Corynebacterium genus microorganism having improved L-amino acid production capacity, and method for producing L-amino acid using same

Abstract

The present invention relates to a Corynebacterium genus microorganism having an improved L-amino acid production capacity and a method for producing an L-amino acid using the same, the Corynebacterium genus microorganism being weakened or inactivated by the activity of a carotenoid biosynthase, whereby the production yield of the L-amino acid can be improved as compared to the parent strain, and more particularly, to a Corynebacterium genus microorganism having an improved L-amino acid production capacity and to a method for producing an L-amino acid using the same.

Description

Technical Field

[0001] The present invention relates to a Corynebacterium microorganism having improved L-amino acid production ability and a method for producing L-amino acids using the same. Background Art

[0002] Amino acids are the basic building blocks of proteins that make up the body. They are divided into essential amino acids that must be taken in through food because they are not synthesized or difficult to synthesize in the body, and non-essential amino acids that can be synthesized through metabolism in the body. Naturally synthesized L-amino acids are used not only as amino acid enhancers, but also as raw materials in various fields such as food, health functional foods, pharmaceuticals, and cosmetics.

[0003] L-amino acid utilizes the microorganism obtained under the natural state or the variant microorganism changed in the mode of improving its amino acid production capacity and is industrially produced by fermentation. In recent years, in order to improve the production efficiency of L-amino acid, with microorganisms such as Escherichia coli and Corynebacterium as the object, the gene recombination technology is applicable. When utilizing the gene recombination technology, by increasing the activity of the enzyme participating in the biosynthesis of amino acids or suppressing the feedback brought by the produced L-amino acid, the amino acid production capacity can be improved. In addition, the expression of the gene that discharges amino acids can be regulated to improve the amino acid production capacity. U.S. Patent No. 5,972,663 describes artificially overexpressing genes such as mex, bmr, qacA, etc. of multiple strains of Escherichia coli to increase the production capacity of L-cysteine, L-cystine, N-ethynylserine, and thiazolidine derivatives. European Patent No. 1,016,710 describes artificially overexpressing genes yahN, yeaS, yfiK, and yggA of Escherichia strains to increase the production capacity of L-glutamate, L-lysine, L-threonine, L-alanine, L-histidine, L-proline, L-arginine, L-valine, and L-isoleucine. Korean Patent No. 10-1023925 describes artificially overexpressing gene yddG of Escherichia strains to increase the production capacity of L-tryptophan and L-phenylalanine.

[0004] However, since various proteins such as enzymes, transcription factors, and transport proteins are involved in the biosynthetic pathway of amino acids, a great deal of research is still required to develop recombinant microorganisms or mutants with improved L-amino acid production capabilities.

[0005] Prior art literature

[0006] Patent Literature

[0007] U.S. Patent No. 5972663

[0008] European Patent No. 1016710

[0009] Korean Patent No. 10-1023925 Summary of the invention

[0010] An object of the present invention is to provide a Corynebacterium microorganism having an improved L-amino acid production ability.

[0011] Another object of the present invention is to provide a method for producing L-amino acids using the above-mentioned microorganism.

[0012] One embodiment of the present invention provides a Corynebacterium microorganism in which the activity of a carotenoid biosynthesis enzyme is weakened or inactivated and the L-amino acid production ability is improved.

[0013] The "carotenoid biosynthetic enzyme" used in the present invention refers to an enzyme involved in the carotenoid biosynthetic pathway. Corynebacterium glutamicum contains glycosylated C50 carotenoid decaprenoxanthin as a yellow pigment, which is synthesized from isopentenyl pyrophosphate generated in the non-mevalonate pathway, and undergoes farnesyl pyrophosphate, geranylgeranylpyrophosphate, lycopene and flavuxanthin. It is known that genes such as crtE, crtB, crtB2, crtI, crtEb, crtYe / f are involved in such a carotenoid biosynthetic pathway.

[0014] The crtB gene encodes phytoene synthase, and is a cluster comprising Cgl0626 (geranylgeranyl pyrophosphate synthase), Cgl0624 (phytoene / squalene synthetase) and Cgl0623 (phytoene dehydrogenase and related proteins).

[0015] The crtB2 gene encodes phytoene synthase, and is a cluster comprising Cgl2433 (phytoene / squalene synthetase) gene, Cgl2432 (phytoene dehydrogenase and related proteins) gene and Cgl2431 (phytoene dehydrogenase and related proteins) gene.

[0016] The carotenoid biosynthetic enzyme in the present invention is encoded by a gene constituting the crtB gene, the crtB2 gene or the crtB / crtB2 gene cluster and may be a polypeptide having phytoene synthase activity, but is not limited thereto.

[0017] The nucleic acid sequence and protein sequence information of the above-mentioned carotenoid biosynthetic enzymes can be obtained from known sequence databases (eg, GenBank, UniProt).

[0018] The "weakened activity" used in the present invention refers to the expression level of the gene encoding the target enzyme, transcription factor, transport protein, etc., compared with the original microorganism, i.e., the wild-type strain or the strain before transformation. Such weakening of activity includes the following situations: the activity of the protein itself is reduced compared with the activity of the protein possessed by the original microorganism by nucleotide substitution, insertion, deletion or a combination thereof of the encoding gene, and the overall protein activity level in the cell is lower than that of the wild-type strain or the strain before transformation by hindering the expression of the gene encoding it or hindering translation, and also includes the combination thereof.

[0019] According to a specific example of the present invention, the weakening of the activity of the carotenoid biosynthetic enzyme may be achieved by inserting, replacing, deleting, or a combination thereof, all or part of the gene encoding the carotenoid biosynthetic enzyme.

[0020] The term "inactivation" used in the present invention refers to the following situations: the expression of genes encoding proteins such as enzymes, transcription factors, and transport proteins is not expressed at all compared with the original microorganism, i.e., the wild-type strain or the strain before transformation, or even if expressed, it has no activity.

[0021] According to one specific example of the present invention, the gene encoding the carotenoid biosynthesis enzyme may be at least one selected from Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431.

[0022] According to a specific example of the present invention, the Cgl0626 gene comprises the base sequence of SEQ ID NO:1, and can encode the amino acid sequence of SEQ ID NO:2. In addition, the Cgl0624 gene comprises the base sequence of SEQ ID NO:3, and can encode the amino acid sequence of SEQ ID NO:4. In addition, the Cgl0623 gene comprises the base sequence of SEQ ID NO:5, and can encode the amino acid sequence of SEQ ID NO:6. In addition, the Cgl2433 gene comprises the base sequence of SEQ ID NO:7, and can encode the amino acid sequence of SEQ ID NO:8. In addition, the Cgl2432 gene comprises the base sequence of SEQ ID NO:9, and can encode the amino acid sequence of SEQ ID NO:10. In addition, the Cgl2431 gene comprises the base sequence of SEQ ID NO:11, and can encode the amino acid sequence of SEQ ID NO:12.

[0023] The base sequence or amino acid sequence of the carotenoid biosynthetic enzyme according to the present invention can be composed of or must contain a sequence having 70% or more, 80% or more, 90% or more, 98% or more, 99% or more, or 99.9% or more homology with the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 or the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, and can have the original function.

[0024] As used in the present invention, "productivity is improved" means that the productivity of L-amino acids is increased compared to the parent strain. The parent strain refers to a wild type or mutant strain that is the object of mutation, including a target that is directly mutated or a target that is transformed by a recombinant vector, etc. In the present invention, the parent strain may be a wild-type Corynebacterium microorganism or strain or a Corynebacterium microorganism or strain mutated from the wild type.

[0025] The above-mentioned Corynebacterium microorganism may be any known microorganism in the art, for example, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium spp. singulare), Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudocorynebacterium pseudopelargi) or Corynebacterium flavescens, but is not limited thereto.

[0026] According to a specific example of the present invention, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0027] The Corynebacterium microorganism having improved L-amino acid productivity according to the present invention can improve L-amino acid productivity by weakening or inactivating the activity of carotenoid biosynthesis enzyme.

[0028] According to a specific example of the present invention, the Corynebacterium microorganism having improved L-amino acid production ability may be formed by lacking one or more of Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431 encoding carotenoid biosynthetic enzymes.

[0029] Specifically, the above-mentioned Corynebacterium microorganism with improved L-amino acid production ability shows an increased L-amino acid production ability compared to the parent strain, and in particular, the L-amino acid production amount can be increased by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 250%, 5%, 90%, 95% or 100%, or can be increased by 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times, but is not limited to this.

[0030] According to a specific example of the present invention, the above-mentioned L-amino acid can be one or more selected from L-alanine, L-isoleucine, L-valine, L-leucine, L-methionine, L-asparagine, L-cysteine, L-glutamine, L-serine, L-threonine, L-phenylalanine, L-tryptophan, L-tyrosine, L-aspartic acid, L-glutamic acid, L-arginine, L-histidine, L-lysine, L-glycine, L-proline and L-citrulline.

[0031] For example, the L-amino acid may be L-arginine, L-citrulline, L-lysine or L-glutamine, but is not limited thereto.

[0032] The Corynebacterium microorganism according to one embodiment of the present invention can be realized by using a recombinant vector in which a gene encoding a carotenoid biosynthesis enzyme is deleted from a parent strain.

[0033] "Carrier (vector)" used in the present invention refers to all types of nucleic acid sequence transport structures used as a means for delivering and expressing target genes to host cells. Unless otherwise indicated, it can be indicated that the nucleic acid sequence carried is inserted into the host cell gene for expression and / or expressed independently. Such a carrier includes the necessary regulatory elements that are operably connected in order to express the gene insert, "operably linked (operably linked)" refers to that the target gene and its regulatory sequence are functionally combined with each other and connected in a manner that can carry out gene expression, and "regulatory elements" include promoter sequences for implementing transcription, arbitrary operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating the termination of transcription and translation.

[0034] The vector used in the present invention is not particularly limited as long as it can be replicated in the host cell, and any vector known in the art can be used. As an example of the above-mentioned vector, a plasmid, a cosmid, a virus and a phage in a natural state or a recombinant state can be cited. For example, as a phage vector or a cosmid vector, there are pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc., and as a plasmid vector, there are pBR system, pUC system, pBluescriptII system, pGEM system, pTZ system, pCL system and pET system, etc., but it is not limited thereto.

[0035] The above-mentioned vector can be representatively constructed as a vector for cloning or a vector for expression. The vector for expression can use conventional vectors used in the art for expressing foreign genes or proteins in plants, animals or microorganisms, and can be constructed by various methods well known in the art.

[0036] The "recombinant vector" used in the present invention can be constructed with prokaryotic cells or eukaryotic cells as hosts, can be replicated independently of the genome of the host cell, or can be sutured to the genome itself. The above-mentioned host cells are capable of replicating the vector and may include a replication origin as a specific base sequence for starting replication. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, a strong promoter (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that can enable transcription generally includes a ribosome binding site for the initiation of translation and a transcription / translation termination sequence. When eukaryotic cells are used as hosts, the replication origins initiated in the eukaryotic cells contained in the vector include f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin, but are not limited thereto. In addition, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV) can be used, and usually have a polyadenylation sequence as a transcription termination sequence.

[0037] The above-mentioned recombinant vector may include a selection marker, which is used to screen transformants (host cells) transformed by the vector. In the culture medium treated with the above-mentioned selection marker, only cells expressing the selection marker can survive, so transformed cells can be screened. As representative examples, the above-mentioned selection markers include ampicillin, kanamycin, streptomycin, chloramphenicol, etc., but are not limited thereto.

[0038] By inserting the above-mentioned recombinant vector into a host cell, a transformant can be prepared, and the above-mentioned transformant can be obtained by introducing the recombinant vector into a suitable host cell. The host cell is a cell that can stably and continuously clone or express the above-mentioned expression vector, and any host cell known in the art can also be used.

[0039] When prokaryotic cells are transformed to produce recombinant microorganisms, Escherichia coli such as E. coli DH5α, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, and E. coli XL1-Blue; Corynebacterium strains; Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis; various enteric bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and Pseudomonas can be used as host cells, but are not limited to these.

[0040] When eukaryotic cells are transformed to produce recombinant microorganisms, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells can be used as host cells, for example, Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc., but are not limited to these.

[0041] As used in the present invention, "transformation" refers to the phenomenon of introducing foreign DNA into host cells to cause artificial genetic changes, and "transformant" refers to a host cell into which foreign DNA is introduced and which stably maintains the expression of the target gene.

[0042] In the above transformation, a suitable vector introduction technique is selected according to the host cell, so that the target gene or the recombinant vector containing the same can be expressed in the host cell. For example, the vector introduction can be carried out by electroporation, heat shock, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof, but is not limited thereto. As long as the transformed gene can be expressed in the host cell, it can be included, without being limited to being inserted into the chromosome of the host cell or being located outside the chromosome.

[0043] The transformant includes cells transfected, transformed or infected with the recombinant vector according to the present invention in vivo or in vitro, and can be used as the same term as a recombinant host cell, a recombinant cell or a recombinant microorganism.

[0044] The gene inserted into the recombinant vector for transformation of the present invention can be replaced into a host cell such as a microorganism of the genus Corynebacterium by homologous recombination and crossover.

[0045] According to a specific example of the present invention, the host cell may be a microorganism of the genus Corynebacterium, for example, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

[0046] Another aspect of the present invention provides a method for producing L-amino acids, comprising the steps of: culturing the above-mentioned Corynebacterium microorganism in a culture medium; and recovering the L-amino acids from the above-mentioned microorganism or the culture medium in which the microorganism is cultured.

[0047] The above-mentioned culture can be carried out according to suitable culture medium and culture conditions known in the art, and those skilled in the art can easily adjust the culture medium and culture conditions for use. Specifically, the above-mentioned culture medium can be a liquid culture medium, but is not limited thereto. The culture method can include, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof, but is not limited thereto.

[0048] According to a specific example of the present invention, the above-mentioned culture medium must meet the requirements of specific strains in a suitable manner and can be appropriately deformed by those skilled in the art. Regarding the culture medium of Corynebacterium microorganisms, reference can be made to known literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981), but it is not limited thereto.

[0049] According to a specific example of the present invention, the culture medium may contain various carbon sources, nitrogen sources and trace element components. As the carbon source that can be used, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, etc. are included; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; fatty acids such as palmitic acid, stearic acid, linoleic acid; alcohols such as glycerol and ethanol; organic acids such as acetic acid. These substances can be used alone or in the form of a mixture, but are not limited to this. As the nitrogen source that can be used, peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be included. Nitrogen sources can also be used alone or in the form of a mixture, but are not limited to this. As a source of phosphorus that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts can be included, but are not limited to this. In addition, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate required for growth, but is not limited to this. In addition, essential growth substances such as amino acids and vitamins may be included. In addition, precursors suitable for the culture medium may be used. The above-mentioned culture medium or individual components may be added to the culture solution in batches or continuously in a suitable manner during the culture process, but are not limited thereto.

[0050] According to a specific example of the present invention, during the culture process, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microbial culture solution in an appropriate manner to adjust the pH of the culture solution. In addition, during the culture process, a defoaming agent such as fatty acid polyethylene glycol ester can be used to suppress the generation of bubbles. Further, in order to maintain the aerobic state of the culture solution, oxygen or oxygen-containing gas (such as air) can be injected into the culture solution. The temperature of the culture solution can generally be 20°C to 45°C, for example, 25°C to 40°C. The culture time can continue until the desired production of the useful substance is obtained, for example, it can be 10 to 160 hours.

[0051] According to a specific example of the present invention, in the above-mentioned step of recovering L-amino acids from the cultured Corynebacterium microorganism and the culture medium for culturing the above-mentioned microorganism, the produced L-amino acids can be collected or recovered from the culture medium according to the culture method and using a suitable method known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion) and other methods can be used, but are not limited thereto.

[0052] According to a specific example of the present invention, in the above-mentioned step of recovering L-amino acids, the culture medium may be centrifuged at a low speed to remove biomass, and the obtained supernatant may be separated by ion exchange chromatography.

[0053] According to a specific example of the present invention, the step of recovering L-amino acids may include a step of purifying L-amino acids.

[0054] According to a specific example of the present invention, the above-mentioned L-amino acid can be one or more selected from L-alanine, L-isoleucine, L-valine, L-leucine, L-methionine, L-asparagine, L-cysteine, L-glutamine, L-serine, L-threonine, L-phenylalanine, L-tryptophan, L-tyrosine, L-aspartic acid, L-glutamic acid, L-arginine, L-histidine, L-lysine, L-glycine, L-proline and L-citrulline.

[0055] For example, the L-amino acid may be L-arginine, L-citrulline, L-lysine or L-glutamine, but is not limited thereto.

[0056] The Corynebacterium microorganism according to the present invention can improve the production yield of L-amino acids as compared with the parent strain by weakening or inactivating the activity of carotenoid biosynthesis enzymes. DETAILED DESCRIPTION

[0057] The present invention will be described in more detail below, but such description is provided for illustrative purposes only to help understand the present invention, and the scope of the present invention is not limited to such illustrative description.

[0058] Example 1. Preparation of L-arginine-producing strain with disrupted carotenoid biosynthetic enzyme gene

[0059] In order to prepare a strain in which one or more of the genes Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431 constituting carotenoid biosynthetic enzymes in an L-arginine-producing strain derived from Corynebacterium glutamicum was disrupted, Corynebacterium glutamicum 14GR (KCCM13219P) and Escherichia coli DH5a (HIT Competent cells) as L-arginine-producing strains were used. TM , Cat No.RH618).

[0060] The above-mentioned Corynebacterium glutamicum 14GR was prepared in a mixture of 1L of distilled water, 10.5g of 98% glucose, 1g of beef extract, 4g of yeast extract, 2g of polypeptone, 2g of NaCl and 40g of (NH 4 ) 2 SO 4 The cells were cultured in ARG-broth (pH 7.2) at 30°C.

[0061] The above-mentioned E. coli DH5a was cultured at 37°C in an LB medium composed of 1 L of distilled water, 10.0 g of tryptone, 10.0 g of NaCl, and 5.0 g of yeast extract.

[0062] The antibiotic kanamycin (kanamycin) used was a product of Sigma.

[0063] DNA sequencing analysis and gene synthesis were commissioned to MacroGene Co., Ltd.

[0064] 1-1. Recombinant vector

[0065] A recombinant vector was prepared for disrupting one or more of the genes Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431 constituting carotenoid biosynthetic enzymes. On the genome of Corynebacterium glutamicum, the 600-650 bp portion of the left arm and the 600-650 bp portion of the right arm were amplified by PCR based on the start codon and stop codon of each gene, and after being connected by the overlap PCR method, they were cloned into the pk19mobsacB (ATCC, 87098) vector. In order to prepare the above-mentioned plasmid, each gene fragment was amplified using the primers in Table 1 below.

[0066]

Table 1

[0067]

[0068] PCR was performed using the above primers under the following conditions. Using a thermocycler (TP600, TAKARABIO Inc., Japan), 1 pM of oligonucleotides and 10 ng of chromosomal DNA of Corynebacterium glutamicum ATCC 13032 were used as templates in a reaction solution to which 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) was added, and 25 to 30 cycles were performed in the presence of 1 unit of pfu-X DNA polymerase mixture (Solgent). Regarding the PCR implementation conditions, it was performed under the conditions of (i) denaturation step: 94°C for 30 seconds, (ii) annealing step: 58°C for 30 seconds, and (iii) extension step: 72°C for 1 to 2 minutes (giving 2 minutes of polymerization time per 1 kb).

[0069] The gene fragments prepared as described above were cloned into the pk19mobsacB vector using self-assembly cloning. The vector was transformed into E. coli DH5a, smeared on LB-agar plates containing 50 μg / ml of kanamycin, and cultured at 37°C for 24 hours. The resulting colonies were isolated, and after confirming whether the insert was correctly present in the vector, the vector was isolated and used for the recombination of the Corynebacterium glutamicum strain.

[0070] As a common process in the above method, the amplification of the corresponding gene is amplified by the PCR method from the genomic DNA of Corynebacterium glutamicum ATCC13032, and according to the strategy, it is inserted into the pk19mobsacB vector by the self-assembled cloning method and screened in E.coli DH5a. Chromosomal base substitution is to amplify the genes of each fragment separately, and the target DNA fragment is manufactured by stacked PCR. In gene manipulation, Ex Taq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR amplification enzymes, and NEB products were used for various restriction enzymes and DNA modification enzymes, and they were used according to the supplied buffer and scheme.

[0071] The Cgl0626 and Cgl0624 disrupted recombinant vectors produced by such a method were named CD1, the Cgl0623 disrupted recombinant vector was named CD2, the Cgl2433 disrupted recombinant vector was named CD3, and the Cgl2432 and Cgl2431 disrupted recombinant vectors were named CD4.

[0072] 1-2. Mutant strains of Corynebacterium glutamicum

[0073] The mutant strains were made using the cloned vectors CD1 to CD4. The strain made by destroying the recombinant vector CD1 using Cgl0626 and Cgl0624 was named DR1, the strain made by destroying the recombinant vector CD2 using Cgl0623 was named DR2, the strain made by destroying the recombinant vector CD3 using Cgl2433 was named DR3, and the strain made by destroying the recombinant vector CD4 using Cgl2432 and Cgl2431 was named DR4. The process of making each recombinant strain is as follows.

[0074] The cloning vector was prepared in a manner such that the final concentration was 1 μg / μl or more, and the electroporation method was used for Corynebacterium glutamicum 14GR (reference document [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]) to induce recombination once. At this time, the electroporated strain was smeared on an agar medium containing 50 μg / μl of kanamycin, and after the colonies were isolated, it was confirmed by PCR and base sequence analysis whether the induction position on the genome was correctly inserted. In order to induce recombination twice again in the strain isolated in this way, it was inoculated in a liquid culture medium, cultured overnight or more, smeared on an agar medium containing 10% sucrose (sucrose), and colonies were isolated. After confirming whether there was resistance to kanamycin in the finally isolated colonies, it was confirmed by base sequence analysis whether the gene was destroyed in the strain without antibiotic resistance (reference document [Schafer et al., Gene 145 (1994) 69-73]). Finally, L-arginine-producing Corynebacterium glutamicum mutant strains DR1, DR2, DR3, and DR4 were prepared in which the gene encoding the carotenoid biosynthesis enzyme was disrupted.

[0075] Experimental Example 1. Evaluation of L-arginine production ability of L-arginine-producing strains in which carotenoid biosynthesis enzyme genes were disrupted

[0076] The L-arginine-producing abilities of the parent strain, Corynebacterium glutamicum 14GR, and the L-arginine-producing Corynebacterium glutamicum DR1, DR2, DR3, and DR4 prepared in Example 1 were evaluated.

[0077] Each strain was patched in a shake flask solid seed medium and cultured at 30°C for 24 hours. The cultured colonies were inoculated into 10 ml of a shake flask titer medium and cultured at 32°C and 200 rpm for 30 hours. The components of the culture medium used here are shown in Table 2 below. After the culture was completed, the culture solution was diluted 50 times with distilled water, filtered with a 0.45 μm filter, and the L-arginine production was analyzed using a high performance liquid chromatograph (HPLC) (agilent technologies 1260infinity, agilent technologies) equipped with a column (Dionex IonPacTM CS12A) and an ultraviolet detector (195 mm), and the results are shown in Table 3 below.

[0078]

Table 2

[0079]

[0080]

Table 3

[0081] strain <![CDATA[OD 610 ]]> L-Arginine(%) Yield (%) 14GR 40.0 2.1 26.4 DR1 37.7 2.5 31.3 DR2 38.0 2.4 30.0 DR3 37.1 2.7 33.6 DR4 38.2 2.4 30.0

[0082] As shown in Table 3 above, it was confirmed that the genes encoding carotenoid biosynthesis enzymes in Corynebacterium glutamicum DR1, DR2, DR3 and DR4 were disrupted, thereby increasing the L-arginine production by about 14.3 to 28.6% and the L-arginine production yield by about 3.6 to 7.2% compared with Corynebacterium glutamicum 14GR as the parent strain.

[0083] Example 2. Preparation of L-citrulline-producing strain with disrupted carotenoid biosynthetic enzyme gene

[0084] In order to prepare a strain in which one or more of the genes Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431 constituting carotenoid biosynthetic enzymes in an L-citrulline-producing strain derived from Corynebacterium glutamicum were disrupted, Corynebacterium glutamicum CT4 and E. coli DH5a (HIT Competent cells) as L-citrulline-producing strains were used. TM , Cat No.RH618).

[0085] The above-mentioned Corynebacterium glutamicum CT4 is a strain for L-citrulline production in which the activities of ornithine carbamoyltransferase and carbamoyl phosphate synthetase are enhanced in order to overproduce citrulline (refer to Korean Patent Application No. 10-2019-0151321), and the composition is 1L of distilled water, 10.5% glucose, 1g of beef extract, 4g of yeast extract, 2g of polypeptone, 2g of NaCl, 40g of (NH4) 2 SO 4 The cells were cultured in a medium containing 20 g of agar at 30°C.

[0086] Other than this, the culture conditions of E. coli DH5a and the method for producing recombinant vectors CD1 to CD4 were the same as those in Example 1-1.

[0087] The mutant strains were made using the cloned vectors CD1 to CD4. The strain made by destroying the recombinant vector CD1 using Cgl0626 and Cgl0624 was named DC1, the strain made by destroying the recombinant vector CD2 using Cgl0623 was named DC2, the strain made by destroying the recombinant vector CD3 using Cgl2433 was named DC3, and the strain made by destroying the recombinant vector CD4 using Cgl2432 and Cgl2431 was named DC4. The process of making each recombinant strain is as follows.

[0088] The cloning vector was prepared in a manner such that the final concentration was 1 μg / μl or more, and the electroporation method was used for Corynebacterium glutamicum CT4 (reference document [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]) to induce recombination once. At this time, the electroporated strain was coated on an agar medium containing 50 μg / μl of kanamycin, and after the colonies were isolated, it was confirmed by PCR and base sequence analysis whether the induction position on the genome was correctly inserted. In order to induce recombination twice again in the strain isolated in this way, it was inoculated in a liquid culture medium, cultured overnight or more, and smeared on an agar medium containing 10% sucrose (sucrose), and the colonies were isolated. After confirming whether there was resistance to kanamycin in the finally isolated colonies, it was confirmed by base sequence analysis whether the gene was destroyed in the strain without antibiotic resistance (reference document [Schafer et al., Gene 145 (1994) 69-73]). Finally, the genes encoding carotenoid biosynthesis enzymes were disrupted and L-citrulline-producing Corynebacterium glutamicum mutants DC1, DC2, DC3, and DC4 were prepared.

[0089] Experimental Example 2. Evaluation of L-citrulline production ability of L-citrulline-producing strains with disrupted carotenoid biosynthesis enzyme genes

[0090] The L-citrulline-producing abilities of the parent strain, Corynebacterium glutamicum CT4, and the L-citrulline-producing Corynebacterium glutamicum DC1, DC2, DC3, and DC4 prepared in Example 2 were evaluated.

[0091] Each strain was patched in a shake flask solid seed medium and cultured at 30°C for 24 hours. The cultured colonies were inoculated into 10 ml of a shake flask titer medium and cultured at 30°C and 180 rpm for 30 hours. The components of the medium used here are shown in Table 4 below. After the culture was completed, the culture solution was diluted 50 times with distilled water, filtered with a 0.45 μm filter, and the L-citrulline production was analyzed using a high performance liquid chromatograph (HPLC) (agilent technologies 1260infinity, agilent technologies) equipped with a column (Dionex IonPacTM CS12A) and an ultraviolet detector (195 mm). The results are shown in Table 5 below.

[0092]

Table 4

[0093]

[0094]

Table 5

[0095] strain <![CDATA[OD 610 ]]> L-Citrulline (%) Yield (%) CT4 35.1 1.2 12.2 DC1 33.5 1.6 16.2 DC2 33.9 1.5 14.8 DC3 33.2 1.7 17.2 DC4 33.8 1.5 15.2

[0096] As shown in Table 5 above, it was confirmed that the genes encoding carotenoid biosynthesis enzymes in Corynebacterium glutamicum DC1, DC2, DC3 and DC4 were disrupted, thereby increasing the L-citrulline production by about 25.0 to 41.7% and the L-citrulline production yield by about 2.6 to 5.0%p compared to the parent strain Corynebacterium glutamicum CT4.

[0097] Example 3. Preparation of L-lysine-producing strain with disrupted carotenoid biosynthetic enzyme gene

[0098] In order to prepare a strain in which one or more of the Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431 genes constituting carotenoid biosynthetic enzymes in an L-lysine-producing strain derived from Corynebacterium glutamicum were disrupted, Corynebacterium glutamicum DS1 (KCCM12969P) and E. coli DH5a (HIT Competent cells, Cgl2433, Cg618) were used as L-lysine-producing strains.

[0099] The above-mentioned Corynebacterium glutamicum DS1 was prepared in a mixture of 1 L of distilled water, 5 g of 98% glucose, 5 g of beef extract, 4 g of yeast extract, 5 g of polypeptone, 2 g of NaCl, 40 g of (NH 4 )2 SO 4 The cells were cultured in a medium (pH 7.2) containing 20 g of agar at 30°C.

[0100] Other than this, the culture conditions of E. coli DH5a and the method for producing recombinant vectors CD1 to CD4 were the same as those in Example 1-1.

[0101] Mutant strains were produced using the produced cloning vectors CD1 to CD4. The strain produced by destroying the recombinant vector CD1 using Cgl0626 and Cgl0624 was named DK1, the strain produced by destroying the recombinant vector CD2 using Cgl0623 was named DK2, the strain produced by destroying the recombinant vector CD3 using Cgl2433 was named DK3, and the strain produced by destroying the recombinant vector CD4 using Cgl2432 and Cgl2431 was named DK4. The process of producing each recombinant strain is as follows.

[0102] The cloning vector was prepared in such a way that the final concentration was 1 μg / μl or more, and the electroporation method (reference [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]) was used to induce recombination once in Corynebacterium glutamicum DS1. At this time, the electroporated strain was coated on an agar medium containing 50 μg / μl of kanamycin, and after the colonies were isolated, PCR and base sequence analysis were used to confirm whether the induction position on the genome was correctly inserted. In order to induce recombination twice again in the strain isolated in this way, it was inoculated in a liquid culture medium, cultured overnight or more, and smeared on an agar medium containing 10% sucrose to isolate colonies. After confirming whether the colonies finally isolated had resistance to kanamycin, base sequence analysis was used to confirm whether the gene was disrupted in strains without antibiotic resistance (see reference [Schafer et al., Gene 145 (1994) 69-73]. Finally, L-lysine-producing Corynebacterium glutamicum mutant strains DK1, DK2, DK3 and DK4 were produced in which the gene encoding the carotenoid biosynthesis enzyme was disrupted.

[0103] Experimental Example 3. Evaluation of L-lysine production capacity of L-lysine-producing strains in which carotenoid biosynthesis enzyme genes were disrupted

[0104] The L-lysine production abilities of the parent strain, Corynebacterium glutamicum DS1, and the L-lysine-producing Corynebacterium glutamicum DK1, DK2, DK3, and DK4 prepared in Example 3 were evaluated.

[0105] Each strain was patched in a shake flask solid seed medium and cultured at 30°C for 24 hours. The cultured colonies were inoculated into 10 ml of a shake flask titer medium and cultured at 30°C and 180 rpm for 28 hours. The components of the culture medium used here are shown in Table 6 below. After the culture was completed, the culture solution was diluted 10 times with distilled water, filtered with a 0.45 μm filter, and the L-lysine production was analyzed using a high performance liquid chromatograph (HPLC) (agilent technologies 1260infinity, agilent technologies) equipped with a column (Dionex IonPacTM CS12A) and an ultraviolet detector (195 mm), and the results are shown in Table 7 below.

[0106]

Table 6

[0107]

[0108]

Table 7

[0109] strain <![CDATA[OD 610 ]]> L-Lysine(%) Yield (%) DS1 32 6.0 60.0 DK1 30 6.6 66.6 DK2 30 6.4 64.1 DK3 28 6.7 67.1 DK4 31 6.4 64.0

[0110] As shown in Table 7 above, it was confirmed that the genes encoding carotenoid biosynthesis enzymes were disrupted in Corynebacterium glutamicum DK1, DK2, DK3 and DK4, thereby increasing L-lysine production by approximately 6.7 to 11.7% and L-lysine production yield by 4.0 to 7.1% compared to the parent strain Corynebacterium glutamicum DS1.

[0111] Example 4. Preparation of L-glutamine-producing strain with disrupted carotenoid biosynthetic enzyme gene

[0112] In order to prepare a strain in which one or more of the genes Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431 constituting carotenoid biosynthetic enzymes in an L-glutamine-producing strain derived from Corynebacterium glutamicum were disrupted, Corynebacterium glutamicum DQ3-6 (KCCM13398P) and Escherichia coli DH5a (HIT Competent cells) as L-glutamine-producing strains were used. TM , Cat No.RH618).

[0113] The above-mentioned Corynebacterium glutamicum DQ3-6 was prepared in a mixture of 1 L of distilled water, 10.5 g of 98% glucose, 1 g of beef extract, 4 g of yeast extract, 2 g of polypeptone, 2 g of NaCl, 5 g of urea, 50 mg of alanine and 40 g of (NH4) 2 SO 4 The cells were cultured in broth medium (pH 7.2) at 30°C.

[0114] Other than this, the culture conditions of E. coli DH5a and the method for producing recombinant vectors CD1 to CD4 were the same as those in Example 1-1.

[0115] The mutant strains were made using the cloned vectors CD1 to CD4. The strain made by destroying the recombinant vector CD1 using Cgl0626 and Cgl0624 was named DE1, the strain made by destroying the recombinant vector CD2 using Cgl0623 was named DE2, the strain made by destroying the recombinant vector CD3 using Cgl2433 was named DE3, and the strain made by destroying the recombinant vector CD4 using Cgl2432 and Cgl2431 was named DE4. The process of making each recombinant strain is as follows.

[0116] The cloning vector was prepared so that the final concentration was 1 μg / μl or more, and the electroporation method was used for Corynebacterium glutamicum DQ3-6 (reference [Tauch et al., FEMS Microbiology letters 123 (1994) 343-347]) to induce recombination once. At this time, the electroporated strain was spread on an agar medium containing 50 μg / μl of kanamycin, and after the colonies were isolated, it was confirmed by PCR and base sequence analysis whether the induction position on the genome was correctly inserted. In order to induce recombination twice again in the strain isolated in this way, it was inoculated in a liquid culture medium, cultured overnight or more, spread on an agar medium containing 10% sucrose, and colonies were isolated. After confirming whether there was resistance to kanamycin in the finally isolated colonies, it was confirmed by base sequence analysis whether the gene was destroyed in the strain without antibiotic resistance (reference [Schafer et al., Gene 145 (1994) 69-73]). Finally, the genes encoding carotenoid biosynthesis enzymes were disrupted and L-glutamine-producing Corynebacterium glutamicum mutant strains DE1, DE2, DE3, and DE4 were prepared.

[0117] Experimental Example 4. Evaluation of L-glutamine production capacity of L-glutamine-producing strains in which carotenoid biosynthesis enzyme genes were disrupted

[0118] The L-glutamine production abilities of the parent strain, Corynebacterium glutamicum DQ3-6, and the L-glutamine-producing Corynebacterium glutamicum DE1, DE2, DE3, and DE4 prepared in Example 4 were evaluated.

[0119] Each strain was patched in a shake flask solid seed medium and cultured at 30°C for 24 hours. The cultured colonies were inoculated into 10 ml of a shake flask titer medium and cultured at 30°C and 160 rpm for 72 hours. The components of the culture medium used here are shown in Table 8 below. After the culture was completed, the culture solution was diluted 100 times with distilled water, filtered with a 0.45 μm filter, and the L-glutamine production was analyzed using a high performance liquid chromatograph (HPLC) (agilent technologies 1260infinity, agilent technologies) equipped with a column (Dionex IonPacTM CS12A) and an ultraviolet detector (195 mm). The results are shown in Table 9 below.

[0120]

Table 8

[0121]

[0122]

Table 9

[0123] strain <![CDATA[OD 610 ]]> L-Glutamine (%) Yield (%) DQ3-6 25.6 2.9 29.4 DE1 24.3 3.3 32.8 DE2 25.1 3.3 32.6 DE3 24.2 3.4 33.5 DE4 24.8 3.5 32.6

[0124] As shown in Table 9 above, it was confirmed that the genes encoding carotenoid biosynthesis enzymes were disrupted in Corynebacterium glutamicum DE1, DE2, DE3 and DE4, thereby increasing the L-glutamine production by about 13.8 to 20.7% and the L-glutamine production yield by about 3.2 to 4.1% compared with Corynebacterium glutamicum DQ3-6 as the parent strain.

[0125] Based on the above results, the activity of carotenoid biosynthetic enzymes encoded by genes constituting the crtB gene, crtB2 gene or crtB / crtB2 gene cluster in Corynebacterium microorganisms is weakened or inactivated, thereby increasing the production amount and production yield of L-amino acids represented by L-arginine, L-citrulline, L-lysine and L-glutamine compared to the microorganisms before mutation.

[0126] So far, the present invention has been studied around its preferred embodiments. It will be appreciated by those skilled in the art that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a restrictive perspective. The scope of the present invention is shown in the claims rather than in the above description, and should be interpreted as all differences within the scope of their equivalents are included in the present invention.

[0127]

Collection Information

[0128] Name of depository institution: Korea Collection of Microorganisms (KCCM)

[0129] Accession number: KCCM13219P

[0130] Collection date: 20220629

[0131] Classification and nomenclature of biological material: Corynebacterium glutamicum.

[0132] Name of depository institution: Korea Collection of Microorganisms (KCCM)

[0133] Accession number: KCCM12969P

[0134] Collection date: 20210402

[0135] Classification and nomenclature of biological materials: Corynebacterium glutamicum.

[0136] Name of depository institution: Korea Collection of Microorganisms (KCCM)

[0137] Accession number: KCCM13398P

[0138] Collection date: 20230926

[0139] Classification and nomenclature of biological material: Corynebacterium glutamicum.

Claims

1. A Corynebacterium microorganism, wherein the activity of a carotenoid biosynthetic enzyme is weakened or inactivated and the L-amino acid production capacity is improved.

2. The Corynebacterium microorganism according to claim 1, wherein The weakening of the activity of the carotenoid biosynthetic enzyme means that all or part of the gene encoding the carotenoid biosynthetic enzyme is inserted, replaced, deleted, or a combination thereof.

3. The Corynebacterium microorganism according to claim 2, wherein The gene encoding the carotenoid biosynthetic enzyme is at least one selected from the group consisting of Cgl0626, Cgl0624, Cgl0623, Cgl2433, Cgl2432 and Cgl2431.

4. The Corynebacterium microorganism according to claim 3, wherein The Cgl0626 gene comprises the base sequence of SEQ ID NO: 1, The Cgl0624 gene comprises the base sequence of SEQ ID NO: 3, The Cgl0623 gene comprises the base sequence of SEQ ID NO: 5, The Cgl2433 gene comprises the base sequence of SEQ ID NO: 7, The Cgl2432 gene comprises the base sequence of SEQ ID NO: 9, and The Cgl2431 gene comprises the base sequence of SEQ ID NO:

11.

5. The Corynebacterium microorganism according to claim 1, wherein The Corynebacterium microorganism is Corynebacterium glutamicum.

6. The Corynebacterium microorganism according to claim 1, wherein The L-amino acid is one or more selected from L-alanine, L-isoleucine, L-valine, L-leucine, L-methionine, L-asparagine, L-cysteine, L-glutamine, L-serine, L-threonine, L-phenylalanine, L-tryptophan, L-tyrosine, L-aspartic acid, L-glutamic acid, L-arginine, L-histidine, L-lysine, L-glycine, L-proline and L-citrulline.

7. A method for producing L-amino acids, comprising the following steps: A step of culturing the Corynebacterium microorganism according to claim 1 in a culture medium; as well as A step of recovering L-amino acid from the microorganism or the medium in which the microorganism is cultured.

8. The method for producing L-amino acids according to claim 7, wherein The L-amino acid is one or more selected from L-alanine, L-isoleucine, L-valine, L-leucine, L-methionine, L-asparagine, L-cysteine, L-glutamine, L-serine, L-threonine, L-phenylalanine, L-tryptophan, L-tyrosine, L-aspartic acid, L-glutamic acid, L-arginine, L-histidine, L-lysine, L-glycine, L-proline and L-citrulline.

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