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  • C12Y202/01—Transketolases and transaldolases (2.2.1)
  • C12Y202/01006—Acetolactate synthase (2.2.1.6)

Definitions

• the present invention relates to novel acetohydroxy acid synthetase variants and their uses, and more particularly to acetohydroxy acid synthetase variants, microorganisms containing them, or a method for producing L-branched chain amino acids using the same.
• Branched chain amino acids such as L-valine, L-leucine and L-isoleucine are known to play an important role as an energy source in exercise and to increase protein in an individual and are used in medicines and foods. Because branched chain amino acids use the same enzymes for similar biosynthetic processes, it is difficult to produce one branched chain amino acid on an industrial scale through fermentation. In the production of branched chain amino acids, the role of acetohydroxy acid synthase, which is the first enzyme of branched chain amino acid biosynthesis, is the most important. However, previous researches on this matter have mainly focused on small subunits (acetohydroxy acid synthase small subunit (Protein Expr Purif. 2015 May; 109: 106-12., US2014-0335574, US2009-496475, US2006-303888, US2008-245610), related studies Is very scarce.
• Acetohydroxyacid synthase plays a role in producing acetolactic acid from two molecules of pyruvic acid and in producing 2-aceto-2-hydroxy-butyrate from ketobutyric acid and pyruvic acid It is an enzyme that plays a role.
• the acetohydroxy acid synthase catalyzes the decarboyxlation of pyruvate and the condensation reaction with other pyruvic acid molecules to produce acetolactic acid, a precursor of valine and leucine, or a dicarboxylic acid derivative of pyruvic acid, It can catalyze the condensation reaction with 2-ketobutyrate to produce acetohydroxybutyrate, which is a precursor of isoleucine.
• acetohydroxy acid synthases are very important enzymes involved in the initial process of L-branched chain amino acid biosynthesis.
• the present inventors have developed mutants of acetohydroxy acid synthase, specifically, acetohydroxoic acid synthase small subunit mutants. Thus, it was confirmed that the L-branched chain amino acid can be produced from the microorganism containing the mutant at a high yield, and the present application was completed.
• One object of the present application is to provide an acetohydroxy acid synthase mutant.
• Another object of the present invention is to provide a polynucleotide encoding said acetohydroxy acid synthetase variant, a vector comprising said polynucleotide, and a transformant into which said vector is introduced.
• the acetohydroxy acid synthetase variant according to the present application significantly increases the L-branched chain amino acid producing ability of the microorganism when the activity is introduced into the microorganism, and thus can be widely used for mass production of L-branched chain amino acids .
• One aspect of the present application for achieving the above object is to provide a method for producing an acetolactate synthase large-sized subunit (IlvB protein), wherein the threonine at the amino acid sequence position 96 of the acetolactate synthase large- Or an acetohydroxy acid synthase mutant in which tryptophan at amino acid sequence position 503 is replaced with another amino acid other than tryptophan or both threonine at amino acid sequence position 96 and tryptophan at position 503 are substituted with another amino acid.
• IlvB protein acetolactate synthase large-sized subunit
• the large subunit of the acetohydroxy acid synthase may have the amino acid sequence shown in SEQ ID NO: 1. More specifically, the acetohydroxy acid synthetase variant is one in which the 96th threonine or tryptophan from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid , Or an acetohydroxy acid synthetase variant in which both the 96th threonine and the 503th tryptophan are replaced with different amino acids.
• acetohydroxy acid synthase is an enzyme involved in the biosynthesis of L-branched chain amino acids and may be involved in the first step of biosynthesis of L-branched chain amino acids. Specifically, the acetohydroxy acid synthase catalyzes the decarboyxlation of pyruvate and the condensation reaction with other pyruvic acid molecules to produce acetolactate, which is a precursor of valine, or the dicarboxylation of pyruvic acid and 2-keto It can catalyze the condensation reaction with 2-ketobutyrate to produce acetohydroxybutyrate, which is a precursor of isoleucine.
• the reaction catalyzed by acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B starts from acetolactate.
• L-valine is biosynthesized on a sequential basis.
• L-leucine is biosynthesized by sequentially catalyzed reactions catalyzed by isopropylmalate isomerase, 3-isopropylmalate dehydrogenase, and transaminase B, respectively.
• Acetohydroxy acid synthetase is encoded by two genes, ilvB and ilvN, the ilvB gene is a large subunit (IlvB) of acetohydroxysultainase, the ilvN gene is a small molecule of acetohydroxyacid synthase And a small subunit (IlvN), respectively.
• the acetohydroxy acid synthase may be derived from a microorganism belonging to the genus Corynebacterium, and specifically may be derived from Corynebacterium glutamicum . More specifically, the acetohydroxy acid synthetase large subunit has not only the amino acid sequence shown in SEQ ID NO: 1 but also the amino acid sequence having 70% or more, specifically 80% or more, more specifically 85% or more, More preferably 90% or more, more specifically 95% or more homology or identity, and has IlvB protein activity.
• a polynucleotide encoding a protein having an IlvB protein activity can change the amino acid sequence of the protein expressed from the coding region in consideration of the codon preference in the organism to which the protein is expressed due to degeneracy of the codon
• the amino acid sequence of SEQ ID NO: 1 may be any of the nucleotide sequences coding for the nucleotide sequence of SEQ ID NO: 2, but may be specifically the nucleotide sequence of SEQ ID NO: 2.
• acetohydroxy acid synthetase variant in the present application means a protein in which one or more amino acids are mutated (e.g., added, removed or substituted) on the amino acid sequence of the above-mentioned acetohydroxy acid synthase protein.
• the acetohydroxy acid synthetase variant is a protein in which the activity of the acetohydroxy acid synthase protein is increased by mutation of the present application in comparison with that before wild-type or deformation.
• Variations in this application are generally known methods known in the art as methods for improving enzymes and may be used without limitation, including strategies such as rational design and directed evolution.
• a rational design strategy may involve site-directed mutagenesis or site-specific mutagenesis of amino acids at specific sites, and methods that induce random mutagenesis . It may also be mutated without external manipulation by a natural mutation.
• the acetohydroxy acid synthetase variant may be isolated, recombinant protein, or non-naturally occurring. However, it is not limited thereto.
• the acetohydroxy acid synthetase variants of the present application include, but are not limited to, the 96th threonine or the 503th tryptophan from the N-terminus of the IlvB protein having the amino acid sequence of SEQ ID NO: 1, May be a mutated IlvB protein or may be an IlvB protein in which the 96th threonine and 503th tryptophan are simultaneously substituted with another amino acid.
• the 96th threonine is substituted with serine, cystein or alanine
• the 503th tryptophan is substituted with glutamine, asparagine or leucine.
• RTI ID 0.0 > IlvB < / RTI >
• an amino acid sequence in which a part of the amino acid sequence is deleted, modified, substituted or added is also the same as or equivalent to the acetohydroxy acid synthetase variant of the present application Is included in the scope of the present application.
• acetohydroxy acid synthetase variants of the present application includes acetohydroxy acid synthetase subunit mutant itself having the above-described mutation, acetohydroxyacetic acid mutant containing the acetohydroxyacid synthase large subunit variant Acid synthase, or an acetohydroxy acid synthase having both a small subunit variant of acetohydroxysultaine and a small subunit, but is not particularly limited thereto.
• the substituted amino acid in the examples of the present application is merely a representative example showing the effect of the present application, and the scope of the present application is not limited to the examples, and the 96th threonine may be substituted with amino acids other than threonine , It is obvious that the effect corresponding to the effect described in the embodiment can be expected when the 503th tryptophan is replaced with another amino acid other than tryptophan or the 96th threonine and the 503th tryptophan are replaced with different amino acids.
• the acetohydroxy acid synthetase variant of the present application may have the amino acid sequence shown in any one of SEQ ID NOS: 28 to 33, but is not limited thereto.
• Homology or identity refers to the degree of association with two given amino acid sequences or nucleotide sequences and can be expressed as a percentage.
• Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and default gap penalties established by the program used can be used together.
• Substantially homologous or identical sequences generally have at least about 50%, 60%, 70%, 80% or 90% of the length of the sequence or the entire length of the sequence under moderate or high stringency conditions can be hybridized under stringent conditions.
• Polynucleotides containing degenerate codons instead of codons in hybridizing polynucleotides are also contemplated.
• BLAST Altschul, [S.] : 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)
• BLAST or ClustalW, of the National Center for Biotechnology Information Database can be used to determine homology, similarity, or identity.
• the homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2: 482, for example, in Needleman et al. (1970), J Mol Biol. 48: 443, by comparing the sequence information using a GAP computer program.
• the GAP program defines the total number of symbols in the shorter of the two sequences, divided by the number of similar aligned symbols (ie, nucleotides or amino acids).
• the default parameters for the GAP program are (1) a linear comparison matrix (containing 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp.
• Another aspect of the present application is a polynucleotide encoding an acetohydroxy acid synthase variant of the present application.
• polynucleotide in the present application has the meaning including DNA and RNA molecules, and the nucleotide, which is a basic constituent unit thereof, includes not only a natural nucleotide but also an analogue in which a sugar or base site is modified.
• the polynucleotide may be a polynucleotide isolated from a cell or an artificially synthesized polynucleotide, but is not limited thereto.
• the polynucleotide encoding the acetohydroxy acid synthetase variant of the present application may be included without limitation as long as it is a nucleotide sequence encoding a protein having the acetohydroxy acid synthetase mutant activity of the present application.
• the polynucleotide may have various modifications to the coding region within a range that does not change the amino acid sequence of the protein, due to codon degeneracy or a codon preferred in the organism to which the protein is to be expressed .
• the nucleotide sequence encoding the amino acid sequence of SEQ ID NOS: 28 to 33 may be any of the nucleotide sequences shown in SEQ ID NOS: 28 to 33, but may be a nucleotide sequence having any of the nucleotide sequences of SEQ ID NOS: 34 to 39, for example. Also, 70%, 75%, 80%, 85% of the sequences are deleted due to codon degeneracy, as long as they have substantially acetohydroxy acid synthase activity including mutations of the present application. Polynucleotides having 90%, 95%, 97%, or 99% or more homology or identity.
• a probe which can be prepared from a known gene sequence for example, a hydrolidase under stringent conditions with a complementary sequence to all or part of the above base sequence, so that the activity of the protein consisting of the amino acid sequence of SEQ ID NOS: May be included without limitation as long as it encodes a protein.
• stringent conditions means conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., J. Sambrook et al., Sangdong).
• Hybridization of genes having a homology or identity of 99% or more and hybridization of genes having less homology or homology with each other or hybridization with normal hybridization conditions such as 60 ° C, 1 x SSC, 0.1 Specifically at a salt concentration and a temperature corresponding to 0.1% SDS, specifically at 60 ⁇ ⁇ , 0.1 ⁇ SSC, 0.1% SDS, more specifically 68 ⁇ ⁇ , 0.1 ⁇ SSC, 0.1% SDS, It is possible to enumerate the conditions for washing.
• Hybridization requires that two nucleic acids have a complementary sequence, although mismatches between bases are possible, depending on the severity of hybridization.
• complementary is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other.
• adenosine is complementary to thymine and cytosine is complementary to guanine.
• the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the entire sequence.
• polynucleotides having homology or identity can be detected using hybridization conditions that include a hybridization step at a Tm value of 55 ° C and using the conditions described above.
• the Tm value may be 60 ° C, 63 ° C, or 65 ° C, but is not limited thereto and may be suitably adjusted by those skilled in the art according to the purpose.
• Suitable stringency to hybridize polynucleotides depends on the length and complementarity of the polynucleotide and the variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).
• Another embodiment of the present application is a vector comprising a polynucleotide encoding a mutated acetohydroxy acid synthetase variant of the present application.
• vector refers to any medium for cloning and / or transfer of a base into a host cell.
• a vector can be a replicon that can bring about the replication of a joined fragment of another DNA fragment.
• Replication unit refers to any genetic unit that functions in vivo as an autonomous unit of DNA replication, i.e., replicable by self-regulation. Specifically, it may be a plasmid, phage, cosmid, chromosome, or virus in a natural or recombinant state.
• pWE15, M13, ⁇ MBL3, ⁇ MBL4, ⁇ IXII, ⁇ ASHII, ⁇ APII, ⁇ t10, ⁇ t11, Charon4A and Charon21A can be used as the phage vector or cosmid vector, and as the plasmid vector, pBR, pUC, pBluescriptII, pGEM system, pTZ system, pCL system, pET system, or the like can be used.
• the vector usable in the present application is not particularly limited, and known expression vectors can be used.
• the vector may include a transposon or an artificial chromosome.
• the vector is not particularly limited as long as it contains a polynucleotide encoding an acetohydroxy acid synthetase variant of the present application, but may be a mammalian cell (such as a human, a monkey, a rabbit, a rat, a hamster,
• the host cell may be a vector capable of replicating and / or expressing the nucleic acid molecule in a eukaryotic or prokaryotic cell comprising a plant cell, a yeast cell, an insect cell or a bacterial cell (for example, Escherichia coli, etc.)
• the vector may be operably linked to a suitable promoter so that the polynucleotide can be expressed in a cell and comprising at least one selectable marker.
• operably linked also means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding the protein of interest of the present application.
• Another aspect of the present application is a transformant into which the vector of the present application has been introduced.
• the transformant is not particularly limited, but any transformable cell can be included as long as the vector can be introduced to express the acetohydroxy acid synthetase variant of the present application.
• bacterial cells such as transformed Escherichia, Corynebacterium, Streptomyces, Brevibacterium, Serratia, Propidensia, Salmonella typhimurium; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowmanella cells, HT-1080, BHK Animal hamster kidney cells, human hamster kidney cells, HEK (human embryonic kidney cells, PERC.6 (human retinal cells), or plant cells.
• Another embodiment of the present application is a microorganism producing the L-branched chain amino acid, wherein the vector contains the acetohydroxy acid synthetase variant or a polynucleotide encoding the mutant.
• L-branched chain amino acid refers to an amino acid having a branched alkyl group in the side chain and includes valine, leucine and isoleucine.
• the L-branched chain amino acid may be, but is not limited to, L-valine or L-leucine.
• microorganism includes both wild-type microorganisms and microorganisms in which natural or artificially genetically modified microorganisms are included, and a specific mechanism is weakened due to the insertion of an external gene, Or enhanced microorganisms. Refers to any microorganism capable of expressing an acetohydroxy acid synthetase variant of the present application.
• it may be a microorganism belonging to the genus Corynebacterium, and more specifically, a microorganism belonging to the genus Corynebacterium glutamicum, 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 so on. More specifically, Corynebacterium glutamicum, but is not limited thereto.
• microorganism producing L-branched chain amino acid in the present application means a microorganism capable of producing L-branched chain amino acid through natural type or mutation and specifically refers to non-natural occurring microorganisms. But are not limited to, recombinant microorganisms.
• the microorganism producing the L-branched chain amino acid includes the acetohydroxy acid synthetase variant of the present application or a vector containing a polynucleotide encoding the variant, and is a vector containing a wild-type microorganism, a natural acetohydroxy acid
• the ability to produce L-branched chain amino acids can be significantly increased as compared with microorganisms containing a synthase protein, non-modified microorganisms containing an acetohydroxy acid synthase protein, or microorganisms not containing an acetohydroxy acid synthase protein.
• Another aspect of the present application relates to a method for producing L-branched chain amino acids, comprising culturing a microorganism producing the L-branched chain amino acid of the present application; And recovering the L-branched chain amino acid from the microorganism or medium obtained in the above step.
• cultivation in the present application means cultivation of microorganisms under moderately artificially controlled environmental conditions.
• the method for producing L-branched chain amino acids using microorganisms having L-branched chain amino acid producing ability in the present application can be carried out by a method widely known in the art. Specifically, the culturing can be carried out continuously in a batch process, an injection batch, or a fed batch or repeated fed batch process, but is not limited thereto.
• the medium used for the culture should meet the requirements of the particular strain in an appropriate manner.
• culture media for Corynebacterium spp. Strains are known (see, for example, Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington, D.C., USA, 1981).
• Sugar sources that may be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, , Fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid.
• nitrogen sources examples include 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.
• the nitrogen source may also be used individually or as a mixture, but is not limited thereto.
• the number of people that can be used may include potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts.
• the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth.
• essential growth materials such as amino acids and vitamins can be used.
• suitable precursors may be used in the culture medium.
• the above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method. However, it is not limited thereto.
• Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.
• bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester.
• An oxygen or oxygen-containing gas e.g., air
• the temperature of the culture may be 20 to 45 ⁇ , specifically 25 to 40 ⁇ .
• Cultivation can continue until the desired amount of L-branched chain amino acid is obtained.
• the incubation time can be 10 to 160 hours.
• the L-branched chain amino acid may be released into the culture medium or contained in the cell. However, it is not limited thereto.
• Methods for recovering L-branched chain amino acids from microorganisms or media can be performed using suitable methods known in the art. For example, centrifugation, filtration, treatment with crystallization protein precipitant (salting-out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography , And HPLC, and a combination of these methods.
• the present invention is not limited to these examples.
• the step of recovering the L-branched chain amino acid may comprise an additional purification step and may be carried out using any suitable method known in the art.
• Example 1 Construction of a DNA library encoding mutant acetohydroxy acid synthase using the artificial mutation method
• a vector library for primary cross-linking in a chromosome was prepared by the following method in order to obtain an acetohydroxy acid synthetase mutant.
• Error-prone PCR was performed on ilvB gene (SEQ ID NO: 2) encoding acetohydroxy acid synthase (SEQ ID NO: 1) derived from Corynebacterium glutamicum ATCC14067 to randomly introduce base substitution mutations Gt ; ilvB < / RTI > mutants (2395 bp).
• the error-prone PCR was performed using the GenemorphII Random Mutagenesis Kit (Stratagene), and Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4) were used as a template for Corynebacterium glutamicum ATCC14067 genomic DNA .
• Primer 1 (SEQ ID NO: 3): 5'-AACCG GTATC GACAA TCCAA T -3 '
• Primer 2 (SEQ ID NO: 4): 5'-GGGTC TCTCC TTATG CCTC -3 '
• PCR conditions were denaturation at 96 ° C for 30 seconds; Annealing 53 ⁇ , 30 seconds; And the polymerization reaction was repeated 30 times at 72 ⁇ for 2 minutes.
• the amplified gene fragment was ligated to a pCR2.1-TOPO vector (hereinafter referred to as 'pCR2.1') using a pCR2.1-TOPO TA cloning kit (Invitrogen) and transformed into E. coli DH5 ⁇ to obtain kanamycin (25 mg / ). ≪ / RTI > After selection of 20 transformed colonies, plasmids were obtained and the nucleotide sequences were analyzed. As a result, it was confirmed that mutations were introduced at different positions with a frequency of 2.1 mutations / kb. Approximately 20,000 transformed E. coli colonies were picked to extract the plasmid and named it the pCR2.1-ilvB (mt) library.
• mt pCR2.1-ilvB
• a plasmid having a wild type ilvB gene for use as a control was prepared .
• Corynebacterium glutamicum ATCC14067 genomic DNA was used as a template using primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) under the same conditions as above.
• PfuUltra High-Fidelity DNA polymerase (Stratagene) was used as the polymerase.
• the constructed plasmid was named pCR2.1-ilvB (WT).
• KCCM11201P was prepared ilvB deficient strain for introducing the pCR2.1-ilvB (mt) to the library (the Republic of Korea Patent No. 10-1117022) strain as the parent strain.
• Primer 3 (SEQ ID NO: 5): 5'-GCGTC TAGAG ACTTG CACGA GGAAA CG-3 '
• Primer 4 (SEQ ID NO: 6): 5'-CAGCC AAGTC CCTCA GAATT GATGT AGCAA TTATC C -3 '
• Primer 5 (SEQ ID NO: 7): 5'-GGATA ATTGC TACAT CAATT CTGAG GGACT TGGCT G -3 '
• Primer 6 (SEQ ID NO: 8): 5'-GCGTC TAGAA CCACA GAGTC TGGAG CC -3 '
• the PCR conditions were denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and polymerization at 72 ° C for 30 seconds. The reaction was carried out at 72 ° C for 7 minutes.
• PCR was performed with primers 3 (SEQ ID NO: 5) and 6 (SEQ ID NO: 8) using the amplified SEQ ID NOS: 9 and 10 as a template.
• the PCR conditions were denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and polymerization at 72 ° C for 60 seconds.
• the reaction was carried out at 72 ° C for 7 minutes.
• ilvB fragment a DNA fragment of SEQ ID NO: 11 (hereinafter ilvB fragment) was amplified with 1407 bp in which a DNA fragment containing the forward portion of the ilvB gene and a DNA fragment containing the 3 'end were ligated .
• the pDZ vector (Korean Patent No. 10-0924065), which can not be cloned in Corynebacterium glutamicum, and the amplified ilvB fragment were treated with a restriction enzyme Xba and then ligated using a DNA joining enzyme, To obtain a plasmid and named it pDZ-ilvB.
• pDZ-ilvB was transformed into Corynebacterium glutamicum KCCM 11201P by the electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52: 541-545), and then kanamycin 25 mg / , L-leucine, and L-isoleucine were each obtained in a selection medium containing 2 mM each.
• the ilvB gene was inactivated by the ilvB fragment inserted in the genome through a secondary recombination process (cross-over), and this strain was named KCCM11201P ilvB .
• the prepared pCR2.1-ilvB (mt) library was transformed by homologous chromosome recombination and plated on a composite plate medium containing kanamycin (25 mg / L) Colonies were obtained and the respective colonies were named KCCM11201P ilvB / pCR2.1- ilvB ( mst) -1 to KCCM11201P ilvB / pCR2.1-ilvB ( mt) -10000.
• KCCM11201P ilvB / pCR2.1-ilvB (WT) was transformed into KCCM11201P ilvB strain to produce a control strain, which was named KCCM11201P ilvB / pCR2.1-ilvB (WT).
• the selected 213 strains were repeatedly subjected to the ninhydrin reaction in the same manner as above, and 60 strains having improved L-amino acid production ability were selected for KCCM 11201P ilvB / pCR2.1 -ilvB ( WT) strain.
• the culture medium components were analyzed by the following method.
• nucleotide sequences of the ilvB gene were analyzed in order to confirm the random mutations introduced into the acetohydroxy acid synthases of the two strains selected in Example 4 above. PCR was performed using primer 7 (SEQ ID NO: 12) and primer 8 (SEQ ID NO: 13) to determine the nucleotide sequence.
• Primer 7 (SEQ ID NO: 12): 5'-CGCTT GATAA TACGC ATG-3 '
• Primer 8 (SEQ ID NO: 13): 5'- GAACA TACCT GATAC GCG -3 '
• mutant ilvB gene was confirmed by comparing the obtained mutant ilvB gene fragments with the wild type ilvB gene sequence of SEQ ID NO: 2 through the nucleotide sequence analysis of each mutant type ilvB gene fragment, thereby obtaining mutant acetohydroxy acid synthase protein was confirmed.
• Information on the mutated acetohydroxyacid synthase proteins of the two strains selected is shown in Table 2 below.
• Example 6 Construction of a vector for introduction of an acetohydroxy acid synthase mutation
• Primer 9 (SEQ ID NO: 14): 5'-CGCTC TAGAC AAGCA GGTTG AGGTT CC -3 '
• Primer 10 (SEQ ID NO: 15): 5'-CGCTC TAGAC ACGAG GTTGA ATGCG CG-3 '
• Primer 11 (SEQ ID NO: 16): 5'-CGCTC TAGAC CCTCG ACAAC ACTCA CC -3 '
• Primer 12 (SEQ ID NO: 17): 5'-CGCTC TAGAT GCCAT CAAGG TGGTG AC-3 '
• Two kinds of gene fragments amplified by PCR were treated with restriction enzyme Xba to obtain respective DNA fragments.
• the DNA fragments were ligated to a pDZ vector for introduction of chromosome with restriction enzyme Xba end, transformed into E. coli DH5 ⁇ , and kanamycin (25 mg / L). < / RTI >
• Two novel mutation introduction vectors prepared in Example 6 were transformed into Corynebacterium glutamicum KCCM11201P, an L-valine producing strain, by two-step homologous chromosome recombination. Then, the strain into which the ilvB mutation was introduced on the chromosome was selected by base sequence analysis, and the strains into which the ilvB mutation was introduced were designated as KCCM11201P :: ilvB (W503Q) and KCCM11201P :: ilvB (T96S), respectively. Then, pDZ-ilvB (T96S) of the mutation introduction vector was transformed into the prepared strain KCCM11201P :: ilvB (W503Q). Then, the strains into which both ilvB mutations on the chromosome were introduced were designated as KCCM11201P :: ilvB (W503Q / T96S).
• the cells were cultured in the same manner as in Example 4, from which the concentration of L-valine was analyzed (Table 3).
• KCCM11201P strain-derived mutant acetohydroxy acid synthase introduced strain L-valine production concentration (g / l) Strain Batch 1 Batch 2 Batch 3 Average Control group KCCM11201P 2.9 2.8 2.8 2.8 One 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 3.4
• KCCM11201P increased the productivity of L-valine by a maximum of 17.8% compared to the parent strain
• the two strains increased the productivity of L-valine by 21.4% compared to the parent strain.
• the mutant of the acetohydroxysultainase synthetase of the present invention is the first enzyme in the biosynthesis pathway of L-branched chain amino acid, and thus it is also influenced not only by the production ability of L-valine but also by the production ability of L-isoleucine and L-leucine Expected.
• KCCM11201P :: ilvB (W503Q) and KCCM11201P :: ilvB (T96S), which are the L-valine improved strains, as Corynebacterium glutamicum KCJ-0793 and KCJ-0796, KCCM) on January 25, 2016, and have been granted accession numbers KCCM11809P and KCCM11810P.
• L-valine over-expression over-expression vector was prepared from L-valine producing strain, Corynebacterium glutamicum KCCM11201P.
• the L-valine biosynthetic overexpression vector containing the DNA coding for the mutated acetohydroxy acid synthase from each of KCCM11201P :: ilvB (W503Q) and KCCM11201P :: ilvB (T96S) prepared in Example 7 was prepared .
• Primer 13 (SEQ ID NO: 18) inserted with a BamH restriction site at the 5'-end and primer 14 (SEQ ID NO: 19) inserted with the Xba restriction site at the 3'-end were synthesized for the construction of the vector.
• primer 14 (SEQ ID NO: 19) inserted with the Xba restriction site at the 3'-end were synthesized for the construction of the vector.
• the PCR conditions were denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and polymerization at 72 ° C for 4 minutes.
• the polymerisation reaction was carried out at 72 ° C for 7 minutes.
• Primer 13 (SEQ ID NO: 18): 5'-CGAGG ATCCA ACCGG TATCG ACAAT CCAAT -3 '
• Primer 14 (SEQ ID NO: 19): 5'-CTGTC TAGAA ATCGT GGGAG TTAAA CTCGC -3 '
• the two DNA fragments amplified by the PCR were treated with restriction enzymes BamH and Xba to obtain respective DNA fragments.
• the DNA fragments were ligated to overexpression vector pECCG117 having restriction enzyme BamH and Xba terminus and then transformed into E. coli DH5 ⁇ And plated on LB solid medium containing kanamycin (25 mg / l).
• the gene is inserted object by PCR using a commonly known plasmid extraction was obtained a plasmid in accordance with the transition into the ilvB gene of this plasmid respectively pECCG117-ilvBN, pECCG117-ilvB (W503Q) N, pECCG117-ilvB (T96S) N.
• Example 9 Construction of L-Valine Biosynthetic Overexpression Vector Containing DNA Encoding Acetohydroxyacid Synthase Substituted with Other Amino Acids at the Same Mutation Site
• the 96th amino acid is an amino acid other than threonine or serine, and the 503th amino acid is tryptophan or an amino acid other than glutamine Was prepared.
• the substituted amino acid is a representative example of the amino acid that can be substituted, but is not limited thereto.
• the chromosome of Corynebacterium glutamicum KCCM11201P strain was used as a template and primers 13 (SEQ ID NO: 18) and 15 (SEQ ID NO: 20), primer 16 (SEQ ID NO: 21) and primer 14 No. 19) was used to amplify a DNA fragment of about 2041 bp having a BamH restriction enzyme site at the 5 'end and a DNA fragment of 1055 bp having an Xba restriction enzyme site at the 3' end.
• the PCR conditions were denaturation at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and polymerization at 72 ° C for 2 minutes.
• the polymerisation reaction was carried out at 72 ° C for 7 minutes.
• Primer 15 (SEQ ID NO: 20): 5'-CTTCA TAGAA TAGGG TCTGG TTTTG GCGAA CCATG CCCAG -3 '
• Primer 16 (SEQ ID NO: 21): 5'- CTGGG CATGG TTCGC CAAAA CCAGA CCCTA TTCTA TGAAG -3 '
• PCR was performed using primer 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19) using the two amplified DNA fragments as a template.
• the PCR conditions were denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and polymerization at 72 ° C for 4 minutes.
• the polymerisation reaction was carried out at 72 ° C for 7 minutes.
• Primer 13 (SEQ ID NO: 18), Primer 17 (SEQ ID NO: 22), Primer 18 (SEQ ID NO: 23) and Primer 14 (SEQ ID NO: 19) were used as templates in the same manner as the chromosome of Corynebacterium glutamicum KCCM11201P strain PCR was performed to amplify a DNA fragment of about 2041 bp having a BamH restriction enzyme site at the 5 'end and a DNA fragment of 1055 bp having an Xba restriction enzyme site at the 3' end.
• Primer 17 (SEQ ID NO: 22): 5'-CTTCA TAGAA TAGGG TCTGC AGTTG GCGAA CCATG CCCAG -3 '
• Primer 18 (SEQ ID NO: 23): 5'-CTGGG CATGG TTCGC CAACT GCAGA CCCTA TTCTA TGAAG -3 '
• PCR was performed using primer 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19) using the two amplified DNA fragments as a template.
• primers 13 (SEQ ID NO: 18) and 19 (SEQ ID NO: 24), primer 20 (SEQ ID NO: 25) and primer 14 (SEQ ID NO: 19) were amplified by using the chromosome of Corynebacterium glutamicum KCCM11201P strain as a template PCR was performed to amplify a DNA fragment of about 819 bp having a BamH restriction enzyme site at the 5 'end and a DNA fragment of 2276 bp having an Xba restriction enzyme site at the 3' end.
• Primer 19 (SEQ ID NO: 24): 5'-GGTTG CGCCT GGGCC AGATG CTGCA ATGCA GACGC CAAC -3 '
• Primer 20 (SEQ ID NO: 25): 5'-GTTGG CGTCT GCATT GCAGC ATCTG GCCCA GGCGC AACC -3 '
• PCR was performed using primer 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19) using the two amplified DNA fragments as a template.
• Primer 21 (SEQ ID NO: 26): 5'-GGTTG CGCCT GGGCC AGAGC ATGCA ATGCA GACGC CAAC -3 '
• Primer 22 (SEQ ID NO: 27): 5'-GTTGG CGTCT GCATT GCATG CTCTG GCCCA GGCGC AACC -3 '
• PCR was performed using primer 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19) using the two amplified DNA fragments as a template.
• Example 8 the above 4 mutant gene fragments amplified by PCR were treated with restriction enzymes BamH and Xba to obtain respective DNA fragments, which were then ligated to the overexpressed vector pECCG117 having restriction enzyme BamH and Xba terminus , Transformed into Escherichia coli DH5 ⁇ , and plated on LB solid medium containing kanamycin (25 mg / L).
• Example 10 Production of wild-type mutant acetohydroxy acid synthase-introduced strain and comparison of L-valine production ability
• the vector When the vector is transformed, it has kanamycin resistance. Therefore, the transformation is confirmed by the growth of
• each strain was inoculated into a 250 ml corn-baffle flask containing 25 ml of the production medium in the same manner as the culture medium used in Example 4, and cultured at 30 DEG C for 72 hours with shaking at 200 rpm Respectively.
• the concentration of L-valine was analyzed using HPLC (Table 4).
• Example 11 Production of mutant strains of introduced acetohydroxy acid synthase and comparison of L-leucine production ability
• Example 6 Two novel mutation introduction vectors prepared in Example 6 were respectively subjected to two-step homologous chromosomal recombination to obtain Corynebacterium glutamicum KCCM11661P (Korean Patent Application No. 10-2015-0119785 , Korean Patent Publication No. 10-2017-0024653). Then, the strain into which the ilvB mutation was introduced on the chromosome was selected by base sequence analysis, and the strains into which the ilvB mutation was introduced were designated as KCCM11661P :: ilvB (W503Q) and KCCM11661P :: ilvB (T96S), respectively.
• Corynebacterium glutamicum KCCM11661P was obtained by the following method as a mutant strain derived from Corynebacterium glutamicum ATCC 14067 having resistance to norleucine (NL).
• Corynebacterium glutamicum ATCC 14067 was cultivated in an activated medium for 16 hours, and the activated strain was inoculated into a seed medium sterilized at 121 ° C for 5 minutes and cultured for 14 hours, and then 5 ml of the culture medium was recovered .
• the recovered culture was washed with 100 mM citric acid buffer, NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) was added to a final concentration of 200 mg / L, And washed with 100 mM phosphate buffer.
• the mortality rate of the strains treated with NTG was 85%.
• NTG-treated strains were cultured in a medium containing NL at a final concentration of 20 mM, 30 mL, 40 mM, and 50 mM And then cultured at 30 DEG C for 5 days to obtain NL resistant mutants.
• NL Norleucine
• Juice 1% polypeptone 1%, sodium chloride 0.5%, yeast extract 1%, agar 2%, pH 7.2
• glucose 1.0% of glucose, 0.4% of ammonium sulfate, 0.04% of magnesium sulfate, 0.1% of potassium phosphate, 0.1% of urea, 0.001% of thiamine, 200 ⁇ g of biotin, 2% of agar, pH 7.0
• the mutant obtained by the above method was named Corynebacterium glutamicum KCJ-24 and deposited on January 22, 2015 with the Korea Microorganism Conservation Center under the Budapest Treaty, No. KCCM11661P.
• KCCM11661P :: ilvB (W503Q) and KCCM11661P :: ilvB (T96S) were cultured in the same manner as in Example 4, and the concentration of L-leucine was analyzed therefrom (Table 5).
• Example 12 Preparation of mutant strains of KCCM11662P-derived mutant of acetohydroxy acid synthase and comparison of L-leucine production ability
• Example 6 Two novel mutation introduction vectors prepared in Example 6 were recombinantly subjected to two-step homologous chromosomes to generate Corynebacterium glutamicum KCCM11662P (Korean Patent Application No. 10-2015-0119785, Korean Patent Publication No. 10-2017-0024653). Then, the strains into which the ilvB mutations were introduced on the chromosome were selected by base sequence analysis, and the strains into which the ilvB mutation was introduced were designated as KCCM11662P :: ilvB (W503Q) and KCCM11662P :: ilvB (T96S), respectively.
• Corynebacterium glutamicum KCCM11662P was obtained by the following method as a mutant strain derived from Corynebacterium glutamicum ATCC 13869 having resistance to norleucine (NL).
• Corynebacterium glutamicum ATCC 13869 was used as a parent strain and cultured in the same manner as in the method for obtaining KCCM11662P of Example 11, finally obtaining an NL resistant mutant strain.
• the mutant obtained by the above method was named Corynebacterium glutamicum KCJ-28, and deposited with the Korean Society for Microbiological Protection, International Depositary under the Budapest Treaty on January 22, 2015, No. KCCM11662P.
• KCCM11662P :: ilvB (W503Q) and KCCM11662P :: ilvB (T96S) were cultured in the same manner as in Example 4, and the concentration of L-leucine was analyzed therefrom (Table 6).
• the two novel mutant introduction strains (KCCM11662P :: ilvB (W503Q) and KCCM11662P :: ilvB (T96S)) increased the productivity of L-leucine up to 13.3% compared to the parent strain.

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