KR100686561B1 - Corynebacterium ammoniagenes cjxcv19 kccm-10692p having low activity of degradation of guanine 5'- monophosphate and high-throughput screening method for mutant - Google Patents

Abstract

A microorganism Corynebacterium ammoniagenes CJXCV19(KCCM-10692P) having low activity of degradation of guanine 5'-monophosphate(GMP) and a high-throughput screening method of the same microorganism are provided to reduce screening time, and produce guanine 5'-monophosphate(GMP) with higher yield from XMP(5'-xanthylic acid). The high-throughput screening method of a mutant of Corynebacterium ammoniagenes CJFXT0301(KCCM-10530) comprises the steps of: culturing the mutants of Corynebacterium ammoniagenes CJFXT0301(KCCM-10530) in a mini-well; inducing conversion reaction of the cultured mutants of Corynebacterium ammoniagenes CJFXT0301(KCCM-10530) mutant into guanine monophosphate(GMP); measuring the concentration of guanine monophosphate(GMP) in the conversion reaction and selecting the mutant showing absorbance of 0.8 at 290 nm when a parent strain shows absorbance of 0.8 or more at 290 nm, wherein the mutant of Corynebacterium ammoniagenes CJFXT0301(KCCM-10530) is Corynebacterium ammoniagenes CJXCV19(KCCM-10692P) which produces XMP(5'-xanthylic acid) and converts it into guanine 5'-monophosphate(GMP) with higher conversion rate.

Description

Corynebacterium ammoniagenes CJXCV19 KCCM-10692P having low activity of degradation of guanine 5'- monophosphate and high-throughput screening method for mutant}

1 is a diagram showing the results of measuring the cell concentration at the absorbance of 526nm using a multi-well reader after mini-scale culture of Corynebacterium ammonia genes wild wine,

Figure 2 is a diagram showing the results of measuring the cell concentration at the absorbance of 526nm using a multi-well reader after small-scale culture of Corynebacterium ammonia genes CJXFT0301 KCCM-10530 strain,

3 is a diagram showing the results of 5'-guanylic acid (GMP) degradation or conversion reaction in a small scale.

The present invention is a high-speed screening method of mutant strains having GMP low degradation or low conversion activity among the mutants causing mutations in microorganisms, mutant strain Corynebacterium ammonia genes selected by the method CJXCV19 KCCM-10692P and GMP production using the same It is about a method.

5'-Guanylic Acid (GMP), along with 5'-Inosine acid (inosine 5'-monophosphate, IMP), is widely used as a food seasoning additive. 5'-Guanylic acid is known to taste mushrooms by itself, but is primarily known to enhance the flavor of monosodium-glutamine acid (MSG). This property is particularly strong when used with 5'-inosinic acid (IMP).

Known methods for producing 5'-guanylic acid are firstly a method of enzymatically degrading ribonucleic acid (RNA) extracted from yeast cells, and secondly, a method of directly fermenting 5'-guanylic acid (GMP) by microbial fermentation. Chemical phosphorylation of guanosine produced by microbial fermentation method; fourth, phosphorylation of guanosine produced by microbial fermentation method; and fifth, 5'-xanthyl acid (XMP) produced by microbial fermentation method The method of converting to 5'-guanylic acid using the 6th method and the method of converting 5'-xanthyl acid (XMP) produced by the microbial fermentation method to 5'-guanylic acid using E. coli are mentioned. Among the above methods, the first method has a problem in supply and economics of raw materials, and the second method has a disadvantage in that the yield is low due to the problem of 5'-guanylic acid (GMP) cell membrane permeability. It is becoming. In particular, a sixth method, 5'-xanthyl acid (XMP), has been widely used.

5'-Xanthyl acid (XMP) is produced using Corynebacterium ammonia genes and 5'-xanthyl acid (XMP) is converted to 5'-guanylic acid (GMP) by adding XMP aminase from E. coli. It is a one-step reaction to produce GMP by aging, but at the same time, it consumes ATP to produce AMP and pyrophosphate (PPi). Corynebacterium ammonia genes have the activity of synthesizing ATP using glucose, so by setting appropriate conversion reaction conditions, ATP synthesis and XMP aminase reaction occur simultaneously. The method using the cells themselves as an enzyme source has the advantage of supplying a large number of enzyme groups, but at the same time has the disadvantage that undesirable side reactions occur.

Side reactions that are problematic in the conversion to 5′-guanylic acid (GMP) include degradation of the product or conversion to other substances. In order to increase the conversion of the reaction, it is necessary to develop a strain with or without weakening or conversion of 5′-guanylic acid (GMP) produced while maintaining ATP regeneration.

Fast screening is a method of screening for having a desired trait among thousands of mutants in a short time, and an appropriate screening method must be performed to accomplish this. The general mutant screening method developed until now has been subjected to primary screening through mutant strains surviving on a solid medium containing a specific medicine (analogue), followed by incubation using a triangular flask and analysis. This method requires a lot of time and labor because of the culture and analysis using the Erlenmeyer flask, and it was difficult to analyze all the numerous variants.

Therefore, there was a need for a method of culturing and analyzing thousands of many variants at once, one of which is using a mini-well. This is because a small volume can cultivate a large number of cells and can be simultaneously analyzed by an ELISA reader. To this end, suitable culture conditions must be established so that cells can grow well in small volumes, and at the same time, a screening method capable of distinguishing characteristics is required.

Accordingly, the present inventors established a high-speed screening method, and by using the same, by improving the existing Corynebacterium ammonia genes CJXFT0301 KCCM-10530, 5'-guanylic acid (GMP) is a trait that can be degraded or converted low The present invention was completed by identifying mutants with significantly increased productivity of 5'-guanylic acid (GMP).

It is an object of the present invention to provide Corynebacterium ammonia genes CJXCV19 KCCM-10692P, which is a mutant of Corynebacterium ammonia genes CJFXT0301 KCCM 10530. In addition, to provide a high-speed screening method of mutant strain inhibited degradation or conversion activity of GMP.

In order to achieve the above object, the present invention comprises a first step of causing microbial mutation; A second step of culturing the mutant induced by the step 1 mutation in a mini-well; A third step of inducing GMP degradation and conversion of the mutants cultured in the mini-well; It provides a high-speed selection method of mutant strains having a low GMP degradation or low conversion activity characterized in that the fourth step of measuring the amount of GMP reduction in the conversion reaction conditions of the third step.

In addition, the present invention is a variant of Corynebacterium ammonia genes CJFXT0301 KCCM-10530, Corynebacterium ammonia genes CJXCV19 KCCM-10692P having GMP low degradation or low conversion activity selected by the high-speed screening method of claim 1 To provide.

It provides a method for producing GMP by using the Corynebacterium ammonia genes CJXCV19 KCCM-10692P.

Hereinafter, the present invention will be described in detail.

The present invention comprises a first step of causing microbial mutations in order to rapidly select mutants having low degradation or low conversion activity of GMP; A second step of culturing the mutant induced by the step 1 mutation in a mini-well; A third step of inducing GMP degradation and conversion of the mutants cultured in the mini-well; It provides a high-speed selection method of mutant strain having a low GMP degradation or low conversion activity characterized in that the fourth step of measuring the amount of GMP reduction in the conversion reaction conditions of the third step.

In the first step of the high-speed screening method of the mutant strain, the microorganism may be applied to all microorganisms, preferably to microorganisms capable of producing XMP, more preferably to Corynebacterium ammonia genes, mutagenesis induced The use of a mutagenesis agent such as UV irradiation, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), and the like can cause variation in accordance with conventional methods. The variants mutated by the method may be used to select the variants securing the parent strain activity using a medium for selecting the parent strain to maintain the activity of the parent strain.

In the second step of the high-speed screening method of the mutant strain, the mutant strains selected in the first stage are incubated in a mini-well, and the culture conditions in the mini-well may be most varied through various combinations such as medium conditions, incubation time, inoculation method Appropriate conditions can be established and the best conditions can then be identified by checking the cell concentration (Abs.562 nm) and CV (coefficient of variance = dispersion / average * 100%) values.

In the case of the third step, the transformation reaction cultured in the mini-well is added to the reaction additive for the conversion reaction from XMP to GMP and GMP to perform the conversion reaction while stirring at 20-60 ℃, preferably 40-50 ℃ In the case of the fourth step, it is a method of rapidly selecting mutants having low GMP degradation or low conversion activity by measuring the amount of GMP reduction under GMP conversion reaction conditions.

In addition, the present invention using the high-speed selection method of the mutant strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530 strain as a parent strain Corynebacterium ammonia genes CJXCV19 KCCM- having low GMP degradation or low conversion activity Provide 10692P.

The Corynebacterium ammonia genes CJXFT0301 KCCM-10530 strain is a strain capable of producing XMP with high yield while retaining adenine requirement, guanine requirement, lysozyme sensitivity, proline analog resistance, glutamine analog resistance and tryptophan analog resistance. However, due to the low conversion rate from XMP to GMP, it is not suitable for GMP production.

Therefore, using the above-mentioned Corynebacterium ammonia genes CJXFT0301 KCCM-10530 strain as a parent strain, UV irradiation, a conventional method using a mutagenesis agent such as N-methyl-N`-nitro -N-nitrosoguanidine (NTG) According to the present invention provides a Corynebacterium ammonia gene CJXCV19 KCCM-10692P having a high conversion rate from XMP to GMP selected by the high-speed screening method of the present invention, and having a low degradation or low conversion activity of the resulting GMP.

The mutant of the present invention not only can maintain the activity of the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530 to produce XMP in high yield, but also has GMP low degradation or low conversion activity, and GMP in GMP It is a strain capable of producing GMP with high yield and high concentration due to improved conversion rate.

Accordingly, the present invention provides a GMP production method using a variant strain Corynebacterium ammonia genes CJXCV19 KCCM-10692P.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only examples of the present invention, and the present invention is not limited thereto.

Reference Example  One. Badge composition

1-1. Proline analogues and glutamine analogues added Minimum badge

Glucose 20g / L, Potassium phosphate 1g / L, Dipotassium phosphate 1g / L, Urea 2g / L, Ammonium sulfate 3g / L, Magnesium sulfate 1g / L, Calcium chloride 100mg / L, Iron sulfate 20mg / L, Sulfuric acid Manganese 10mg / L, Zinc sulfate 10mg / L, Biotin 30ug / L, Thiarate 0.1mg / L, Copper sulfate 0.8mg / L, Adenine 20mg / L, Guanine 20mg / L, Proline analog 100mg / L, Glutamine analog 100mg / L Formulated and adjusted to pH 7.2.

1-2. Mini-scale culture medium

10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2.5 g / L sodium chloride, 3 g / L urea, 200 mg / L adenine, 200 mg / L guanine, adjusted to pH 7.2.

1-3. 5’- In Xanthanic Acid (XMP)  5’- To guanylic acid (GMP)  Conversion reaction additive

Phytic acid (1.8 g / L), MgSO ₄ 4.8 g / L, Nimine (3 ml / L), adenine 100 mg / L, Na ₂ HPO ₄ 7.7 g / L, glucose 46 g / L.

Example  1. Preparation of Strains

Corynebacterium ammonia genes CJXFT0301 KCCM-10530 was prepared as a parent strain for the production of mutant strains.

Experimental Example  1. Establishment of small scale culture conditions

Medium conditions, incubation time, inoculation to establish conditions for normal culture of Corynebacterium ammonia genes wild strain and parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530 at a mini-scale Various combinations of methods and the like have established the most suitable conditions.

The wild line exhibited an average cell concentration (Abs.562 nm) of 0.29 diluted 50-fold after 2 days of culture and a standard deviation of 0.025. CV (coefficient of variance = variance / average * 100%) value, which is an index for measuring the validity as a high-speed screening method, was found to be 8.9% and was a suitable condition (see FIG. 1).

In addition, in the case of the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530, the average cell growth concentration (Abs.562nm) 0.26, standard deviation 0.020, And CV value was confirmed to represent 7.7% (see Fig. 2).

Thus, culture conditions at small scale suitable for both wild and mutant strains could be established.

Experimental Example  2. Establishment of conditions for high-speed screening for accurate experiments and measurement of 5′-guanylic acid degradation or conversion reactions

Strains with different characteristics were performed to find the conditions of high-speed screening for accurate experiments and measurement of degradation or conversion reactions of 5′-guanylic acid at small scale. Corynebacterium ammonia genes wild and Corynebacterium ammonia genes CJXFT0301 KCCM-10530 and 5'-guanylic acid free control was selected.

A total of three strains were stirred at 42 ° C. with the reaction additive of Reference Example 1-3 and 5′-guanylic acid (GMP), which are conversion conditions at small scale. We calculated the time with the largest difference between the three types using time as a variable. After the reaction, the cells were precipitated, the supernatant was taken 50 times, diluted, and the change amount of 5′-guanylic acid (GMP) was measured using a multi-well reader (Abs. 290 nm).

As a result of the experiment, it was possible to establish a clear condition between the wild and mutant strains (see FIG. 3). Compared with wild wines, the parent strain, Corynebacterium ammonia genes CJXFT0301 KCCM-10530, has already reduced the 5'-guanylic acid (GMP) degradation ability, but the difference was about 3 times, but the degradation activity was still higher than that of the control group without degradation. Many remained. Increasing the reaction time and dilution factor was found to show a larger difference.

Experimental Example  3. Variation Corynebacterium Ammonia Genes CJXCV19  making

Corynebacterium ammonia genes CJXFT0301 KCCM-10530 prepared in Example 1 to obtain a 5'-guanylic acid (GMP) low degradation or low conversion variants as a parent strain, N-methyl -N`- which is a mutagenesis agent Variant libraries were prepared according to conventional methods using nitro-N-nitrosoguanidine (NTG).

The mutant library was plated in a minimal medium to which the proline and glutamine analogues of Reference Example 1-1, the parental strain selection medium, were added to secure colonies.

The obtained colonies were picked and transferred using Q-bot (Cat X8000) to transfer them to mini-wells (mini-well, 96well Deep Plate-Dome, Cat 90061, Bioneer, Inc.). Incubated for 48 hours in the culture medium. The culture is incubated with the evaporation, the temperature, the culture medium HT-MegaGrow ^TM stirring incubator (HT-MegaGrow ^TM Shaking Incubator, Cat. A-4080, Bioneer Co., Ltd.) to adjust the oxygen supply to the culture of the mutant safe.

Thereafter, the additive of Reference Example 1-3 and 5 g / L of GMP were added thereto, followed by stirring at 42 ° C. for 4 hours to perform a conversion reaction. In order to measure the concentration of GMP before and after the conversion reaction, the highest concentration after GMP reaction was selected by measuring the value at Abs.290 nm using a multi-well reader (Cat.MDK050315-1, Molecular Devices). .

When the parent strain showed a value of 0.6 at Abs.290 nm, the variants showing values of about 0.8 or more were selected. The selected variants were subjected to Erlenmeyer flask fermentation in the following manner.

5 ml of seed medium (30 g / L glucose, 15 g / L peptone, 15 g / L yeast extract, 2.5 g / L sodium chloride, 3 g / L urea, 150 mg / L adenine, guanine 150 mg / L, pH 7.2) is dispensed into an 18 mm diameter test tube After autoclaving according to the conventional method, the inoculated strain was used and the culture was shaken at 180 ° C. for 18 hours at 30 ° C. to be used as a seed culture solution. Main medium in fermentation medium (glucose 60g / L, magnesium sulfate 10g / L, iron sulfate 20mg / L, zinc sulfate 10mg / L, manganese sulfate 10mg / L, adenine 30mg / L, guanine 30mg / L, biotin 100ug / L, Copper sulfate 1mg / L, thiamine hydrochloride 5mg / L, calcium chloride 10mg / L, pH 7.2) and starch medium (10 g / L potassium phosphate, 10 g / L potassium phosphate, urea 7 g / L, ammonium sulfate 5 g / L) After autoclaving according to the conventional method, 29mL and 10mL were respectively dispensed into a 500mL shake-type Erlenmeyer flask which was autoclaved in advance, and 1mL of the seed culture solution was inoculated and then incubated for 90 hours. The rotation speed was adjusted to 200 rpm and temperature 30 degreeC.

After the Erlenmeyer flask culture, the concentration of 5'-xanthyl acid was maintained or high was finally selected and named Corynebacterium ammonia genes CJXCV19, which was deposited on November 9, 2005 at the Korea Microorganism Conservation Center and deposited. The number is KCCM-10692P.

After completion of the Erlenmeyer platter culture, the accumulation amount of 5'-xanthyl acid in the medium was 30.1 g / L in the case of the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530, and the variant strain Corynebacterium ammonia of the present invention. The case of Gennes CJXCV19 KCCM-10692P was confirmed to be 32.2g / L. (The cumulative concentration of 5'-xanthyl acid was expressed as 5'-sodium xanthate.7H2O.)

Experimental Example  4. 5’- To guanylic acid (GMP)  Conversion reaction

The conversion reaction to 5'-guanylic acid (GMP) was carried out in 40 mL of the 5'-xanthyl acid (XMP) Erlenmeyer flask fermentation broth of Experimental Example 3, E. coli fermentation broth producing 2.8 mL of the reaction additive of Reference Example 1-3 and XMP aminase 4.2. The addition of mL was carried out for 24 hours at 42 ℃, 170 rpm conversion.

As a result of the experiment, the parent strain Corynebacterium ammonia genes CJXFT0301 KCCM-10530 showed a conversion rate of 75% to 22.5 g / L of 5'-guanylic acid (GMP) in the culture medium, The mutant strain Corynebacterium ammonia angenes CJXCV19 KCCM-10692P was found to have a conversion rate of 81% at 26.1g / L.

As described above, the present invention provides a high-speed screening method of mutant strains having GMP low degradation or low conversion activity among the mutant strains causing microorganisms, and the strain strain Corynebacterium ammonia genes CJXCV19 selected by the method It provides KCCM-10692P and GMP production method using the same.

The high-speed screening method according to the present invention is a method that can easily and easily select a mutant strain having a characteristic to be selected by culturing a number of variants at a time and easy to analyze. In addition, Corynebacterium ammonia genes CJXCV19 KCCM-10692P selected by the above method possesses all of the characteristics of the parent strain Corynebacterium ammonia genes CJFXT0301 KCCM-10530, while producing high yield of XMP, GMP has a low degradation or low conversion activity, and the conversion rate from XMP to GMP is also improved and can be produced in high yield GMP. Therefore, the strain can be used to produce GMP with high yield and high concentration.

Claims (3)

Culturing the mutant of Corynebacterium ammoniagenes CJFXT0301 KCCM-10530 in a mini-well; Inducing a conversion of mutants cultured in the mini-well to 5'-guanylic acid (Guanine monophosphate (GMP)); Corynebacterium ammonia gene ( Coynebacterium ) characterized in that the step of measuring the concentration of 5'- guanylic acid in the conversion reaction step of selecting a mutant strain exhibiting a value of 0.8 or more when the absorbance of the parent strain at 290nm is 0.6 ammoniagenes ) CJFXT0301 KCCM-10530 High-speed screening method for mutant strains. Corynebacterium ammoniagenes CJFXT0301 KCCM-10530, a variant of Corynebacterium ammoniagenes that produces 5'-xanthyl acid and converts 5'-xanthyl acid to 5'-guanylic acid compared to the parent strain Corynebacterium ammoniagenes CJXCV19 KCCM-10692P. A method for producing 5'-guanylic acid using Corynebacterium ammoniagenes CJXCV19 KCCM-10692P.

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