KR20190037224A - Acetohydroxy acid synthase variant, microorganism comprising thereof, and method of producing L-branced-chained amino acid using the same - Google Patents

Abstract

The present invention relates to a novel acetohydroxy acid synthase variant; microorganisms comprising the same; or a method of producing L-branched-chain amino acids using the same. According to the present invention, by using the microorganisms comprising the acetohydroxy acid synthase variant, L-branched-chain amino acids can be produced with high yield.

Description

[Acetohydroxy acid synthase variant, microorganism comprising thereof, and method of producing L-branced-chained amino acid using the same}

The present application relates to a novel acetohydroxy acid synthase variant and its use, and specifically, to an acetohydroxy acid synthase variant, a microorganism comprising the same, or a method of producing an L-branched amino acid using the same.

Branched-chain amino acids, that is, L-valine, L-leucine, and L-isoleucine, act to increase protein in individuals, and are known to play an important role as an energy source during exercise, and are used in medicines and foods. Since branched chain amino acids use the same enzyme for similar biosynthetic processes, it is difficult to manufacture one branched chain amino acid through fermentation on an industrial scale. In the manufacture of branched chain amino acids, the role of acetohydroxy acid synthase, the first enzyme in branched chain amino acid biosynthesis, is the most important, but previous studies on this have mainly been conducted on acetohydroxy acid synthase small subunit. ; IlvN protein), most of the studies related to feedback release due to mutations (Protein Expr Purif. 2015 May;109:106-12., US2014-0335574, US2009-496475, US2006-303888, US2008-245610), related studies Is very scarce.

Acetohydroxy acid synthase plays a role in producing acetolactic acid from two molecules of pyruvate, and producing aceto-2-hydroxy-butyrate from ketobutyric acid and pyruvate. It is an enzyme that plays a role. The acetohydroxy acid synthase catalyzes decarboyxlation of pyruvate and condensation reaction with other pyruvate molecules to produce acetolactic acid, a precursor of valine and leucine, or decarboxylation of pyruvate and 2-keto. Acetohydroxybutyrate, a precursor of isoleucine, can be produced by catalyzing the condensation reaction with 2-ketobutyrate. Thus, acetohydroxy acid synthase is a very important enzyme involved in the initial process of L-branched amino acid biosynthesis.

Under this background, the present applicants have endeavored to effectively produce L-branched chain amino acids, and as a result, have developed a variant of acetohydroxy acid synthase, specifically a large subunit variant of acetohydroxy acid synthase. Accordingly, it was confirmed that L-branched chain amino acids can be produced in high yield from microorganisms containing the mutant, and the present application was completed.

One object of the present application is to provide an acetohydroxy acid synthase variant.

Another object of the present application is to provide a polynucleotide encoding the acetohydroxy acid synthase variant, a vector including the polynucleotide, and a transformant into which the vector is introduced.

Another object of the present application is to provide a microorganism comprising the acetohydroxy acid synthase variant, or into which the vector is introduced, to produce an L-branched amino acid.

Another object of the present application is the step of culturing a microorganism that produces the L-branched amino acid in a medium; And recovering the L-branched chain amino acid from the microorganism or a medium thereof.

One aspect of the present application for achieving the above object is that the threonine at amino acid sequence position 96 of the acetohydroxy acid synthase large subunit (IlvB protein) is an amino acid other than threonine. Or, tryptophan at amino acid sequence position 503 is substituted with an amino acid other than tryptophan, or threonine at amino acid sequence position 96 and tryptophan at position 503 are both substituted with other amino acids.

Specifically, the large subunit of the acetohydroxy acid synthase may have an amino acid sequence represented by SEQ ID NO: 1. More specifically, the acetohydroxy acid synthase variant is that in the amino acid sequence shown in SEQ ID NO: 1, 96 th threonine or 503 th tryptophan from its N-terminus is substituted with another amino acid. Or, the 96 th threonine and the 503 th tryptophan may be all substituted with different amino acids, and may be an acetohydroxy acid synthase variant.

In the present application, the term "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, acetohydroxy acid synthase catalyzes decarboyxlation of pyruvate and condensation reaction with other pyruvate molecules to produce acetolactic acid, a precursor of valine, or decarboxylation of pyruvate and 2-keto. Acetohydroxybutyrate, a precursor of isoleucine, can be produced by catalyzing the condensation reaction with 2-ketobutyrate. Specifically, starting from acetolactic acid, a reaction catalyzed by acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B is performed. L-valine is biosynthesized when passed sequentially. In addition, from acetolactic acid, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, 2-isopropylmalate synthase, and isopropylmalic acid L-leucine is biosynthesized through sequential reactions catalyzed by isomerase (isopropylmalate isomerase), 3-isopropylmalate dehydrogenase (3-isopropylmalate dehydrogenase), and transaminase B (transaminase B). On the other hand, starting from acetohydroxybutyrate, a reaction catalyzed by acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. L- isoleucine is biosynthesized through sequentially. Therefore, it is an important enzyme in the biosynthetic pathway of L-branched chain amino acids.

Acetohydroxy acid synthase is encoded by two genes, ilvB and ilvN, ilvB gene is a large subunit of acetohydroxy acid synthase (IlvB), and ilvN gene is a small acetohydroxy acid synthase. Each small subunit (IlvN) is coded.

In the present application, the acetohydroxy acid synthase may be derived from a microorganism of the genus Corynebacterium, and specifically, may be derived from Corynebacterium glutamicum. More specifically, the acetohydroxy acid synthase large subunit is 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically with the amino acid sequence described in SEQ ID NO: 1 as well as the sequence. Any protein with 95% or more homology and IlvB protein activity may be included without limitation. In addition, the polynucleotide encoding the protein having the IlvB protein activity changes the amino acid sequence of the protein expressed from the coding region in consideration of the preferred codon in the organism to express the protein due to the degeneracy of the codon. Since various modifications can be made to the coding region within the range not specified, the amino acid sequence of SEQ ID NO: 1 may be included without limitation as long as it is a coding nucleotide sequence, but may be specifically encoded by the nucleotide sequence of SEQ ID NO: 2.

In the present application, "acetohydroxy acid synthase variant" refers to a protein in which one or more amino acids in the amino acid sequence of the acetohydroxy acid synthase protein have been mutated (eg, added, removed or substituted). Specifically, the acetohydroxy acid synthase variant is a protein in which the activity of the acetohydroxy acid synthase protein is efficiently increased compared to wild type or before modification by the mutation of the present application. In the present application, mutations are generally methods for improving enzymes, and known methods known in the art may be used without limitation, and there are strategies such as rational design and directed evolution. For example, rational design strategies include site-directed mutagenesis or site-specific mutagensis of amino acids at specific positions, and induced evolution strategies include methods of causing random mutagenesis. There is this. In addition, it may be mutated without external manipulation by natural mutation. Specifically, the acetohydroxy acid synthase variant may be isolated, recombinant protein, or non-naturally occurring. However, it is not limited thereto.

The acetohydroxy acid synthase variant of the present application is not limited thereto, but specifically, 96 th threonine or 503 th tryptophan from the N-terminus of the IlvB protein having the amino acid sequence shown in SEQ ID NO: 1 This mutated IlvB protein may be, or, although not limited thereto, may be an IlvB protein in which 96 th threonine and 503 th tryptophan are simultaneously substituted with other amino acids. For example, the 96th threonine is substituted with serine, cysteine, or alanine, or the 503th tryptophan is substituted with glutamine, asparagine, or leucine. It may be an IlvB protein. In addition, when the 96th or 503th amino acid is substituted with another amino acid and at the same time, some of the amino acid sequences have an amino acid sequence that is deleted, modified, substituted or added, it is the same as or corresponding to the acetohydroxy acid synthase variant of the present application. It is obvious that it is included in the scope of the present application if it exhibits the activity to be performed.

In addition, in the category of the acetohydroxy acid synthase variant of the present application, the acetohydroxy acid synthase large subunit variant itself, the acetohydroxy acid synthase large subunit variant having the above-described mutation, is included in the acetohydroxy acid synthase variant. Acid synthase, or acetohydroxy acid synthase An acetohydroxy acid synthase having both a large subunit variant and a small subunit is included, but is not particularly limited thereto.

In the present application, it was confirmed that the production of L-branched chain amino acids increased by substituting the 96th and 503th amino acids of the acetohydroxy acid synthase protein with various other amino acids, thereby increasing the production of L-branched chain amino acids. In the mutation of the synthase protein, it was confirmed that the 96th and 503th positions are important positions. However, the amino acid substituted in the examples of the present application is only 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 96 th threonine is an amino acid other than threonine. As a result, it is apparent that when the 503 th tryptophan is substituted with an amino acid other than tryptophan, or the 96 th threonine and the 503 th tryptophan are all substituted with other amino acids, an effect corresponding to the effect described in the Examples can be expected.

In addition, the acetohydroxy acid synthase variant of the present application may have an amino acid sequence described in any one of SEQ ID NOs: 28 to 33, but is not limited thereto. In addition, as long as it has substantially the same or corresponding activity as the acetohydroxy acid synthase variant, including the variant of the present application, 70%, 80%, 90%, 95%, or 99% or more homology with the above sequences. Branches can include, without limitation, polypeptides. In the present application, the term "homology" refers to the percent of identity between two polynucleotide or polypeptide moieties. Homology between sequences from one moiety to another can be determined by known art techniques. Its homologous sequence having the same or similar activity as a given amino acid sequence or base sequence is indicated as "% homology". For example, using standard software that calculates parameters such as score, identity and similarity, specifically BLAST 2.0, or by using hybridization experiments under defined stringent conditions. It can be confirmed by comparing. As used herein, the term "strict conditions" means conditions that allow specific hybridization between polynucleotides. Appropriate hybridization conditions to be defined are within the scope of the technology, and methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

Another aspect of the present application is a polynucleotide encoding the acetohydroxy acid synthase variant of the present application.

In the present application, the term "polynucleotide" has the meaning of including DNA and RNA molecules, and the nucleotide, which is a basic structural unit thereof, includes not only natural nucleotides but also analogs with modified sugar or base moieties. In the present application, 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 synthase variant of the present application may be included without limitation as long as it is a nucleotide sequence encoding the protein having the acetohydroxy acid synthase variant activity of the present application. Specifically, the polynucleotide is subjected to various modifications in the coding region within a range that does not change the amino acid sequence of the protein due to the codon degeneracy or in consideration of the preferred codon in the organism to express the protein. I can. Any nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 28 to 33 may be included without limitation, but for a specific example, it may have a nucleotide sequence described in any one of SEQ ID NOs: 34 to 39. In addition, as long as it has substantially the activity of acetohydroxy acid synthase, including the mutation of the present application, due to the degeneracy of the codon, 70%, 75%, 80%, 85% of the above sequences. Polynucleotides having a homology of 90%, 95%, 97%, or 99% or more may be included without limitation.

Or a probe that can be prepared from a known gene sequence, for example, a complementary sequence for all or a part of the nucleotide sequence and hydride under stringent conditions, and the activity of the protein consisting of the amino acid sequence of SEQ ID NOs: 28 to 33 Branches may be included without limitation as long as they are sequences encoding proteins. The "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., homolog). For example, among genes with high homology, genes with homology of 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, and particularly 99% or more Under conditions that hybridize to each other and do not hybridize to genes with lower homology, or to wash conditions for general Southern hybridization, 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1 % SDS, more specifically 68° C., 0.1×SSC, and the conditions for washing once, specifically two to three times at a salt concentration and temperature corresponding to 0.1% SDS can be listed. Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence. Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by a person skilled in the art according to the purpose. The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

Another aspect of the present application is a vector comprising a polynucleotide encoding the modified aceto hydroxy acid synthase variant of the present application.

In the present application, the term "vector" refers to any medium for cloning and/or transfer of a base into a host cell. The vector may be a replicon capable of binding other DNA fragments to bring about replication of the bound fragment. "Replication unit" refers to any genetic unit that functions as an autologous unit of DNA replication in vivo, ie, capable of replicating by its own regulation. Specifically, it may be a natural or recombinant plasmid, phage, cosmid, chromosome, or virus. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc. can be used as a phage vector or a cosmid vector, and as a plasmid vector, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used. The vector usable in the present application is not particularly limited, and a known expression vector may be used. In addition, the vector may contain a transposon or an artificial chromosome.

In the present application, the vector is not particularly limited as long as it contains the polynucleotide encoding the acetohydroxy acid synthase variant of the present application, but mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.) , Plant cells, yeast cells, insect cells or bacterial cells (eg, E. coli, etc.) in eukaryotic or prokaryotic cells, including a vector capable of replicating and / or expressing the nucleic acid molecule, specifically a host It is operably linked to an appropriate promoter so that the polynucleotide can be expressed in cells, and may be a vector including at least one selection marker.

In addition, the term "operably linked" in the above means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a protein of interest of the present application and the gene sequence are functionally linked.

Another aspect of the present application is a transformant into which the vector of the present application has been introduced.

In the present application, the transformant is not particularly limited thereto, but all transformable cells may be included as long as the vector is introduced to express the acetohydroxy acid synthase variant of the present application. Bacterial cells, such as specifically transformed Escherichia genus, Corynebacterium genus, Streptomyces, Brevibacterium genus, Serratia genus, Providencia genus, Salmonella typhimurium, etc.; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK ( Animal cells, such as baby hamster kidney cells, baby hamster kidney cells, HEK (human embryonic kidney cells, PERC.6 (human retinal cells), etc.; or plant cells.

Another aspect of the present application is a microorganism comprising the acetohydroxy acid synthase variant or into which a vector including a polynucleotide encoding the variant has been introduced, producing an L-branched amino acid.

In the present application, the term "L-branched amino acid" refers to an amino acid having a branched alkyl group in the side chain, and includes valine, leucine, and isoleucine. Specifically, in the present application, the L-branched amino acid may be L-valine or L-leucine, but is not limited thereto.

In the present application, the "microorganism" includes all wild-type microorganisms or microorganisms that have undergone natural or artificial genetic modification, and a specific mechanism is weakened due to reasons such as insertion of an external gene or enhancement or attenuation of the activity of an intrinsic gene. It is a concept that includes all microorganisms that have become or fortified. Refers to all microorganisms capable of expressing the acetohydroxy acid synthase variant of the present application, specifically, may be a microorganism of the genus Corynebacterium, and 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) Etc. More specifically, it is Corynebacterium glutamicum, but is not limited thereto.

In the present application, the term "a microorganism producing L-branched chain amino acids" refers to a microorganism having the ability to produce L-branched chain amino acids through a natural type or mutation, and specifically non-natural occuring It may be a recombinant microorganism, but is not limited thereto. The microorganism producing the L-branched amino acid is a vector comprising the acetohydroxy acid synthase variant of the present application or a vector containing a polynucleotide encoding the variant has been introduced, and a wild-type microorganism, a natural acetohydroxy acid Compared to a microorganism comprising a synthase protein, an unmodified microorganism including an acetohydroxy acid synthase protein, or a microorganism not containing acetohydroxy acid synthase protein, the ability to produce L-branched amino acids may be significantly increased.

Another aspect of the present application, the step of culturing a microorganism that produces 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.

In the present application, the term "culture" means to grow microorganisms under appropriately artificially controlled environmental conditions. In the present application, the L-branched amino acid production method using a microorganism having L-branched chain amino acid production capability may be performed using a method widely known in the art. Specifically, the culture may be continuously cultured in a batch process, an injection batch or a repeated fed batch process, but is not limited thereto.

The medium used for cultivation must meet the requirements of the specific strain in an appropriate manner. For example, culture media for strains of the genus Corynebacterium are known (eg, Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981). Sugar sources that can 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, stearic acid. , Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto. Nitrogen sources that may be used may include peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture, but is not limited thereto. Personnel that may be used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate required for growth. Additionally, essential growth substances such as amino acids and vitamins can be used. In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added batchwise or continuously in a manner appropriate to the culture during the cultivation process. 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 an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (eg, air) may be injected into the culture to maintain an aerobic condition. The temperature of the culture may be 20 to 45 °C, specifically 25 to 40 °C. The cultivation can be continued until the maximum amount of production of the desired L-branched amino acid is obtained. For this purpose, the incubation time may be 10 to 160 hours. The L-branched amino acid may be excreted into the culture medium or contained in the cell. However, it is not limited thereto.

The method of recovering the L-branched amino acid from the microorganism or medium can be carried out using a suitable method known in the art. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salt-in method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography Various chromatography such as, HPLC, and methods thereof may be used in combination, but are not limited to these examples. In addition, the step of recovering the L-branched amino acid may include an additional purification step, and may be performed using a suitable method known in the art.

The acetohydroxy acid synthase variant according to the present application significantly increases the ability of the microorganism to produce L-branched chain amino acids when its activity is introduced into a microorganism, and thus can be widely used for mass production of L-branched chain amino acids. .

Hereinafter, the present application will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited to these examples.

Example 1: Preparation of DNA library encoding mutated acetohydroxy acid synthase using artificial mutation method

In this example, in order to obtain an acetohydroxy acid synthase variant, a vector library for primary cross-insertion in a chromosome was constructed by the following method. Error-prone PCR was performed on the ilvB gene (SEQ ID No. 2) encoding acetohydroxy acid synthase (SEQ ID No. 1) derived from Corynebacterium glutamicum ATCC14067, and base substitution mutations were randomly introduced. The ilvB gene variants (2395bp) were obtained. Error-prone PCR was performed using GenemorphII Random Mutagenesis Kit (Stratagene), and primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) were used using Corynebacterium glutamicum ATCC14067 genomic DNA as a template. .

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'

0 to 3.5 mutations per 1 kb were introduced into the amplified gene fragment, and PCR conditions were denaturation 96° C., 30 seconds; Annealing 53° C., 30 seconds; And polymerization reaction at 72° C. for 2 minutes were repeated 30 times.

The amplified gene fragment was ligated to a pCR2.1-TOPO vector (hereinafter'pCR2.1') using a pCR2.1-TOPO TA cloning kit (Invitrogen), transformed into E. coli DH5α, and kanamycin (25 mg/ℓ). ) Was spread on LB solid medium containing. After selecting 20 transformed colonies, plasmids were obtained, and as a result of nucleotide sequence analysis, it was confirmed that mutations were introduced at different locations with a frequency of 2.1 mutations/kb. About 20,000 transformed E. coli colonies were taken to extract a plasmid, which was named pCR2.1-ilvB(mt) library.

In addition, a plasmid having a wild-type ilvB gene was prepared for use as a control. Using primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4), Corynebacterium glutamicum ATCC14067 genomic DNA as a template was subjected to PCR under the same conditions as described above. As the polymerase, PfuUltra High-Fidelity DNA polymerase (Stratagene) was used. The produced plasmid was named pCR2.1-ilvB (WT).

Example 2: ilvB Production of defective strains

Using the KCCM11201P (Korean Patent No. 10-1117022) strain as a parent strain, an ilvB- deficient strain for introducing the pCR2.1-ilvB(mt) library was prepared.

To construct the ilvB deletion vector, 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: 6) using the chromosome of wild-type Corynebacterium glutamicum ATCC14067 as a template PCR was performed using number 8).

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'

PCR conditions were denatured 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 were repeated 30 times, followed by polymerization at 72°C for 7 minutes.

As a result, a DNA fragment of 731 bp of SEQ ID NO: 9 and a DNA fragment of SEQ ID NO: 10 of 712 bp were obtained each including a portion in front of the ilvB gene promoter and the 3'end of the ilvB gene.

Using the amplified SEQ ID NO: 9 and SEQ ID NO: 10 as templates, PCR was performed with primer 3 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 8). PCR conditions were denatured at 95° C. for 5 minutes, then denaturing at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 60 seconds were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.

As a result, the DNA fragment of SEQ ID NO: 11 (hereinafter, ilvB fragment) of 1407 bp to which the DNA fragment including the promoter front portion of the ilvB gene and the DNA fragment including the 3'end were linked was amplified.

In Corynebacterium glutamicum, the pDZ vector (Korean Patent Registration No. 10-0924065) and the amplified ilvB fragment were treated with restriction enzyme Xba, ligated using a DNA conjugation enzyme, and then cloned. By doing so, a plasmid was obtained, which was named pDZ-ilvB.

pDZ-ilvB was transformed into Corynebacterium glutamicum KCCM11201P by electric pulse method (Appl. Microbiol. Biothcenol. (1999) 52:541-545), respectively, and then kanamycin 25 mg/l and L-valine , L-leucine and L-isoleucine were obtained in a selection medium containing 2 mM each. A strain in which the ilvB gene was inactivated was obtained by the ilvB fragment inserted into the genome through the second recombination process (cross-over), and this was designated as KCCM11201P ilvB.

Example 3: Acetohydroxy acid synthase mutant strain library preparation and L-amino acid production ability increase strain selection

Using the prepared KCCM11201P ilvB strain as a parent strain, the prepared pCR2.1-ilvB(mt) library was transformed by homologous chromosome recombination, and then spread on a complex plate medium containing kanamycin (25 mg/L) to about 10,000. It has secured the colonies, and named each of the colonies from a KCCM11201P ilvB /pCR2.1-ilvB(mt)-1 to KCCM11201P ilvB /pCR2.1-ilvB(mt)-10000.

In addition, the prepared pCR2.1-ilvB (WT) vector was transformed into the KCCM11201P ilvB strain to prepare a control strain, and was named KCCM11201P ilvB /pCR2.1-ilvB (WT).

<Composite flat plate (pH 7.0)>

Glucose 10 g, peptone 10 g, beef extract 5 g, yeast extract 5 g, brain heart infusion 18.5 g, NaCl 2.5 g, urea 2 g, sorbitol 91 g, agar 20 g (distilled water 1 Liters)

About 10,000 colonies obtained were inoculated into 300 µl of each selection medium and incubated in a 96-deep well plate at 32° C. and 1000 rpm for about 24 hours. In order to analyze the production amount of L-amino acid produced in the culture medium, the ninhydrin method was used (J. Biol. Chem. 1948. 176:367-388). After the incubation was completed, 10 µl of the culture supernatant and 190 µl of the ninhadrin reaction solution were reacted at 65°C for 30 minutes, and the absorbance was measured with a spectrophotometer at a wavelength of 570 nm, and the control KCCM11201P ilvB /pCR2.1-ilvB ( WT) about 213 colonies of mutant strains showing an absorbance increased by 10% or more compared to the absorbance of the strain were selected. Other colonies showed similar or decreased absorbance compared to the control.

<Selection medium (pH 8.0)>

Glucose 10 g, ammonium sulfate 5.5 g, magnesium sulfate 7 hydrate 1.2 g, monobasic potassium 0.8 g, dibasic potassium 16.4 g, biotin 100 μg, thiamine-HCl 1000 μg, pantothenate calcium 2000 μg, nicotinamide 2000 μg ( 1 liter of distilled water)

The selected 213 strains were repeatedly subjected to ninhydrin reaction in the same manner as described above, and the top 60 strains having improved L-amino acid production ability compared to the KCCM11201P ilvB / pCR2.1-ilvB (WT) strain were selected.

Example 4: Confirmation of L-valine production ability of acetohydroxy acid synthase mutant strain library selection line

In order to compare the L-valine production ability of the 60 strains selected in Example 3, the culture medium components were analyzed by culturing in the following manner.

Each strain was inoculated with platinum in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours and 200 rpm. The concentration of L-valine was analyzed using HPLC.

<Production medium (pH 7.0)>

Glucose 100 g, ammonium sulfate 40 g, soy protein 2.5 g, corn steep solids) 5 g, urea 3 g, dibasic potassium 1 g, magnesium sulfate 7 hydrated 0.5 g, biotin 100 ㎍, thiamine-HCl 1000 ㎍, calcium pantothenate 2000 ㎍, nicotinamide 3000 ㎍, calcium carbonate 30 g (based on 1 liter of distilled water)

Among the 60 strains, two strains with improved L-valine concentration were selected and the above culture and analysis were repeated, and the analyzed concentration of L-valine is shown in Table 1 below. The remaining 58 strains showed a result that the concentration of L-valine decreased.

Selected two kinds of KCCM11201P ilvB /pCR2.1-ilvB(mt) L-valine production concentration Strain L-valine (g/ℓ) Batch 1 Batch 2 Batch 3 Average Control KCCM11201P ilvB /pCR2.1-ilvB(WT) 2.7 2.9 2.9 2.8 One KCCM11201P ilvB /pCR2.1-ilvB(mt)-5602 3.1 3.5 3.4 3.3 2 KCCM11201P ilvB /pCR2.1-ilvB(mt)-7131 2.9 3.3 3.1 3.1

As a result of L-valine concentration analysis, it was confirmed that the L-valine yield of the two kinds of strains increased by up to 20.7% compared to the control KCCM11201P ilvB /pCR2.1-ilvB (WT) strain.

Example 5: Acetohydroxy acid synthase mutant strain library selection strain ilvB Genetic Variation Identification

In order to confirm the random mutation introduced into the acetohydroxy acid synthase of the two strains selected in Example 4, the nucleotide sequence of the ilvB gene was analyzed. PCR was performed using primers 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'

Through the nucleotide sequence analysis of each of the obtained mutant ilvB gene fragments, the nucleotide sequence of the mutant ilvB gene was confirmed by comparing it with the wild type ilvB gene nucleotide sequence of SEQ ID NO: 2, through which the mutated acetohydroxy acid synthase protein The amino acid sequence of was confirmed. Information on the mutated acetohydroxy acid synthase proteins of the two selected strains is shown in Table 2 below.

Selection 2 KCCM11201P/pCR2.1-ilvB(mt) mutated
Acetohydroxy acid synthase protein information Strain Acetohydroxy acid synthase amino acid mutation KCCM11201P ilvB /pCR2.1-ilvB(mt)-5602 W503Q KCCM11201P ilvB /pCR2.1-ilvB(mt)-7131 T96S

Example 6: Construction of a vector for introducing mutant acetohydroxy acid synthase

In order to confirm the effect of the mutated acetohydroxy acid synthase protein identified in Example 5, a vector capable of introducing it onto the chromosome was constructed.

Primer 9 (SEQ ID NO: 14) and primer 10 (SEQ ID NO: 15), and primers 11 (SEQ ID NO: 16) and primer 12 (SEQ ID NO: 17) with Xba restriction enzyme sites inserted at the 5'end based on the identified nucleotide sequence Was synthesized. Using this primer pair, PCR was performed on each of the two selected chromosomes as a template to amplify two mutant ilvB gene fragments. PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 2 minutes were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.

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 gene fragments amplified by PCR were treated with restriction enzyme Xba to obtain respective DNA fragments, ligated to pDZ vector for chromosome introduction with restriction enzyme Xba end, and transformed into E. coli DH5α, followed by kanamycin (25 mg/ℓ) was spread on LB solid medium.

After selecting a colony transformed with a vector into which the target gene was inserted through PCR, a plasmid was obtained using a commonly known plasmid extraction method, and according to the mutations inserted into the ilvB gene of this plasmid, pDZ-ilvB (W503Q), respectively, It was named pDZ-ilvB (T96S).

Example 7: KCCM11201P-derived acetohydroxy acid synthase mutant introduction strain construction and L-valine production ability comparison

The two new mutant introduction vectors prepared in Example 6 were transformed into L-valine-producing strain, Corynebacterium glutamicum KCCM11201P, by two-step homologous chromosome recombination, respectively. Thereafter, the strain into which the ilvB mutation was introduced on the chromosome was selected by sequencing analysis, and the strain into which the ilvB mutation was introduced was designated as KCCM11201P:: ilvB (W503Q) and KCCM11201P:: ilvB (T96S), respectively. And pDZ-ilvB (T96S) of the mutant introduction vectors was transformed into the prepared strain KCCM11201P:: ilvB (W503Q). Thereafter, the strain into which both kinds of ilvB mutations on the chromosome were introduced was named as KCCM11201P:: ilvB (W503Q/T96S), respectively.

Culture was carried out in the same manner as in Example 4, and the concentration of L-valine was analyzed therefrom (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 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

The two new mutant introduced strains (KCCM11201P:: ilvB (W503Q), KCCM11201P:: ilvB (T96S)) increased the L-valine production capacity by up to 17.8% compared to the parent strain, and both strains were introduced (KCCM11201P: : ilvB (W503Q/T96S)) increased L-valine production capacity by 21.4% compared to the parent strain.

Accordingly, since the variant of the large subunit of acetohydroxy acid synthase of the present invention is the first enzyme in the biosynthetic pathway of L-branched amino acids, it affects not only L-valine, but also the increase in the production capacity of L-isoleucine and L-leucine. It is expected to go crazy.

The present inventors named the strains having improved L-valine, KCCM11201P:: ilvB (W503Q) and KCCM11201P:: ilvB (T96S), as Corynebacterium glutamicum KCJ-0793 and KCJ-0796, and the Korea Microbial Conservation Center ( KCCM) on January 25, 2016, and were given accession numbers KCCM11809P and KCCM11810P.

Example 8: Construction of L-valine biosynthetic overexpression vector containing DNA encoding mutated acetohydroxy acid synthase

As a control, an L-valine biosynthetic overexpression vector was constructed from the L-valine producing strain, Corynebacterium glutamicum KCCM11201P. In addition, each of the KCCM11201P:: ilvB (W503Q), KCCM11201P:: ilvB (T96S) prepared in Example 7 containing the DNA encoding the mutated acetohydroxy acid synthase was prepared L-valine biosynthetic overexpression vector. .

For the construction of the vector, primer 13 (SEQ ID NO: 18) with a BamH restriction enzyme site inserted at the 5'end and primer 14 (SEQ ID NO: 19) with an Xba restriction enzyme site inserted at the 3'end were synthesized. Using this primer pair, the chromosomes of L-valine-producing strain Corynebacterium glutamicum KCCM11201P and KCCM11201P prepared in Example 7 above:: ilvB (W503Q), KCCM11201P:: ilvB (T96S) were used as templates, respectively. PCR was performed to amplify two mutant ilvBN gene fragments. PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 4 minutes were repeated 30 times, followed by polymerization 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 gene fragments amplified by the PCR were treated with restriction enzymes BamH and Xba to obtain respective DNA fragments, and then ligated to the overexpression vector pECCG117 having restriction enzymes BamH and Xba ends, and transformed into E. coli DH5α. It was spread on LB solid medium containing kanamycin (25 mg/ℓ).

After selecting the colonies transformed with the vector into which the target gene was inserted through PCR, a plasmid was obtained using a commonly known plasmid extraction method, and pECCG117-ilvBN, pECCG117-ilvB, respectively, depending on the mutations inserted into the ilvB gene of this plasmid. It was named (W503Q)N, pECCG117-ilvB(T96S)N.

Example 9: Construction of L-valine biosynthetic overexpression vector containing DNA encoding acetohydroxy acid synthase substituted with another amino acid at the same mutant position

In the mutated acetohydroxy acid synthase protein identified in Example 5, in order to confirm the effect of the mutation position, the 96th amino acid is an amino acid other than threonine or serine, and the 503th amino acid is an amino acid other than tryptophan or glutamine. A vector containing the mutation substituted with was constructed.

Specifically, from Corynebacterium glutamicum KCCM11201P, an L-valine-producing strain, a variant in which the 503th amino acid of acetohydroxy acid synthase is substituted with asparagine or leucine, or the 96th amino acid is substituted with alanine or cysteine. An L-valine biosynthetic overexpression vector containing the mutation of was constructed. The substituted amino acids are only representative examples of amino acids that can be substituted, but are not limited thereto.

For the construction of the vector, first, primer 13 (SEQ ID NO: 18) and primer 15 (SEQ ID NO: 20), primer 16 (SEQ ID NO: 21) and primer 14 (SEQ ID NO: 21) using the chromosome of the Corynebacterium glutamicum KCCM11201P strain as a template PCR was performed using No. 19) 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. PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 2 minutes were repeated 30 times, followed by polymerization 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'

Then, using the two amplified DNA fragments as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19). PCR conditions were denatured at 94° C. for 5 minutes, then denatured at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 4 minutes were repeated 30 times, followed by polymerization at 72° C. for 7 minutes.

As a result, an ilvBN gene fragment containing a mutation in the form in which the 503th amino acid of acetohydroxy acid synthase was substituted with asparagine was obtained.

In the same way, primer 13 (SEQ ID NO: 18) and primer 17 (SEQ ID NO: 22), primer 18 (SEQ ID NO: 23) and primer 14 (SEQ ID NO: 19) were used as templates using the chromosome of the Corynebacterium glutamicum KCCM11201P strain. PCR was performed using a DNA fragment of about 2041 bp having a BamH restriction enzyme site at the 5'end and a 1055 bp DNA fragment 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'

Then, using the two amplified DNA fragments as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19).

As a result, an ilvBN gene fragment containing a mutation in which the 503th amino acid of acetohydroxy acid synthase was substituted with leucine was obtained.

In the same way, primer 13 (SEQ ID NO: 18) and primer 19 (SEQ ID NO: 24), primer 20 (SEQ ID NO: 25) and primer 14 (SEQ ID NO: 19) were used as templates using the chromosome of the Corynebacterium glutamicum KCCM11201P strain. PCR was performed using a DNA fragment of about 819 bp having a BamH restriction enzyme site at the 5'end and a 2276 bp DNA fragment 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'

Then, using the two amplified DNA fragments as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19).

As a result, an ilvBN gene fragment containing a mutation in which the 96th amino acid of acetohydroxy acid synthase was substituted with alanine was obtained.

In the same way, primer 13 (SEQ ID NO: 18) and primer 21 (SEQ ID NO: 26), primer 22 (SEQ ID NO: 27) and primer 14 (SEQ ID NO: 19) were used as templates using the chromosome of the Corynebacterium glutamicum KCCM11201P strain. PCR was performed using a DNA fragment of about 819 bp having a BamH restriction enzyme site at the 5'end and a 2276 bp DNA fragment having an Xba restriction enzyme site at the 3'end.

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'

Then, using the two amplified DNA fragments as a template, PCR was performed with primers 13 (SEQ ID NO: 18) and primer 14 (SEQ ID NO: 19).

As a result, an ilvBN gene fragment containing a mutation in which the 96th amino acid of acetohydroxy acid synthase was substituted with cysteine was obtained.

In the same manner as in Example 8, the four mutant gene fragments amplified by PCR were treated with restriction enzymes BamH and Xba to obtain respective DNA fragments, and then the overexpression vector pECCG117 having restriction enzymes BamH and Xba ends. After linking to E. coli DH5α was transformed and plated on LB solid medium containing kanamycin (25 mg/ℓ).

After selecting the colonies transformed with the vector into which the target gene was inserted through PCR, a plasmid was obtained using a conventionally known plasmid extraction method, and pECCG117-ilvB (W503N) in order according to the mutations inserted into the ilvB gene of this plasmid. )N, pECCG117-ilvB(W503L)N, pECCG117-ilvB(T96A)N, and pECCG117-ilvB(T96C)N.

Example 10: Preparation of wild-type-derived mutated acetohydroxy acid synthase-introducing strain and comparison of L-valine production ability

L-valine biosynthetic overexpression vectors pECCG117-ilvBN, pECCG117-ilvB(W503Q)N, pECCG117-ilvB(T96S)N and pECCG117-ilvB(W503N)N, pECCG117-ilvB(W503L) prepared in Examples 8 and 9 )N, pECCG117-ilvB(T96A)N, and pECCG117-ilvB(T96C)N were inserted into Corynebacterium glutamicum wild type strain ATCC13032 by electroporation, respectively. The produced strains are Corynebacterium glutamicum ATCC13032::pECCG117-ilvBN, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(W503Q)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB, respectively. (T96S)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(W503N)N, Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(W503L)N, Corynebacterium glutamicum ATCC13032: Named as :pECCG117-ilvB(T96A)N and Corynebacterium glutamicum ATCC13032::pECCG117-ilvB(T96C)N. When the vector was transformed, it became resistant to kanamycin, so the transformation was confirmed through growth in a medium containing kanamycin at a concentration of 25 mg/L.

To evaluate the produced strain, in the same manner as the medium used in Example 4, each strain was inoculated into a 250 mL corner-baffle flask containing 25 mL of the production medium, and cultured with shaking at 30° C. for 72 hours and 200 rpm. I did. The concentration of L-valine was analyzed using HPLC (Table 4).

Wild type origin Mutated L-valine production concentration of acetohydroxy acid synthase-introduced strain Strain L-valine (g/ℓ) Batch 1 Batch 2 Batch 3 Average Control ATCC13032::pECCG117-ilvBN 0.1 0.1 0 0.1 One 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

As a result, it was confirmed that the new mutation in which the 96th or 503th amino acid of acetohydroxy acid synthase was substituted with another amino acid increased L-valine production capacity by up to 700% compared to the control group. From this, the importance of the 96 th 503 th position was confirmed, and it is expected to affect the production capacity of not only L-valine but also other branched chain amino acids.

Example 11: Preparation of mutated acetohydroxy acid synthase-introducing strain and comparison of L-leucine production ability

In order to confirm whether the variant of the large subunit of acetohydroxy acid synthase of the present application has an effect on the increase in the production capacity of other L-branched amino acids, as another example of the L-branched chain amino acid, the production capacity of L-leucine Confirmed.

Specifically, Corynebacterium glutamicum KCCM11661P (Korean Patent Application No. 10-2015-0119785), which is an L-leucine-producing strain, was carried out by recombining the two new mutant introduction vectors prepared in Example 6 each by two-step homologous chromosome recombination. , Korean Patent Application Publication No. 10-2017-0024653). Thereafter, the strain into which the ilvB mutation was introduced on the chromosome was selected by sequencing analysis, and the strain into which the ilvB mutation was introduced was named as KCCM11661P:: ilvB (W503Q) and KCCM11661P:: ilvB (T96S), respectively.

The Corynebacterium glutamicum KCCM11661P is a mutant derived from Corynebacterium glutamicum ATCC 14067 having resistance to norleucine (NL), and was obtained by the following method.

Specifically, Corynebacterium glutamicum ATCC 14067 was cultured in an activation 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 solution was recovered. . After washing the recovered culture medium with 100 mM citric buffer, NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) was added to a final concentration of 200 mg/L, and then treated for 20 minutes. And washed with 100 mM phosphate buffer. The mortality rate was 85% as a result of calculating the mortality rate by spreading the strain treated with NTG on a minimal medium. In order to obtain a mutant strain resistant to norleucine (NL), which is a derivative of L-leucine, the NTG-treated strain was added so that the final concentrations of NL were 20 mM, 30 mL, 40 mM, and 50 mM, respectively. Plated on a medium and cultured at 30° C. for 5 days to obtain NL-resistant mutant strains.

<Activation badge>

Juicy 1%, Polypeptone 1%, Sodium Chloride 0.5%, Yeast Extract 1%, Agar 2%, pH 7.2

<Seed medium>

Glucose 5%, Bactopeptone 1%, Sodium Chloride 0.25%, Yeast Extract 1%, Urea 0.4%, pH 7.2

<minimum badge>

Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium phosphate 0.1%, urea 0.1%, thiamine 0.00 1%, biotin 200 ㎍/L, agar 2%, pH 7.0

The mutant strain obtained by the above method was named Corynebacterium glutamicum KCJ-24, and as of January 22, 2015, it was deposited with the Korea Microbial Conservation Center, an international depository under the Budapest Treaty, and entrusted with each. It has been assigned the number KCCM11661P.

The 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).

Concentration of L-leucine production of strains introduced with mutant acetohydroxy acid synthase derived from KCCM11661P strain (g/ℓ) Strain Batch 1 Batch 2 Batch 3 Average Control KCCM11661P 2.7 2.6 2.9 2.7 One KCCM11661P:: ilvB (W503Q) 3.1 3.3 3.3 3.2 2 KCCM11661P P:: ilvB (T96S) 3.0 3.2 3.1 3.1

Two new mutant introduced strains (KCCM11661P:: ilvB (W503Q), KCCM11661P:: ilvB (T96S)) increased L-leucine production capacity by up to 26.9% compared to the parent strain.

Example 12: KCCM11662P-derived mutated acetohydroxy acid synthase-introduced strain construction and L-leucine production ability comparison

Corynebacterium glutamicum KCCM11662P (Korean Patent Application No. 10-2015-0119785, which is another L-leucine-producing strain, each of the two new mutant introduction vectors prepared in Example 6 by two-step homologous chromosome recombination, Korean Patent Application Publication No. 10-2017-0024653). Thereafter, the strain into which the ilvB mutation was introduced on the chromosome was selected by sequencing analysis, and the strain into which the ilvB mutation was introduced was designated as KCCM11662P:: ilvB (W503Q) and KCCM11662P:: ilvB (T96S), respectively.

The Corynebacterium glutamicum KCCM11662P was obtained by the following method as a mutant derived from Corynebacterium glutamicum ATCC 13869 having resistance to norleucine (NL).

Specifically, using Corynebacterium glutamicum ATCC 13869 as a parent strain, it was cultured in the same manner as in the method of obtaining KCCM11662P of Example 11, and finally an NL-resistant mutant strain was obtained.

The mutant strain obtained by the above method was named Corynebacterium glutamicum KCJ-28, and as of January 22, 2015, it was deposited with the Korea Microbial Conservation Center, an international depository under the Budapest Treaty, and entrusted with each. Has been assigned the number KCCM11662P

The 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).

L-leucine production concentration of strains introduced with mutated acetohydroxy acid synthase derived from KCCM11662P strain (g/ℓ) Strain Batch 1 Batch 2 Batch 3 Average Control KCCM11662P 3.1 3.0 3.1 3.1 One KCCM11662P:: ilvB (W503Q) 3.5 3.4 3.3 3.4 2 KCCM11662P P:: ilvB (T96S) 3.3 3.3 3.2 3.3

The two new mutant introduced strains (KCCM11662P:: ilvB (W503Q), KCCM11662P:: ilvB (T96S)) showed a maximum 13.3% increase in L-leucine production capacity compared to the parent strain.

From the above description, those skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof.

Korea Microorganism Conservation Center (overseas) KCCM11809P 20160125 Korea Microorganism Conservation Center (overseas) KCCM11810P 20160125

<110> CJ CheilJedang Corporation <120> Acetohydroxy acid synthase variant, microorganism comprising thereof, and method of producing L-branced-chained amino acid using the same <130> KPA151321-KR-D1 <160> 39 <170> KopatentIn 2.0 <210> 1 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> acetohydroxy acid synthase <400> 1 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Thr 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Trp Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 2 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> acetohydroxy acid synthase <400> 2 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcaacctc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaatggc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 3 aaccggtatc gacaatccaa t 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 4 gggtctctcc ttatgcctc 19 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 5 gcgtctagag acttgcacga ggaaacg 27 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer 4 <400> 6 cagccaagtc cctcagaatt gatgtagcaa ttatcc 36 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer 5 <400> 7 ggataattgc tacatcaatt ctgagggact tggctg 36 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 6 <400> 8 gcgtctagaa ccacagagtc tggagcc 27 <210> 9 <211> 731 <212> DNA <213> Artificial Sequence <220> <223> ilvB 5'fragment <400> 9 gcgtctagag acttgcacga ggaaacgcat ggtgaccatc cacggcatgc cgaccaccgt 60 tggcggtgtg acttccgtgg ctgggtgcac atcgagttca ccgaggattt ccggtgcgat 120 tttctcctgg ccaagtgcgc gacgagtcgc agcttcagag cgcgcgatga catctgtgat 180 gttttcagaa cccaacatgg ggatcggaat aacgacaacc gcacgcgacc agttattaga 240 attgttgatg cacactttcg ccgtggagtt ggggatgatc acggtctctt gtgcaatcgt 300 gcgaattttg gtcgcgcgca tggtgatctc aatgacggtg ccttcgacaa cgatgccgtt 360 gccctcaaaa cgcacccagt cacccacgcc gaattgcttt tccgtcagga tgaaaaatcc 420 ggccaagaag tccgcaacaa tcgactgcgc accaaggcca atggcagctg acgcaatggt 480 tgccggaatc gcagcgcccg cgagagagaa accaaaagcc tgcatcgcgg agacggcaag 540 catgaaaaac gccacaattt gcgcgatata aacgccaacg ccagcgaacg cgagctggtt 600 cttagtggtg tccgcatcgg ctgcagactc cactcgccgc ttgataatac gcatggccag 660 tcggccgata cgtggaatca aaaacgccaa gaccaggata attgctacat caattctgag 720 ggacttggct g 731 <210> 10 <211> 712 <212> DNA <213> Artificial Sequence <220> <223> ilvB 3'fragment <400> 10 ggataattgc tacatcaatt ctgagggact tggctgtgtt gccatccgcg tcaccaaagc 60 ggaggaagta ctgccagcca tccaaaaggc tcgagagatc aacgaccgcc cagtagtcat 120 cgacttcatc gtcggtgaag acgcacaggt atggccaatg gtgtctgctg gatcatccaa 180 ctccgatatc cagtacgcac tcggattgcg cccattcttt gatggtgatg aatctgcagc 240 agaagatcct gccgacattc acgaagccgt cagcgacatt gatgccgccg ttgaatcgac 300 cgaggcataa ggagagaccc aagatggcta attctgacgt cacccgccac atcctgtccg 360 tactcgttca ggacgtagac ggaatcattt cccgcgtatc aggtatgttc acccgacgcg 420 cattcaacct cgtgtccctc gtgtctgtaa agaccgaaac actcggcatc aaccgcatca 480 cggttgttgt cgacgccgac gagctcaaca ttgagcagat caccaagcag ctcaacaagc 540 tgatccccgt gctcaaagtc gtgcgacttg atgaagagac caccatcgcc cgcgcaatca 600 tgctggttaa ggtctctgcg gatagcacca accgtccgca gatcgtcgac gccgcgaaca 660 tcttccgcgc ccgagtcgtc gacgtggctc cagactctgt ggttctagac gc 712 <210> 11 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> ilvB 5'+ 3'fragment <400> 11 gcgtctagag acttgcacga ggaaacgcat ggtgaccatc cacggcatgc cgaccaccgt 60 tggcggtgtg acttccgtgg ctgggtgcac atcgagttca ccgaggattt ccggtgcgat 120 tttctcctgg ccaagtgcgc gacgagtcgc agcttcagag cgcgcgatga catctgtgat 180 gttttcagaa cccaacatgg ggatcggaat aacgacaacc gcacgcgacc agttattaga 240 attgttgatg cacactttcg ccgtggagtt ggggatgatc acggtctctt gtgcaatcgt 300 gcgaattttg gtcgcgcgca tggtgatctc aatgacggtg ccttcgacaa cgatgccgtt 360 gccctcaaaa cgcacccagt cacccacgcc gaattgcttt tccgtcagga tgaaaaatcc 420 ggccaagaag tccgcaacaa tcgactgcgc accaaggcca atggcagctg acgcaatggt 480 tgccggaatc gcagcgcccg cgagagagaa accaaaagcc tgcatcgcgg agacggcaag 540 catgaaaaac gccacaattt gcgcgatata aacgccaacg ccagcgaacg cgagctggtt 600 cttagtggtg tccgcatcgg ctgcagactc cactcgccgc ttgataatac gcatggccag 660 tcggccgata cgtggaatca aaaacgccaa gaccaggata attgctacat caattctgag 720 ggacttggct gtgttgccat ccgcgtcacc aaagcggagg aagtactgcc agccatccaa 780 aaggctcgag agatcaacga ccgcccagta gtcatcgact tcatcgtcgg tgaagacgca 840 caggtatggc caatggtgtc tgctggatca tccaactccg atatccagta cgcactcgga 900 ttgcgcccat tctttgatgg tgatgaatct gcagcagaag atcctgccga cattcacgaa 960 gccgtcagcg acattgatgc cgccgttgaa tcgaccgagg cataaggaga gacccaagat 1020 ggctaattct gacgtcaccc gccacatcct gtccgtactc gttcaggacg tagacggaat 1080 catttcccgc gtatcaggta tgttcacccg acgcgcattc aacctcgtgt ccctcgtgtc 1140 tgtaaagacc gaaacactcg gcatcaaccg catcacggtt gttgtcgacg ccgacgagct 1200 caacattgag cagatcacca agcagctcaa caagctgatc cccgtgctca aagtcgtgcg 1260 acttgatgaa gagaccacca tcgcccgcgc aatcatgctg gttaaggtct ctgcggatag 1320 caccaaccgt ccgcagatcg tcgacgccgc gaacatcttc cgcgcccgag tcgtcgacgt 1380 ggctccagac tctgtggttc tagacgc 1407 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer 7 <400> 12 cgcttgataa tacgcatg 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer 8 <400> 13 gaacatacct gatacgcg 18 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 9 <400> 14 cgctctagac aagcaggttg aggttcc 27 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 10 <400> 15 cgctctagac acgaggttga atgcgcg 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 11 <400> 16 cgctctagac cctcgacaac actcacc 27 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 12 <400> 17 cgctctagat gccatcaagg tggtgac 27 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 13 <400> 18 cgaggatcca accggtatcg acaatccaat 30 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 14 <400> 19 ctgtctagaa atcgtgggag ttaaactcgc 30 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer 15 <400> 20 cttcatagaa tagggtctgg ttttggcgaa ccatgcccag 40 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer 16 <400> 21 ctgggcatgg ttcgccaaaa ccagacccta ttctatgaag 40 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer 17 <400> 22 cttcatagaa tagggtctgc agttggcgaa ccatgcccag 40 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer 18 <400> 23 ctgggcatgg ttcgccaact gcagacccta ttctatgaag 40 <210> 24 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer 19 <400> 24 ggttgcgcct gggccagatg ctgcaatgca gacgccaac 39 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer 20 <400> 25 gttggcgtct gcattgcagc atctggccca ggcgcaacc 39 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer 21 <400> 26 ggttgcgcct gggccagagc atgcaatgca gacgccaac 39 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer 22 <400> 27 gttggcgtct gcattgcatg ctctggccca ggcgcaacc 39 <210> 28 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> T96S <400> 28 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Ser 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Trp Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 29 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> T96A <400> 29 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Ala 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Trp Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 30 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> T96C <400> 30 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Cys 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Trp Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 31 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> W503Q <400> 31 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Thr 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Gln Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 32 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> W503N <400> 32 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Thr 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Asn Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 33 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> W503L <400> 33 Met Asn Val Ala Ala Ser Gln Gln Pro Thr Pro Ala Thr Val Ala Ser 1 5 10 15 Arg Gly Arg Ser Ala Ala Pro Glu Arg Met Thr Gly Ala Gln Ala Ile 20 25 30 Val Arg Ser Leu Glu Glu Leu Asn Ala Asp Ile Val Phe Gly Ile Pro 35 40 45 Gly Gly Ala Val Leu Pro Val Tyr Asp Pro Leu Tyr Ser Ser Thr Lys 50 55 60 Val Arg His Val Leu Val Arg His Glu Gln Gly Ala Gly His Ala Ala 65 70 75 80 Thr Gly Tyr Ala Gln Val Thr Gly Arg Val Gly Val Cys Ile Ala Thr 85 90 95 Ser Gly Pro Gly Ala Thr Asn Leu Val Thr Pro Ile Ala Asp Ala Asn 100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Gly Ser Gly 115 120 125 Leu Leu Gly Thr Asp Ala Phe Gln Glu Ala Asp Ile Arg Gly Ile Thr 130 135 140 Met Pro Val Thr Lys His Asn Phe Met Val Thr Asp Pro Asn Asp Ile 145 150 155 160 Pro Gln Ala Leu Ala Glu Ala Phe His Leu Ala Ile Thr Gly Arg Pro 165 170 175 Gly Pro Val Leu Val Asp Ile Pro Lys Asp Val Gln Asn Ala Glu Leu 180 185 190 Asp Phe Val Trp Pro Pro Lys Ile Asp Leu Pro Gly Tyr Arg Pro Val 195 200 205 Ser Thr Pro His Ala Arg Gln Ile Glu Gln Ala Val Lys Leu Ile Gly 210 215 220 Glu Ala Lys Lys Pro Val Leu Tyr Ile Gly Gly Gly Val Ile Lys Ala 225 230 235 240 Asp Ala His Glu Glu Leu Arg Ala Phe Ala Glu Tyr Thr Gly Ile Pro 245 250 255 Val Val Thr Thr Leu Met Ala Leu Gly Thr Phe Pro Glu Ser His Glu 260 265 270 Leu His Met Gly Met Pro Gly Met His Gly Thr Val Ser Ala Val Gly 275 280 285 Ala Leu Gln Arg Ser Asp Leu Leu Ile Ala Ile Gly Ser Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly Asp Val Asp Thr Phe Ala Pro Asp Ala Lys Ile 305 310 315 320 Ile His Ala Asp Ile Asp Pro Ala Glu Ile Gly Lys Ile Lys Gln Val 325 330 335 Glu Val Pro Ile Val Gly Asp Ala Arg Glu Val Leu Ala Arg Leu Leu 340 345 350 Glu Thr Thr Lys Ala Ser Lys Ala Glu Thr Glu Asp Ile Ser Glu Trp 355 360 365 Val Asp Tyr Leu Lys Gly Leu Lys Ala Arg Phe Pro Arg Gly Tyr Asp 370 375 380 Glu Gln Pro Gly Asp Leu Leu Ala Pro Gln Phe Val Ile Glu Thr Leu 385 390 395 400 Ser Lys Glu Val Gly Pro Asp Ala Ile Tyr Cys Ala Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala Gln Phe Val Asp Phe Glu Lys Pro Arg Thr 420 425 430 Trp Leu Asn Ser Gly Gly Leu Gly Thr Met Gly Tyr Ala Val Pro Ala 435 440 445 Ala Leu Gly Ala Lys Ala Gly Ala Pro Asp Lys Glu Val Trp Ala Ile 450 455 460 Asp Gly Asp Gly Cys Phe Gln Met Thr Asn Gln Glu Leu Thr Thr Ala 465 470 475 480 Ala Val Glu Gly Phe Pro Ile Lys Ile Ala Leu Ile Asn Asn Gly Asn 485 490 495 Leu Gly Met Val Arg Gln Leu Gln Thr Leu Phe Tyr Glu Gly Arg Tyr 500 505 510 Ser Asn Thr Lys Leu Arg Asn Gln Gly Glu Tyr Met Pro Asp Phe Val 515 520 525 Thr Leu Ser Glu Gly Leu Gly Cys Val Ala Ile Arg Val Thr Lys Ala 530 535 540 Glu Glu Val Leu Pro Ala Ile Gln Lys Ala Arg Glu Ile Asn Asp Arg 545 550 555 560 Pro Val Val Ile Asp Phe Ile Val Gly Glu Asp Ala Gln Val Trp Pro 565 570 575 Met Val Ser Ala Gly Ser Ser Asn Ser Asp Ile Gln Tyr Ala Leu Gly 580 585 590 Leu Arg Pro Phe Phe Asp Gly Asp Glu Ser Ala Ala Glu Asp Pro Ala 595 600 605 Asp Ile His Glu Ala Val Ser Asp Ile Asp Ala Ala Val Glu Ser Thr 610 615 620 Glu Ala 625 <210> 34 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> T96S <400> 34 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcatcctc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaatggc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881 <210> 35 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> T96A <400> 35 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcagcatc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaatggc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881 <210> 36 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> T96C <400> 36 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcatgctc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaatggc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881 <210> 37 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> W503Q <400> 37 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcaacctc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaacagc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881 <210> 38 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> W503N <400> 38 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcaacctc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaaaacc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881 <210> 39 <211> 1881 <212> DNA <213> Artificial Sequence <220> <223> W503L <400> 39 gtgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60 gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120 gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180 tcctccacaa aggtgcgcca cgtcctggtg cgccacgagc agggcgcagg ccacgcagca 240 accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcaacctc tggcccaggc 300 gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360 atcaccggcc aggtcggaag tggcctgctg ggtaccgatg ctttccagga agccgatatc 420 cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccgaccc caacgacatt 480 ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg ccctgttctg 540 gtggatattc ctaaggatgt ccaaaacgct gaattggatt tcgtctggcc accaaagatc 600 gacctgccag gctaccgccc agtttctact ccgcatgctc gacagattga gcaggctgtc 660 aaactgatcg gtgaagccaa aaagccagtc ctttacattg gcggcggcgt tatcaaggct 720 gatgcacacg aagaactgcg tgcatttgct gagtacaccg gcatcccagt tgtcaccacc 780 ttgatggcat tgggtacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840 cacggcaccg tgtccgctgt tggcgcactg cagcgcagtg acctgctgat tgctatcggt 900 tcccgcttcg acgaccgcgt caccggtgac gttgacacct tcgcacctga tgccaagatc 960 attcacgctg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020 gtgggcgatg cccgcgaggt tcttgctcgt ctgctggaaa ccaccaaggc aagcaaggca 1080 gagaccgagg acatctccga gtgggttgat tacctcaagg gcctcaaggc acgtttccca 1140 cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200 tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260 gcagctcagt tcgttgactt tgaaaagcca cgcacctggc tcaactctgg cggcctgggc 1320 accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380 gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440 gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggcatggtt 1500 cgccaactgc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560 ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620 gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680 ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740 ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800 gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860 gttgaatcga ccgaggcata a 1881

Claims (12)

The amino acid sequence of the acetolactate synthase large subunit (IlvB protein) of the acetohydroxy acid synthase of SEQ ID NO: 1 was substituted with an amino acid other than tryptophan, and the acetohydroxy acid synthase Mutant.
The acetohydroxy acid synthase variant according to claim 1, wherein the other amino acid is glutamine, asparagine, or leucine.
The acetohydroxy acid synthetase variant of claim 1, wherein the acetohydroxy acid synthetase variant is composed of the amino acid sequence of any one of SEQ ID NO: 31 to SEQ ID NO: 33.
A polynucleotide encoding an acetohydroxy acid synthetase variant of any one of claims 1 to 3.
A vector comprising a polynucleotide encoding an acetohydroxy acid synthase variant of any of claims 1 to 3.
A transformant into which the vector of claim 5 has been introduced.
An acetohydroxy acid synthetase variant of any one of claims 1 to 3; A polynucleotide encoding said variant; And a vector comprising the polynucleotide, wherein the microorganism produces an L-branched chain amino acid.
The microorganism according to claim 7, wherein the microorganism is Corynebacterium sp., A microorganism producing L-branched chain amino acid.
9. The microorganism according to claim 8, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum, which produces an L-branched chain amino acid.
8. The microorganism according to claim 7, wherein the L-branched chain amino acid is L-valine or L-leucine.
(a) culturing a microorganism producing the L-branched chain amino acid of claim 7 in a medium; And
(b) recovering the L-branched chain amino acid from the microorganism or medium of step (a).
13. The method of claim 12, wherein the L-branched chain amino acid is L-valine, or L-leucine.