BR122022018007B1 - MICROORGANISM OF THE GENUS CORYNEBACTERIUM AND METHOD OF PREPARATION, COMPOSITION TO PRODUCE L-LEUCINE AND METHOD OF PRODUCTION - Google Patents

Abstract

The present disclosure relates to an innovative modified polypeptide having isopropylmalate synthase activity, a polynucleotide encoding the same, a microorganism comprising the polypeptide, and a method for producing L-leucine by culturing the microorganism.

Description

FIELD OF TECHNIQUE

[001] The present disclosure relates to an innovative modified polypeptide having isopropylmalate synthase activity, a polynucleotide encoding the same, a microorganism comprising the polypeptide and a method for producing L-leucine by culturing the microorganism.

BACKGROUND OF THE INVENTION

[002] L-Leucine is an essential amino acid, one that is costly and widely used in medicine, food, food additives, industrial chemicals, etc. Furthermore, L-leucine is mainly produced with the use of a microorganism. Fermentation of branched-chain amino acids that include L-leucine is carried out primarily by a microorganism of the genus Escherichia or a microorganism of the genus Corynebacterium, known to biosynthesize 2-ketoisocaproate as a precursor from pyruvic acid through several steps (Korean Patent Nos. 10-0220018 and 10-0438146).

[003] Isopropylmalate synthase (hereinafter referred to as "IPMS"), which is an enzyme involved in leucine biosynthesis, is an enzyme of the first step in leucine biosynthesis, which converts 2-ketoisovalerate, produced during the valine biosynthetic pathway, to isopropylmalate, which allows the biosynthesis of leucine instead of valine, and thus IPMS is an important enzyme in the process of leucine biosynthesis. However, IPMS undergoes feedback inhibition by L-leucine, which is an end product, or derivatives thereof. Consequently, although there is a variety of prior art relevant to IPMS variants that release feedback inhibition for the purpose of producing a high concentration of leucine (U.S. Patent Publication Application 2015-0079641 and U.S. Patent 6,403,342), research still continues. to discover better variants.

REVELATION TECHNIQUE PROBLEM

[004] The present inventors endeavored to develop an IPMS variant that can be used for the production of L-leucine with a high concentration, and as a result, the present inventors have developed an innovative IPMS variant. It was confirmed that the variant released feedback inhibition by L-leucine, which is an end product, and increased an activity thereof so that the variant is able to produce L-leucine in a high yield from a microorganism which contains the same, thereby completing the present disclosure.

TECHNIQUE SOLUTION

[005] An object of the present disclosure is to provide an innovative modified polypeptide that has isopropylmalate synthase activity.

[006] Another object of the present disclosure is to provide a polynucleotide that encodes the modified polypeptide.

[007] Yet another object of the present disclosure is to provide a microorganism of the genus Corynebacterium that produces L-leucine, containing the polypeptide.

[008] Yet another object of the present disclosure is to provide a method for producing L-leucine by culturing the microorganism in a medium.

ADVANTAGEOUS EFFECTS

[009] The innovative modified polypeptide having an isopropylmalate synthase activity is a polypeptide in which the activity is increased compared to that of wild-type and feedback inhibition by L-leucine is released, and thus L-leucine can be produced in a high yield with the use of such a modified polypeptide.

BEST WAY TO CARRY OUT THE INVENTION

[010] To achieve the above objects, one aspect of the present disclosure provides an innovative modified polypeptide having an isopropylmalate synthase activity. The innovative modified polypeptide can be a modified polypeptide that has an isopropylmalate synthase activity, wherein arginine at position 558 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is replaced by a amino acid residue other than arginine, or glycinine at position 561 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid residue other than glycinine. The modified polypeptide of the present disclosure not only has an activity superior to that of a polypeptide of SEQ ID NO: 1 which has an isopropylmalate synthase activity, but also has a characteristic that feedback inhibition by L-leucine is released.

[011] As used herein, the term "isopropylmalate synthase" refers to an enzyme that converts 2-ketoisovalerate to isopropylmalate, which is a precursor of L-leucine, by reaction with acetyl-CoA. The isopropylmalate synthase of the present disclosure can be included as long as the enzyme has the converting activity irrespective of a microorganism origin. Specifically, isopropylmalate synthase may be an enzyme derived from a microorganism of the genus Corynebacterium. More specifically, the isopropylmalate synthase can be an isopropylmalate synthase derived from Corynebacterium glutamicum, and specifically, it can include the amino acid sequence of SEQ ID NO: 1, but without limitation thereto. In addition, the isopropylmalate synthase can include a polypeptide that has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 1. example, it is obvious that an amino acid sequence having such homology and exhibiting an effect corresponding to that of isopropylmalate synthase can be included within the scope of the present disclosure even if it has an amino acid sequence in which some of the sequences are deleted, modified , replaced or added.

[012] As used herein, the term "increase in isopropylmalate synthase activity" refers to an increase in activity converting to isopropylmalate. Therefore, the modified polypeptide of the present disclosure has a superior level of isopropylmalate converting activity compared to a polypeptide of SEQ ID NO: 1 that has isopropylmalate synthase activity. Isopropylmalate conversion activity can be confirmed directly by measuring the level of isopropylmalate produced, or it can be confirmed indirectly by measuring the level of CoA produced. As used herein, the term "increase in activity" may be used in combination with "increased activity". Furthermore, isopropylmalate is a precursor of L-leucine and therefore, the use of the modified polypeptide of the present disclosure results in producing a higher level of L-leucine compared to a polypeptide of SEQ ID NO: 1 which has isopropylmalate synthase activity .

[013] Furthermore, unlike a polypeptide of SEQ ID NO: 1 that has isopropylmalate synthase activity, the modified polypeptide of the present disclosure can be characterized by the fact that feedback inhibition by L-leucine, which is a final product, or a derivative thereof, is released. As used herein, the term "feedback inhibition" refers to the inhibition of a reaction in the initial state of an enzyme system by an end product in the enzyme system. For the purposes of the present disclosure, feedback inhibition can be feedback inhibition in which L-leucine or a derivative thereof inhibits isopropylmalate synthase activity, which means the first step of the biosynthetic pathway, but is not limited thereto. . Therefore, when the feedback inhibition of isopropylmalate synthase is released, the productivity of L-leucine can be increased compared to the case of not releasing it.

[014] As used herein, the term “modification”, “modified” or “variant” refers to a culture or an individual that shows a hereditary or non-hereditary alternation in a stabilized phenotype. Specifically, the term "variant" may be intended to mean a variant in which its activity is efficiently increased due to the amino acid sequence corresponding to isopropylmalate synthase derived from Corynebacterium glutamicum being modified compared to the wild type, a variant in which feedback inhibition by L-leucine or a derivative thereof is released, or a variant in which the increase in activity and feedback inhibition are both released.

[015] Specifically, the modified polypeptide of the present disclosure, which has isopropylmalate synthase activity, may be a modified polypeptide that has isopropylmalate synthase activity, wherein arginine, an amino acid at position 558 from an N -terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, is replaced with an amino acid residue other than arginine, or glycinine, an amino acid residue at position 561 from an N-terminus of a polypeptide that consists of the amino acid sequence of SEQ ID NO: 1, is replaced by an amino acid residue other than glycinine. Amino acid other than arginine may include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, glycinine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid and glutamic acid; and the amino acid other than glycinine may include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid and glutamic acid; but amino acids are not limited to those. More specifically, the modified polypeptide can be a modified polypeptide, in which arginine, an amino acid residue at position 558 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, is replaced by histidine , alanine, or glutamine; or glycinine, an amino acid residue at position 561 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, is substituted with, but is not limited to, aspartic acid, arginine or tyrosine. Furthermore, the modified polypeptide can be one in which the arginine at position 558 is replaced by histidine, alanine or glutamine; and the glycinine at position 561 is replaced by but not limited to aspartic acid, arginine or tyrosine. More specifically, the modified polypeptide can include an amino acid sequence of any one of SEQ ID NO: 21 through SEQ ID NO: 35.

[016] In addition, the modified polypeptide can include a polypeptide that has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of any one of SEQ ID NO: 21 to SEQ ID NO: 35. For example, it is obvious that a variant enzyme having an amino acid sequence in which some of the sequences are deleted, modified, substituted or added while the modified amino acid sequence corresponding to the sequence of amino acids at positions 558 and/or 561 is fixed, it should also fall within the scope of the present disclosure provided that the amino acid sequence has the above homology and exhibits an effect corresponding to that of isopropylmalate synthase. On the other hand, positions 558 and 561, which are specific modification positions, refer to positions that are determined based on the N-terminus in the amino acid sequence of SEQ ID NO: 1, and therefore the fact that such positions are determined by considering the number of amino acids that are added or deleted from the N-terminus of SEQ ID NO: 1 is obvious to a person of ordinary skill in the art, and thus also belongs within the scope of the present revelation. For example, leuA, which is the gene encoding isopropylmalate synthase, was represented by SEQ ID NO: 1 which consists of 616 amino acids. However, in some references, the translational initiation code is indicated 35 amino acids downstream of the leuA gene sequence, i.e., a gene consisting of 581 amino acids. In that case, the 558th amino acid is interpreted as the 523rd amino acid and the 561st amino acid as the 526th amino acid and is therefore included within the scope of the present disclosure.

[017] As used herein, the term "homology" refers to a percent identity between two polynucleotide or polypeptide chemical moieties. Sequence homology from one chemical moiety to another chemical moiety can be determined by technology known in the art. For example, homology can be determined by directly providing sequence information, i.e., parameters such as score, identity, similarity, etc., of two polynucleotide molecules or two polypeptide molecules using a computer program. easily accessible (Example: BLAST 2.0). Furthermore, homology between polynucleotides can be determined by hybridizing polynucleotides under the condition of forming a stable double strand between the homologous regions, disassembling with a specific single-stranded nuclease, followed by size determination of the disassembled fragments.

[018] Another aspect of the present disclosure provides a polynucleotide encoding the modified polypeptide.

[019] The polynucleotide may be a polynucleotide encoding a modified polypeptide having isopropylmalate synthase activity, wherein arginine, an amino acid at position 558 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, is substituted with an amino acid residue other than arginine, or glycinine, an amino acid residue at position 561 from an N-terminus of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, is replaced with another amino acid residue other than glycinine. Specifically, a polynucleotide that encodes a polypeptide that includes the amino acid sequence of SEQ ID Nos: 21 to 35 and that has isopropylmalate synthase activity; a modified polypeptide that has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to the above polypeptide; or which encodes a modified polypeptide having isopropylmalate synthase activity, in which some of the sequences are deleted, modified, substituted or added while the amino acid sequence is modified at positions 558 and/or 561, which are specific modification positions in the above polypeptide is fixed may be included without limitation. Alternatively, the probe which may be prepared from a known gene sequence, for example a sequence encoding a protein having Isopropylmalate synthase activity, by hybridizing a sequence complementary to all or part of the above nucleotide sequence under conditions stringent, may be included without limitation.

[020] As used herein, the term "stringent conditions" refers to conditions under which a so-called hybrid is formed while non-specific hybrids are not formed. Examples of such conditions include conditions under which genes that have high degrees of homology, such as genes that have a homology of 80% or more, specifically, 90% or more, more specifically, 95% or more, additionally specifically, 97% or more, and being the most specific, 99% or more, hybridize to each other while genes that have low degrees of homology do not hybridize to each other, or conditions under which genes are washed 1 time, and specifically 2 and 3 times, at a temperature and salt concentration equivalent to 60°C, 1xSSC, and 0.1% SDS, specifically, 60°C, 0.1xSSC, and 0.1% SDS, and more specifically, 68° C, 0.1xSSC, and 0.1% SDS, which are common Southern hybridization wash conditions (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y. (2001)).

[021] The probe used in hybridization may be a part of the sequence complementary to the nucleotide sequence. Such a probe can be constructed by PCR using an oligonucleotide prepared on the basis of a known sequence as a primer and a gene fragment containing such a nucleotide sequence as a template. For example, a gene fragment that is about 300 bp in length can be used as a probe. More specifically, in the case of using a probe that has a length (about 300 bp), 50°C, 2xSSC, and 0.1% SDS can be suggested for the hybridization wash conditions.

[022] On the other hand, the polynucleotide may be a polynucleotide having a nucleotide sequence of any one of SEQ ID NO: 36 to SEQ ID NO: 50, and it is obvious that the polynucleotide also includes a polynucleotide that can be translated into the polypeptide modified by codon degeneracy.

[023] Yet another aspect of the present disclosure is to provide a microorganism of the genus Corynebacterium that produces L-leucine containing the modified polypeptide.

[024] In the present disclosure, the microorganism can include any one of an artificially produced microorganism through processing or a naturally occurring microorganism.

[025] As used herein, the term "transformation" refers to introducing a gene into a host cell for expression. In the present disclosure, the transformation method includes any method that introduces a gene into a cell and can be performed by selecting a suitable standard technique known in the art. Examples of the transformation method are electroporation, calcium phosphate coprecipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposome, heat shock method, etc., but are not limited to these.

[026] The gene to be transformed may include both a form inserted into the chromosome of a host cell and a form located outside the chromosome, provided that it can be expressed in the host cell. Furthermore, the gene includes both DNA and RNA as a polynucleotide capable of encoding a polypeptide, and any gene that can be introduced and expressed in the host cell can be used without limitation. For example, the gene can be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct that contains all the elements required for self-expression. The expression cassette usually includes a promoter operably linked to the gene, a transcription termination signal, ribosome binding sites, and a translational termination signal. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the gene can either be introduced into a host cell itself or in a form of a polynucleotide construct, i.e. a form of a vector, and operably linked to sequences required for expression in the host cell.

[027] As used herein, the term "vector" refers to any carrier for cloning and/or transferring nucleotides to a host cell. A vector can be a replicon to allow replication of fragments combined with other DNA fragments. “Replicon” refers to any genetic unit that acts as a self-replicator even for in vivo DNA replication, that is, replicable by self-regulation (eg, plasmid, phage, cosmid, chromosome, and virus). The term "vector" can include viral and non-viral carriers for introducing nucleotides into a host cell in vitro, ex vivo or in vivo, and can also include minispherical DNA. For example, the vector can be a plasmid lacking a bacterial DNA sequence. Removal of bacterial DNA sequences that are rich in CpG area has been conducted to reduce silencing of transgene expression and to promote more continuous expression from a plasmid DNA vector (eg, Ehrhardt, A. et al. (2003) Hum Gene Ther 10:215 to 225; Yet, N.S. (2002) MoI Ther 5:731 to 738; Chen, Z.Y. et al. (2004) Gene Ther 11:856 to 64). The term "vector" can also include a transposon such as Sleeping Beauty ( Izsvak et al. J. Mol. Biol. 302:93-102 (2000 )), or an artificial chromosome. Examples of the vector typically used can be natural or recombinant plasmid, cosmid, virus and bacteriophage. For example, such as phage vector or cosmid vector, pWE15, M13, ÀMBL3, ÀMBL4, ÀIXII, ÀASHII, ÀAPII, Àt10, Àt11, Charon4A, Charon21A, etc. can be used. Also, such as plasmid vector, pDZ type, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type, pET type, etc. can be used. Specifically, the pECCG117 vector can be used. The vector that can be used in the present disclosure is not particularly limited, and the known expression/replacement vector can be used.

[028] In addition, the vector may be a recombinant vector that may additionally include several antibiotic resistance genes.

[029] As used herein, the term "antibiotic resistance gene" refers to a gene that has resistance to antibiotics, and the cells comprising that gene survive even in the environment treated with the corresponding antibiotic. Therefore, the antibiotic resistance gene can be used effectively as a selection marker for large-scale production of plasmids in microorganisms such as E. coli, etc. In the present invention, as the antibiotic resistance gene is not a factor that significantly affects the expression efficiency that is obtained by an optimal combination of vector components which is the key feature of the present invention, any common antibiotic resistance gene can be be used as a selection marker without limitation. Specifically, genes for resistance against ampicillin, tetracycline, kanamycin, chloramphenicol, streptomycin, or neomycin can be used.

[030] As used herein, the term "operably linked" refers to the operable linkage of a regulatory sequence for nucleotide expression with a nucleotide sequence encoding a target protein to perform its general function, thereby affecting the expression of a coding nucleotide sequence. Operable ligation with a vector can be performed using an art-known gene recombination technique, and site-specific DNA cleavage and ligation can be performed using an art-known restriction enzyme and ligase.

[031] As used herein, the term "host cell into which a vector is introduced (transformed)" refers to a cell transformed with a vector that has a gene encoding one or more target proteins. The host cell may include any of a prokaryotic microorganism and a eukaryotic microorganism provided that the microorganism includes a modified polypeptide capable of producing isopropylmalate synthase by introducing the above vector. For example, the strain of microorganism that belongs to the genera of Escherichia, Erwinia, Serratia, Providencia, Corynebacterium and Brevibacterium can be included. An example of the microorganism of the genus Corynebacterium can be Corynebacterium glutamicum, but it is not limited thereto.

[032] The microorganism of the genus Corynebacterium that produces L-leucine, which is capable of expressing the modified polypeptide that has isopropylmalate synthase activity, includes all microorganisms capable of expressing the modified polypeptide by various known methods besides the introduction of a vector.

[033] Yet another aspect of the present disclosure provides a method for producing L-leucine, comprising: (a) culturing the microorganism of the genus Corynebacterium that produces L-leucine; and (b) recovering L-leucine from the cultured microorganism or the cultured medium.

[034] As used herein, the term "culture" refers to the cultivation of the microorganism under appropriately controlled environmental conditions. The cultivation process of the present disclosure can be carried out depending on a suitable culture medium and condition known in the art. Such a cultivation process can be easily adjusted and used by a person of ordinary skill in the art depending on the strain to be selected. Specifically, the culture can be a batch type, a continuous type and a fed batch type, but it is not limited to these.

[035] The carbon sources contained in the medium may 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, coconut oil, etc.; 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 materials may be used singly or in combinations thereof, but are not limited thereto. Nitrogen sources contained in the medium may include organic nitrogen sources, such as peptone, yeast extract, gravy, malt extract, milocin and soy; and sources of inorganic nitrogen, such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources can be used alone or in combination, but are not limited to that. Sources of phosphorus contained in the medium may include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts. Furthermore, metal salts such as magnesium sulfate or iron sulfate may be contained. In addition, amino acids, vitamins, suitable precursors, etc. may be contained. These media or precursors can be added in a batch culture process or a continuous culture process to but are not limited to one culture.

[036] The pH of the culture can be adjusted during cultivation by adding an appropriate compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid, and the generation of foam can be inhibited during cultivation with the use of an antifoaming agent such as polyglycolic fatty acid ester. In order to maintain aerobic conditions of the culture, oxygen or gas containing oxygen may be injected into the culture. In order to maintain anaerobic and microaerobic conditions, no gas can be injected or nitrogen, hydrogen, or carbon dioxide can be injected. Culture temperature can be 27°C to 37°C, and specifically 30°C to 35°C, but is not limited to that. The cultivation period can be continuous as long as the desired amount of useful material is recovered, and preferably for 10 to 100 hours, but the cultivation period is not limited to this.

[037] The step of recovering L-leucine produced in the culturing step of the present disclosure can collect the desired L-leucine from the microorganism or the medium using a suitable method known in the art depending on culture methods. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. can be used, and a suitable method known in the art can be used to recover the desired L-leucine from the medium or the microorganism. Furthermore, the above recovery step may include a purification process.

METHOD OF CARRYING OUT THE INVENTION

[038] Below in the present document, the present disclosure will be described in detail with exemplary embodiments attached. However, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure.

EXAMPLE 1: CONFIRMATION OF LEUINE NUCLEOTIDE SEQUENCE OF LEUCINE PRODUCING MICROORGANISM KCCM11661P

[039] Corynebacterium glutamicum ATCC14067 was inoculated into a seeding medium that has the ingredients described below at 121 °C for 15 minutes, cultured for 13 hours, and then 25 ml of the culture medium was recovered. The recovered culture medium was washed with a 100 mM citrate buffer and treated with N-methyl-N-nitro-N-nitrosoguanidine (NTG) for 30 minutes to a final concentration of 400 µg/ml. Thereafter, the resultant was washed with a 100 mM phosphate buffer. The mortality rate of the NTG-treated strains was determined to be 99.6% as a result of staining the strains on a minimal medium that has the ingredients described below. In order to obtain norleucine (NL) resistant variants, NTG treated strains were stained in minimal media with final concentrations of 20 mM, 40 mM and 50 mM, cultured at 30 °C for 5 days and then NL were obtained.

SOWING MEDIUM

[040] Glucose (20 g), peptone (10 g), yeast extract (5 g), urea (1.5 g), KH2PO4 (4 g), K2HPO4 (8 g), MgSO4-7HaO (0.5 g), biotin (100 μg), thiamine hydrochloride (1,000 μg), calcium pantothenic acid (2,000 μg), and nicotinamide (2,000 μg; based on 1 L of distilled water), pH 7 ,0

PRODUCTION MEANS

[041] Glucose (100 g), (NH4)2SO4 (40 g), soy protein (2.5 g), corn wash water concentrates (5 g), urea (3 g), KH2PO4 (1 g ), MgSO4^7H2O (0.5 g), biotin (100 μg), thiamine hydrochloride (1,000 μg), calcium pantothenic acid (2,000 μg), nicotinamide (3,000 μg), and CaCO3 ( 30 g; based on 1 l of distilled water), pH 7.0

[042] The variants obtained by the above method were designated as Corynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28 and deposited to the Korean Microorganism Culture Center, an international depository authority, on January 22, 2015, under the Budapest Treaty, and as a result, Corynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28 were deposited under Accession Nos. KCCM11661P and KCCM11662P, respectively. Corynebacterium glutamicum KCJ-24 and Corynebacterium glutamicum KCJ-28 produced L-leucine at a concentration of 2.7 g/l and 3.1 g/l, respectively. Therefore, it was confirmed that the productivity of L-leucine produced from the variants was 10-fold higher than that of the wild type.

[043] Furthermore, an attempt was made to confirm whether the leuA variation encoding isopropylmalate synthase (IPMS) occurred in the KCCM11661P variant. The amino acid sequence (SEQ ID NO: 1) of wild-type leuA was confirmed by reference to Genebank WP_003863358.1. The chromosomal DNA of the variant was amplified using a polymerase chain reaction (hereinafter referred to as 'PCR') method. Although it is known that the leuA gene consists of 616 amino acids, in some references it is published that the translational initiation code is indicated 35 amino acids downstream of the leuA gene sequence, and thus the leuA gene consists of 581 amino acids. In this case, the position number that indicates the corresponding amino acid variation may vary. Therefore, in cases where the leuA gene is considered to consist of 581 amino acids, the position of variation is further indicated in parentheses.

[044] Specifically, PCR was performed using the chromosomal DNA of the variant as a template and using primers from SEQ ID Nos: 3 and 4 under the following conditions: denaturation at 94 °C for 1 minute; annealing at 58 °C for 30 seconds; and polymerization at 72 °C for 2 minutes using Taq DNA polymerase. This PCR was repeated a total of 28 times to amplify a fragment of about 2700 base pairs. The nucleotide sequence of the fragment was analyzed using the same primer, and as a result, it was confirmed that G, which is the 1673rd nucleotide of leuA in KCCM11661P, was replaced by A. This result implies that arginine, which is the 558th (or 523rd; hereinafter referred to as just the 558th) amino acid, is replaced by histidine. Furthermore, it was also confirmed that GC, which are the 1682nd and 1683rd nucleotides, were replaced by AT. This result also implies that glycinin, which is the 561st (or 526th, hereinafter referred to as just the 561st) amino acid, is replaced by aspartic acid.

EXAMPLE 2: PRODUCTION OF IPMS VARIANT REPLACEMENT VECTOR

[045] In order to produce a vector containing the modified nucleotide sequence confirmed in Example 1, PCR was performed using the chromosomal DNA of the above variant as a template and using primers of SEQ ID Nos: 5 and 6 under the following conditions: denaturation at 94 °C for 1 minute; annealing at 58 °C for 30 seconds; and polymerization at 72 °C for 1 minute using Pfu DNA polymerase. This PCR was repeated a total of 25 times to amplify a fragment of about 1460 base pairs with SalI and XbaI restriction enzyme sites. The amplified fragment was treated with restriction enzymes, SalI and XbaI, and then pDZ-leuA (R558H, G561D) was prepared by ligation with the pDZ vector (Korean Patent No:10-0924065 and International Patent Publication No. 2008033001) treated with the same enzymes. Furthermore, in order to prepare a vector with each variation, ATCC14067 was used as a template, and then 2 fragments were amplified using primers 5 and 7, and primers 8 and 6, respectively. PCR was performed using the two prepared fragments as templates under the following conditions: denaturation at 94 °C for 1 minute; annealing at 58 °C for 30 seconds; and polymerization at 72 °C for 1 minute using Pfu DNA polymerase. This PCR was repeated a total of 25 times to amplify a fragment of about 1460 base pairs with SalI and XbaI restriction enzyme sites. The amplified fragment was treated with restriction enzymes SalI and XbaI and then pDZ-leuA (R558H) was prepared by ligation with pDZ treated with the same enzymes. pDZ-leuA (G561D) was prepared using primers 5 and 9, and primers 10 and 6 by the same method as above.

EXAMPLE 3: PRODUCTION OF IPMS VARIANT REPLACEMENT CEPA

[046] Corynebacterium glutamicum ATCC14067 was used as a parent strain in order to prepare a strain containing the modified leuA nucleotide sequence which was found in the above modified strain.

[047] Corynebacterium glutamicum ATCC14067 was transformed with vectors pDZ-leuA (R558H), pDZ-leuA (G561D) and pDZ-leuA (R558H, G561D), which were prepared in Example 2 by electroporation. Each of the strains prepared by secondary crossing was designated as 14067::leuA (R558H), 14067::leuA (G561D) and 14067::leuA (R558H, G561D). In order to confirm that the leuA nucleotide was replaced, PCR was performed using primers from SEQ ID Nos: 3 and 4 under the following conditions: denaturation at 94 °C for 1 minute; annealing at 58 °C for 30 seconds; and polymerization at 72 °C for 2 minutes using Taq DNA polymerase. This PCR was repeated a total of 28 times to amplify a fragment of about 2700 base pairs. Thereafter, the leuA nucleotide substitution was confirmed by analyzing the nucleotide sequence with the same primer.

[048] The strain, 14067::leuA (R558H, G561D) that was transformed with the vector pDZ-leuA (R558H, G561D), was designated as KCJ-0148, and deposited for the Korean Microorganism Culture Center in January 25, 2016, and as a result, the strain was deposited under Accession in KCCM11811P.

EXAMPLE 4: PRODUCTION OF L-LEUCINE AS REPLACEMENT OF IPMS VARIANT STRAIN

[049] In order to produce L-leucine from Corynebacterium glutamicum 14067::leuA (R558H), 14067::leuA (G561D) and 14067::leuA (R558H, G561D), which were prepared in Example 3, it was Cultivation was carried out in the following way.

[050] One platinum cycle of each of the parent strain, Corynebacterium glutamicum ATCC14067, and Corynebacterium glutamicum strains 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D) prepared was inoculated into an Erlenmeyer flask with recessed corners (250 ml) containing a production medium (25 ml). Thereafter, L-leucine was produced by incubation in a shaking water bath at 30°C at a rate of 200 rpm for 60 hours.

[051] After completion of the incubation, the amount of L-leucine produced was measured by high-performance liquid chromatography. The concentration of L-leucine in the culture medium for each experimental strain is shown in Table 1 below. TABLE 1 L-LEUCINE PRODUCTION IN IPMS VARIANT REPLACEMENT STRAIN

[052] As shown in Table 1 above, it was confirmed that the L-leucine productivity of the strains that produce L-leucine, Corynebacterium glutamicum 14067::leuA (R558H), 14067::leuA (G561D), and 14067::leuA (R558H, G561D), which have the R558H, G561D, or R558H/G561D variation in the leuA gene, was increased about 12- to 25-fold compared to that of the parent strain, Corynebacterium glutamicum ATCC14067.

EXAMPLE 5: IPMS VARIANT OVEREXPRESSION VECTOR PRODUCTION

[053] In order to produce an expression vector containing the modified nucleotide sequence confirmed in Example 1, PCR was performed using ATCC14067 and the chromosomal DNA of the 3 variants prepared in Example 3 as templates and using primers from SEQ ID Nos: 11 and 12 under the following conditions: denaturing at 94 °C for 1 minute; annealing at 58 °C for 30 seconds; and polymerization at 72 °C for 1 minute using Pfu DNA polymerase. This PCR was repeated a total of 25 times to amplify a fragment of about 2050 base pairs with NdeI and XbaI restriction enzyme sites. The amplified fragment was treated with restriction enzymes, NdeI and XbaI, and then expression vectors p117_PCJ7-leuA (WT), p117_PCJ7-leuA (R558H), p117_PCJ7-leuA (G561D), and p117_PCJ7-leuA (R558H, G561D ) were prepared by ligation using p117_PCJ7 in which a PCJ7 promoter was inserted into the pECCG117 vector (Biotechnology letters Vol. 13, No. 10, p. 721 to 726 (1991)) treated with the same enzymes. The PCJ7 promoter is a promoter that enhances gene expression, and is publicly known in Korean Patent No. 10-0620092 and International Patent Publication No. 2006-065095.

EXAMPLE 6: PRODUCTION OF TRANSFORMED CEPA WITH IPMS VARIANT OVEREXPRESSION VECTOR

[054] In order to produce a strain transformed with an overexpression vector containing the modified leuA nucleotide sequence prepared in Example 5, the parent strain, which is wild-type Corynebacterium glutamicum ATCC14067, and the leucine-producing strains KCCM11661P and KCCM11662P were used.

[055] Each of the vectors p117_PCJ7-leuA (WT), p117_PCJ7-leuA (R558H), p117_PCJ7-leuA (G561D), and p117_PCJ7-leuA (R558H, G561D), prepared in Example 5, was transformed with Corynebacterium glutamicum ATCC14067, KCCM11661P and KCCM11662P by electroporation. As a result, 14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D), 14067::p117_PCJ7-leuA (R558H,G561D); KCCM11661P::p117_PCJ7-leuA (WT), KCCM11661P::p117_PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D), KCCM11661P::p117_PCJ7-leuA (R558H, G561D) ); and KCCM11662P::p117_PCJ7-leuA (WT), KCCM11662P::p117_PCJ7-leuA (R558H), KCCM11662P::p117_PCJ7-leuA (G561D), KCCM11662P::p117_PCJ7-leuA (R558H, G561D ) were produced.

EXAMPLE 7: L-LEUCINE PRODUCTION IN TRANSFORMED STRAIN WITH IPMS VARIANT OVEREXPRESSION VECTOR

[056] In order to produce L-leucine from strains that produce L-leucine, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D ), 14067::p117_PCJ7-leuA (R558H, G561D); KCCM11661P::p117_PCJ7-leuA (WT), KCCM11661P::117PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D), KCCM11661P::p117_PCJ7-leuA (R558H, G561D); and KCCM11662P::p117_PCJ7-leuA (WT), KCCM11662P::p117_PCJ7-leuA (R558H), KCCM11662P::p117_PCJ7-leuA (G561D), KCCM11662P::p117_PCJ7-leuA (R558H, G561 D), which were produced in Example 6 , cultivation was carried out as follows.

[057] One cycle of platinum from each of the parent strains, Corynebacterium glutamicum ATCC14067, KCCM11661P and KCCM11662P, and the strains produced in Example 6 was inoculated into an Erlenmeyer flask with recessed corners (250 ml) containing a production medium ( 25ml). Thereafter, L-leucine was produced by incubation in a shaking water bath at 30°C at a rate of 200 rpm for 60 hours.

[058] After completion of the incubation, the amount of L-leucine produced was measured by high performance liquid chromatography. The concentration of L-leucine in the culture medium for each experimental strain is shown in Table 2 below. TABLE 2 L-LEUCINE PRODUCTION IN IPMS VARIANT OVEREXPRESSION STRAIN

[059] As shown in Table 2 above, it was confirmed that L-leucine production of strains producing L-leucine, 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D) and 14067:: p117_PCJ7-leuA (R558H, G561D), which were transformed with the overexpression vector containing leuA gene variation in strain ATCC14067, was increased 45- to 98-fold compared to that of the parent strain ATCC14067; L-leucine production from strains producing L-leucine, KCCM11661P::p117_PCJ7-leuA (R558H), KCCM11661P::p117_PCJ7-leuA (G561D) and KCCM11661P::p117_PCJ7-leuA (R558H, G561D), which were transformed with the overexpression vector containing leuA gene variation in the KCCM11661P strain, it was increased 2.3- to 4.5-fold compared to that of the parent strain KCCM11661P; and that L-leucine production from strains producing L-leucine, KCCM11662P::p117_PCJ7-leuA (R558H), KCCM11662P::p117_PCJ7-leuA (G561D) and KCCM11662P::p117_PCJ7-leuA (R558H,G561D), which were transformed with the overexpression vector containing leuA gene variation in the KCKCM11662P strain, it was increased 2- to 4.2-fold compared to that of the parent strain KCCM11662P.

EXAMPLE 8: MEASUREMENT OF ISOPROPYLMALATE SYNTHASE ACTIVITY IN TRANSFORMED STRAIN WITH LEUA OVEREXPRESSION VECTOR

[060] In order to measure isopropylmalate synthase activity in strains that produce L-leucine, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT), 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA ( G561D) and 14067::p117_PCJ7-leuA (R558H, G561D), produced in Example 6, experiments were performed as follows.

[061] One platinum cycle of each of the 4 strains above was inoculated into an Erlenmeyer flask with recessed corners (250 ml) containing the seeding medium (25 ml). After that, the results were incubated in a water bath with stirring at 30 °C at a rate of 200 rpm for 16 hours. Upon completion of the incubation, the culture medium was centrifuged to discard the supernatant, the pellet was washed and mixed with a lysis buffer, and the cells were pulverized with a droplet homogenizer. Proteins present in the lysate were quantified according to the Bradford assay, and isopropylmalate synthase activity was measured by measuring the CoA produced when protein-containing lysate (100 μg/ml) was used. The results of measuring isopropylmalate synthase activity in each strain are shown in Table 3 below. TABLE 3

[062] In order to confirm the degree of leucine feedback inhibition release in the enzyme, isopropylmalate synthase activity was measured by measuring the CoA produced when protein-containing lysate (100 μg/ml) was used under the condition to which leucine (3 g/l) was added. The results of measuring isopropylmalate synthase activity in each strain are shown in Table 4 below. TABLE 4

[063] As shown in Tables 3 and 4 above, it was confirmed that the isopropylmalate synthase activity of the strains that produce L-leucine, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558H), 14067::p117_PCJ7-leuA (G561D) and 14067::p117_PCJ7-leuA (R558H, G561D), which were transformed with the vector expressing the IPMS variant, was increased 1.05-fold, 1.3-fold, and 3.2-fold, respectively, compared to to that of the control, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (WT). Furthermore, strains producing L-leucine maintained their IPMS activity at 61%, 70% and 89%, respectively, even when leucine (2 g/l) was added, confirming that feedback inhibition by leucine was released.

EXAMPLE 9: VECTOR PRODUCTION TO IMPROVE ISOPROPYLMALATE SYNTHASE VARIANT (IPMS)

[064] In Examples 4, 7 and 8, since it was confirmed that the 558th and 561st amino acids in the amino acid sequence (SEQ ID NO: 1) of isopropylmalate synthase were important sites for the activity of the IPMS variant enzyme, An attempt was made to confirm whether enzyme activity was increased or whether feedback inhibition was further released when substituted with an amino acid other than the amino acids in the variant. Therefore, an attempt was made to prepare a variant substituted by an amino acid from other amino acid groups capable of causing structural variations.

[065] A variant in which the 558th amino acid, arginine, was replaced by alanine (Ala) or glutamine (Gln) was prepared. The p117_PCJ7-leuA (R558A) vector, in which the 558th amino acid is replaced by alanine (Ala), and the p117_PCJ7-leuA (R558Q) vector, in which the 558th amino acid is replaced by glutamine (Gln), were prepared using of a site-directed mutagenesis method and using the vector p117_PCJ7-leuA (R558H) as a template, the primers of SEQ ID Nos: 13 and 14, and the pair of primers of SEQ ID Nos: 15 and 16.

[066] A variant in which the 561st amino acid, glycinine, was replaced by arginine (Arg) or tyrosine (Tyr) was prepared. The vector p117_PCJ7-leuA (G561R), in which the 561st amino acid is replaced by arginine (Arg), and the vector p117_PCJ7-leuA (G561Y), in which the 561st amino acid is replaced by tyrosine (Tyr), were obtained using of a site-directed mutagenesis method and using p117_PCJ7-leuA (G561D) as a template, the primers of SEQ ID Nos: 17 and 18, and the pair of primers of SEQ ID Nos: 19 and 20.

EXAMPLE 10: PRODUCTION OF STRAIN INTO WHICH MODIFIED ISOPROPYLMALATE VARIANT IS INTRODUCED

[067] In order to prepare a strain transformed with an expression vector containing the modified leuA nucleotide sequence prepared in Example 9, wild type Corynebacterium glutamicum ATCC14067 was used as a parent strain.

[068] Each of the vectors, p117_PCJ7-leuA (R558A), p117_PCJ7-leuA (R558Q), p117_PCJ7-leuA (G561R) and p117_PCJ7-leuA (G561Y), which were prepared in Example 9, was transformed into Corynebacterium glutamicum ATCC14067 by electroporation to prepare 14067::p117_PCJ7-leuA (R558A), 14067::p117_PCJ7-leuA (R558Q), 14067::p117_PCJ7-leuA (G561R), and 14067::p117_PCJ7-leuA (G561Y).

EXAMPLE 11: PRODUCTION OF L-LEUCINE IN STRAIN INTO WHICH VARIANT MODIFIED BY ISOPROPYLMALATE SYNTHASE IS INTRODUCED

[069] In order to produce L-leucine from strains that produce L-leucine, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558A), 14067::p117_PCJ7-leuA (R558Q), 14067::p117_PCJ7-leuA (G561R ) and 14067::p117_PCJ7-leuA (G561Y), which were prepared in Example 10, cultivation was carried out in the following manner.

[070] One cycle of platinum each of the parent strain, Corynebacterium glutamicum ATCC14067 and the above 4 strains was inoculated into an Erlenmeyer flask with recessed corners (250 ml) containing a production medium (25 ml). Thereafter, L-leucine was produced by incubation in a shaking water bath at 30°C at a rate of 200 rpm for 60 hours.

[071] After completion of the incubation, the amount of L-leucine produced was measured by high-performance liquid chromatography. The concentration of L-leucine in the culture medium for each experimental strain is shown in Table 5 below. TABLE 5 L-LEUCINE PRODUCTION IN IPMS VARIANT OVEREXPRESSION STRAIN

[072] As shown in Table 5 above, it was confirmed that the L-leucine productivity of the L-leucine producing strains, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (R558A) and 14067::p117_PCJ7-leuA (R558Q), was increased 32- to 38-fold compared to the parent strain, Corynebacterium glutamicum ATCC14067.

[073] Furthermore, it was confirmed that the L-leucine productivity of the strains that produce L-leucine, Corynebacterium glutamicum 14067::p117_PCJ7-leuA (G561R) and 14067::p117_PCJ7-leuA (G561Y), was increased by about 36 - a 40-fold compared to that of the parent strain, Corynebacterium glutamicum ATCC14067.

[074] Based on the above results, it was confirmed that the 558th and 561st amino acids in the amino acid sequence (SEQ ID NO: 1) of isopropylmalate synthase were important sites for the IPMS variant enzyme activity, and that even when each one of the 558th and 561st amino acids of the wild-type IPMS protein was replaced by histidine and aspartic acid, respectively, the productivity of L-leucine was remarkably increased in the strain that has such a modification.

[075] While the present disclosure has been described with reference to particular illustrative embodiments, it will be understood by those skilled in the art to whom the present disclosure pertains that the present disclosure may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the present disclosure. Therefore, the embodiments described above are considered to be illustrative in all respects and not restrictive. Furthermore, the scope of the present disclosure is defined by the appended claims and not the detailed description, and it is to be understood that all modifications or variations derived from the meanings and scope of the present disclosure and equivalents thereof are included within the scope of the appended claims.

Claims (8)

1. A microorganism of the genus Corynebacterium comprising a modified polypeptide that has improved isopropylmalate synthase activity compared to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1, in which the glycine at position 561 a from an N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is replaced by aspartic acid encoded by SEQ ID NO: 39 or a degenerate sequence thereof, wherein the microorganism shows a synthase activity of isopropylmalate improved compared to its parent strain by expression of the modified polypeptide; wherein the microorganism has resistance to norleucine or leucine; wherein the promoter of the polynucleotide encoding the modified polypeptide in the microorganism is replaced with a promoter capable of increasing expression compared to the native promoter; and wherein the microorganism is capable of producing L-leucine from the modified leuA gene. 2. Microorganism according to claim 1, characterized in that the promoter capable of increasing expression is the PCJ7 promoter. 3. Microorganism according to claim 1, characterized in that the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. 4. Composition for producing L-leucine characterized in that it comprises a microorganism of the genus Corynebacterium that has an improved activity of a modified polypeptide that has an activity of isopropylmalate synthase, in which glycine at position 561 from an N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is replaced by aspartic acid encoded by SEQ ID NO: 39 or a degenerate sequence thereof, wherein the promoter of the polynucleotide encoding the modified polypeptide in the microorganism is replaced by a promoter capable of increasing expression compared to the native promoter. 5. Composition for producing L-leucine, characterized in that it comprises a vector, wherein the vector comprises a polynucleotide of SEQ ID NO: 39 or a degenerate sequence thereof that encodes a modified polypeptide that has isopropylmalate synthase activity, in which glycine at position 561 from an N-terminus of a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid; and wherein the promoter of the polynucleotide in the vector is replaced with a promoter capable of increasing expression compared to the native promoter. 6. Composition according to claim 4, characterized in that the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. 7. Method for preparing a microorganism of the genus Corynebacterium that produces L-leucine, characterized in that it comprises introducing a polynucleotide of SEQ ID NO: 39 or degenerate sequence thereof or vector, which comprises a polynucleotide of SEQ ID NO: 39 or sequence degenerate form thereof encoding a modified polypeptide having an isopropylmalate synthase activity, in which the glycine at position 561 from an N-terminus of the polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 is replaced with acid aspartic, in a microorganism of the genus Corynebacterium, where the microorganism is resistant to norleucine or leucine; and wherein the polynucleotide promoter in the polynucleotide or vector is replaced with a promoter capable of increasing expression compared to the native promoter. 8. Method for producing L-leucine, comprising: (a) cultivating a microorganism comprising a modified polypeptide that has isopropylmalate synthase activity, in which the glycine at position 561 from an N-terminus of the polypeptide which consists of an amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid; or a microorganism in which the polynucleotide of SEQ ID NO: 39 or degenerate sequence thereof or vector comprises the polynucleotide of SEQ ID NO: 39 or degenerate sequence thereof, in a medium for producing L-leucine; and (b) recovering L-leucine from the cultured microorganism or cultured medium, wherein the promoter of the polynucleotide encoding the modified polypeptide in the microorganism is replaced with a promoter capable of increasing expression compared to the promoter native; or wherein the polynucleotide promoter in the polynucleotide or vector is replaced with a promoter capable of increasing expression compared to the native promoter.