Microorganisms with increased glycine production ability and method for producing fermentation composition using the same

Abstract

A microorganism with increased glycine-producing ability and a method for producing a fermentation composition using the same are provided. [Solution] We provide a Corynebacterium microorganism in which the activity of ATP phosphoribosyltransferase (HisG) is enhanced, more specifically, in which a mutation is introduced into HisG to increase glycine production ability, a method for producing a fermentation composition containing glycine and glutamic acid using the same, and the fermentation composition.

Description

[Technical field]

[0001] The present application relates to a microorganism having increased glycine-producing ability and a method for producing a fermentation composition using the same, and more particularly to a Corynebacterium microorganism having increased glycine-producing ability by introducing a mutation into HisG, a method for producing a fermentation composition containing glycine and glutamic acid using the same, and the fermentation composition. [Background technology]

[0002] L-amino acids are the basic building blocks of proteins and are used as important raw materials for pharmaceuticals, food additives, animal feed, nutrients, insecticides, and fungicides. Among them, L-glutamic acid is a representative amino acid produced by fermentation, and has a unique taste (umami), making it one of the important amino acids that is widely used in the food industry, pharmaceuticals, and other animal feed fields. Glycine is mainly used as a seasoning in the food industry to add sweetness, and is used together with natural seasonings to enhance the taste. It is also used for antioxidant and buffering effects, and is used in medicine as infusions, antacids, comprehensive amino acid preparations, and nutritional supplements.

[0003] Conventional methods for producing the amino acids include fermentation methods using microorganisms such as Brevibacterium and Corynebacterium (Non-Patent Document 1), as well as Escherichia coli, Bacillus, Streptomyces, Penicillium, Klebsiella, Erwinia, and Pantoea (Patent Documents 1 and 2). Additionally, the amino acids are also produced industrially via synthesis methods such as the monochloroacetic acid method and the Strecker method.

[0004] In addition, various studies for efficient amino acid production, for example, efforts are being made to develop microorganisms and fermentation process technologies for highly efficient amino acid production. Specifically, target substance-specific approaches have been developed, such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains or removing genes unnecessary for amino acid biosynthesis (Patent Documents 3 and 4), and in addition to these methods, methods for removing genes not involved in amino acid production and methods for removing genes whose specific functions in amino acid production are unknown are also being utilized. However, there is still an emerging need for research into methods for efficient and high-yield production of amino acids. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 3,220,929 [Patent Document 2] U.S. Patent No. 6,682,912 [Patent Document 3] Korean Patent Registration No. 10-0924065 [Patent Document 4] Korean Patent Registration No. 10-1208480 [Patent Document 5] Korean Patent Registration No. 10-0292299 [Patent Document 6] International Patent Publication No. 2008-033001 [Non-patent literature]

[0006] [Non-Patent Document 1] Amino Acid Fermentation, Gakkai Shuppan Center: 195-215, 1986 [Non-Patent Document 2] Microb Biotechnol. 2013 Mar; 6(2):103-117. [Non-Patent Document 3] Karlin &Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873(1993) [Non-Patent Document 4] Methods Enzymol., 183, 63, 1990 [Non-Patent Document 5] Sambrook et al., supra, 9.50-9.51, 11.7-11.8 [Non-Patent Document 6] Appl. Microbiol. Biothcenol.,1999,52:541-545 Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have endeavored to develop a method for simultaneously producing multiple amino acids, and have confirmed that when HisG activity is enhanced in a glutamic acid-producing strain compared to a parent strain, the ability to produce glycine can be improved while maintaining the ability to produce glutamic acid, and have thus completed the present application. [Means for solving the problem]

[0008] One object of the present application is to develop a Corynebacterium tuberculosis strain having enhanced ATP phosphoribosyltransferase (HisG) activity and increased glycine production capacity. The present invention aims to provide a microorganism of the genus Cerium.

[0009] Another object of the present application is to provide a method for producing a fermentation composition containing glycine and glutamic acid, comprising the steps of culturing the Corynebacterium microorganism in a medium and fermenting the same.

[0010] Another object of the present application is to provide a fermented composition produced by said method. Effect of the Invention

[0011] The HisG mutation of the present application can be introduced into a microorganism to produce glutamic acid and glycine simultaneously, which can be useful for amino acid production. In addition, the amount of glutamic acid and glycine in the fermented product can be adjusted to improve the taste and palatability of the fermented product, and the fermented liquid and seasoning material products using the same can be applied. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, any combination of various elements disclosed in the present application belongs to the category of the present application. Also, the specific descriptions described below are not considered to limit the category of the present application.

[0013] In order to achieve the above object, one aspect of the present application provides a method for the preparation of a mitochondrial protein having enhanced activity of ATP phosphoribosyltransferase (HisG), A Corynebacterium sp. microorganism having increased glycine productivity is provided.

[0014] Specifically, the present invention relates to a Corynebacterium sp. microorganism having an increased ability to produce glycine, the microorganism including ATP phosphoribosyltransferase (HisG) in which the 233rd amino acid in the amino acid sequence represented by SEQ ID NO: 4 is replaced with histidine (H). Can provide things.

[0015] Specifically, the 233rd amino acid in the amino acid sequence shown in SEQ ID NO:4 is replaced with histidine (H) and the 235th amino acid is replaced with glutamine (Q). ) can be provided. In this application, "ATP phosphoribosyltransferase" ", also named "HisG", refers to an enzyme involved in the histidine synthesis pathway. The histidine synthesis pathway consists of a total of nine enzymes (HisG-HisE-HisI-HisA-HisH-HisB-HisC-HisN-HisD), of which HisG constitutes one step.

[0016] The HisG has been known to be involved in histidine synthesis, but its relationship with glycine synthesis has not been reported. The relationship between the two was not known and was first discovered by the present inventors. More specifically, the increase in the amount of glycine produced is due to the enhancement of HisG activity, and in particular, HisG is subject to feedback inhibition by the product histidine, but in the present application, a mutation that relieves histidine feedback inhibition is introduced, and the associated increase in the amount of glycine produced and the maintenance of the amount of glutamic acid produced are first discovered by the present inventors.

[0017] In the present application, "enhancing HisG activity" means that the activity of HisG enzyme is increased from the intrinsic activity, which is the activity state of the enzyme that Corynebacterium microorganism has in the natural state. Examples of methods for enhancing HisG activity include i) a method of increasing the copy number of the base sequence encoding the enzyme by further inserting a polynucleotide containing a base sequence encoding HisG into a chromosome or a method of introducing a polynucleotide containing a base sequence encoding HisG into a vector system, ii) a method of enhancing the promoter of the hisG gene, for example, a method of replacing it with a strong promoter or a method of introducing a mutation into the promoter, and iii) a method of mutating the gene to an enzyme with strong activity. Specifically, in the present application, the 233rd amino acid of the HisG amino acid sequence represented by SEQ ID NO: 4 may be substituted with histidine for glycine, or the 233rd amino acid of the HisG amino acid sequence represented by SEQ ID NO: 4 may be substituted with histidine and the 235th amino acid of the HisG sequence represented by SEQ ID NO: 4 may be substituted with glutamine for threonine. Accordingly, a Corynebacterium microorganism containing such a mutated HisG may have a significantly increased glycine producing ability while maintaining almost no effect on glutamic acid bioactivity. The increase in glycine producing ability may be increased compared to the variant of the present application, i.e., a microorganism having HisG without the substitution.

[0018] As another example, the promoter of the HisG enzyme may be mutated to a stronger promoter than the endogenous promoter through mutation or substitution. An improved promoter having a base substitution mutation or a heterologous promoter may be linked in place of the promoter of the endogenous enzyme, and examples of the heterologous promoter include, but are not limited to, the cj7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA promoter, and aceB promoter.

[0019] In addition, since the hisG gene is composed of an operon with the hisE gene, the activity of the HisG enzyme can be enhanced through overexpression of hisG due to mutation or substitution of the promoter sequence of the hisEG gene. More specifically, the mutation of the promoter sequence of the hisEG gene can be achieved by substituting the 53rd and 55th nucleotides of the polynucleotide sequence of SEQ ID NO: 1 with T, or by substituting the 53rd and 55th nucleotides of the polynucleotide sequence with T and the 60th nucleotide with G, thereby enhancing the activity of the HisG enzyme with a stronger promoter than the endogenous promoter. Looking at a research paper on the promoter sequence of Corynebacterium glutamicum (Non-Patent Document 2), a number of transcription factors were identified through RNA sequencing (RNA-seq). The location of the transcriptional start point (TSP) and promoter can be identified. Here, the RAN of the wild-type Corynebacterium glutamicum strain ATCC13869 was The promoter sequence of the hisEG gene was confirmed through a sequencing experiment, and the endogenous (native) HisEG overexpression was induced via promoter modification. One method of endogenous promoter modification is to approach the consensus sequence via mutation of the -35 and -10 regions of the promoter of Corynebacterium glutamicum. When the sequence of the −10 region (TATA box) of the G gene promoter is mutated to approach a consensus sequence, the promoter may be transformed into a stronger promoter than the endogenous promoter.

[0020] Specifically, the ATP phosphoribosyltransferase contained in the Corynebacterium microorganism may consist of the amino acid sequence of SEQ ID NO: 5, or the ATP phosphoribosyltransferase may consist of the amino acid sequence of SEQ ID NO: 6.

[0021] The amino acid sequences of the present application may be modified by conventional mutagenesis techniques, such as directed evolution and site-directed mutagenesis. The shape may be modified by any of the following methods.

[0022] Therefore, the ATP phosphoribosyltransferase may include HisG containing an amino acid sequence having at least 60% or more, specifically 70% or more, more specifically 80% or more, even more specifically 83% or more, 84% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 97% or more homology to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. As a sequence having homology with the sequence, an amino acid sequence having substantially the same or corresponding biological activity as the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, even if the sequence has an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added, it is self-evident that the present application includes such a category.

[0023] In this regard, the term "glutamic acid" (L-glutamate) is a type of amino acid and is classified as a non-essential amino acid. It is known as the most common excitatory neurotransmitter in the central nervous system and is also said to have a umami taste, and its monosodium salt (monosodium glutamate: MSG) has been developed and is widely used as a seasoning. It is generally produced through the fermentation of microorganisms that produce glutamic acid.

[0024] The term "glycine" is a colorless crystalline amino acid that has a sweet taste. It is also called glycine. It is mainly used as a food seasoning, and in medicine, it is also used as an infusion, antacid, comprehensive amino acid preparation, and nutritional supplement. It is generally produced by industrial synthesis methods such as the monochloroacetic acid method and the Strecker method, but the synthesis method requires optical resolution because D- and L-amino acids are mixed. Therefore, it is necessary to produce glycine by fermentation, which has various advantages such as mild reaction conditions, enabling mass production in a short time, environmental friendliness, and biodegradability of the produced substances.

[0025] In the present application, the term "homology" refers to the degree of identity with a given amino acid or nucleotide sequence, which may be expressed as a percentage. Herein, a homologous sequence having the same or similar activity as a given amino acid or nucleotide sequence is expressed as "% homology". Homology to said amino acid or nucleotide sequence can be determined, for example, using the literature algorithm BLAST [see Non-Patent Document 3] or Pears The determination may be made using FASTA by on (see Non-Patent Document 4). Based on these algorithms BLAST, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0026] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., Non-Patent Document 5). For example, highly homologous genes have a homology of 60% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly specifically 99% or more. or conditions for washing in a normal Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, The conditions may include washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 68°C, 0.1xSSC, 0.1% SDS. Hybridization requires that two nucleotides have complementary sequences, even if mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present application may also include isolated polynucleotide fragments that are complementary to the entire sequence, as well as substantially similar polynucleotide sequences.

[0027] Specifically, the homologous polynucleotides may be detected using the above-mentioned hybridization conditions including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and the variables are well known in the art (see Non-Patent Document 5).

[0028] In this application, the term "microorganism" refers to a concept that includes wild-type microorganisms and all microorganisms that have been genetically modified, either naturally or artificially, and that includes all microorganisms in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or weakening the activity of an endogenous gene.

[0029] In the present application, the microorganism may contain the ATP phosphoribosyltransferase. The ATP phosphoribosyltransferase may be transformed with a vector. Therefore, the HisG gene may be introduced into the microorganism, but is not limited thereto. In addition, as long as the microorganism can express the HisG gene, it does not matter whether the gene encoding the HisG gene is located on the chromosome or extrachromosomally.

[0030] In the present application, the term "vector" may refer to an artificial DNA molecule carrying genetic material capable of expressing a gene of interest in a compatible host, and may also refer to a DNA construct comprising the nucleotide sequence of the gene encoding HisG.

[0031] The vector used in the present application is not particularly limited as long as it can be expressed in a host cell, and any vector known in the art may be used to transform the host cell. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages, either naturally occurring or recombinant.

[0032] For example, pWE15, M13, λLB3, λBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET may be used as plasmid vectors.

[0033] Alternatively, the polynucleotide encoding HisG of the present application may be introduced into the chromosome of the host cell via a vector for chromosomal insertion, such as, but not limited to, pECCG117, pDZ, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, and pXMJ19 vectors.

[0034] Alternatively, insertion of the polynucleotide into a chromosome may be accomplished by any method known in the art, such as homologous recombination.

[0035] Since the vector of the present application can be inserted into a chromosome by homologous recombination, it may further include a selection marker for confirming whether the chromosome has been inserted. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether or not a polynucleotide has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein may be used. In an environment treated with a selective agent, the selection marker is expressed. The transformed cells can then be selected for survival or displaying some other phenotype.

[0036] In the present application, the term "transformation" refers to introducing a vector containing the polynucleotide or a gene encoding HisG into a host cell so that the gene and HisG can be expressed in the host cell. Furthermore, the transformed gene may include all of these cases, regardless of whether it is located on the chromosome of the host cell or extrachromosomally, as long as the target gene can be expressed in the host cell.

[0037] The transformation method includes all methods for introducing the gene into cells, and depending on the host cell, a suitable standard technique may be selected as known in the art. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride precipitation, etc. These include, but are not limited to, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE dextran method, cationic liposome method, and lithium acetate DMSO method.

[0038] In the present application, the microorganism may include, without limitation, any microorganism in which HisG of the present application can be introduced to increase the productivity of glycine.

[0039] Specifically, the microorganism may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum or Corynebacterium flavum, and most specifically, Corynebacterium glutamicum, but is not limited thereto.

[0040] Another aspect of the present application provides a method for producing a fermentation composition containing glycine and glutamic acid, comprising the steps of culturing the Corynebacterium microorganism in a medium and fermenting the same.

[0041] Another aspect of the present application provides a fermentation composition produced by the method.

[0042] The fermented composition may have an increased content of glycine.

[0043] The microorganism is as described above.

[0044] In the present application, the term "culture" means growing a microorganism under suitable artificially controlled environmental conditions. The method for producing a target substance using the microorganism in the present application may be carried out using a method widely known in the art. Specifically, the culture may be carried out by a batch process, a fed batch or a repeated fed batch process. The culture may be, but is not limited to, a continuous culture. The medium used for the culture should meet the requirements of the particular strain in an appropriate manner. Culture media for Corynebacterium strains are known (see, for example, Manual of Methods for General Bacteriology by the American Society for Bacteriology, Washington DC, USA, 1981).

[0045] Sugar sources used in the medium include, but are not limited to, sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, etc. These materials may be used individually or in mixtures.

[0046] The nitrogen sources that may be used include, but are not limited to, peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources may also be used individually or in mixtures and are not limited thereto.

[0047] The phosphorus sources used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain metal salts such as magnesium sulfate or ferrous sulfate required for growth. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins may be used. Also, suitable precursors may be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch or continuous manner by an appropriate method during the culture process.

[0048] During the cultivation of the microorganism, the pH of the culture may be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, or ammonia, or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner. Also, foam formation may be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture to maintain an aerobic state.

[0049] The temperature of the culture (medium) may usually be 20° C. to 45° C., specifically, 25° C. to 40° C. The culture may be continued until a desired amount of the target substance is produced, specifically, for 10 to 160 hours.

[0050] The target substance may be recovered from the culture (medium) by separation using a conventional method known in the art. These separation methods include centrifugation, filtration, chromatography, and crystallization. For example, the culture may be centrifuged at low speed to remove the biomass, and the resulting supernatant may be separated via ion exchange chromatography, but is not limited thereto. Alternatively, the target substance may be recovered from the culture (medium) without a separate purification step by performing a bacterial cell separation and filtration step. Alternatively, the recovery step may further include a purification step.

[0051] In the present application, the term "the fermented composition" refers to a composition obtained by culturing the microorganism of the present application. Furthermore, the fermented composition may include a liquid or powdered composition obtained after culturing the microorganism and then performing an appropriate post-treatment process. In this case, the appropriate post-treatment process may include, for example, a process of culturing the microorganism, a process of removing bacteria, a process of concentrating, a process of filtering, and a process of mixing with a carrier, and may further include an additional drying process. Depending on the case, the post-treatment process may not include a purification process. The fermented composition includes a composition in which the content of glycine is increased while maintaining a certain level of glutamic acid by culturing the microorganism of the present application, thereby providing an optimal taste.

[0052] In addition, the term "the fermented composition" does not exclude seasoning products (e.g., soup powder products, snack seasoning products, etc.) that contain the liquid or powdered composition. Furthermore, as long as the "fermented composition" includes a composition obtained by culturing the microorganism of the present application, it does not exclude the case where the composition further includes a substance obtained by a non-fermentative process and / or another substance obtained by a non-natural process.

[0053] The present application will be described in more detail with reference to examples below. However, these examples are for illustrative purposes only and the scope of the present application is not limited to these examples.

[0054] Example 1. Introduction of mutations into KFCC11074 strain to increase glycine production and confirmation of glutamic acid and glycine production of mutant KFCC11074 Example 1-1. Construction of a vector with a mutation introduced In order to confirm the effect of increasing glycine production by enhancing HisG activity in glutamate-producing strains, we created strains with mutations in the promoter of the HisEG gene and strains with mutations that relieved histidine feedback inhibition, and confirmed their glycine production ability.

[0055] Meanwhile, the hisE and hisG genes are composed of an operon, and these genes are involved in the histidine biosynthesis pathway. In particular, since HisG is subject to feedback inhibition by the product histidine, it was confirmed whether the glycine production ability of the strain would increase when the activity of the HisG gene was increased by introducing a mutation that released the feedback inhibition. Here, a HisEG promoter mutation and a HisG histidine feedback inhibition release mutation were introduced into the KFCC11074 strain (Patent Document 5), which is known as a glutamic acid producing strain. Specifically, a gene replacement vector was constructed to replace the 53rd and 55th nucleotides of the polynucleotide sequence of SEQ ID NO: 1 containing the HisEG promoter with T, replace the 53rd and 55th nucleotides of the polynucleotide sequence with T, and replace the 60th nucleotide with G.

[0056] In addition, gene replacement vectors were constructed to replace glycine (Gly / G) at position 233 of the HisG amino acid sequence represented by SEQ ID NO:4 with histidine (His / H), and to replace glycine (Gly / G) and threonine (Thr / T) at positions 233 and 235 with histidine (His / H) and glutamine (Gln / Q). Gene fragments for constructing each replacement vector were obtained by PCR using ATCC13869 genomic DNA as a template. Based on the information on the Bacterium glutamicum (ATCC13869) gene and the surrounding nucleotide sequence, the respective primers were constructed. The PCR conditions for constructing the HisEG promoter replacement vector were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated 30 times, and polymerization reaction at 72°C for 5 minutes. More specifically, a 500 bp polynucleotide was obtained by amplified using primers of SEQ ID NOs: 7 and 8, and a 500 bp polynucleotide was obtained by amplified using primers of SEQ ID NOs: 9 and 10. The two obtained gene fragments were ligated to a pDZ vector (Patent Documents 3 and 6) cut with the restriction enzyme SalI using an infusion enzyme, to construct two gene replacement vectors containing the hisEG promoter, which were named "pDZ-hisEG-pro-2mt". A 500 bp polynucleotide was also obtained by amplified using primers of SEQ ID NOs: 7 and 11, and a 500 bp polynucleotide was also obtained by amplified using primers of SEQ ID NOs: 10 and 12. The two obtained gene fragments were ligated to a pDZ vector (Patent Documents 3 and 6) cut with the restriction enzyme SalI using an infusion enzyme to prepare a gene replacement vector containing the hisEG promoter, which was named "pDZ-hisEG-pro-3mt". The primer sequences used to prepare the vector are shown in Table 1 below.

[0057] A gene replacement vector was constructed to replace the 233rd, 233rd, and 235th amino acids of the HisG amino acid sequence with H, H, and Q, respectively. Specifically, the PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization reaction at 72°C for 5 minutes. A 722 bp polynucleotide was amplified using primers of SEQ ID NOs: 13 and 14, and a 798 bp polynucleotide was amplified using primers of SEQ ID NOs: 15 and 16. The two obtained gene fragments were ligated to a pDZ vector (Patent Documents 3 and 6) cut with the restriction enzyme SalI using an infusion enzyme to construct a 1.5 kbp gene replacement vector containing a polynucleotide containing a HisG (G233H) mutation, which was named "pDZ-hisG (G233H)". In addition, a 722 bp polynucleotide was obtained by amplified using primers of SEQ ID NO: 13 and 17, and a 798 bp polynucleotide was obtained by amplified using primers of SEQ ID NO: 16 and 18. The two obtained gene fragments were ligated to a pDZ vector (Patent Documents 3 and 6) cut with a restriction enzyme SalI using an infusion enzyme to prepare a 1.5 kbp gene replacement vector containing a polynucleotide containing a HisG (G233H / T235Q) mutation, which was named "pDZ-hisG (G233H / T235Q)". The primer sequence information used to prepare the vector is shown in Table 1 below.

[0058] [Table 1] Example 1-2. Construction of mutant KFCC11074 and confirmation of glutamic acid and glycine production The HisEG promoter replacement vectors pDZ-hisEG-pro-2mt and pDZ-hisEG-pro-3mt and the HisG gene replacement vectors pDZ-hisG(G233H) and pDZ-hisG(G233H / T235Q) prepared in Example 1-1 were introduced into the KFCC11074 strain by electroporation. t)_hisEG”, “KFCC11074_Pro(3mt)_hisEG”, “KFC "C11074_hisG(G233H)" and "KFCC11074_hisG(G233H / T235Q)" were produced.

[0059] Specifically, the strains were constructed through transformation (Non-Patent Document 6), and the vector was inserted into the chromosome through recombination of homologous sequences, and selected from agar nutrient medium containing 25 mg / L kanamycin. The selected primary strains were then subjected to a second crossover to select strains into which the target mutation had been introduced. The presence or absence of mutations (substitutions) in the transformed strains was confirmed by sequencing after performing PCR using the primer pair of SEQ ID NOs: 7 and 10 and the primer pair of SEQ ID NOs: 13 and 16, respectively.

[0060] Then, the selected strains, KFCC11074_Pro(2mt)_hisEG, KFCC11074_Pro(3mt)_hisEG, and KFCC11074_hisG(G233H ) and KFCC11074_hisG(G233H / T235Q) were smeared on nutrient medium. The culture was performed at 30°C for 16 hours. Then, 25 ml of the fermentation medium, which had been autoclaved at 121°C for 15 minutes, was dispensed into a 250 ml shaking Erlenmeyer flask, inoculated with the strain cultured in the nutrient medium, and cultured for 48 hours. The culture conditions were adjusted to a rotation speed of 200 rpm, a temperature of 37°C, and a pH of 8.0. The compositions of the nutrient medium and fermentation medium were as follows:

[0061] Nutrient medium: Glucose 1%, meat juice 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2 Fermentation medium: Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium monophosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10mg / L, biotin 0.3mg / L, Thiamine hydrochloride 0.2 mg / L After the cultivation was completed, the amounts of L-glutamic acid and glycine produced were measured by a method using HPLC, and the measurement results are shown in Table 2 below.

[0062] [Table 2] As shown in Table 2, the concentrations of L-glutamic acid produced by the mutant Corynebacterium glutamicum strains KFCC11074_Pro(2mt)_hisEG, KFCC11074_Pro(3mt)_hisEG, KFCC11074_hisG(G233H) and KFCC11074_hisG(G233H / T235Q) were similar to those produced by the unmutated Corynebacterium glutamicum strain KFCC11074. On the other hand, the KFCC11074_Pro(2mt)_hisEG and KFCC11074_ It was confirmed that the concentrations of glycine produced by the Pro(3mt)_hisEG and KFCC11074_hisG(G233H) strains were increased by 33 mg / L, 44 mg / L, and 45 mg / L, respectively, compared to the concentration of glycine produced by the KFCC11074 strain. In particular, the glycine concentration was increased by 2.0 mg / L in the case of the KFCC11074_hisG(G233H / T235Q) strain. A significant increase was observed at 68 mg / L. That is, it was confirmed that the above mutation, which includes the HisEG promoter mutation and the release of HisG feedback inhibition, significantly increases the glycine production ability of the microorganism while maintaining the L-glutamic acid production ability with almost no effect. Example 2. Confirmation of glutamic acid and glycine production amounts of ATCC13869 strains into which mutations have been introduced In order to confirm whether the above mutations have an effect of increasing glycine production without affecting glutamic acid production in the wild-type Corynebacterium glutamicum ATCC13869 strain, an ATCC13869-based strain into which the mutations were introduced was constructed.

[0063] The HisEG promoter replacement vectors pDZ-hisEG-pro-2mt and pDZ-hisEG-pro-3mt and the HisG gene replacement vectors pDZ-hisG(G233H) and pDZ-hisG(G233H / T235Q) prepared in Example 1-1 were introduced into the ATCC13869 strain by electroporation. mt)_hisEG", "ATCC13869_Pro(3mt)_hisEG", "AT "CC13869_hisG(G233H)" and "ATCC13869_hisG(G233H / T235Q)" were produced.

[0064] Specifically, the strains were prepared through transformation (Non-Patent Document 6), and the vector was inserted into the chromosome through recombination of homologous sequences, and selected from an agar nutrient medium containing 25 mg / L kanamycin. The selected primary strains were then subjected to a second cross-over to select strains into which the target mutation was introduced. The presence or absence of mutation (substitution) in the final transformed strain was confirmed through sequencing after PCR using the primer pair of SEQ ID NOs: 7 and 10 and the primer pair of SEQ ID NOs: 13 and 16, respectively.

[0065] Each colony was subcultured in nutrient medium and then cultured in fermentation medium for 5 hours. Then, 25% Tween 40 was added to each medium at a concentration of 0.4%, and each The colonies were re-cultured for 32 hours.

[0066] Nutrient medium: Glucose 1%, meat juice 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2 Fermentation medium: Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium monophosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10mg / L, biotin 0.3mg / L, Thiamine hydrochloride 0.2 mg / L Each colony was cultured under the above conditions, and the L-glutamic acid concentration was measured using YSI and the glycine concentration was measured using HPLC. The measured L-glutamic acid and glycine concentrations are shown in Table 3 below.

[0067] [Table 3] As shown in Table 3, wild-type Corynebacterium glutamicum (ATCC13869 ATCC13869_Pro(2mt)_hisEG, ATCC13869_Pro(3mt)_hisEG, ATCC13869_hisG(G233H), and L-glutamate produced by the ATCC13869_hisG(G233H / T235Q) strain The concentrations of L-glutamic acid produced by the ATCC13869 strain were similar to those of L-glutamic acid produced by the ATCC13869 strain, while the concentrations of glycine were all increased.

[0068] In other words, it was again confirmed that the above mutations, including the HisEG promoter mutation and the release of HisG feedback inhibition, significantly increased the microorganism's ability to produce glycine while maintaining its L-glutamic acid production ability with little effect. Meanwhile, the ATCC13869_hisG(G233H) and ATCC13869_hisG(G233H / T235Q) strains were deposited with the Korea Center for Microorganisms (KCCM), a depository institution under the Budapest Treaty, on March 14, 2019 under the strain name “CA02-9216” and The DNA was deposited internationally under the accession numbers "CA02-9217" and "CA02-9217" and assigned the accession numbers "KCCM12458P" and "KCCM12459P".

[0069] Example 6. Preparation of fermented composition for the production of seasoning material products As described above, it was confirmed that the HisG activity-enhanced strain increases the glycine production ability without affecting the L-glutamic acid production ability. Therefore, a fermentation composition was prepared using the Corynebacterium sp. microorganism with enhanced HisG activity according to the present invention.

[0070] For example, the product was produced using glutamic acid, a well-known seasoning ingredient, as the main ingredient, and the fermentation strain and fermentation process were adjusted to increase the by-product ingredients of other seasoning ingredients in order to enhance the rich flavor composition. A fermentation composition was produced in a 5 L fermenter using a strain containing both of the above mutations, including the HisEG promoter mutation and the release of HisG feedback inhibition.

[0071] All ingredients used in the preparation of the culture medium were food-grade. Primary seed medium: A primary seed medium containing 1% glucose, 1% yeast extract, 1% peptone, 0.1% ammonium sulfate, 0.25% sodium chloride, 0.15% potassium phosphate monobasic, and 0.15% potassium phosphate dibasic was prepared with a pH of 8.0. Secondary seed medium: 4.6% organic raw sugar of 98.5% purity, 0.05% magnesium sulfate, 0.5% yeast extract, 0.2% potassium phosphate monobasic, 0.002% ferrous sulfate, 1 mg / L biotin, 2 mg / L thiamine hydrochloride and a small amount of antifoaming agent, pH 7.2. A secondary seed medium was prepared.

[0072] Fermentation medium: A fermentation medium was prepared containing 4% 98.5% pure organic raw sugar, 0.03% magnesium sulfate, 1% yeast extract, 0.22% phosphoric acid, 0.4% potassium hydroxide, 0.2 mg / L biotin, 0.6 mg / L thiamine hydrochloride, 0.002% manganese sulfate, 0.002% ferrous sulfate, 0.002% zinc sulfate, 0.0006% copper sulfate and a small amount of antifoaming agent, with a pH of 7.4.

[0073] 50 ml of the primary seed medium was dispensed into a 500 ml shaking Erlenmeyer flask, autoclaved at 121°C for 20 minutes, cooled, and then inoculated with the strains and cultured with shaking at 200 rpm and 30°C for 5 to 7 hours.

[0074] 0.25 L of the secondary seed medium was prepared in a 1.5 L test fermentation tank and incubated at 121° C. for 20 After sterilizing under pressure for 10 minutes and cooling, 50 ml of the primary seed culture was inoculated and rotated at 90 The culture was performed at 0 times per minute and 31.5°C for 15 hours.

[0075] 0.25 L of the fermentation medium was prepared in a 5 L test fermenter, sterilized under pressure at 121°C for 20 minutes, and cooled. Then, 0.26 L of the secondary seed culture was inoculated and cultured at 30 to 34°C with a rotation speed of 900 rpm.

[0076] While culturing under the above conditions, the pH of the fermentation liquid during the culture of Corynebacterium glutamicum was continuously adjusted to a range of 7.0 to 7.4 using 28% ammonia water. When the residual sugar concentration during the culture reached 0.5 to 1.5%, sterilized organic raw sugar was added from time to time, and the culture was continued until the total amount of added sugar reached 30 to 34% of the fermentation liquid volume.

[0077] [Table 4] As a result, as shown in Table 4, there was little difference in the amount of glutamic acid produced between the two strains, and the amount of glutamic acid produced by the mutant Corynebacterium glutamicum KFCC11074_hisG( It was confirmed that the glycine content in the fermentation broth produced by the G233H / T235Q)_Pro(3mt)_hisEG strain was significantly increased.

[0078] In the case of the fermentation composition using a 3 kL fermenter, there was little difference in the amount of glutamic acid produced between the two strains, and the amount of glutamic acid produced by the mutant Corynebacterium glutamicum KFCC11074_ hisG(G233H / T235Q)_Pro(3mt)_hisEG strain is KFCC1 Although there was no significant difference in glutamic acid production compared to strain 1074 (64.2 g / L glycine content was significantly increased (0.2g / L vs 3.2g / L) I confirmed that.

[0079] From the above description, a person skilled in the art to which the present application pertains can understand that the present application can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it should be understood that the above described embodiments are illustrative in all respects and are not limiting. The scope of the present application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below and their equivalent concepts from the above detailed description. TIFF0007607613000005.tif234170TIFF0007607613000006.tif234170

Claims

1. A method for preparing glycine, comprising: fermenting a microorganism for producing glycine by culturing the microorganism in a medium; and measuring the amount of glycine produced in the resulting fermented medium, The microorganism for producing glycine is Corynebacterium glutamicum having an ATP phosphoribosyltransferase (HisG) in which the 233rd amino acid of the amino acid sequence of SEQ ID NO: 4 is substituted with histidine (H) or the 233rd and 235th amino acids of the amino acid sequence of SEQ ID NO: 4 are substituted with histidine (H) and glutamine (Q), respectively, and having increased glycine producing ability.

2. The method of claim 1, wherein the ATP phosphoribosyltransferase consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

6.

3. 2. The method of claim 1, wherein the fermented composition contains an increased amount of glycine compared to a fermented composition prepared by Corynebacterium glutamicum having an unmodified ATP phosphoribosyltransferase (HisG).

4. 10. The method of claim 1, further comprising the steps of removing bacterial cells, concentrating, filtering, mixing with a carrier, or drying.

5. The method of claim 1 , wherein the fermented composition further contains glutamic acid.

6. A composition for producing glycine, comprising Corynebacterium glutamicum having enhanced ATP phosphoribosyltransferase (HisG) activity, A composition comprising the ATP phosphoribosyltransferase, wherein the 233rd amino acid in the amino acid sequence of SEQ ID NO: 4 is substituted with histidine (H).

7. A composition for producing glycine, comprising Corynebacterium glutamicum having enhanced ATP phosphoribosyltransferase (HisG) activity, A composition comprising the ATP phosphoribosyltransferase, in which the 233rd amino acid in the amino acid sequence of SEQ ID NO: 4 is replaced with histidine (H) and the 235th amino acid is replaced with glutamine (Q).

8. The composition of claim 6 , wherein the ATP phosphoribosyltransferase consists of the amino acid sequence of SEQ ID NO:

5.

9. The composition of claim 7 , wherein the ATP phosphoribosyltransferase consists of the amino acid sequence of SEQ ID NO:6.

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