WO2009154320A1

WO2009154320A1 - Aureobasidium pullulans np-1221 kctc 11265bp and a producing method for exo-type beta-glucan using the same

Info

Publication number: WO2009154320A1
Application number: PCT/KR2008/004148
Authority: WO
Inventors: Byung-Dae Yoon; Min-Soo Kim; Chan-Sun Park; Keug-Hyun Ahn; Byung-Kwan Kang
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2008-06-19
Filing date: 2008-07-15
Publication date: 2009-12-23
Also published as: KR100993577B1; KR20090131815A

Abstract

The present invention relates to novel microorganisms which produce not pullulan but exo-type beta-glucan and a method of producing exo-type b-glucan using the same.

Description

[DESCRIPTION] [Invention Title]

AUREOBASIDIUM PULLULANS NP-1221 KCTC 11265BP AND A PRODUCING METHOD FOR EXO-TYPE BETA-GLUCAN USING THE SAME

[Technical Field]

<i> The present invention relates to new aureobasidium pullulans strains producing exo-type beta-glucan and a method of producing the exo-type beta- glucan using the same.

<2>

[Background Art]

<3> In recent years, beta-glucans (b-glucans) have received attention for various applications in light of excellent biological regulation functions such as lipid metabolism enhancement, intestinal disorder relieving action and suppression of blood sugar level increase, antitumor activity or immune adjuvant activity. Beta-glucans are generally contained in microorganisms, fungi and the cell walls of plants to constitute the cell walls of these organisms. Beta-glucans have a structure which mainly contains a glucose polymer having at least two kinds of bl→2, 1→3, 1→4, 1→6 D-glucopyranose linkages. Beta-glucans, which are capable of improving immunological activity in animals, are found in various edible mushrooms, yeasts, barley, oats, etc. In particular, beta-glucans contained in fruiting bodies or mycelia of fungi (mushrooms) have high immune-enhancing activity, and some polymers extracted from the fruiting body of pyogo mushrooms, such as lentinan, are used as medical products. However, since the content or molecular weight of beta- glucans contained in the fruiting bodies or mycelia of fungi (mushrooms) varies considerably depending on growing or cultivation conditions, extracted beta-glucans do not have uniform quality due to, for example, mixing of high and low molecular weight beta-glucans, so that they are not applicable in practice.

<4> On the other hand, the cell wall of microorganisms contains a large amount of beta-glucans and is significant as a beta-glucan source. In particular, yeast, lactobacillus and Aureobasidium derived beta-glucans are known as useful food materials due to their excellent stability. However, beta-glucans contained in the cell wall of the microorganisms also contain a variety of impurities, causing difficulty in separation and purification. Further, since these beta-glucans are insoluble in water, it is difficult to produce highly effective water-soluble beta-glucans.

<5> Agrobacterium species (sp.), Macrophomopsis sp., AlcaIigenes sp., and Aureobasidium pullulans have been reported as microorganisms producing beta- glucans so far (Agaric. Biol. Chem. 47(6), 1167-1172 (1983)).

<6> It has been revealed that high molecular weight materials produced by microorganisms have a variety of structures and properties, and some exhibit exclusive physiological activities and comparative physical properties, which have never been found in vegetable polymers. Microorganism derived beta- glucans exhibit superior anticancer activity (Wagner et al . , 1988) with respect to various kinds of cancers, and antibacterial and antiviral activities (Bonn & BeMi Her, 1995) through enhancement of immunity. Further, the microorganism derived beta-glucans provide excellent wound healing and skin restoration effects (Jamas et al., 1991), which lead to antiinflammatory and anti-aging effects. Besides, various pharmacological effects of the beta-glucans, such as blood pressure reduction (Chang & Miles, 1991), blood sugar level reduction (Chang & Miles, 1987), and the like, have been reported in conferences, thereby increasing expectation of their applications to pharmaceutical industries, dietary supplements, cosmetic industries, etc. Furthermore, in light of their excellent viscosity, gel formability, thermal stability, pH stability and emulsification activity (Hamada, 1987), beta- glucans have received attention for application to food, cosmetics, medical products, and a variety of industries.

<7> However, beta-glucans are produced along with alpha-glucans (i.e. pullulan) by microorganisms. Here, it has been reported that pullulan does not exhibit physiological activity such as enhancement of immunological activity. Therefore, it is necessary to process the mixed glucans with expensive pullulanase for a long duration to remove the pullulan, followed by purification to produce highly pure beta-glucans which have enhanced immunological activity. Accordingly, there is a need for development of microorganisms capable of producing only pure beta-glucans for achieving cost reduction while ensuring advantages by the use of pure beta-glucans.

<8>

[Disclosure] [Technical Problem] <9> The present invention is directed to solve the problems as described above, and an aspect of the present invention is to provide a novel strain which produces natural exo-type beta-glucans with excellent yield and simple purification. <io> Another aspect of the present invention is to provide a novel strain which produces only beta-glucans having physiological activities by inactivating pullulan synthetase, i.e. enzyme producing alpha-glucans. <ii> A further aspect of the present invention is to provide a method of effectively producing exo-type beta-glucans having excellent immunological activity using the novel strain. <i2> Yet another aspect of the present invention provides immunity enhancement compositions, dietary supplements, and cosmetic compositions containing exo-type beta-glucans obtained by the method.

<13>

[Technical Solution]

<i4> The inventors of the present invention have studied for many years to obtain pullulan synthetase involved in biosynthesis of pullulan from Aureobasidium pullulans and to inactivate the pullulan synthetase using a gene disruption method, thereby producing a recombinant microorganism, Aureobasidium pullulans NP-1221, which produces only pure beta-glucans. Specifically, according to an embodiment of the present invention, a vector for removing pullulan synthetase gene is prepared and injected into Aureobasidium pullulans to develop a novel recombinant microorganism, that is, Aureobasidium pullulans NP-1221, from which pullulan synthetase is inactivated. In order to complete the present invention, Aureobasidium pullulans NP-1221 was submitted under Accession No. KCTC11265BP to the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology, on January 18, 2008.

<15>

[Advantageous Effects]

<16> A novel strain of the present invention, Aureobasidium pullulans NP- 1221 KCTC 11265BP, produces pure exo-type beta-glucans, which exhibit superior solubility in water to facilitate mass production. Thus, the exo- type beta-glucans produced by the novel strain are available in a variety of applications to medical products, dietary supplements and cosmetic compositions.

<17>

[Description of Drawings] <18> Fig. 1 is a picture of a parental strain (left) and a transformed novel strain NP-1221 (right) of Aureobasidium pullulans according to the present invention;

<19> Fig. 2 is an agarose gel picture of a gene transformed by PCR, <20> M: lkb + ladder markers <2i> 1: PCR-amplified product showing an increase in size of Exon 1 of a

PuIS gene by insertion of a hygromycin cassette in an Aureobasidium pullulans

NP-1221 genome, <22> 2: PCR-amplified product of Exon 1 of a pure PuIS gene in a parental strain genome; <23> Fig. 3 is an agarose gel DNA picture of a TH cassette and PCR amplification of an exon of a PuIS gene involved in synthesis of pullulan in the parental Aureobasidium pullulans', <24> Fig. 4 is a diagram and agarose gel DNA pictures illustrating PCR amplification of a TEF promoter and a hygromycin-resistant gene Hyg which form the TH cassette; <25> Fig. 5 is an agarose gel DNA picture and a diagram illustrating the conjugation of Exon 1, the cassette, and Exon 2;

<26> Fig. 6 is a diagram of a recombinant plasmid DNA, pKBR103, and an agarose gel DNA picture to confirm cloning; and

<27> Fig. 7 is a chromatogram illustrating HPLC analysis of polysaccharides produced by the novel strain, Aureobasidium pullulans NP-1221, reacted with laminarinase (top), a chromatogram illustrating HPLC analysis of polysaccharides produced by the novel strain, Aureobasidium pullulans NP- 1221, reacted with pullulanase (middle), and a chromatogram illustrating HPLC analysis of polysaccharides produced by the parental strain, Aureobasidium pullulans IMS-822 KCTC BP11179, reacted with pullulanase (bottom).

<28>

[Best Mode]

<29> The above and other aspects and features of the present invention can be accomplished by the provision of a vector in which pullulan synthetase is inactivated.

<30> Further, the present invention provides a strain transformed with the vector in which the pullulan synthetase is inactivated.

<3i> Further, the present invention provides a novel strain which produces pure beta-glucans, Aureobasidium pullulans NP-1221 KCTC 11265BP, transformed with the vector in which the pullulan synthetase is inactivated.

<32> Furthermore, the present invention provides a method of producing beta- glucans using a strain in which the pullulan synthetase is inactivated. The strain with the inactivated pullulan synthetase may include Aureobasidium pullulans NP-1221 KCTC 11265BP.

<33> In accordance with an embodiment of the present invention, an exo-type beta-glucan producing strain is provided, in which the pullulan synthetase involved in biosynthesis of pullulan is inactivated.

<34> The strain may be Aureobasidium pullulans NP-1221 KCTC 11265BP.

<35> In accordance with another embodiment of the present invention, there is provided a recombinant vector for producing the exo-type beta-glucan producing strain, in which PuIS gene, involved in biosynthesis of pullulan, is inactivated.

<36> The PuIS gene may be inactivated by a gene disruption method.

<37> The PuIS gene may be inactivated by disrupting at least one of two exons of the PuIS gene.

<38> The PuIS gene may be inactivated by inserting a cassette into at least one exon of the PuIS gene, the cassette comprising a TEF promoter and an antibiotic-resistant gene linked with the TEF promoter.

<39> In accordance with a further embodiment of the present invention, there is provided a method of preparing an exo-type beta-glucan producing strain in which a PuIS gene, involved in biosynthesis of pullulan, is inactivated by transforming an exo-type beta-glucan producing strain with the recombinant vector.

<40> The exo-type beta-glucan producing strain, in which the PuIS gene involved in the biosynthesis of the pullulan is inactivated may be Aureobasidium pullulaπs NP-1221 KCTC 11265BP.

<4i> In accordance with yet another embodiment of the present invention, there is provided a method of producing exo-type beta-glucan by culturing the exo-type beta-glucan producing strain with the gene involved in biosynthesis of pullulan being inactivated.

<42> In accordance with yet another embodiment of the present invention, there is provided a pharmaceutical composition for immunity enhancement including a cultured fluid, a supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain, and having an enhanced immune activity through activation of immune cells, in which the PuIS gene being inactivated.

<43> In accordance with yet another embodiment of the present invention, there is provided a dietary supplement including a cultured fluid, a supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain, in which the PuIS gene being inactivated.

<44> In accordance with yet another embodiment of the present invention, there is provided a skin preparation including a cosmetic composition, which includes a cultured fluid, a supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain, in which the PuIS gene being inactivated.

<45> In accordance with yet another embodiment of the present invention, there is provided a feed composition for an animal including a cultured fluid, supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain, in which the PuIS gene involved in biosynthesis of pullulan being inactivated.

<46> Beta-glucan produced by the foregoing method may be used in the form of a cultured fluid or a dried substance thereof without any purification for pharmaceutical compositions for immunity enhancement, dietary supplements, skin preparations containing cosmetic compositions, and feed compositions for animals.

<47> The present invention may provide a pharmaceutical composition for immunity enhancement which contains the beta-glucan as an effective element.

<48> The pharmaceutical composition may include beta-glucan in an amount of 0.01 to 99.9%, and more preferably in an amount of 0.1 to 90%. However, the present invention is not limited thereto, and the content of beta-glucan can be changed according to a patient's condition and the kind and severity of disease.

<49> The pharmaceutical composition containing beta-glucan may further include carriers, excipients and diluents, which are generally employed in the preparation of a pharmaceutical composition.

<50> The pharmaceutical composition containing beta-glucan may be formed into oral administration forms, such as powder, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols^'. topical applications; suppositories; and sterile injection solutions according to methods known in the art. Examples of the carriers, excipients and diluents for the composition containing beta-glucan may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may be prepared into dosage forms using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. Solid dosage forms for oral administration include tablets, pills, powder, granules and capsules, and are prepared through composition mixing with one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to the excipients, lubricants such as magnesium styrate and talc may be added. Liquid dosage forms for oral administration are exemplified by suspensions, internal solutions, emulsions, syrups, etc., and may include simple diluents, such as water and liquid paraffin, as well as wetting agents, sweetening agents, fragrances, and preservatives. Dosage forms for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, suppositories, etc. For formulation of non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil or injectable esters such as ethyl oleate, may be used. As a base for suppositories, witepsol, macrogol, Tween 61, cocoa oil, laurinic acid, and glycerogelatine are useful.

<5i> A dosage of the pharmaceutical composition containing beta-glucan depends on a patient s condition and weight, the severity of a disease, a drug type, and an administration route and period, but may be suitably determined by those skilled in the art. For a desired effect, however, the appropriate dosage of beta-glucan of the present invention is 0.01mg/kg to 10g/kg a day and, preferably, lmg/kg to lg/kg, and may be administrated once or several separate times. The dosage does not limit the scope of the present invention.

<52> The composition according to the present invention can be administered through various routes to mammals such as mice, rats, livestock and humans, for example, oral routes or rectal, intravenous, intramuscular, subcutaneous, transdermal and cerebrovascular injections.

<53> The present invention may provide a dietary supplement composition which contains beta-glucan having immunity enhancement effect.

<54> The composition containing beta-glucan may be variously used in drugs, food and drinks for immunity enhancement. Beta-glucan of the present invention may be added to various kinds of food, drinks, gum, tea, vitamin complexes, and dietary supplements in the form of powder, granules, tablets, capsules or drinks.

<55> Since beta-glucan of the present invention is nontoxic and presents substantially no side effects, it is safe even in long-term administration.

<56> Beta-glucan of the present invention may be added to food or drinks for immunity enhancement. Here, a dose of beta-glucan in food or drinks may generally be 0.01 to 15 percent by weight (wt.%) with respect to the total weight in case of the dietary supplement composition of the present invention, and it may be 0.02 to 1Og, preferably 0.3 to Ig, per 100ml in the case of beverage compositions.

<57> The beverage composition of the present invention is not limited to a specific liquid ingredient except that it essentially contains the beta- glucan at the instructed proportion and may further include a variety of flavors or carbohydrates as are found in other drinks. The carbohydrates include monosaccharides, e.g., glucose and fructose, disaccharides, e.g., maltose and sucrose, polysaccharides, e.g., dextrin and cyclodextrin, and sugar alcohols, e.g., xylitol, sorbitol and erythritol. In addition, the flavors include natural flavors (thaumatin and stevia extracts, e.g., rebaudioside A, glycyrrhizin, etc.) and artificial flavors (saccharine, aspartame, etc.). The natural carbohydrates are generally added at a ratio of about 1 to 2Og, preferably about 5 to 12g, per 100ml of the composition of the present invention.

<58> In addition, the composition of the present invention may contain a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents or natural flavoring agents, coloring agents and improving agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, an organic acid, a protective colloidal adhesive, a pH controlling agent, a stabilizer, a preservative, glycerin, alcohol, carbonizing agents used in carbonated beverages, etc. Other components may include fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage. The components can be used independently or in combination. The ratio of the components is not critical, but may be generally in the range of 0 to 20 parts by weight per 100 parts by weight of the composition of the present invention.

<59> The present invention also provides a skin preparation composition including a cosmetic composition which contains the beta-glucan. The skin preparation composition may be formed into any type of application by a method known in the art. In preparing a cream application, for example, an oil-in-water (0/W) or water-in-oil (W/0) cream base includes the beta-glucan of the present invention, employing perfume, a chelating agent, a coloring agent, an antioxidant, a preservative, etc. as necessary or using protein, minerals, and vitamins to improve properties.

<60> In addition, the beta-glucan of the present invention may also be employed for cosmetics, and may be mixed with a pH controlling agent, perfume, an emulsifying agent, and a preservative, if necessary, to be formed into cosmetic waters, gels, water-soluble powders, fat-soluble powders, water-soluble liquids, creams or serums by a method known in the art.

<6i> If the beta-glucan of the present invention is used for an immunity enhancement composition, a dietary supplement composition, a skin preparation composition, such as cosmetics, and a feed composition, it functions to induce differentiation of dendritic cells and to activate immune cells, thereby enhancing immunity. When added in an amount of 5μg/ml or more to the immunity enhancement composition, the beta-glucan provides superior effects. If the concentration of beta-glucan is less than 5/_g/ml , immunity enhancement effect is insignificant. The beta-glucan may be added to a concentration of 5 to lOOOμg/ml to the immunity enhancement composition. Over 1000//g/ml , immunity enhancement does not increase significantly for the amount of beta- glucan, which is not efficient.

<62> If the beta-glucan is employed for a dietary supplement or cosmetic composition, it exhibits immune activity enhancement and can be usefully employed. The added amount thereof can vary according to products, but is not limited thereto.

<63> In one desirable embodiment, For the dietary supplement, the beta- glucan may be added to a concentration of 0.005 to 2 wt.% with respect to the total weight. In one desirable embodiment, For the cosmetic composition, it may be added to a concentration of 0.001 to 2 wt.% with respect to the total weight. Further, in one desirable embodiment, for the feed composition, the beta-glucan may be added to a concentration of 0.005 to 2 wt.% with respect to the total weight. Immunity enhancement is insignificant below the lower limit. On the other hand, if the amount of beta-glucan exceeds the upper limit, the beta-glucan does not provide significant improvement of effect by an increase in amount of beta-glucan, which results in cost increase.

<64> Hereinafter, the present invention will be described in more detail.

<65> The inventor of the present invention developed a strain producing pure exo-type beta-glucan, which has excellent yield and enables facile purification. The novel strain of the present invention, Aureobasidium pullulans NP-1221 KCTC 1265BP, contains pullulan synthetase being inactivated to produce pure beta-glucan. The beta-glucan produced by the novel strain of the present invention, Aureobasidium pullulans NP-1221 KCTC 1265BP, exhibits excellent enhancement of immunological activity, so that it is useful for an immunity enhancement composition, dietary supplement, or feed composition.

<66> The inventors developed the strain with the foregoing features, named Aureobasidium pullulans NP-1221, and submitted the strain to the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology, on January 18, 2008 (Accession No. KCTC11265BP).

<67> The strain produces exo-type beta-glucan, which has been identified as culturing well in a liquid medium prepared with 1.0 to 5.0 wt.% of sugar, 0.01 to 0.05 wt .% of sodium nitrate, 0.1 to 1.0 wt.% of potassium phosphate dibasic, 0.05 to 0.50 wt.% of potassium chloride, 0.02 to 0.2 wt.% of magnesium sulfate, and 0.001 to 0.01 wt.% of ferrous sulfate Monohydrate.

<68> As an example of culture of the strain of the present invention, the liquid medium is sterilized with steam under pressure in a fermentation tank at 121°C or more for 30 to 60 minutes and is cooled to 30°C. The stain is inoculated into the liquid medium and cultured in the tank at 30°C, an air supply of 0.5 to 1.0 wm, and an agitation rate of 150 to 300 rpm.

<69> Then, the cultured product may be purified by freeze-drying or centrifugal separation to obtain beta-glucan. For example, the strain culturing broth is centrifuged to remove fungus bodies, and is then dried to provide purified beta-glucan or can be used as it is. The beta-glucan produced by the strain of the present invention has a molecular weight of

5

8.9x10 Daltons, which is the same as the parental strain.

<70> Further, the inventors analyzed how the beta-glucan, produced by the novel strain of the present invention, Aureobasidium pullulans NP-1221, effects differentiation and activation of dendrite cells, which, among in- vivo immune cells, serve a key role in controlling the immune system. Dendrite cells are obtained by treating a red blood cell concentrate from the blood bank with a red blood cell (RBC) lysis buffer to remove red blood cells and isolating monocytes using monocyte isolation kits. The dendrite cells are cultured in a 6-well micro-plate with a medium (10% FBS, RPM1640) including Granulocyte-macrophage colony-stimulating factor (GM-CSF, 1000 U/ml) and IL-4 (1000 U/ml). Four days later, ImI of a medium and the same amount of GM-CSF and IL-4 are added to the cultured product. On the sixth day, it is treated with beta-glucan and cultured in a C02 incubator at 37°C for 24 hours. Then, the differentiation of the dendrite cells is analyzed. The result proves that the dendrite cells serving immune functions are actively differentiated in the presence of beta-glucan, which identifies that the beta-glucan induces the differentiation of the dendrite cells to enhance immunity functions in- vivo (data omitted). The result corresponds to the immunity enhancement of the beta-glucan obtained by the parental strain, Aureobasidium pullulans NP- 1221 (refer to Korean Patent Application No. 10-2008-57124).

<71>

[Mode for Invent ionl

<72> Next, the present invention will be described with reference to some examples. However, it should be noted that that the present invention is not limited to the examples of the present invention disclosed below but can be implemented in various ways.

<73>

<74> Example l: Analysis and Acquisition of Gene Involved in Biosynthesis of Pullulan

<75> The inventors identified a PuIS gene involved in biosynthesis of Pullulan produced by Aureobasidium pullulans through the National Center for Biotechnology Information, USA, as deposited under Accession No. AF470615.

<76> The PuIS gene includes 1816 base pairs (bp) in total, and includes two exons. The first exon includes 209-488 bps, and the second exon includes 1070-1242 bps. The inventors amplified the first exon via polymerase chain reaction (PCR).

<77> An oligomer employed for the PCR amplification was PuISlF 5^'-

-3^* and PuISlR 5^'-

and PulS2F 5^V-

-3^' and PulS2R 5^'- In PCR, genomic DNA of Aureobasidium

pullulans was used as a template. The PCR products were a left portion and a right portion on the center of the first exon, which were named Exon 1 and Exon 2, respectively. Then, an antibiotic-resistance genetic cassette was inserted thereto.

<78>

<79> Example 2^: Preparation of Cassette for Disruption of Target Gene <80> In the present invention, the disruption of a target gene was carried out by homologous recombination which involves a genetic exchange by attraction between two identical strands of DNA. An exon of the target gene was amplified at the right and left sides, after which a cassette having an antibiotic-resistant gene was linked in the middle of the target gene. In detail, with overlapping PCR, the cassette was linked to the 3^' portion of the PCR product of the left exon portion, and the 3^' portion of the cassette was linked to the right exon portion. In the preparation of the cassette, the amplification of the antibiotic-resistance gene via PCR and a promoter functioning in Aureobasidiυm pullulans were required. Thus, TEF was amplified by PCR, which was identified to actively function as a promoter in Aureobasidium sp. Further, a hygromycin phosphotransferase gene, that is, an antibiotic-resistant gene controlling the synthesis of hygromycin in Aureobasidium sp, was amplified.

<8i> In PCR of the promoter, a template was a pFA6a-mRFP-KanMX6 plasmid DNA, and oligomers were

and TEFR 5^'-

In the amplification of the antibiotic- resistant gene, a template was genomic DNA of E. coli, and oligomers were HygF

and HygR 5^'-

Further, overlapping PCR was carried out to link the antibiotic-resistant gene amplified via PCR, Hyg, to the TEF promoter and the 3^' portion of the promoter. In the overlapping PCR, the respective amplified products were used as templates, because fifteen bps of the HygF oligomer to the 5^' portion have a complementary sequence to the TEFR oligomer. That is, if the promoter and antibiotic-resistant gene are reacted as templates, complementary portions thereof are combined. Then, the combined product was amplified into one product using the TEFF and HygR oligomers. As a result, a TH PCR product with TEF-Hyg was obtained as a cassette.

<82>

<83> Example 3: PCR for Conjugation of Exon of Target Gene and Antibiotic Cassette

<84> As described above, the inventors of the present invention amplified right and left sides of the first exon on the middle via PCR to disrupt the first exon of the two exons of the target gene. The PCR products are combinable with antibiotic cassettes, wherein the left amplified product (Exon 1) has a 3^'-terminal complementary DNA sequence and the right amplified product (Exon 2) has a 5^'-terminal complementary DNA sequence. First, the left portion was conjugated with the TH antibiotic cassette via overlapping PCR. A template was the left amplified product (Exon 1) and the cassette, and oligomers for amplification were PuISlF and HygR. Then, another overlapping PCR was carried out with the amplified product, in which Exon 1 was conjugated with the cassette, and the right amplified product (Exon 2) as templates. The oligomers were PuISlF and PulS22R. As a result, a product of Exon 1 + TH cassette + Exon 2 to disrupt a target portion was produced, which was named "ETHE". Then, the product was cloned to a T vector.

<85>

<86> Example 4: Cloning of Recombinant Gene Using T Vector <87> The PCR product forms a tail of adenine (A) bases at the 3^' terminal and the 5' terminal, which are inclined to form a bond with thymine (T). Thus, the inventors of the present invention conducted cloning using pBTl.l (Biosolution Co.) T-vector, which contains thymine bases and clones the adenine tail of the amplified product. First, the amplified product, i.e., ETHE (Exonl + cassette + Exon2), the T-vector, a ligation buffer, ligase, and triple distilled water were mixed to conjugate the amplified product and the vector. After thorough conjugation over 2 hours or more, the product was transformed to an E. coli host using the conjugated DNA. Since the T-vector contains an ampici 11 in-resistant gene, a transformed strain can be isolated using an antibiotic. Further, it can be chromatically sorted using X-gal (5- bromo-4-chloro-3-indolyl-b-D-galactopyranoside) and IPTG (Isopropyl b-D- thiogalactopyranoside). The transformation was conducted by a heat shock method, by which DNAs are introduced into cells via high temperature. The product was cultured in a medium respectively containing ampici 11 in, X~gal or IPTG for 16 hours at 37°C . Then, normal-colored colonies excluding blue colonies were sorted to obtain recombinant DNAs.

<88> <89> Example 5: Analysis and Acquisition of Cloned Recombinant Plasmid DNA <90> Normal-colored colonies observed on the plate were inoculated into a liquid medium, to which 5ml of ampicillin had been added. The product was sufficiently cultured for 12 hours at 37°C and 200 rpm, after which DNAs were extracted from the bacteria. The extraction was conducted using a DNA extraction kit. The DNAs were amplified to identify whether ETHE is or not in the DNA using PCR. And, the size of total DNA, vector, and ETHE region were detected using PCR and restriction enzymes. The DNAs were treated with XbI to identify their whole size. FTo identify the sizes and other properties of the vector and ETHE, Xbal and Hindi 11 were used to observe properties of DNA fragments. As a result, all were proved to be normal, and the recombinant plasmid DNAs were named pKBR103.

<91>

<92> Example 6: Transformation of Aureobasidium Pullulans using Recombinant Plasmid DNA

<93> Aureobasidium pullulans was transformed with the recombinant plasmid pKBR193 produced in the present invention. In the transformation, the DNAs were inserted into spheroplasts, prepared from Aureobasidium sp., by polyethylene glycol (PEG), and hygromycin was used as a marker to sort transformed bodies. Estimated transformed colonies, found on the plate, underwent replica plating for 10 generations to disrupt gene owing to homologous recombination of complementary base pairs and to induce the insertion of a hygromycin cassette. A finally obtained strain was identified by PCR, which proved that the size of genes increased by the insertion of the cassette.

<94> As a result, it can be seen from Fig. 1 that the strain was structurally and genetically comparable with the parental strain.

<95>

<96> Example 7: Cultural and Physiological Properties of Transformed Aureobasidium Pullulans

<97> Cultural and physiological properties of the transformed novel strain, Aureobasidium pullulans NP-1221, were analyzed. As for cultural properties, the transformed novel strain could use only glucose, sucrose and maltose as a carbon source, as listed in Table 1. Consequently, it was verified that NP- 1221 exhibited the same cultural and physiological properties except for pullulan synthetic ability.

<98>

<99>

Aureobasidium Pullulans

<ioo> The parental strain, Aureobasidium pullulans IMS-822, and the transformed novel strain, Aureobasidium pullulans NP-1221, were inoculated into a Cazpeck medium and cultured for three days at 30°C , after which the product was centrifuged at 4000 rpm to remove the cells to collect ImI of the supernatant. The supernatant was treated with pullulanase and laminarinase and reacted for 24 hours. Then, separated sugar was analyzed by high pressure liquid chromatography (HPLC). A reaction product of pullulanase, maltotriose, was detected in sugar produced by the parental strain, while no reaction product of pullulanase was found in the transformed strain. Accordingly, it was confirmed that the novel strain did not produce pullulan. Further, the separated sugar was analyzed with laminarinase which exclusively cleaves 1,3- beta-linkage, thereby verifying glucose and gentiobiose, which confirmed polysaccharides only having beta-linkages produced from mutants.

<101>

[Industrial Applicability]

<iO2> The present invention provides a novel strain which produces exo~type beta-glucan enabling facile purification by suppressing the biosynthesis of pullulan. Accordingly, beta-glucan allowing simple purification can be mass produced using the novel strain applicable to a variety of applications, such as pharmaceutical compositions, cosmetics, food, feeds, etc.

<103>

[Sequence List Text]

<iO4> The nucleotide sequences attached to the present invention relates to primers for amplifying two exons of a pullulan biosynthetic gene and primers for amplifying a promoter.

Claims

[CLAIMS] [Claim 1]

An exo-type beta-glucan producing strain inactivating a PuIS gene involved in biosynthesis of pullulan. [Claim 2]

The exo-type beta-glucan producing strain according to claim 1, wherein the strain is Aureobasidium pullulans NP-1221 KCTC 11265BP. [Claim 3]

A method of producing exo-type beta-glucan by culturing the exo-type beta-glucan producing strain according to claim 1 or 2. [Claim 4]

A recombinant vector for producing the exo-type beta-glucan producing strain according to claim 1 or 2, the strain inactivating a PuIS gene involved in biosynthesis of pullulan. [Claim 5]

The recombinant vector according to claim 4, where the PuIS gene is inactivated by a gene disruption method. [Claim 6]

The recombinant vector according to claim 5, wherein the PuIS gene is inactivated by disrupting at least one of two exons of the PuIS gene. [Claim 7]

The recombinant vector according to claim 6, wherein the PuIS gene is inactivated by inserting a cassette into at least one exon of the PuIS gene, the cassette comprising a TEF promoter and an antibiotic-resistance gene linked with the TEF promoter. [Claim 8]

A method of preparing an exo-type beta-glucan producing strain inactivating a PuIS gene involved in biosynthesis of pullulan by transforming an exo-type beta-glucan producing strain with the recombinant vector according to any one of claims 4 to 7. [Claim 9] The method of preparing the exo-type beta-glucan producing strain according to claim 8, wherein the exo-type beta-glucan producing strain inactivating the PuIS gene involved in the biosynthesis of the pullulan is Aureobasidium pullulans NP-1221 KCTC 11265BP. [Claim 10]

A medical composition for immunity enhancement comprising a cultured fluid, a supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain according to claim 1 or 2, and having an enhanced immune activity through activation of immune cells, the strain inactivating a PuIS gene involved in biosynthesis of pullulan. [Claim 11]

A dietary supplement comprising a cultured fluid, a supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain according to claim 1 or 2, the strain inactivating a PuIS gene involved in biosynthesis of pullulan. [Claim 12]

A dermatologic preparation comprising a cultured fluid, a supernatant or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain according to claim 1 or 2, the strain inactivating a PuIS gene involved in biosynthesis of pullulan. [Claim 13]

A feed composition for an animal comprising: a cultured fluid or a dried substance thereof obtained by culturing the exo-type beta-glucan producing strain according to claim 1 or 2, the strain inactivating a PuIS gene involved in biosynthesis of pullulan.