Title of the invention: Microorganism producing L-amino acid with enhanced activity of alpha-glucosidase and method for producing L-amino acid using the same
Technical field
[One]
The present application relates to a microorganism producing L-amino acid with enhanced activity of α-glucosidase and a method for producing L-amino acid using the same.
[2]
Background
[3]
L-amino acids are used in animal feed, pharmaceuticals, and cosmetics industries, and are mainly produced by fermentation using strains of the genus Corynebacterium or strains of the genus Escherichia. For the production of L-amino acids, various studies such as the development of highly efficient production strains and fermentation process technology have been conducted. Specifically, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-amino acid biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (Republic of Korea Patent Registration No. 10-0838038 arc).
[4]
On the other hand, in order to increase the ability of microorganisms to produce a target product, several studies have been conducted to increase sugar utilization, and there has been a continuing need for efficient medium composition and microorganism growth. For example, the use of cellobiose is increased through overexpression of the ascB or chbF gene, and mutant microorganisms that can simultaneously use cellobiose and other sugars such as xylose, mannose, and galactose are produced, and biofuels are produced using this Technology has been reported (Korean Patent Registration No. 10-1484108). However, continuing research is needed on the relationship between microbial sugar utilization and L-amino acid productivity.
[5]
Detailed description of the invention
Technical challenge
[6]
The present inventors introduced isomaltose, α-glucosidase, which is known to degrade maltose, into a strain of the genus Corynebacterium. Surprisingly, even without adding isomaltose and maltose in the medium, the target product, L- This application was completed by confirming the effect of improving the yield of amino acids.
[7]
Means of solving the task
[8]
One object of the present application is to provide a microorganism of the genus Corynebacterium ( Corynebacterium sp. ) that produces L-amino acids with enhanced activity of α-glucosidase .
[9]
Another object of the present application is culturing the microorganism in a medium; And it is to provide a method for producing L-amino acid comprising the step of recovering the L-amino acid from the cultured medium or microorganism.
[10]
Another object of the present application is to provide a method for increasing L-amino acid production, comprising enhancing expression of α-glucosidase in microorganisms.
[11]
Another object of the present application is to provide a use of α-glucosidase to increase L-amino acid production.
[12]
Effects of the Invention
[13]
The microorganisms of the genus Corynebacterium producing L-amino acids of the present application have enhanced α-glucosidase activity, and thus the yield of L-amino acid production is improved. Therefore, the microorganism can be very usefully used for the production of L-amino acid.
[14]
Brief description of the drawing
[15]
Figure 1 shows the results of SDS-PAGE confirming the expression of α-glucosidase in Corynebacterium glutamicum strain.
[16]
Best mode for carrying out the invention
[17]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below.
[18]
[19]
One aspect of the present application for achieving the above object is a microorganism of the genus Corynebacterium ( Corynebacterium sp. ) producing L-amino acids with enhanced activity of α-glucosidase . The microorganisms of the genus Corynebacterium of the present application have enhanced L-amino acid production ability by enhancing the activity of α-glucosidase. Therefore, the microorganism of the genus Corynebacterium of the present application can be very usefully used in the production of L-amino acids.
[20]
[21]
In the present application, the term "α-glucosidase" is a type of glucosidase that decomposes sugar into glucose, and in particular, refers to an enzyme having the property of decomposing α(1→4) bonds. . In the present application, α-glucosidase may be a protein having α-glucosidase activity encoded by the aglA gene, but it has an activity corresponding to glucosidase and its activity is enhanced in microorganisms in the genus Corynebacterium. As long as it can improve the production ability of L-amino acid, it is not particularly limited to its kind. Proteins having α-glucosidase activity encoded by the aglA gene are known to have isomaltose or maltose degrading activity (Glycobiology. 2010 Nov; 20(11)), and to the α-glucosidase. Information about the information can be easily obtained by a person skilled in the art through a known database (eg, NCBI, UniProt, etc.). In the present application, α-glucosidase is α-glucosidase derived from Bifidobacterium adolescentis , Erwinia amylovora , or Saccharomyces cerevisiae . And, specifically, Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) may be derived from α-glucosidase, but is not limited thereto. The α-glucosidase derived from Bifidobacterium adolescentis presented as an example in the present application is not limited thereto, but may be a protein including the amino acid sequence of SEQ ID NO: 1. The α-glucosidase derived from Erwinia amylovora is not limited thereto, but may be a protein including the amino acid sequence of SEQ ID NO: 28. The α-glucosidase derived from Saccharomyces cerevisiae is not limited thereto, but may be a protein comprising the amino acid sequence of SEQ ID NO: 29. The protein comprising the amino acid sequence of SEQ ID NO: 1 may be used interchangeably with a protein having the amino acid sequence of SEQ ID NO: 1 and a protein consisting of the amino acid sequence of SEQ ID NO: 1.
[22]
In addition, even if it is described in the present application as'a protein or polypeptide comprising an amino acid sequence described in a specific sequence number', if it has the same or corresponding activity as a polypeptide containing the amino acid sequence of the sequence number, some sequences are It is obvious that proteins having amino acid sequences that have been deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. For example, if it has the same or corresponding activity as the polypeptide containing the amino acid sequence of the corresponding sequence number, the addition of a sequence that does not change the function of the protein before or after the amino acid sequence, mutations that may occur naturally, and its potential It does not exclude silent mutations or conservative substitutions, and it is obvious that even if such sequence additions or mutations are present, they fall within the scope of the present application. In addition, if it has the same or corresponding activity as the polypeptide containing the amino acid sequence of the corresponding sequence number, 80% or more, specifically, 90% or more, more specifically, 95% or more, than the amino acid sequence of the sequence number. More specifically, an amino acid sequence having a homology or identity of 99% or more may be included within the scope of the present application.
[23]
[24]
For example, the protein having α-glucosidase activity in the present application is the amino acid sequence of α-glucosidase derived from Bifidobacterium adolescentis (SEQ ID NO: 1), Erwinia amylobora ( Erwinia amylovora ) derived from α-glucosidase amino acid sequence (SEQ ID NO: 28), or Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) derived from α-glucosidase amino acid sequence (SEQ ID NO: 29) It can be a protein. Α-glucosidase of the present application shows corresponding efficacy as α-glucosidase, and the α-glucosidase of the present application is a protein whose activity is enhanced in microorganisms in Corynebacterium to improve the production ability of L-amino acids. It is obvious that it is included in the protein having the activity of sidase. Specifically, as long as the α-glucosidase of the present application exhibits the activity of α-glucosidase and enhances the activity of the microorganisms in the genus Corynebacterium to improve the production capacity of L-amino acids, the above SEQ ID NO: 1 , SEQ ID NO: 28, or the amino acid sequence of the amino acid sequence of SEQ ID NO: 29 and 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 99% or more of the amino acid sequence having homology or identity It may be included within the scope of the application.
[25]
In the present application, the term "homology or identity" means the degree to which two given amino acid sequences or base sequences are related, and may be expressed as a percentage. In addition, the homology and identity can often be used interchangeably.
[26]
The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and the default gap penalty established by the program used can be used together. Substantially, homologous or identical sequences are generally in moderate or high stringent conditions along at least about 50%, 60%, 70%, 80% or 90% of the sequence or full-length. (stringent conditions) can be hybridized. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotide.
[27]
Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: Can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Alternatively, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids). Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073), for example, BLAST of the National Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity or identity.
[28]
The homology, similarity, or identity of a polynucleotide or polypeptide can be found in, for example, Smith and Waterman, Adv. Appl. As known in Math (1981) 2:482, for example, Needleman et al. (1970), J Mol Biol. 48: It can be determined by comparing sequence information using a GAP computer program such as 443. In summary, the GAP program is defined as the total number of symbols in the shorter of the two sequences, divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). The default parameters for the GAP program are (1) a monolithic comparison matrix (contains a value of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. As disclosed by 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); And (3) no penalty for end gaps. Thus, as used in this application, the term “homology” or “identity” refers to relevance between sequences.
[29]
In the present application, the term "conservative substitution" means replacing one amino acid with another amino acid having similar structural and/or chemical properties. The variant may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions can generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; Negatively charged (acidic) amino acids include glutamic acid and arpartic acid; Aromatic amino acids include phenylalanine, tryptophan and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.
[30]
[31]
In addition, the polynucleotide sequence encoding the α-glucosidase exhibits the activity of α-glucosidase, and its activity is enhanced in microorganisms of the genus Corynebacterium, thereby encoding a protein that improves the production ability of L-amino acids. It may be a polynucleotide sequence. For example, α-glucosidase derived from Bifidobacterium adolescentis (SEQ ID NO: 1), α-glucosidase derived from Erwinia amylovora (SEQ ID NO: 28), Saka access to my Celebi jiae ( Saccharomyces cerevisiae) May be a polynucleotide sequence encoding the derived α-glucosidase (SEQ ID NO: 29), but is not limited thereto. For example, it may have the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 30, and the nucleotide sequence of SEQ ID NO: 31, but the nucleotide sequence may be modified in the coding region due to the degeneracy of the codon, and the base Various modifications can be made to the coding region within a range that does not change the amino acid sequence in consideration of the codons preferred by the organism to express the sequence. It may be a polynucleotide comprising a polynucleotide sequence encoding the protein or a polynucleotide sequence having at least 80%, 90%, 95%, or 99% homology or identity thereto. In addition, if a polynucleotide sequence encoding a protein having such homology or identity and exhibiting substantially the same or corresponding efficacy as the protein, a polynucleotide sequence in which some sequences are deleted, modified, substituted or added are also within the scope of the present application. Inclusion is self-evident.
[32]
Or a probe that can be prepared from a known gene sequence, for example, a protein having the activity of α-glucosidase of the present application by hydride under stringent conditions with a complementary sequence for all or part of the polynucleotide sequence It may be included without limitation as long as it is a sequence encoding. The "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. These conditions are described in (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989;). For example, among genes with high homology, genes with homology of 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, and particularly 99% or more Under conditions that hybridize to each other and do not hybridize to genes with lower homology, or to wash conditions for general Southern hybridization at 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1 At a salt concentration and temperature corresponding to% SDS, more specifically 68° C., 0.1×SSC, and 0.1% SDS, the conditions for washing once, specifically, two to three times, can be enumerated. Hybridization requires that two polynucleotides have a complementary sequence, although a mismatch between bases is possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between bases of nucleotides capable of hybridizing to 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 substantially similar nucleotide sequences as well as isolated nucleotide fragments that are complementary to the entire sequence. Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 ℃, 63 ℃ or 65 ℃, It is not limited thereto, and may be appropriately adjusted by a person skilled in the art according to the purpose. The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[33]
[34]
In the present application, the term "enhancement of activity" means that the activity of a protein exhibited by the original microorganism in its natural state or in a pre-mutation state, that is, compared with the intrinsic activity, means that the activity is increased, and the activity of a specific protein is The concept also includes the introduction of an activity that imparts its activity by introducing the protein into a microorganism that does not have it. The "intrinsic activity" refers to an active state of a protein originally exhibited by a microorganism in its natural state or in an unmutated state.
[35]
Specifically, the "enhancement of activity" is not particularly limited thereto, but includes not only increasing the activity of the protein itself to induce an effect that is more than the original function, but also increasing the intrinsic gene activity, an intrinsic gene due to internal or external factors The activity may be increased by amplification, introduction of a gene from the outside, replacement or modification of a promoter, and an increase in enzyme activity due to mutation. For example, increasing the intracellular copy number of the gene encoding the protein, a method of modifying the gene expression control sequence encoding the polypeptide, encoding the polypeptide on the chromosome with a mutated gene to increase the polypeptide activity A method of modifying the gene encoding the polypeptide on the chromosome by inducing a mutation in the gene on the chromosome encoding the polypeptide so as to replace the gene or to enhance the activity of the polypeptide. It can be carried out by a method of inserting, etc., but is not limited to the method described above.
[36]
In the above, the increase in the copy number of the gene is not particularly limited thereto, but may be performed in a form operably linked to a vector, or may be performed by being inserted into a chromosome in a host cell. Specifically, a vector capable of replicating and functioning irrespective of the host, in which the polynucleotide encoding the protein of the present application is operably linked, may be introduced into a host cell. Alternatively, a vector capable of inserting the polynucleotide into a chromosome in a host cell to which the polynucleotide is operably linked may be introduced into a chromosome of a host cell. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, by homologous recombination. Since the vector of the present application can be inserted into a chromosome by causing homologous recombination, it may further include a selection marker for confirming whether the chromosome is inserted. The selection marker is for selecting cells transformed with a vector and confirming the insertion of the desired polynucleotide, and confers a selectable phenotype such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins. Markers may be used, but are not limited thereto. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, and thus transformed cells can be selected.
[37]
In the present application, the term "vector" may be a DNA preparation containing a polynucleotide sequence encoding the target protein operably linked to an appropriate expression control sequence so that the target protein can be expressed in a suitable host. The expression control sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling termination of transcription and translation, It is not limited thereto. Vectors can be transformed into a suitable host cell and then replicated or function independently of the host genome, or can be integrated into the genome itself. The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or a cosmid vector, and as a plasmid vector, pDZ system, pBR system, pUC system , pBluescriptII system, pGEM system, pTZ system, pCL system, pET system, etc. can be used.
[38]
In addition, the vector may include a polynucleotide sequence encoding a signal peptide. In the present application, the term "signal peptide" refers to a protein capable of secreting a protein of interest to the outside of a cell, which may act by being expressed in a fused state with a gene encoding the target protein or in a separate state. In the present application, the type of the signal peptide is not particularly limited as long as it can be secreted outside the cell while maintaining the function of the target protein. For example, CgR0949, NCgl2101, CgR1834 and ST2 (respectively SEQ ID NOs: 14 to 17) presented as an example of a signal peptide in the present application may be used, and furthermore, a person skilled in the art selects an appropriate known signal peptide to obtain α-glucose. It can be used for the purpose of secretion and expression of sidase.
[39]
In the present application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression, but is not limited thereto. The expression cassette may generally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell.
[40]
In addition, the term "operably linked" in the above means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a protein of interest of the present application and the gene sequence are functionally linked.
[41]
Next, modifying the expression control sequence to increase the expression of the polynucleotide is not particularly limited thereto, but deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or It may be performed by inducing a mutation in the sequence in combination, or by replacing with a nucleic acid sequence having a stronger activity. Specifically, it can be performed by replacing it with a strong promoter. The expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, and the like.
[42]
A strong promoter may be connected to the top of the polynucleotide expression unit instead of the original promoter, but is not limited thereto. Examples of known strong promoters include cj1 to cj7 promoters (Korea Patent Publication No. 0620092), spl1,7 or 13 promoters (Korea Patent Publication No. 1787170), PgapA promoter, lac promoter, trp promoter, trc promoter, tac promoter , Lambda phage PR promoter, PL promoter and tet promoter may be included.
[43]
In addition, modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but mutations in the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence, or a combination thereof to further enhance the activity of the polynucleotide sequence It can be carried out by inducing or by replacement with a polynucleotide sequence modified to have a stronger activity. However, it is not limited thereto.
[44]
Such enhancement of protein activity is generally 1%, 10%, 25% based on the activity or concentration in the wild-type protein or the initial microbial strain, or the activity or concentration thereof appears to have no activity of the corresponding protein. %, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, may increase up to 1000% or 2000%, but is not limited thereto.
[45]
[46]
As described above, the microorganisms in the genus Corynebacterium of the present application may improve the ability to produce L-amino acids by enhancing the activity of α-glucosidase. Therefore, the microorganism of the genus Corynebacterium of the present application can be used for the production of L-amino acids.
[47]
In the present application, the term "L-amino acid" generally refers to a basic structural unit of a protein constituting the body of an organism in which an amino group and a carboxyl group are bonded to the same carbon atom. The L-amino acid is, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine. , L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and any one selected from the group consisting of L-valine It can be more than that. In addition, the L-amino acid may be, for example, an L-amino acid derived from L-aspartic acid in the biosynthetic pathway of microorganisms, and may be an L-amino acid biosynthesized using L-aspartic acid as a substrate or intermediate. The L-amino acid derived from L-aspartic acid may be any one or more selected from the group consisting of L-lysine, L-threonine, and L-isoleucine, as a more specific example, but is not limited thereto.
[48]
In the present application, "a microorganism producing L-amino acid" may be a microorganism capable of producing and accumulating L-amino acid from a carbon source in a medium. Microorganism producing the L- amino acid, but not the type is not particularly limited, Enterobacter ( Enterbacter ) genus Escherichia ( Escherichia ), An air Winiah ( Erwinia ) genus, Serratia marcescens ( Serratia ) genus Pseudomonas ( Pseudomonas ) It may be a microorganism belonging to the genus, Providencia genus, Corynebacterium genus and Brevibacterium genus. More specifically , it may be a microorganism belonging to the genus Corynebacterium . In the present application, the "microorganisms of the genus Corynebacterium" specifically refers to Corynebacterium glutamicum , Corynebacterium ammoniagenes), Brevibacterium Lactobacillus buffer momentum ( Brevibacterium lactofermentum ), Breda thinning pan rim Solarium Plastic ( Brevibacterium flavu m), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens ), etc. However, it is not necessarily limited thereto. More specifically, the microorganism producing the L-amino acid may be Corynebacterium glutamicum, but is not limited thereto.
[49]
The microorganisms of the genus Corynebacterium with enhanced α-glucosidase activity of the present application can produce L-amino acids with a higher L-amino acid production yield than the microorganism before the protein activity is enhanced, that is, unmodified microorganism have.
[50]
Another aspect of the present application is a method for producing L-amino acids, comprising culturing a microorganism of the genus Corynebacterium that produces L-amino acids with enhanced activity of the α-glucosidase in a medium.
[51]
The method for producing the L-amino acid may further include recovering the L-amino acid from the cultured medium or microorganism.
[52]
Microorganisms and L-amino acids with enhanced activity of α-glucosidase are as described above.
[53]
In the present application, the term "culture" means growing the microorganism in an appropriately controlled environmental condition. The cultivation process of the present application may be performed in a suitable medium and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art according to the selected strain. Specifically, the culture may be a batch type, continuous type, and fed-batch type, but is not limited thereto.
[54]
As a carbon source contained in the medium, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, Fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid may be included, and these substances may be used individually or as a mixture, but are not limited thereto. As nitrogen sources contained in the medium, organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, anmonium carbonate, and ammonium nitrate are It may be included, and these nitrogen sources may be used alone or in combination. However, it is not limited thereto. The number of personnel included in the medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt, but is not limited thereto. In addition, the medium may contain a metal salt such as magnesium sulfate or iron sulfate, and other amino acids, vitamins, and suitable precursors may be included. These media or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.
[55]
The pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture in an appropriate manner during the culture. In addition, during cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas may be injected into the culture to maintain the aerobic state of the culture, and no gas or nitrogen, hydrogen or carbon dioxide gas may be injected to maintain the anaerobic and microaerobic state. The temperature of the culture may be usually 25 to 40 °C, specifically 27 to 35 °C. The cultivation period may be continued until the production amount of the desired useful substance is obtained, and specifically, may be 10 to 100 hours. However, it is not limited thereto.
[56]
The present application can further recover and/or purify the L-amino acid produced in the culturing step, and the method is a suitable method known in the art according to a cultivation method, for example, a batch, continuous, or fed-batch culture method. The desired L-amino acid can be recovered from the medium by using the method, but is not limited thereto. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, and the like may be used, and a desired L-amino acid may be recovered from the cultured medium or microorganism using a suitable method known in the art.
[57]
[58]
Another aspect of the present application provides a method for increasing L-amino acid production, comprising enhancing the activity of α-glucosidase in microorganisms.
[59]
Another aspect of the present application provides the use of α-glucosidase to increase L-amino acid production.
[60]
The'increased L-amino acid production' refers to increasing the L-amino acid production ability to produce L-amino acid with a higher L-amino acid production yield than the microorganism before the protein activity is enhanced, that is, an unmodified microorganism I can.
[61]
Mode for carrying out the invention
[62]
Hereinafter, the present application will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited to these examples.
[63]
[64]
Example 1: Construction of a vector for introducing α-glucosidase gene
[65]
[66]
In order to confirm the effect of the α-glucosidase aglA gene, as an example, the aglA gene (SEQ ID NO: 2) derived from Bifidobacterium adolescentis was used in the strain of Corynebacterium glutamicum . A vector for insertion on the chromosome was constructed. In order to amplify the PgapA promoter derived from Corynebacterium clutamicum, a primer designed to insert an EcoRⅠ restriction enzyme site at the 5'end of the PgapA promoter (SEQ ID NO: 3) and an NdeI restriction enzyme site designed to be inserted at the 3'end A primer (SEQ ID NO: 4) was synthesized. As a result, PgapA promoter DNA fragments containing an EcoRI restriction enzyme site at the 5'end and an NdeI restriction enzyme site at the 3'end were obtained, respectively. PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 30 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.
[67]
[68]
PgapA promoter amplification primer
[69]
Forward: 5'-TCA GAATTC TTGGGATTACCATTGAAGCC-3' (SEQ ID NO: 3)
[70]
Reverse: 5'-TCA CATATG GTGTCTCCTCTAAAGATTGT-3' (SEQ ID NO: 4)
[71]
[72]
Primer designed to insert the NdeI restriction enzyme site and the signal peptide for protein secretion at the initiation codon position to amplify the ORF of the aglA gene derived from Bifidobacterium adolescence based on the reported nucleotide sequence (sequence Numbers 5 to 8) and a primer designed to include a Spe I restriction enzyme site at the bottom of the stop codon (SEQ ID NO: 9) were synthesized.
[73]
Signal peptides were exemplified by selecting four proteins (SEQ ID NOs: 14 to 17, respectively) as proteins that help AglA enzyme to go out of cells well. The primer sequence and the amino acid sequence of the signal peptide are as follows (Table 1).
[74]
[75]
[Table 1]
aglA ORF amplification primer Forward direction with SP1 5'-TCA CAT ATG caaataaaccgccgaggcttcttaaaagccaccgcaggacttgccactatcggcgctgccagcatgtttatgccaaaggccaacgcccttggagca ACGAATTTCAATCGTTCCA -3' (SEQ ID NO: 5)
Forward direction with SP2 5'-TCA CAT ATG CATTCAAAGGAAGAGTTAACAGTGCGTAAAGGAATTTCCCGCGTCCTCTCGGTAGCGGTTGCTAGTTCAATCGGATTCGGAACTGTACTGACAGGCACCGGCATCGCAGCAGCTCAAGAC ACGAATTTCAATCGTTCCA -3' (SEQ ID NO: 6)
Forward direction with SP3 5'-TCA CAT ATG CGTAAGTTCCGCAATACTGCAATCGCACTGGTTTCAGCTGCTGCTATCTCCCTCGGTGGAGTTACTGCTGCAACCGCTCAGGAAGCT ACGAATTTCAATCGTTCCA -3' (SEQ ID NO: 7)
Forward direction with SP4 5'-TCA CAT ATG AAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTTCTATTGCTACAAACGCGTACGCT ACGAATTTCAATCGTTCCA -3' (SEQ ID NO: 8)
Reverse 5'-TCA ACTAGT TCA GAGCTGAATCACGACTC-3' (SEQ ID NO: 9)
Mall ORF amplification primer Forward direction with SP3 5'-TCA CAT ATG CGTAAGTTCCGCAATACTGCAATCGCACTGGTTTCAGCTGCTGCTATCTCCCTCGGTGGAGTTACTGCTGCAACCGCTCAGGAAGCT TCAGGCATCAAACTTTCTTC -3' (SEQ ID NO: 10)
Reverse 5'-TCA ACTAGT TCA ATTTAGCCTATAGATAC-3' (SEQ ID NO: 11)
Primer for Ima1 ORF amplification Forward direction with SP3 5'-TCA CAT ATG CGTAAGTTCCGCAATACTGCAATCGCACTGGTTTCAGCTGCTGCTATCTCCCTCGGTGGAGTTACTGCTGCAACCGCTCAGGAAGCT ACTATTTCTTCTGCACATCC -3' (SEQ ID NO: 12)
Reverse 5'-TCA ACTAGT TCA TTCGCTGATATATATTCTT-3' (SEQ ID NO: 13)
Signal peptide SP1 CgR0949 MQINRRGFLKATAGLATIGAASMFMPKANALGA (SEQ ID NO: 14)
SP2 NCgl2101 MHSKEELTVRKGISRVLSVAVASSIGFGTVLTGTGIAAAQD (SEQ ID NO: 15)
SP3 CgR1834 MRKFRNTAIALVSAAAISLGGVTAATAQEA (SEQ ID NO: 16)
SP4 ST2 MKKNIAFLLASMFVFSIATNAYA (SEQ ID NO: 17)
[76]
Using the genomic DNA of Bifidobacterium adolescentis as a template, an aglA ORF fragment including an NdeI restriction enzyme site and each signal peptide at the 5'end and a Spe I restriction enzyme site at the 3'end was obtained. PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 2 minutes were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.
[77]
[78]
After the four PCR amplification products were treated with restriction enzymes at both ends, pDZ vector (Korean Patent Registration No. 10-0924065) was ligated with a DNA fragment obtained by treatment with restriction enzymes EcoRI and SalI, and pDZ-PgapA -SP1-aglA( B.al ), pDZ-PgapA-SP2-aglA( B.al ), pDZ-PgapA-SP3-aglA( B.al ) and pDZ-PgapA-SP4-aglA( B.al ) vectors. Was produced.
[79]
[80]
In addition, in order to prepare a microorganism having enhanced activity of another α-glucosidase, an α-glucosidase gene derived from the following strain was obtained. Specifically, using the EAMY_1858 (malL) genome of Erwinia amylovora CFBP1430 and the IMA1 genome DNA of Saccharomyces cerevisiae as templates, the NdeI restriction site and each A mall, Ima1 ORF fragment containing a signal peptide and a Spe I restriction enzyme site at the 3'end was obtained. PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 2 minutes were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.
[81]
After the two PCR amplification products were treated with restriction enzymes at both ends, pDZ vector (Korean Patent Registration No. 10-0924065) was ligated with a DNA fragment obtained by treatment with restriction enzymes EcoRI and SalI, and pDZ-PgapA malL--SP3 (E .am ), pDZ-PgapA-SP3-Ima1 ( S.ce was produced) vector.
[82]
[83]
Example 2: Preparation of microorganisms into which α-glucosidase was introduced
[84]
[85]
In order to introduce the gene encoding α-glucosidase into the Corynebacterium glutamicum strain, each of the six vectors produced in Example 1 was used as a Corynebacterium glutamicum lysine producer KCCM11016P (the microorganism It is have been published KFCC10881, the material deposited in the Budapest Treaty servant international deposition feedback given by the Accession number KCCM11016P, Republic of Korea Patent No. electric pulse method (Van der Rest the number 10-0159812) et al ., Appl. Microbiol. Biotechnol. 52:541-545, 1999) and selected colonies into which each gene was introduced by homologous chromosome recombination . In order to select colonies by PCR method, primers of SEQ ID NOs: 18 and 19 were used.
[86]
[87]
Primer for confirming the introduction of aglA gene
[88]
Forward: 5'-GACCATTTATTCGCAACTGTG-3' (SEQ ID NO: 18)
[89]
Reverse: 5'-TCTGCAAGGCGTTCGGAATT-3' (SEQ ID NO: 19)
[90]
[91]
Each of the transformed strains KCCM11016P::PgapA-SP1-aglA ( B.al ), KCCM11016P::PgapA-SP2-aglA ( B.al ), KCCM11016P::PgapA-SP3-aglA ( B.al ) KCCM11016P: : PgapA-SP4-aglA ( B.al ), KCCM11016P :: PgapA-SP3-malL (E .am ), and KCCM11016P :: PgapA-SP3-Ima1 ( S.ce was named).
[92]
[93]
Example 3: Confirmation of protein expression in lysine-producing microorganisms into which α-glucosidase was introduced
[94]
[95]
The parent strain, Corynebacterium glutamicum KCCM11016P, was used as a control, and KCCM11016P::PgapA-SP1-aglA ( B.al ), KCCM11016P::PgapA-SP2-aglA ( B.al ) prepared in Example 2 was used as a control. , KCCM11016P :: PgapA-SP3-aglA ( B.al ) KCCM11016P :: PgapA-SP4-aglA ( B.al ), KCCM11016P :: PgapA-SP3-malL (E .am ), and KCCM11016P :: PgapA-SP3- After culturing 6 species of Ima1 ( S.ce ) by the method shown in Example 4 below, the supernatant was obtained by high-speed centrifugation. Using a part of the obtained supernatant, the expression of α-glucosidase enzyme in the culture medium was measured by the SDS-PAGE method. As a result, the protein expressed at the 70 Kda position could be confirmed (FIG. 1).
[96]
[97]
Example 4: Evaluation of L-amino acid production ability of lysine-producing microorganisms introduced with α-glucosidase
[98]
[99]
The parent strain, Corynebacterium glutamicum KCCM11016P, was used as a control, and KCCM11016P::PgapA-SP1-aglA ( B.al ), KCCM11016P::PgapA-SP2-aglA ( B.al ) prepared in Example 2 was used as a control. , KCCM11016P::PgapA-SP3-aglA( B.al ), KCCM11016P::PgapA-SP4-aglA( B.al ), KCCM11016P::PgapA-SP3-malL( E.am ), and KCCM11016P::PgapA-SP3 -Ima1 ( S.ce ) 6 species were cultured for a certain period of time by the following method, and then the lysine concentration was measured, and the results are shown in Table 2. First, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of a seed medium, and cultured with shaking at 30° C. for 20 hours and 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of the production medium, followed by incubation with shaking at 32° C. for 72 hours and 200 rpm. Compositions of the species medium and production medium are as follows, respectively. After completion of the culture, the concentration of L-lysine was measured by HPLC (Waters 2478).
[100]
[101]

[102]
Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH 2 PO 4 4 g, K 2 HPO 4 8 g, MgSO 4 7H 2 O 0.5 g, biotin 100 µg, thiamine HCl 1000 µg, Calcium-pantothenic acid 2000 ㎍, nicotinamide 2000 ㎍ (based on 1 liter of distilled water)
[103]
[104]

[105]
Glucose 100 g, (NH 4 ) 2 SO 4 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH 2 PO 4 1 g, MgSO 4 7H 2 O 0.5 g, Biotin 100 ㎍, Thiamine HCl 1000 ㎍, calcium-pantothenic acid 2000 ㎍, nicotinamide 3000 ㎍, CaCO 3 30 g (based on 1 liter of distilled water)
[106]
[107]
[Table 2]
strain No. Lysine concentration (g/L) Lysine average concentration (g/L)
KCCM11016P One 36.6 36.8
2 36.4
3 37.5
KCCM11016P::PgapA-SP1-aglA(B.al) One 40.0 39.1
2 38.5
3 38.9
KCCM11016P::PgapA-SP2-aglA(B.al) One 39.0 38.7
2 38.4
3 38.7
KCCM11016P::PgapA-SP3-aglA(B.al) One 39.8 40.4
2 41.9
3 39.5
KCCM11016P::PgapA-SP4-aglA(B.al) One 39.3 38.8
2 38.5
3 38.6
KCCM11016P::PgapA-SP3-malL(E.am) One 38.1 38.4
2 38.5
3 38.6
KCCM11016P::PgapA-SP3-ima1(S.ce) One 37.9 38.0
2 38.1
3 38.1
[108]
From the above results, it was found that the lysine-producing ability was increased compared to the control in all six lysine-producing strains into which the α-glucosidase expression ability was introduced. In particular, KCCM11016P::PgapA-SP3-aglA( B.al ) showed the highest productivity increase rate. In the culture of microorganisms, it is a very meaningful result that the production capacity of lysine increases from at least 3.2% to at most 9.7% due to the regulation of the activity of genes other than the biosynthetic pathway. In addition, as it was confirmed that the L-amino acid production capacity was increased without adding isomaltose and maltose, which are expected to be utilized as a substrate, in the medium, L-glucosidase activity of the present application was increased. The effect of increasing the amino acid production ability was confirmed. In addition, from the above results, it is expected to show a higher increase rate when an appropriate signal peptide is used by a person skilled in the art's selection.
[109]
[110]
The prepared KCCM11016P::PgapA-SP2-aglA ( B.al ) was named Corynebacterium glutamicum CA01-7523, and as of March 5, 2018, the Korea Microbial Conservation Center (KCCM), an international depository under the Budapest Treaty. ) And was given accession number KCCM12228P.
[111]
[112]
Example 5: Preparation of a CJ3P strain into which α-glucosidase was introduced and analysis of lysine production ability
[113]
[114]
In order to confirm whether the same effect as the above is also in the strain belonging to the other Corynebacterium glutamicum that produces L-lysine, three kinds of mutations in the wild-type Corynebacterium glutamicum ATCC13032 strain [pyc (P458S), hom (V59A), lysC (T311I)] was introduced to have L-lysine-producing ability for Corynebacterium glutamicum CJ3P (Binder et al. Genome Biology 2012, 13:R40) strain, Example 2 PgapA-SP3-aglA ( B.al ) was introduced in the same manner as described above to prepare a strain into which α-glucosidase was introduced. The produced strain was named CJ3P::PgapA-SP3-aglA ( B.al ). The control group CJ3P strain and CJ3P::PgapA-SP3-aglA ( B.al ) were cultured in the same manner as in Example 4, and the lysine-producing ability was analyzed and shown in Table 3 below.
[115]
[116]
[Table 3] Analysis of lysine production capacity
strain No. Lysine concentration (g/L) Lysine average concentration (g/L)
CJ3P One 8.0 8.0
2 7.6
3 8.4
CJ3P::PgapA-SP3-aglA(B.al) One 8.6 8.7
2 8.3
3 9.1
[117]
As a result of lysine concentration analysis, it was confirmed that the lysine yield was increased in the strain into which α-glucosidase was introduced. In addition, in the culture of microorganisms, it is a very meaningful result that the lysine production capacity increases by 8.8% due to the regulation of the activity of genes other than the biosynthetic pathway. In addition, it is expected to show a higher rate of increase when an appropriate signal peptide is used according to the selection of a person skilled in the art.
[118]
[119]
Example 6: Preparation of threonine-producing strain into which α-glucosidase was introduced and analysis of threonine-producing ability
[120]
[121]
In order to clearly confirm the change in L-threonine production ability due to the introduction of α-glucosidase, homoserine dehydrogena that produces homoserine, a common intermediate of L-threonine and L-isoleucine biosynthetic pathways. It was strengthened by introducing a mutation into the gene encoding the homoserin dehydrogenase. Specifically, the CJ3P::PgapA-SP3-aglA ( B.al ) strain used in Example 5 previously known hom (G378E) mutation (R. Winkels, S. et al., Appl. Microbiol. Biotechnol. 45) , 612-620, 1996) was prepared. In addition, a strain in which only the hom (G378E) mutation was introduced into CJ3P was also prepared as a control. Recombinant vectors for mutagenesis were constructed in the following manner.
[122]
In order to construct a vector introducing hom (G378E), first, genomic DNA extracted from the wild-type Corynebacterium glutamicum ATCC13032 strain was used as a template, 5'from the 1131 to 1134th position of the hom gene back and forth, respectively, about 600 bp away. The primers (SEQ ID NOs: 20 and 21) in which the restriction enzyme XbaI recognition site was inserted into the fragment and the 3'fragment were synthesized. In addition, primers (SEQ ID NOs: 22 and 23) for substituting the base sequence of the hom gene were synthesized. The pDZ-hom (G378E) plasmid was constructed in a form in which DNA fragments (600 bp each) located at the 5'and 3'ends of the hom gene were linked to a pDZ vector (Korean Patent Registration No. 10-0924065).
[123]
[124]
Primer for XbaI recognition site insertion
[125]
5'fragment: 5'- TCCTCTAGACTGGTCGCCTGATGTTCTAC -3' (SEQ ID NO: 20)
[126]
3'fragment: 5'- GACTCTAGATTAGTCCCTTTCGAGGCGGA -3' (SEQ ID NO: 21)
[127]
[128]
Primer for hom gene replacement
[129]
5'- GCCAAAACCTCCACGCGATC -3' (SEQ ID NO: 22)
[130]
5'-ATCGCGTGGAGGTTTTGGCT -3' (SEQ ID NO: 23)
[131]
[132]
Using the chromosome of the wild-type strain as a template, a 5'end gene fragment was produced through PCR using primers (SEQ ID NOs: 20 and 22). PCR conditions were denatured at 94° C. for 2 minutes, then denatured at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40 seconds were repeated 30 times, followed by polymerization at 72° C. for 10 minutes. In the same way, a gene fragment located at the 3'end of the hom gene was produced through PCR using primers (SEQ ID NOs: 21 and 23). The amplified DNA fragment was purified using Quiagen's PCR Purification kit, and then used as an insert DNA fragment for vector construction. Meanwhile, after treatment with restriction enzyme XbaI, the pDZ vector heat-treated at 65° C. for 20 minutes and the inserted DNA fragment amplified through the above PCR were ligated using an Infusion Cloning Kit, transformed into E. coli DH5α, and kanamycin (25 mg/l ) Was spread on the conjugated LB solid medium. After selecting a colony transformed with a vector into which the target gene is inserted through PCR using the primers of SEQ ID NOs: 20 and 21, a plasmid was obtained through a commonly known plasmid extraction method, and the base substitution mutation of hom (G378E) was obtained from The vector pDZ-hom (G378E) was constructed for introduction into the phase.
[133]
[134]
Then, by introducing the prepared pDZ-hom (G378E) vector into CJ3P and CJ3P::PgapA-SP3-aglA ( B.al ) strains in the same manner as in Example 2, CJ3P::hom (G378E) and CJ3P::PgapA-SP3-aglA( B.al )-hom(G378E) strain was obtained. The obtained two strains were cultured in the same manner as in Example 4, and the threonine production concentration was analyzed and shown in Table 4 below.
[135]
[136]
[Table 4] Threonine production concentration
strain No. Thr concentration (g/L) Thr average concentration (g/L)
CJ3P::hom(G378E) One 1.1 1.23
2 1.5
3 1.1
CJ3P::PgapA-SP3-aglA(B.al)-hom(G378E) One 1.4 1.60
2 1.8
3 1.6
[137]
As a result of threonine concentration analysis, it was confirmed that the threonine concentration was increased in the strain into which α-glucosidase was introduced. In the cultivation of microorganisms, it is a very meaningful result that the production capacity of threonine increases by 30% due to the regulation of the activity of genes other than the biosynthetic pathway. In addition, it is expected to show a higher rate of increase when an appropriate signal peptide is used according to the selection of a person skilled in the art.
[138]
[139]
Example 7: Preparation of isoleucine-producing strain into which α-glucosidase was introduced and analysis of isoleucine-producing ability
[140]
[141]
In order to confirm the effect of the introduction of α-glucosidase on the L-isoleucine production ability, a mutation was introduced into the gene encoding the known threonine dehydratase and enhanced. Specifically, CJ3P used in Example 6:: PgapA-SP3-aglA ( B.al )-hom (G378E) strain known ilvA (V323A) mutation (S. Morbach et al., Appl. Enviro. Microbiol., 62(12): 4345-4351, 1996) was prepared. In addition, a strain in which only the ilvA (V323A) mutation was introduced into CJ3P::hom (G378E) as its control was also produced. Recombinant vectors for mutagenesis were constructed in the following manner.
[142]
[143]
In order to construct a vector introducing ilvA (V323A), first, genomic DNA extracted from the wild-type Corynebacterium glutamicum ATCC13032 strain was used as a template at positions 966 to 969 of the hom gene, respectively, at a position approximately 600 bp back and forth. The primers (SEQ ID NOs: 24 and 25) in which the restriction enzyme XbaI recognition site was inserted into the fragment and the 3'fragment were synthesized. In addition, primers (SEQ ID NOs: 26 and 27) for substituting the base sequence of the ilvA gene were synthesized. The pDZ-ilvA (V323A) plasmid was constructed in a form in which DNA fragments (600 bp each) located at the 5'and 3'ends of the ilvA gene were linked to a pDZ vector (Korean Patent Registration No. 10-0924065).
[144]
[145]
Primer for XbaI recognition site insertion
[146]
5'fragment: 5'-ACGGATCCCAGACTCCAAAGCAAAAGCG -3' (SEQ ID NO: 24)
[147]
3'fragment: 5'- ACGGATCCAACCAAACTTGCTCACACTC -3' (SEQ ID NO: 25)
[148]
[149]
ilvA gene replacement primer
[150]
5'- ACACCACGGCAGAACCAGGTGCAAAGGACA -3' (SEQ ID NO: 26)
[151]
5'-CTGGTTCTGCCGTGGTGTGCATCATCTCTG -3' (SEQ ID NO: 27)
[152]
[153]
Using the chromosome of the wild-type strain as a template, a 5'end gene fragment was produced through PCR using primers (SEQ ID NOs: 24 and 26). PCR conditions were denatured at 94° C. for 2 minutes, then denatured at 94° C. for 1 minute, annealing at 56° C. for 1 minute, and polymerization at 72° C. for 40 seconds were repeated 30 times, followed by polymerization at 72° C. for 10 minutes. In the same way, a gene fragment located at the 3'end of the ilvA gene was produced through PCR using primers (SEQ ID NOs: 25 and 27). The amplified DNA fragment was purified using Quiagen's PCR Purification kit, and then used as an insert DNA fragment for vector construction. Meanwhile, after treatment with restriction enzyme XbaI, the pDZ vector heat-treated at 65° C. for 20 minutes and the inserted DNA fragment amplified through the above PCR were ligated using an Infusion Cloning Kit, transformed into E. coli DH5α, and kanamycin (25 mg/l ) Was spread on the conjugated LB solid medium. After selecting the colony transformed with the vector into which the target gene is inserted through PCR using the primers of SEQ ID NOs: 24 and 25, the ilvA (V323A) mutation was introduced into the chromosome by obtaining a plasmid through a commonly known plasmid extraction method. The following vector pDZ-ilvA (V323A) was constructed.
[154]
[155]
Then, the prepared pDZ-ilvA (V323A) vector was introduced into CJ3P::hom (G378E) and CJ3P:: PgapA-SP3-aglA ( B.al )-hom (G378E) strain in the same manner as in Example 2. Thus, CJ3P::hom(G378E)-ilvA(V323A) and CJ3P::PgapA-SP3-aglA( B.al )-hom(G378E)-ilvA(V323A) were obtained. The obtained two strains were cultured in the same manner as in Example 4, and the isoleucine production concentration was analyzed, and the results are shown in Table 5 below.
[156]
[157]
[Table 5] Isoleucine production concentration
strain No. Ile concentration (g/L) Ile average concentration (g/L)
CJ3P::hom(G378E)-ilvA(V323A) One 0.12 0.10
2 0.10
3 0.09
CJ3P::PgapA-SP3-aglA(B.al)-hom(G378E)-ilvA(V323A) One 0.15 0.13
2 0.11
3 0.13
[158]
As shown in the results of Table 5, it was confirmed that the isoleucine concentration was increased in the strain into which α-glucosidase was introduced. In the culture of microorganisms, it is a very meaningful result that the production capacity of isoleucine increases by 30% due to the regulation of the activity of genes other than the biosynthetic pathway. In addition, it is expected to show a higher rate of increase when an appropriate signal peptide is used according to the selection of a person skilled in the art.
[159]
[160]
From the above description, those skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.
[161]

Claims
[Claim 1]
A microorganism of the genus of Corynebacterium that produces L-amino acids with enhanced α-glucosidase activity .
[Claim 2]
The method of claim 1 wherein the α- glucosidase is Bifidobacterium Adolfo LES sentiseu ( Bifidobacterium adolescentis ), a control See Winiah amyl ( Erwinia amylovora ), or a saccharide as MY process three Levy jiae ( Saccharomyces cerevisiae a) pedigree microbe.
[Claim 3]
The microorganism according to claim 1, wherein the α-glucosidase is encoded by the aglA gene.
[Claim 4]
The microorganism according to claim 1, wherein the α-glucosidase is a protein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 28, or SEQ ID NO: 29.
[Claim 5]
The microorganism of claim 1, wherein the L-amino acid is at least one selected from the group consisting of L-lysine, L-threonine, and L-isoleucine.
[Claim 6]
The microorganism according to claim 1, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum .
[Claim 7]
α- glucosidase genus Corynebacterium, which produce L- amino acids enhance the activity of (the genus of Corynebacterium ), the production method of the L- amino acid which comprises culturing a microorganism in a culture medium.
[Claim 8]
The method of claim 7, wherein the method further comprises recovering L-amino acid from the cultured medium or microorganism.
[Claim 9]
The method of claim 7, wherein the α- glucosidase is Bifidobacterium Adolfo LES sentiseu ( Bifidobacterium adolescentis ), a control See Winiah amyl ( Erwinia amylovora ), or a saccharide as MY process three Levy jiae ( Saccharomyces cerevisiae a) pedigree Method for the production of L-amino acids.
[Claim 10]
The method of claim 7, wherein the α-glucosidase is encoded by the aglA gene.
[Claim 11]
The method of claim 7, wherein the α-glucosidase is a protein comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 28, or SEQ ID NO: 29.
[Claim 12]
The method of claim 7, wherein the L-amino acid is at least one selected from the group consisting of L-lysine, L-threonine, and L-isoleucine.
[Claim 13]
A method of increasing L-amino acid production comprising enhancing α-glucosidase expression in a microorganism.
[Claim 14]
Use of α-glucosidase to increase L-amino acid production.