively low salt conditions from the SWCN16 strain, when confirming the ectoine production in MA (2% NaCl), MA+2% NaCl (4% NaCl), and MA+4% NaCl (6% NaCl) media, It was confirmed that the synthesis amount of ectoin increased as the NaCl concentration increased. As a result of checking the amount of ectoin synthesis in MA+4% NaCl medium using Halomonas elongata, a commercially used strain as a control, both the SWCN16 strain (33.8 mg/L) and the control (30.3 mg/L) were cultured in the culture medium. The highest amount of ectoin synthesis was shown on day 1 (FIG. 2).

In order to confirm the phylogenetic position of the SWCN16 strain, which has excellent ectoin productivity under low salt conditions, a phylogeny was confirmed based on 16s rRNA gene information, and as a result, it was confirmed that it belongs to the Aestuariispira genus of the Rhodobacteraceae family (FIG. 3).

<Example 2> Search for ectoin synthase of novel strains

In order to search for the ectoin enzyme of the new strain that synthesizes ectoin, genome sequencing was performed, and genes related to ectoin synthesis were identified. As a result, L-2,4-diaminobutyric acid acetyltransferase (ectA), diaminobutyrate-2-oxoglutarate transaminase (ectB), and L-ectoine synthase (ectC) genes involved in ectoin synthesis were identified. It was confirmed that they were located and had sizes of 561 bp, 1281 bp, and 408 bp, respectively (FIG. 4).

In order to confirm the novelty of this enzyme, the amino acid sequence of each enzyme was compared with the amino acid sequence of each enzyme of related species ( Thalassospira mesophila JCM18969 and T. marina KCTC62333) and control ( Halomonas elongata DSM 2581). As a result of the phylogenetic analysis of the SWCN16 strain, the related species grouped into the same group did not have ectoin synthetase, and the strains of the genus Thalassospira among the relatively adjacent strains were compared. The ectA gene of strain SWCN16 showed 61.4% and 62.9% homology with the ectA enzymes of T. mesophila JCM18969 and T. marina KCTC62333, respectively, and 44.4% homology with the ectA enzyme of H. elongata . The ectB gene of strain SWCN16 showed 66.7% and 68.1% homology to the ectB enzymes of T. mesophila JCM18969 and T. marina KCTC62333, respectively, and 58.5% homology to the ectB enzyme of H. elongata. The ectC gene of strain SWCN16 showed 64.9% and 67.2% homology with the ectC enzymes of T. mesophila JCM18969 and T. marina KCTC62333, respectively, and 56.8% homology with the ectC enzyme of H. elongata (Fig. 5). ).

<Example 3> Heterologous expression of novel ectoin synthase in E. coli

In order to mass-produce ectoin using the novel ectoin synthetase obtained from the SWCN16 strain, the novel ectoin synthetase was expressed in E. coli. The novel ectoin synthetase gene was obtained through PCR using specific primers (Table 1) in a cluster (ectABC) state from the gDNA of the SWCN16 strain, and was inserted using the restriction enzymes NheI and HindIII to express in the pET21a vector. Since it has been reported that the amount of enzyme and the ratio of each enzyme play an important role in the efficiency of ectoin synthesis, E. coli RBS (5'-AAGGAG-3') in front of each gene to increase enzyme expression and change the relative ratio of enzymes Alternatively, ectABC (RBS) to which 5'-GAGGAG-3') was added and ectBAC (RBS) to which its gene sequence was changed were also obtained through PCR using specific primers (Table 1), respectively. inserted into.

- Primers used for amplification of genes related to ectoin biosynthesis -

Name Sequences (5’→3’) Product ectABC_F ATG GCTAGC ATGCCTGATGGCACC ectABC ectABC_R CGC AAGCTT GTTAGGCGTTCTCT ectABC-A_F GGAGATATACAT ATG GCTAGCATGCCTGATGGCACCGTACG ectABC(RBS) ectABC-A_R TCAATCTCTCTCCTCGAATTTCTGACAGGCCG ectABC-B_F AATTC GAGGAG AGAGATTG ATG ACAGTATTTCAAC ectABC-B_R TATCTCCTTCGTTTGCTCAAATCAATGTCCGG ectABC-C_F TTGAGCAAACG AAGGAG ATAGACCA ATG ATCGT ectABC-C_R CTCGAGTGCGGCCGCAAGCTTGTTAGGCGTTCTCTTTTTCCAGA ectBAC-B_F GGAGATATACAT ATG GCTAGCATGACAGTATTTCAACGCCGCG ectBAC (RBS) ectBAC-B_R GTCTCCTCTTTTATTCCGCCGCAGCCGC ectBAC-A_F GGCGGAATAAAA GAGGAG ACACTTAAG ATG CCTG ectBAC-A_R CTCCTTCGTTTTAGGCGACATCCTCCAGAG ectBAC-C_F TGTCGCCTAAAACG AAGGAG ATAGACCA ATG ATCG ectBAC-C_R CTCGAGTGCGGCCGCAAGCTTGTTAGGCGTTCTCTTTTTCC

The structure of each ectoin synthase gene is shown in FIG. 6A. The plasmid into which each of these genes was inserted was transformed into E. coli BL21, and enzyme expression was induced using 1mM IPTG for 8 hours. Afterwards, only the cells were collected and disrupted by ultrasonic waves in PBS buffer. Phosphorus synthase was confirmed through SDS-PAGE (FIG. 6B). In the enzyme expression using the ectoin synthase gene cluster ectABC, EctA showed a protein band as a result of SDS-PAGE, but the expression of the other two enzymes, EctB and EctC, was insignificant, making it difficult to identify. When E. coli RBS was added in front of each enzyme to increase the expression level of the enzyme, it was confirmed that the protein band increased in SDS-PAGE. Since the expression level of EctB, which is located and requires a large amount, was significantly increased, ectBAC (RBS) strain was used for ectoin synthesis.

<Example 4> Exploration of optimal conditions for ectoin production

The ectBAC+RBS strain was cultured to induce enzyme expression in the same manner as above, and ectoin bioconversion conditions were explored therefrom. Strains expressing ectA, ectB, and ectC were recovered, washed three times with sterilized purified water, and then suspended in ectoin bioconversion buffer to a final OD value of 10. The ectoin bioconversion buffer basically had 50 mM KCl, 50 mM sodium aspartate, and 100 mM glycerol added to 100 mM sodium phosphate buffer (pH 7.0), and the concentration of sodium aspartate (50, 100, 200, 300 mM ), concentrations of KCl (0, 50, 100, 200, 300 mM), pH of sodium phosphate buffer (6.0, 6.5, 7.0, 7.5, 8.0), and reaction temperatures of 25, 30, 35 and 40 °C, respectively. As a result, it was confirmed that 200 mM sodium aspartate, 200 mM KCl, pH 7 sodium phosphate buffer, and a reaction temperature of 30 ° C were optimal (FIG. 7).

Based on the optimal conditions for ectoin bioconversion, the amount of ectoin conversion was measured while culturing for 33 hours. As a result, intracellular ectoin was almost saturated at 9 hours, and extracellular ectoin steadily increased, showing a maximum production of 1.67 g/L at 33 hours (FIG. 8).

<110> KRIBB <120> Novel ectoine synthase and uses thereof <130> M20-7801 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 186 <212> PRT <213> artificial sequence <220> <223> SWCN16_EctA <400> 1 Met Pro Asp Gly Thr Val Arg Val Met Lys Arg Ala Ala Lys Gly Gly 1 5 10 15 Phe Thr Phe Arg Lys Pro Ala Thr Asp Asp Gly Thr Ala Ile His Glu 20 25 30 Leu Ile Ala Arg Cys Arg Pro Leu Asp Glu Asn Ser Ile Tyr Cys Asn 35 40 45 Leu Leu Gln Cys Ser His Phe Ala Asp Thr Cys Val Val Ala Glu Glu 50 55 60 Gly Gly Glu Ile Arg Gly Phe Val Ser Gly Tyr Ile Val Pro Asp Ala 65 70 75 80 Arg Glu Arg Leu Phe Ile Trp Gln Val Ala Val Ala Pro Glu Ala Arg 85 90 95 Gly Asn Ser Leu Gly Arg Arg Met Ile Leu Glu Ile Leu Asn Arg Pro 100 105 110 Glu Gln Gly Asp Ile Thr Glu Leu His Thr Ser Ile Thr Pro Asp Asn 115 120 125 Lys Pro Ser Gln Gly Val Phe Ser Ser Val Ala Asp Phe Leu Asp Ala 130 135 140 Asp Val Glu Gln Lys Val Leu Phe His Glu Asp Asp His Phe Asp Gly 145 150 155 160 Gln Gln Lys Thr Glu Ile Leu Trp Glu Ile Gly Pro Phe Asp Met Glu 165 170 175 Glu Ala Asp Arg Pro Leu Glu Asp Val Ala 180 185 <210> 2 <211> 426 <212> PRT <213> artificial sequence <220> <223> SWCN16_EctB <400> 2 Met Thr Val Phe Gln Arg Arg Glu Ser Glu Val Gln Ser Tyr Ala Arg 1 5 10 15 Ala Phe Pro Val Val Phe Asp Gln Ala Asp Gly Ala Arg Leu Thr Asp 20 25 30 Arg Asp Gly Arg Ser Tyr Leu Asp Phe Leu Ala Gly Ala Gly Thr Leu 35 40 45 Asn Tyr Gly His Asn His Pro Lys Leu Arg Asp Ala Leu Val Asp Tyr 50 55 60 Ile Gln Ser Gly Gly Ile Thr His Gly Leu Asp Met His Thr Glu Ala 65 70 75 80 Lys Glu Lys Phe Leu Glu Thr Phe Glu Glu Val Ile Leu Ala Pro Arg 85 90 95 Glu Met Asp His Val Val Gln Phe Thr Gly Pro Thr Gly Ala Asn Ala 100 105 110 Val Glu Ala Ala Met Lys Leu Ala Arg Gln Val Lys Gly Arg Asn Asn 115 120 125 Ile Ile Ala Phe Thr Asn Gly Phe His Gly Val Thr Leu Gly Ala Leu 130 135 140 Ala Ala Thr Gly Asn Gln His His Arg Gly Ala Ala Gly Val Ala Leu 145 150 155 160 Thr Gly Val Asp Arg Met Pro Phe Asp Gly Tyr Met Gly Asp Gly Tyr 165 170 175 Asp Thr Thr Asp Tyr Leu Asp Gln Val Leu Ser Asp Ala Ser Ser Gly 180 185 190 Val Glu Arg Pro Ala Ala Val Ile Val Glu Cys Val Gln Gly Glu Gly 195 200 205 Gly Leu Lys Ala Ala Asp Phe Gly Trp Leu Arg His Leu Glu Ala Val 210 215 220 Cys Arg Lys His Asp Ile Leu Leu Ile Val Asp Asp Ile Gln Ala Gly 225 230 235 240 Cys Gly Arg Thr Gly Thr Phe Phe Ser Phe Glu Pro Ala Gly Ile Lys 245 250 255 Pro Asp Ile Ile Thr Leu Ser Lys Ser Leu Ser Gly Phe Gly Leu Pro 260 265 270 Phe Ala Val Thr Leu Ile Arg Lys Asp Leu Asp Ala Trp Val Pro Gly 275 280 285 Ala His Asn Gly Thr Phe Arg Gly Asn Asn His Ala Phe Val Thr Ala 290 295 300 Ala Thr Ala Leu Asp Leu Phe Trp Arg Asp Gly Lys Phe Ala Glu Ser 305 310 315 320 Val Arg Glu Lys Gly Asp Tyr Leu Thr Asn Arg Phe Arg Arg Ile Ala 325 330 335 Glu Arg Phe Pro Glu Leu Gly Leu Lys Leu Arg Gly Arg Gly Leu Met 340 345 350 Gln Gly Leu Val Tyr Pro Asp Gly Asp Leu Thr Gly Ala Ile Cys Lys 355 360 365 Glu Cys Phe Lys Asn Gly Leu Ile Ile Glu Thr Ser Gly Pro Asn Asp 370 375 380 Glu Val Ile Lys Cys Leu Cys Pro Leu Ile Ile Asp Glu Ala Asp Leu 385 390 395 400 Ala Lys Gly Leu Asp Ile Leu Glu Glu Ala Thr Ala Thr Val Leu Ala 405 410 415 Arg Gln Ala Ser Gln Ala Ala Ala Ala Glu 420 425 <210> 3 <211> 135 <212> PRT <213> artificial sequence <220> <223> SWCN16_EctC <400> 3 Met Ile Val Arg Thr Leu Glu Asn Cys Lys Asn Ser Ser Arg His Val 1 5 10 15 Val Thr Glu Thr Trp Glu Ser Thr Arg Met Leu Leu Ser Asp Asp Gln 20 25 30 Met Gly Phe Ser Phe His Ile Thr Thr Ile Tyr Ala Gly Thr Glu Thr 35 40 45 Pro Ile Trp Tyr Lys Asn His Leu Glu Ser Val Tyr Cys Ile Ser Gly 50 55 60 Glu Gly Glu Val Glu Thr Leu Asp Asp Gly Lys Val Tyr Gln Leu Lys 65 70 75 80 Pro Gly Thr Leu Tyr Ile Leu Asp Lys Asn Asp Arg His Leu Leu Arg 85 90 95 Gly Lys Thr Asp Ile Thr Phe Ala Cys Val Phe Asn Pro Pro Leu Asn 100 105 110 Gly Arg Glu Val His Asp Glu Asp Gly Ala Tyr Gln Leu Asn Glu Ala 115 120 125 Pro Leu Glu Lys Glu Asn Ala 130 135

Claims (8)

Aestuarispira species comprising at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 ( Ectoin synthetase isolated from Aestuariispira sp.) SWCN16 strain. Aestu comprising a nucleotide sequence encoding at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain Aestuarispira species comprising at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 ( Aestuariispira sp.) Composition for producing ectoin containing ectoin synthetase isolated from SWCN16 strain Aestu comprising a nucleotide sequence encoding at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 A composition for producing ectoin containing a vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain The method according to claim 3 or 4, further comprising at least one selected from the group consisting of sodium aspartate and KCl; pH is selected from 6 to 8 conditions; and a temperature condition selected from 25 to 40 ° C.; Aestu comprising a nucleotide sequence encoding at least one ectoin synthetase selected from the group consisting of ectoin synthetase A of SEQ ID NO: 1, ectoin synthetase B of SEQ ID NO: 2, and ectoin synthetase C of SEQ ID NO: 3 Preparing a vector overexpressing ectoin synthetase isolated from Aestuariispira sp. SWCN16 strain;
transduction into an ectoin-producing strain; and
Ectoin production method comprising the step of culturing. 7. The method according to claim 6, wherein the ectoin-producing strain is E. coli. The method of claim 6, further comprising at least one selected from the group consisting of sodium aspartate and KCl; pH is selected from 6 to 8; And the temperature is selected from 25 to 40 ℃; Ectoin production method characterized in that it further comprises at least one step selected from the group consisting of

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