The present application relates to novel hydroxy-acetoacetic acid synthase variants and relates to their use, specifically acetonitrile hydroxy acid synthase variants, methods of producing microbial L- or branched-chain amino acid using the same comprising the same.
[2]
BACKGROUND
[3]
Branched-chain amino acids, i.e., L- valine, L- leucine, L- isoleucine, and acts to increase the protein in the object, it is known to play an important role as an energy source when exercising, etc. are used pharmaceuticals, foods. Branched-chain amino acids are There is difficult to manufacture through fermentation one of the branched chain amino acids on an industrial scale due to the use of the same enzymes in a similar synthesis process. In the production of branched-chain amino acids, branched-chain amino acids first enzyme acetonitrile hydroxy acid synthase (acetohydroxy acid synthase) serve one, previous studies mainly small subunit (acetohydroxy acid of this most important in the biosynthesis synthase small subunit R 106-12, US2014-0335574, US2009-496475, US2006-303888, US2008-245610), related:; 109; IlvN protein), most studies related to the feedback off due to the variation (protein Expr Purif 2015 of May. the situation is very tight.
[4]
Acetonitrile hydroxy acid synthase is generating acetonitrile-hydroxybutyric acid (2-aceto-2-hydroxy-butyrate) from the role, and the keto acid (ketobutyric acid) and pyruvic acid to generate the acetoacetate lactate (acetolactic acid) from two molecules of pyruvic acid an enzyme that plays a role. The acetoacetate-hydroxy acid synthase is pyruvic acid (pyruvate) of dicarboxylic reubok misfire (decarboyxlation) and to catalyze the condensation of pyruvic acid and other molecules of the valine and leucine precursor of acetoacetate lactic acid or pyruvic acid produced in the digital camera reubok misfire and the 2-keto the condensation reaction with butyrate (2-ketobutyrate) to produce the precursor can be the acetoacetate-hydroxybutyrate of isoleucine catalyst. Therefore, acetonitrile hydroxy acid synthase is an important enzyme involved in the initial phase of L- branched chain amino acid biosynthesis.
[5]
Detailed Description of the Invention
SUMMARY
[6]
The present applicants have developed the results sought, acetonitrile hydroxy acid synthase variants of, in particular acetonitrile hydroxy acid synthase large subunit mutants in order to effectively produce the branched-chain amino acid L-. Thus, in a high yield from a microorganism containing the mutant confirmed that can produce L- branched-chain amino acids, and completed the present application.
[7]
Problem solving means
[8]
One object of the present application is to provide an acetonitrile-hydroxy acid synthase (acetohydroxy acid synthase) mutant.
[9]
Another object of the present application is to provide the acetonide-hydroxy acid synthase and vector transformant with the vector is introduced, including a polynucleotide, the polynucleotide encoding the variants.
[10]
It is another object of the present application is to provide a microorganism containing or producing said vector is introduced, L- branched-chain amino acids to the acetonide-hydroxy acid synthase variants.
[11]
It is another object of the present application includes the steps of a microorganism producing the branched-chain amino acid L- cultivated in a culture medium; And to provide a branched-chain amino acid producing L- recovering the L- branched chain amino acids from the microorganism or its culture medium.
[12]
Effects of the Invention
[13]
Acetonitrile hydroxy acid synthase variants according to the present application that when the active introduction of the microorganism is increased significantly because the L- branched chain amino acid-producing ability of the microorganism, and can be widely used in the mass production of L- branched-chain amino acid .
[14]
Best Mode for Carrying Out the Invention
[15]
One aspect of the present application for achieving the above object, the large subunit of acetonitrile-hydroxy acid synthase; other amino acid other than Leo non Leo non-used write amino acid sequence position 96 times of (acetolactate synthase large subunit IlvB protein) substituted or is a single amino acid sequence position 503 tryptophan or substituted by another amino acid other than tryptophan or an amino acid sequence position 96 single-threonine and 503 single tryptophan all is replaced with a different amino acid, acetoacetic hydroxy acid synthase variants with.
[16]
Specifically, the large subunit of the acetonitrile-hydroxy acid synthase can have an amino acid sequence represented by SEQ ID NO: 1. In more detail, the acetonitrile-hydroxy acid synthase variants in the amino acid sequence shown in SEQ ID NO: 1, Su 96th from its N- terminal non-Leo (threonine) or 503rd tryptophan (tryptophan) is substituted to another amino acid may be, or 96th threonine and tryptophan are all the 503rd, acetonitrile hydroxy acid synthase variants replaced with another amino acid.
[17]
The term "acetonide hydroxy acid synthase (acetohydroxy acid synthase)" in the present application may be involved in the first step in the biosynthesis, branched-chain amino acid L- of the enzymes relating to the biosynthesis of branched-chain amino acid L-. Specifically, acetonitrile hydroxy acid synthase is pyruvic acid (pyruvate) of dicarboxylic reubok misfire (decarboyxlation) and to catalyze the condensation reaction of pyruvic acid with another molecule to produce lactic acid in acetonitrile, or the valine precursor dicarboxylic reubok misfire of pyruvic acid and 2-keto the condensation reaction with butyrate (2-ketobutyrate) to produce the precursor can be the acetoacetate-hydroxybutyrate of isoleucine catalyst. Specifically, the starting and acetonitrile hydroxy acid isopropyl merori reductase by Ajay (acetohydroxy acid isomeroreductase), dihydroxy acid-dihydroxy Alpharetta claim (dihydroxy acid dehydratase), transaminase B (transaminase B) a catalytic reaction from acetoacetate lactate It is L- valine biosynthesis geochimyeon sequentially. In addition, the acetonitrile from the acetonitrile-hydroxy lactic acid isobutyl merori reductase kinase (acetohydroxy acid isomeroreductase), dihydroxy acid-dihydroxy Alpharetta claim (dihydroxy acid dehydratase), 2- isopropyl malate synthase (2-isopropylmalate synthase), isopropyl malate the iso camera (isopropylmalate isomerase), 3- di-isopropyl malate dehydrogenase (3-isopropylmalate dehydrogenase) is the L- leucine biosynthesis by, transaminase B (transaminase B) geochimyeon the catalyst reaction in order. Meanwhile, Acetonitrile hydroxy Starting from the butyrate-acetoacetate-hydroxy acid isopropyl merori reductase kinase (acetohydroxy acid isomeroreductase), dihydroxy acid-dihydroxy Alpharetta claim sequentially a catalytic reaction by the (dihydroxy acid dehydratase), transaminase B (transaminase B) the L- isoleucine biosynthesis is a geochimyeon. Thus, a key enzyme in the biosynthetic pathway of branched chain amino acids L-.
[18]
Acetonitrile hydroxy acid synthase is encoded by the ilvB and ilvN, two genes, ilvB gene large subunit of acetonitrile-hydroxy acid synthase; the (large subunit IlvB), ilvN gene is small in acetonitrile hydroxy acid synthase subunits; a (small subunit IlvN) each coding.
[19]
Acetonitrile hydroxy acid synthase in the present application it may be a genus Corynebacterium microorganism-derived, specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum may be) derived. More specifically, the acetonide-hydroxy acid synthase large subunit is above as well as the amino acid sequence shown in SEQ ID NO: 1 sequence with at least 70%, specifically 80% or more, more specifically, more specifically at least 85% at least 90%, even more specifically, if having a 95% or greater homology or identity IlvB protein comprising the protein activity can be included without limitation. Further, the polynucleotide encoding a protein of IlvB protein activity, taking into account the codon due to the degeneracy (degeneracy) of the codons preferred in the organisms intended to be expressing the protein, changing the amino acid sequence of the protein expressed from the coding region it does not in the scope various modifications may be made to the coding region amino acid sequence of SEQ ID nO: 1 is nucleotide sequence encoding can include, without limitation, but there may be specifically SEQ ID nO: 2 encoded by the nucleotide sequence of.
[20]
[21]
"Acetonide hydroxy acid synthase variants" in this application refers to a protein wherein the acetoacetate-hydroxy acid synthase The amino acid sequence of the one or more amino acid mutations in the protein (e. G., Added, removed, or replaced). More specifically, the acetonide-hydroxy acid synthase variant is a protein of acetonitrile-hydroxy acid synthase protein by the mutation of the present application, its activity is effectively increased as compared to wild-type or modified before. Variations in the present application are generally known industry methods known in the art as a way to improve the enzymes can be used without limitation, and thus has a rational design strategies such as (rational design) and induced evolution (directed evolution). For example, a rationally designed strategy where the amino acid in a particular location - and a method of specifying mutations introduced (site-directed mutagenesis, or site-specific mutagensis), induce the evolution strategy method to cause a random variation (random mutagenesis), etc. there is. In addition, it can be mutated without the operation of the outside by natural mutation. More specifically, the acetonide-hydroxy acid synthase variants may be separated or not, the recombinant protein, may be in a non-naturally occurring. However, without being limited thereto.
[22]
Acetonitrile hydroxy acid synthase variants of the present application include, but are not limited to, specifically, 96th threonine (threonine) or 503rd tryptophan from the N- terminus of IlvB protein having the amino acid sequence shown in SEQ ID NO: 1 (tryptophan) this may be a mutated IlvB protein, or, but are not limited thereto, and may be 96th threonine and tryptophan 503rd the same time replaced by a different amino acid IlvB protein. Examples substituted with the 96th threonine-serine (serine), cysteine ​​(cystein) or alanine (alanine) of it, or that the 503rd tryptophan, glutamine (glutamine) substituted with, asparagine (asparagine) or leucine (leucine) the IlvB may be proteins. In the case as soon the the 96th or 503rd amino acid substituted by another amino acid, some of the same time the amino acid sequence sequence having a deletion, modified, substituted or added in the amino acid sequence same with acetonitrile-hydroxy acid synthase variants of the present application, or equivalent if that shows the activity it is obvious that within the scope of the present application.
[23]
In addition, in having the above-described variation scope of acetonitrile-hydroxy acid synthase variants of the present application acetonitrile hydroxy acid synthase large subunit mutants itself, acetonitrile hydroxy containing the acetoacetate-hydroxy acid synthase large subunit mutants It includes, but are acid synthase, or acetonitrile-hydroxy acid synthase acetonitrile hydroxy acid synthase having both a large subunit and a small subunit variant, is not particularly limited.
[24]
[25]
The present application in acetonitrile hydroxy acid synthase by substituting the 96th and 503rd amino acids of the protein kinase in a variety of different amino acids L- confirmed that the branched-chain amino acid production is increased, acetonitrile associated with increased L- branched-chain amino acid producing hydroxy acids synthase was identified as the 96 th and 503 th position is an important position in the mutant protein. However, the amino acid substitution in the embodiment of the present application is not limited to the embodiment scope of this application that only illustrate typical one that shows the effect of the present application, another amino acid other than the 96th write Leo write the non-Leo Nin , it is apparent that the 503 th tryptophan with another amino acid other than tryptophan, or threonine and the 96th that all of the 503 th tryptophan can be expected an effect corresponding to the effect described in example If replaced by another amino acid.
[26]
Also, acetoacetate-hydroxy acid synthase variants of the present application may have the amino acid sequence described by any one of SEQ ID NO: 28 to 33, but is not limited thereto. Further, substantially having an activity of the same or correspond to the acetonide-hydroxy acid synthase variants, including the mutations of the present application, each of the sequences with 70%, 80%, 85%, 90%, 95% or 99% a polypeptide having a homology or identity can include, without limitation.
[27]
[28]
Homology (homology) or identities (identity) is related to the degree of two given amino acid sequence or nucleotide sequence and can be expressed as a percentage.
[29]
Terms homology and identity can often be used interchangeably.
[30]
A conserved (conserved) polynucleotide or sequence homology or identity of polypeptides can be used with the default gap penalties established by the program that is determined by the standard arrangement algorithm used. In practice, it has a homology or (homologous) or the same (identical) sequences are generally sequences in whole or in total at least about 50% of the length, 60%, 70%, 80% or intermediate or high stringent conditions in accordance with the 90% It may be hybrid in (stringent conditions). Containing degenerate codons instead of the codons in a hybrid polynucleotide polynucleotides are also contemplated.
[31]
Whether any two polynucleotide or polypeptide sequence of the has the homology, similarity or identity include, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: by using the default parameters as in 2444 may be determined using known computer algorithms such as the "FASTA" program. Alternatively, only the needles of the EMBOSS package program, as is done in (EMBOSS: 276-277: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet 16..) (Version 5.0.0 or later) needle-only-flavor (Needleman-Wunsch) algorithm (.. Needleman and Wunsch, 1970, J. Mol Biol 48: 443-453) can be determined is used. (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED,.] Academic Press, San Diego, 1994, and [CARILLO ETA /.] (1988) SIAM J Applied Math 48: 1073 contains) for example, you can use the BLAST, ClustalW or the National Center for Biotechnology information database to determine the homology, similarity or identity.
[32]
Polynucleotides or polypeptides of homology, similarity or identity, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2: 482, as is known in, e.g., Needleman et al. (1970), J Mol Biol.48: can be determined by using the GAP computer program, such as 443 compares the sequence information. In summary, GAP program defines a value obtained by dividing the total number of symbols in the shorter of the two sequences from the number, the arrangement similar to sign (i.e., nucleotides or amino acids). The default parameters for the GAP program include: (1) one binary comparison matrix (1 and ratio to the identity-by containing a value of 0 for identity) and Schwartz and Dayhoff, eds, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. As disclosed by 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: the weighted comparison matrix of 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) addition of 0.10 penalty for each symbol in each gap, and the penalty of 3.0 for each gap (or gap opening penalty 10, gap extension penalty 0.5); And (3) may include no penalty for end gaps. Thus, as used herein, the term "homology" or "identity" represents the relevance (relevance) between the sequences.
[33]
Another aspect of the present application, a polynucleotide encoding an acetonitrile-hydroxy acid synthase variants of the present application.
[34]
The term "polynucleotide" in the present application has a meaning that includes DNA and RNA molecules, its basic structural units of nucleotides are also included, as well as natural nucleotides, analogues (analogue) the sugar or base portion is deformed. In this application it is not the polynucleotide may be a polynucleotide or an artificially synthesized polynucleotide separated from the cells and thus limiting.
[35]
Polynucleotide encoding an acetonitrile-hydroxy acid synthase variants of the present application, if the nucleotide sequence encoding a protein of acetonitrile-hydroxy acid synthase activity mutant of the present application can be included without limitation. Specifically, the polynucleotides due to the degeneracy (degeneracy) of the codon or in consideration of the codon-preferred organisms intended to be expressing the protein, within a range that does not alter the amino acid sequence of the protein made various changes to the coding region can. The sequence number may be included without limitation if the nucleotide sequence encoding the amino acid sequence of 28 to 33, it may be one having a specific nucleotide sequence, for example, by any one of SEQ ID NO: 34 to the substrate 39. Further, due to the above, the degeneracy of the codon that has a substantially acetonitrile hydroxy acid synthase activity of the, including the mutations of the present application, each of the sequences and of 70%, 75%, 80%, 85%. A polynucleotide having at least 90%, 95%, 97%, or 99% homology or identity can include, without limitation.
[36]
Or which can be prepared from a known gene sequence probe, e.g., a complementary sequence and to under stringent conditions Hydride Chemistry, protein activity consisting of SEQ ID NO: 28 to 33 amino acid sequence of all or part of the nucleotide sequence It has can be included, without limitation, if the sequence encoding the protein. Refers to conditions that permit the specific hybridization between the "stringent condition" is a polynucleotide. These conditions are described in detail in the literature (e.g., J. Sambrook et al., Above). For example, homology or identity with the high genetic each other, more than 80%, specifically at least 85%, more specifically at least 90%, more specifically at least 95%, more specifically more than 97%, in particular concrete with the hybridization between genes having at least 99% homology or identity, and rather homology or identity with low gene together do not hybridization conditions, or a 60 ℃ ordinary washing condition of Southern hybridization, 1 × SSC, 0.1 % SDS, specifically 60 ℃, 0.1 × SSC, 0.1% SDS, more specifically, at a salt concentration and temperature corresponding to 68 ℃, 0.1 × SSC, 0.1% SDS, 1 times, specifically, twice or three times can enumerate the conditions for cleaning. Hybridization Although the mismatch (mismatch) between the base be possible depending on the stringency of hybridization, though, requires that the two nucleic acids having a complementary sequence. The term, "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. therefore, This application is also similar as well as a nucleic acid sequence substantially complementary to an isolated nucleic acid fragment comprising the sequence throughout. Specifically, a polynucleotide having a homology or identity can be detected using hybridization conditions comprising a hybridization step at Tm value of 55 ℃ using the above-described conditions. Further, the Tm value can be 60 ℃, 63 ℃ or 65 ℃. However, it is not limited to, it can be properly adjusted by those skilled in the art according to the purpose. Appropriate stringency for hybridizing polynucleotide is dependent on the degree of complementarity and the length of the polynucleotide and variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).
[37]
Another aspect of the present application, a vector comprising a polynucleotide encoding a mutated acetonitrile hydroxy acid synthase variants of the present application.
[38]
Term in this application, the terms "vector" refers to any vehicle for the cloning and / or transformation of the base into a host cell. The vector may be a different DNA fragment the replicon (replicon) that may lead to replication of the fragments coupled in combination. A "replicon" is DNA replication in vivo self functioning as a unit, that is, refers to a replicable, any genetic units by the control of itself. More specifically, it may be a natural condition or a condition of a recombinant plasmid, phage, cosmid, chromosome, virus. For example, a phage vector or a cosmid vector and the like pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A and Charon21A, pBR series, pUC system, pBluescriptII the system as a plasmid vector, It may be used based pGEM, pTZ-based, such as pCL and pET-based system. The available vector herein is not particularly limited and may be a known expression vector. It may also include transposons, or an artificial chromosome in said vector.
[39]
In this application vector is a not particularly limited including a polynucleotide encoding an acetonitrile-hydroxy acid synthase variants of this application, mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.) , may be a plant cell, yeast cell, insect cell or bacterial cell vector that can replicate and / or express the nucleic acid molecule in a eukaryotic or prokaryotic cell (e. g., E. coli, etc.), specifically a host in the cell connected operably to an appropriate promoter, such that the polynucleotide is to be expressed, can be a vector that includes at least one selectable marker.
[40]
Further, means that in the term, and the promoter sequence "operably linked" to initiate the expectation and mediate transcription of the polynucleotide encoding the target protein of the present application is the gene sequence is functionally connected.
[41]
[42]
Another aspect of the present application, is a transgenic vector is introduced in this application.
[43]
Transformant in this application is not particularly limited, if the vector is introduced to express the acetonitrile-hydroxy acid synthase variants of the present application may include a switch all transformed cells possible. Specifically transformed to Escherichia genus, the genus Corynebacterium, Streptomyces MRS, Brevibacterium genus, Serratia genus, Providencia genus, Salmonella typhimurium, such as bacterial cells; Yeast cells; Fungal cells, such as blood Chiapas pastoris; Draw Joe Pilar, insect cells such as Spodoptera Sf9 cells Terra; CHO (Chinese hamster ovary cells, chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymph subregion (human lymphoblastoid), COS, NSO (mouse myeloma), 293T, bow melanoma cells, HT-1080, BHK ( baby hamster kidney cells, baby hamster kidney cells), HEK (human embryonic kidney cells (human embryonic kidney cells), PERC.6 (human retina cells), animal cells and the like; or may be a plant cell.
[44]
[45]
Another aspect of the present application is a microorganism in which the acetonitrile hydroxy include hydroxy acid synthase mutant, or a vector production cost, L- branched-chain amino acids comprising introducing a polynucleotide encoding the variants.
[46]
The term refers to, amino acids with a branched alkyl group in the side chain is "L- branched chain amino acid" in the present application, valine, leucine and isoleucine include. Specifically, the L- branched-chain amino acids in the present application is not intended to be, but may be L- or L- valine, leucine, limited.
[47]
The "microorganism" in the present application a specific mechanism is weakened due to a cause such as a wild-type microorganism and, and naturally or artificially include all of the microorganisms takes place the genetic modification, the foreign gene inserted, or that the activity of the intrinsic gene enhanced or weakened or a concept that includes both enhanced microbes. It refers to any microorganism capable of expressing acetonitrile hydroxy acid synthase variants of the present application, and specifically may be a genus Corynebacterium microorganism, more specifically, Corynebacterium glutamicum, Corynebacterium ammonia to Ness, Brevibacterium Lactobacillus buffer momentum ( Brevibacterium lactofermentum ), Brevibacterium Plastic pan ( Brevibacterium flavum ), Corynebacterium thermo amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens ) and the like. Still more specifically not from Corynebacterium glutamicum, or limited to this.
[48]
In the present application by the term, "L- branched microorganism producing the amino acid" is meant wild-type or mutant microorganism with L- branched chain amino acid-producing ability and, (non-natural occuring) caused by non-naturally specifically It may be a recombinant microorganism, but is not limited thereto. Microorganism producing the branched chain amino acid is L- acetonitrile hydroxy acid synthase that contains the mutant kinase, or vector is introduced, including a polynucleotide encoding the variant, the wild-type microorganism, native acetonitrile-hydroxy acid of the present application synthase may be significantly increased ability L- branched-chain amino acid production compared with the non-modified microorganism or a microorganism that does not contain acetonitrile hydroxy acid synthase proteins, including microorganisms, acetonitrile hydroxy acid synthase protein containing protein.
[49]
[50]
Another aspect of the present application, the method comprising culturing a microorganism producing L- branched chain amino acids of the present application; And a method for the branched-chain amino acid producing L- recovering the L- branched-chain amino acids from the microorganism or the medium obtained in the above step.
[51]
Term in this application, the terms "culture" is meant that the growth in the environmental conditions which control the microorganisms with artificially appropriately. L- branched-chain amino acid production method using microorganisms having the L- branched chain amino acid-producing ability in the present application can be carried out using methods well known in the art. Specifically, the culture is not however be cultured in a continuous manner to the injection batch process a batch process, a batch or repeated injection (fed batch or repeated fed batch process), this limit.
[52]
The medium used to culture should meet the requirements of the particular strains in a suitable manner. For example, known is a culture medium for Corynebacterium spp (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981). Which can be used members include glucose, saccharose, lactose, paroxetine lactose, maltose, starch, cellulose and and carbohydrates, soybean oil, sunflower oil as per, castor oil, oils and fats such as coconut oil, palmitic acid, stearic acid, , an alcohol such as a fatty acid, glycerol and ethanol, such as linoleic acid, may be included the organic acids such as acetic acid. These materials may be used separately or as mixtures, without being limited thereto. The nitrogen source which can be used may include a peptone, yeast extract, gravy, 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. In addition, sources of nitrogen can be used separately or as mixtures, without being limited thereto. Personnel to that may be used include phosphate, sodium or potassium phosphate or the corresponding potassium susoyi-containing salts can be included. Also, culture media may contain metal salts such as magnesium sulfate or iron sulfate needed for growth. The essential growth substances, such as addition, amino acids and vitamins can be used. Also may be used, suitable precursors to the culture medium. The above described material may be added in a batch or continuous system by a suitable method to the culture in culture. However, without being limited thereto.
[53]
With an acid compound such as phosphoric acid or sulfuric acid or a basic compound such as sodium hydroxide, potassium hydroxide, ammonia in a suitable manner it is possible to adjust the pH of water culture. It is also possible to use anti-foaming agents such as fatty acid polyglycol ester can suppress foam generation. To maintain aerobic conditions, oxygen or oxygen into the culture-containing gas can be injected (for example, air). Incubation temperature of water from 20 to 45 ℃, may be specifically 25 to 40 ℃. Culture can be continued until the production of the desired L- branched-chain amino acid is obtained at the maximum. For this purpose the incubation time may be from 10 to 160 hours. L- branched-chain amino acids can be contained in, or released into the culture medium, cells. However, without being limited thereto.
[54]
[55]
The method for recovering a branched-chain L- amino acid from the medium or the microorganism may be performed using suitable methods known in the art. For example, centrifugation, filtration, crystallization, protein precipitant treatment with (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography various types of chromatography, such as, HPLC, and may be used in combination of these methods, it is not limited to these examples. Also recovering the L- branched-chain amino acid may include a further purification step, it may be performed using a suitable method known in the art.
[56]
[57]
Mode for the Invention
[58]
The present application in more detail described below through an embodiment. It is, however, not intended that the scope of the present application are for explaining the present application by way of example only to the examples.
[59]
[60]
Example 1: Production DNA library encoding the acetoacetate-hydroxy acid synthase (acetohydroxy acid synthase) variation using artificial mutation method
[61]
[62]
In the present embodiment it was produced acetonitrile hydroxy acid synthase vector library For the first cross-chromosomal insert in the following manner in order to obtain a mutant. Corynebacterium glutamicum ATCC14067 acetonitrile hydroxy acid synthase (SEQ ID NO: 1) the encryption of derived ilvB gene (SEQ ID NO: 2) introduced in the Error-prone PCR method by performing the base substitution mutations randomly target the ilvB to obtain the mutant gene (2395bp). An Error-prone PCR is GenemorphII was performed using Random Mutagenesis Kit (Stratagene), Corynebacterium glutamicum ATCC14067 genomic DNA subject to a template primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) was used .
[63]
[64]
Primer 1 (SEQ ID NO: 3): 5'- AACCG GTATC GACAA TCCAA T -3 '
[65]
Primer (SEQ ID NO: 4): 5'- GGGTC TCTCC TTATG CCTC -3 '
[66]
[67]
Was allowed variation is 10 to 3.5 dog introduced per kb within the amplified gene fragment, PCR conditions were denaturation 96 ℃, 30 seconds; ℃ annealing 53, 30 seconds; And the polymerization reaction 72 ℃, was repeated for 2 minutes 30 times.
[68]
The amplified gene fragment by using the pCR2.1-TOPO TA cloning kit (Invitrogen 社) was connected to a pCR2.1-TOPO vector (hereinafter referred to as 'pCR2.1'), and transformed into E. coli DH5α kanamycin (25 mg / ℓ ) it was plated on the LB solid medium containing. After transfection selection of the conversion 20 kinds of colonies was confirmed that the obtained plasmid is introduced by shifting the different locations as a result of 2.1 mutations / kb frequency analysis of the nucleotide sequence. Taking the approximately 20,000 transformed E. coli colony was extracted plasmid, and named it as pCR2.1-ilvB (mt) library.
[69]
In addition, wild-type for use as a control ilvB to prepare a plasmid having the gene. Using primers 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) PCR was as Corynebacterium glutamicum ATCC14067 genomic DNA under the same conditions as described above as a template. Polymerase was used PfuUltra High-Fidelity DNA Polymerase (Stratagene). A production plasmid was named as pCR2.1-ilvB (WT).
[70]
[71]
Example 2: ilvB deletion strain produced
[72]
[73]
KCCM11201P for introducing the pCR2.1-ilvB (mt) to the library (the Republic of Korea Patent No. 10-1117022) strain as the parent strain ilvB prepare a defect strain.
[74]
ilvB deletion vectors with the wild type Corynebacterium glutamicum ATCC14067 Chromosomal template in order to produce a primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6), primer 5 (SEQ ID NO: 7) and primer 6 (SEQ ID NO: PCR using a No. 8) was carried out.
[75]
[76]
Primer 3 (SEQ ID NO: 5): 5'- GCGTC TAGAG ACTTG CACGA GGAAA CG -3 '
[77]
Primer 4 (SEQ ID NO: 6): 5'- CAGCC AAGTC CCTCA GAATT GATGT AGCAA TTATC C -3 '
[78]
Primer 5 (SEQ ID NO: 7): 5'- GGATA ATTGC TACAT CAATT CTGAG GGACT TGGCT G -3 '
[79]
Primer 6 (SEQ ID NO: 8): 5'- GCGTC TAGAA CCACA GAGTC TGGAG CC -3 '
[80]
[81]
PCR conditions were carried out for 5 min denaturation at 95 ℃, 95 ℃ was repeated 30 seconds denaturation, annealing 55 ℃ 30 seconds, 30 seconds 72 ℃ polymerization 30 times, in 72 ℃ polymerization 7 minutes of reaction.
[82]
As a result ilvB gene promoter front and ilvB to give the gene 3 'of the DNA fragment of 731bp SEQ ID NO: 9 and SEQ ID NO: 10 of the 712bp DNA fragment containing the terminal, respectively.
[83]
And the amplified SEQ ID NO: 9 and SEQ ID NO: 10 as a template, PCR was performed using the primer 3 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 8). PCR conditions were carried out for 5 min denaturation at 95 ℃, 95 ℃ was repeated 30 seconds denaturation, annealing 55 ℃ 30 seconds, 60 seconds, 72 ℃ polymerization 30 times, in 72 ℃ polymerization 7 minutes of reaction.
[84]
As a result, ilvB DNA fragment containing the promoter, the early part of the gene and the 3 'DNA fragment is attached SEQ ID NO: 11, a DNA fragment of 1407bp including a terminal (the ilvB fragment) was amplified.
[85]
Corynebacterium glutamicum pDZ vector replication can not be in the (Republic of Korea Patent No. 10-0924065 call) and the amplified ilvB treated fragment with restriction enzyme Xba back, after the connection is established by using a DNA joining enzymes, cloning was obtained by this plasmid was named as pDZ-ilvB.
[86]
pDZ-ilvB the electric pulse method in Corynebacterium glutamicum KCCM11201P: After each transformed with (Appl Microbiol Biothcenol (1999) 52 ... 541-545) kanamycin (kanamycin) 25 mg / ℓ, and L- valine to obtain a transformant strain in selective media containing 2 mM each by a L- leucine, L- isoleucine. Inserted in the genome into a secondary recombination (cross-over) ilvB by short ilvB was obtained a strain gene is inactivated, thereby KCCM11201P ilvB was named.
[87]
[88]
Example 3: The acetonide-hydroxy acid synthase mutant library produced L- amino acid-producing ability and increased strain Screening
[89]
[90]
The production of KCCM11201P ilvB about 10,000 and transformed by the production of pCR2.1-ilvB (mt) library, the strain with the parent strain homologous recombination, and chromosome spread on the composite plate medium containing the kanamycin (25 mg / L) has secured the colonies, each colony KCCM11201P ilvB from /pCR2.1-ilvB(mt)-1 KCCM11201P ilvB was named to /pCR2.1-ilvB(mt)-10000.
[91]
In addition, the production of pCR2.1-ilvB (WT) vector KCCM11201P ilvB by transforming the strain was the control strain produced, KCCM11201P ilvB was named /pCR2.1-ilvB(WT).
[92]
[93]

[94]
Glucose 10 g, peptone 10 g, beef extract 5 g, yeast extract 5 g, brain heart leachate (brain heart infusion) 18.5 g, NaCl 2.5 g, urea 2 g, sorbitol (sorbitol) 91 g, agar 20 g (distilled water 1 liter)
[95]
[96]
Was inoculated with approximately 10,000 colonies were obtained on selection medium for each 300 ㎕ 32 ℃ in 96-deep-well plates, and cultured for about 24 hours with 1000 rpm. To analyze the production of L- amino acid-producing culture was used in the ninhydrin method (J. Biol Chem 1948. 176:. . 367-388). After the incubation is completed, the culture supernatant 10 ㎕ and non-hard-lean reaction solution was measured for absorbance in a spectrophotometer (spectrophotometer) at a wavelength of 570 nm after the 190 ㎕ at 65 ℃ 30 minutes of reaction and control KCCM11201P ilvB /pCR2.1-ilvB ( WT) were selected mutant strain of about 213 colonies were visible in an increased absorbance at least 10% compared to the absorbance of the strain. More colony are indicated a similar or decreased compared to the control absorbance.
[97]
[98]

[99]
Glucose 10 g, ammonium sulfate 5.5 g, magnesium sulfate heptahydrate 1.2 g, the first potassium phosphate 0.8 g, potassium phosphate dibasic 16.4 g, biotin 100 ㎍, Thiamine -HCl 1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide 2000 ㎍ ( 1 liter of distilled water)
[100]
[101]
Strains selected 213 has been carried out repeatedly ninhydrin reaction in the same manner as described above, KCCM11201P ilvB /pCR2.1-ilvB(WT) strain capability than L- amino acid-producing strains were selected for improved top 60 thereof.
[102]
[103]
Example 4: acetonitrile-hydroxy acid synthase mutant library screening confirmation note L- valine-producing ability
[104]
[105]
And to compare the L- valine-producing ability of the 60 strains selected in Example 3 were cultured in the following ways: a culture solution ingredient.
[106]
Producing 250 ㎖ corner medium containing 25 ㎖ - each strain bapeul to flask 1, and the platinum is in the inoculation, 30 ℃ for 72 hours, then this was cultured with shaking at 200 rpm. By HPLC analysis, the concentration of L- valine.
[107]
[108]

[109]
Glucose 100 g, ammonium sulfate 40 g, soy protein 2.5 g, corn steep solids Corn Steep Solids) 5 g, urea 3 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.5 g, biotin 100 ㎍, Thiamine -HCl 1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide 3000 ㎍, 30 g calcium carbonate (in 1 liter of distilled water)
[110]
[111]
Was of the 60 strains is L- valine concentration selected strains improved two kinds of performing the cultivation, and repeating analysis, the analyzed concentration of L- valine is to be as given in Table 1. The remaining 58 strains of the species showed a result that the concentration of L- valine rather decline.
[112]
[113]
TABLE 1
KCCM11201P of the selected two kinds of ilvB /pCR2.1-ilvB(mt) L- valine production levels
Strain L- Valine (g / ℓ)
Placed one Placed second Placed third Average
Controls KCCM11201PilvB/pCR2.1-ilvB(WT) 2.7 2.9 2.9 2.8
1 KCCM11201PilvB/pCR2.1-ilvB(mt)-5602 3.1 3.5 3.4 3.3
2 KCCM11201PilvB/pCR2.1-ilvB(mt)-7131 2.9 3.3 3.1 3.1

[114]
[115]
[116]
L- valine concentration analysis, the two above screening care L- valine yield control KCCM11201P ilvB confirmed that the increase up to 20.7% compared to /pCR2.1-ilvB(WT) strain.
[117]
[118]
Example 5: acetonitrile-hydroxy acid synthase mutant library screening care ilvB gene mutation confirmed
[119]
[120]
In order to determine the random mutations introduced to the Example 4-hydroxy-acetoacetic acid of the selected two kinds of strain synthase ilvB the base sequence of the gene were analyzed. A PCR using primers 7 (SEQ ID NO: 12) and primer 8 (SEQ ID NO: 13) to determine the nucleotide sequence was carried out.
[121]
[122]
Primer 7 (SEQ ID NO: 12): 5'- CGCTT GATAA TACGC ATG -3 '
[123]
Primer 8 (SEQ ID NO: 13): 5'- GAACA TACCT GATAC GCG -3 '
[124]
[125]
The securing each variant ilvB through the sequencing of the gene fragment, wild-type of SEQ ID NO: 2 ilvB compared to the gene sequence variants ilvB has confirmed that the nucleotide sequence of the gene, which the acetoacetate-hydroxy acid synthase protein variants through of the amino acid sequence was confirmed. Information on the variation of the selected strains two kinds acetonitrile hydroxy acid synthase protein is shown in Table 2 below.
[126]
[127]
TABLE 2
Selecting two kinds KCCM11201P / pCR2.1-ilvB (mt) mutated acetonitrile hydroxy acid synthase protein information
Strain Acetonitrile hydroxy acid synthase amino acid mutation
KCCM11201P ilvB /pCR2.1-ilvB(mt)-5602 W503Q
KCCM11201P ilvB /pCR2.1-ilvB(mt)-7131 T96S

[128]
[129]
Example 6: Vector production for introduction of acetoacetate-hydroxy acid synthase mutant
[130]
[131]
In order to confirm the effect of the mutated acetonitrile hydroxy acid synthase protein identified in the Example 5 was produced in a vector capable of introducing it into a chromosome.
[132]
Inserting an Xba restriction site at the 5 'terminus on the basis of the identified base sequence primer 9 (SEQ ID NO: 14) and primer 10 (SEQ ID NO: 15) and primer 11 (SEQ ID NO: 16) and primer 12 (SEQ ID NO: 17) It was synthesized. Using this primer pair, by performing a PCR to chromosome of the selected two kinds of each template variants of two ilvB was amplified gene fragment. PCR conditions were carried out for 5 min denaturation at 94 ℃, after repeating 30 times a 94 ℃ 30 cho denaturation, annealing 56 ℃ cho 30, 72 ℃ 2 bun polymerization, polymerization at 72 ℃ 7 minutes of reaction.
[133]
[134]
Primer 9 (SEQ ID NO: 14): 5'- CGCTC TAGAC AAGCA GGTTG AGGTT CC -3 '
[135]
Primer 10 (SEQ ID NO: 15): 5'- CGCTC TAGAC ACGAG GTTGA ATGCG CG -3 '
[136]
Primer 11 (SEQ ID NO: 16): 5'- CGCTC TAGAC CCTCG ACAAC ACTCA CC -3 '
[137]
Primer 12 (SEQ ID NO: 17): 5'- CGCTC TAGAT GCCAT CAAGG TGGTG AC -3 '
[138]
[139]
After the treatment, a gene fragment of the two kinds of amplified by PCR with restriction enzymes Xba obtain the respective DNA fragment, and this restriction enzyme Xba transformed after E. coli DH5α is connected to the pDZ vector for chromosomal introduced having a terminal kanamycin (25 mg / ℓ) were plated on the LB solid medium containing.
[140]
After the gene purpose by PCR screening for the conversion colonies transfected with the inserted vector typically has attained a plasmid using the known plasmid extraction of the plasmid ilvB each pDZ-ilvB (W503Q) based on the variation into the gene, It was named pDZ- ilvB (T96S).
[141]
[142]
Example 7: KCCM11201P derived acetonitrile hydroxy acid synthase mutation introduced strain produced and L- valine-producing ability compared
[143]
[144]
It was transformed in the sixth embodiment a novel mutation step 2 of two or introduced vector, respectively prepared in the chromosome by homologous recombination of L- valine-producing strain Corynebacterium glutamicum KCCM11201P. After that on the chromosomal ilvB were selected by the mutant strain is introduced into the sequence analysis, the ilvB the mutated strains are introduced respectively KCCM11201P :: ilvB (W503Q) and KCCM11201P :: ilvB was named (T96S). And the mutant strain pDZ- ilvB KCCM11201P the produce (T96S) :: introduction of vector ilvB was transformed to (W503Q). Then the strain variation ilvB 2 all paper introduced on the chromosome, respectively KCCM11201P :: ilvB was named (W503Q / T96S).
[145]
[146]
Carried out by culturing in the same manner as in Example 4, was analyzed for the concentration of L- valine therefrom (Table 3).
[147]
[148]
TABLE 3
Mutated strains derived KCCM11201P acetonitrile hydroxy acid synthase introduced strain produced L- valine concentration (g / ℓ)
Strain Placed one Placed second Placed third Average
Controls KCCM11201P 2.9 2.8 2.8 2.8
1 KCCM11201P::ilvB(W503Q) 3.3 3.2 3.3 3.3
2 KCCM11201P::ilvB(T96S) 3.2 3.0 3.1 3.1
3 KCCM11201P::ilvB(W503Q/T96S) 3.3 3.4 3.4 3.4

[149]
[150]
New mutagenic strains of two (KCCM11201P :: ilvB (W503Q), KCCM11201P :: ilvB (T96S)) is an increase of up to 17.8% L- valine production ability compared to the parent strain, two kinds of variations are both introduced strain (KCCM11201P: : ilvB (W503Q / T96S)) it was increased by 21.4% L- valine production ability compared to the parent strain.
[151]
Thus, acetonitrile hydroxy acid synthase variants of the large subunit of the present invention because the effect on the first enzyme in the biosynthetic pathway of branched chain L- amino acids, as well as L- valine, L- isoleucine producing ability and increase in the L- leucine It is expected to have.
[152]
The present inventors have found that the L- valine improved strain KCCM11201P :: ilvB (W503Q) and KCCM11201P :: ilvB were La (T96S) a Corynebacterium glutamicum KCJ-0793 and-0796 KCJ named, Korea Culture Center microorganism ( the KCCM) with accession dated 25 January 2016 and was given an accession number and KCCM11809P KCCM11810P.
[153]
[154]
Example 8: Preparation of containing the DNA encoding the mutated acetonitrile hydroxy acid synthase L- valine biosynthesis overexpression vector
[155]
[156]
It was produced L- valine biosynthesis overexpression vector from L- valine-producing strain of Corynebacterium glutamicum KCCM11201P as a control. In addition, the Example 7 by respective KCCM11201P prepared :: ilvB (W503Q), KCCM11201P :: ilvB was prepared (T96S) the acetoacetate-hydroxy acid synthase L- valine biosynthesis overexpression vector that contains the DNA encoding a mutant from .
[157]
[158]
The "BamH restriction site primer 13 insert (SEQ ID NO: 18) and the third terminal, a primer 14 inserted into the Xba restriction site at the terminal thereof (SEQ ID NO: 19) 5 for the production of the vector was prepared. Using this primer pair, L- valine-producing strain of Corynebacterium glutamicum KCCM11201P, and KCCM11201P Example 7 A prepared in :: ilvB (W503Q), KCCM11201P :: ilvB into the Chromosome of each mold (T96S) by performing PCR variants of two ilvBN was amplified gene fragment. PCR conditions were carried out for 5 min denaturation at 94 ℃, it was repeated 30 times 4 minutes 72 ℃ 94 ℃ 30 cho denaturation, 56 ℃ 30 cho annealing, polymerization, and polymerization at 72 ℃ 7 minutes of reaction.
[159]
[160]
Primer 13 (SEQ ID NO: 18): 5'- CGAGG ATCCA ACCGG TATCG ACAAT CCAAT -3 '
[161]
Primer 14 (SEQ ID NO: 19): 5'- CTGTC TAGAA ATCGT GGGAG TTAAA CTCGC -3 '
[162]
[163]
After obtaining each of the DNA fragment by treating the gene segments of the two kinds of amplification by the PCR with restriction enzyme BamH with Xba, and then connect it to the over-expression vector pECCG117 with restriction enzymes BamH and Xba end and transformed into E. coli DH5α kanamycin (25 mg / ℓ) were plated on the LB solid medium containing.
[164]
After selecting the conversion colonies transfected with the vector the gene is inserted object by PCR using a commonly known plasmid extraction were obtained The plasmid of the plasmid ilvB according to the variation into the gene, respectively pECCG117-ilvBN, pECCG117-ilvB (W503Q) was named as N, pECCG117-ilvB (T96S) N.
[165]
[166]
Example 9: Production of L- valine biosynthesis overexpression vector that contains DNA that codes the same acetonitrile-hydroxy acid synthase substitution mutation in position by another amino acid
[167]
[168]
In the mutated acetonitrile hydroxy acid synthase protein identified in the Example 5, to determine the effect of mutation position, 96th amino acid as threonine, or an amino acid other than serine, 503rd amino acid is an amino acid other than tryptophan or glutamine a vector comprising a substituted mutation was produced.
[169]
Specifically, L- valine-producing strain of Corynebacterium glutamicum from acetonitrile-hydroxy acid synthase is 503 amino acid form of the variation or 96th amino acid of the type substituted by asparagine, leucine or the dehydratase is substituted with alanine or cysteine ​​KCCM11201P It was produced in the mutant overexpression vector containing the L- valine biosynthesis. The substituted amino acid is not only a typical example of amino acids that can be substituted, limited.
[170]
[171]
The vector produced, first, Corynebacterium glutamicum primers with chromosomal template of KCCM11201P strain 13 for (SEQ ID NO: 18) and primer 15 (SEQ ID NO: 20), primer 16 (SEQ ID NO: 21) and primer 14 (SEQ ID NO: No. 19) to perform a PCR, it was amplified in a 1055bp DNA fragment having an Xba restriction site in 'DNA fragment of about 2041bp and 3 with BamH restriction sites at the terminal, the terminal 5 used. PCR conditions were carried out for 5 min denaturation at 94 ℃, after repeating 30 times a 94 ℃ 30 cho denaturation, annealing 56 ℃ cho 30, 72 ℃ 2 bun polymerization, polymerization at 72 ℃ 7 minutes of reaction.
[172]
[173]
Primer 15 (SEQ ID NO: 20): 5'- CTTCA TAGAA TAGGG TCTGG TTTTG GCGAA CCATG CCCAG -3 '
[174]
Primer 16 (SEQ ID NO: 21): 5'- CTGGG CATGG TTCGC CAAAA CCAGA CCCTA TTCTA TGAAG -3 '
[175]
[176]
Then, the two DNA fragments amplified as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19). PCR conditions were carried out for 5 min denaturation at 94 ℃, it was repeated 30 times 4 minutes 72 ℃ 94 ℃ 30 cho denaturation, 56 ℃ 30 cho annealing, polymerization, and polymerization at 72 ℃ 7 minutes of reaction.
[177]
As a result, a 503 amino acid in acetonitrile-hydroxy acid synthase that contains a mutation of the type substituted with asparagine ilvBN to give a gene fragment.
[178]
In the same way, the Corynebacterium glutamicum primer 13 the chromosome as a template for KCCM11201P strain (SEQ ID NO: 18) and primer 17 (SEQ ID NO: 22), primer 18 (SEQ ID NO: 23) and primer 14 (SEQ ID NO: 19) used to perform the PCR, it was amplified in a 1055bp DNA fragment having an Xba restriction site in 'DNA fragment of about 2041bp and 3 with BamH restriction sites at the terminal, the terminal 5.
[179]
[180]
Primer 17 (SEQ ID NO: 22): 5'- CTTCA TAGAA TAGGG TCTGC AGTTG GCGAA CCATG CCCAG -3 '
[181]
Primer 18 (SEQ ID NO: 23): 5'- CTGGG CATGG TTCGC CAACT GCAGA CCCTA TTCTA TGAAG -3 '
[182]
[183]
Then, the two DNA fragments amplified as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19).
[184]
As a result, containing the variation of the type substituted with the 503rd amino acid in acetonitrile-hydroxy acid synthase leucine ilvBN to give a gene fragment.
[185]
[186]
In the same way, the Corynebacterium glutamicum primer 13 the chromosome as a template for KCCM11201P strain (SEQ ID NO: 18) and primer 19 (SEQ ID NO: 24), primer 20 (SEQ ID NO: 25) and primer 14 (SEQ ID NO: 19) used to perform the PCR, it was amplified in a 2276bp DNA fragment having an Xba restriction site in the "DNA fragment 3 of about 819bp with the BamH restriction site at the terminal, the terminal 5.
[187]
[188]
Primer 19 (SEQ ID NO: 24): 5'- GGTTG CGCCT GGGCC AGATG CTGCA ATGCA GACGC CAAC -3 '
[189]
Primer 20 (SEQ ID NO: 25): 5'- GTTGG CGTCT GCATT GCAGC ATCTG GCCCA GGCGC AACC -3 '
[190]
[191]
Then, the two DNA fragments amplified as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19).
[192]
As a result, the 96th amino acid in acetonitrile-hydroxy acid synthase that contains a mutation of the type substituted with alanine ilvBN to give a gene fragment.
[193]
[194]
In the same way, the Corynebacterium glutamicum primer 13 the chromosome as a template for KCCM11201P strain (SEQ ID NO: 18) and primer 21 (SEQ ID NO: 26), primer 22 (SEQ ID NO: 27) and primer 14 (SEQ ID NO: 19) used to perform the PCR, it was amplified in a 2276bp DNA fragment having an Xba restriction site in the "DNA fragment 3 of about 819bp with the BamH restriction site at the terminal, the terminal 5.
[195]
[196]
Primer 21 (SEQ ID NO: 26): 5'- GGTTG CGCCT GGGCC AGAGC ATGCA ATGCA GACGC CAAC -3 '
[197]
Primer 22 (SEQ ID NO: 27): 5'- GTTGG CGTCT GCATT GCATG CTCTG GCCCA GGCGC AACC -3 '
[198]
[199]
Then, the two DNA fragments amplified as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19).
[200]
As a result, the 96th amino acid in acetonitrile-hydroxy acid synthase that contains a mutation of the type substituted with cysteine ilvBN to give a gene fragment.
[201]
[202]
Carried out in the same manner as in Example 8, and then processes the above mentioned four mutant gene fragment of amplified by PCR with a restriction enzyme BamH and Xba obtained each DNA fragment, over-expression with this restriction enzyme BamH and Xba end vector pECCG117 after connecting to transform the E. coli DH5α, which was plated on LB solid medium containing kanamycin (25 mg / ℓ).
[203]
After the gene purpose by PCR screening for the conversion colonies transfected with the inserted vector typically has attained a plasmid using the known plasmid extraction of the plasmid ilvB in each sequence in accordance with the variation into the gene pECCG117-ilvB (W503N ) of N, named pECCG117-ilvB (W503L) N, pECCG117-ilvB (T96A) N, pECCG117-ilvB (T96C) N.
[204]
[205]
Example 10: Preparation of the wild type-derived mutant acetonitrile hydroxy acid synthase introduced strain compared production and L- valine-producing ability
[206]
[207]
Example 8 and carrying a L- valine biosynthesis overexpression vector prepared in Example 9 pECCG117-ilvBN, pECCG117-ilvB (W503Q) N, pECCG117-ilvB (T96S) N and pECCG117-ilvB (W503N) N, pECCG117-ilvB (W503L ) were each inserted in the N, pECCG117-ilvB (T96A) N, electroporation the pECCG117-ilvB (T96C) N a Corynebacterium glutamicum wild-type strain ATCC13032. The production strain, respectively from Corynebacterium glutamicum ATCC13032 :: pECCG117-ilvBN, Corynebacterium glutamicum ATCC13032 :: pECCG117-ilvB (W503Q) N, Corynebacterium glutamicum ATCC13032 :: pECCG117-ilvB (T96S) N, Corynebacterium glutamicum ATCC13032 :: pECCG117-ilvB (W503N) N, Corynebacterium glutamicum ATCC13032 :: pECCG117-ilvB (W503L) N, Corynebacterium glutamicum ATCC13032: : pECCG117-ilvB (T96A) were named as N and Corynebacterium glutamicum ATCC13032 :: pECCG117-ilvB (T96C) N. If the vector is transformed, so have the kanamycin resistance was confirmed that transformed through growth whether in a kanamycin containing a 25 mg / ℓ concentration in the culture medium.
[208]
In the same way as the medium used in the 4 to conducted to evaluate the production strain, 250 ㎖ corner containing a production medium 25 ㎖ - at 30 ℃ inoculated, and each strain in bapeul flask for 72 hours, shaking culture at 200 rpm It was. By HPLC analysis, the concentration of L- valine (Table 4).
[209]
[210]
TABLE 4
Wild-type-derived mutated acetonitrile hydroxy acid synthase introduced L- valine production levels of strain
Strain L- Valine (g / ℓ)
Placed one Placed second Placed third Average
Controls ATCC13032::pECCG117-ilvBN 0.1 0.1 0 0.1
1 ATCC13032::pECCG117-ilvB(W503Q)N 0.8 0.8 0.7 0.8
2 ATCC13032::pECCG117-ilvB(T96S)N 0.4 0.5 0.5 0.5
3 ATCC13032::pECCG117-ilvB(W503N)N 0.7 0.6 0.5 0.6
4 ATCC13032::pECCG117-ilvB(W503L)N 0.7 0.7 0.5 0.5
5 ATCC13032::pECCG117-ilvB(T96A)N 0.2 0.3 0.2 0.2
6 ATCC13032::pECCG117-ilvB(T96C)N 0.4 0.3 0.5 0.4

[211]
[212]
As a result, the 96th or 503rd amino acids are substituted with other amino acids of the novel mutations acetonitrile hydroxy acid synthase was confirmed that the increase up to 700% capability L- valine production compared to the control group. From this, it was confirmed the importance of the 96th 503 th position, and, it is expected that only L- valine not have an influence also neunge production of other branched amino acids.
[213]
[214]
Example 11 Mutated acetonitrile hydroxy acid synthase introduced strain produced and L- leucine-producing ability compared
[215]
[216]
For the present application a variant of acetonitrile-hydroxy acid synthase large subunit to identify affects to increase the production capacity of other branched amino acids L-, L- isoleucine producing ability of a still another example of a branched chain amino acid L- confirmed.
[217]
Specifically, the sixth embodiment a novel mutation step 2 of two or introduction vector produced in each of L- leucine-producing strain by homologous recombination chromosome of Corynebacterium glutamicum KCCM11661P (Republic of Korea Patent Application No. 10-2015-0119785 No. , it was transformed in the Republic of Korea Patent Publication No. 10-2017-0024653 call). After that on the chromosomal ilvB were selected by the mutant strain is introduced into the sequence analysis, the ilvB the mutated strains are introduced respectively KCCM11661P :: ilvB (W503Q) and KCCM11661P :: ilvB was named (T96S).
[218]
The Corynebacterium glutamicum KCCM11661P was obtained by the following method as norleucine Corynebacterium glutamicum ATCC 14067 derived from mutants having a resistance to (Norleucine, NL).
[219]
After the culture, the culture solution 5 ㎖ was recovered during specifically, Corynebacterium glutamicum ATCC was inoculated a strain active 14 067 to the culture in the activation medium for 16 hours at 121 ℃ a seed medium sterilized 5 minutes 14 hours . The collected culture solution 100 mM citrate buffer solution (citric buffer) to a three and then, after the addition of NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) so to have a final concentration of 200 mg / L, processing for 20 minutes and washed with 100 mM phosphate buffer solution (phosphate buffer). To smear the strains treated with NTG in a minimal medium results mortality rate ever calculating the mortality rate was 85%. In order to obtain a mutant strain resistant to norleucine (Norleucine, NL) for the derivatives of L- leucine, least cost to be the final concentration of the NTG-treated strains NL 20 mM, 30mL, 40 mM and 50 mM respectively was added smeared on the medium, and cultured at 30 ℃ 5 ilgan acquires NL-resistant mutants.
[220]

[221]
Juicy 1%, polypeptone 1%, 0.5% sodium chloride, 1% yeast extract, 2% agar, pH 7.2
[222]

[223]
Glucose 5%, Bacto peptone 1%, sodium chloride 0.25%, 1% yeast extract, urea 0.4%, pH 7.2
[224]

[225]
Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium phosphate 0.1%, 1, 0.1% urea, thiamine 0.00 to 1%, 2% ㎍ biotin 200 / L, agar, pH 7.0
[226]
[227]
Mutant obtained by the above method is Corynebacterium glutamicum KCJ-24 (Corynebacterium glutamicum KCJ-24) termed was, as of January 2015 22 Date of the accession to the international deposit agency, Korea Culture Center of Microorganisms under the Budapest Treaty, each trustee It was given the number KCCM11661P.
[228]
[229]
The KCCM11661P :: ilvB (W503Q) and KCCM11661P :: ilvB to the (T96S) carried cultured in the same manner as in Example 4, was analyzed for the concentration of L- leucine therefrom (Table 5).
[230]
[231]
Table 5
L- leucine producing strain derived from the concentration of KCCM11661P mutated acetonitrile hydroxy acid synthase introduced strain (g / ℓ)
Strain Placed one Placed second Placed third Average
Controls KCCM11661P 2.7 2.6 2.9 2.7
1 KCCM11661P::ilvB(W503Q) 3.1 3.3 3.3 3.2
2 KCCM11661P P::ilvB(T96S) 3.0 3.2 3.1 3.1

[232]
[233]
Two kinds of new mutagenic strains (KCCM11661P :: ilvB (W503Q), KCCM11661P :: ilvB (T96S)) was increased up to 26.9% L- leucine production ability compared to the parent strain.
[234]
[235]
Example 12: KCCM11662P derived mutated acetonitrile hydroxy acid synthase introduced strain produced and L- leucine-producing ability compared
[236]
[237]
Example novel mutation step 2 of two or introduction vector produced in each of six different L- leucine-producing strain by homologous recombination chromosome of Corynebacterium glutamicum KCCM11662P (Republic of Korea Patent Application No. 10-2015-0119785 call, It was transformed in the Republic of Korea Patent Publication No. 10-2017-0024653 call). After that on the chromosomal ilvB were selected by the mutant strain is introduced into the sequence analysis, the ilvB the mutated strains are introduced respectively KCCM11662P :: ilvB (W503Q) and KCCM11662P :: ilvB was named (T96S).
[238]
The Corynebacterium glutamicum KCCM11662P was obtained by the following method as norleucine Corynebacterium glutamicum ATCC 13869 derived from mutants having a resistance to (Norleucine, NL).
[239]
Specifically, Corynebacterium glutamicum ATCC 13869 by the Tommy to the parent strain glues, were cultured in the same way to obtain KCCM11662P in Example 11, was finally obtained the NL-resistant mutants.
[240]
Mutant obtained by the above method is Corynebacterium glutamicum KCJ-28 (Corynebacterium glutamicum KCJ-28) termed was, as of January 2015 22 Date of the accession to the international deposit agency, Korea Culture Center of Microorganisms under the Budapest Treaty, each trustee It was given the number KCCM11662P.
[241]
[242]
The KCCM11662P :: ilvB (W503Q) and KCCM11662P :: ilvB to the (T96S) carried cultured in Example 4 in the same manner, was analyzed, the concentration of L- leucine therefrom (Table 6).
[243]
[244]
TABLE 6
L- leucine producing strain derived from the concentration of KCCM11662P mutated acetonitrile hydroxy acid synthase introduced strain (g / ℓ)
Strain Placed one Placed second Placed third Average
Controls KCCM11662P 3.1 3.0 3.1 3.1
1 KCCM11662P::ilvB(W503Q) 3.5 3.4 3.3 3.4
2 KCCM11662P P::ilvB(T96S) 3.3 3.3 3.2 3.3

[245]
[246]
New mutagenic state (KCCM11662P :: of the two above ilvB (W503Q), KCCM11662P :: ilvB (T96S)) was increased up to 13.3% L- leucine production ability compared to the parent strain.
[247]
[248]
From the above description, those skilled in the filed will appreciate that this application without changing the technical spirit or essential features may be embodied in other specific forms. In this regard, the embodiments described above are only to be understood as exemplary rather than limiting in all aspects. The scope of the present application should be construed as the meaning and scope, and all such modifications as derived from the equivalent concepts of the claims to be described later, rather than the description above within the scope of the present application.
[249]
[250]
[251]
[252]
[253]

WE Claims

[Claim 1]
Acetonitrile hydroxy acid synthase large subunit of (acetolactate synthase large subunit; IlvB protein) amino acid sequence position 96 threonine are substituted with another amino acid other than threonine one of the 503 single amino acid sequence position tryptophan other than tryptophan the or substituted with other amino acids, or amino acid sequence position 96 and threonine 503 tryptophan both single one has been substituted by another amino acid, acetoacetic hydroxy acid synthase variants.
[Claim 2]
The method of claim 1, wherein the protein is IlvB, acetonitrile hydroxy acid synthase variant having an amino acid sequence represented by SEQ ID NO: 1.
[Claim 3]
The method of claim 1 wherein the 96th threonine-serine (serine), the, acetonitrile hydroxy acid synthase variants replaced with cysteine ​​(cysteine) or an alanine (alanine).
[Claim 4]
The method of claim 1, wherein the 503rd tryptophan, glutamine (glutamine), asparagine (asparagine), or a leucine, acetonitrile hydroxy acid synthase variants substituted with (leucine).
[Claim 5]
The method of claim 1, wherein the acetoacetate-hydroxy acid synthase variant has an amino acid sequence described by any one of SEQ ID NO: 28 to SEQ ID NO: 33, acetonitrile hydroxy acid synthase variants.
[Claim 6]
Any one of claims 1 to 5 any one of the polynucleotides encoding the acetoacetate-hydroxy acid synthase variants of the term.
[Claim 7]
Any one of claims 1 to 5, wherein in any one of the vector comprising a polynucleotide encoding an acetonitrile-hydroxy acid synthase variants.
[Claim 8]
The transformant of claim 7 vector is introduced.
[Claim 9]
Any one of claims 1 to 5, wherein in any one of the acetoacetate-hydroxy acid synthase comprises a mutant kinase, or claim 7 microorganism vector is introduced production cost, L- branched-chain amino acids.
[Claim 10]
10. The method of claim 9, wherein the microorganism is the genus Corynebacterium ( Corynebacterium sp.) Which, L- branched-chain amino acid producing micro-organisms.
[Claim 11]
11. The method of claim 10, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ) which, microorganism producing L- branched-chain amino acids.
[Claim 12]
10. The method of claim 9, wherein the branched chain amino acid is L- L- valine, L- or a leucine, L- branched-chain amino acid producing micro-organisms.
[Claim 13]
(A) the microorganism of claim 9 to 12 for producing any one of the amino acids of the L- branched wherein culturing in a medium; And (b), L- branched-chain amino acid production method that includes the recovery of L- branched-chain amino acid from the medium or the microorganism of step (a).
[Claim 14]
The method of claim 13 wherein the branched-chain amino acid is L- L- valine, L- or a leucine, L- branched-chain amino acid production method.