Title of Invention: A CORYNEBACTERIA STRAIN HAVING ENHANCED 5'-XANTHOSINE MONOPHOSPHATE PRODUCTIVITY AND A METHOD OF PRODUCING 5'-XANTHOSINE MONOPHOSPHATE USING THE SAME Technical Field

[1] The present invention relates to a recombinant vector, having the structure shown in the cleavage map of FIG. 1 which carries a gene of SEQ ID NO. 7 and a Corynebacteria strain, transformed with the recombinant vector, having higher than the endogenous malate dehydrogenase activity. Also, the present invention is concerned with a method of producing 5'-xanthosine monophosphate using the Corynebacteria strain.

[2]

Background Art

[3] 5'-Guanosine monophosphate (hereinafter referred to as "GMP") is a food additive widely used as a flavor enhancer, like inosine monophosphate (hereinafter referred to as "IMP"). GMP elicits an umami taste and its use is dependent on monosodium glutamate (MSG) also being used. It is often used in synergy with IMP to increase the intensity of the umami taste of MSG.

[4] Examples of the methods for the preparation of GMP known thus far include (1) the enzymatic degradation of yeast RNA, (2) direct microorganism fermentation to GMP,

(3) microorganism fermentation to guanosine, followed by chemical phosphorylation,

(4) microorganism fermentation to guanosine, followed by enzymatic phosphorylation,

(5) microorganism fermentation to xanthosine 5 '-monophosphate (hereinafter referred to as "XMP"), followed by conversion into GMP by a corynebacteria strain, and (6) microorganism fermentation to XMP, followed by conversion of XMP into GMP by Escherichia coli which has aminase activity. Of them, method (1) has difficulties of material supply and is economically non-beneficial and method (2) suffers from the disadvantage of being of low yield due to the membrane permeability of GMP. Thus, the other methods are widely used in industrial applications.

[5] For the method in which XMP is produced and converted into GMP, it is critical to increase XMP productivity. For example, Korean Patent Application No. 10-1991-018016 discloses an XMP aminase-inactive strain capable of producing XMP in high yield, which is semi-auxotrophic for adenine and guanine, tolerant of guanosine analoges and very susceptible to lysozyme, an enzyme which destroying cell walls. Korean Patent Application No. 10-2001-000513 discloses a strain of Corynebacterium ammoniagenes which can directly accumulate XMP at high concentration in a culture medium and a method of producing XMP using the same. The strain is prepared by irradiating the mother microorganism with UV light, treating with the mutagen N- methyl-N'-nitro-N-nitrosoguanidine (NTG), and selecting a mutant tolerant of norvaline, an analog of valine which affects the biosynthesis of XMP. Korean Patent Application No. 10-2008-006537 describes a method of increasing XMP yield in which purN and purH, genes involved in the biosynthesis of XMP, are modified.

[6] On the other hand, ATP productivity leads to XMP yield because ATP is involved in the biosynthesis of XMP. Also, The activity of malate dehydrogenase has a great influence on the production of ATP. However, nowhere are microorganisms and methods which are designed to enhance malate dehydrogenase activity for increasing XMP production yields mentioned in previous documents.

[7]

[8] Keeping in the mind that it is important to increase the ATP productivity of the

XMP-producing strain, the present inventors conducted intensive research and found a gene which is responsible for the increase. Also, it was found that a Corynebacteria strain transformed with a recombinant vector carrying two copies of the gene in tandem, could produce XMP in high yield.

[9]

Disclosure of Invention

Technical Problem

[10] It is therefore an object of the present invention to provide a Corynebacteria strain having higher than the endogenous malate dehydrogenase activity. [11] It is another object of the present invention to provide a recombinant vector having the structure shown in the cleavage map of FIG. 1 which carries the gene of SEQ ID

NO. 7. [12] It is a further object of the present invention to provide a Corynebacteria strain transformed with the recombinant vector. [13] It is still a further object of the present invention to provide a method of producing

XMP by culturing the transformed microorganism and obtaining XMP from the culture. [14]

Solution to Problem [15] In accordance with an aspect thereof, the present invention is directed to a corynebacteria strain with enhancements over endogenous malate dehydrogenase activity. Preferably, the corynebacteria strain of the present invention is modified to have malate dehydrogenase activity higher than endogenous activity, resulting in an enhancement in XMP productivity.

[16]

[17] The term "malate dehydrogenase", as used herein, means an enzyme that catalyzes the conversion of malate into oxaloacetate by dehydrogenation. This enzyme is found in a very broad spectrum of living organisms with the accompaniment of lactate dehydrogenase and requires DPN and NAD as cofactors for its activity, these usually accompanying lactate dehydrogenase. In the corynebacteria strain according to the present invention, the malate dehydrogenase activity is increased to produce XMP in higher yield.

[18]

[19] As used herein, the term "endogenous activity" is intended to refer to the enzyme activity of interest in a wild-type microorganism. The term "higher than endogenous activity" means increased enzyme activity compared to the activity of the endogenous variety, whether resulting from an activity increase by the enzyme itself or by an endogenous gene or a foreign gene. For example, an increase in enzyme activity may be achieved by any method well known in the art, including, but not limited to, increasing or decreasing the number of gene copies, replacing, modifying or mutating a promoter of interest, etc.

[20] The target enzyme malate dehydrogenase whose activity is sought to be increased according to the present invention is encoded by the mqo gene of Corynebacteria. As long as it is biologically identical or correspondends to the mqo gene, any derivative or analog may be used in the present invention. That is, if its activity is substantially the same as or similar to that of the mqo gene, any gene falling within the range of the mqo gene is useful in the present invention. Advantageously, the gene useful in the present invention shares at least 70%, more preferably at least 80%, even more preferably at least 90%, even far more preferably at least 95% and most preferably at least 98% homology with the sequence of the mqo gene. More advantageously, the malate dehydrogenase is encoded by the nucleotide sequence of SEQ ID NO.: 7. The increase of gene copies can be achieved by the introduction of exogenous genes and/or the amplification of endogenous genes. The number of gene copies may be readily determined by those skilled in the art according to need and purpose. The amplification of the endogenous gene can also be conducted using a method known in the art, for example, by culturing in a suitable selective medium under pressure. In a preferred example, a vector carrying a gene coding for malate dehydrogenase is introduced into a corynebacteria strain to generate a transformed microorganism with an enhancement over the endogenous activity.

[21] [22] As long as it is known in the art and belongs to the corynebacteria genus, any strain may be used in the present invention without limitation. Preferably, examples of the corynebacteria strain useful in the present invention include Corynebacterium ammo- niagenes, Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum, but are not limited thereto. In detail, among the corynebacteria strains are Corynebacterium ammoniagenes ATCC6872, Corynebacterium thermoaminogenes FERM BP- 1539, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum R, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and derivatives thereof. Preferred is Corynebacterium ammoniagenes KCJ-1304 transformed from Corynebacterium ammoniagenes KCCM-10530.

[23]

[24] In accordance with another aspect thereof, the present invention is directed into a recombinant vector having the structure shown in the cleavage map of FIG. 1, which carries the gene of SEQ ID NO. 7.

[25] The gene of SEQ ID NO. 7 has a wild-type nucleotide sequence of an mqo gene from

Corynebacteria. However, it is obvious that a recombinant vector which carries a derivative or analog of the mqo gene is also useful in the present invention as long as the derivative or analog is biologically identical or correspondends to the mqo gene, as mentioned above. As used herein, the term "mqo gene", an abbreviation of "malate:quinone oxidoreductase" gene, means a gene coding for malate oxaloacetate which functions to oxidize malate to oxaloacetate.

[26] In a preferred embodiment, the present invention provides a recombinant vector carrying the mqo gene of SEQ ID NO. 7. As long as it carries an mqo gene, any usual recombinant vector can be employed in the present invention without limitation. Preferable is pDZ-mqo. In a preferred example of the present invention, the mqo gene containing SEQ ID NO. 7 was employed to construct a recombinant pDZ-mqo vector (see FIG. 1).

[27]

[28] In accordance with a further aspect thereof, the present invention is directed to a

Corynebacteria strain transformed with a recombinant vector carrying the mqo gene.

[29] The corynebacteria-derived mqo gene may be used using a transformation method known in the art without limitation. Preferably, the mqo gene is cloned in a vector for use in transformation into cells.

[30]

[31] Any method may be employed for transformation if it is known in the art. As used herein, the term "transformation" is the genetic alteration of a cell resulting from the uptake, genomic incorporation and expression of foreign DNA. Typical transformation methods include CaCl ₂ precipitation, a Hanahan method in which the effect of CaCl ₂ precipitation is improved in combination with DMSO (dimethyl sulfoxide), electro- poration, calcium phosphate transfection, protoplast fusion, silicon carbide fiber- mediated transformation, agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation/inhibition-mediated transformation. Transformation with pDZ-mqo in accordance with the present invention is not limited to the examples of transformation, but can be achieved using any method known in the art without limitation.

[32]

[33] The term "vector", as used herein, means a DNA molecule used as a vehicle to transfer foreign genetic material into a suitable host cell and is a DNA construct containing regulatory elements which allow a transgene to do recombination with a host genome. Preferably, the recombinant vector carrying the mqo vector in accordance the present invention may have the structure shown in the cleavage map of FIG. 1. The vector represented by the cleavage map of FIG. 1 may be introduced into corynebacteria sequentially or simultaneously. In accordance with a preferred embodiment of the present invention, the recombinant vector is transformed into Corynebacterium ammoniagenes KCCM- 10530 which is then cultured in a selective medium to allow two copies of the mqo gene to incorporate into the genome of the host through homologous recombination, resulting in the generation of a Corynebacterium ammoniagenes mutant, named Corynebacterium ammoniagenes KCJ- 1304. It was deposited with accession number KCCM10972P.

[34]

[35] Corynebacterium ammoniagenes KCJ- 1304 has two copies of mqo gene incorporated into the genome of KCCM- 10530 resulting from the introduction thereinto of pDZ- mqo having the structure shown in the cleavage map of FIG. 1 and the homologous recombination of two copies of mqo gene with the endogenous gene.

[36]

[37] In accordance with a further aspect thereof, the present invention is directed to a method of producing XMP, comprising: culturing the transformed corynebacteria strain and obtaining XMP from the culture. In the present invention, XMP is produced in higher yield by the direct fermentation of the transformed microorganism. Preferably, the transformed microorganism is the corynebacteria strain which is enhanced in malate dehydrogenase activity over the endogenous activity. The mqo gene of the recombinant vector is preferably incorporated into the genome of the transformed microorganism which can thus produce XMP in high yield. In a preferred embodiment, the microorganism is Corynebacterium ammoniagenes KCCM10972P.

[38]

[39] Any medium known in the art may be used without limitation in culturing the strain capable of producing XMP. Preferably, the medium contains glucose as a carbon source and optionally various other carbon sources. For use in culturing a microorganism of interest, a medium must meet requirements for the growth of the microorganism. Culture media for corynebacteria strains are known in the art (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D. C, USA, 1981). Examples of the carbon sources useful for corynebacteria strains include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, etc., oils and lipids such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linolenic acid, etc., alcohols such as glycerol, ethanol, etc., and organic acids such as acetic acid. These carbon sources may be used individually or in combination. Organic materials such as peptone, yeast extract, beef extract, malt extract, corn steep liquor, soybean, etc., urea, and inorganic compounds such as ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be used individually or in combination as nitrogen sources in the medium. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or corresponding sodium salts may be useful as phosphorus sources. In addition, the culture medium may contain metal salts such as magnesium sulfate, iron sulfate, etc. Further, amino acids and/or vitamins may be required as essential elements. The culture medium may also contain suitable precursors. These materials may be added to a medium in a batch manner or continuous manner.

[40] The culture medium may be adjusted in pH by basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, etc. or acid compounds such as phosphoric acid or sulfuric acid. An antifoaming agent such as fatty acid polyglycol ester may be used to prevent the generation of bubbles during the culturing. The medium may be aerated with oxygen or oxygen-containing gas (e.g., air) to maintain an aerobic condition or with nitrogen, hydrogen or carbon dioxide gas to maintain an anaerobic condition. Culturing temperature is usually maintained at 2O ⁰ C ~ 45 ⁰ C, and preferably at 3O ⁰ C ~ 35 ⁰ C. Culturing is continued until the maximum amount of XMP is obtained. In this regard, a time period of from 10 to 160 hours is required.

[41] The 5XMP thus produced may be secreted into the culture medium or remain within the cell. The method of producing XMP in accordance with the present invention comprises recovering XMP from the cells or the culture medium. For the recovery of XMP from cells or culture media, any method well known in the art may be utilized. Examples of such methods include filtration, anionic exchange chromatography, crystallization, and HPLC, but are not limited thereto.

[42]

[43] As used herein, the term "5'-xanthosine monophosphate" is an intermediate in nucleic acid biosynthesis and is of physiological significance in animals and plants. Thus, it finds applications in a variety of fields including the food industry, the pharmaceutical industry and the medical industry. Xanthosine monophosphate are food additives used as nucleic acid-based flavor enhancers to provide the taste of mushroom particularly in synergy with MSG. XMP is an intermediate in purine metabolism, formed from IMP, forming GMP. When using the transformed microorganism in accordance with the present invention, XMP can be produced in a high yield which is found to be improved by about 6% compared to conventional microorganisms. [44]

Advantageous Effects of Invention

[45] Thanks to improved malate dehydrogenase activity, the transformed corynebacteria strain of the present invention produces XMP in higher yield than do conventional microorganisms.

[46]

Brief Description of Drawings

[47] FIG. 1 is a schematic diagram showing the structure of the recombinant vector pDZ- mqo in which two copies of an mqo gene are inserted into a pDZ vector.

[48]

Mode for the Invention

[49] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

[50]

[51] EXAMPLE 1: Cloning of the XMP-Producing Strain Corynebacterium ammo- niagenes KCCM- 10530-Derived mqo and Construction of Recombinant Vector (pDZ-mqo) for Genomic Incorporation

[52]

[53] The nucleotide sequence of the mqo gene (NCBI ID_3345228) was obtained from data from the NIH GenBank. Based on the sequence, two pairs of primers (SEQ ID NOS. 1 to 4) were synthesized.

[54]

[55] While the genome of Corynebacterium KCCM- 10530 served as a template, PCR was conducted in the presence of the high-fidelity DNA polymerase PfuUltra™ (Stratagene) using the primers of SEQ ID NOS. 1 to 4, with 25 cycles of denaturing at 95 ⁰ C for 30 sec, annealing at 55 ⁰ C for 30 sec and extension at 68 ⁰ C for 2 min. The PCR products thus obtained were two copies of the mqo gene, each 2.1 kb long (mqo-A, mqo-B), which were amplified using two sets of SEQ ID NOS: 1 and 2, and SEQ ID NOS: 3 and 4, respectively.

[56]

[57] SEQ ID NO: 1; gctctagaATCGGTCATTCCATGAACCC

[58] SEQ ID NO: 2; cgcggatccCATCGATATCGCCAACTCCA

[59] SEQ ID NO: 3; cgcggatccATCGGTCATTCC ATGAACCC

[60] SEQ ID NO: 4; gctctagaCATCGATATCGCCAACTCCA

[61]

[62] After being treated with suitable restriction enzymes (mqo-A: Xbal + BamHI, mqo-

B: BamHI + Xbal), the PCR products mqo-A and mqo-B were inserted into the pDZ vector which was previously treated with Xbal and shrimp alkaline phosphotase, through the three-piece junction (see Korean Patent Application No. 10-2007-94433). Finally, a recombinant pDZ-mqo vector in which two copies of the mqo gene were cloned in tandem was obtained. FIG. 1 is a schematic diagram showing the structure of the recombinant pDZ-mqo vector for incorporation into Corynebacterium genome.

[63]

[64] EXAMPLE 2: Generation of an mqo-Inserted Strain

[65]

[66] The pDZ-mqo vector construct was transformed into the KCCM- 10530 strain and subjected to homologous recombination with the genome to insert one mqo gene copy at a position adjacent to the mqo gene on the genome. Thus, a novel XMP-producing strain, named Corynebacterium ammoniagenes KCJ- 1304, which had two copies of the mqo gene on the genome thereof, was obtained. The insertion of two copies of the mqo gene in tandem was identified using PCR using a set of primers (SEQ ID NOS. 5 and 6) which targeted nucleotide sequences upstream and downstream of the two copies of the mqo gene.

[67]

[68] SEQ ID NO. 5: CTTTTCGATGACGCCCAA

[69] SEQ ID NO. 6: CCACTTTATCGGGTGAGACCA

[70]

[71] EXAMPLE 3: Malate Dehydrogenase Activity of the mqo-Inserted Strain

[72]

[73] The XMP-producing Corynebacterium ammoniagenes KCJ- 1304 prepared in

Example 2 was assayed for malate dehydrogenase activity as follows. The strain was inoculated into a medium containing 10g/f bactopeptone, 5gll bacto-beef extract, 5gll bacto-yeast extract, 2.5gll NaCl, 50 mg/f adenine, and 50 mg/f guanine and incubated at 3O ⁰ C for 12 hrs until OD 10 was obtained. 10 mL of the cell culture was recovered, washed twice with buffer comprising 50 mM HEPES, 1OmM potassium acetate, 1OmM CaCl ₂ and 1OmM MgCl ₂ , and suspended in 1 mL of the same buffer. After in- terraption using a sonicator, the cell lysate was centrifuged. The supernatant was re- centrifuged to give a pellet which was then suspended in 100 μL of buffer. 10 μL of this suspension was used as an enzyme solution. A reaction buffer was prepared by mixing 5OmM HEPES, 1OmM potassium acetate and 50 μM 2,6-dichloroindolphenol (C12Ind). C12Ind was thawed and mixed just before reaction. To 980 μL of the reaction mixture were added 10 μL of 100 mM malate as a substrate and 10 μL of the enzyme solution, followed by incubation at 3O ⁰ C for 15 min with shaking. The enzyme activity was determined by measuring the concentration of reduced Cl ₂ lnd. Cl ₂ lnd had an absorption coefficient of 22 cm- ImM-I at 600 nm.

[74]

[75] Table 1

[Table 1] [Table ]

[76]

[77] As shown in Table 1, KCJ- 1304 was observed to increase in malate dehydrogenase activity by 30.6% compared to the mother strain KCCM- 10530.

[78]

[79] EXAMPLE 4: XMP Production of the mqo-Inserted Strain

[80]

[81] The XMP-producing strain Corynebacterium ammoniagenes KCJ- 1304 prepared in

Example 2 was cultured to produce XMP as follows. The mother strain Corynebacterium ammoniagenes KCCM-10530 and the mutant KCJ-1304 were inoculated into respective 14 mL tubes, each containing 3 mL of the following seed medium, and incubated at 3O ⁰ C for 20 hrs with shaking at 200 rpm. Then, the seed cultures were added in an amount of 0.4 mL to 32 mL of the following production medium (24 mL of main medium + 8 mL of medium A) in respective 250 mL corner- baffle flasks, followed by shake culturing at 3O ⁰ C and 230 rpm for 96 hrs. Thereafter, the production of 5'-XMP was quantitatively measured using HPLC. The XMP amounts produced from Corynebacterium ammogenes KCCM-10530 and KCJ-1304 are given in Table 2, below.

[82]

[83] Table 2 [Table 2] [Table ]

[84] [85] Seed Medium: glucose 30g/f, peptone 15g/f, yeast extract 15g/f, NaCl 2.5g/£, urea 3g/f, adenine 150 mg/f, guanine 150 mg/f, pH 7.2

[86] Production Medium (main): glucose 80g/f, magnesium sulfate 10g/f , ferrous sulfate 20 mg/f, zinc sulfate 10 mg/f , manganese sulfate 10 mg/f, adenine 30 mg/f , guanine 30 mg/f, biotin 100 βglt, copper sulfate 1 mg/f, thiamine chloride 5 mg/f , calcium chloride 10 mg/£ pH 7.2

[87] Production Medium (medium A): monopotassium phosphate 10g/f , dipotassium phosphate 10g/f , urea 7g/£, ammonium sulfate 5gll

[88] [89] As is apparent from the data of Table 2, KCJ- 1304 was found to increase in XMP production by 1.7 g/1, corresponding to 5.9% increase, compared to the mother strain KCCM- 10530.

[90] [91] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[92]

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

To. CJ Cheiljedang Corporation 500 5-GA NAMDAEMUN-RO RECEIPT IN THE CASE OF AN ORIGINAL CHUNG-KU, SEOUL issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY REPUBLIC OF KOREA identified at the bottom of this page

the AUTHORITY:

DESIGNATION

identified under I above, deposit) ¹

of person(s) having the power

status of intern authority was acquired; where a deposit made outside the Budapest Treaty after the acquisition of the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authouity. Form BP/4 Sole page

— Ef^πi'gBaE-ΪEi