Microorganism with improved glycine productivity and method for producing glycine using the same

Abstract

This invention relates to a Corynebacterium species mutant strain with improved glycine productivity compared to a parent strain, obtained by irradiation mutagenesis, a method for preparing the strain, and a method for producing glycine using the strain. This Corynebacterium species mutant strain exhibits improved glycine productivity compared to the parent strain, and therefore, when used, glycine can be produced efficiently and in high quantities.

Description

Microorganism with improved glycine productivity and method for preparing glycine using the same The present invention relates to a microorganism having improved glycine productivity and a method for producing glycine using the same. Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2023-0037523 (filed on March 22, 2023, and the entire content disclosed in the corresponding patent application document is incorporated herein by reference). Glycine is an important amino acid that is widely used in fields such as chemistry, food, medicine, etc. Glycine is an amino acid that produces a slight sweet taste, and it has been particularly used as a flavoring agent and nutritional supplement in the food industry. Based on 2020, it is estimated that the global glycine market is 70 million USD or higher, and it is expected to show an annual growth rate of about 4%. Generally, glycine is obtained by chemical synthesis or protein hydrolysis. Chemical synthesis is achieved through the reaction of chloroacetic acid, trichloroethylene, tetrachloroethane, and ammonia or Strecker synthesis, and protein hydrolysis is not commonly used industrially. Most industrial production has the following problems: it is completed by chemical synthesis, and high-temperature / high-pressure processes are necessary, and reactants that are toxic to the human body are used. Glycine fermentation by microorganisms is carried out under conditions of room temperature and atmospheric pressure, and a process using a safe carbon source is recommended. Naturally, microorganisms use the L-serine biosynthesis pathway or the L-threonine degradation pathway to form glycine. However, the production of glycine using natural strains is limited because of the low production concentration. Accordingly, efforts are being made to develop microorganisms or fermentation process technologies that produce glycine with high efficiency. In particular, methods such as enhancing the activity of enzymes involved in the amino acid synthesis pathway of Corynebacterium species microorganisms (US 11661616 B2) have been used. However, the need for research on methods that can produce glycine with high efficiency and high yield still exists. In this context, the inventors of the present invention have diligently developed a microorganism for producing glycine that has a higher production effect compared to conventional strains, and as a result, have found the fact that a specific Corynebacterium mutant strain produces glycine in high yield, thereby completing the present invention. Technical problem A specific example of the present invention provides a Corynebacterium species mutant strain having improved glycine productivity compared to a parent strain, which is a mutant strain obtained by mutating the parent strain Corynebacterium glutamicum ATCC13032 strain. Another specific example of the present invention provides a method for preparing a mutant strain having improved glycine productivity compared to a parental strain, which comprises: 1) irradiating the parental strain Corynebacterium glutamicum ATCC13032 with radiation to mutate it; and 2) culturing the mutant strain of step 1) in a medium to select a mutant strain having improved glycine productivity. Other specific examples of the present invention provide a composition for producing glycine, which comprises the microorganism having improved glycine productivity. Other specific examples of the present invention provide a method for producing glycine, which comprises culturing the microorganism having improved glycine productivity in a medium. Other specific examples of the present invention provide a use of the microorganism having improved glycine productivity, which is for glycine production. Technical solution This is explained in detail as follows. On the other hand, each description and specific example disclosed in this application can also be applied to each other description and specific example. In other words, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the scope of this application should not be regarded as limited by the specific descriptions below. In addition, those of ordinary skill in the art to which the invention pertains can identify or confirm many equivalents of the specific examples of this application described in this application only by using ordinary experiments. In addition, it is intended to incorporate these equivalents into this application. The present invention provides a microorganism having improved glycine productivity. In the present invention, screening was carried out to discover mutant strains highly productive of glycine. As a result, it was confirmed that 3 mutant strains prepared by irradiating the Corynebacterium glutamicum ATCC13032 strain with radiation have improved glycine productivity compared to the ATCC13032 strain. The present invention provides a mutant strain of the genus Corynebacterium having improved glycine productivity compared to a parental strain, which is a mutant strain obtained by mutating the parental strain Corynebacterium glutamicum ATCC13032. The microorganism can be a mutant strain of Corynebacterium glutamicum. The mutant strain of Corynebacterium glutamicum can be any one selected from the group consisting of: Corynebacterium glutamicum CJ2004 strain (deposit number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (deposit number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (deposit number KCCM13319P). In this description, the term "glycine" is an amino acid, which is a colorless crystal that produces a sweet taste and is named glycine, and refers to having the chemical formula "C 2 H 5 NO 2 amino acids of " In this description, the term "productivity" means an ability to produce glycine, and can be used interchangeably with "production", and can be confirmed by measuring the concentration of glycine contained in the culture solution after culturing a strain that produces glycine. The mutation method can be carried out by various methods known in the corresponding field, and a method of a physical or chemical mutagenesis method can be used. For example, as a physical mutation method applicable to the present invention, a method of irradiating gamma rays or ultraviolet rays can be used, but is not limited thereto. In addition, as a chemical mutation method, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethyl methanesulfonate, mustard compounds, hydrazine, and nitrite can be used, but are not limited thereto. The mutation for preparing the mutant strain can be by a method of irradiating radiation. The radiation used to cause the mutation can be gamma rays or ultraviolet rays, and preferably gamma rays. The radiation can have a dose of 3.0 kGy to 5.0 kGy, 3.1 kGy to 4.9 kGy, 3.2 kGy to 4.8 kGy, 3.3 kGy to 4.7 kGy, 3.4 kGy to 4.6 kGy, 3.5 kGy to 4.5 kGy, 3.6 kGy to 4.4 kGy, 3.7 kGy to 4.3 kGy, 3.8 kGy to 4.2 kGy, 3.9 kGy to 4.1 kGy per hour, and preferably 4.0 kGy, but is not limited thereto. The mutant strain can have an increased glycine productivity of 20% or higher compared to the parent strain, and can have an increase of 22% or higher thereof, and can have an increase of 25% or higher thereof, and can have an increase of 30% or higher thereof, and can have an increase of 40% or higher thereof, and can have an increase of 50% or higher thereof, and can have an increase of 60% or higher thereof, and can have an increase of 70% or higher thereof. The parent strain can be Corynebacterium glutamicum ATCC13032, but is not limited thereto. In addition, the present invention provides a method for preparing a mutant strain having improved glycine productivity compared to a parent strain, which comprises 1) irradiating the parent strain Corynebacterium glutamicum ATCC13032 with radiation to mutate it; and 2) culturing the mutant strain of step 1) in a medium to select a mutant strain having improved glycine productivity. The radiation used to cause the mutation can be gamma rays or ultraviolet rays, and preferably gamma rays. The radiation may have a dose of 3.0 kGy to 5.0 kGy, 3.1 kGy to 4.9 kGy, 3.2 kGy to 4.8 kGy, 3.3 kGy to 4.7 kGy, 3.4 kGy to 4.6 kGy, 3.5 kGy to 4.5 kGy, 3.6 kGy to 4.4 kGy, 3.7 kGy to 4.3 kGy, 3.8 kGy to 4.2 kGy, 3.9 kGy to 4.1 kGy, preferably 4.0 kGy, but not limited thereto. During the mutagenesis period, the parent strain is affected by mutagenic factors under the condition of leaving a certain scale of living organisms. These conditions vary based on the type of mutagenic factors and the amount of mutations caused by these mutagenic factors in living organisms at a certain mortality rate (kill rate). For example, in the case of gamma rays, the mortality rate may leave about 0.00001% to 20% of the starting population, and in the case of NTG, the kill rate may leave about 10% to 50% of the starting population, and the occurrence of mutations caused by nitrite may leave 0.01% to 0.1% of the starting population, but not limited thereto. In addition, the present invention provides a composition for producing glycine, which comprises a Corynebacterium species mutant strain having improved glycine productivity compared to the parent strain, and the mutant strain is obtained by mutating the parent strain Corynebacterium glutamicum ATCC13032. The microorganism may be a Corynebacterium glutamicum mutant strain. The Corynebacterium glutamicum mutant strain may be any one selected from the group consisting of: Corynebacterium glutamicum CJ2004 strain (deposit number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (deposit number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (deposit number KCCM13319P). In one specific example of the present invention, it was confirmed that Corynebacterium glutamicum CJ2004 strain (deposit number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (deposit number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (deposit number KCCM13319P) (which are obtained by mutating the parent strain Corynebacterium glutamicum ATCC13032) produce glycine at a high concentration. Therefore, the composition containing the mutant strain can produce glycine at a high concentration. In addition, the present invention provides a method for producing glycine, which comprises culturing a Corynebacterium species mutant strain having improved glycine productivity compared to the parent strain, and the Corynebacterium species mutant strain is obtained by mutating the parent strain Corynebacterium glutamicum ATCC13032. The method may further include recovering glycine from the cultured microorganism, culture product, or both after the culture. The term "culture" as used herein means growing a microorganism under appropriate artificially controlled environmental conditions. The method for culturing a microorganism of the present invention can be carried out using a culture method of Corynebacterium glutamicum widely known in the art. In particular, specific examples of the culture method include batch culture, continuous culture, and fed-batch culture, but are not limited thereto. These various methods are disclosed in, for example, "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp138-176, 1991), etc. The term "culture product" as used herein means a substance containing a culture medium in which a microorganism is growing or has grown under appropriate artificially controlled environmental conditions. Narrowly, these culture products may include the grown microorganisms, but broadly, they can be included. These "culture products" include various substances discharged into the culture medium created for culturing the microorganism together with the culture medium components during the growth period by the microorganism, and particularly the target substances, including glycine. The culture medium for culturing should meet the needs of a specific strain in an appropriate manner. Culture media for strains of the genus Corynebacterium are known. For example, the microorganism of the present application can be cultured in a common culture medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc. under aerobic conditions with adjustment of temperature, pH, etc. Then, as the carbon source, carbohydrates such as glucose, fructose, and sucrose, amino acids such as glutamic acid and cysteine, etc. are included. In particular, natural organic nutrients such as starch hydrolysates and molasses can be used, and preferably they can be carbohydrates such as glucose, fructose, sterilized pretreated molasses (i.e., molasses converted into reducing sugar), etc., and other carbon sources can be used in appropriate amounts differently without limitation, but are not limited thereto. As the nitrogen source, inorganic nitrogen sources such as ammonia and amino acids such as glutamic acid and cysteine, and organic nitrogen sources such as peptone, meat extract, yeast extract, etc. can be used. These nitrogen sources can be used alone or in combination, but are not limited thereto. In this culture medium, as the phosphorus source, phosphates, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate or corresponding nitrogen-containing salts can be used, but are not limited thereto. As inorganic compounds, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium chloride, etc. can be used, and in addition, amino acids, vitamins, and appropriate precursors, etc. can be included. These culture media or precursors can be added to the culture product in a batch or continuous manner, but are not limited thereto. During the cultivation period, the pH of the culture product can be adjusted by adding compounds such as potassium hydroxide, ammonia, and phosphate by an appropriate method. In addition, during the cultivation period, an antifoaming agent such as polyethylene glycol fatty acid ester can be used to suppress the generation of bubbles. In addition, in order to maintain the aerobic state of the culture product, oxygen or an oxygen-containing gas can be injected into the culture products. The temperature of the culture products can be 27°C to 37°C, particularly 30°C to 33°C. The cultivation period can continue until the desired useful substance in the desired production amount is obtained, and particularly it can be 20 to 120 hours. The recovered glycine can collect the target glycine from the culture medium, the culture solution, or the microorganism according to the cultivation method by an appropriate method known in the technical field to which the invention pertains. For example, the recovery can be carried out by a method selected from at least one of the following: centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc., but is not limited thereto. The method for producing glycine can further include purification before the recovery, simultaneously with the recovery, or after the recovery. Advantageous effects The mutant strain of the Corynebacterium species of the present invention is a microorganism having improved glycine productivity compared to the parent strain through mutagenesis. Accordingly, by the method for producing glycine using the mutant strain of the Corynebacterium species of the present invention, glycine can be produced with high efficiency and high yield. Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples. Examples 1. Selection of mutant strains by artificial mutagenesis method 1-1. Establishment of conditions for irradiating gamma rays to obtain mutant strains having improved glycine productivity In order to obtain a microbial mutant strain having improved glycine productivity, microbial mutagenesis was induced using the following method. For inducing mutagenesis, a physical method, the gamma ray irradiation method, was applied. High-energy gamma rays emitted from the radiation source Co-60 flow along the chromosome and randomly cause changes in the base sequence, such as base substitution, deletion, insertion, etc. The amount of change in the base sequence is proportional to the irradiation intensity of the gamma rays. Therefore, a high amount of gamma rays not only cause mutations at a high ratio, but also increase the mortality rate (kill rate) of the strains. In order to sufficiently obtain a high-quality mutant library with high diversity at a high mutation rate, the conditions for irradiating gamma rays for Corynebacterium strains (recipients of gamma ray irradiation) were established in advance. Using the high-dose gamma-ray irradiation device of Nordion Company located at the Advanced Radiation Technology Institute of the Korea Atomic Energy Research Institute, gamma rays with intensities of 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 kGy / hr were irradiated for 1 hour on a 30 mL inoculated culture medium with an absorbance value of 6.09 at 562 nm, and then 100 μl of the gamma-ray irradiated solution diluted at dilution ratios of 10 0 、10 -1 、10 -2 、10 -3 、10 -4 were respectively smeared on the activation medium, and the plate medium on which the gamma-ray irradiated solution was smeared was cultured in a 30 °C stationary incubator for 48 hours, and then the number of individual colonies produced was counted to calculate the mortality rate (kill rate) caused by each irradiation condition. Based on the CFU / mL of gamma-ray irradiation conditions of 0, 0.5, 1, 2, 3, 4, 5, 7.5, 10 kGy / hr, they were respectively 3.6 X 10 8 、6.9 X 10 7 、9.8 X 10 6 、2.7 X 10 5 、2.1 X 10 3 、3.0 X 10 1 、0、0、0, and the mortality rates calculated from them were 0%, 81.00467%, 97.31540%, 99.92589%, 99.99942%, 99.99999%, 100%, 100%, 100% respectively. Considering that no colonies could be obtained because all colonies were killed under the gamma-ray irradiation condition with an intensity of 5 kGy / hr, an intensity of 4 kGy / hr was established as the gamma-ray irradiation condition for obtaining a high-quality mutant library with high mutation diversity through gamma-ray irradiation of Corynebacterium strains. 1-2. Selection of mutant strains with improved glycine productivity To obtain a microorganism with high glycine productivity, for Corynebacterium glutamicum ATCC13032 strain, a mutant library based on gamma-ray irradiation was constructed and high glycine productivity mutants were screened. For gamma-ray irradiation of ATCC13032 strain, it was cultured in a 500 mL flask containing 50 mL of inoculum medium in a shaking incubator at 30 °C for 24 hours, and a cultured solution with an absorbance value of 7.84 at 562 nm was obtained, and then it was diluted so that the absorbance value at 562 nm using the inoculum medium was 6.00. Using a high-dose gamma-ray irradiation device located at the Advanced Radiation Technology Institute of the Korea Atomic Energy Research Institute, gamma rays at 4 kGy / hr were irradiated to 30 mL of the diluted solution for 1 hour, and then the crude solution of the irradiated solution was separated and 100 μl of each was spread on the activation medium of 100 90 X 15 mm Petri dishes. Then, the Petri dishes were cultured in a stationary incubator at 30 °C for 48 hours to obtain a total of about 40,000 individual colonies, and for the stability of the obtained mutants, all the individual colonies produced in all the Petri dishes were recovered, suspended in a 20% glycerol solution, and then in 1.5 mL microtubes at 1 mL per time, and stored in an ultra-low temperature freezer at -80 °C. When screening for high glycine productivity mutant strains, the 20% glycerol suspension taken out from the ultra-low temperature freezer and thawed at room temperature was continuously diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 using saline, and then the diluted solutions corresponding to each dilution factor were spread on the activation medium at 100 μl each, and they were reactivated in the form of individual colonies by culturing in a stationary incubator at 30 °C for 24 hours. The reactivated individual colonies were inoculated into a 96-deep well plate, where 350 μl of production medium corresponding to a filling rate of 17% per well was used, aliquots were taken using a Qpix420 device (a colony picker from Molecular Devices), and the non-gamma-ray irradiated ATCC13032 strain was inoculated into 4 wells per plate on the same plate and used as a control group to screen for high productivity mutants. The 96-deep well plates inoculated with the control strain and mutants were sealed with the breathable sealant mark2 from Azenta, and then cultured in a Multitron device (a shaking incubator from Infors-HT) at 30 °C and 1,000 rpm for 48 hours. The 96-deep well plates cultured for 48 hours were centrifuged in a 5810R device (a centrifuge from Eppendorf) at 15 °C and 4,000 rpm for 20 minutes. Then, for NIR spectroscopy analysis, using a Biomek i5 (a liquid handling device from Beckman Coulter), 100 μl of the cultured supernatant in which the microbial cells had been separated was transferred to a 96-well black polystyrene microplate from Corning. After that, individual analysis data for each well were obtained by applying the NIR spectroscopy method produced by the Analysis & Quality Department of CJ First Sugar Biotechnology Research Institute, and a selection logic with a 20% or higher improvement in glycine concentration compared to the control group was applied, and 48 strains were initially selected from 10,032 mutant strains. For the 48 selected strains, culturing was carried out by the same method as above, and finally, the best 3 strains with high glycine concentration were selected. The mutant strains obtained by this method were named Corynebacterium glutamicum CJ2004, CJ2029, and CJ2049, and were deposited at the Korean Culture Center of Microorganisms, an international depository institution under the Budapest Treaty, on January 6, 2023, and were given the deposit numbers KCCM13317P, KCCM13318P, and KCCM13319P, respectively. The compositions of the culture media used in Example 1 and Example 2 are as follows. < Activation medium > Beef extract 5 g / L, polypeptone 10 g / L, yeast extract 5 g / L, urea 2 g / L, sodium chloride (NaCl) 2.5 g / L, agar 20 g / L, glucose 10 g / L, 10N sodium hydroxide (NaOH) 100 μl / L < Seed culture medium > Glucose (anhydrous glucose) 20 g / L, polypeptone 10 g / L, yeast extract 10 g / L, ammonium sulfate ((NH 4 ) 2 SO 4) 10 g / L, urea 1.5 g / L, potassium dihydrogen phosphate (KH 2 PO 4 ) 5.2 g / L, dipotassium hydrogen phosphate (K 2 HPO 4 ) 10.7 g / L, d-biotin 1.8 mg / L, thiamine-HCl 9 mg / L, CAPA 9 mg / L, NCA 60 mg / L, magnesium sulfate (MgSO 4 ) 0.5 g / L < Production medium > Calcium carbonate (CaCO 3 ) 30 g / L, sucrose 50 g / L, MgSO 4 0.6 g / L, (NH 4 ) 2 SO 4 20 g / L, KH 2 PO 4 1 g / L, yeast extract 5 g / L, d-biotin 0.05 mg / L, thiamine-HCl 0.1 mg / L, MnSO 4 18 μg / L, FeSO 4 18 μg / L, ZnSO 4 0.9 μg / L, CuSO 4 0.9 μg / L Example 2. Evaluation of glycine productivity of mutant strains producing glycine To confirm the glycine productivity of Corynebacterium glutamicum CJ2004, CJ2029, and CJ2049 strains obtained in Example 1 above, they were cultured by the following method. Specifically, after inoculating Corynebacterium glutamicum ATCC13032 strain (which is the parental strain) and the three mutant strains into a 250 ml flask with a buffer at the corner containing 25 ml of the seed culture medium, they were cultured with shaking at 200 rpm at 30°C for 20 hours to obtain the cultured seed solution. Then, 1 ml of the cultured seed solution was inoculated into a 250 ml flask with a buffer at the corner containing 24 ml of the production culture medium, and they were cultured at 200 rpm at 30°C for 72 hours to produce glycine. After completion of the culture, the concentration of glycine contained in the cultured solution was measured using high performance liquid chromatography (HPLC) to measure the glycine production amount of each strain. The results obtained are shown in Table 1 below. Table 1 Comparison of glycine productivity of Corynebacterium glutamicum CJ2004 strain, CJ2029 strain and CJ2049 strain As a result, as shown in Table 1, it was confirmed that the parental strain Corynebacterium glutamicum ATCC13032 strain produced glycine at a concentration of 110 mg / L, while the mutant Corynebacterium glutamicum CJ2004 strain, CJ2029 strain and CJ2049 strain according to the present invention produced glycine at concentrations of 135 mg / L, 192 mg / L and 138 mg / L, respectively, and thus their glycine productivity increased by about 22% to 74% compared to the parental strain. This result means that the three strains of the Corynebacterium glutamicum mutant strain according to the present invention can produce glycine with high efficiency and high yield. None None Depositing institution name: Korean Culture Center of Microorganisms (KCCM) Deposit number: KCCM13317P Deposit date: 20230106 Depositing institution name: Korean Culture Center of Microorganisms (KCCM) Deposit number: KCCM13318P Deposit date: 20230106 Depositing institution name: Korean Culture Center of Microorganisms (KCCM) Deposit number: KCCM13319P Deposit date: 20230106

Claims

1. A strain of Corynebacterium glutamicum CJ2029, which is registered under the accession number KCCM13318P.

2. The strain of claim 1, wherein the strain has 40% or more of improved glycine productivity compared to the parent strain Corynebacterium glutamicum ATCC13032.

3. A composition for the production of glycine, comprising the strain as claimed in claim 1.

4. A method for producing glycine, comprising culturing the strain of claim 1 in a culture medium.