KR970005914B1 - Process for gmp by recombinant microorganism - Google Patents

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Description

Method for preparing 5'-guanylic acid by recombinant microorganism

Figure 1 shows the cleavage map of the restriction enzymes Sal I, Xba I, Pst I of pGH 16 and its formation steps.

The present invention converts 5'-xanthyl acid (hereinafter referred to as XMP) into 5'-guanylic acid (hereinafter referred to as GMP) using a microorganism enriched in 5'-xanthyl acid aminase by recombinant genetic engineering. It relates to a method of manufacturing GMP. GMP, a kind of purine-based nucleic acid, is widely known as 5'-inosinic acid (hereinafter referred to as IMP) and XMP as a nucleic acid-based seasoning having excellent taste. Conventional methods of manufacturing GMP include 1) hydrolyzing ribonucleic acid in yeast by enzymes such as ribonuclease, phosphodiesterase, and 2) producing by microorganisms. There are many methods of phosphorylating guanosine, but these methods have many disadvantages such as problems of raw material supply, low yield, and low yield. 3) Direct fermentation by microbial mutants, but the synthetic route of in vivo GMP is very complicated. Since the byproducts other than the target product are produced, and the final product GMP cell membrane permeability is very low, the amount of accumulation in the medium is small, so the method of adding surfactants or preservatives should be used. Known as 4) A method of converting fermentation from xanthine or xanthosine has been announced, but the supply of precursors is difficult. 5) There is also a method of producing GMP by mixing and culturing two kinds of microorganisms, but since two kinds of microorganisms must be cultured at the same time, it is not easy to control the culture, and in particular, by-products other than GMP are generated, and thus it cannot be a preferable manufacturing method. . 6) Recently, a two-step production method of accumulating XMP in a culture medium using microbial mutant strains and incubating mutant strains fortified with 5'-xanthyl acid aminase converts XMP to GMP is widely used. Publication 39069/71, Japanese Patent Publication 78697/84, Korean Patent Publication 78-344)

5'-xanthyl acid aminase has the following reactor field.

To obtain a mutant strain enriched with 5'-xanthyl acid aminase, XMP can be transferred to GMP by giving microorganisms resistance to decoinine, a substance that specifically inhibits 5'-xanthyl acid aminase. It has been announced that it can enhance the conversion activity. In E. coli, 5'-inosinic acid dehydrogenase (IMP dehydrogenase, guaB) and 5'-xanthyl acid aminase (XMP aminase, guaA), enzymes involved in the synthesis of GMP, form an operon. It is reported that the sequence consists of the sequence of operator, guaB, and guaA (J. Bacteriol. 115, 992-1002 (1973)), and in the case of E. coli coding for 5'-xanthyl acid aminase. An example has been reported for the production of conversion from XMP to GMP using a transformant cloned with a guaA gene (Japanese Patent Publication No. 224498/85, Agric. Biol. Chem. 50, 1879-1884 (1986)). However, there have been no reports on the cloning and use of 5'-xanthyl acid aminase genes from coryneform bacteria such as Corynebacterium and Brevibacterium, which are widely used for the production of amino acids and nucleic acids. The present invention developed a gene encoding 5'-xanthyl acid aminase from Brebibacterium ammoniagenes BA 17-2 strain, which is a strain of the invention, which is enhanced with 5'-xanthyl acid aminase. The recombinant plasmid containing the genetic information of this 5'-xanthyl acid aminase was transformed into a suitable strain by cloning using the E. colibacterium shuttle vector pECCG 117 of the present invention to determine the titer of 5'-xanthyl acid aminase. The present invention relates to a method for producing GMP by a recombinant microorganism which improves and converts XMP into GMP more efficiently using a transfection line having an improved 5'-xanthyl acid aminase titer. The present inventors isolated the cryptic plasmid PCG 1 from the Corynebacterium glutamicum ATCC 13058 strain for the development of a host-vector system of coryneform glutamate producing bacteria and then cloned the strain of Escherichia coli. By combining with the cloning vector pACYC 177, a pECCG 1, a shuttle vector capable of transforming and expressing both strains of Corynebacterium and E. coli, was constructed, and the ampicillin resistance gene portion was removed from the pECCG 1. A multi-cloning site of the plasmid pBluescript II KS ⁺ was introduced to produce a shuttle vector, pECCG 117, which is stably expressed in strains of the genus Corynebacterium, and applied for a patent (Korean Patent Application 89-5569 and 90-9199), this shuttle vector pECCG 117 is not only a strain of the genus Corynebacterium, but also Brevibac, a kind of coryneform glutamate producing bacteria. It was confirmed that the expression and stability is maintained even in the strain of the terriium was used in the present invention, the strains and plasmids used in the present invention are shown in Table 1.

Cultivation of enzyme bacteria giving 5'-xanthyl acid aminase for conversion from XMP to GMP by a transformant carrying a recombinant plasmid can be carried out by a known method. That is, the transformants are cultured in a conventional culture medium containing a carbon source, a nitrogen source, an inorganic compound, amino acids, vitamins and other nutritionally demanding substances under aerobic conditions while controlling the temperature, pH, etc., and the enzyme source for the conversion reaction. Used as As the carbon source, various carbohydrates such as glucose, fructose, storose, maltose, mannose and molasses, various organic acids such as pyruvic acid, fumaric acid, lactic acid and acetic acid can be used, and nitrogen sources include ammonia, ammonium chloride, Various inorganic and organic ammonium salts such as ammonium sulfate, ammonium carbonate and ammonium acetate, urea and other nitrogen-containing substances and peptones, NZ-amines, meat extracts, yeast extracts, corn steep liquors, casein hydrolysates, fish or degradation products thereof, Degreased soy cakes, or degradation products thereof may be used. As the inorganic compound, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium carbonate and the like may be used, and vitamins, amino acids and nutrient-forming bases may be added as necessary. have. The culturing is carried out under aerobic conditions, for example, by shaking culture or aeration stirring, preferably at a temperature of 20 to 40 ° C. The pH of the medium is preferably maintained near neutral during incubation, and the cells obtained by incubation for 10-50 hours or centrifuged are used as the enzyme source for the conversion reaction. XMP is converted to GMP when XMP solution or XMP fermentation broth is added to enzyme-containing solution and reacted for 24 hours to 48 hours while adjusting pH to 7-8 with ammonia water or ammonia gas at 30-50 ℃. . The generated GMP was recovered by an ion exchange resin method (eg Dowex, etc.) according to a known method. Therefore, by using microorganisms transformed by recombinant plasmids containing genes with genetic information on the enzymatic synthesis of 5'-xanthyl acid aminase derived from Brevibacterium ammonia gene, a glutamate producing coryneform bacterium, GMP can be produced more efficiently than methods. The present invention will be described in detail as follows, and the genetic manipulation methods used herein mainly include molecular cloning (A laboratory manual, 2nd ed., J. Sambrook, EF, Fritch, T., Maniatis) and molecular cloning techniques. Although performed according to the method of (Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152, SI, Berger, AR, Kimme), the Brevibacterium ammonia genes ATCC 6872 / pGH 16 used in the present invention The strain was deposited with the Korea Microorganism Conservation Center on July 10, 1991. (Accession No. KCCM 10008)

Example 1

5'-Xanthylate Aminase Gene Cloning

Chromosome DNA of Brevibacterium ammonia genes BA 17-2 and pECCG 117 isolated from Brevibacterium ammonia genes ATCC 6872 / pECCG 117 BA 17-2 strains were cultured at 32 ° C. for 18 hours in C medium having the following composition The cells were collected by centrifugation, washed with 10 ml of 1 mM Tris buffer (pH 8.0), and then 40 ml of lysozyme reaction buffer (10 mM Tris (pH 8.0), 5 mM sodium chloride, 1 mM ethylenediamine-4-acetic acid (EDTA), 0.5 M Sucrose, 10 mg / ml lysozyme) and then treated at 37 ° C. for 90 minutes. The cells were recovered by centrifugation, suspended in 10 ml of sucrose and lysozyme excluding lysozyme, and 0.6 ml of 0.5 M EDTA and 4.4 ml of 4% SDS were added to digest the cells. Add 1 g of cesium chloride per ml, mix 2 ml of ethidium bromide concentrate (10 mg / ml), and centrifuge the mixture at 48 ° C and 50,000 rpm for 48 hours using an ultra-centrifuge. The chromosomal DNA was taken using a syringe. Treatment with isopropyl alcohol 3-4 times to remove ethidium bromide, followed by dialysis in TE buffer (10 mM Tris (pH 8.0), 1 mM EDTA) for 24 hours to separate and purify the chromosomal DNA. Shuttle vector pECCG 117 was isolated and purified from the transformant ATCC 6872 / pECCG 117 according to the molecular cloning procedure, at which time kanamycin was added to the culture medium to 50μg / ml.

C medium: yeast extract 10g / I, peptone 10g / I, sodium chloride 2.5g / I, urea 3g / I; pH 7.2

Preparation of Genomic Library

Purified chromosomal DNA from BA 17-2 strain was purified by restriction enzyme Mbo 1 at 37 ° C., 15-30 in medium salt restriction enzyme buffer (50 mM sodium chloride, 10 mM Tris (pH 7.5), 10 mM magnesium chloride, 1 mM dithiothitol). After partial digestion, the restriction enzyme was inactivated by heat treatment, followed by electrophoresis on a 0.7% low-melting point agarose gel, and DNA fragments of 3 to 8 kb were obtained. . This was heated for 10 minutes at 68 ° C with water to dissolve the gel, mixed with phenol saturated with the same volume of Tris buffer (pH 8.0), centrifuged to obtain a supernatant, and then precipitated with chloroform and precipitated with ethanol. Used as. Plasmid pECCG 117 was used as a restriction enzyme BamHI for high concentration salt restriction enzyme buffer (100 mM sodium chloride, 50 mM Tris (pH 7.5), 10 mM magnesium chloride, 1 mM dithiothitol) for isolation of the target gene, 5'-xanthyl acid aminase gene. Complete cleavage by treatment at 37 ° C. for 2 hours, treatment with calf intestine phosphatase, and mixing with chromosomal DNA fragments of 3 to 8 kb BA 17-2 strain followed by ligase buffer solution (0.5 M Tris, pH 7.4). ), 0.1 M magnesium chloride, 0.1 M dithiothreitol, 10 mM spermidine, 10 mM ATP, 1 mg / ml bovine serum albumin) was used to prepare a gene library by reacting with T4 DNA ligase at 14 ° C. for 16 hours.

Gene Cloning of 5'-Xanthyl Acid Aminase

Brevibacterium ammonia genes D 1550-40 strain incubated for 14 to 15 hours in C medium containing adenine and guanine was inoculated in 1 L of the same medium so that absorbance (600 nm) was 0.1 or less, followed by shaking culture at 32 ° C. Then, when the absorbance reached 0.3, penicillin G was added to 0.3 U / ml, and cultured until the absorbance reached 0.6, and then used for transformation. The cells were recovered by centrifugation and then once with 1 L of 1 mM N- (2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid) buffer and once with 500 ml of cooled sterile deionized distilled water. After washing twice with 20 ml of 10% glycerol solution, it was suspended and used in 2-3 ml of 10% glycerol solution. This suspension can be divided into suitable portions and rapidly frozen in dry ice and stored at -70 ° C for use for about one month without decreasing the frequency of transformation. After slowly dissolving the frozen cell suspension in ice, add an appropriate amount of gene library DNA to it, mix it well, apply the electric field shock with a field shock device (Bio-Rad, Gene Pulser), and then suspend it in 1 ml C medium. After 1 hour incubation at 32 ℃ washed with sterile physiological saline and then plated on at least agar plate medium of the following composition containing 100mg / L adenine and 50μg / ml kanamycin and cultured for 1 to 5 days transformants Could get

* Minimum agar plate medium: Glucose 10g / ℓ, Ammonium sulfate 2g / ℓ, Urea 2g / ℓ, Potassium dihydrogen phosphate 0.2g / ℓ, Potassium dihydrogen phosphate 0.2g / ℓ, Magnesium sulfate 0.1g / ℓ, Calcium chloride 0.1 g / l, d-biotin 100μg / l, thiamine hydrochloride 100μg / l, borax 88mg / l, ammonium molybdate 36mg / l, zinc sulfate 8.8mg / l, copper sulfate 270mg / l, manganese chloride 7.7mg / l, chloride Ferrous 970 mg / l, agar 20 g / l; pH 7.2

Example 2

Expression of 5'-Xanthyl Acid Aminase in Brevibacterium Transformants

Plasmid DNA was isolated from the transformants obtained in Example 1 by a conventional method to obtain plasmid pGH 16 containing 6.5 kb of chromosomal DNA. The recombinant plasmid was converted into Brevibacterium ammonia genes ATCC 6872 and BA 17-2 and D 1550-40 strains were transformed to determine the enzymatic activity of 5′-xanthyl acid aminase of the transformants. To obtain the enzyme solution, the transformants were grown from C medium (if necessary, 50 μg / ml of kanamycin and small amounts of adenine and guanine) were grown to logarithmic growth, followed by centrifugation to recover the cells and an appropriate amount of 0.1M Tris buffer (pH7). The supernatant obtained by suspending in .3) and pulverizing the cells with an ultrasonic cell grinding machine (ultrasonicator) and centrifuging was used as the enzyme solution. An appropriate amount of the enzyme solution thus obtained was added to 2.4 ml of 5'-xanthyl acid aminase reaction solution (0.16 M Tris (ph 7.6), 4 mM ATP, 16 mM magnesium chloride, 25 mM XMP, 0.33 m ammonium sulfate) After reacting for 30 minutes to 1 hour, 30 ml of 3.5% trichloroacetic acid was added and centrifuged to measure the absorbance of the supernatant at 290 nm. As a result, the titer of 5'-xanthyl acid aminase was not expressed in D 1550-40 strain, whereas the titer of 5'-xanthyl acid aminase was expressed in transformant D 1550-40 / pGH 16. In the case of ATCC 6872 / pGH 16 and BA 17-2 / pGH 16, the specific enzyme activity of 5'-xanthyl acid aminase was increased by 14 and 10 times, respectively. , Detailed results are listed in Table 2.

* The unit (U) of enzyme activity was expressed as an increase in absorbance at 290 nm per unit time after the reaction.

Example 3

Expression of 5'-xanthyl acid aminase in transformants of the genus Corynebacterium and Escherichia

The transformants Corynebacterium glutamicum ATCC 13032 / pGH 16 and Escherichia coli W 3110 / pGH 16 obtained by transforming the recombinant plasmid pGH 16 were prepared in the same manner as in Example 1 to obtain 5'-xanthyl acid aminase. Enzyme titer analysis was performed. As a result, as shown in Table 1, it was expressed in strains of Corynebacterium and E. coli, and the non-enzyme titers were confirmed to be increased by about 5 and 2 times, respectively.

Example 4

Conversion of XMP to GMP by Transformants

C medium was used as a seed culture medium, and 30 ml each of 500 ml shaking Erlenmeyer flasks was autoclaved at 120 ° C. for 15 minutes, and 16 cells of transformed BA 17-2 / pGH were inoculated with platinum for 24 hours at 30 ° C. The culture was used as the seed culture solution. In addition, 1.5 liter of the fermentation broth was added to a 5 L fermenter, followed by autoclaving at 120 ° C. for 30 minutes, and the seed culture solution was inoculated at a 2% capacity and incubated at 30 ° C. and pH 7.0 for 3 days. At this time, the rotation speed was 750rpm, the air addition speed was 1vvm. XMP was added at a final concentration of 20 mg / ml to the enzyme-containing culture medium, and the reaction was carried out for 24 hours while maintaining the pH at 7.0 to 8.0 at 35 to 50 ° C. At this time, the parent strain BA 17-2 was performed as a control. . The start of the conversion reaction was started by adding an appropriate amount of surfactant (SDS, Hyamine, etc.). The conversion rate of GMP over time during the reaction is shown in Table 3. As shown in Table 4, the rate of the conversion reaction increased about three times and the conversion rate also improved from 67% to 50%.

* Fermentation medium: 100g / l glucose, 10g / l potassium phosphate 1,2g, 10g / l magnesium sulfate, 0.1g / l calcium chloride, 10mg / l iron sulfate, 2mg / l manganese sulfate, 10g / yeast extract l, casein hydrolyzate 10 g / l, d-biotin 30 μg / l, adenine 10 mg / l, pH 8.5

Claims (1)

Coryneform glutamate producing microorganisms containing the 5'-xanthylate aminase gene cloned from Brevibacterium ammonia genes and transformed with the expression vehicle pGH 16 that are cloned and expressed in coryneform glutamate producing bacteria and E. coli Non-bacterium ammonia genes ATCC 6872 / pGH 16- phosphorus KCCM-10008. 2. Coryneform glutamate producing microorganisms containing the 5'-xanthyl acid aminase gene cloned from Brevibacterium ammonia genes and transformed with the expression vehicle pGH 16 which can be cloned and expressed in coryneform glutamate producing bacteria and E. coli. -Brevibacterium ammonia gene ATCC 6872 / pGH 16- KCCM-10008 incubated in a culture medium, converting XMP to GMP, and the method of producing GMP, characterized in that to recover GMP therefrom.