Corynebacterium glutamicum mutant strain with improved l-lysine production ability and method for producing l-lysine using the same

Abstract

To provide a Corynebacterium glutamicum mutant strain with improved L-lysine production ability.SOLUTION: The present invention relates to a Corynebacterium glutamicum mutant strain with improved L-lysine production ability and a method for producing L-lysine using the same. The mutant strain can improve the L-lysine production yield compared to a parent strain by increasing or enhancing the expression of a gene encrypting aspartate semialdehyde dehydrogenase.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a Corynebacterium glutamicum mutant strain having improved L-lysine-producing ability and a method for producing L-lysine using the same. [Background technology]

[0002] L-lysine is an essential amino acid that cannot be synthesized in the human or animal body and must be supplied from an external source. It is generally produced by fermentation using microorganisms such as bacteria or yeast. L-lysine can be produced by using wild-type strains obtained in nature or mutant strains modified to improve its L-lysine production ability. Recently, in order to improve the efficiency of L-lysine production, genetic engineering has been applied to microorganisms such as Escherichia coli and Corynebacterium, which are commonly used to produce L-amino acids and other useful substances, to develop a variety of recombinant strains or mutants with excellent L-lysine production ability and methods for producing L-lysine using the same.

[0003] According to Korean Patent Registration Nos. 10-0838038 and 10-2139806, L-lysine production ability can be improved by changing the nucleotide sequence or amino acid sequence of a gene encoding a protein containing an enzyme involved in L-lysine production to increase expression of the gene or by removing unnecessary genes. In addition, Korean Patent Publication No. 10-2020-0026881 discloses a method for changing the existing promoter of a gene encoding an enzyme involved in L-lysine production to a promoter with strong activity in order to increase expression of the gene.

[0004] As described above, various methods for increasing L-lysine productivity have been developed. However, since there are several dozen types of proteins, such as enzymes, transcription factors, and transport proteins, that are directly and indirectly related to L-lysine production, much research is still needed to determine whether L-lysine productivity can be increased by changing the activity of these proteins. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent No. 10-0838038 [Patent Document 2] Korean Patent No. 10-2139806 [Patent Document 3] Korean Patent Publication No. 10-2020-0026881 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a Corynebacterium glutamicum mutant strain with improved L-lysine productivity.

[0007] Another object of the present invention is to provide a method for producing L-lysine using the mutant strain. [Means for solving the problem]

[0008] The present inventors conducted research to develop a new mutant strain of Corynebacterium glutamicum with improved L-lysine production ability, and as a result, they found that substitution of an amino acid at a specific position in the amino acid sequence of the asd gene, which encodes aspartate semialdehyde dehydrogenase involved in the L-lysine biosynthesis pathway, increases the amount of L-lysine produced, thereby completing the present invention.

[0009] One aspect of the present invention provides a Corynebacterium glutamicum mutant strain having enhanced L-lysine productivity due to enhanced aspartate semialdehyde dehydrogenase activity.

[0010] As used herein, the term "aspartate-semialdehyde dehydrogenase" refers to an enzyme involved in the third step of the L-lysine biosynthesis pathway, which catalyzes the reaction of producing aspartate 4-semialdehyde from aspartyl phosphate using NADPH.

[0011] According to one embodiment of the present invention, the aspartate semialdehyde dehydrogenase may be derived from a strain of the genus Corynebacterium. Specifically, the Corynebacterium strains include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uteri ... Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenesThe bacterium may be, but is not limited to, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0012] As used herein, "enhanced activity" means that the expression of a gene encoding a protein such as a target enzyme, transcription factor, or transport protein is newly introduced or increased, resulting in an increased expression level compared to a wild-type strain or a strain before transformation. Such activity enhancement includes cases where the activity of a protein itself is increased compared to the activity of the protein originally possessed by a microorganism due to nucleotide substitution, insertion, deletion, or a combination thereof that encodes the gene, as well as cases where the overall level of enzymatic activity within the cell is higher than that of a wild-type strain or a strain before transformation due to increased expression or translation of the gene encoding the protein, etc., in combination with these.

[0013] According to one embodiment of the present invention, the activity of aspartate semialdehyde dehydrogenase may be enhanced by site-specific mutagenesis of a gene encoding aspartate semialdehyde dehydrogenase.

[0014] According to one embodiment of the present invention, the gene encoding the aspartate semialdehyde dehydrogenase may be represented by the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2.

[0015] According to one embodiment of the present invention, the activity of the aspartate semialdehyde dehydrogenase may be enhanced by substituting one or more amino acids in the region of amino acids 10 to 100 in the amino acid sequence of the gene encoding the aspartate semialdehyde dehydrogenase.

[0016] More specifically, the genetic mutation in the present invention may be a consecutive or discontinuous substitution of one or more amino acids in the region of amino acids 10 to 100, preferably one, two, three, four, or five amino acids in the region of amino acids 20 to 90, 30 to 80, 30 to 50, or 60 to 80.

[0017] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new amino acid sequence of the asd gene encoding aspartate semialdehyde dehydrogenase was obtained by substituting alanine (Ala) at position 35 with glycine (Gly), serine (Ser) at position 39 with glutamic acid (Glu), and alanine (Ala) at position 78 in the amino acid sequence of the asd gene of a Corynebacterium glutamicum strain. Such a Corynebacterium glutamicum mutant strain may contain an asd gene with the amino acid sequence of SEQ ID NO: 3.

[0018] According to another embodiment of the present invention, a Corynebacterium glutamicum mutant strain having a new amino acid sequence of the asd gene encoding aspartate semialdehyde dehydrogenase was obtained by substituting alanine (Ala) at position 35 with glycine (Gly), arginine (Arg) at position 38 with leucine (Ile), serine (Ser) at position 39 with glutamic acid (Glu), and alanine (Ala) at position 78 in the amino acid sequence of the asd gene of a Corynebacterium glutamicum strain. Such a Corynebacterium glutamicum mutant strain may contain an asd gene with the amino acid sequence of SEQ ID NO: 5.

[0019] As used herein, "improved productivity" means increased L-lysine productivity compared to a parent strain. The parent strain refers to a wild-type or mutant strain that can be mutated, and includes strains that can be directly mutated or transformed with a recombinant vector. In the present invention, the parent strain may be a wild-type Corynebacterium glutamicum strain or a strain mutated from the wild type.

[0020] According to one embodiment of the present invention, the parent strain may be a Corynebacterium glutamicum strain deposited at the Korean Culture Center of Microorganisms under accession number KCCM12969P on April 2, 2021, as a mutant strain in which mutations have been induced in the sequences of genes involved in lysine production (e.g., lysC, zwf, and hom genes) (hereinafter referred to as the "C. glutamicum DS1 strain").

[0021] According to one embodiment of the present invention, the Corynebacterium glutamicum mutant strain having improved L-lysine productivity exhibits increased L-lysine productivity compared to the parent strain due to the inclusion of an amino acid mutation in the asd gene encoding aspartate semialdehyde dehydrogenase. In particular, the L-lysine production amount is increased by 3% or more, specifically 3 to 40%, and more specifically 5 to 30%, compared to the parent strain, and the mutant strain can produce 65 to 90 g of L-lysine, preferably 67 to 80 g of L-lysine per liter of strain culture medium.

[0022] According to one embodiment of the present invention, a Corynebacterium glutamicum mutant can be produced by a recombinant vector containing a mutant in which the amino acid sequence of the gene encoding aspartate semialdehyde dehydrogenase is partially substituted in the parent strain.

[0023] As used herein, the term "part" does not mean the entire amino acid sequence, nucleotide sequence, or polynucleotide sequence, and may be, but is not limited to, 1 to 300, preferably 1 to 100, and more preferably 1 to 50.

[0024] The term "mutant" used in the present invention refers to a mutant in which one or more amino acids are substituted in the region of amino acids 10 to 100 in the amino acid sequence of the aspartate semialdehyde dehydrogenase gene involved in the biosynthesis of L-lysine.

[0025] According to one embodiment of the present invention, the mutant in which the amino acids at positions 35, 39, and 78 in the amino acid sequence of the aspartate semialdehyde dehydrogenase gene are substituted may have the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 4.

[0026] According to one embodiment of the present invention, the mutant in which the amino acids at positions 35, 38, 39, and 78 in the amino acid sequence of the aspartate semialdehyde dehydrogenase gene are substituted may have the amino acid sequence of SEQ ID NO: 5 or the nucleotide sequence of SEQ ID NO: 6.

[0027] As used herein, the term "vector" refers to an expression vector capable of expressing a target protein in a suitable host cell, and refers to a gene construct containing the necessary regulatory elements operably linked to allow expression of a gene insert. Here, "operably linked" means that the gene to be expressed and its regulatory sequences are functionally linked to each other in a manner that allows expression of the gene. "Regulatory elements" include a promoter for transcription, an optional operator sequence for transcription control, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence for controlling the termination of transcription and translation. Examples of such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors.

[0028] The "recombinant vector" used in the present invention can replicate independently of the genome of a suitable host cell after being transformed into the host cell, or can be sewn into the genome itself. In this case, the "suitable host cell" is one in which the vector can replicate and which contains an origin of replication, which is a specific base sequence from which replication is initiated.

[0029] For the transformation, a suitable vector introduction technique can be selected depending on the host cell, allowing the gene of interest to be expressed in the host cell. For example, vector introduction can be performed by electroporation, heat shock, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene may be inserted into the host cell's chromosome or located extrachromosomally, as long as it can be expressed in the host cell.

[0030] The host cell includes cells transfected, transformed, or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell containing a recombinant vector of the invention may be a recombinant host cell, recombinant cell, or recombinant microorganism.

[0031] Furthermore, the recombinant vector of the present invention may contain a selection marker, which is used to select transformants (host cells) transformed with the vector, and only cells expressing the selection marker can survive in a medium treated with the selection marker, allowing for the selection of transformed cells. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0032] The gene inserted into the recombinant vector for transformation of the present invention is transposed into a host cell such as a microorganism of the genus Corynebacterium by homologous crossover.

[0033] According to one embodiment of the present invention, the host cell may be a strain of the genus Corynebacterium, for example, the Corynebacterium glutamicum DS1 strain.

[0034] Another aspect of the present invention provides a method for producing L-lysine, comprising: a) culturing the Corynebacterium glutamicum mutant strain in a medium; and b) recovering L-lysine from the mutant strain or the medium in which the mutant strain has been cultured.

[0035] The culture may be performed using an appropriate medium and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be, but is not limited to, a liquid medium. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0036] According to one embodiment of the present invention, the medium should be appropriately adapted to meet the requirements of a specific strain and can be modified by a person skilled in the art. Culture media for Corynebacterium strains can be found in known literature (Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington DC, USA, 1981), but are not limited thereto.

[0037] According to one embodiment of the present invention, the medium can contain a variety of carbon sources, nitrogen sources, and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures, but are not limited to these. Usable nitrogen sources include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean malt, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources can also be used individually or in mixtures, but are not limited to these. Usable phosphorus sources can include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain, but is not limited to, metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Other essential growth substances, such as amino acids and vitamins, may also be included. Appropriate precursors can also be used for the culture medium. The medium or individual components may be added to the culture solution in an appropriate manner during the culture process, either batchwise or continuously, but are not limited to these.

[0038] According to one embodiment of the present invention, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microbial culture medium in an appropriate manner during cultivation. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Additionally, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture medium to maintain an aerobic state in the culture medium. The temperature of the culture medium can generally be 20°C to 45°C, for example, 25°C to 40°C. The cultivation period can be continued until a desired amount of useful substance is produced, and can be, for example, 10 to 160 hours.

[0039] According to one embodiment of the present invention, the step of recovering L-lysine from the cultured mutant strain and the medium in which the mutant strain has been cultured can be carried out by collecting or recovering the produced L-lysine from the medium using a suitable method known in the art depending on the culture method, including, but not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion).

[0040] According to one embodiment of the present invention, the step of recovering lysine can be carried out by centrifuging the culture medium at low speed to remove the biomass, and separating the resulting supernatant by ion exchange chromatography.

[0041] According to one embodiment of the present invention, the step of recovering L-lysine can include a step of purifying L-lysine. [Effects of the Invention]

[0042] The Corynebacterium glutamicum mutant strain according to the present invention can improve the L-lysine production yield compared to the parent strain by increasing or enhancing the expression of the gene encoding aspartate semialdehyde dehydrogenase. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 shows the structure of the pCGI (asd A35G / S39E / A78V) vector containing an asd gene in which the 35th amino acid in the amino acid sequence of the asd gene has been substituted from alanine to glycine, the 39th amino acid from serine to glutamic acid, and the 78th amino acid from alanine to valine, according to one embodiment of the present invention. [Figure 2]FIG. 1 shows the structure of the pCGI (asd A35G / R38L / S39E / A78V) vector containing an asd gene in which the 35th amino acid in the amino acid sequence of the asd gene has been substituted from alanine to glycine, the 38th amino acid from arginine to leucine, the 39th amino acid from serine to glutamic acid, and the 78th amino acid from alanine to valine, according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention will be described in more detail below. However, such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0045] Example 1. Preparation of Corynebacterium glutamicum mutant strains To produce a Corynebacterium glutamicum mutant strain with enhanced aspartate semialdehyde dehydrogenase activity, we used Corynebacterium glutamicum DS1 and E. coli DH5a (HIT Competent Cells). TM , Cat No. RH618) was used.

[0046] The Corynebacterium glutamicum DS1 was cultured at 30°C in CM-broth medium (pH 6.8) containing 5 g of glucose, 2.5 g of NaCl, 5.0 g of yeast extract, 1.0 g of urea, 10.0 g of polypeptone, and 5.0 g of beef extract in 1 L of distilled water.

[0047] The E. coli DH5a was cultured at 37°C on LB medium containing 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract in 1 L of distilled water.

[0048] The antibiotics kanamycin and streptomycin were manufactured by Sigma, and DNA sequencing analysis was performed by Macrogen Corporation.

[0049] 1-1. Construction of recombinant vectors To enhance the activity of aspartate semialdehyde dehydrogenase, which is involved in the lysine biosynthesis pathway, and thereby increase lysine productivity, an aspartate semialdehyde dehydrogenase enhancement gene was introduced into the strain. The method used in this example involved specific mutation of the asd gene, which encodes aspartate semialdehyde dehydrogenase, to increase expression. The amino acid sequence of the asd gene was substituted by substituting glycine (alanine) at position 35, glutamic acid (serine) at position 39, and valine (alanine) at position 78. A 1,000-bp segment of the left arm and a 1,785-bp segment of the right arm were amplified by PCR from the C. glutamicum genome, centered on the amino acids 35, 39, and 78 of the asd gene. The resulting fragments were then ligated by overlap PCR and cloned into the recombinant vector pCGI (see Kim et al., Journal of Microbiological Methods 84 (2011) 128-130). The resulting plasmid was designated pCGI(asd A35G / S39E / A78V) (see Figure 1). The primers listed in Table 1 below were used to amplify each gene fragment to construct the plasmid.

[0050] [Table 1]

[0051] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, Takara Bio Inc., Japan), 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added to the reaction mixture. 1 pM of oligonucleotides and 10 ng of Corynebacterium glutamicum DS1 chromosomal DNA were used as the template. 30 cycles were performed in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent). PCR conditions were: (i) denaturation step: 94°C for 30 seconds; (ii) annealing step: 58°C for 30 seconds; and (iii) extension step: 72°C for 1–2 minutes (allowing 2 minutes of polymerization time per 1 kb fragment).

[0052] The gene fragment thus prepared was cloned into the pCGI vector using self-assembly cloning. The vector was transformed into E. coli DH5a, spread onto an LB-agar plate containing 50 μg / ml kanamycin, and cultured at 37°C for 24 hours. The resulting colonies were isolated to confirm that the insert was correctly present in the vector. The vector was then isolated and used to recombine Corynebacterium glutamicum strains.

[0053] A common step in these methods was to amplify the gene of interest by PCR from Corynebacterium glutamicum ATCC13032 genomic DNA, insert it into the pCGI vector using a self-assembled cloning strategy, and select it from E. coli DH5a. Chromosomal base substitution was performed by amplifying each gene fragment individually and then using overlap PCR to generate the target DNA fragment. PCR amplification enzymes used for gene recombination were Ex Taq polymerase (Takara) and Pfu polymerase (Solgent), and various restriction enzymes and DNA modifying enzymes were from NEB, using the supplied buffers and protocols.

[0054] 1-2. Production of mutant strains The pCGI(asd A35G / S39E / A78V) vector was used to generate the mutant strain DS4. The vector was prepared at a final concentration of 1 μg / μl or higher and the Corynebacterium glutamicum DS1 strain was electroporated (see Tauch et al., FEMS Microbiology Letters 123 (1994) 343-347) to induce primary recombination. The electroporated strain was then plated onto CM-agar plates containing 20 μg / μl kanamycin to isolate colonies. PCR and nucleotide sequence analysis were used to confirm proper insertion into the induced site on the genome. The isolated strain was then inoculated into CM-agar liquid medium containing streptomycin and cultured overnight. The colonies were then plated onto agar plates containing the same concentration of streptomycin to isolate secondary recombination. Finally, the isolated colonies were checked for resistance to kanamycin, and then the strains without antibiotic resistance were examined by nucleotide sequence analysis to determine whether a mutation had been introduced into the asd gene (see reference [Schafer et al., Gene 145 (1994) 69-73]). Finally, a Corynebacterium glutamicum mutant (DS4) carrying the mutated asd gene was obtained.

[0055] Example 2. Preparation of Corynebacterium glutamicum mutant strains A Corynebacterium glutamicum mutant strain was prepared in the same manner as in Example 1, except that the 35th amino acid in the amino acid sequence of the asd gene was substituted from alanine to glycine, the 38th amino acid from arginine to leucine, the 39th amino acid from serine to glutamic acid, and the 78th amino acid from alanine to valine. To construct the plasmid, the primers listed in Table 1 were used to amplify each gene fragment, and the resulting plasmid, pCGI(asd A35G / R38L / S39E / A78V) vector (see Figure 2), was used to construct the mutant strain DS4-1. Finally, a Corynebacterium glutamicum mutant (DS4-1) carrying the mutant asd gene was obtained.

[0056] Experimental Example 1: Comparison of L-lysine productivity among mutant strains The L-lysine productivity of the parent strain Corynebacterium glutamicum DS1 strain was compared with that of the lysine-producing mutant strains DS4 and DS4-1 prepared in Examples 1 and 2.

[0057] Each strain was inoculated into a 100 ml flask containing 10 ml of lysine medium having the composition shown in Table 2 below, and cultured with shaking at 180 rpm at 30°C for 48 hours. After the culture was completed, the amount of L-lysine produced was measured by HPLC (Shimazu, Japan) for lysine analysis, and the results are shown in Table 3.

[0058] [Table 2]

[0059] [Table 3]

[0060] As shown in Table 3, the Corynebacterium glutamicum mutant strains DS4 and DS4-1 were found to have optimal amino acids substituted at specific positions in the amino acid sequence of the asd gene (amino acids 35, 39, and 78, or amino acids 35, 38, 39, and 78) to enhance the lysine biosynthetic pathway. This resulted in approximately 10.0% and 5.7% increases in L-lysine productivity, respectively, compared to the parent strain, Corynebacterium glutamicum DS1. These results demonstrate that enhanced expression of the asd gene enhances the supply of lysine precursors, thereby improving the strain's L-lysine production ability.

[0061] The present invention has been described above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

Claims

1. A mutant strain of Corynebacterium glutamicum having enhanced L-lysine productivity due to enhanced aspartate semialdehyde dehydrogenase activity.

2. The Corynebacterium glutamicum mutant according to claim 1 , wherein the activity of aspartate semialdehyde dehydrogenase is enhanced by introducing a site-specific mutation into a gene encoding aspartate semialdehyde dehydrogenase.

3. The Corynebacterium glutamicum mutant according to claim 2 , wherein the gene encoding the aspartate semialdehyde dehydrogenase is represented by the amino acid sequence of SEQ ID NO:

1.

4. The Corynebacterium glutamicum mutant strain according to claim 1 , wherein the mutant strain comprises the amino acid sequence represented by SEQ ID NO: 3 or 5.

5. a) culturing the mutant strain of claim 1 in a medium; and b) recovering L-lysine from the mutant strain or the medium in which the mutant strain has been cultured.

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