Microorganisms with improved glycine-producing ability and method for producing glycine using the same

Abstract

The present invention relates to a Corynebacterium mutant strain that has improved glycine-producing ability compared to a parent strain through mutagenesis using irradiation, a method for producing the strain, and a method for producing glycine using the strain.The Corynebacterium mutant strain has improved glycine-producing ability compared to a parent strain, and glycine can be produced with high efficiency and high yield using the strain.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2023-0037523, filed March 22, 2023, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a microorganism having improved glycine-producing ability and a method for producing glycine using the same. [Background technology]

[0003] Glycine is an important amino acid with a wide range of applications in fields such as chemistry, food, and medicine. Glycine is an amino acid that imparts a mild sweet taste and is used as a flavoring agent and nutritional supplement, particularly in the food industry. The global glycine market is valued at over USD 700 million as of 2020 and is expected to grow at an annual rate of approximately 4%.

[0004] Glycine is generally obtained by chemical synthesis or protein hydrolysis. Chemical synthesis is carried out through the reaction of chloroacetic acid, trichloroethylene, tetrachloroethane, and ammonia, or through the Strecker synthesis, while protein hydrolysis is not widely used industrially. Most industrial production is carried out through chemical synthesis, which requires high-temperature / high-pressure processes and has the disadvantage of using substrates that are toxic to the human body.

[0005] Microbial glycine fermentation can be performed at room temperature and atmospheric pressure, and offers a process that utilizes a safe carbon source. In nature, microorganisms form glycine using the L-serine biosynthetic pathway or the L-threonine catabolic pathway. However, glycine production using naturally occurring strains is limited by low production concentrations.

[0006] Therefore, efforts are being made to develop highly efficient glycine-producing microorganisms and fermentation process technologies. Specifically, a method for enhancing the activity of enzymes involved in the amino acid synthesis pathway in Corynebacterium microorganisms (US 11661616 B2) has been utilized. However, there is still a growing need for research into methods that can produce glycine efficiently and with high yields.

[0007] Under these circumstances, the present inventors have made extensive efforts to develop a glycine-producing microorganism having a higher production efficiency than conventional strains, and as a result have discovered that a specific Corynebacterium mutant strain produces glycine in a high yield, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] One example of the present invention provides a mutant strain of the genus Corynebacterium obtained by mutating the parent strain Corynebacterium glutamicum ATCC13032, which has improved glycine production ability compared to the parent strain.

[0009] Another example of the present invention is 1) irradiating the parent strain Corynebacterium glutamicum ATCC 13032 to cause mutation; and 2) A method for producing a mutant strain having improved glycine productivity compared to a parent strain, comprising culturing the mutated strain of step 1) in a medium and selecting a mutant strain having improved glycine productivity.

[0010] Another embodiment of the present invention provides a composition for producing glycine, comprising the microorganism having improved glycine-producing ability.

[0011] Another embodiment of the present invention provides a method for producing glycine, comprising culturing the microorganism having improved glycine-producing ability in a medium.

[0012] Another embodiment of the present invention provides a use of the microorganism having improved glycine-producing ability for producing glycine. [Means for solving the problem]

[0013] This will be described in detail below. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below should not be construed as limiting the scope of this application. Furthermore, those skilled in the art will be able to recognize or ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of this application described herein. Furthermore, such equivalents are intended to be encompassed by this application. Furthermore, such equivalents are intended to be encompassed by this application.

[0014] The present invention provides a microorganism with improved glycine-producing ability.

[0015] In this study, we conducted screening to identify mutant strains that produce high levels of glycine. As a result, we confirmed that three mutant strains produced by irradiating Corynebacterium glutamicum ATCC13032 strain with radiation showed improved glycine productivity compared to ATCC13032 strain.

[0016] The present invention provides a mutant strain of the genus Corynebacterium obtained by mutating a parent strain, Corynebacterium glutamicum ATCC13032, and having improved glycine productivity compared to the parent strain.

[0017] The microorganism may be a mutant strain of Corynebacterium glutamicum.

[0018] The Corynebacterium glutamicum mutant strain may be any one strain selected from the group consisting of Corynebacterium glutamicum CJ2004 strain (accession number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (accession number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (accession number KCCM13319P).

[0019] As used herein, the term "glycine" refers to a colorless, crystalline amino acid that has a sweet taste, also known as glycine, and has the chemical formula "C2H5NO2."

[0020] As used herein, the term "production ability" refers to the ability to produce glycine and may be used interchangeably with "productivity." This can be confirmed by culturing a glycine-producing strain and then measuring the concentration of glycine in the culture medium.

[0021] The mutation can be carried out by various means known in the art, including physical or chemical mutagenesis. For example, a physical mutation suitable for the present invention can be irradiated with gamma rays or ultraviolet light, but is not limited thereto. Chemical mutations can be carried out using, but are not limited to, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethyl methanesulfonate, mustard compounds, hydrazine, and nitrous acid.

[0022] The mutation to produce the mutant strain can be by irradiation.

[0023] The radiation for inducing the mutation may be gamma rays or ultraviolet rays, preferably gamma rays.

[0024] The radiation may be administered at 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 per hour, but is not limited thereto.

[0025] The mutant strain may have a glycine productivity that is increased by 20% or more, 22% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more compared to the parent strain.

[0026] The parent strain may be, but is not limited to, Corynebacterium glutamicum ATCC13032.

[0027] The present invention also provides 1) irradiating the parent strain Corynebacterium glutamicum ATCC 13032 to cause mutation; and 2) A method for producing a mutant strain of Corynebacterium sp. having improved glycine production ability compared to the parent strain, comprising the steps of culturing the mutated strain of step 1) in a medium and selecting a mutant strain having improved glycine production ability.

[0028] The radiation may be gamma rays or ultraviolet rays, preferably gamma rays.

[0029] The radiation may be administered at 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 per hour, but is not limited thereto.

[0030] During mutagenesis, the parent strain is affected by a mutagen under conditions sufficient to leave surviving bacteria of a specific size. These conditions vary depending on the type of mutagen and depend on the amount of mutations the mutagen induces in surviving bacteria at a certain mortality rate (kill rate). For example, in the case of gamma rays, the kill rate can be approximately 0.00001% to 20% of the starting population; in the case of NTG, the kill rate can be approximately 10% to 50% of the starting population; and in the case of mutations induced by nitrite, the kill rate can be approximately 0.01% to 0.1% of the starting population, but these are not intended to be limiting.

[0031] The present invention also provides a composition for producing glycine, comprising a mutant strain of the genus Corynebacterium obtained by mutating a parent strain, Corynebacterium glutamicum ATCC13032, and having improved glycine production ability compared to the parent strain.

[0032] The microorganism may be a mutant strain of Corynebacterium glutamicum.

[0033] The Corynebacterium glutamicum mutant strain may be any one strain selected from the group consisting of Corynebacterium glutamicum CJ2004 strain (accession number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (accession number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (accession number KCCM13319P).

[0034] In one embodiment of the present invention, mutant strains obtained by mutating the parent strain Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum CJ2004 (Accession No. KCCM13317P), Corynebacterium glutamicum CJ2029 (Accession No. KCCM13318P), and Corynebacterium glutamicum CJ2049 (Accession No. KCCM13319P), were confirmed to produce high concentrations of glycine. Therefore, high concentrations of glycine can be produced using compositions containing these mutant strains.

[0035] The present invention also provides a method for producing glycine, which includes culturing in a medium a mutant strain of the genus Corynebacterium, which is obtained by mutating a parent strain, Corynebacterium glutamicum ATCC13032, and has improved glycine-producing ability compared to the parent strain.

[0036] The method may further comprise, after the culturing step, recovering glycine from the cultured microorganism, the culture, or both.

[0037] As used herein, the term "culturing" refers to growing a microorganism under appropriate artificially controlled environmental conditions. The method for culturing the microorganism of the present invention can be carried out using a culture method for Corynebacterium glutamicum that is widely known in the art. Specifically, examples of the culture method include, but are not limited to, batch culture, continuous culture, and fed-batch culture. Such various methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

[0038] As used herein, the term "culture" refers to a substance containing a medium in which microorganisms are growing or have completed growing under appropriately artificially controlled environmental conditions. In a narrow sense, the term "culture" does not include the grown microorganisms, but in a broad sense it can be included. The "culture" includes medium components prepared for microbial culture as well as various substances released into the medium by the microorganisms during growth, specifically the target substance glycine.

[0039] The culture medium used for cultivation should be appropriately tailored to meet the requirements of the specific strain. Culture media for Corynebacterium strains are well known. For example, the microorganisms of the present application can be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. under aerobic conditions while controlling temperature and pH. Carbon sources include carbohydrates such as glucose, fructose, and sucrose, and amino acids such as glutamic acid and cysteine. Natural organic nutrient sources such as starch hydrolysates and molasses can be used, with carbohydrates such as glucose, fructose, and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) being preferred. Various other carbon sources can be used in appropriate amounts without limitation, but are not limited to these. Nitrogen sources include inorganic nitrogen sources such as ammonia; amino acids such as glutamic acid and cysteine; and organic nitrogen sources such as peptone, meat extract, and yeast extract. These nitrogen sources can be used alone or in combination, but are not limited to these. The medium may contain, but is not limited to, phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt as a phosphorus source. Inorganic compounds may include magnesium sulfate, iron sulfate, manganese sulfate, calcium chloride, and other compounds, including amino acids, vitamins, and appropriate precursors. These media or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

[0040] During cultivation, compounds such as potassium hydroxide, ammonia, and phosphoric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. Additionally, antifoaming agents such as fatty acid polyglycol esters can be used to suppress foam formation during cultivation. Oxygen or oxygen-containing gas can be injected into the culture to maintain an aerobic state. The culture temperature is 27°C to 37°C, specifically 30°C to 33°C. The cultivation period can continue until the desired amount of useful substances is produced, specifically 20 to 120 hours.

[0041] The glycine recovery step can be performed by collecting the target glycine from the culture medium, culture solution, or microorganism using a suitable method known in the art depending on the culture method. For example, the recovery step can be performed by one or more methods selected from, but not limited to, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method for producing glycine can additionally include a purification step before, simultaneously with, or after the recovery step. [Effects of the Invention]

[0042] The Corynebacterium mutant strain of the present invention is a microorganism whose glycine-producing ability is improved by mutagenesis compared to that of a parent strain. Therefore, the method for producing glycine of the present invention using the Corynebacterium mutant strain can produce glycine with high efficiency and high yield. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention will be described in more detail below with reference to the following examples, but these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0044] Example 1. Selection of mutant strains through artificial mutation 1-1. Establishment of gamma-ray irradiation conditions to obtain mutant strains with improved glycine production To obtain a microbial mutant strain with improved glycine production ability, the following method was used to induce microbial mutation.

[0045] To induce mutations, gamma ray irradiation, a physical method, was used. High-energy gamma rays emitted from the Co-60 radiation source flow along the chromosome, randomly inducing changes in base sequences such as base substitutions, deletions, and insertions. Because the level of changes in base sequences is proportional to the intensity of gamma ray irradiation, high-level gamma rays induce a high rate of mutations while also increasing the mortality rate (killing rate) of the strain. In order to ensure a sufficient amount of high-quality mutant library with a high diversity of high mutation rates, we first established the gamma ray irradiation conditions for the corynebacterium strain to be irradiated.

[0046] Using Nordion's high-level gamma ray irradiation equipment installed at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute, 30 mL of seed medium culture solution with an absorbance of 6.09 at 562 nm was irradiated with gamma rays at intensities of 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 10 kGy / hr for 1 hour, and then 10 kGy / hr was added to the activation medium. 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 100 μl of gamma-irradiated solution diluted to the dilution ratio was smeared on each plate, and the plate medium on which the gamma-irradiated solution was smeared was cultured in a stationary incubator at 30°C for 48 hours, after which the number of individual colonies formed was counted and the mortality rate (killing rate) for each irradiation condition was calculated. The CFU / mL corresponding to the gamma-irradiation conditions of 0, 0.5, 1, 2, 3, 4, 5, and 7.510 kGy / hr was 3.6X10 8 , 6.9X10 7 , 9.8X10 6 , 2.7X10 5 , 2.1X10 3 , 3.0X10 1, 0, 0, 0, and the calculated mortality rates were 0%, 81.00467%, 97.31540%, 99.92589%, 99.99942%, 99.99999%, 100%, 100%, and 100%, respectively. Considering that gamma ray irradiation at a strength of 5 kGy / hr results in complete death and failure to secure a colony, a gamma ray irradiation strength of 4 kGy / hr was established as the gamma ray irradiation condition for securing a high-quality mutation library with high mutation diversity through gamma ray irradiation of a corynebacterium strain.

[0047] 1-2. Selection of mutant strains with improved glycine production ability To identify a microorganism with high glycine production, we constructed a gamma-irradiation-based mutation library for Corynebacterium glutamicum ATCC13032 and screened for high-glycine-producing mutants. For gamma irradiation of ATCC13032, 50 mL of seed medium was cultured in a 500 mL flask in a shaking incubator at 30°C for 24 hours to obtain a culture medium with an absorbance of 7.84 at 562 nm. The culture medium was then diluted with the seed medium to an absorbance of 6.00 at 562 nm. 30 mL of the diluted solution was irradiated with 4 kGy / hr of gamma rays for 1 hour using a high-level gamma-irradiation device installed at the Advanced Radiation Research Center of the Korea Atomic Energy Research Institute. Then, 100 μl of the irradiated stock solution was spread onto 100 90 x 15 mm Petri dishes containing the activation medium. The Petri dishes were then cultured in a stationary incubator at 30°C for 48 hours to obtain a total of approximately 40,000 individual colonies. To ensure the stability of the mutants, all individual colonies formed in all Petri dishes were collected and suspended in a 20% glycerol solution. The resulting suspension was then divided into 1 mL aliquots in 1.5 mL microtubes and stored in a deep freezer at -80°C.

[0048] In the screening of glycine hyper-producing mutants, a 20% glycerol suspension was taken out of the ultra-low temperature freezer and thawed at room temperature. -1 , 10 -2 , 10 -3, 10 -4 , 10 -5 After serial dilution, 100 μl of each dilution was plated onto activation medium and cultured in a stationary incubator at 30°C for 24 hours to reactivate individual colonies. The reactivated individual colonies were inoculated into 96 deep well plates containing 350 μl of production medium, equivalent to a filling rate of 17%, using a Molecular Devices Qpix420 colony picker. The ATCC13032 strain, which had not been gamma-irradiated, was inoculated into four wells per plate on the same plate to serve as a control for screening high-glycine-producing mutants.

[0049] The 96-deep-well plates inoculated with the control strain and mutants were sealed using an Azenta Gas Permeable Seal Mark 2 and then incubated for 48 hours at 30°C and 1,000 rpm in an Infors-HT Multitron shaking incubator. After 48 hours of incubation, the 96-deep-well plates were centrifuged for 20 minutes at 15°C and 4,000 rpm in an Eppendorf Centrifuge 5810R. Then, 100 μl of the culture supernatant containing the isolated bacterial cells was transferred to a Corning 96-well black polystyrene microplate using a Beckman Coulter Biomek i5 liquid handler for NIR spectrometry analysis. The Analysis & Quality Department of CJ CheilJedang's Bio Technology Research Institute then applied the NIR Spectrometry developed by the company to obtain individual analysis data for each well, and selected 48 strains from the 10,032 mutant strains by applying a selection logic that increased glycine concentration by 20% or more compared to the control group.The 48 selected strains were cultured in the same manner as above, and the top three strains with the highest glycine concentration were finally selected.

[0050] The mutant strains obtained in the above manner were named Corynebacterium glutamicum CJ2004, CJ2029, and CJ2049, and were deposited with the Korea Microorganism Collection, an international depository under the Budapest Treaty, on January 6, 2023, and assigned accession numbers KCCM13317P, KCCM13318P, and KCCM13319P, respectively.

[0051] The compositions of the media used in Examples 1 and 2 are as follows:

[0052] <Activation medium> Beef extract 5g / L, Polypeptone 10g / L, Yeast extract 5g / L, Urea 2g / L, Sodium chloride (NaCl) 2.5g / L, Agar 20g / L, Glucose 10g / L, 10N sodium hydroxide (NaOH) 100μl / L

[0053] <Seed medium> Glucose (anhydrous glucose) 20g / L, polypeptone 10g / L, yeast extract 10g / L, ammonium sulfate [(NH4)2SO4] 10g / L, urea 1.5g / L, potassium phosphate monobasic (KH2PO4) 5.2g / L, potassium phosphate dibasic (K2HPO4) 10.7g / L, d-biotin 1.8mg / L, thiamine-HCl 9mg / L, CAPA 9mg / L, NCA 60mg / L, magnesium sulfate (MgSO4) 0.5g / L

[0054] <Production medium> Calcium carbonate (CaCO3) 30g / L, sucrose 50g / L, MgSO4 0.6g / L, (NH4)2SO4 20g / L, KH2PO4 1g / L, yeast extract 5g / L, d-biotin 0.05mg / L, thiamine-HCl 0.1mg / L, MnSO4 18μg / L, FeSO4 18μg / L, ZnSO4 0.9μg / L, CuSO4 0.9μg / L

[0055] Example 2. Evaluation of glycine productivity of glycine-producing mutant strains To confirm the glycine productivity of the Corynebacterium glutamicum strains CJ2004, CJ2029 and CJ2049 obtained in Example 1, they were cultured as follows.

[0056] Specifically, the parent strain Corynebacterium glutamicum ATCC 13032 and the three mutant strains were inoculated into 250 ml corner baffle flasks containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm to obtain seed cultures. Then, 1 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 24 ml of the production medium and cultured at 30°C for 72 hours with shaking at 200 rpm to produce glycine. After the culture was completed, the glycine concentration in the culture medium was measured using high-performance liquid chromatography (HPLC) to determine the glycine production amount of each strain. The results are shown in Table 1 below.

[0057] [Table 1]

[0058] As a result, as shown in Table 1, the parent strain, Corynebacterium glutamicum ATCC13032, produced glycine at a concentration of 110 mg / L, while the mutant strains of Corynebacterium glutamicum CJ2004, CJ2029, and CJ2049 according to the present invention produced glycine at concentrations of 135 mg / L, 192 mg / L, and 138 mg / L, respectively, demonstrating an increase in glycine productivity of about 22% to 74% compared to the parent strain.

[0059] The above results indicate that the three mutant strains of Corynebacterium glutamicum according to the present invention are capable of producing glycine with high efficiency and high yield.

[0060] [Accession number] Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13317P Date of acceptance: 20230106 Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13318P Date of acceptance: 20230106 Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM133179P Date of acceptance: 20230106 [Table 2] [Table 3] [Table 4]

Claims

1. A mutant strain of the genus Corynebacterium obtained by mutating a parent strain, Corynebacterium glutamicum ATCC13032, has improved glycine productivity compared to the parent strain.

2. The mutant strain of the genus Corynebacterium according to claim 1, wherein the mutant strain of the genus Corynebacterium is Corynebacterium glutamicum.

3. 2. The Corynebacterium mutant strain according to claim 1, wherein the Corynebacterium mutant strain is any one selected from the group consisting of Corynebacterium glutamicum CJ2004 strain (accession number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (accession number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (accession number KCCM13319P).

4. 2. The Corynebacterium mutant according to claim 1, wherein the mutant has glycine productivity improved by 20% or more compared to the parent strain, Corynebacterium glutamicum ATCC13032.

5. A composition for producing glycine, comprising the Corynebacterium mutant strain according to claim 1.

6. 6. The composition for producing glycine according to claim 5, wherein the Corynebacterium mutant strain is any one selected from the group consisting of Corynebacterium glutamicum CJ2004 strain (accession number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (accession number KCCM133178P), and Corynebacterium glutamicum CJ2049 strain (accession number KCCM13319P).

7. A method for producing glycine, comprising culturing the Corynebacterium mutant strain of claim 1 in a medium.

8. 8. The method for producing glycine according to claim 7, wherein the Corynebacterium mutant strain is any one selected from the group consisting of Corynebacterium glutamicum CJ2004 strain (accession number KCCM13317P), Corynebacterium glutamicum CJ2029 strain (accession number KCCM13318P), and Corynebacterium glutamicum CJ2049 strain (accession number KCCM13319P).

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