阅读说明：本技术 具有增加的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法 (Microorganism having increased glycine production ability and method for producing fermentation composition using the same ) 是由 李智软 张真淑 金亨俊 尹炳勋 崔先亨 崔允祯 于 2019-03-27 设计创作，主要内容包括：本申请涉及具有增强的甘氨酸生产能力的微生物和利用其生产发酵组合物的方法,和更具体地,涉及将突变引入到HisG中而具有增加的甘氨酸生产能力的棒杆菌属的微生物,利用其制备包含甘氨酸和谷氨酸的发酵组合物的方法,以及发酵组合物。(The present application relates to a microorganism having enhanced glycine productivity and a method of producing a fermentation composition using the same, and more particularly, to a microorganism of corynebacterium having increased glycine productivity by introducing a mutation into HisG, a method of preparing a fermentation composition comprising glycine and glutamic acid using the same, and a fermentation composition.)

1. A microorganism of the genus Corynebacterium having an increased glycine producing ability, wherein the activity of ATP phosphoribosyltransferase (HisG) is enhanced.

2. The microorganism according to claim 1, wherein the amino acid at position 233 of the amino acid sequence of SEQ ID NO. 4 is substituted with histidine (H) in the ATP phosphoribosyltransferase.

3. The microorganism according to claim 1, wherein amino acids 233 and 235 of the amino acid sequence of SEQ ID NO 4 are substituted with histidine (H) and glutamine (Q), respectively, in the ATP phosphoribosyltransferase.

4. The microorganism of claim 2, wherein the ATP phosphoribosyltransferase consists of the amino acid sequence of SEQ ID NO 5.

5. The microorganism of claim 3, wherein the ATP phosphoribosyltransferase consists of the amino acid sequence of SEQ ID NO 6.

6. The microorganism according to any one of claims 1 to 5, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum (Corynebacterium glutamicum).

7. A method for producing a fermentation composition comprising glycine and glutamic acid, the method comprising performing fermentation by culturing the microorganism of the genus Corynebacterium of any one of claims 1 to 5 in a medium.

8. A fermentation composition prepared by the method of claim 7.

Technical Field

The present disclosure relates to a microorganism having increased glycine-producing ability and a method of producing a fermentation composition using the same, and more particularly, to a microorganism of Corynebacterium (genus Corynebacterium) having increased glycine-producing ability due to introduction of a mutation in HisG, a method of preparing a fermentation composition comprising glycine and glutamic acid using the microorganism of Corynebacterium, and a fermentation composition.

Background

L-amino acid is a basic structural unit of protein and is used as important materials such as medical raw materials, food additives, animal feeds, nutritional supplements, agricultural chemicals, bactericides and the like, wherein L-glutamic acid is a representative amino acid produced by fermentation and has a characteristic, unique taste (umami taste), and thus is an important amino acid widely used in the food field as well as the medical field and other animal feed fields.

Typical methods for producing Amino acids include Fermentation methods using microorganisms of the genus Brevibacterium (genus Brevibacterium) or Corynebacterium (Amino Acid Fermentation, Gakkai Shuppancenter:195-215,1986) or using microorganisms of the genus Escherichia (Escherichia coli) or Bacillus (genus Bacillus), Streptomyces (genus Streptomyces), Penicillium (genus Penicillium), Klebsiella (genus Klebsiella), Erwinia (genus Erwinia), Pantoea (genus Pantoea), and the like (U.S. Pat. Nos. 3,220,929 and 6,682,912). In addition, such amino acids are also produced by industrial methods using synthetic processes such as monochloroacetic acid method, Strecker method, and the like.

In addition, various studies have been made for the purpose of efficiently producing amino acids; for example, efforts have been made to develop microbial or fermentation process technologies for the efficient production of amino acids. Specifically, specific approaches to the target substances (specific aptamers) have been developed, such as enhancement of expression of genes encoding enzymes involved in amino acid biosynthesis in strains of Corynebacterium or deletion of genes unnecessary for amino acid biosynthesis (Korean patent Nos. 10-0924065 and 1208480). In addition to these methods, a method of removing a gene not involved in amino acid production and a method of removing a gene whose specific function of producing amino acids is unknown are also utilized. However, there is still a growing need to develop efficient methods for producing amino acids in high yields.

Disclosure of Invention

Technical problem

The present inventors have made an effort to develop a method capable of producing several amino acids simultaneously, and as a result, they confirmed that when HisG activity of a glutamic acid-producing microorganism is enhanced as compared to HisG activity of its parent strain, glycine productivity can be improved while maintaining glutamic acid productivity, thereby completing the present disclosure.

Technical scheme

It is an object of the present disclosure to provide a microorganism of the genus Corynebacterium having increased glycine production ability, in which the activity of ATP phosphoribosyltransferase (HisG) is enhanced.

It is another object of the present disclosure to provide a method of preparing a fermentation composition comprising glycine and glutamic acid, which comprises fermenting by culturing a microorganism of the genus corynebacterium.

It is yet another object of the present disclosure to provide a fermentation composition prepared by the above method.

Advantageous effects

Since the HisG mutation of the present disclosure can be introduced into a microorganism and simultaneously produce glutamic acid and glycine, it can be effectively used for the production of amino acids. In addition, the present disclosure can improve the taste and palatability of the fermentation product by adjusting the amount of glutamic acid and glycine in the fermentation product, for the preparation of the fermentation broth and its use in flavoring products.

Detailed Description

Hereinafter, the present disclosure will be described in detail. Meanwhile, each description and embodiment disclosed in the present disclosure may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure are within the scope of this disclosure. Further, the specific description disclosed below should not be construed as limiting the scope of the disclosure.

To achieve the above objects, one aspect of the present disclosure provides a microorganism of the genus corynebacterium having increased glycine production ability, in which activity of ATP phosphoribosyltransferase (HisG) is enhanced.

Specifically, a microorganism having an increased glycine-producing ability, wherein the 233 rd amino acid of the amino acid sequence of SEQ ID NO. 4 is substituted with histidine (H) in ATP phosphoribosyltransferase, can be provided.

In addition, specifically, a microorganism having an increased glycine-producing ability in which amino acids 233 and 235 of the amino acid sequence of SEQ ID NO:4 are substituted with histidine (H) and glutamine (Q), respectively, in ATP phosphoribosyltransferase can be provided.

As used herein, the term "ATP phosphoribosyltransferase," also known as "HisG," refers to an enzyme involved in the histidine synthesis pathway. The histidine synthesis pathway consists of a total of 9 enzymes (HisG-HisE-HisI-HisA-HisH-HisB-HisC-HisN-HisD), and HisG constitutes its first step.

HisG is known to be involved in histidine production, but its relationship to glycine production is not known and was first identified by the present inventors. More specifically, the present inventors have confirmed for the first time that the amount of glycine production can be increased by enhancing the activity of HisG. In particular, HisG is subject to feedback inhibition by the product histidine, and in the present disclosure, a mutation is introduced (histidine feedback inhibition is released), and as a result, the effects of increasing the amount of glycine production and maintaining the amount of glutamic acid are determined for the first time by the present inventors.

As used herein, the term "enhancement of HisG activity" means that the activity of HisG enzyme is increased compared to the endogenous activity possessed by a microorganism of the genus corynebacterium in its natural state. Examples of the method of increasing HisG activity may include: (i) a method of increasing the copy number of a nucleotide sequence encoding an enzyme by further inserting a polynucleotide containing a nucleotide sequence encoding HisG into a chromosome, or by introducing a polynucleotide containing a nucleotide sequence encoding HisG into a vector system, or the like; (ii) a method of enhancing the hisG gene promoter (e.g., substitution with a stronger promoter, introduction of a mutation on the promoter, etc.); (iii) a method of modifying the enzyme to have a higher activity by gene mutation, and the like.

Specifically, in the present disclosure, amino acid 233 (i.e., glycine) of the HisG amino acid sequence of SEQ ID NO. 4 may be substituted with histidine; or in the HisG amino acid sequence of SEQ ID NO. 4, the 233 rd amino acid (i.e., glycine) may be substituted with histidine and the 235 th amino acid (i.e., threonine) may be substituted with glutamine. Therefore, a microorganism of the genus corynebacterium comprising the modified HisG described above can significantly increase glycine-producing ability while maintaining glutamic acid-producing ability without any adverse effect thereon. The increase in glycine productivity may mean that glycine productivity is increased as compared to a microorganism having HisG without the modification of the present disclosure (i.e., HisG without the above mutation).

In another embodiment, a promoter of HisG enzyme may be modified to be stronger than a native promoter through mutation or substitution, a modified promoter having nucleotide substitution mutation or a heterologous promoter may be linked instead of an endogenous enzyme promoter, and examples of the heterologous promoter may include cj7 promoter, lysCP1 promoter, EF-Tu promoter, groE L promoter, aceA promoter, aceB promoter, etc., but the heterologous promoter is not limited thereto.

In addition, since the hisG gene is composed of the hisE gene and the operon, the activity of the hisG enzyme can be enhanced by overexpression of hisG through mutation or substitution of the promoter sequence of the hisEG gene. More specifically, a promoter stronger than the native promoter prepared by mutation in the promoter sequence of hisEG gene can be used to enhance the activity of HisG enzyme, in which nucleotides 53 and 55 are substituted with T in the nucleotide sequence of SEQ ID NO: 1; or replacing nucleotides 53 and 55 with T and 60 with G. Reviewing the literature on the promoter sequence study of Corynebacterium glutamicum (Microb Biotechnol.2013Mar; 6(2): 103-. Thus, the present inventors have confirmed the promoter sequence of the hisEG gene through RNA-seq experiments of ATCC13869 strain, and in addition, have attempted to induce overexpression of the promoter sequence of the hisEG gene through mutation of its native promoter. As a method for modifying a natural promoter, the nucleotide sequences at positions-35 and-10 from the promoter region of Corynebacterium glutamicum may be modified so that the modified promoter sequence becomes close to the consensus sequence. In particular, when the sequence of the-10 region (TATA box) from the promoter sequence of the hisEG gene is modified to be close to the consensus sequence, the promoter may be modified to be a stronger promoter than the natural promoter.

Specifically, ATP phosphoribosyltransferase included in a microorganism of the genus Corynebacterium may consist of the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6.

In addition, the amino acid sequences of the present disclosure can be modified by known mutagenesis methods, such as directed evolution, site-directed mutagenesis, and the like.

Thus, the ATP phosphoribosyltransferase can include HisG, which includes a nucleotide sequence that is at least 60%, specifically at least 70%, more specifically at least 80%, and even more specifically at least 83%, at least 84%, at least 88%, at least 90%, at least 93%, at least 95%, or at least 97% homologous to the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6. Obviously, any amino acid sequence having such homology (in which a part of the sequence is deleted, modified, substituted or added) is also within the scope of the present disclosure, as long as the resulting amino acid sequence has a biological activity substantially identical to or corresponding to the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6.

In particular, the term "L-glutamic acid (L-glutamic acid or L-glutamate)" refers to an amino acid classified as a non-essential amino acid L-glutamic acid is known to be the most common excitatory neurotransmitter in the central nervous system, furthermore, since L-glutamic acid has an umami taste, monosodium glutamate (MSG) has been developed therefrom and is widely used as an odorant, it is generally produced by fermentation of L-glutamic acid-producing microorganisms.

In addition, the term "glycine" means an amino acid having colorless crystalline form and sweetness, glycine is mainly used as a taste enhancer for food, and in the medical field, it is used for infusion solutions, antacids, polyamino acid preparations, nutritional supplements, in general, glycine is prepared by an industrial synthetic method such as monochloroacetic acid method, Strecker method, etc. however, since a mixture of D-form and L-form amino acids is produced when the amino acid is prepared using the synthetic method, there is an inconvenience that optical resolution is necessary.

As used herein, the term "homology" may indicate the degree of match to a given amino acid sequence or nucleotide sequence and may be expressed as a percentage (%). in the present disclosure homologous sequences having the same or similar activity as a given amino acid sequence or nucleotide sequence are expressed as "% homology". homology to an amino acid sequence or nucleotide sequence may be determined by, for example, the algorithm B L AST (see Karlin and Altschul, pro. natl. acad. sci. usa,90,5873(1993)) or FASTA (see Pearson, Methods enzymol.,183,63,1990) based on this algorithm B L, the programs B L ASTN and B L ASTX (see http:// www.ncbi.nlm.nih.gov) have been developed.

As used herein, the term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides, such stringent conditions are specifically described in the literature (e.g., j. sambrook et al.) for example, stringent conditions may include conditions under which genes having high homology (e.g., 60% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and even more specifically 99% or more) can hybridize to each other, but genes having lower homology than the same cannot hybridize to each other, or conditions under which conventional Southern hybridization (i.e., conditions corresponding to 60 ℃,1 ×, 0.1% SDS, specifically 60 ℃, 0.1 ×, SSC 0.1% SDS, and more specifically at 68 ℃, 0.1 × SSC, 0.1% salt concentration and temperature are washed once, more specifically two or three times.) although mismatches between bases are possible depending on the degree of stringency, the hybridization between nucleotides requires that the two nucleotides have complementary sequences.

In particular, T at 55 ℃ is used under the above conditions_mThe hybridization conditions of the hybridization step under the values can detect polynucleotides having homology. In addition, T_mThe value may be 60 ℃,63 ℃ or 65 ℃, but is not limited thereto. Those skilled in the art can appropriately adjust T according to their purpose_mThe value is obtained. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al, supra, 9.50-9.51, 11.7-11.8).

As used herein, the term "microorganism" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and it may be a microorganism having a specific attenuation or enhancement mechanism due to insertion of an exogenous gene or enhancement or attenuation of activity of an endogenous gene.

In the present disclosure, the microorganism may comprise ATP phosphoribosyltransferase. In addition, ATP phosphoribosyltransferase may be introduced into a microorganism by transformation via a vector, but the transformation method is not limited thereto. Furthermore, as long as HisG can be expressed in a microorganism, it does not matter whether the gene encoding HisG is located on the chromosome or extrachromosomally.

As used herein, the term "vector" is an artificial DNA molecule having genetic material capable of expressing a gene of interest in a suitable host, and may refer to a DNA construct comprising the nucleotide sequence of a gene encoding HisG.

The vector used in the present disclosure is not particularly limited as long as the vector can be expressed in a host cell, and any vector known in the art may be used to transform the host cell. Examples of conventional vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages.

For example, as the phage vector or cosmid vector, pWE15, M13, λ L B3, λ B L4, λ IXII, λ ASHII, λ APII, λ t10, λ t11, Charon4A, Charon21A and the like can be used, and as the plasmid vector, plasmid vectors based on pBR, pUC, pBluescriptII, pGEM, pTZ, pC L, pET and the like can be used.

For example, vectors pECCG117, pDZ, pACYC177, pACYC184, pC L, pUC19, pBR322, pMW118, pCC1BAC, pCES208, pXMJ19, and the like can be used, but are not limited thereto.

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

Since the vector of the present disclosure can be inserted into a chromosome by inducing homologous recombination, a selection marker may be additionally included to confirm whether the gene is successfully inserted into the chromosome. The selection marker is used to screen cells transformed with the vector, in other words, to determine whether the polynucleotide is inserted. Markers that provide a selectable phenotype, such as drug resistance, auxotrophy, resistance to toxic agents, or expression of surface proteins, may be used. In the context of treatment with a selection agent, only cells expressing the selection marker survive, or exhibit a different phenotype, and thus successfully transformed cells can be selected in this way.

As used herein, the term "transformation" refers to the introduction of a polynucleotide comprising or a gene encoding HisG into a host cell to allow the gene and HisG to be expressed in the host cell. Further, as long as the gene of interest can be expressed in the host cell, it does not matter whether the transformed gene is located on the chromosome of the host cell or extrachromosomally, and both cases are included.

The transformation method may include all methods of introducing a gene into a cell, and may be performed by selecting an appropriate standard technique known in the art according to a host cell. For example, suitable standard techniques may be selected from electroporation, calcium phosphate (CaPO)₄) Precipitate, calcium chloride (CaCl)₂) Precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, and lithium acetate-DMSO technology, but suitable standard techniques are not limited thereto.

In the present disclosure, the microorganism may be any microorganism, without limitation, in which HisG of the present disclosure is introduced and thus glycine production ability is increased.

Specifically, the microorganism may be a microorganism of the genus Corynebacterium; more specifically, Corynebacterium glutamicum or Brevibacterium flavum (Brevibacterium flavum); and most specifically, Corynebacterium glutamicum, but the microorganism is not limited thereto.

Another aspect of the present disclosure provides a method for preparing a fermentation composition, which includes fermenting by culturing a microorganism of the genus corynebacterium in a medium.

Yet another aspect of the present disclosure provides a fermented composition prepared by the above method.

The fermentation composition may be a composition wherein the amount of glycine is increased.

The microorganisms are as described above.

As used herein, the term "culturing" refers to culturing a microorganism under artificially controlled environmental conditions. In the present disclosure, the method for producing a substance of interest using a microorganism can be performed by a method widely known in the art. Specifically, the cultivation may be performed in a batch process or a continuous process (e.g., a fed-batch process or a repeated fed-batch process), but is not limited thereto. The medium used for the cultivation must meet the requirements of the particular strain used. Suitable media for culturing Corynebacterium strains are known in the art (e.g., Manual of Methods for General Bacteriology by the American Society for Bacteriology, Washington D.C., USA, 1981).

Carbon sources that can be used for the medium may be sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, starch, and cellulose); oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil); fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid); alcohols (e.g., glycerol and ethanol); and organic acids (e.g., acetic acid). These may be used alone or in combination, but the manner of use is not limited thereto.

Examples of the nitrogen source that can be used include peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate). These nitrogen sources may also be used alone or in combination, but the manner of use is not limited thereto.

Phosphorus sources that may be used in the medium may include dipotassium hydrogen phosphate, potassium dihydrogen phosphate, or the corresponding sodium-containing salts. In addition, the medium may contain metal salts necessary for cell growth. Finally, substances essential for growth (e.g., amino acids and vitamins) may be used in addition to the above substances. Further, a precursor suitable for the medium may be used. The above-mentioned raw materials may be sufficiently supplied to the culture in a batch or continuous manner.

During the cultivation of the microorganism, the pH of the culture can be adjusted by means of suitable basic compounds (e.g., sodium hydroxide, potassium hydroxide or ammonia), or acidic compounds (e.g., phosphoric acid or sulfuric acid). Foaming can be adjusted by anti-foaming agents (e.g., fatty acid polyglycol esters). Aerobic conditions of the culture can be maintained by introducing oxygen or oxygen-containing gas (e.g., air).

The temperature of the culture (medium) may generally be in the range of 20 ℃ to 45 ℃, specifically 25 ℃ to 40 ℃. The cultivation may be continued until a desired production amount of the objective substance is obtained, and specifically 10 to 160 hours.

The recovery of the objective substance from the culture (culture medium) can be carried out by a conventional separation method known in the art. For the separation method, methods such as centrifugation, filtration, chromatography, crystallization, and the like can be used. For example, a supernatant obtained by centrifuging the medium at a low speed to remove the biomass may be separated by ion exchange chromatography, but the separation method is not limited thereto. In the replaceable method, the objective substance can be recovered by performing a process of separating and filtering bacterial cells from the culture product (culture medium) without an additional purification process. In another alternative method, the recovering step may further comprise a purification process.

As used herein, the term "fermentation composition" refers to a composition obtained by culturing a microorganism of the present disclosure. Furthermore, the fermentation composition may comprise a composition in liquid or powder form obtained after suitable post-treatment after culturing the microorganism. In particular, suitable post-treatment processes may include, for example, a process of culturing microorganisms, a process of removing bacterial cells, a concentration process, a filtration process, and a process of mixing carriers, and may further include a drying process. In some cases, the post-treatment process may not include a purification process. The fermented composition obtained by culturing the microorganism of the present invention contains an increased amount of glycine while maintaining a certain level of glutamic acid production, thus making it possible to provide the best taste.

In addition, "fermented composition" does not exclude flavored products (e.g., powdered soup products, snack flavored products, etc.) containing the composition in liquid or powder form. In addition, the "fermented composition" does not exclude the case where a substance obtained by a non-fermentation process and/or other substances obtained by a non-natural process are further included, as long as the composition obtained by culturing the microorganism of the present disclosure is included therein.