BACKGROUND
[3]
PDHC (pyruvate dehydrogenase multienzyme complex) is pyruvic acid (pyruvate) that is generated in the process as an enzyme that serves to convert acetyl -CoA (acetyl-CoA), an important enzyme for determining the inflow of carbon into TCA path. PDHC consists of pyruvate dehydrogenase (Pyruvate dehydrogenase, E1p), dihydro-lipoic amide acetyl transferase dehydratase (Dihydrolipoamide acetyltransferase, E2p), dihydro-lipoic amide dehydrogenase (dihydrolipoamide dehydrogenase, E3p). Among them, E1p enzyme aceE is encoded by the gene. The aceE gene defects and weakening bar but known for L- lysine-producing variations in the L- lysine-producing strain according to (Blombach et al , Appl Microbiol Biotechnol, 76:.... 615, 2007; Buchholz J et al ,. . Appl Environ Microbiol, 79 (18 ): 5566-75, 2013), the bar can not report on E1p variants that can increase the L- amino acid-producing ability.
[4]
Detailed Description of the Invention
SUMMARY
[5]
That the present inventors to produce L- amino acid in a high yield from a microorganism which has developed the results sought example, E1p variant to develop a microorganism which can be used with high concentrations of L- amino acid-producing mutants and E1p this end, including the mutant OK, and it completed the present invention.
[6]
Problem solving means
[7]
One object of the present application is to provide a novel pyruvate dehydrogenase (pyruvate dehydrogenase) variants.
[8]
It is another object of the present application is to provide a polynucleotide coding for the variant.
[9]
It is another object of the present application is to provide a microorganism of the genus Corynebacterium having, L- amino acid-producing ability, including the pyruvate dehydrogenase mutant.
[10]
It is another object of the present application provides a method comprising: (a) producing an L- amino acid to the genus Corynebacterium microorganism having, L- amino acid-producing ability, including the pyruvate dehydrogenase mutant cultured in a culture medium; And (b) to provide a process for producing, L- amino acid comprising the step of recovering the L- amino acid from the medium or the microorganism.
[11]
Effects of the Invention
[12]
Via the pyruvate dehydrogenase mutant of the present application can be obtained with the enzyme activity is attenuated. According to this use of the strains containing the pyruvate dehydrogenase activity of the mutant is weakened, it can produce L- amino acids than the L- amino acid-producing strains containing the wild-type pyruvate dehydrogenase protein with high efficiency. In addition, in the case of using the above strain, the pyruvate dehydrogenase deficiency, unlike the strain, can lead to an effective production of L- amino acids while not substantially inhibit the growth of the strain. For example, lysine can be obtained in the feed production cost savings when producing L- lysine with a high efficiency, such as bars, as required herein is industrially mass-produced as the essential amino acids for animal feed.
[13]
Best Mode for Carrying Out the Invention
[14]
One embodiment for implementing the present application, is in SEQ ID NO: 1 to 190 times, 205 times or 415 times to 440 times the amino acid region having one or more amino acid mutations in the amino acid region, pyruvate dehydrogenase (pyruvate dehydrogenase) variants.
[15]
[16]
The term herein, the term "pyruvate dehydrogenase (pyruvate dehydrogenase)" is one of the enzymes constituting a (pyruvate dehydrogenase multienzyme complex) PDHC, involved in the conversion of pyruvate to acetyl -CoA. Herein are but are not particularly limited as long as having the pyruvate Di art active dehydrogenase kinase, the genus Corynebacterium microorganism, specifically, Corynebacterium glutamicum may first come to pyruvate-dihydro-derived, but limited to, It is not. For example, the pyruvate dehydrogenase is SEQ ID NO: amino acid sequence, or the 75% or more of 1, specifically 80%, more specifically more than 85%, still more specifically 90%, 91%, 92% , 93%, and may be an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% homology. E1p protein having the amino acid sequence of SEQ ID NO: 1 having the polynucleotide sequence of SEQ ID NO: 2 aceE , but may be encoded by the gene, but is not limited thereto. Also, if the amino acid sequence having a biological activity substantially the same as or correspond to the protein of SEQ ID NO: 1 as a sequence having homology with said sequence, if some sequence having a deletion, modified, substituted or added in the amino acid sequence also in the present included in the category are self-evident. If a polynucleotide sequence encoding a pyruvate dehydrogenase herein may be included in the scope of the present application. For example, the SEQ ID NO: 2 in the polynucleotide sequence, or the 75% or more, specifically, even more specifically more than 80%, more specifically at least 85%, 90%, 91%, 92%, 93%, 94 %, it may be a polynucleotide sequence having 95%, 96%, 97%, 98%, or at least 99% homology. In addition, the polynucleotide sequence encoding the protein, taking into account the codon-preferred organisms intended to express the result or the protein to the degeneracy (degeneracy) of the codon, which do not change the amino acid sequence of the protein expressed from the coding region the coding region to the extent various modifications may be made.
[17]
[18]
Pyruvate dehydrogenase mutant according to the present is greater than or equal to 1 in SEQ ID NO: 1-190 times to 205 times the amino acid moiety or 415 times to 440 times the amino acid portion, at least 2, 3 or more, 4 or more, 5 or more, at least 6, 7 or more , 8 may have more, or 9 or 10 or more amino acid mutations.
[19]
Here, the pyruvate dehydrogenase mutant according to the present application may be one having a 190 to 205 times more than once in the amino acid portion, at least 2, at least 3 or 4 or more amino acid mutations in SEQ ID NO: 1. Specifically, the amino acid variation at times 190 times to 205 amino acids of the SEQ ID NO: 1 region can be selected from the group consisting of amino acids 190 times, 195 times, 199 times and 201 times, but is not limited to this.
[20]
Amino acid mutation in the SEQ ID No. 1 190 times to 205 amino acids in the region are, and at least one amino acid of the 190 times to 205 times the amino acid portion may be substituted with other amino acids, more specifically, 190 times more, It may be that one or more of 195 times, 199 times and 201 times the amino acid amino acid substitutions of amino acids of a different kind, and more specifically replacement of the histidine in the substituted (E190V), 195 times glutamine to valine at one 190 glutamic acid ( Q195H), at time 199 be at Lee Sangil one proline is substituted in the (P199S) and 201 times of the tyrosine-serine selected from the group consisting of substituted (Y201A) to alanine, but is not limited to a specific.
[21]
In addition, the pyruvate dehydrogenase mutant according to the present application may have one or more, 2 or more, 3 or more, 4 or more, 5 or more 6 or more amino acid mutations in the area 415 times to 440 times. Specifically, the amino acid variations from 415 times to 440 times the amino acid portion of the SEQ ID NO: 1 may be one selected from the group consisting of 418 times, 428 times, 432 times, 435 times and 438 amino acids.
[22]
Amino acid mutation in the SEQ ID NO: 1 of 415 times to 440 times the amino acid portion may be one in which one or more amino acids among the 415 times to 440 times the amino acid region substituted with amino acids of different types, specifically, 418 times, 428 times, 432 times, and may be substituted by 435 times and 438 times, at least one amino acid other amino acids in the amino acid, and more specifically replacement of the alanine at No. 418 tyrosine in the substituted (Y418H), asparagine 428 times of the histidine than (N428A), substitution of the glutamic acid at the 432 times glutamine (Q432E), 432 times substitutions in substituted (Q432A), 435 times lysine to alanine glutamine to proline in substitution (K435A) and 438 times leucine to alanine (L438P ), but as can be one or more selected from the group consisting of, but is not particularly limited.
[23]
[24]
More specifically, the pyruvate dehydrogenase mutant may have an amino acid sequence selected from the group consisting of SEQ ID NO: 14 to 33.
[25]
The pyruvate dehydrogenase variants, as well as proteins having the amino acid sequence shown in SEQ ID NO: 14 to 33, wherein the amino acid sequence with at least 75%, specifically at least 80%, more specifically at least 85%, even more specifically, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or a variant having at least 99% homologous, substantially as compared to the wild-type pyruvate dehydrogenase activity If the inhibition can be included without limitation. If the amino acid sequence having the biological activity of the same substantially in SEQ ID NO: protein having the amino acid sequence shown as 14 to 33 or equivalent as a sequence having such homology, if some sequence having a deletion, modified, substituted or added in the amino acid sequence also included is apparent within the scope of the present application.
[26]
The term used herein, "homology" refers to the percent of identity between two polynucleotide or polypeptide base ET. Sequence homology between from one mode to the other one of the parent ET ET castle may be determined by the known art. For example, it can be determined by directly aligning the sequence information between two polynucleotide molecules, or two polypeptide molecules by using a homology retrieving possible to align the sequence information and computer program easily. The computer program may be such as BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc). In addition, polynucleotide homology between After hybridizing polynucleotide under conditions forming a stable duplexes between homologous regions, single-can be determined by determining the amount of the decomposed fragment was digested zero-specific nuclease-stranded.
[27]
[28]
Another aspect of the present application is to provide a polynucleotide coding for the pyruvate dehydrogenase mutant.
[29]
[30]
It is as described above for the pyruvate dehydrogenase mutant. Specifically, the pyruvate dehydrogenase polynucleotide encoding a dehydratase variant, if a polynucleotide encoding a protein having the amino acid sequence shown in SEQ ID NO: 14 to 33 may be included within the scope of the present application. In addition to this, at least 75% to the polynucleotide sequence, specifically 80%, more specifically more than 85%, still more specifically 90%, 91%, 92%, 93%, 94%, 95%, having at least 96%, 97%, 98%, or 99% homology can be a polynucleotide.
[31]
Herein, the term "polynucleotide" is a nucleotide unit is that of a polymer of nucleotides long by covalent bonds that result in a chain shape, generally it refers to a predetermined length or more DNA or RNA strands, and coding for the pyruvate dehydrogenase variants herein It means a polynucleotide. The polynucleotide may have a different nucleotide sequence, encoding the same amino acid sequence due to the degeneracy (genetic code degeneracy) of the genetic code. In addition, codons to optimize expression in accordance with the type of host cells - may have the optimized sequence.
[32]
[33]
Another specific aspect of the present application is a microorganism of the genus Corynebacterium having, L- amino acid-producing ability, including the pyruvate dehydrogenase mutant.
[34]
[35]
The microorganisms specifically may be a by mutant transformed by a vector comprising a polynucleotide containing or encoding the pyruvate dehydrogenase mutants of the pyruvate dehydrogenase of claim variant.
[36]
The term used herein, "vector" refers to any vehicle for the cloning and / or transformation of the base into a host cell. The vector may be a different DNA fragment the replicon (replicon) that may lead to replication of the fragments coupled in combination. A "replicon" is DNA replication in vivo self functioning as a unit, that is, refers to a possible duplicate by modulation of themselves, any genetic units (e. G., Plasmid, phage, cosmid, chromosome, virus) . In the present application as long as it can replicate in a host vector is not particularly limited and can utilize any vector known in the art. Vector used in the production of the recombinant vector may be a natural condition or a condition of a recombinant plasmid, cosmid, virus and bacteriophage. For example, the phage vector or cosmid vector, and as the like pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and Charon21A, pDZ vector, pBR series, pUC series as a plasmid vector It may be used based pBluescriptII, pGEM series, pTZ-based, such as pCL and pET-based system. The available vector is not particularly limited and may be a known expression vector. Specifically, the pDZ (Patent Registration No. 10-0924065 No. Korea; is included is incorporated in its entirety in the present invention) can be used, but is not limited to this.
[37]
By introducing the term "transgenic" is a gene herein in a host cell will to allow to express in a host cell, the transformed gene if it can be expressed in the host cell is located in addition to insertion or chromosome chromosome of the host cell that it can be incorporated without any restriction.
[38]
The gene can be introduced into a host cell in the form of there is expressed by itself in the expression cassette (expression cassette) A polynucleotide construct containing all the elements required. The expression cassette typically comprises a promoter that is operably linked to the gene, transcription termination signal, ribosome binding site and translation termination signal. The expression cassette may be an expression vector form a self-replicable. Also, the gene that is introduced into the host cell itself or in the form of the polynucleotide structure, but may be, which is possibly connected with the operation sequence necessary for the expression in a host cell is not limited thereto.
[39]
The microorganisms may be included which would prokaryotic microorganisms and eukaryotic microorganisms, if the art pyruvate dehydrogenase transformed so that it contains a protein of the mutant kinase activity or protein expression per art. For example, Escherichia ( Escherichia ), An air Winiah ( Erwinia ) genus, Serratia marcescens ( Serratia ) genus, Providencia ( Providencia ) genus, Enterobacter bacteria ( Enterobacteria ) genus, Salmonella ( Salmonella ) genus Streptomyces ( Streptomyces ), genus Pseudomonas ( Pseudomonas ) genus Brevibacterium ( Brevibacterium ) in or Corynebacterium ( Corynebacterium may include a microbial strain, such as) in. Specifically, a microorganism belonging to the genus Corynebacterium, more specifically, Corynebacterium glutamicum ( Corynebacterium glutamicum may be a), and the like.
[40]
In addition, pyruvate dehydrogenase kinase mutants of the present protein is a D, L- amino acids do not significantly inhibit the growth of cells as compared with microorganisms, including the wild-type pyruvate dehydrogenase protein, if contained in the microorganism having the L- amino acid-producing ability it is possible to improve the production capacity.
[41]
The term herein, "L- amino acid" is to include all L- amino acids that can be produced via the pyruvate (Pyruvate), specifically acetyl Koei (acetyl CoA) to pyruvate in a biosynthetic pathway from various carbon sources It may be a L- amino acid that bypasses the step of switching. More specifically, L- lysine, L- threonine, L- methionine, L- isoleucine, L- valine, and includes the L- leucine, L- alanine or, even more specifically, L- lysine or L- It can be a valine.
[42]
The microorganism producing L- amino acid comprises both a prokaryotic or eukaryotic microorganism strain capable of producing L- amino acid in the organism, the type of microorganism which can be applied. Examples are as described above. Microorganism producing the amino acid is L- if having L- amino acid-producing ability include, without limitation of the type of micro-organisms and includes both forms of the wild-type strain and the recombinant strain.
[43]
[44]
For example, the genus Corynebacterium microorganism which may have an L- lysine-producing ability, or is a mutant strain having resistance to L- lysine analog, the activity of L- lysine biosynthesis-related protein is modified to be the bibyeon enhanced compared to the strain can. Specifically, in the case of improving L- lysine biosynthesis-related gene expression 1 or more species, such expression enhancement can be achieved by introduction of a mutation for improving the replacement or modification of the sequence, such as gene amplification, a promoter or initiation codon, such as expression However, not limited to this.
[45]
In addition, examples of the L- lysine biosynthesis-related genes, and include a gene which is located in the L- lysine biosynthetic pathway, specifically, the Diffie-dihydro choline acid synthase gene (dapA), aspartate kinase SAT gene (lysC), di dihydro Diffie choline acid reductase gene (dapB), diaminopimelic acid Cartesian decarboxylase gene (lysA), diaminopimelic acid dehydrogenase gene (ddh), phosphorylase a play pyruvic acid decarboxylase gene (ppc), Aspergillus aspartate semi-aldehyde dehydrogenase can be mentioned the gene (asd), O Spartan genes (aspB), pyruvate acid decarboxylase (Pyc), but are not limited to these. Further, Kane transformer present on the pentose phosphate pathway Tora claim (tkt). However, and the like, and the like.
[46]
On the other hand, the L- genus Corynebacterium microorganism may have a lysine-producing ability can be represented, but the L- lysine-producing ability by mutation to include the well-known in the art relating to the production of L- lysine, but not limited thereto.
[47]
[48]
The L- threonine is a microorganism having production capacity, especially but not limited to, methionine requirement, threonine analogue resistance, resistance, isoleucine analogue resistance and / or analogue of methionine for lysine analogs for the for microorganisms may be resistant. Methionine analogues include, for example, D, L- on thionine, Nord leucine, α- methyl-methionine, L- methionine, and -D, may be one or more compounds selected from the group consisting of L- sulfoxide citizen, used Leo non analogs α- -β- amino-hydroxy valeric acid and D, L- threonine hydroxy may be one or more compounds selected from the group consisting of roksa formate, lysine analogue is S- (2- aminoethyl) -L - it may be one or more compounds selected from the group consisting of cysteine and δ- -L- lysine methyl. In addition, L- threonine producing ability are microorganisms having an L- threonine biosynthesis intermediate oxaloacetic acetate (OAA) a phosphonate to play pyruvate (PEP) involved in changing the activity of a protein PckA weakened or that inactivation of the microorganism, or oxaloacetic from acetate switching to aspartate lysC with a TyrR to inhibit gene attenuated or inactivated microorganism, or involved in glucose influx of galP of the GalR for inhibiting the expression of genes weakened or inactivated microorganisms but it can include, but is not limited thereto.
[49]
Microorganism having L- isoleucine producing ability, the microorganism, or with resistance to L- isoleucine or derivative thereof may be a genetic modification is L- isoleucine or feedback inhibition by derivatives thereof to be released. Examples of the L- isoleucine derivatives of 4-thiazol isoleucine (4-thiaisoleucine, thiaile) or isoleucine - but are a hydroxy roksa formate (isoleucine-hydroxamate, ileHx), but is not limited thereto.
[50]
[51]
Microorganism having an L- valine-producing ability is, L- valine or is a microorganism having resistance to its derivative enzyme on the biosynthetic pathway of L- valine can be a genetically modified microorganism that feedback is turned off by the L- valine or derivatives thereof . Examples include but are microorganisms containing the, acetonitrile hydroxy acid synthase variants feedback inhibition is released on L- valine, but are not limited thereto. In addition, the microorganisms can be mutated to enhance L- expression of valine operon. Examples The polynucleotide sequence coding for all or part of the leader peptide in the control region of the L- valine operon is deficiency of L- Valine O Perrin expression-enhanced (Republic of Korea Patent Publication No. 10-2014-0111421 call; may include specification as a reference, but not limited to) it may be a.
[52]
[53]
Another specific aspect of the present application is a step to produce L- amino acid by culturing a microorganism of the genus Corynebacterium having the L- amino acid-producing ability, including the pyruvate dehydrogenase variant (a) in a medium; And (b) a method for producing, L- lysine recovering the L- amino acid from the medium or the microorganism.
[54]
It is as described above for the like genus Corynebacterium microorganism having the L- amino acid-producing ability.
[55]
The term herein, the term "culture" is meant that the growth in the environmental conditions which control the microorganisms with artificially appropriately. Culturing process of the present application may be made in accordance with the appropriate culture medium and culture conditions known in the art. Specific incubation temperature, incubation time and conditions, including pH of the medium may be performed according to methods known to the general knowledge of one of ordinary skill in the art, or conventional, and therefore can be properly adjusted. Specifically, these known culture methods are described in [Chmiel; Bioprozesstechnik 1. Einfuhrung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren und periphere Einrichtungen is described in detail in (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)]. Further, batch culture (batch culture), is how the culture continuous culture (cintinuous culture), and fed-batch culture (fed-batch culture) may include, specifically, a batch process or injection batch or repeated injection batch process (fed batch or repeated fed batch process), but can be cultured in a continuous manner, without being limited thereto.
[56]
The medium used to culture should meet the requirements of the particular strains in a suitable manner. As a carbon source that can be used in the medium is glucose, saccharose, lactose, paroxetine lactose, maltose, starch, cellulose and and carbohydrates, soybean oil, sunflower oil as per, castor oil, oils and fats such as coconut oil, palmitic Although the acid, stearic acid, linoleic acid, such as fatty acids, glycerol, ethanol and like alcohols, such as organic acid such as acetic acid can include, without being limited thereto. These materials may be used separately or as mixtures, and the like. The nitrogen source which can be used may include a peptone, yeast extract, gravy, malt extract, corn steep liquor, such as soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, and , nitrogen also can be used separately or as mixtures, and the like. Personnel to that may be used include phosphate, sodium or potassium phosphate or the corresponding potassium susoyi-containing salt may be included, etc., and the like. Also, culture media may contain metal salts such as magnesium sulfate or iron sulfate needed for growth. Finally, the required growth substances such as amino acids and vitamins can be used in addition to the above materials. Also may be used, suitable precursors to the culture medium. The above described material may be added in batch or in continuous mode by an appropriate method to culture in culture, but is not limited to this.
[57]
In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid was added in an appropriate manner in the culture during cultivation, it is possible to adjust the pH of water culture. During cultivation it is possible to suppress the air bubbles generated by using antifoaming agents such as fatty acid glycol esters. Also, it can be injected without injection of the gas or of nitrogen, hydrogen or carbon dioxide gas in order to maintain the culture, oxygen or oxygen-containing gas injected into or anaerobic conditions and Miho group into the water in order to maintain aerobic condition of water culture. Incubation temperature of water is usually 27 ℃ to 37 ℃, specifically 30 ℃ to 35 ℃. The incubation period may be continued until it is obtained the production of the desired useful substance, and specifically may be from 10 to 100 hours. L- amino acid or is released into the culture medium, there may be contained in the microorganism.
[58]
Further, a method of producing L- amino acids of the present application is a method of recovering an L- amino acid from the cultured microorganisms or culture medium are well known in the art. The L- amino acid recovery methods, filtration, anion exchange chromatography, crystallization and HPLC, but such can be used, not limited to these examples.
[59]
Mode for the Invention
[60]
And in more details below the present invention through the embodiments. It is, however, not intended that the scope of the present invention are for the purpose of illustrating the invention by way of example only to the examples.
[61]
[62]
Example 1: Production E1p mutant libraries using artificial mutations law
[63]
[64]
In this embodiment, to prepare a vector for library insertion within the first cross-chromosome in the following manner in order to obtain a variant E1p. Corynebacterium glutamicum of the ATCC13032-derived E1p (SEQ ID NO: 1) encryption of aceE of the targeting gene (SEQ ID NO: 2) performing a Error-prone PCR method base substitution mutation is randomly introduced into the aceE gene mutant ( to obtain a 2852bp). Error-prone PCR was done using the GenemorphII Random Mutagenesis Kit (Stratagene), was used for Corynebacterium glutamicum ATCC13032 genomic DNA primers as a template 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4).
[65]
[66]
Primer 1 (SEQ ID NO: 3): 5'-TGGGA CCGGG AAACC GGG-3 '
[67]
Primer (SEQ ID NO: 4): 5'-GATTT ATCTG TCCCT TGA-3 '
[68]
[69]
Was allowed variation is introduced from 0 to 3.5 per dog 1kb within the amplified gene fragment, PCR conditions were denaturation 96 ℃, 30 seconds; ℃ annealing 53, 30 seconds; And the polymerization reaction 72 ℃, was repeated for 2 minutes 30 times.
[70]
Was connected to the amplified gene fragment to pCR2.1-TOPO TA cloning kit (Invitrogen) pCR2.1-TOPO vector (hereinafter referred to as 'pCR2.1') ​​by using, by transforming into E. coli DH5α kanamycin (25mg / L) is It was plated on the LB solid medium containing. After transfection selection of the conversion 20 colonies species obtained plasmid was confirmed that the variation in different locations as a result of 1.4 mutations / kb frequency analysis of the polynucleotide sequence introduced. Taking the approximately 20,000 transformed E. coli colony was extracted plasmid, and named it as pCR2.1-aceE (mt) library.
[71]
In addition, wild-type for use as a control aceE to prepare a plasmid having the gene. Using primers 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) PCR was as Corynebacterium glutamicum ATCC13032 genomic DNA under the same conditions as described above as a template. Polymerase was used PfuUltra High-Fidelity DNA Polymerase (Stratagene). A production plasmid was named as pCR2.1-aceE (WT).
[72]
[73]
Example 2: aceE deficient production strain
[74]
[75]
KCCM11016P (the microorganism was published KFCC10881, the material deposited in the Budapest Treaty servant international deposition, received an accession number given by KCCM11016P, Korea Patent Registration No. 10-0159812 call) to a strain with the parental strain pCR2.1-aceE for introducing the (mt) library aceE the defective strain was produced.
[76]
aceE defective vector to the wild type Corynebacterium glutamicum ATCC13032 Chromosomal template in order to produce a primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6), primer 5 (SEQ ID NO: 7) and primer 6 (SEQ ID NO: PCR using a No. 8) was carried out.
[77]
[78]
Primer 3 (SEQ ID NO: 5): 5'-GCAGG TCGAC TCTAG ATGCG ATTCG CGTCA AACGT G-3 '
[79]
Primer 4 (SEQ ID NO: 6): 5'-GTCCC TTGAG GTGAT GTGAA TCCAT CCACT-3 '
[80]
Primer 5 (SEQ ID NO: 7): 5'-AGTGG ATGGA TTCAC ATCAC CTCAA GGGAC-3 '
[81]
Primer 6 (SEQ ID NO: 8): 5'-CCGGG GATCC TCTAG ACGAA GCGCC GTGAG CAATT C-3 '
[82]
[83]
PCR conditions were carried out for 5 min denaturation at 95 ℃, 95 ℃ was repeated 30 seconds denaturation, annealing 55 ℃ 30 seconds, 30 seconds 72 ℃ polymerization 30 times, in 72 ℃ polymerization 7 minutes of reaction.
[84]
As a result aceE to give the SEQ ID NO: 9 and SEQ ID NO: 10 538bp of 521bp containing the gene 5 'end and 3' end respectively.
[85]
[86]
And the amplified SEQ ID NO: 9 and SEQ ID NO: 10 as a template, PCR was performed using the primer 3 (SEQ ID NO: 5) and primer 6 (SEQ ID NO: 8). PCR conditions were carried out for 5 min denaturation at 95 ℃, 95 ℃ was repeated 30 seconds denaturation, annealing 55 ℃ 30 seconds, 60 seconds, 72 ℃ polymerization 30 times, in 72 ℃ polymerization 7 minutes of reaction.
[87]
As a result, aceE 5 of the gene & apos; end and the 3 'end is connected 1029bp of SEQ ID NO: 11 (hereinafter, △ aceE ) was amplified.
[88]
[89]
Corynebacterium glutamicum pDZ vector replication can not be in the (Korea Patent Registration No. 10-0924065 call) and the amplified △ aceE after treatment the fragments with restriction enzymes XbaⅠ, after connecting with a DNA joining enzymes, cloning the plasmid was obtained by this pDZ- △ aceE was named.
[90]
pDZ- △ aceE electric pulse method in which the L- lysine-producing strain Corynebacterium glutamicum KCCM11016P: After each transformed with (Appl Microbiol Biothcenol (1999) 52 ... 541-545) kanamycin (kanamycin) 25mg / L to obtain the conversion strain transformed in a selection medium containing. Secondary recombination the DNA fragment inserted into the genome by △ (cross-over) aceE by aceE was obtained a strain gene is inactivated, KCCM11016P △ aceE was named.
[91]
[92]
Example 3: E1p artificial mutant library production and L- lysine -producing ability increased strain Screening
[93]
[94]
The production of KCCM11016P △ aceE subject to strain as the parent strain produced transformed by the pCR2.1-aceE (mt) library homologous chromosomal recombination and kanamycin (25mg / L) of about 10,000 to spread on plate medium containing the compound has secured the colonies, each colony to KCCM11016P △ aceE was named from /pCR2.1- aceE (mt) -1 to KCCM11016P / pCR2.1- aceE (mt) -10000 .
[95]
In addition, the production of pCR2.1- aceE (WT) vector KCCM11016P △ aceE by transforming the strain was the control strain produced, KCCM11016P △ aceE was named / pCR2.1- aceE (WT).
[96]
[97]

[98]
포도당 10g, Peptone 10g, Beef extract 5g, Yeast extract 5g, Brain Heart Infusion 18.5g, NaCl 2.5g, Urea 2g, Sorbitiol 91g, Agar 20g(증류수 1 리터 기준)
[99]
[100]
Was inoculated 10,000 colonies secured respectively to selection medium in a 96-deep-well plate in 300μL 32 ℃, and cultured for about 24 hours to 1000rpm. To analyze the production of L- lysine production in the culture medium was used for ninhydrin method (J. Biol Chem 1948. 176:. . 367-388). After the culture supernatants with 10μl hard non-lean solution after the reaction was 190μl in 65 ℃ 30 minutes of reaction as a spectrophotometer measuring the absorbance at a wavelength of 570 nm, and the culture is completed, the control KCCM11016P △ aceE / pCR2.1-aceE (WT) absorbance of the strain and it was compared to the selected mutant strain of 256 colonies were seen the increased absorbance over 10%. More colony are indicated a similar or decreased compared to the control absorbance.
[101]
[102]

[103]
Glucose 10g, (NH 4 ) 2 SO 4 5.5g, MgSO 4 · 7H 2 O 1.2g, KH 2 PO 4 0.8g, K 2 HPO 4 16.4g, 100㎍ biotin, thiamine HCl 1000㎍, calcium pantothenate 2000㎍ , nicotinamide 2000㎍ (in 1 liter of distilled water)
[104]
[105]
Strains selected 256 has been carried out repeatedly ninhydrin reaction in the same manner as described above, KCCM11016P △ aceE / pCR2.1-aceE (WT) strains were selected over the L- lysine-producing ability improved strain top 53 thereof.
[106]
[107]
Example 4: E1p artificial mutant library screening careful check L- lysine production capacity
[108]
[109]
And to compare the L- lysine-producing ability of the 53 strains selected in Example 3 were cultured in the following ways: a culture solution ingredient.
[110]
Seed medium 250 ㎖ corner containing 25㎖ - in bapeul of each strain was inoculated into a flask, 30 ℃ for 20 hours, then this was cultured with shaking in 200rpm. Lysine production medium containing 250㎖ corner 24㎖ - from inoculated with seed culture of the 1㎖ bapeul flask and 30 ℃ for 72 hours, then this was cultured with shaking at 200rpm. By HPLC analysis, the concentration of L- lysine.
[111]
[112]
<종 배지 (pH 7.0)>
[113]
Glucose 20g, Peptone 10g, Yeast extract 5g, Urea 1.5g, KH 2 PO 4 4g, K 2 HPO 4 8g, MgSO 4 · 7H 2 O 0.5g, 100㎍ biotin, thiamine HCl 1000㎍, calcium pantothenate 2000㎍, nicotinamide 2000㎍ (in 1 liter of distilled water)
[114]
[115]

[116]
Glucose 100g, (NH 4 ) 2 SO 4 40g, 2.5g of soy protein, corn steep solids (Corn Steep Solids) 5g, 3g Urea, KH 2 PO 4 1g, MgSO 4 · 7H 2 O 0.5g, 100㎍ biotin, thiamine 1000㎍ hydrochloride, calcium pantothenate 2000㎍, nicotinamide 3000㎍, CaCO 3 30g (per liter of distilled water).
[117]
[118]
It was among the 53 strains screened L- lysine concentration Top 10 strain carry out the culture and repeating analysis, the analyzed concentration of L- lysine is shown in Table 1.
[119]
Table 1 [Table 1]
The selected 10 kinds KCCM11016P △ aceE / pCR2 .1- aceE (mt) L- lysine production levels
Strain L- lysine (g / L)
Placed one Placed second Placed third Average
Controls KCCM11016P △ various /pCR2.1-aceE(WT) 42.1 41.9 41.7 41.9
1 KCCM11016P △ various /pCR2.1-aceE(mt)-1235 45.1 46.2 45.8 45.7
2 KCCM11016P △ various /pCR2.1-aceE(mt)-1542 45.6 46.1 44.9 45.5
3 KCCM11016P △ various /pCR2.1-aceE(mt)-3152 46.1 45.7 46.0 45.9
4 KCCM11016P △ various /pCR2.1-aceE(mt)-5013 44.5 45.1 45.7 45.1
5 KCCM11016P △ various /pCR2.1-aceE(mt)-5312 45.9 44.9 46.1 45.6
6 KCCM11016P △ various /pCR2.1-aceE(mt)-6001 44.8 45.7 45.4 45.3
7 KCCM11016P △ various /pCR2.1-aceE(mt)-7139 46.1 46.3 45.9 46.1
8 KCCM11016P △ various /pCR2.1-aceE(mt)-8264 46.6 48.1 47.3 47.3
9 KCCM11016P △ various /pCR2.1-aceE(mt)-9174 45.6 45.1 44.8 45.2
10 KCCM11016P △ various /pCR2.1-aceE(mt)-9586 46.1 46.6 45.9 46.2

[120]
L- lysine concentration analysis result, 10 kinds of selective attention L- lysine yield control KCCM11016P △ aceE was confirmed that /pCR2.1-aceE (WT) over increased up to 22% strain.
[121]
[122]
Example 5: E1p artificial mutant library screening Attention aceE mutation confirmed
[123]
[124]
In order to determine the mutagenic in E1p of the 10 strains selected in Example 4 aceE the polynucleotide sequence of the mutant was analyzed. The PCR was carried out using primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4), to determine the polynucleotide sequence.
[125]
[126]
Primer 1 (SEQ ID NO: 3): 5'-TGGGA CCGGG AAACC GGG-3 '
[127]
Primer (SEQ ID NO: 4): 5'-GATTT ATCTG TCCCT TGA-3 '
[128]
[129]
The securing each variant aceE through a polynucleotide sequence analysis of the gene segment variants aceE confirm the polynucleotide sequence of the gene, and was compared to the polynucleotide sequence of SEQ ID NO: 2, This was confirmed that the amino acid sequence of the variant E1p. E1p variation information of the amino acid sequence of the selected strains is shown in Table 2.
[130]
Table 2 [Table 2]
Selected 10 kinds KCCM11016P / pCR2.1-aceE (mt) E1p amino acid mutations
Strain E1p amino acid mutations
KCCM11016P△aceE /pCR2.1-aceE(mt)-1235 Q432E
KCCM11016P△aceE /pCR2.1-aceE(mt)-1542 E190V
KCCM11016P△aceE /pCR2.1-aceE(mt)-3152 L438P
KCCM11016P△aceE /pCR2.1-aceE(mt)-5013 Q195H
KCCM11016P△aceE /pCR2.1-aceE(mt)-5312 P199S
KCCM11016P△aceE /pCR2.1-aceE(mt)-6001 K435A
KCCM11016P△aceE /pCR2.1-aceE(mt)-7139 Q432A
KCCM11016P△aceE /pCR2.1-aceE(mt)-8264 Y418H
KCCM11016P△aceE /pCR2.1-aceE(mt)-9174 N428A
KCCM11016P△aceE /pCR2.1-aceE(mt)-9586 Y201A

[131]
[132]
Example 6: E1p vector construction for introducing chromosomal mutations
[133]
[134]
In order to determine the variation E1p Effects confirmed in Example 5 to prepare a vector capable of introducing it into a chromosome.
[135]
To the 5 'restriction site to insert the XbaⅠ terminal primer 9 (SEQ ID NO: 12) and 3' restriction site primer XbaⅠ 10 insert (SEQ ID NO: 13) at the terminal based on the reported polynucleotide sequence it was synthesized. It is by using a pair of primers, and the amplification of chromosome of the selected 10 kinds of performing a PCR as templates respectively, and 10 kinds of mutation type aceE (mt) gene fragment. PCR conditions were carried out for 5 min denaturation at 94 ℃, after repeating 30 times a 94 ℃ 30 cho denaturation, annealing 56 ℃ cho 30, 72 ℃ 2 bun polymerization, polymerization at 72 ℃ 7 minutes of reaction.
[136]
[137]
Primer 9 (SEQ ID NO: 12): 5'-AATCT AGATG GGACC GGGAA ACCGG G-3 '
[138]
Primer 10 (SEQ ID NO: 13): 5'-AATCT AGAGA TTTAT CTGTC CCTTG A-3 '
[139]
[140]
After the treatment, a gene fragment of the 10 kinds of amplified by PCR with a restriction enzyme XbaⅠ obtain each DNA fragment, this restriction XbaⅠ transformed after E. coli DH5α is connected to the pDZ vector for chromosomal introduced with the terminal and kanamycin (25mg / L) were plated on the LB solid medium containing.
[141]
[142]
Transfected with a vector a gene is inserted object by PCR, evaluate whether the transition colonies after using a commonly known plasmid extraction was obtained a plasmid in accordance with the transition into the E1p of this plasmid respectively pDZ-E1p (Q432E), pDZ -E1p (E190V), pDZ-E1p (L438P), pDZ-E1p (Q195H), pDZ-E1p (P199S), pDZ-E1p (K435A), pDZ-E1p (Q432A), pDZ-E1p (Y418H), pDZ- It was named E1p (N428A), pDZ-E1p (Y201A).
[143]
[144]
Example 7: KCCM11016P derived E1p mutagenic strains produced and L- lysine -producing ability compared
[145]
[146]
Example 6 using the 10 kinds of vectors for introduction of novel mutant produced in step through a second homologous chromosomal recombination was transformed to L- lysine-producing strain Corynebacterium glutamicum KCCM11016P. After that the polynucleotide sequence was analyzed by screening a strain E1p mutation is introduced on the chromosome via, respectively KCCM11016P :: E1p (Q432E) in accordance with the insertion mutation E1p, KCCM11016P :: E1p (E190V), KCCM11016P :: E1p (L438P) , KCCM11016P :: E1p (Q195H), KCCM11016P :: E1p (P199S), KCCM11016P :: E1p (K435A), KCCM11016P :: E1p (Q432A), KCCM11016P :: E1p (Y418H), KCCM11016P :: E1p (N428A), KCCM11016P It was named :: E1p (Y201A).
[147]
[148]
Example 4 and cultured in the same manner, was analyzed for the concentration of said L- lysine therefrom to measure the residual sugar concentration after the culture started 18 hours to measure the growth of the production strain [Table 3].
[149]
Table 3 [Table 3]
Per medium KCCM11016P strain introduced mutations derived E1p state (g / L), and L- lysine-producing concentration (g / L)
Strain Placed one Placed second Placed third Average
Medium sugar L- lysine Medium sugar L- lysine Medium sugar L- lysine Medium sugar L- lysine
Controls KCCM11016P 35.8 42.8 34.5 41.6 35.1 43.1 35.1 42.5
1 KCCM11016P::E1p(Q432E) 41.2 46.2 40.5 45.8 42.1 45.2 41.3 45.7
2 KCCM11016P::E1p(E190V) 43.2 45.2 41.8 45.8 43 46.7 42.7 45.9
3 KCCM11016P::E1p(L438P) 40.5 45.1 41.1 45.9 40.6 45.8 40.7 45.6
4 KCCM11016P::E1p(Q195H) 38.1 44.5 37.5 45.2 38.5 44.9 38.0 44.9
5 KCCM11016P::E1p(P199S) 44.1 46.2 43.5 45.9 44.8 44.8 43.8 45.6
6 KCCM11016P::E1p(K435A) 44.1 45.2 43.5 45.1 42.9 46.5 43.5 45.6
7 KCCM11016P::E1p(Q432A) 40.5 46.5 38.9 46.1 40.3 45.8 39.9 46.1
8 KCCM11016P::E1p(Y418H) 38.5 47.5 37.6 46.6 39.1 46.5 38.4 46.9
9 KCCM11016P::E1p(Y201A) 42.5 45.7 43.1 44.6 40.5 45.8 42.0 45.4
10 KCCM11016P::E1p(N428A) 39.8 46.5 40.1 47.9 40.3 46.2 40.1 46.9

[150]
신규 변이주 10종(KCCM11016P::E1p(Q432E), KCCM11016P::E1p(E190V), KCCM11016P::E1p(L438P), KCCM11016P::E1p(Q195H), KCCM11016P::E1p(P199S), KCCM11016P::E1p(K435A), KCCM11016P::E1p(Q432A), KCCM11016P::E1p(Y418H), KCCM11016P::E1p(Y201A), KCCM11016P::E1p(N428A))은 모균주 대비 당소모 속도가 소폭 하락하고 라이신 생산능은 최대 10% 증가하였다.
[151]
[152]
Thus, the present inventors Budapest Treaty on the capability of producing L- lysine improved strain representative strains of KCCM11016P :: E1p (N428A) in Corynebacterium glutamicum "CA01-2289" was named LA, October 2014 23rd accession to the international deposit under the agency Korea Culture Center of microorganisms (KCCM) and were given an accession number KCCM11590P.
[153]
[154]
Analysis results, E1p mutation (E1p (Q432E) of 10 species (SEQ ID NO: 14), E1p (E190V) (SEQ ID NO: 15), E1p (L438P) (SEQ ID NO: 16), E1p (Q195H) (SEQ ID NO: 17), E1p (P199S) (SEQ ID NO: 18), E1p (K435A) (SEQ ID NO: 19), E1p (Q432A) (SEQ ID NO: 20), E1p (Y418H) (SEQ ID NO: 21), E1p (Y201A) (SEQ ID NO: 22), E1p (N428A) (SEQ ID NO: 23)) was the concentration is divided into two groups (190 amino acid residues from amino acid residues 201 and 418 th 438 th amino acid residue from the amino acid residues of the protein E1p) distribution.
[155]
[156]
Mutant belonging to each group are a strain comprising a combination 10 kinds (KCCM11016P :: E1p (E190V, Q195H), KCCM11016P :: E1p (E190V, P199S), KCCM11016P :: E1p (Q195H, P199S), KCCM11016P :: E1p (E190V , Y201A), KCCM11016P :: E1p (Q195H, Y201A), KCCM11016P :: E1p (P199S, Y201A), KCCM11016P :: E1p (N428A, Q432E), KCCM11016P :: E1p (N428A, K435A), KCCM11016P :: E1p (Y418H , K435A), KCCM11016P :: E1p (Y418H, Q432A) a was produced, the lysine-producing ability was determined in the same manner as in Table 4.
[157]
Table 4 [Table 4]
Per medium KCCM11016P strain introduced mutations derived E1p combined state (g / L), and L- lysine-producing concentration (g / L)
Strain Placed one Placed second Placed third Average
Medium sugar L- lysine Medium sugar L- lysine Medium sugar L- lysine Medium sugar L- lysine
Controls KCCM11016P 35.2 42.1 34.6 42.5 35.6 42.5 35.1 42.4
1 KCCM11016P::E1p(E190V, Q195H) 47.1 47.1 46.5 48.1 46 43.1 46.5 46.1
2 KCCM11016P::E1p(E190V, P199S) 45.2 45.2 46.3 46.1 46 45 45.8 45.4
3 KCCM11016P::E1p(Q195H, P199S) 46.2 49.8 45.7 48.7 44 48.5 45.3 49.0
4 KCCM11016P::E1p(E190V, Y201A) 42.8 46.1 43.1 45.7 42.5 46 42.8 45.9
5 KCCM11016P::E1p(Q195H, Y201A) 48.3 45.3 47.5 47.1 47 46.2 47.6 46.2
6 KCCM11016P::E1p(P199S, Y201A) 39.1 45.3 38.6 45.9 39.2 45.6 39.0 45.6
7 KCCM11016P::E1p(N428A, Q432E) 45.9 48.1 47.1 49.2 46.5 49.3 46.5 48.9
8 KCCM11016P::E1p(N428A, K435A) 46.1 47.9 45.8 48.3 46.7 47.9 46.2 48.0
9 KCCM11016P::E1p(Y418H, K435A) 38.2 45.9 37.6 45.7 39.1 46 38.3 45.9
10 KCCM11016P::E1p(Y418H, Q432A) 41.2 46.1 40.9 45.1 39.9 47.2 40.7 46.1

[158]
As shown in Table, the introduction of new mutant combinations week 10 kinds exhausted speed is slightly lower compared to each parent strain and the lysine-producing ability was increased up to 15.6%. This means that may represent a larger-lysine-producing ability increased effectiveness when introduced in combination than when introducing new mutations one member.
[159]
[160]
The results, E1p new mutant 10 species of proteins is an effective mutation sikineunde greatly increased compared to strain mode to stand-lysine-producing ability do not reduce significantly the sugar consumption rate, since 190th amino acid residues from the 205th amino acid residue or the 415th amino acid residue the 440th amino acid residue shows that a major part illustrating the effect.
[161]
[162]
Example 8: E1p pyruvate for mutant dehydrogenase complex ( Pyruvate Dehydrogenase Complex, PDHC) activity measurement
[163]
[164]
The methods reported in prior literature (Schreiner et al, J. Bacteriol 187 :.. 6005, 2005) for the PDHC active targeting the selected strains were determined over. KCCM11016P, KCCM11016P △ used as a control aceE 10 species and the selected strains (KCCM11016P :: E1p (Q432E), KCCM11016P :: E1p (E190V), KCCM11016P :: E1p (L438P), KCCM11016P :: E1p (Q195H), KCCM11016P :: E1p (P199S), KCCM11016P :: E1p ( K435A), KCCM11016P :: E1p (Q432A), KCCM11016P :: E1p (Y418H), KCCM11016P :: E1p (Y201A), KCCM11016P :: E1p the specified (N428A) in example 4 jong was inoculated into the medium 25mL, were cultured to late log phase. after the cells are harvested by centrifugation, 100mM Tris-HCl (pH 7.2, 3mM L-cysteine, 10mM MgCl 2 ) was washed two times with buffer solution, the same end were suspended in the buffer solution 2mL. the cell suspension to a common glass beads Vortex after the crushing for 10 min physical singbeop, two times of centrifugal separation and recovery, PDHC enzyme activity measurement of the supernatant through a (13,000rpm, 4 ℃, 30 min.) crude enzyme solution (crude extract) was used as a. PDHC enzyme activity measurement reaction solution (MgCl for enzyme activity measurement for for 2 10mM, 3mM cysteine, 2mM NAD, Thiamine pyrophosphate (thiamine diphosphate) 0.9mM, chlorpromazine (chlorpromazine) 0.25mM, pyruvic acid (pyruvate) 6mM, CoA 0.2 mM in Tris-HCl buffer solution (pH 7.2)) followed by the addition of crude enzyme solution in 0.95mL 0.05mL was reacted at 30 ℃. PDHC activation unit (unit) was defined as the number one consumption μmol NADH per minute, to measure enzymatic activity results shown in Table 5.
[165]
Table 5 Table 5
PDHC enzyme activity (%), measured
Strain PDHC activity (%)
KCCM11016P 100
KCCM11016P△aceE 0
KCCM11016P::E1p(Q432E) 39
KCCM11016P::E1p(E190V) 56
KCCM11016P::E1p(L438P) 53
KCCM11016P::E1p(Q195H) 55
KCCM11016P::E1p(P199S) 51
KCCM11016P::E1p(K435A) 53
KCCM11016P::E1p(Q432A) 42
KCCM11016P::E1p(Y418H) 36
KCCM11016P::E1p(Y201A) 46
KCCM11016P::E1p(N428A) 35

[166]
Introducing new mutations care PDHC activity exhibited 35% to 56% of the activity compared to the parental strain.
[167]
[168]
Example 9: aceE L- lysine-producing ability compared with the strain deficient
[169]
[170]
Example 2 aceE strain deficient in the production KCCM11016P △ aceE and selected 10 kinds of strains (KCCM11016P :: E1p (Q432E), KCCM11016P :: E1p (E190V), KCCM11016P :: E1p (L438P), KCCM11016P :: E1p (Q195H), KCCM11016P :: E1p (P199S), KCCM11016P :: E1p (K435A), KCCM11016P :: E1p (Q432A), KCCM11016P :: E1p (Y418H), KCCM11016P :: E1p (Y201A), compared to KCCM11016P :: E1p (N428A)) using the following medium containing ammonium acetate, and cultured in the same manner as in example 4 to evaluate. By HPLC culture starts to measure the growth rate of the strains produced were analyzed for the concentration of L- lysine was measured 18 hours after the residual sugar concentration [Table 6].
[171]
[172]

[173]
Glucose 100g, CH 3 COONH 3 5g, (NH 4 ) 2 SO 4 40g, 2.5g of soy protein, corn steep solids (Corn Steep Solids) 5g, 3g Urea, KH 2 PO 4 1g, MgSO 4 · 7H 2 O 0.5g , 100㎍ biotin, thiamine hydrochloride 1000㎍, calcium pantothenate 2000㎍, nicotinamide 3000㎍, CaCO 3 30g (per liter of distilled water)
[174]
Table 6 [Table 6]
Per KCCM11016P intermediate strains derived E1p mutagenic state and aceE defect state (g / L), and L- lysine-producing concentration (g / L)
Strain Placed one Placed second Placed third Average
Medium sugar L- lysine Medium sugar L- lysine Medium sugar L- lysine Medium sugar L- lysine
Controls KCCM11016P 45.7 40.9 45.6 42.1 46.7 41.3 46.0 41.4
Experimental KCCM11016P△aceE 67.1 45.6 66.8 45.2 68.2 45.4 67.4 45.4
1 KCCM11016P::E1p(Q432E) 48.2 43.7 46.5 44.2 47.5 43.4 47.4 43.8
2 KCCM11016P::E1p(E190V) 49.7 44.6 47.8 43.8 48.1 44.4 48.5 44.3
3 KCCM11016P::E1p(L438P) 47.9 45.3 48.9 43.2 47.5 44.6 48.1 44.4
4 KCCM11016P::E1p(Q195H) 46.7 42.9 46.3 43.6 45.6 43.0 46.2 43.2
5 KCCM11016P::E1p(P199S) 50.4 45.2 50.6 45.1 51.2 44.6 50.7 45.0
6 KCCM11016P::E1p(K435A) 51.3 44.9 50.9 45.2 52.7 45.0 51.6 45.0
7 KCCM11016P::E1p(Q432A) 48.9 45.6 47.8 46.0 47.9 45.9 48.2 45.8
8 KCCM11016P::E1p(Y418H) 44.6 45.8 45.7 46.5 47.3 46.3 45.9 46.2
9 KCCM11016P::E1p(Y201A) 47.6 43.8 46.7 44.5 48.7 43.7 47.7 44.0
10 KCCM11016P::E1p(N428A) 49.7 46.2 47.8 45.2 48.3 45.7 48.6 45.7

[175]
KCCM11016P △ aceE strain were increased by 9.6% lysine-producing ability compared to the parent strain, the growth rate was significantly slower. On the other hand mutagenic strain exhibited a growth rate and yield of lysine consumption rate per degree similar to the conditions without addition of the ammonium acetate.
[176]
[177]
The results indicate that lysine yield is increased, but the growth rate is significantly mutagenic aceE strain of the present invention compared to the aceE deficient strain characteristics appear to fall can produce lysine in a high yield without a significant difference in the growth rate.
[178]
[179]
[180]
Example 10: KFCC10750 derived E1p mutagenic strains produced and L- lysine -producing ability compared
[181]
[182]
Corynebacterium-in other strains belonging to Tommy glutamicum to determine the effect of the introduction of new mutations 10 kinds by using the same procedure as in Example 7 L- lysine-producing strain Corynebacterium glutamicum KFCC10750 (the microorganisms have been published in KFCC10750 be re-deposited at the International Depositary authority under the Budapest Treaty, has received a grant KCCM11347P. Korea Patent production of the strains introduced each of the 10 species of mutant E1p No. 10-0073610) and KFCC10750 :: E1p (Q432E), KFCC10750 :: E1p (E190V), KFCC10750 :: E1p (L438P), KFCC10750 :: E1p (Q195H), KFCC10750 :: E1p (P199S), KFCC10750 :: E1p (K435A), KFCC10750 :: E1p (Q432A ), it was named KFCC10750 :: E1p (Y418H), KFCC10750 :: E1p (Y201A), KFCC10750 :: E1p (N428A). By culturing a strain of 11, including strain KFCC10750 as a control in the same manner as in Example 4, was analyzed for the concentration of L- lysine therefrom (Table 7).
[183]
Table 7 [Table 7]
KFCC10750 strains derived E1p mutagenic week L- lysine production levels
Strain L- lysine (g / L)
Placed one Placed second Placed third Average
Controls KFCC10750 38.8 38.1 37.9 38.3
1 KFCC10750::E1p(Q432E) 43.1 42.8 43.5 43.1
2 KFCC10750::E1p(E190V) 41.2 42.1 41.8 41.7
3 KFCC10750::E1p(L438P) 40.9 41.3 41.5 41.2
4 KFCC10750:E1p(Q195H) 41.6 42.3 41.8 41.9
5 KFCC10750::E1p(P199S) 42 42.4 41.9 42.1
6 KFCC10750 :: E1p (K435A) 41.8 42.1 42.2 42.0
7 KFCC10750::E1p(Q432A) 40.2 41.1 41.2 40.8
8 KFCC10750::E1p(Y418H) 44.5 44.9 44.8 44.7
9 KFCC10750::E1p(Y201A) 40.9 41 41.1 41.0
10 KFCC10750 :: E1p (N428A) 44.5 44.1 45.8 44.8

[184]
As a result, the introduction of new mutations Note 10 species was confirmed to be increased up to 17% L- lysine-producing ability compared to the parent strain.
[185]
[186]
Example 11: KCCM10770P derived E1p mutagenic strains produced and L- lysine -producing ability compared
[187]
[188]
Corynebacterium-in other strains belonging to Tommy glutamicum in order to confirm the effect of the new 10 kinds of variations using the same procedure as in Example 7 L- lysine-producing strain Corynebacterium glutamicum KCCM10770P (Korea register making the Patent No. 10-0924065), the strain E1p mutation is introduced into and KCCM10770P :: E1p (Q432E), KCCM10770P :: E1p (E190V), KCCM10770P :: E1p (L438P), KCCM10770P :: E1p (Q195H), KCCM10770P :: E1p (P199S), KCCM10770P :: E1p (K435A), was named KCCM10770P :: E1p (Q432A), KCCM10770P :: E1p (Y418H), KCCM10770P :: E1p (Y201A), KCCM10770P :: E1p (N428A). Of Example 4 and cultured in the same way, it was analyzed for the concentration of L- lysine therefrom Table 8.
[189]
Table 8 [Table 8]
KCCM10770P strains derived E1p mutagenic week L- lysine production capacity
Strain L- lysine (g / L)
Placed one Placed second Placed third Average
Controls KCCM10770P 48.1 47.5 47.8 47.8
1 KCCM10770P::E1p(Q432E) 53.3 53.4 54.1 53.6
2 KCCM10770P :: e1p (E190V) 51.2 52.1 52.8 52.0
3 KCCM10770P::E1p(L438P) 51.5 51.3 50.7 51.2
4 KCCM10770P::E1p(Q195H) 51.9 52.1 52 52.0
5 KCCM10770P :: E1p (P199S) 51.3 52.1 52.7 52.0
6 KCCM10770P :: E1p (K435A) 52.4 52.6 51.4 52.1
7 KCCM10770P :: E1p (Q432A) 50.2 51.2 50 50.5
8 KCCM10770P :: E1p (Y418H) 55.8 54.5 54.1 54.8
9 KCCM10770P :: E1p (Y201A) 49.9 50.3 50.7 50.3
10 KCCM10770P :: E1p (N428A) 56.1 55.7 55.9 55.9

[190]
As a result, the introduction of new mutations Note 10 species was confirmed to be increased up to 17% L- lysine-producing ability compared to the parent strain.
[191]
[192]
Example 12: CJ3P derived E1p mutagenic strains produced and L- lysine-producing ability compared
[193]
[194]
Corynebacterium glutamicum is effective in other strains also L- lysine-producing strain in the same manner as in Example 7 to determine belonging to Corynebacterium glutamicum CJ3P (Binder et al., Genome Biology 2012 , 13: R40) E1p mutation produced a strain introduced, and CJ3P :: E1p (Q432E) to, CJ3P :: E1p (E190V), CJ3P :: E1p (L438P), CJ3P :: E1p (Q195H), CJ3P: : E1p (P199S), CJ3P :: E1p (K435A), was named CJ3P :: E1p (Q432A), CJ3P :: E1p (Y418H), CJ3P :: E1p (Y201A), CJ3P :: E1p (N428A). Carried out by culturing in the same manner as in Example 4, was analyzed for the concentration of L- lysine therefrom Table 9.
[195]
Table 9 [Table 9]
CJ3P strains derived E1p mutagenic week L- lysine production levels
Strain L- lysine (g / L)
Placed one Placed second Placed third Average
Controls Kg3P 8.2 8.3 8 8.2
1 CJ3P :: E1p (Q432E) 9.5 9.3 9.4 9.4
2 CJ3P::E1p(E190V) 8.8 9 8.7 8.8
3 CJ3P :: E1p (L438P) 9.1 8.7 8.9 8.9
4 CJ3P :: E1p (Q195H) 9.2 8.9 9 9.0
5 CJ3P::E1p(P199S) 9.3 9 9.1 9.1
6 CJ3P :: E1p (K435A) 9.1 9 8.9 9.0
7 CJ3P :: E1p (Q432A) 8.7 8.9 8.8 8.8
8 Kg3P :: Alp (Y4l8H) 9.6 9.6 9.5 9.6
9 CJ3P::E1p(Y201A) 8.7 8.7 8.9 8.8
10 CJ3P :: E1p (N428A) 9.7 9.8 9.8 9.8

[196]
As a result, 10 kinds of the main introducing new mutations were identified that increased up to 19.5% L- lysine-producing ability compared to the parent strain.
[197]
The result is a new secure the 10 species of E1p mutation (E1p (Q432E), E1p (E190V), E1p (L438P), E1p (Q195H), E1p (P199S), E1p (K435A), E1p (Q432A), E1p (Y418H ), E1p (Y201A), E1p (N428A)) are respectively mean a good to excellent effect L- lysine-producing ability increases.
[198]
[199]
Example 13: KCCM11201P derived E1p mutagenic strains produced and L- valine -producing ability compared
[200]
[201]
Corynebacterium glutamicum other amino acids produced in order to confirm the effect of the selected 10 strains E1p variation, the L- valine-producing strain in the same manner as in Example 7, belonging to Corynebacterium glutamicum KCCM11201P ( Korea Patent registration No. 10-1117022 produce a call), a strain E1p mutation is introduced into and KCCM11201P :: E1p (Q432E), KCCM11201P :: E1p (E190V), KCCM11201P :: E1p (L438P), KCCM11201P :: E1p (Q195H) , named KCCM11201P :: E1p (P199S), KCCM11201P :: E1p (K435A), KCCM11201P :: E1p (Q432A), KCCM11201P :: E1p (Y418H), KCCM11201P :: E1p (Y201A), KCCM11201P :: E1p (N428A) It was.
[202]
To 250 ㎖ corner containing a specified L- valine production medium 25㎖ in order to evaluate the production strain inoculated with each strain in bapeul flask, and at 30 ℃ for 72 hours, then this was cultured with shaking in 200rpm. By HPLC analysis, the concentration of L- valine. [Table 10].
[203]
[204]

[205]
Glucose 50g, (NH 4 ) 2 SO 4 20g, corn steep liquor (Corn Steep liquid) 20g, KH 2 PO 4 1g, MgSO 4 · 7H 2 O 0.5g, biotin 200㎍, CaCO 3 30g (per liter of distilled water) .
[206]
Table 10 [Table 10]
KCCM11201P derived E1p mutagenic week L- valine production levels
Strain L- Valine (g / L)
Placed one Placed second Average
Controls KCCM11201P 2.8 2.8 2.8
1 KCCM11201P::E1p(Q432E) 3.1 3.0 3.1
2 KCCM11201P::E1p(E190V) 3.3 3.2 3.3
3 KCCM11201P::E1p(L438P) 3.2 3.2 3.2
4 KCCM11201P::E1p(Q195H) 3.0 3.1 3.1
5 KCCM11201P::E1p(P199S) 3.1 3.3 3.2
6 KCCM11201P::E1p(K435A) 3.1 3.1 3.1
7 KCCM11201P::E1p(Q432A) 3.2 3.2 3.2
8 KCCM11201P::E1p(Y418H) 3.3 3.4 3.4
9 KCCM11201P::E1p(Y201A) 3.0 3.0 3.0
10 KCCM11201P::E1p(N428A) 3.4 3.4 3.4

[207]
As a result, the introduction of new mutations Note 10 species was confirmed to be increased up to 21% L- valine production ability compared to the parent strain.
[208]
[209]
Example 14: Preparation of the wild type-derived strains produced E1p mutagenic and L- valine-producing ability compared
[210]
[211]
Example In the embodiment 13 of the 10 kinds of mutation E1p screened 7 for variations four types of showing a high L- valine yield increase effect, in order to reconfirm the Corey-valine-producing ability in the same manner as in Example 7 Four tumefaciens Solarium was produced in a strain E1p mutation is introduced into the glutamicum ATCC13032 and named ATCC13032 :: E1p (E190V), ATCC13032 :: E1p (L438P), ATCC13032 :: E1p (Y418H), ATCC13032 :: E1p (N428A) .
[212]
After transfection the L- valine biosynthesis overexpression vector-pECCG117 DvalS (Republic of Korea Patent Publication No. 10-2014-0111421 call) to each of the strains to determine the L- valine-producing ability of the strain as electroporation kanamycin (kanamycin ) obtain a transformant strain in 25mg / L containing a selection medium, and ATCC13032 :: E1p (E190V) _DvalS, ATCC13032 :: E1p (L438P) _DvalS, ATCC13032 :: E1p (Y418H) _DvalS, ATCC13032 :: E1p (N428A) It was named _DvalS.
[213]
By culturing in the same manner as the above strain as in Example 13, was analyzed, the concentration of L- valine therefrom (Table 11).
[214]
Table 11 [Table 11]
Derived from wild-type E1p mutagenic week L- valine production levels
Strain L- Valine (g / l)
Placed one Placed second Average
ATCC13032 0.1 0.1 0.1
Controls ATCC13032_DvalS 1.3 1.2 1.3
1 ATCC13032::E1p(E190V) 0.1 0.1 0.1
2 ATCC13032::E1p(E190V)_DvalS 1.5 1.7 1.6
3 ATCC13032 :: E1p (L438P) 0.1 0.1 0.1
4 ATCC13032::E1p(L438P)_DvalS 1.4 1.4 1.4
5 ATCC13032::E1p(Y418H) 0.1 0.1 0.1
6 ATCC13032::E1p(Y418H)_DvalS 1.8 1.7 1.8
7 ATCC13032 :: E1p (N428A) 0.1 0.1 0.1
8 ATCC13032::E1p(N428A)_DvalS 1.9 1.7 1.8

[215]
As a result, the introduction of new mutations Note 4 species was confirmed to be increased up to 38% L- valine production ability compared to the control group.
[216]
[217]
From the above description, those skilled in the art will appreciate that may be embodied in other specific forms of the present invention without changing the technical spirit or essential characteristics. The embodiments described above In this regard, the examples should be understood as illustrative and not be limiting in all aspects. The scope of the invention should be construed to be hitting the meaning and scope of the claims to be described later, rather than the foregoing description, all such modifications as derived from the equivalent concept within the scope of the invention.

Claims
[Claim 1]
In SEQ ID NO: 1 to 190 times, 205 times or 415 times to 440 times the amino acid region having one or more amino acid mutations in the amino acid region, pyruvate dehydrogenase mutant.
[Claim 2]
The method of claim 1, wherein pyruvate dehydrogenase mutants that amino acid mutations at 190 times to 205 times the amino acid portion of the SEQ ID NO: 1 is selected from the group consisting of one times 190, 195 times, and # 201 199 amino acids .
[Claim 3]
The method of claim 1, wherein the amino acid mutation in the SEQ ID No. 1 190 times to 205 amino acid region, the substitution of the valine at 190 times glutamate (E190V), substitution of a histidine in 195 times glutamine (Q195H), 199 times in the substituted (P199S) and 201 times in the tyrosine-proline with serine is selected from the group consisting of substituted (Y201A) of the alanine, pyruvate dehydrogenase mutant.
[Claim 4]
The method of claim 1, wherein the amino acid mutation in the SEQ ID NO amino acid region in the first 415 times to 440 times is 418 times, 428 times, 432 times, 435 times, and the 438 number is selected from the group consisting of amino acids, pyruvate dehydrogenase dehydratase mutants.
[Claim 5]
The method of claim 1 wherein the substitution in substituted (Y418H), asparagine 428 times in the amino acid mutation in the SEQ ID NO: 1 of 415 times to 440 times the amino acid portion is 418 times tyrosine to histidine to alanine (N428A), 432 times glutamine in is selected from the group consisting of substituted (Q432E), substituted (Q432A), substituted (L438P) to a proline in 435 times lysine in substitution (K435A) and 438 times leucine to alanine to alanine at the 432 times of glutamine to glutamic acid a, pyruvate dehydrogenase mutant.
[Claim 6]
The method of claim 1, wherein the pyruvate dehydrogenase is a variant amino acid sequence of the kinase, pyruvate dehydrogenase mutant amino acid sequence that is selected from the group consisting of SEQ ID NO: 14 to 33.
[Claim 7]
Any one of claims 1 to 6, a polynucleotide encoding any one of the pyruvate dehydrogenase mutant of the claims.
[Claim 8]
Claim 1 to claim any one of the pyruvate dehydrogenase of 6 genus Corynebacterium microorganism having the containing dehydratase variant, L- amino acid-producing ability.
[Claim 9]
The method of claim 8, wherein said microorganism of the genus Corynebacterium is Corynebacterium glutamicum of, the genus Corynebacterium microorganism having the L- amino acid-producing ability.
[Claim 10]
(A) culturing a microorganism of the genus Corynebacterium with claim 8 L- amino acid-producing ability in a culture medium phase to produce L- amino acid; And (b) a method of producing, L- amino acid comprising the step of recovering the L- amino acid from the medium or the microorganism.