RU2822660C1 - Microorganisms producing l-valine, and method of producing l-valine using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a microorganism producing L-valine, having enhanced activity of dihydroxy acid dehydratase compared to endogenous activity. Also, the microorganism has any one or more of the combinations selected from reduced activity of transaminase C compared to endogenous activity, reduced activity of pyruvate dehydrogenase compared to endogenous activity and reduced activity of citrate synthase compared to endogenous activity. Also disclosed is a method of producing L-valine using said microorganism.

EFFECT: invention enables to obtain L-valine with high output.

15 cl, 11 tbl, 2 ex

Description

Field of invention

The present invention relates to a microorganism that produces L-valine and a method for producing L-valine using this microorganism.

Prior Art

L-Valine, which is a branched chain amino acid, is biosynthesized in microorganisms starting with pyruvic acid through acetolactic acid, dihydroxyisovaleric acid and ketoisovaleric acid. These intermediate metabolites are formed in a reaction catalyzed by acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase and transaminase B. However, these enzymes are also involved in the biosynthesis of L-isoleucine, starting with ketobutyric acid and pyruvic acid, and L-leucine is also biosynthesized from the intermediate metabolite, which is ketoisovaleric acid, through 2-isopropyl malic acid, 3-isopropyl malic acid and ketoisocaproic acid. Thus, since the enzymes used in the biosynthetic pathways of branched chain amino acids, i.e. L-valine, L-isoleucine and L-leucine are identical; it is known that industrial production of one type of branched chain amino acid through fermentation is difficult. In addition, feedback inhibition occurs on the part of the final product L-valine or its derivatives, which makes industrial mass production of L-valine difficult.

However, to date, studies have been conducted on the method of producing valine by feedback inhibition (US Patent No. 10457919), but there have been no studies on increasing the ability to produce valine by combining enzymes with increased or decreased activity, in accordance with the description of the present invention .

Description of the invention

Technical problem

Accordingly, the inventors of the present invention have conducted continuous research on an effective method for producing L-valine, and as a result, it has been confirmed that a microorganism having enhanced dihydroxy acid dehydratase activity; decreased transaminase C activity; weakened activity of pyruvate dehydrogenase; decreased citrate synthase activity; or a combination thereof, has superior ability to produce L-valine compared to the wild type microorganism, thus completing the present invention.

Technical solution

One of the objects of the present invention is to provide an L-valine producing microorganism having enhanced dihydroxy acid dehydratase activity; and any one or more than one of the combinations selected from (1)-(3) below:

(1) decreased transaminase C activity;

(2) weakened pyruvate dehydrogenase activity;

(3) reduced citrate synthase activity.

Another object of the present invention is to provide a method for producing L-valine, comprising growing a microorganism.

Favorable Actions

By growing the L-valine-producing microorganism according to the description of the present invention, L-valine can be produced in high yield. Accordingly, the effects of convenience of obtaining and reducing cost of obtaining from an industrial aspect can be expected.

Detailed Description of Preferred Embodiments

Next, a description of the present invention will be described in detail.

At the same time, each description and embodiment disclosed herein may be applied in different descriptions and embodiments herein with respect to the general properties. That is, all combinations of the various components disclosed herein are included within the scope of the present invention. Moreover, the scope of the description of the present invention should not be limited to the specific description below.

Additionally, those skilled in the art may be able to understand or confirm, using purely conventional experimental means, many equivalents to specific aspects of the invention described herein. Moreover, it is also intended that these equivalents be included in the description of the present invention.

In one aspect of the present invention there is provided a microorganism that produces L-valine having enhanced dihydroxy acid dehydratase activity; and any one or more than one of the combinations selected from (1)-(3) below:

(1) decreased transaminase C activity;

(2) weakened pyruvate dehydrogenase activity; And

(3) reduced citrate synthase activity.

In one embodiment, the valine-producing microorganism may be a microorganism having increased dihydroxy acid dehydratase activity and decreased transaminase C activity.

In yet another embodiment, the valine-producing microorganism may be a microorganism having enhanced dihydroxy acid dehydratase activity and reduced pyruvate dehydrogenase activity.

In yet another embodiment, the valine-producing microorganism may be a microorganism having enhanced dihydroxy acid dehydratase activity and reduced citrate synthase activity.

In yet another embodiment, the valine-producing microorganism may be a microorganism having enhanced dihydroxy acid dehydratase activity and reduced citrate synthase activity, and further having reduced transaminase C activity or reduced pyruvate dehydrogenase activity.

As used herein, the term "dihydroxy acid dehydratase" is an enzyme involved in the synthesis of the valine precursor ketoisovalerate in the L-valine biosynthetic pathway, starting from pyruvate through acetolactate, dihydroxyisovalerate to ketoisovalerate. In the present invention, it is possible to promote the production of L-valine by enhancing the activity of dihydroxy acid dehydratase, and thus increasing the synthesis of ketoisovalerate.

As used herein, the term "transaminase C" is an enzyme involved in the pathway for the synthesis of L-alanine from pyruvate.

As used herein, the term "pyruvate dehydrogenase" is an enzyme involved in the synthesis of acetyl-coA from pyruvic acid.

As used herein, the term “citrate synthase” is an enzyme that synthesizes citrate from acetyl-coA.

As used herein, the term "amplification" means that the activity of the protein is increased relative to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, up-regulation, overexpression, increase, and the like. In particular, activation, amplification, up-regulation, overexpression and amplification can include both cases in which activity is demonstrated that was not initially detected, or activity is enhanced relative to endogenous activity or activity before modification. "Endogenous activity" refers to the activity of a particular polypeptide initially present in the parent strain before transformation, or in an unmodified microorganism when the property is altered by genetic modification caused by natural or artificial factors, and can be used interchangeably with "activity before modification". “Amplification,” “upregulation,” “overexpression,” or “increase” in the activity of a polypeptide relative to its endogenous activity means that the activity and/or concentration (level of expression) of the polypeptide is increased relative to the particular polypeptide originally present in the parent strain before transformation or in an unmodified microorganism.

Enhancement can be achieved by introducing a foreign polypeptide or by enhancing the activity and/or increasing the concentration (expression level) of an endogenous polypeptide. Whether the dihydroxy acid dehydratase activity is enhanced or not can be confirmed by increasing the level of the polypeptide, the level of expression, or the amount of product excreted from the polypeptide.

Enhancing the activity of the dihydroxy acid dehydratase regulator can be achieved by using various methods well known in the art, and these methods are not limited provided that they enhance the activity of the target polypeptide equal to the activity of the microorganism before modification. In particular, genetic engineering and/or protein engineering may be used, well known to those skilled in the art, which are methods conventional in molecular biology, but the method is not limited to them (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al.

In particular, enhancing the activity of dihydroxy acid dehydratase in accordance with the description of the present invention can be achieved by:

1) increasing the number of copies of the polynucleotide encoding the polypeptide in cells;

2) replacing the sequence that regulates the expression of the gene encoding the polypeptide in the chromosome with a sequence that has stronger activity;

3) modification of the nucleotide sequence encoding the start codon or 5'-UTR of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of the polypeptide, such that the activity of the polypeptide is enhanced;

5) modifying a polynucleotide sequence encoding a polypeptide such that the activity of the polypeptide is enhanced (eg, by modifying the polynucleotide sequence of a polypeptide gene to encode a polypeptide modified so as to enhance the activity of the polypeptide);

6) introduction of a foreign polypeptide demonstrating the activity of the polypeptide, or a foreign polynucleotide encoding it;

7) optimization of the codon of the polynucleotide encoding the polypeptide;

8) analysis of the tertiary structure of the polypeptide and, thus, selection and modification of the exposed site or its chemical modification; or

9) combinations of two or more than two selected from, but not limited to, (1)-(8) above.

More specifically,

1) A method of increasing the copy number of a polynucleotide in a cell encoding a polypeptide can be achieved by introducing a vector that is operably linked to the polynucleotide encoding the polypeptide and is capable of replicating and functioning independently of a host cell in a host cell. Alternatively, this method can be achieved by introducing one copy or two copies of polynucleotides encoding the polypeptide into the host cell chromosome. Introduction into a chromosome can be accomplished by introducing, but not limited to, a vector capable of inserting a polynucleotide into the chromosome of a host cell into the host cell. The vector is as described above.

2) A method of replacing the expression control region (or expression control sequence) of a gene encoding a polypeptide in a chromosome with a sequence having strong activity can be achieved, for example, by inducing a modification in the sequence by deletion, insertion, non-conservative or conservative substitution, or any combination thereof, to further enhance the activity of the expression regulatory region, or by replacing the sequence with a sequence having stronger activity. The expression regulating region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription or translation termination, and the like. In one example, the method may include, but is not limited to, replacing the original promoter with a strong promoter.

Examples of a known strong promoter may include the CJ1-CJ7 promoters (US Patent No. US 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 promoter (sm3) (US Patent No. US 10584338 B2), promoter 02 (US Patent No. US 10273491 B2), tkt promoter and uscA promoter, but are not limited to.

3) A method for modifying a nucleotide sequence encoding a start codon or the 5'-UTR of a gene transcript encoding a polypeptide can be achieved, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a higher level of expression of the polypeptide than the endogenous start codon, but is not limited to this.

4) and 5) Methods of modifying an amino acid sequence or a polynucleotide sequence can be achieved by inducing a modification in the sequence by deletion, insertion, non-conservative or conservative substitution of an amino acid sequence of a polypeptide or a polynucleotide sequence encoding a polypeptide, or any combination thereof to enhance the activity of the polypeptide or by replacing the sequence with, but not limited to, an amino acid sequence or a polynucleotide sequence modified so as to enhance activity. The vector used herein may further include a selection marker to confirm chromosomal integration. The selection marker is as described above.

6) The method of introducing a foreign polynucleotide exhibiting the activity of a polypeptide can be achieved by introducing into a cell a foreign polynucleotide encoding a polypeptide that exhibits activity identical/similar to that of the polypeptide. The foreign polynucleotide may be used without limitation regardless of its origin or sequence, provided that it exhibits activity identical/similar to that of the polypeptide. Administration can be accomplished by those skilled in the art by appropriate selection of a transformation method known in the art, and expression of the introduced polynucleotide in a host cell causes production of the polypeptide, thereby increasing its activity.

7) A method for optimizing the codon of a polynucleotide encoding a polypeptide can be achieved by optimizing the codon of an endogenous polynucleotide to enhance transcription or translation in a host cell, or by optimizing its codons so as to enable optimized transcription and translation of a foreign polynucleotide to be achieved in a cell. owner.

In addition, 8) a method for analyzing the tertiary structure of a polypeptide and thus selecting and modifying the exposed site or chemically modifying it can be achieved, for example, by comparing the sequence information of the analyzed polypeptide with a database that stores sequence information of known proteins, to identify candidate template proteins according to the degree of sequence similarity, and thus confirm the structure based on the information, thereby selecting and transforming or modifying the exposed site that is intended to be modified or chemically modified.

A vector as described herein is a DNA molecule used as an intermediary for the artificial transfer of foreign genetic material into other cells, and may include a DNA construct containing the nucleotide sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression regulatory region (expression control sequence) such that it is capable of expressing the target polypeptide in a suitable host cell. The expression regulatory region may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating transcription termination and translation. Once transformed into a suitable host cell, the vector can replicate or function independently of the host genome or can be integrated into its genome.

The vector used in the description of the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A, etc. can be used as a phage vector or cosmid vector, and pDZ-based vectors can be used as a plasmid vector , pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. In particular, the vectors pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC and the like can be used.

In one example, a polynucleotide encoding a desired polypeptide can be inserted into a chromosome using an intracellular chromosome insertion vector. The insertion of a polynucleotide into a chromosome can be carried out without limitation using any method known in the art, for example, using homologous recombination. The vector may further include a selectable marker to confirm chromosomal integration. The selectable marker is intended to select cells transformed with the vector, that is, to confirm that the desired nucleic acid molecule has been inserted, and markers can be used to provide selective phenotypes such as drug resistance, nutrient requirements, resistance to cytotoxic agents and expression of surface polypeptides. When treated with a selection agent, only cells expressing the selectable markers can survive or exhibit other phenotypes in the environment, and thus transformed cells can be selected.

As used herein, the term “transformation” refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may be present in a form integrated into the chromosome of the host cell, or in a form located outside the chromosome, provided that the polypeptide is expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it does not matter whether the transformed polynucleotide is integrated into the chromosome of the host cell or located extrachromosomally, and both cases can be included. In addition, the polynucleotide may include DNA and/or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, provided that it can be introduced into and expressed in a host cell. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct that includes all elements necessary for its autonomous expression. Typically, the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into a host cell in its original form and is operably linked to, but is not limited to, a sequence required for expression in the host cell.

Additionally, as used herein, the term “operably linked” means that the gene sequence is operably linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein as described herein.

Such an increase in protein activity may mean that the activity of the corresponding protein is enhanced, which it does not initially possess, or its activity or concentration is generally increased by 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, and a maximum of 1000% or 2000% or more, based on, but not limited to, the activity or concentration of the wild type protein or the original microbial strain.

In particular, increased dihydroxy acid dehydratase activity may mean that the dihydroxy acid dehydratase activity of the microorganism is enhanced relative to that of a wild-type microorganism, a pre-modified microorganism, or a microorganism having an unmodified protein, thereby increasing the synthesis of ketoisovalerate, which is a precursor to L- valine, from dihydroxyisovalerate, resulting in increased production of L-valine.

As used herein, the term "reduction" is a comprehensive concept that includes the event in which the activity of a protein present in a microorganism in its natural state or before modification is weakened or eliminated (removed), i.e. compared to endogenous activity or one copy of a gene encoding a protein in a cell, and can mean that the activity is 0% or more to 100% or less.

Such "downregulation" of a protein means that the activity of the protein itself is eliminated or does less than its original function, but is not specifically limited to this. That is, activity reduction specifically includes both “elimination of activity” and “attenuation of activity.”

“Elimination of activity” may mean that the enzyme or protein is not expressed at all compared to a natural wild-type strain, which is the parent strain or a strain having an unmodified protein, or its activity is not detectable even when the enzyme or protein is expressed.

In the present invention, elimination of activity can be achieved by using various methods well known in the art. Examples of these methods include: 1) elimination of a fragment or entire gene encoding a protein; 2) modification of the sequence of the gene encoding the protein, such that the activity of the protein is eliminated or weakened; 3) introduction of an antisense oligonucleotide (for example, antisense RNA), which complementarily binds to the transcript of the gene encoding the protein; 4) adding a sequence complementary to the Shine-Dalgarno (SD) sequence to the upstream end of the SD sequence of the protein-coding gene to form a secondary structure, thereby inhibiting ribosomal attachment; 5) reverse transcription engineering (RTE), in which a promoter, which is to be reversibly transcribed, is added at the 3' end of the open reading frame (ORF) of the protein-coding gene sequence; or a combination thereof, but is not specifically limited thereto.

Additionally, "attenuation" may mean that an effect less than the original function is achieved and can be achieved by methods including: eliminating a portion of a protein-encoding gene on a chromosome; replacement of a gene encoding a protein on a chromosome with a mutant gene, such that the activity of the protein is reduced; introducing a mutation into the expression-regulating sequence of a gene encoding a protein on the chromosome; replacement of the expression-regulating sequence of a gene encoding a protein with a sequence having weak activity (for example, replacement of a gene promoter with a promoter that is weaker than the endogenous promoter), etc., but not limited to them, and known methods of weakening can be used without limitation activity.

Such a decrease in protein activity may mean that the activity of the corresponding protein is eliminated or its activity or concentration is generally reduced by 0%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90%, 95%, 98%, 99% relative to, but not limited to, the activity or concentration in the wild-type protein or parent strain of the microbe.

In particular, the transaminase C activity of a microorganism may be reduced compared to the activity of a natural wild-type strain, a parental strain before mutation, or a strain having an unmodified protein. In one embodiment, the microorganism may lack transaminase C protein activity due to deletion of the transaminase C gene, or may have reduced activity because the sequence encoding the start codon of the transaminase C gene is modified to GTG, thereby reducing the expression of transaminase C protein.

Additionally, in particular, the activity of pyruvate dehydrogenase in a microorganism may be reduced compared to the activity of a natural wild-type strain, a parental strain before mutation, or a strain having an unmodified protein. In one embodiment, the microorganism may lack pyruvate dehydrogenase protein activity due to deletion of the pyruvate dehydrogenase gene, or may have reduced activity because the sequence encoding the start codon of the pyruvate dehydrogenase gene is modified to GTG, thereby reducing pyruvate dehydrogenase protein expression. In yet another embodiment, because the pyruvate dehydrogenase gene sequence is mutated in a microorganism, the microorganism may express a pyruvate dehydrogenase mutant having reduced activity compared to that of the wild-type protein, in which the amino acid corresponding to ^{the 432nd} or ^435th position from the N-terminus of the amino acid sequence SEQ ID NO : 3 replaced with another amino acid. In particular, a mutant of pyruvate dehydrogenase having reduced activity compared to that of the wild-type protein may include, but is not limited to, SEQ ID NO: 5 or SEQ ID NO: 6.

In addition, in particular, the activity of citrate synthase in a microorganism may be reduced compared to the activity of a natural wild-type strain, a parental strain before mutation, or a strain having an unmodified protein. In one embodiment, the microorganism may lack citrate synthase protein activity due to deletion of the citrate synthase gene, or may have reduced activity because the sequence encoding the start codon of the citrate synthase gene is modified to GTG, thereby reducing expression of the citrate synthase protein. In yet another embodiment, because the citrate synthase gene sequence is mutated in a microorganism, the microorganism may express a mutant citrate synthase having reduced activity compared to that of the wild-type protein, in which the amino acid corresponding to ^{position 241} or 312 from the N-terminus of the amino acid sequence SEQ ID NO : 4 replaced with another amino acid. In particular, a citrate synthase mutant having reduced activity compared to that of the wild-type protein may include, but is not limited to, SEQ ID NO: 7 or SEQ ID NO: 8.

The expression "replacement with another amino acid" is not limited, provided that the amino acid before the substitution is replaced with another amino acid. In particular, it may be replaced by any amino acid selected from lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine , arginine and glutamine.

More specifically, the pyruvate dehydrogenase having reduced activity may be a pyruvate dehydrogenase in which the amino acid corresponding to ^{the 432nd} or ^435th position from the N-terminus of the amino acid sequence SEQ ID NO: 3 is replaced by a non-polar amino acid.

Additionally, more specifically, the citrate synthase having reduced activity may be a citrate synthase in which the amino acid corresponding to position ²⁴¹ or ³¹² from the N-terminus of the amino acid sequence SEQ ID NO: 4 is replaced by a polar or non-polar amino acid.

As used herein, the term “corresponding” refers to an amino acid residue at a position specified in a protein or peptide, or an amino acid residue that is similar, identical or homologous to a residue specified in a protein or peptide. As used herein, “corresponding region” generally refers to a similar or corresponding position in a related protein or reference protein.

In describing the present invention, specific numbering of amino acid residue positions in the protein used herein may be used. For example, it is possible to renumber the positions of amino acid residues in a protein as described herein to corresponding positions by aligning the sequence of the protein as described herein with the desired protein being compared.

As used herein, the term “homology” or “identity” refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. Often the terms homology and identity can be used interchangeably with each other.

The homology or sequence identity of conserved polynucleotides or polypeptides can be determined by a standard alignment algorithm and can be coupled with a sequence gap penalty set in the program being used. Substantially homologous or identical sequences can generally hybridize to entire sequences or portions of sequences under moderately stringent or very stringent conditions. It is understood that hybridization with polynucleotides containing a common codon or degenerate codons in the polynucleotides being hybridized is also included.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can, for example, be determined using a known computer algorithm such as the FASTA program (Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444) using default parameters. Alternatively, they can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is carried out using the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite , Rice et al., 2000, Trends Genet. 16:276 277) (preferably version 5.0.0 or later) (GCG software package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)) , BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or National Center for Biotechnology Information (NCBI) ClustalW.

Homology, similarity or identity between polynucleotides or polypeptides can be determined, for example, by comparing sequence information using, for example, a GAP computer program such as Needleman et al., (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In general, the GAP program determines homology, similarity, or identity by dividing the number of aligned identical characters (i.e., nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing a value of 1 if identical and 0 if not identical) and a weighted comparison matrix according to Gribskov, et al. (1986), Nucl. Acids Res. 14:6745, as described in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or EDNAFULL substitution matrix (EMBOSS NCBI NUC4.4 version); (2) a penalty of 3.0 for each omission and an additional penalty of 0.10 for each character in each omission (or a penalty of 10 and penalty for extending skips 0.5) and (3) no penalty for end skips.

As used herein, the term “variant” refers to a protein having one or more amino acids that differ from the described sequence by conservative substitutions and/or modifications such that the functions or properties of the protein are retained. Variants differ from sequences identified by substitution, deletion, or insertion of multiple amino acids. Such variants can generally be identified by modifying one or more of the above amino acid sequences of the protein and by evaluating the properties of the modified protein. That is, the ability of the variants may be enhanced, unchanged, or reduced compared to the native protein. Other variants may include variants in which the fragment is removed from the N- and/or C-terminus of the mature protein. The term “variant” may be used interchangeably with terms such as modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, and the like, provided that the terms are used to denote variation, but the terms are not limited to them. For the purpose of describing the present invention, a variant may be, but is not limited to, variants in which the activity of the protein is reduced or weakened compared to the activity of the naturally occurring wild-type or unmodified protein.

As used herein, the term “conservative substitution” refers to the replacement of an amino acid with another amino acid having similar structural and/or chemical properties. Such an amino acid substitution can generally occur on the basis of similarity in polarity, charge (basic, acidic), solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residue.

In addition, the variant may also include deletion or addition of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus involved in co-translational or post-translational protein transfer. In addition, the polypeptide may also be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

As used herein, the term “L-valine-producing microorganism” refers to a microorganism capable of producing an excess amount of L-valine from a carbon source in the medium compared to a wild-type microorganism or an unmodified microorganism. In addition, the L-valine-producing microorganism may be a recombinant microorganism. In particular, the microorganism is not particularly limited to its type as long as it can produce L-valine, and may be a microorganism of the Enterobacter genus, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus and Brevibacterium genus. More specifically, it may be a microorganism of the genus Corynebacterium or the genus Escherichia.

More specifically, the microorganism of the genus Escherichia may be Escherichia coli, and the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum, and any microorganism of the genus Corynebacterium or the genus Escherichia capable of increasing the production of L-valine into which enhanced dihydroxy acid dehydratase activity is introduced can be included; and any one or more combinations selected from reduced transaminase C activity; decreased pyruvate dehydrogenase activity; or decreased citrate synthase activity.

A parent strain of an L-valine-producing microorganism that has been modified to have enhanced dihydroxy acid dehydratase activity; and any one or more than one of the combinations selected from (1)-(3) below:

(1) decreased transaminase C activity;

(2) weakened pyruvate dehydrogenase activity; And

(3) reduced citrate synthase activity, is not particularly limited provided that it is an L-valine producing microorganism.

The L-valine producing microorganism may itself be a naturally occurring microorganism or a microorganism having an improved ability to produce L-valine by insertion of a gene associated with an extrinsic L-valine production mechanism or by enhancing or decreasing (weakening or suppressing) the activity of an endogenous gene.

In one embodiment, the microorganism may be an L-valine-producing microorganism having increased dihydroxy acid dehydratase activity compared to the wild-type strain or pre-mutation parent strain, and reduced transaminase C activity compared to the wild-type or pre-mutation parent strain. In particular, dihydroxy acid dehydratase may be encoded by the ilvD gene, and transaminase C may be encoded by the avtA gene. The genes may be derived from, but are not limited to, Corynebacterium glutamicum.

In yet another embodiment, the microorganism may be an L-valine-producing microorganism having enhanced dihydroxy acid dehydratase activity compared to the wild-type strain or pre-mutation parent strain, and reduced pyruvate dehydrogenase activity compared to the wild-type strain or pre-mutation parent strain. In particular, dihydroxy acid dehydratase may be encoded by the ilvD gene, and pyruvate dehydrogenase may be encoded by the aceE gene. The genes may be derived from, but are not limited to, Corynebacterium glutamicum.

In yet another embodiment, the microorganism may be an L-valine-producing microorganism having enhanced dihydroxy acid dehydratase activity compared to the wild-type strain or pre-mutation parent strain and reduced citrate synthase activity compared to the wild-type strain or pre-mutation parent strain. In particular, dihydroxy acid dehydratase may be encoded by the ilvD gene, and citrate synthase may be encoded by the gltA gene. The genes may be derived from, but are not limited to, Corynebacterium glutamicum.

In addition to the above microorganism, it may be an L-valine-producing microorganism in which transaminase C activity is reduced or pyruvate dehydrogenase activity is reduced compared with the wild-type strain or the parent strain before the mutation, or may be an L-valine-producing microorganism in which transaminase C activity is reduced and pyruvate dehydrogenase activity is reduced compared to the wild type strain or the parental strain before the mutation.

In yet another aspect of the present invention, there is provided a method for producing L-valine, comprising: cultivating an L-valine producing microorganism that has enhanced dihydroxy acid dehydratase activity; and any one or more than one of the combinations selected from (1)-(3) below:

(1) decreased transaminase C activity;

(2) weakened pyruvate dehydrogenase activity; And

(3) reduced citrate synthase activity.

"L-valine" as used herein may include not only L-valine itself, but also a salt thereof.

As used herein, the term "culture" means that the microorganism is grown under appropriately controlled environmental conditions. The cultivation process in accordance with the description of the present invention can be carried out in a suitable culture medium and growth conditions known in the art. Such a cultivation process can be easily adjusted for use by one skilled in the art according to the strain selected. In particular, the cultivation may be, but is not limited to, batch cultivation, continuous cultivation, and fed-culture cultivation.

As used herein, the term "medium" refers to a mixture of substances that contains as a main ingredient the nutrients required for the cultivation of a microorganism, and it provides nutrients and growth factors, as well as water, which are necessary for life and growth. In particular, the medium and other culture conditions used for culturing the microorganism in accordance with the description of the present invention may be any medium used for conventional cultivation of microorganisms without any particular limitation. However, the microorganism in accordance with the description of the present invention can be grown under aerobic conditions in a conventional environment containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and/or vitamin, with adjustments in temperature, pH, and the like. .

In the present invention, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose and the like; sugar alcohols such as mannitol, sorbitol and the like; organic acids such as pyruvic acid, lactic acid, citric acid and the like; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source may include natural organic nutrients such as starch hydrolyzate, molasses, raw molasses, rice bran, cassava, sugar cane pomace and liquid corn extract, and the like. In particular, carbohydrates such as glucose and sterilized pre-processed molasses (ie molasses converted to reduced sugars) can be used, and in addition, various other carbon sources in suitable amounts can be used without limitation. These carbon sources may be used alone or in combination of two or more of them, but are not limited to this.

The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate and the like; amino acids such as glutamic acid, methionine, glutamine and the like; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, liquid corn extract, casein hydrolysate, fish or its degradation products, defatted soybean meal or its degradation product, and the like. These nitrogen sources may be used alone or in combination of two or more of them, but are not limited to this.

The phosphorus source may include potassium dihydrogen phosphate, potassium hydrogen phosphate, or appropriate sodium containing salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and/or appropriate precursors may be included. These components or precursors may be added to the medium in batches or in a continuous manner, but these phosphorus sources are not limited to this.

In the present invention, the pH of the medium can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like, as appropriate during the cultivation of the microorganism. In addition, during culture, foam formation can be prevented by using an antifoaming agent such as a polyglycol fatty acid ester. In addition, oxygen or an oxygen-containing gas may be introduced into the medium to maintain aerobic conditions in the medium; or nitrogen, hydrogen or carbon dioxide may be introduced to maintain anaerobic and microaerobic conditions without forcing gas into it, but the gas is not limited to them.

The ambient temperature may be in the range of 20°C to 45°C, in particular, but not limited to 30°C to 37°C. Cultivation may be carried out until the desired amount of nutrients produced is obtained, particularly for, but not limited to, about 10 to 100 hours.

L-valine produced by cultivation in accordance with the description of the present invention can be released into the medium or remain in the cells.

The method for producing L-valine in accordance with the description of the present invention may further include the step of preparing a microorganism in accordance with the description of the present invention, the step of preparing a medium for culturing the strain, or a combination thereof (regardless of the sequence, in any sequence), for example, before the growth step.

The method for producing L-valine as described herein may further include the step of isolating L-valine from a culture medium or microorganism as described herein. An isolation step may further be included after the culture step.

In the isolation step, the desired L-valine can be collected using a microorganism culture method as described in the present invention, for example, using a suitable method known in the art according to a batch culture method, a continuous culture method or a fed-batch culture method. For example, methods such as centrifugation, filtration, treatment with a protein precipitating agent (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography can be used. , ion exchange chromatography and affinity chromatography, etc., HPLC (high performance liquid chromatography) or a combination thereof, and the desired L-valine can be isolated from the culture medium or microorganism using suitable methods known in the art.

In addition, the method for producing L-valine in accordance with the description of the present invention may further include a purification step, which can be carried out using a suitable method known in the art. In one example, when the method for producing L-valine in accordance with the description of the present invention includes both an isolation step and a purification step, then the isolation step and the purification step can be carried out continuously or intermittently regardless of the sequence, or can be carried out simultaneously, or may be combined into one step, but the method is not limited to this.

In the method according to the description of the present invention, a polynucleotide, vector, microorganism, L-valine, etc. are as described in other aspects above.

In yet another aspect of the present invention, there is provided a method of increasing the ability to produce L-valine, comprising introducing a modification into a microorganism that is characterized by enhanced dihydroxy acid dehydratase activity; and any one or more than one of the combinations selected from (1)-(3) below:

(1) decreased transaminase C activity;

(2) weakened pyruvate dehydrogenase activity; And

(3) reduced citrate synthase activity.

Another aspect of the present invention provides the use of a microorganism having enhanced dihydroxy acid dehydratase activity; and any one or more of the combinations selected from (1)-(3) below:

(1) decreased transaminase C activity;

(2) weakened pyruvate dehydrogenase activity; And

(3) reduced citrate synthase activity when producing L-valine.

Method for carrying out the invention

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are merely preferred examples given for illustrative purposes, and thus it is not intended that the scope of the description of the present invention be limited to these examples.

Example 1: Construction of a Valyl-derived strain and its evaluation

One type of mutation [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467] were introduced into wild-type Corynebacterium glutamicum strains ATCC14067 and ATCC13869 to create a strain with enhanced ability to produce L-valine.

Specifically, genomic DNA from wild-type Corynebacterium glutamicum strain ATCC 14067 was extracted using the G-spin Total DNA Mini Kit (intron, cat. no. 17045) according to the protocol provided in the kit. PCR (polymerase chain reaction) was carried out using a pair of primers in accordance with SEQ ID NO: 9 and 10 and a pair of primers in accordance with SEQ ID NO: 11 and 12, based on genomic DNA as a template to obtain a 537 bp gene fragment ., respectively. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes.

PCR with overlapping primers was carried out based on the two fragments obtained above as a template using the primer pair according to SEQ ID NO: 9 and 12 to obtain a PCR product of 1044 bp in length. (hereinafter referred to as “fragment 2 with introduced mutation”).

The thus obtained fragment 2 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct a vector containing fragment 2 with the introduced mutation. The vector for introducing the A42V mutation into the ilvN gene is named pDZ-ilvN(A42V).

pDZ-ilvN(A42V) was then transformed into each of the wild-type Corynebacterium glutamicum strains ATCC14067 and ATCC13869 to induce homologous recombination in the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains into which the vector was introduced into the chromosome were selected by recombination of homologous sequences in a medium containing 25 mg/l kanamycin.

Gene fragments were then amplified from the Corynebacterium glutamicum transformants selected above by PCR using the primer pair of SEQ ID NOs: 18 and 21, and the introduction of mutation was confirmed by sequencing analysis. The recombinant strains were named Corynebacterium glutamicum CJ7V and CJ8V, respectively.

The experiment to determine the fermentation titer was carried out on the basis of wild-type Corynebacterium glutamicum strains ATCC14067 and ATCC13869, and strains CJ7V and CJ8V constructed above. Each strain was subcultured in growth medium and then inoculated into a 250 ml corner flask containing 25 ml of production medium and grown with stirring at 30°C for 72 hours at 200 rpm. L-valine concentrations were then analyzed using HPLC (High Performance Liquid Chromatography) and the L-valine concentrations analyzed are presented in Table 2 below.

Nutrient medium (pH 7.2)

10 g glucose, 5 g meat broth, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (based on 1 liter of distilled water).

Production medium (pH 7.0)

100 g glucose, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g KH ₂ PO ₄ , 0.5 g MgSO ₄ ⋅ 7H ₂ O, 100 μg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide and 30 g CaCO ₃ (based on 1 liter of distilled water).

In accordance with the above results, it was confirmed that the ability to produce L-valine was increased in strains CJ7V and CJ8V into which the ilvN(A42V) gene mutation was introduced, compared with wild-type Corynebacterium glutamicum strains ATCC 14067 and ATCC 13,869.

Example 2: Construction of strains with high valine-producing ability and their evaluation

Example 2-1. Construction of strains with enhanced valine biosynthesis gene ilvD and their evaluation

The pDZ-Pcj7-ilvD vector to replace the ilvD gene promoter was constructed using primer pairs according to SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, and SEQ ID NOs: 17 and 18 to construct a highly valine-producing strain an ability in which the expression of the valine biosynthesis gene ilvD is enhanced.

Specifically, to construct pDZ-Pcj7-ilvD, PCR was performed using primer pairs according to SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, and SEQ ID NOs: 17 and 18, based on the genomic DNA of the wild strain type Corynebacterium glutamicum ATCC 14067 as a template to obtain, respectively, a gene fragment. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes.

PCR was carried out with overlapping primers based on the fragments obtained above as a template using the primer pair according to SEQ ID NO: 13 and 18 to obtain fragment 3 with the introduced mutation. The thus obtained fragment 3 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct a vector which was named pDZ-Pcj7-ilvD.

The above constructed pDZ-Pcj7-ilvD was then transformed into each of CJ7V, CJ8V and KCCM11201P, which are valine-derived strains, to induce homologous recombination in the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545 , 1999). Strains into which the vector was introduced into the chromosome by recombination of homologous sequences were selected in a medium containing 25 mg/l kanamycin. Gene fragments were then amplified from the Corynebacterium glutamicum transformants selected above by PCR using the primer pair of SEQ ID NOs: 13 and 18, and the introduction of the mutation was confirmed by gene sequencing analysis. The selected recombinant strains were named Corynebacterium glutamicum CJ7V:ilvD, CJ8V:ilvD, KCCM11201P:ilvD, respectively. The fermentation titer of the selected strains with the enhanced ilvD gene was determined in the same way as in example 1, and the results are presented below.

According to the above results, when ilvD, which is one of the valine biosynthesis genes, was enhanced, it was then confirmed that the ability to produce valine in all valine-producing strains CJ7V, CJ8V and KCCM11201P was increased.

Example 2-2. Construction of avtA deletion and alg attenuated strains and their evaluation

To construct strains with a high ability to produce valine, an attempt was made to weaken (by modifying the start codon of the avtA gene with GTG) or delete avtA. Accordingly, a vector for constructing a strain with attenuated avtA (by modifying the start codon of the avtA gene with GTG) was constructed using primer pairs in accordance with SEQ ID NOs: 19 and 20, and SEQ ID NOs: 21 and 22 below, and the vector for construction eliminating avtA activity using primer pairs according to SEQ ID NOs: 23 and 24 and SEQ ID NOs: 25 and 26 below, which were named pDZ-avtA(Alg) and pDZ-avtA(del), respectively.

Specifically, to construct the avtA attenuated vector (pDZ-avtA(Alg)), PCR was performed using primer pairs according to SEQ ID NOs: 19 and 20 and SEQ ID NOs: 21 and 22, based on the genomic DNA of the wild-type Corynebacterium strain glutamicum ATCC 14067 as a matrix. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was performed with overlapping primers based on each of the fragments obtained above as a template using the primer pair according to SEQ ID NOs: 19 and 22 to obtain fragment 4 with the introduced mutation. The thus obtained fragment 4 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-avtA(Alg).

To construct the avtA deletion vector (pDZ-avtA(del)), PCR was performed using primer pairs in accordance with SEQ ID NOs: 23 and 24 and SEQ ID NOs: 25 and 26, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC14067 in as a matrix. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was performed with overlapping primers based on each of the fragments obtained above as a template using the primer pair according to SEQ ID NOs: 23 and 26 to obtain fragment 5 with the introduced mutation. The thus obtained fragment 5 with the introduced mutation was treated with the restriction enzyme Xbal (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the vector pDZ-avtA(del).

pDZ-avtA(del) and pDZ-avtA(Alg) were then transformed into each of CJ7V:ilvD, CJ8V:ilvD and KCCM11201P:ilvD, which are valine-producing strains, to induce homologous recombination in the chromosome (van der Rest et al ., Appl Microbiol Biotechnol 52:541-545, 1999). Strains into which vectors were introduced into the chromosome by recombination of homologous sequences were selected in a medium containing 25 mg/l kanamycin.

Gene fragments were then amplified from the Corynebacterium glutamicum transformants selected above by PCR using primer pairs according to SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, and SEQ ID NOs : 25 and 26, and the introduction of the mutation was confirmed by gene sequencing analysis in the same manner as in Example 1. The recombinant strains were named Corynebacterium glutamicum CJ7V:ilvD-avtA(del), CJ7V:ilvD-avtA(alg), CJ8V :ilvD-avtA(del), CJ8V:ilvD-avtA (alg), KCCM11201P:ilvD-avtA(del) and KCCM11201P:ilvD-avtA (alg) as presented below, and titer assessment was carried out in the same manner as in example 1.

Consistent with the results presented above, when avtA was deleted, then the ability to produce valine was enhanced, and when avtA expression was attenuated, then the ability to produce valine was increased in the CJ8V:ilvD-avtA (alg) strain compared to the CJ8V:ilvD strain, showing equal or higher level of ability to produce valine compared to ilvD-enhanced control strains. It was confirmed that the ability to produce valine was enhanced in all cases of ilvD-enhanced strains and avtA deletion strains or avtA-attenuated strains compared with strains CJ7V, CJ8V and KCCM11201P, as presented in Table 4.

Example 2-3. Construction of strains with aceE deletion and strains with attenuated aceE (alg, Q432A, K435A) and their evaluation

To construct strains with a high ability to produce valine, an attempt was made to attenuate or remove aceE. Accordingly, to construct strains with attenuated aceE, a vector for constructing a strain in which the start codon of the aceE gene was modified to GTG was constructed using primer pairs in accordance with SEQ ID NOs: 27 and 28 and SEQ ID NOs: 29 and 30, vector to construct the aceE(Q432A) strain was constructed using primer pairs according to SEQ ID NOs: 27 and 32 and SEQ ID NOs: 33 and 36, and the vector for constructing the aceE(K435A) strain was constructed using primer pairs according to SEQ ID NOs: 27 and 34 and SEQ ID NOs: 35 and 36. Additionally, a vector for constructing an aceE deletion strain (aceE(del)) was constructed using primer pairs according to SEQ ID NOs: 37 and 38 and SEQ ID NO: 39 and 40. The vectors thus constructed were named pDZ-aceE(Alg), pDZ-aceE(Q432A), pDZ-aceE(K435A) and pDZ-aceE(del), respectively.

Specifically, to construct a vector to weaken the start codon of AcE, PCR was performed using primer pairs in accordance with SEQ ID NOs: 27 and 28 and SEQ ID NOs: 29 and 30, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC 14067 as a template. . PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was carried out with overlapping primers based on each of the fragments obtained above as a template using the primer pair according to SEQ ID NO: 27 and 30 to obtain fragment 6 with the introduced mutation. The thus obtained fragment 6 with the introduced mutation was treated with the restriction enzyme Xbal (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-aceE(Alg).

To construct a vector for introducing the aceE(Q432A) mutation, PCR was performed using primer pairs according to SEQ ID NOs: 27 and 28 and SEQ ID NOs: 33 and 36, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC14067 as a template. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was carried out with overlapping primers based on each of the fragments obtained above as a template using the primer pair according to SEQ ID NOs: 27 and 36 to obtain fragment 7 with the introduced mutation. The thus obtained fragment 7 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-aceE(Q432A).

To construct a vector for introducing the aceE(K435A) mutation, PCR was performed using primer pairs in accordance with SEQ ID NOs: 27 and 34 and SEQ ID NOs: 35 and 36, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC14067 as a template. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was performed with overlapping primers based on each of the fragments obtained above as a template using the primer pair according to SEQ ID NOs: 27 and 36 to obtain fragment 8 with the introduced mutation. The thus obtained fragment 8 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct DNA vector pDZ-aceE(K435A).

To introduce the vector used to introduce the aceE(del) mutation, PCR was performed using primer pairs in accordance with SEQ ID NOs: 37 and 38 and SEQ ID NOs: 39 and 40, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC 14067 as matrices. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was carried out with overlapping primers based on each of the fragments obtained above as a template using the primer pair according to SEQ ID NO: 37 and 40 to obtain fragment 9 with the introduced mutation. The thus obtained fragment 9 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-aceE(del).

Then pDZ-aceE(del), pDZ-aceE(Alg), pDZ-aceE(Q432A) and pDZ-aceE(K435A) were transformed into each of CJ7V:ilvD, CJ8V:ilvD and KCCM11201P:ilvD, which are strains producing valine, to induce homologous recombination in the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains into which vectors were introduced into the chromosome by recombination of homologous sequences were selected in a medium containing 25 mg/l kanamycin.

Gene fragments were then amplified from the Corynebacterium glutamicum transformants selected above by PCR using the primer pair of SEQ ID NOs: 27 and 36, and the introduction of the mutation was confirmed by gene sequencing analysis. The recombinant strains were named Corynebacterium glutamicum CJ7V:ilvD-aceE(del), CJ7V:ilvD-aceE (alg), CJ7V:ilvD-aceE(Q432A), CJ7V:ilvD-aceE(K435A), CJ8V:ilvD-aceE(del) , CJ8V:ilvD-aceE (alg), CJ8V:ilvD-aceE(Q432A), CJ8V:ilvD-aceE(K435A), KCCM11201P:ilvD-aceE(del), KCCM11201P:ilvD-aceE(alg), KCCM11201P:ilvD- aceE(Q432A), and KCCM11201P:ilvD-aceE(K435A) as presented below, and titer assessment was carried out in the same manner as in Example 1.

Consistent with the results presented above, when aceE was further deleted in strains having enhanced dihydroxyacid dehydratase (ilvD) activity, then growth rates and sugar consumption were rapidly reduced, resulting in a reduced ability to produce valine. At the same time, in the case of strains having attenuated aceE, it was confirmed that the growth rates and sugar consumption were not significant, and the ability to produce valine increased, although there were differences depending on the level of attenuation.

Example 2-4. Construction of strains with weakened gltA (alg, N241T, M312I) and their evaluation

To construct strains with a high ability to produce valine, an attempt was made to weaken gltA. Accordingly, a vector for constructing a gltA attenuated strain in which the start codon of the gltA gene is modified to GTG was constructed using primer pairs in accordance with SEQ ID NOs: 41 and 42 and SEQ ID NOs: 43 and 44, vector for constructing a gltA strain ( N241T) was constructed using primer pairs according to SEQ ID NOs: 45 and 46 and SEQ ID NOs: 47 and 50, and the gltA(M312I) strain construction vector was constructed using primer pairs according to SEQ ID NOs: 45 and 48 and SEQ ID NOs: 49 and 50. The vectors thus constructed were named pDZ-gltA(Alg), pDZ-gltA(N241T) and pDZ-gltA(M312I), respectively.

To introduce a vector to introduce a mutation weakening the gltA start codon, PCR was performed using primer pairs in accordance with SEQ ID NOs: 41 and 42 and SEQ ID NOs: 43 and 44, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC 14067 as matrices. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was performed with overlapping primers based on each of the fragments (A, B) obtained above as a template using the primer pair according to SEQ ID NOs: 41 and 44 to obtain fragment 10 with the introduced mutation. The thus obtained fragment 10 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-gltA(Alg).

To introduce a vector for introducing the gltA(N241T) mutation, PCR was performed using primer pairs according to SEQ ID NOs: 45 and 46 and SEQ ID NOs: 47 and 50, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC14067 as a template. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was carried out with overlapping primers based on each of the fragments (A, B) obtained above as a template using the primer pair according to SEQ ID NO: 45 and 50 to obtain fragment 11 with the introduced mutation. The thus obtained fragment 11 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-gltA(N241T).

To introduce a vector for introducing the gltA(M312I) mutation, PCR was performed using primer pairs according to SEQ ID NOs: 45 and 48 and SEQ ID NOs: 49 and 50, based on the genomic DNA of the wild-type strain Corynebacterium glutamicum ATCC14067 as a template. PCR conditions were as follows: denaturation at 94°C for 5 minutes and then 25 cycles of denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds and polymerizing at 72°C for 60 seconds, and then polymerizing at 72°C for 7 minutes. PCR was carried out with overlapping primers based on each of the fragments (A, B) obtained above as a template using the primer pair according to SEQ ID NO: 45 and 50 to obtain fragment 12 with the introduced mutation. The thus obtained fragment 12 with the introduced mutation was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated into the pDZ vector, which had been treated with the same restriction enzyme, using T4 ligase (New England Biolabs, Beverly, MA) to construct the DNA vector pDZ-gltA(M312I).

pDZ-gltA(Alg), pDZ-gltA(N241T), and pDZ-gltA(M312I) were then transformed into each of CJ7V:ilvD, CJ8V:ilvD, and KCCM11201P:ilvD, which are valine-producing strains, to induce homologous recombination in chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains into which vectors were introduced into the chromosome by recombination of homologous sequences were selected in a medium containing 25 mg/l kanamycin.

Gene fragments were then amplified from the Corynebacterium glutamicum transformants selected above by PCR using the primer pair of SEQ ID NOs: 45 and 50, and the introduction of the mutation was confirmed by gene sequencing analysis. The recombinant strains were named Corynebacterium glutamicum CJ7V:ilvD-gltA (alg), CJ7V:ilvD-gltA(N241T), CJ7V:ilvD-gltA(M312I), CJ8V:ilvD-gltA(alg), CJ8V:ilvD-gltA(N241T) . 1.

Consistent with the results presented above, when gltA was attenuated in strains having enhanced dihydroxyacid dehydratase (ilvD) activity, it was then confirmed that growth rates and sugar consumption were not significant and the ability to produce valine was increased, although there were differences depending on the level of attenuation. .

Example 2-5. Designing a combination of strains for the performance of effective mutants and their evaluation

Based on the results confirmed in Examples 2-2 to 2-4, an attempt was made to determine whether there was a synergistic effect on the ability to produce valine when different mutations were combined. Vectors pDZ-avtA(del) and pDZ-aceE(K435A) were transformed into each of CJ7V:ilvD-gltA-attenuated(N241T), CJ8V:ilvD-gltA-attenuated(N241T), and KCCM11201P:ilvD-gltA-attenuated(M241T) , which are the valine-producing strains constructed in Example 2-4 (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which vectors were introduced into the chromosome by recombination of homologous sequences were selected in a medium containing 25 mg/l kanamycin.

Gene fragments were then amplified from Corynebacterium glutamicum transformants in which secondary recombination had been completed by PCR using primer pairs according to SEQ ID NOs: 23 and 26 and SEQ ID NOs: 27 and 36, and strains with the introduced mutation were then confirmed by gene sequencing analysis. The recombinant strains were named as follows and titer assessment was carried out in the same manner as in Example 1.

According to the above results, it was confirmed that when the aceE-attenuated strains and the avtA-deletion strains were introduced into the ilvD-enhanced and gltA-attenuated strains, then the growth rates and sugar consumption were at the same level, and the ability to produce valine was further increased.

The pDZ-aceE(K435A) vector was transformed into the KCCM11201P:ilvD-gltA-attenuated (N241T) strain, and the strain into which the ace(K435A) mutation was introduced into the chromosome by recombination of homologous sequences was named CA08-1592. CA08-1592 was deposited with the Korea Center for Microbial Culture (KCCM) under the Treaty of Budapest on July 3, 2020, under the number KCCM12761P.

In accordance with the foregoing, one skilled in the art to whom the description of the present invention is directed will be able to understand that the description of the present invention can be embodied in other specific forms without modification of the technical concepts or essential features of the description of the present invention. Accordingly, the exemplary embodiments disclosed herein are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention. On the contrary, the description of the present invention is intended to cover not only exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments that may be included within the spirit and scope of the description of the present invention as defined in the claims.

--->

LIST OF SEQUENCES

<110>CJ CheilJedang Corporation

<120> MICROORGANISM PRODUCING L-VALINE AND METHOD FOR PRODUCING L-

VALINE WITH ITS USE

<130> OPA21084-PCT

<150> KR 10-2020-0111084

<151> 2020-09-01

<160> 50

<170> KoPatentIn 3.0

<210> 1

<211> 613

<212>PRT

<213> Unknown

<220>

<223> Dihydroxy acid dehydratase Corynebacterium sp

<400> 1

Met Ile Pro Leu Arg Ser Lys Val Thr Thr Val Gly Arg Asn Ala Ala

1 5 10 15

Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Thr Lys Glu Asn Glu Phe

20 25 30

Gly Lys Pro Ile Val Ala Ile Val Asn Ser Tyr Thr Gln Phe Val Pro

35 40 45

Gly His Val His Leu Lys Asn Val Gly Asp Ile Val Ala Asp Ala Val

50 55 60

Arg Lys Ala Gly Gly Val Pro Lys Glu Phe Asn Thr Ile Ala Val Asp

65 70 75 80

Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser

85 90 95

Arg Glu Ile Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Thr

100 105 110

Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly

115 120 125

Met Leu Asn Ala Ala Met Arg Leu Asn Ile Pro Val Val Phe Val Ser

130 135 140

Gly Gly Pro Met Glu Ala Gly Lys Ala Val Val Val Asp Gly Val Ala

145 150 155 160

His Ala Pro Thr Asp Leu Ile Thr Ala Ile Ser Ala Ser Ala Ser Asp

165 170 175

Ala Val Asp Asp Ala Gly Leu Ala Ala Val Glu Ala Ser Ala Cys Pro

180 185 190

Thr Cys Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys

195 200 205

Leu Thr Glu Ala Leu Gly Leu Ser Leu Pro Gly Asn Gly Ser Thr Leu

210 215 220

Ala Thr His Ala Ala Arg Arg Ala Leu Phe Glu Lys Ala Gly Glu Thr

225 230 235 240

Val Val Glu Leu Cys Arg Arg Tyr Tyr Gly Glu Glu Asp Glu Ser Val

245 250 255

Leu Pro Arg Gly Ile Ala Thr Lys Lys Ala Phe Glu Asn Ala Met Ala

260 265 270

Leu Asp Met Ala Met Gly Gly Ser Thr Asn Thr Ile Leu His Ile Leu

275 280 285

Ala Ala Ala Gln Glu Gly Glu Val Asp Phe Asp Leu Ala Asp Ile Asp

290 295 300

Glu Leu Ser Lys Asn Val Pro Cys Leu Ser Lys Val Ala Pro Asn Ser

305 310 315 320

Asp Tyr His Met Glu Asp Val His Arg Ala Gly Gly Ile Pro Ala Leu

325 330 335

Leu Gly Glu Leu Asn Arg Gly Gly Leu Leu Asn Lys Asp Val His Ser

340 345 350

Val His Ser Asn Asp Leu Glu Gly Trp Leu Asp Asp Trp Asp Ile Arg

355 360 365

Ser Gly Lys Thr Thr Glu Val Ala Thr Glu Leu Phe His Ala Ala Pro

370 375 380

Gly Gly Ile Arg Thr Thr Glu Ala Phe Ser Thr Glu Asn Arg Trp Asp

385 390 395 400

Glu Leu Asp Thr Asp Ala Ala Lys Gly Cys Ile Arg Asp Val Glu His

405 410 415

Ala Tyr Thr Ala Asp Gly Gly Leu Val Val Leu Arg Gly Asn Ile Ser

420 425 430

Pro Asp Gly Ala Val Ile Lys Ser Ala Gly Ile Glu Glu Glu Leu Trp

435 440 445

Asn Phe Thr Gly Pro Ala Arg Val Val Glu Ser Gln Glu Glu Ala Val

450 455 460

Ser Val Ile Leu Thr Lys Thr Ile Gln Ala Gly Glu Val Leu Val Val

465 470 475 480

Arg Tyr Glu Gly Pro Ser Gly Gly Pro Gly Met Gln Glu Met Leu His

485 490 495

Pro Thr Ala Phe Leu Lys Gly Ser Gly Leu Gly Lys Lys Cys Ala Leu

500 505 510

Ile Thr Asp Gly Arg Phe Ser Gly Gly Ser Ser Gly Leu Ser Ile Gly

515 520 525

His Val Ser Pro Glu Ala Ala His Gly Gly Val Ile Gly Leu Ile Glu

530 535 540

Asn Gly Asp Ile Val Ser Ile Asp Val His Asn Arg Lys Leu Glu Val

545 550 555 560

Gln Val Ser Asp Glu Glu Leu Gln Arg Arg Arg Asp Ala Met Asn Ala

565 570 575

Ser Glu Lys Pro Trp Gln Pro Val Asn Arg Asn Arg Val Val Thr Lys

580 585 590

Ala Leu Arg Ala Tyr Ala Lys Met Ala Thr Ser Ala Asp Lys Gly Ala

595 600 605

Val Arg Gln Val Asp

610

<210> 2

<211> 383

<212>PRT

<213>Unknown

<220>

<223> Transaminase C Corynebacterium sp

<400> 2

Met Lys Pro Ser Thr Arg Ser Asn Val Gln Pro Phe Arg Val Met Gln

1 5 10 15

Met Leu Asp Arg Val His Arg Arg Arg Arg Glu Gly Lys Asp Thr Ile

20 25 30

Met Phe Cys Ala Gly Gln Pro Ser Thr Gly Ala Pro Glu Ala Val Ile

35 40 45

Glu Glu Ala Glu Ile Ala Leu Arg Ser Gly Pro Leu Gly Tyr Thr Glu

50 55 60

Val Ile Gly Asp Arg Glu Phe Arg Glu Arg Ile Ala Asp Trp His Ser

65 70 75 80

Ala Thr Tyr Asp Val Asp Thr Asn Pro Asp Asn Val Ile Val Thr Thr

85 90 95

Gly Ser Ser Gly Gly Phe Val Ala Ser Phe Ile Ala Thr Leu Asp His

100 105 110

Gly Asp Tyr Val Ala Met Pro Thr Pro Gly Tyr Pro Ala Tyr Arg Asn

115 120 125

Ile Leu Glu Ser Leu Gly Ala Lys Val Leu Asn Leu Arg Cys Thr Ala

130 135 140

Glu Thr Arg Phe Gln Pro Thr Ala Gln Met Leu Glu Glu Leu Pro His

145 150 155 160

Lys Pro Lys Ala Val Ile Val Thr Ser Pro Gly Asn Pro Thr Gly Thr

165 170 175

Ile Ile Asp Pro Glu Glu Leu Glu Arg Ile Ala Lys Trp Cys Asp Asp

180 185 190

Asn Asp Ala Val Leu Ile Ser Asp Glu Asp Tyr His Gly Met Ser Phe

195 200 205

Gly Arg Pro Leu Ala Thr Ala His Gln Phe Ser Lys Asn Ala Ile Val

210 215 220

Val Gly Thr Leu Ser Lys Tyr Phe Ser Met Thr Gly Trp Arg Val Gly

225 230 235 240

Trp Ile Ile Val Pro Asp Glu Leu Val Thr Ser Ile Glu Asn Leu Gln

245 250 255

Ala Ser Leu Ser Leu Cys Ala Pro Ala Ile Gly Gln Ala Ala Gly Arg

260 265 270

Ala Ala Phe Thr Leu Glu Ala Gly Ala Glu Leu Asp Ala His Val Glu

275 280 285

Ala Tyr Arg Glu Ala Arg Glu Val Phe Val Asp Lys Leu Pro Glu Ile

290 295 300

Gly Leu Gly Thr Phe Ala Asp Pro Asp Gly Gly Leu Tyr Leu Trp Val

305 310 315 320

Asp Val Ser Ala Tyr Thr Asp Asp Ser Glu Glu Trp Val Leu Arg Leu

325 330 335

Leu Asp Glu Ala Gly Val Ala Val Ala Pro Gly Val Asp Phe Asp Pro

340 345 350

Glu Glu Gly His Lys Trp Ile Arg Leu Ser Leu Cys Ala Ser Lys Glu

355 360 365

Asp Thr Ile Glu Gly Val Arg Lys Ile Gly Glu Phe Ile Lys Lys

370 375 380

<210> 3

<211> 922

<212>PRT

<213>Unknown

<220>

<223> Pyruvate dehydrogenase Corynebacterium sp

<400> 3

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln

420 425 430

Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro

675 680 685

Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895

Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 4

<211> 437

<212>PRT

<213>Unknown

<220>

<223> Citrate synthase Corynebacterium sp

<400> 4

Met Phe Glu Arg Asp Ile Val Ala Thr Asp Asn Asn Lys Ala Val Leu

1 5 10 15

His Tyr Pro Gly Gly Glu Phe Glu Met Asp Ile Ile Glu Ala Ser Glu

20 25 30

Gly Asn Asn Gly Val Val Leu Gly Lys Met Leu Ser Glu Thr Gly Leu

35 40 45

Ile Thr Phe Asp Pro Gly Tyr Val Ser Thr Gly Ser Thr Glu Ser Lys

50 55 60

Ile Thr Tyr Ile Asp Gly Asp Ala Gly Ile Leu Arg Tyr Arg Gly Tyr

65 70 75 80

Asp Ile Ala Asp Leu Ala Glu Asn Ala Thr Phe Asn Glu Val Ser Tyr

85 90 95

Leu Leu Ile Asn Gly Glu Leu Pro Thr Pro Asp Glu Leu His Lys Phe

100 105 110

Asn Asp Glu Ile Arg His His Thr Leu Leu Asp Glu Asp Phe Lys Ser

115 120 125

Gln Phe Asn Val Phe Pro Arg Asp Ala His Pro Met Ala Thr Leu Ala

130 135 140

Ser Ser Val Asn Ile Leu Ser Thr Tyr Tyr Gln Asp Gln Leu Asn Pro

145 150 155 160

Leu Asp Glu Ala Gln Leu Asp Lys Ala Thr Val Arg Leu Met Ala Lys

165 170 175

Val Pro Met Leu Ala Ala Tyr Ala His Arg Ala Arg Lys Gly Ala Pro

180 185 190

Tyr Met Tyr Pro Asp Asn Ser Leu Asn Ala Arg Glu Asn Phe Leu Arg

195 200 205

Met Met Phe Gly Tyr Pro Thr Glu Pro Tyr Glu Ile Asp Pro Ile Met

210 215 220

Val Lys Ala Leu Asp Lys Leu Leu Ile Leu His Ala Asp His Glu Gln

225 230 235 240

Asn Cys Ser Thr Ser Thr Val Arg Met Ile Gly Ser Ala Gln Ala Asn

245 250 255

Met Phe Val Ser Ile Ala Gly Gly Ile Asn Ala Leu Ser Gly Pro Leu

260 265 270

His Gly Gly Ala Asn Gln Ala Val Leu Glu Met Leu Glu Asp Ile Lys

275 280 285

Asn Asn His Gly Gly Asp Ala Thr Ala Phe Met Asn Lys Val Lys Asn

290 295 300

Lys Glu Asp Gly Val Arg Leu Met Gly Phe Gly His Arg Val Tyr Lys

305 310 315 320

Asn Tyr Asp Pro Arg Ala Ala Ile Val Lys Glu Thr Ala His Glu Ile

325 330 335

Leu Glu His Leu Gly Gly Asp Asp Leu Leu Asp Leu Ala Ile Lys Leu

340 345 350

Glu Glu Ile Ala Leu Ala Asp Asp Tyr Phe Ile Ser Arg Lys Leu Tyr

355 360 365

Pro Asn Val Asp Phe Tyr Thr Gly Leu Ile Tyr Arg Ala Met Gly Phe

370 375 380

Pro Thr Asp Phe Phe Thr Val Leu Phe Ala Ile Gly Arg Leu Pro Gly

385 390 395 400

Trp Ile Ala His Tyr Arg Glu Gln Leu Gly Ala Ala Asp Asn Lys Ile

405 410 415

Asn Arg Pro Arg Gln Val Tyr Thr Gly Asn Glu Ser Arg Lys Leu Val

420 425 430

Pro Arg Glu Glu Arg

435

<210> 5

<211> 922

<212>PRT

<213>Unknown

<220>

<223> Variant of pyruvate dehydrogenase Corynebacterium sp

<400> 5

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Ala

420 425 430

Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro

675 680 685

Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895

Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 6

<211> 922

<212>PRT

<213> Unknown

<220>

<223> Variant of pyruvate dehydrogenase Corynebacterium sp

<400> 6

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln

420 425 430

Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro

675 680 685

Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895

Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 7

<211> 922

<212>PRT

<213>Unknown

<220>

<223> Citrate synthase variant Corynebacterium sp

<400> 7

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln

420 425 430

Met Lys Ala Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro

675 680 685

Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser

885 890 895

Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 8

<211> 437

<212>PRT

<213>Unknown

<220>

<223> Citrate synthase variant Corynebacterium sp

<400> 8

Met Phe Glu Arg Asp Ile Val Ala Thr Asp Asn Asn Lys Ala Val Leu

1 5 10 15

His Tyr Pro Gly Gly Glu Phe Glu Met Asp Ile Ile Glu Ala Ser Glu

20 25 30

Gly Asn Asn Gly Val Val Leu Gly Lys Met Leu Ser Glu Thr Gly Leu

35 40 45

Ile Thr Phe Asp Pro Gly Tyr Val Ser Thr Gly Ser Thr Glu Ser Lys

50 55 60

Ile Thr Tyr Ile Asp Gly Asp Ala Gly Ile Leu Arg Tyr Arg Gly Tyr

65 70 75 80

Asp Ile Ala Asp Leu Ala Glu Asn Ala Thr Phe Asn Glu Val Ser Tyr

85 90 95

Leu Leu Ile Asn Gly Glu Leu Pro Thr Pro Asp Glu Leu His Lys Phe

100 105 110

Asn Asp Glu Ile Arg His His Thr Leu Leu Asp Glu Asp Phe Lys Ser

115 120 125

Gln Phe Asn Val Phe Pro Arg Asp Ala His Pro Met Ala Thr Leu Ala

130 135 140

Ser Ser Val Asn Ile Leu Ser Thr Tyr Tyr Gln Asp Gln Leu Asn Pro

145 150 155 160

Leu Asp Glu Ala Gln Leu Asp Lys Ala Thr Val Arg Leu Met Ala Lys

165 170 175

Val Pro Met Leu Ala Ala Tyr Ala His Arg Ala Arg Lys Gly Ala Pro

180 185 190

Tyr Met Tyr Pro Asp Asn Ser Leu Asn Ala Arg Glu Asn Phe Leu Arg

195 200 205

Met Met Phe Gly Tyr Pro Thr Glu Pro Tyr Glu Ile Asp Pro Ile Met

210 215 220

Val Lys Ala Leu Asp Lys Leu Leu Ile Leu His Ala Asp His Glu Gln

225 230 235 240

Asn Cys Ser Thr Ser Thr Val Arg Met Ile Gly Ser Ala Gln Ala Asn

245 250 255

Met Phe Val Ser Ile Ala Gly Gly Ile Asn Ala Leu Ser Gly Pro Leu

260 265 270

His Gly Gly Ala Asn Gln Ala Val Leu Glu Met Leu Glu Asp Ile Lys

275 280 285

Asn Asn His Gly Gly Asp Ala Thr Ala Phe Met Asn Lys Val Lys Asn

290 295 300

Lys Glu Asp Gly Val Arg Leu Ile Gly Phe Gly His Arg Val Tyr Lys

305 310 315 320

Asn Tyr Asp Pro Arg Ala Ala Ile Val Lys Glu Thr Ala His Glu Ile

325 330 335

Leu Glu His Leu Gly Gly Asp Asp Leu Leu Asp Leu Ala Ile Lys Leu

340 345 350

Glu Glu Ile Ala Leu Ala Asp Asp Tyr Phe Ile Ser Arg Lys Leu Tyr

355 360 365

Pro Asn Val Asp Phe Tyr Thr Gly Leu Ile Tyr Arg Ala Met Gly Phe

370 375 380

Pro Thr Asp Phe Phe Thr Val Leu Phe Ala Ile Gly Arg Leu Pro Gly

385 390 395 400

Trp Ile Ala His Tyr Arg Glu Gln Leu Gly Ala Ala Asp Asn Lys Ile

405 410 415

Asn Arg Pro Arg Gln Val Tyr Thr Gly Asn Glu Ser Arg Lys Leu Val

420 425 430

Pro Arg Glu Glu Arg

435

<210> 9

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 9

aatttctaga ggcagaccct attctatgaa gg 32

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 10

agtgtttcgg tctttacaga cacgagggac 30

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

gtccctcgtg tctgtaaaga ccgaaacact 30

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

aatttctaga cgtgggagtg tcactcgctt gg 32

<210> 13

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

ttcgagctcg gtacccggtc tagagcactt tcgctcgcac c 41

<210> 14

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

gatttgaaaa gcgcatcaga aacatcccag cgctac 36

<210> 15

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

gtagcgctgg gatgtttctg atgcgctttt caaatc 36

<210> 16

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

gaaacactat gatccactt cgttcaaaag tcaccaccgt c 41

<210> 17

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

gacggtggtg acttttgaac gaagtgggat catagtgttt c 41

<210> 18

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

gcatgcctgc aggtcgactc tagagcgtgt gcaacgccgt c 41

<210> 19

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

gctcggtacc cggggatcct ctagaccgct tccttggctg cctgaagatg 50

<210> 20

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

tgcttggctt cacaagagac aagcct 26

<210> 21

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 21

aggcttgtct cttgtgaagc caagca 26

<210> 22

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 22

gcctgcaggt cgacctagat ctagacctca tcagagataa gaacagcatc 50

<210> 23

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 23

gctcggtacc cggggatcct ctagatactc cggtctgctt tatgcaggta 50

<210> 24

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

aactaaccta gtcgcttaag agacaagcct atctgc 36

<210> 25

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

gcagataggc ttgtctctta agcgactagg ttagtt 36

<210> 26

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

gcctgcaggt cgacctagat ctagaaagtg ccacgagcat ttcatcagct 50

<210> 27

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

gctcggtacc cggggatcct ctagataccg tccaaccggt actttgaacc 50

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

ttgatcggcc acttccacac 20

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 29

ggtgtggaag tggccgatca a 21

<210> 30

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 30

gcctgcaggt cgacctagat ctagagatcg tcttcagaaa ggcgaccctc 50

<210> 31

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 31

gctcggtacc cggggatcct ctagaaccgt ggcatcaagg acacctctga 50

<210> 32

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 32

agcttcttca ttgcgtgggt tg 22

<210> 33

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

caacccacgc aatgaagaag ct 22

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

aagcgtcagt gccttcatct g 21

<210> 35

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

ccagatgaag gcactgacgc tt 22

<210> 36

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 36

gcctgcaggt cgacctagat ctagaagatg gagtcaccgg tgcgctggaa 50

<210> 37

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 37

gctcggtacc cggggatcct ctagaacctt tccctggaat tttttccttt 50

<210> 38

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 38

tgtcccttga ggtgatttcc acacctcctg ttggaatg 38

<210> 39

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 39

tccaacagga ggtgtggaaa tcacctcaag ggacaga 37

<210> 40

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 40

gcctgcaggt cgacctagat ctagatgctg cgcggcaagc gccgtggatt 50

<210> 41

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 41

gctcggtacc cggggatcct ctagaaccct gaagctgtca gttcctagca 50

<210> 42

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 42

ccctttcaaa cacatttgtt from 21

<210> 43

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 43

cgaacaaatg tgtttgaaag gg 22

<210> 44

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 44

gcctgcaggt cgacctagat ctagatgcgc ggtgtgcgta cgcagccagc 50

<210> 45

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 45

gctcggtacc cggggatcct ctagatatgt gagcactggc tctaccgagt 50

<210> 46

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 46

aggtggagca ggtctgctcg tg 22

<210> 47

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 47

cacgagcaga cctgctccac ct 22

<210> 48

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 48

tgtccgaagc cgatgaggcg gac 23

<210> 49

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 49

cgtccgcctc atcggcttcg gaca 24

<210> 50

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 50

gcctgcaggt cgacctagat ctagagtcag agattacgag atcttccggt 50

thirty

<---

Claims (18)

1. An L-valine producing microorganism having enhanced dihydroxy acid dehydratase activity compared to endogenous activity and any one or more of the combinations selected from (1) to (3) below: (1) reduced transaminase C activity compared to endogenous activity; (2) weakened pyruvate dehydrogenase activity compared to endogenous activity; And (3) weakened citrate synthase activity compared to endogenous activity. 2. The microorganism according to claim 1, which has enhanced activity of dihydroxy acid dehydratase and reduced activity of transaminase C. 3. The microorganism according to claim 1, which has enhanced activity of dihydroxy acid dehydratase and weakened activity of pyruvate dehydrogenase. 4. The microorganism according to claim 1, which has enhanced activity of dihydroxy acid dehydratase and weakened activity of citrate synthase. 5. The microorganism according to claim 4, additionally having reduced transaminase C activity or weakened pyruvate dehydrogenase activity. 6. Microorganism according to claim 1, where dihydroxy acid dehydratase is encoded by the ilvD gene. 7. The microorganism according to claim 1, where transaminase C is encoded by the avtA gene. 8. The microorganism according to claim 1, where pyruvate dehydrogenase is encoded by the aceE gene. 9. The microorganism according to claim 1, where citrate synthase is encoded by the gltA gene. 10. The microorganism according to claim 1, wherein the weakened pyruvate dehydrogenase is characterized in that the amino acid corresponding to position 432 or 435 relative to the N-terminus of the amino acid sequence SEQ ID NO: 3 is respectively replaced by alanine. 11. The microorganism according to claim 1, wherein the citrate synthase, which has weakened activity compared to endogenous activity, is characterized in that the amino acid corresponding to position 241 or 312 relative to the N-terminus of the amino acid sequence SEQ ID NO: 4 is replaced by threonine or isoleucine, respectively. 12. The microorganism according to claim 1, where the L-valine-producing microorganism is a microorganism of the genus Corynebacterium . 13. The microorganism according to claim 12, where the microorganism of the genus Corynebacterium is Corynebacterium glutamicum . 14. A method for producing L-valine, including cultivating a microorganism according to any one of claims. 1-13. 15. The method according to claim 14, further comprising isolating L-valine from the cultivated microorganism or medium.