RU2805253C1 - New modified polypeptide with reduced citrate synthase activity and method for producing l-amino acid using it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: modified polypeptide with citrate synthase activity, where the amino acid corresponding to position 312 from the N-terminus of the polypeptide, consisting of the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid. The invention also relates to a modified polypeptide having citrate synthase activity and having a sequence homology of 80% or more and less than 100% with the amino acid sequence of SEQ ID NO: 1, where the amino acid corresponding to position 312 from the N-terminus of the polypeptide, consisting of the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid.

EFFECT: invention makes it possible to effectively obtain an L-amino acid using a microorganism containing one of the said modified polypeptides.

14 cl, 15 tbl, 8 ex

Description

Field of invention

The present invention relates to a new modified polypeptide with weakened citrate synthase activity, to a microorganism including this modified polypeptide, and to a method for producing L-amino acids using this microorganism.

Prior Art

The microorganism of the genus Corynebacterium, in particular Corynebacterium glutamicum, is a Gram-positive microorganism that is widely used in the production of L-amino acids and other beneficial substances. To obtain L-amino acids and other beneficial substances, various studies are being carried out aimed at developing microorganisms with highly efficient products and technologies for fermentation processes. For example, approaches that are specific to the desired substance are generally used, such as increasing the expression of genes encoding enzymes involved in the biosynthesis of L-lysine, or deleting genes that are not necessary for biosynthesis (US 8048650 B2).

At the same time, L-amino acids such as L-lysine, L-threonine, L-methionine, L-isoleucine and L-glycine are aspartate-derived amino acids, and the level of biosynthesis of oxaloacetate (i.e., the precursor of aspartate ) may influence the levels of biosynthesis of these L-amino acids.

Citrate synthase (CS) is an enzyme that produces citrate by catalyzing the condensation of acetyl-CoA and oxaloacetate, which are produced during the microbial growth process, and it is also an important enzyme for determining carbon flux in the TCA (tricarboxylic acid cycle) pathway.

Phenotypic changes in L-lysine-producing strains due to deletion of the gltA gene encoding citrate synthase have been previously reported in the literature (Ooyen et al., Biotechnol. Bioeng., 109(8): 2070-2081, 2012). However, these gltA gene deletion strains have the disadvantage that not only their growth is inhibited, but also the rate of sugar accumulation is significantly reduced, thereby leading to low lysine production per unit time. Accordingly, there remains a need for studies that can address both effective enhancement of L-amino acid production capacity and strain growth.

Technical problem

The present inventors have confirmed that when a new modified polypeptide is used in which the citrate synthase activity is weakened to a certain level, then the amount of L-amino acids produced is increased, thus completing the present invention.

Technical solution

One object of the present invention is to provide a modified polypeptide having citrate synthase activity, wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid.

Another object of the present invention is to provide a polynucleotide encoding this modified polypeptide.

Another object of the present invention is to provide a vector comprising this polynucleotide.

Another object of the present invention is to provide an L-amino acid producing microorganism including a modified polypeptide, a polynucleotide encoding the modified polypeptide, or a vector including the polynucleotide.

Another object of the present invention is to provide a method for producing an L-amino acid, comprising culturing in a medium of said microorganism comprising the modified polypeptide, a polynucleotide encoding the modified polypeptide, or a vector comprising the polynucleotide.

Beneficial Effects

When an L-amino acid-producing microorganism of the genus Corynebacterium in which the substrate citrate synthase activity is modified is cultured, then the L-amino acid can be produced in high yield compared to a microorganism bearing an existing unmodified polypeptide.

Detailed Description of Preferred Embodiments

The present invention is described in detail below. At the same time, the corresponding descriptions and embodiments disclosed herein may also be applied to suitably other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention are included within the scope of the present invention. Moreover, the scope of the present invention is not limited to the specific description given below.

In one aspect of the present invention, a modified polypeptide having citrate synthase activity may be provided wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid.

In the present invention, the amino acid sequence of SEQ ID NO: 1 may refer to an amino acid sequence having citrate synthase activity, and in particular may refer to a protein sequence having citrate synthase activity encoded by the gltA gene. The amino acid sequence of SEQ ID NO: 1 can be obtained from GenBank at NCBI (National Center for Biotechnology Information), which is a well-known database. For example, the amino acid sequence of SEQ ID NO: 1 may be derived from, but is not limited to, Corynebacterium glutamicum, and may include any amino acid sequence of a protein having the same activity as that of a protein including, but not limited to, the above amino acid sequence. In addition, the protein having citrate synthase activity of the present invention may be a protein comprising the amino acid sequence of SEQ ID NO: 1, and may include a mutation that may arise due to the insertion of an antisense sequence upstream or downstream of the amino acid sequence of SEQ ID NO: naturally occurring mutation or silent mutation, and one skilled in the art will understand that any protein having the same or corresponding activity as a protein comprising the amino acid sequence of SEQ ID NO: 1 may be included among the proteins having citrate synthase activity according to the present invention. In a specific example, the protein having citrate synthase activity of the present invention may be a protein comprising the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence having homology or identity of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more of the amino acid sequence SEQ ID NO: 1. It is further understood that any protein having an amino acid sequence in which a portion of the amino acid sequence is deleted, modified, substituted or inserted also may be among the proteins targeted for modification according to the present invention, provided that the protein has such homology or identity and exhibits an action corresponding to the action of the above protein.

As used herein, the term "citrate synthase (CS)" refers to the enzyme that produces citrate by catalyzing the condensation of acetyl-CoA and oxaloacetate that are produced during glycolysis of a microorganism, and it is an important enzyme that determines carbon flux in the tricarboxylic acid (TCA) cycle. . In particular, citrate synthase may act as a rate regulator in the first step of the tricarboxylic acid cycle as an enzyme for citrate synthesis. In addition, citrate synthase catalyzes the condensation reaction of a two-carbon acetate moiety from acetyl-CoA and a 4-carbon oxaloacetate molecule to form a 6-carbon acetate. In the present invention, citrate synthase may be used interchangeably with "citrate synthesis enzyme", "CS", "gltA protein" or "gltA".

As used herein, the term “variant” refers to a polypeptide having at least one amino acid sequence that differs from the described sequence by conservative substitutions and/or modifications such that the functions or properties of the protein are retained. Modified polypeptides differ from the identified sequence by substitution, deletion or insertion of several amino acids. Such variants can typically be identified by modifying one of the above polypeptide sequences and by evaluating the properties of the modified polypeptide. That is, the ability of variants can be enhanced, left unchanged, or reduced compared to the native protein. Such variants can generally be identified by modifying the above polypeptide sequences and by assessing the reactivity of the modified polypeptide. In addition, some variants may include variants in which one or more than one fragment, such as an N-terminal leader sequence or transmembrane domain, has been deleted. Other variants may include variants in which a fragment is removed from the N- and/or C-terminus of the mature 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. For example, a variant may have at least one conservative substitution while retaining at least one biological activity. Such an amino acid substitution can generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residue. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, proline, glycine and tryptophan. Typically, a conservative substitution has little or no effect on the activity of the polypeptide produced.

In addition, variants may also include the 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 of a protein 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 “modified polypeptide having citrate synthase activity” refers to a polypeptide having citrate synthase activity that is attenuated relative to the wild type by replacing a portion of the amino acid sequence of the polypeptide having citrate synthase activity. The present invention may refer to a modified polypeptide that can effectively balance carbon flux by modifying at least one amino acid in the amino acid sequence of the polypeptide having citrate synthase activity, and thus its activity is attenuated relative to that of the wild type.

In particular, in various proteins having citrate synthase activity, the modified polypeptide may be a modified polypeptide in which the amino acid corresponding to position 312 in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid. "Other amino acid" may refer to a different amino acid from the amino acid before the substitution, and it may be any amino acid other than the amino acid before the substitution.

More specifically, in various proteins having citrate synthase activity, the modified polypeptide may be a modified polypeptide in which the methionine corresponding to position 312 in the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid. Methionine may be replaced by any one or more amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine , threonine, tryptophan, tyrosine and valine, and more specifically, it may be, but is not limited to, a modified amino acid replaced by isoleucine.

In addition, not only naturally occurring amino acids, but also non-naturally occurring amino acids may be included among the replacement amino acid residues. Non-naturally occurring amino acids may be, for example, D-amino acids, homo-amino acids, beta-homo-amino acids, N-methyl-amino acids, alpha-methyl-amino acids, unusual amino acids (for example, citrulline, naphthylalanine, etc.) , but are not limited to them. At the same time, in the present invention, by the expression “a specific amino acid is replaced” it is meant that an amino acid other than the amino acid before the replacement has been replaced, although the latter does not indicate that a different amino acid has been replaced.

As used herein, the term “corresponding” refers to an amino acid residue at a position specified in a protein or peptide, or to 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 position in a related protein or reference protein.

The present invention may employ specific numbering of amino acid residue positions in the polypeptide used herein. For example, it is possible to renumber the positions of amino acid residues in a polypeptide of the present invention to corresponding positions by aligning the polypeptide sequence of the present invention with the desired polypeptide being compared.

The modified polypeptide having citrate synthase activity proposed in the present invention may have an improved ability to produce L-amino acid compared to the polypeptide before modification by replacing an amino acid at a specific position in the citrate synthase described above.

The modified polypeptide may have 80% or more sequence homology and less than 100% sequence homology with, but is not limited to, the amino acid sequence of SEQ ID NO: 1. In particular, the modified polypeptide of the present invention may have at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO: 1. It is further understood that to the amino acid sequence at position 312, any protein having an amino acid sequence in which part of that amino acid sequence has been subjected to deletion, modification, substitution or insertion may also fall within the scope of the present invention provided that the protein has such homology and exhibits effects corresponding to the effects the above protein.

In addition, in the present invention, although the specification is "a protein or polypeptide having the amino acid sequence of a particular SEQ ID NO", it is understood that any protein that has a deletion, modification, substitution or insertion of a portion of the amino acid sequence can also be used in the present invention, provided that the protein has an activity substantially the same or equivalent to that of a polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO. For example, in the case where a protein or polypeptide has an activity that is the same or equivalent to that of a modified polypeptide, it is possible that a mutation may occur due to the insertion of an antisense sequence below or above the amino acid sequence corresponding to SEQ ID NO, a naturally occurring mutation, or a silent mutation in addition to a modification at position 312 that provides a particular activity, and it is understood that a protein or polypeptide having such a sequence insertion or mutation is also within the scope of the present invention.

A modified polypeptide in which the amino acid corresponding to position 312 in the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid may include the amino acid sequence of SEQ ID NO: 3. More specifically, a modified polypeptide in which a methionine corresponding to position 312 in the amino acid sequence SEQ ID NO: 1, replaced by isoleucine, may consist of the amino acid sequence of SEQ ID NO: 3. In addition, the modified polypeptide may include an amino acid sequence having 80% or more and less than 100% homology with the amino acid sequence of SEQ ID NO: 3, but is not limited to them. In particular, the modified polypeptide of the present invention may include the amino acid sequence of SEQ ID NO: 3 or a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence of SEQ ID NO: 3. It is further understood that any protein having an amino acid sequence in which a portion of the amino acid sequence is subject to deletion, modification, substitution or insertion, in addition to the amino acid sequence at position 312, can also be used in the present invention, provided that that this protein has such homology and includes an amino acid sequence that exhibits an effect corresponding to that of the above protein.

In the case of a microorganism including a modified polypeptide with weakened citrate synthase activity for the purposes of the present invention, it has the feature that the yield of L-amino acid is increased at a sugar consumption rate similar to the sugar consumption rate of the control. Thus, it can be concluded that the amount of L-amino acids produced can be increased by a suitable balance between the carbon flow into the TCA cycle and the consumed amount of oxaloacetate, used as a precursor for L-amino acid biosynthesis, by regulating the activity of citrate synthase.

As used herein, the term “homology” refers to the percentage of identity between two polynucleotide or polypeptide moieties. It may also refer to the degree of match to a given amino acid sequence or nucleotide sequence, and may be expressed as a percentage. In the description of the present invention, a homologous sequence having an activity that is identical or similar to that of a given amino acid sequence or nucleotide sequence may be indicated in terms of "% homology". Homology between sequences from one group to another can be determined using methods known in the art. For example, homology can be confirmed using standard software for calculating parameters such as score, identity and similarity, in particular BLAST 2.0, or by comparing sequences through Southern hybridization experiments under conditions with a certain stringency. Certain suitable hybridization conditions are within the scope of the art, and can be determined using a method well known to one skilled in the art (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

In another aspect of the present invention, there may be provided a polynucleotide encoding a modified polypeptide having citrate synthase activity, wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid.

The amino acid sequence of SEQ ID NO: 1, citrate synthase, and modified polypeptide are as described above.

As used herein, the term "polynucleotide", which is a polymer of nucleotides consisting of nucleotide monomers linked in a chain by covalent bonds, is a DNA or RNA chain of at least a certain length, and more specifically may refer to a fragment of a polynucleotide, encoding option.

The polynucleotide of the present invention may include, without limitation, any polynucleotide sequence encoding a modified polypeptide of the present invention that has citrate synthase activity. In the present invention, the gene encoding the amino acid sequence of citrate synthase may be a gltA gene, and the gene may be derived from, but is not limited to, Corynebacterium glutamicum. Moreover, the gene may be a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, and more specifically, it may be a sequence including, but not limited to, the nucleotide sequence of SEQ ID NO: 2.

In particular, the polynucleotide of the present invention may undergo various modifications in the coding region to the extent that the amino acid sequence of the polypeptide is not altered due to codon degeneracy or to account for codons that are preferred in the organism in which the polypeptide is to be expressed. In particular, any polynucleotide sequence encoding a modified polypeptide in which the amino acid corresponding to position 312 in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid may be included without limitation. For example, the modified polypeptide of the present invention may be, but is not limited to, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. More specifically, the modified polypeptide of the present invention may be a polypeptide that consists of, but is not limited to, the polynucleotide sequence of SEQ ID NO: 4.

Additionally, without limitation, a probe may be included that may be derived from a known gene sequence, for example, any sequence that can hybridize under stringent conditions to a sequence complementary to all or part of a nucleotide sequence, to encode a protein having citrate synthase activity, in which the amino acid at position 312 in the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid. The term "strict conditions" refers to conditions under which specific hybridization between polynucleotides is permitted. Such conditions are specifically described in the literature (eg, J. Sambrook et al., supra). For example, stringent conditions may include conditions in which genes having high homology or identity of 40% or greater, particularly 90% or greater, more particularly 95% or greater, more particularly 97% or greater, even more particularly 99% or greater, hybridize with each other, and genes having homology or identity lower than the above homologies or identities do not hybridize with each other, or the usual washing conditions for Southern hybridization (i.e., washing once, in particular two or three times at salt concentration and temperature corresponding to 60°C, 1 × SSC (sodium citrate and chloride solution), 0.1% SDS (sodium dodecyl sulfate), in particular 60°C, 0.1 × SSC, 0.1% SDS, and more specifically 68°C, 0.1 x SSC, 0.1% SDS).

Hybridization requires that the two nucleic acids contain complementary sequences, although base-pairing errors are possible depending on the stringency of the hybridization conditions. The term "complementary" is used to describe the interaction between nucleotide bases that can hybridize with each other. For example, in the case of DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present invention may include isolated nucleic acid fragments complementary to the entire sequence, as well as to nucleic acid sequences substantially similar thereto.

In particular, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a T _m value of 55°C under the conditions described above. In addition, the T _m value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be suitably adjusted by one skilled in the art depending on the application.

The appropriate stringency of conditions for polynucleotide hybridization depends on the length of the polynucleotides and the degree of complementarity, and these variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

In another aspect of the present invention, a vector may be provided comprising a polynucleotide encoding a modified polypeptide having citrate synthase activity, wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid.

The amino acid sequence of SEQ ID NO: 1, citrate synthase, modified polypeptide and polynucleotide are as described above.

As used herein, the term “vector” refers to a DNA product containing the nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operably linked to a suitable control sequence for expression of the desired polypeptide in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable ribosome binding site on the mRNA, and sequences regulating the termination of transcription and translation. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may itself be integrated into the genome.

The vector used in 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, Charon21A, etc. can be used as a phage vector or cosmid vector, and pBR-based vectors can be used as a plasmid vector, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. In particular, the vectors pCR2.1, pDC, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC and the like can be used.

The vector that can be used in the present invention is not particularly limited, and any known expression vector can be used. In addition, a polynucleotide encoding a desired polypeptide can be inserted into a chromosome using an intracellular chromosomal insertion vector. Incorporation of a polynucleotide into a chromosome can be accomplished without limitation by any method known in the art (eg, homologous recombination). In addition, 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. Markers conferring selective phenotypes such as drug resistance, nutrient requirements, resistance to cytotoxic agents and surface protein expression can be used. When treated with a selection agent, only cells expressing the selectable markers can survive or exhibit other phenotypic traits, 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 desired protein into a host cell such that the protein encoded by the polynucleotide can be 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 and located in the chromosome of the host cell, or located outside the chromosome, and both cases can be included.

In addition, the polynucleotide may include DNA and RNA encoding the desired protein.

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 the necessary elements required for its autonomous expression. An expression cassette may conventionally comprise a promoter operably linked to a polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, without limitation, the polynucleotide can be introduced into a host cell by itself and operably linked to a sequence required for its expression in the host cell. The transformation method includes any method of introducing a polynucleotide into a cell and can be carried out by selecting a suitable standard method known in the art depending on the host cell. For example, the method may include electroporation, calcium phosphate (Ca( _H2PO4 ) ₂ , _CaHPO4 or _Ca3 ( _PO4 ) ₂ ) precipitation, calcium _chloride ( _CaCl2 ) precipitation, microinjection, polyethylene glycol (PEG) method, the DEAE (diethylaminoethyl)-dextran method, the cationic liposome method, the lithium acetate-DMSO (dimethyl sulfoxide) method, and the like, but are not limited to them.

In addition, as used herein, the term "operably linked" may mean that the polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the desired protein of the present invention. Functional linking can be carried out using a gene recombination method known in the art and site-specific DNA cleavage, and linking can be carried out using cleavage and ligation enzymes known in the art, etc., but functional linking is not limited to them.

In yet another aspect of the present invention, there may be provided an L-amino acid producing microorganism comprising a modified polypeptide having citrate synthase activity, wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid, a polynucleotide encoding the modified polypeptide, and a vector including the polynucleotide.

The amino acid sequence of SEQ ID NO: 1, citrate synthase, modified polypeptide, nucleotide and vector are as described above.

The microorganism may be a microorganism that includes, but is not limited to, a polynucleotide encoding a modified polypeptide or that is transformed with a vector including a polynucleotide encoding a modified polypeptide.

In addition, the microorganism may have an improved ability to produce L-amino acids without inhibiting the growth or rate of sugar consumption of the microorganism compared to a microorganism comprising a wild-type polypeptide. Thus, L-amino acid can be obtained in high yield from these microorganisms.

As used herein, the term “microorganism comprising a modified polypeptide” refers to a microorganism that naturally has little ability to produce L-amino acids, or a microorganism that is endowed with the ability to produce L-amino acids, the parent strain of which does not have the ability to produce L-amino acids. In particular, the microorganism is a microorganism expressing a modified polypeptide including at least one amino acid modification in the polypeptide having citrate synthase activity, and the amino acid modification may include substitution of the amino acid corresponding to position 312 from the N-terminus of the amino acid sequence SEQ ID NO: 1, other amino acid. A modified polypeptide having citrate synthase activity expressed by microorganisms may, but is not limited to, have reduced activity.

The microorganism may be a cell or microorganism that includes a polynucleotide encoding the modified polypeptide, or which is transformed with a vector including a polynucleotide encoding the modified polypeptide, such that the modified polypeptide can be expressed. Any host cell or microorganism can be used for the purposes of the present invention, provided that it can produce the L-amino acid by incorporating the modified polypeptide.

As used herein, the term "L-amino acid producing microorganism" includes all natural or artificially genetically modified microorganisms, and it may be a microorganism in which a particular mechanism is enhanced or weakened due to the insertion of a foreign gene or the enhancement or reduction of the activity of an endogenous gene, and for the purpose of L-amino acid production, it may be a microorganism in which genetic modification has been carried out or activity has been weakened. For purposes of the present invention, an L-amino acid producing microorganism may refer to a microorganism that includes a modified polypeptide such that it can produce the desired L-amino acid in excess from a carbon source in the medium compared to a wild type or unmodified microorganism. In the description of the present invention, "L-amino acid-producing microorganism" may be used interchangeably with "microorganism having the ability to produce L-amino acid" or "L-amino acid-producing microorganism".

The L-amino acid produced by the L-amino acid producing microorganism may be any one or more than one selected from the group consisting of, but is not limited to, leucine, lysine, valine, isoleucine and o-acetylhomoserine.

Specific examples of the L-amino acid producing microorganism may include a microorganism strain of the genus Escherichia, Serratia, Erwinia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Corynebacterium, and the like. and, in particular, a microorganism of the genus Corynebacterium, and more specifically, but not limited to Corynebacterium glutamicum.

In particular, the L-amino acid producing microorganism may be strain CJL-8100, which has the ability to produce L-leucine by introducing an isopropyl maleate synthase variant [leuA_(R558H, G561D)] into a microorganism of the genus Corynebacterium, Corynebacterium CJ3P (Binder et al., Genome Biology 2012, 13:R40), which has the ability to produce L-lysine by introducing three mutations [pyc(P458S), hom(V59A), lysC(T311I)] into microorganisms of the genus Corynebacterium, Corynebacterium KCCM11201P, which is a strain producing valine (US 8465962 B2), Corynebacterium KCCM11248P, which is a strain producing L-isoleucine (Korean patent No. 10-1335789), or Corynebacterium glutamicum, which has the ability to produce O-acetylhomoserine due to the deletion of the metB gene encoding cystathionine gamma synthase included into the O-acetylhomoserine degradation pathway, and the metY gene encoding O-acetylhomoserine (thiol)-lyase in the microorganism of the genus Corynebacterium, and by introducing a mutation (L377K) (US 10662450 B2) to release feedback inhibition for L-lysine and L-threonine from the lysC gene encoding aspartate kinase to increase, but is not limited to, the biosynthesis of O-acetylhomoserine. For purposes of the present invention, the L-amino acid producing microorganism may also include a modified polypeptide to increase the ability to produce the desired L-amino acid.

As used herein, "microorganism of the genus Corynebacterium" may include, but is not necessarily limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, and the like. More specifically, the microorganism of the genus Corynebacterium of the present invention may be Corynebacterium glutamicum, in which the L-amino acid yield is increased in the presence of a similar rate of sugar consumption, although the citrate synthase activity is weakened compared to the unmodified microorganism.

In another aspect of the present invention, there may be provided a method for producing L-amino acid, comprising:

cultivation in a microorganism medium comprising a modified polypeptide having citrate synthase activity, wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid, a polynucleotide encoding the modified polypeptide, or a vector comprising this polynucleotide.

The amino acid sequence of SEQ ID NO: 1, citrate synthase, modified polypeptide, polynucleotide, vector and microorganism are as described above.

The method can be easily determined by one skilled in the art using optimized culture conditions and enzymatic activity conditions known in the art. In particular, the microorganism can be cultivated by, but is not particularly limited to, known batch culture, continuous culture, fed-batch culture and the like. In particular, the culture conditions are not particularly limited, but the pH (for example, pH 5 - pH 9, in particular, pH 6 - pH 8, and more specifically, pH 6.8) can be suitably adjusted using a basic compound (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (such as phosphoric acid or sulfuric acid). Aerobic conditions can be maintained by adding oxygen or an oxygen-containing gas mixture to the culture. The temperature in the culture can be maintained at 20°C to 45°C, and specifically at 25°C to 40°C, and cultivation can be carried out for about 10 to 160 hours, but the culture conditions are not limited to them. L-amino acid produced in culture may be secreted into the medium or may remain within the cells.

In addition, the culture medium used contains carbon sources such as, but not limited to, sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (e.g., soybean oil, sunflower oil , peanut butter and coconut oil), fatty acids (eg palmitic acid, stearic acid and linoleic acid), alcohols (eg glycerin and ethanol) and organic acids (eg acetic acid), may be used alone or in combination ; nitrogen sources such as, without limitation, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, liquid corn extract, soybean meal, and urea) or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be used alone or in combination; and sources of potassium, such as, without limitation, potassium dihydrogen phosphate, potassium hydrogen phosphate, or corresponding sodium salts, can be used alone or in combination. In addition, other necessary growth promoting substances, including metal salts (eg, magnesium sulfate or ferrous sulfate), amino acids and vitamins may be contained in the medium, but the necessary growth promoting substances are not limited to them.

The method may further include, but is not limited to, the step of isolating L-amino acids from the culture medium or microorganism after the culture step.

In the method for isolating L-amino acid produced in the culture step, it is possible to collect the desired amino acid from the culture solution using a suitable method known in the art, according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC (high performance liquid chromatography) and the like can be used, and the desired L-amino acid can be isolated from the medium or microorganism using a suitable method known in the art.

In addition, the isolation step may include a purification process, and the purification process may be carried out using a suitable method known in the art. Thus, the released L-amino acid may be in a purified form or a microbial fermentation medium containing the L-amino acid.

The L-amino acid produced may be any one or more amino acids selected from the group consisting of, but is not limited to, leucine, lysine, valine, isoleucine and o-acetylhomocysteine.

In another aspect of the present invention, a composition for producing L-amino acid may be provided, comprising a microorganism of the genus Corynebacterium containing a modified polypeptide having citrate synthase activity, or a culture thereof.

The microorganism may be, but is not limited to, a microorganism of the genus Corynebacterium, or in particular Corynebacterium glutamicum. The microorganism is as described above

A composition for producing L-amino acid may mean a composition capable of producing L-amino acid using a modified polypeptide of the present invention that has citrate synthase activity. The composition may include, without limitation, a modified polypeptide having citrate synthase activity or a configuration capable of controlling a modified polypeptide having citrate synthase activity. The modified polypeptide having citrate synthase activity may be in a form included in a vector so as to express a gene operably linked in the host cell into which it is introduced.

The composition may further include a cryoprotectant or excipient. The cryoprotectant or excipient may be, but is not limited to, a non-natural substance or a natural substance. In yet another embodiment, the cryoprotectant or excipient may be, but is not limited to, a substance that does not naturally come into contact with a microorganism, or a substance that does not naturally coexist with a microorganism.

In another aspect of the present invention, the use of a microorganism of the genus Corynebacterium that includes a modified polypeptide having citrate synthase activity for the production of L-amino acid can be provided.

Method for implementing the invention

Next, the present invention will be described in detail with the help of examples. However, these examples are provided for illustrative purposes only and are not intended to limit the scope of the description of the present invention.

Example 1: Discovery of the gltA mutation

1-1. Construction of a vector including gltA

To construct a gltA mutation library with citrate synthase activity, a recombinant vector containing a portion of gltA was first constructed. The amino acid sequence and nucleotide sequence of wild-type gltA are the same as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. PCR was performed using the chromosomal DNA of the wild-type strain Corynebacterium glutamicum as a template along with primers SEQ ID NO: 5 and SEQ ID NO: 6, and the amplified product was cloned into the E. coli vector pCR2.1 using the TOPO cloning kit (Invitrogen) to obtain pCR-gltA.

1-2. Construction of a library of gltA mutations

A gltA mutation library was constructed based on the vector constructed in Example 1-1. The library was constructed using a reduced fidelity PCR kit (clontech Diversify ^® PCR Random Mutagenesis Kit). The PCR reaction was carried out using SEQ ID NO: 5 and SEQ ID NO: 6 as primers under conditions where mutation can occur. Specifically, PCR was performed by preheating at 94°C for 30 seconds, followed by 25 cycles of denaturation at 94°C for 30 seconds and polymerization at 68°C for one and a half minutes under conditions in which 0 to 3 mutations occurred per 1000 bp The PCR products thus obtained were used as a megaprimer (from 500 ng to 125 ng), which was subjected to 25 cycles of denaturation at 95°C for 50 seconds, annealing at 60°C for 50 seconds, and polymerization at 68°C for 12 minutes, treated with DpnI and transformed into E. coli DH5α, and the transformed E. coli DH5α were plated on solid LB medium (lysogenic medium) containing kanamycin (25 mg/L). 20 types of transformed colonies were selected, and the plasmids obtained from them were analyzed by polynucleotide sequencing. As a result, it was confirmed that mutations were obtained at sites different from each other, with a frequency of 2 mutations/bp. As a result, approximately 20,000 transformed E. coli colonies were selected and plasmids were extracted from them and named the pTOPO-gltA library.

The primers used in this example are presented in Table 1 below.

Example 2: Evaluating the Constructed Library and Selecting Options

The pTOPO-gltA library constructed in Example 1-2 was transformed into Corynebacterium glutamicun ATCC13032 by electroporation, and then plated on culture plates containing 25 mg/L kanamycin to obtain colonies of 10,000 strains into which the mutant gene was inserted, and each colony was named ATCC13032/pTOPO_gltA(mt)1 to ATCC13032/pTOPO_gltA(mt) 10000, respectively.

An assessment of the fermentation titer for each colony to identify colonies with increased leucine production among the resulting 10,000 colonies was carried out as follows.

- Production medium: glucose 100 g, (NH ₄ ) ₂ SO ₄ 40 g, soy protein 2.5 g, corn extract 5 g, urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ ⋅7H ₂ O 0, 5 g, biotin 100 mcg, thiamine HCl 1000 mcg, calcium-pantothenic acid 2000 mcg, nicotinamide 3000 mcg, CaCO ₃ 30 g (based on 1 l of distilled water), pH 7.0

Each colony was inoculated using a platinum loop into a corner septum vial containing 25 μg/ml kanamycin in 25 ml autoclaved production medium and cultured with shaking at 30°C at 200 rpm for 60 hours. After completion of cultivation, the amount of leucine produced was measured by a high-performance liquid chromatography (HPLC) method to select one strain having the most improved leucine-producing ability compared with the wild-type Corynebacterium glutamicum strain. The concentration of leucine produced in the selected strains is presented in Table 2 below.

PCR was then performed to confirm the genetic mutation in the mutant strain using primers SEQ ID NO: 7 and SEQ ID NO: 8 based on strain ATCC13032/pTOPO_gltA(mt)3142, followed by sequencing to compare the gltA gene with wild type ATCC13032. As a result, it was confirmed that the above strain contained a mutation in the gltA gene.

In particular, it was confirmed that G, which was the 936th nucleotide of the gltA gene in strain ATCC13032/pTOPO_gltA(mt)3142, was replaced by C. The latter is a mutation in which the methionine at position 312 in the gltA amino acid sequence is replaced by isoleucine. Thus, the following examples attempt to establish whether this mutation has an effect on the amount of leucine produced in microorganisms of the genus Corynebacterium.

Example 3: Confirmation of the leucine-producing ability of the selected gltA mutant

3-1. Construction of an insertion vector containing the gltA mutation

To introduce the mutation selected in Example 2 into the strain, an attempt was made to construct an insertion vector. The vector for introducing the gltA(M312I) mutation was constructed using the site-directed mutagenesis method. PCR was performed using the chromosomal DNA of the wild type strain Corynebacterium glutamicum as a template along with the primer pair SEQ ID NOs: 9 and 10 and the primer pair SEQ ID NOs: 11 and 12. PCR was performed by denaturation at 94°C for 5 minutes, and then 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for one and a half minutes, and then polymerization at 72°C for 5 minutes. As a result, vector pDC was constructed, in which the resulting gene fragment was cleaved with the restriction enzyme SmaI, and vector pDC-gltA(M312I), in which methionine, which is the 312th amino acid, was replaced by isoleucine by linking and cloning a homologous sequence of 15 nucleotides at the ends of DNA fragments using a fusion enzyme.

3-2. Introduction of a variant into ATCC13032 and its evaluation

The vector pDC-gltA (M312I) constructed in Example 3-1 was transformed into ATCC13032, and the strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in medium containing 25 mg/L kanamycin. The selected primary strain was again subjected to a secondary crossover and a strain was selected into which the mutation of the desired gene was introduced. Introduction of the gltA gene mutation into the finally transformed strain was confirmed by performing PCR using primers SEQ ID NO: 7 and SEQ ID NO: 8, and then analyzing the basic sequence, thereby confirming that the mutation was introduced into the strain. The constructed strain was named ATCC13032_gltA_M312I.

To evaluate the leucine-producing ability of the ATCC13032_gltA_M312I strain constructed above, flask fermentation titer was also assessed. Corynebacterium glutamicum ATCC13032, which is the parent strain, and ATCC13032_gltA_M312I constructed above were each inoculated using a platinum loop into a 250 ml corner baffle flask containing 25 ml of production medium of Example 2, and cultured with shaking at 200 rpm at 30°C for 60 hours to produce leucine. After completion of cultivation, the amount of leucine produced was measured using HPLC. The concentration of leucine in the culture medium for each strain tested is presented in Table 3 below.

Example 4: Confirmation of Leucine-Producing Capacity of Selected gltA Mutation in Leucine-Producing Strains

Although the wild-type strain of the genus Corynebacterium produces leucine, only trace amounts are produced. Accordingly, a leucine-producing strain derived from ATCC13032 was constructed, and an experiment was performed to confirm the leucine-producing ability by introducing a selected mutation. The specific experiment is given below.

4-1. Construction of leucine-producing strain CJL-8100

To construct a high leucine producing strain derived from ATCC13032, an isopropyl malate synthase introduced variant of strain CJL-8100 (hereinafter referred to as “IPMS”) was constructed. The mutation includes a mutation in which G, which is the 1673rd nucleotide of the leuA gene encoding IPMS, is replaced by A, so that arginine, which is the 558th amino acid of the IPMS protein, is replaced by histidine, and a mutation in which GC, which is 1682 The -th and 1683rd nucleotides are replaced by AT, so that glycine, which is the 561st amino acid, is replaced by aspartic acid.

The pDC-leuA (R558H, G561D) vector containing the leuA mutation was transformed into ATCC13032, and the strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in medium containing 25 mg/L kanamycin. The selected primary strain was again subjected to a secondary crossover, and a strain was selected into which the leuA gene mutation was introduced. Introduction of the mutation into the finally transformed strain was confirmed by performing PCR using primers SEQ ID NO: 13 and SEQ ID NO: 14, and then analyzing the basic sequence, thereby confirming that the mutation had been introduced. Strain ATCC13032_leuA_(R558H, G561D) transformed with the vector pDC-leuA (R558H, G561D) was named CJL-8100.

4-2. Introduction of the gltA variant into strain CJL-8100 and its evaluation

CJL-8100, which is a leucine-producing strain, was transformed with the vector pDC-gltA (M312I) constructed in Example 3-1, and a strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg/ l kanamycin. The selected primary strain was again subjected to a secondary crossover and a strain was selected into which the mutation of the desired gene was introduced. The introduction of the gltA gene mutation into the finally transformed strain was confirmed by performing PCR using primers SEQ ID NO: 7 and SEQ ID NO: 8, and then analyzing the basic sequence, thereby confirming that the gltA mutation was introduced into the strain. The constructed CJL8100_gltA_M312I was named CA13-8104 and deposited at the Korea Center for Microbial Culture (KCCM), which is the Authorized International Depository of the Budapest Agreement, on December 20, 2019 under the identification number KCCM 12649P.

The leucine-producing ability of the strain CA13-8104 constructed above was assessed. Culture in the flask was carried out in the same manner as in Example 2, and after completion of the culture, the amount of leucine produced was measured by a method using HPLC, and the culture results are presented in Table 4 below.

As shown in Table 4, it was confirmed that the leucine-producing strain Corynebacterium glutamicum CJL8100 significantly improved the leucine-producing ability compared with the parent strain Corynebacterium glutamicum ATCC13032. In addition, strain CJL8104, a leucine-producing strain resulting from the introduction of the gltA M312I mutation into Corynebacterium glutamicum strain CJL8100, was confirmed to have a 10% improved leucine-producing ability compared with the parental strain CJL8100.

In addition, the leucine-producing strain CJL-8100 was transformed by electroporation with pTOPO-gltA(mt)3142 from the pTOPO-gltA library constructed in Example 1-2, and then the vector-transformed strain was selected in a medium containing 25 mg/L kanamycin . The selected strain was named CJL8100/pTOPO_gltA(mt)3142.

The leucine-producing ability of the above-constructed strain CJL8100/pTOPO_gltA(mt)3142 was assessed. Culture in the flask was carried out in the same manner as in Example 2, and after completion of the culture, the amount of leucine produced was measured by a method using HPLC, and the culture results are presented in Table 5 below.

The results of the above example can confirm that the amino acid at position 312 of the amino acid sequence of gltA, which is a citrate synthase, is an important position for the activity of the gltA enzyme.

Example 5: Construction of the CJ3P strain into which the gltA mutant strain (M312I) was introduced and analysis of the amount of lysine produced

To test whether the effect of changing citrate synthase activity occurs even in a strain belonging to Corynebacterium glutamicum that produces L-lysine, a strain into which the gltA mutation (M312I) was introduced was constructed based on Corynebacterium glutamicum CJ3P (Binder et al., Genome Biology 2012, 13:R40), which has L-lysine-producing ability, by introducing three types of mutations into wild-type strains [rus (P458S), hom (V59A), lysC (T311I)] in the same way as in Example 3. The strain thus constructed was named CJ3P::gltA (M312I). In strain CJ3P, which represents the control group, and in CJ3P::gltA (M312I), the amount of lysine produced was measured as follows. First, each strain was inoculated into a 250-ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Then, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium and cultured with shaking at 200 rpm at 32°C for 72 hours. Compositions of inoculation medium and production medium are presented below. After completion of culture, L-lysine concentration was measured using HPLC (Waters 2478) and the results are presented in Table 6.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ ⋅ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 mcg, calcium pantothenic acid 2000 mcg, nicotinamide 2000 mcg (based on 1 liter of distilled water)

<Production medium (pH 7.0)>

Glucose 100 g, (NH ₄ ) ₂ SO ₄ 40 g, soy protein 2.5 g, corn extract 5 g, urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ ⋅7H ₂ O 0.5 g, biotin 100 mcg, thiamine HCl 1000 mcg, calcium pantothenic acid 2000 mcg, nicotinamide 3000 mcg, CaCO ₃ 30 g (based on 1 liter of distilled water).

As can be seen from Table 6, strain CJ3P::gltA(M312I), in which the gltA(M312I) mutation was introduced into Corynebacterium glutamicum CJ3P, which is a lysine-producing strain, there was an increase in the amount of lysine produced up to 127% compared with the parental strain.

Example 6: Confirmation of the valine-producing ability of a selected gltA mutation in a valine-producing strain

To test whether the selected mutation has an effect on valine, which is a typical branched-chain amino acid like leucine, a valine-producing ability was confirmed by introducing the selected mutation into the valine-producing strain KCCM11201P (US 8465962 B2) of the genus Corynebacterium .

KCCM11201P was transformed with the vector pDC-gltA (M312I) constructed in Example 3-1, and the strain in which the vector was integrated into the chromosome was selected by recombination of the homologous sequence in a medium containing 25 mg/L kanamycin. The selected primary strain was again subjected to a secondary crossover and a strain was selected into which the mutation of the desired gene was introduced. The introduction of the gltA gene mutation into the finally transformed strain was confirmed by performing PCR using primers SEQ ID NO: 7 and SEQ ID NO: 8, and then analyzing the basic sequence, thereby confirming that the gltA mutation was introduced into the strain. The strain constructed in this way was named KCCM11201P - gltA(M312I).

The valine-producing ability of the KCCM11201P-gltA(M312I) strain constructed above was assessed. The culture in the flask was carried out in the same way as in Example 2-2, and after completion of the culture, the amount of valine produced was measured by a method using HPLC, and the culture results are shown in Table 7 below.

As shown in Table 7, it was confirmed that strain KCCM11201P-gltA(M312I), in which the gltA M312I mutation was introduced into Corynebacterium glutamicum strain KCCM11201P, which is a valine-producing strain, had a 7% increased valine-producing capacity compared with KCCM11201P, which is the parent strain.

The above results can confirm that the amino acid at position 312 of the amino acid sequence of gltA, which is a citrate synthase, is an important position for the activity of gltA enzyme.

Example 7: Confirmation of the L-isoleucine-producing ability of a selected gltA mutation in an isoleucine-producing strain

7-1. Construction of an L-isoleucine strain introducing the gltA(M312I) ORF (open reading frame) mutation into the L-isoleucine-producing strain Corynebacterium glutamicum KCCM11248P, and evaluation of its L-isoleucine-producing ability

The strain for which the recombinant plasmid pDC-gltA (M312I) constructed in Example 3-1 was introduced into Corynebacterium glutamicum KCCM11248P, which is an L-isoleucine-producing strain (Korean Patent No. 10-1335789), was obtained by homologous recombination on the chromosome in the same manner as in Example 4 and named KCCM11248P::gltA(M312I). The strains thus constructed were cultured as follows to compare the isoleucine-producing ability.

Each strain was inoculated into a 250 ml corner baffled flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Then, 1 ml of seed medium solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium and cultured with shaking at 200 rpm at 30°C for 48 hours. The compositions of inoculation medium and production medium are presented below.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH ₂ PO ₄ 4 g, K ₂ HPO ₄ 8 g, MgSO ₄ ⋅ 7H ₂ O 0.5 g, biotin 100 μg, thiamine HCl 1000 mcg, calcium pantothenic acid 2000 mcg, nicotinamide 2000 mcg (based on 1 liter of distilled water)

<Production medium (pH 7.0)>

Glucose 50 g, (NH ₄ ) ₂ SO ₄ 12.5 g, soy protein 2.5 g, corn extract 5 g, urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ ⋅7H ₂ O 0.5 g, biotin 100 mcg, thiamine HCl 1000 mcg, calcium-pantothenic acid 2000 mcg, nicotinamide 3000 mcg, CaCO ₃ 30 g (based on 1 liter of distilled water)

After completion of cultivation, L-isoleucine-producing capacity was measured by HPLC. The concentration of L-isoleucine in the culture solution for each of the tested strains is presented in Table 8 below.

As shown in Table 8, it was confirmed that the concentration of L-isoleucine was increased by approximately 36% in KCCM11248P::gltA (M312I), which was introduced with the gltA mutation (M312I), compared to the L-isoleucine-producing strain KCCM11248P. Based on this result, it was confirmed that L-isoleucine-producing capacity could be improved by mutation in the gltA gene (M312I).

The above results indicate that introduction of the gltA mutation (M312I) into an L-isoleucine-producing strain of the genus Corynebacterium is effective in producing L-isoleucine.

7-2. Construction of an L-isoleucine strain with the introduced gltA(M312I) ORF mutation into the wild-type Corynebacterium glutamicum strain ATCC13032, and assessment of its L-isoleucine-producing ability

To confirm the effect of introducing the gltA(M312I) mutation on L-isoleucine-producing ability, a strain was constructed based on the Corynebacterium glutamicum strain ATCC13032 (hereinafter WT), into which the lysC(L377K) variant (Korean patent No. 10-2011994) and the hom( R407H), and the ilvA mutation (V323A) (Appl. Enviro. Microbiol., Dec. 1995, p. 4315-4320) were introduced into the gene encoding a known threonine dehydratase (L-threonine dehydratase) to compare L-isoleucine-producing ability.

PCR was performed using WT chromosomal DNA as a template with primers SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18. The sequences of the primers used are shown in Table 9 below.

PCR was carried out under PCR denaturation conditions at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds. and then polymerization at 72°C for 7 minutes. As a result, a DNA fragment of 509 bp was obtained. to the 5' region above and a DNA fragment of 520 bp. 3' downstream around the lysC gene mutation.

Using the two amplified DNA fragments as templates, PCR was performed with primers SEQ ID NO: 15 and 18 by denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55° C for 30 seconds, and cure at 72°C for 60 seconds, and then cure at 72°C for 7 minutes. As a result, a 1011 bp DNA fragment was amplified containing a mutation in the lysC gene encoding an aspartokinase variant in which the 377th leucine was replaced by lysine.

Vector pDZ (Korean Patent No. 0924065), which cannot replicate in Corynebacterium glutamicum, and 1011 bp DNA fragments. treated with restriction enzyme XbaI, ligated using DNA ligation enzyme and then cloned to obtain a plasmid, which was named pDZ-lysC (L377K).

The above vector pDZ-lysC (L377K) was introduced into the WT strain using the electrical pulse method (Appl. Microbiol. Biothcenol. (1999, 52:541-545)) and then the transformed strain was obtained in selective medium containing 25 mg/ l kanamycin. By secondary crossover, WT::lysC (L377K) was obtained, which is a strain in which a nucleotide mutation was introduced into the lysC gene by inserting a DNA fragment into the chromosome. The gene into which the nucleotide mutation was introduced was finally confirmed by PCR using primers SEQ ID NO: 15 and 18, and then by sequencing, and the sequence was compared with the sequence of the wild type lysC gene.

Additionally, to construct a vector into which hom(R407H) was introduced, PCR was performed using WT genomic DNA as a template with primers SEQ ID NO: 19 and 20 and SEQ ID NO: 21 and 22. The sequences of the primers used are shown in Table 10 below.

PCR was carried out under PCR denaturation conditions at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds. and then polymerization at 72°C for 7 minutes. As a result, a DNA fragment of 220 bp was obtained. to the 5' region above and a DNA fragment of 220 bp. 3' region downstream around the hom gene mutation. Using the two amplified DNA fragments as templates, PCR was carried out using primers with SEQ ID NO: 5 and 8. PCR was carried out under PCR amplification conditions by denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C in for 30 seconds, annealing at 55°C for 30 seconds, and polymerizing at 72°C for 30 seconds, and then polymerizing at 72°C for 7 minutes. As a result, a 440 bp DNA fragment containing a mutation of the hom gene was amplified.

Vector pDZ used above and 440 bp DNA fragments. treated with restriction enzyme XbaI, ligated using DNA ligation enzyme and then cloned to obtain a plasmid, which was named pDZ-hom(R407H).

The above vector pDZ-hom(R407H) was introduced into the WT::lysC(L377K) strain using the electrical pulse method, and then the transformed strain was obtained in selective medium containing 25 mg/L kanamycin. By secondary crossover, WT::lysC(L377K)-hom(R407H) was obtained, which is a strain into which a nucleotide mutation was introduced into the hom gene by inserting a DNA fragment into the chromosome.

A strain in which the recombinant plasmid pDC-gltA (M312I) constructed in Example 3-1 was introduced into the WT::lysC(L377K)-hom (R407H) strain was obtained by homologous recombination on the chromosome in the same manner as in the above Example , and named WT::lysC(L377K)-hom(R407H)-gltA(M312I).

A pair of primers (SEQ ID NOs: 23 and 24) to amplify the 5' region upstream and a pair of primers (SEQ ID NOs: 25 and 26) to amplify the 3' region downstream around the mutation site were designed to construct a vector into which the known ilvA mutation was introduced ( V323A), based on the ilvA gene. Primers with SEQ ID NOs: 23 and 26 were inserted using a BamHI restriction enzyme site (indicated by an underline) at each end, and primers with SEQ ID NOs: 24 and 25 were designed to crossover with each other so as to position mutations by nucleotide sequences. substitutions (indicated by underlining) at designated sites. The primer sequences are presented in Table 11 below.

PCR was carried out using WT chromosomal DNA as a template with primers with SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26. PCR was carried out under PCR denaturation conditions at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 7 minutes. As a result, a DNA fragment of 627 bp was obtained. to the 5' region above and a DNA fragment of 608 bp. 3' region downstream of the ilvA gene mutation.

Using the two amplified DNA fragments as templates, PCR was performed using primers SEQ ID NO: 23 and 26 by denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55 °C for 30 seconds, and polymerize at 72 °C for 60 seconds, and then polymerize at 72 °C for 7 minutes. As a result, a 1217 bp DNA fragment was amplified containing a mutation of the ilvA gene encoding a variant of ilvA in which valine at position 323 was replaced by alanine.

Vector pECCG117 (Korean patent No. 10-0057684) and 1011 bp DNA fragments. treated with the restriction enzyme BamHI, ligated using a DNA ligation enzyme and then cloned to obtain a plasmid, which was named pECCG117-ilvA(V323A).

A strain was constructed in which the vector pECCG117-ilvA(V323A) was introduced into ATCC13032::hom(R407H)-lysC(L377K)-gltA(M312I) of the above example. Additionally, as a control, a strain was also constructed in which only the ilvA mutation (V323A) was introduced into ATCC13032::hom (R407H)-lysC (L377K).

The strains thus constructed were cultured in the same manner as the flask culture method presented in Example 4-1 to analyze the concentration of L-isoleucine in the culture solution, and the results are shown in Table 12 below.

As can be seen from Table 12, it was confirmed that the concentration of L-isoleucine increased by approximately 43% in ATCC13032::hom(R407H)-lysC(L377K)-gltA(M312I)/pECCG117-ilvA(V323A) in which the mutation was introduced gltA (M312I), compared to the wild-type strain ATCC13032::-hom(R407H)-lysC(L377K)/pECCG117-ilvA(V323A).

The above results indicate that introduction of the gltA mutation (M312I) into an L-isoleucine-producing strain of the genus Corynebacterium is effective in producing L-isoleucine.

Example 8: Construction of strains having improved O-acetylhomoserine-producing ability and evaluation of O-acetylhomoserine-producing ability

To study the effect of introducing the gltA(M312I) mutation on O-acetylhomoserine production, O-acetylhomoserine-producing strains were constructed by deleting the metB gene, encoding cystathionine gamma synthase, in the O-acetylhomoserine degradation pathway, and the metY gene, encoding O-acetylhomoserine-( thiol)-lyase in the O-acetylhomoserine degradation pathway, and by introducing a mutation (L377K) (US 10662450 B2) to cause feedback inhibition for L-lysine and L-threonine in the lysC gene (SEQ ID NO: 11), encoding aspartokinase, to increase the biosynthesis of O-acetylhomoserine.

First, for the deletion of the metB gene, the metB gene encoding cystathionine gamma synthase in the O-acetylhomoserine degradation pathway was obtained by PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template. Nucleotide sequence information for the metB gene (NCBI accession number Ncgl2360, SEQ ID NO: 27) was obtained from the US National Institutes of Health (NIH GenBank). Based on this information, primers (SEQ ID NOs: 28 and 29) that include the N-terminal region and the linker region of the metB gene, and primers (SEQ ID NOs: 30 and 31) that include the C-terminal region and the linker region were synthesized. The primer sequences are presented in Table 13 below.

PCR was performed using ATCC13032 chromosomal DNA as a template with primers SEQ ID NOs: 28 and 29 and SEQ ID NOs: 30 and 31. PfuUltra™ High Confidence DNA Polymerase (Stratagene) was used as the polymerase, and PCR was performed by repeating 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute. The result was a 558 bp amplified gene, which includes the N-terminal region and the linker region of the metB gene, and a 527 bp amplified gene, which includes the C-terminal region and the linker region of the metB gene.

PCR was performed using the two amplified DNA genes obtained above as templates by repeating 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, and after adding primers with SEQ ID NO: 2 and 5, the polymerization reaction was further repeated 20 times. As a result, a 1064 bp inactivation cassette was obtained, including the N-terminus-linker-C-terminus of the metB gene. The resulting gene was treated with restriction enzymes XbaI and SalI, at the end of the PCR fragment obtained by PCR, and ligated into the vector pDZ (US 9109242 B2), treated with restriction enzymes XbaI and SalI and cloned into the final construct, the recombinant vector pDZ-ΔmetB, into which the metB inactivation cassette was cloned.

The vector pDZ-ΔmetB thus constructed was transformed into ATCC13032 using an electrical pulse method, and ATCC13032ΔmetB in which the metB gene was inactivated in the chromosome was obtained through a second crossover process. The inactivated metB gene was finally confirmed by comparison with ATCC13032, in which the metB gene was not inactivated, after PCR performed using primers with SEQ ID NOs: 28 and 31.

To delete the metY gene, which encodes an enzyme involved in another O-acetylhomoserine degradation pathway, the metY gene, encoding an O-acetylhomoserine(thiol)-lyase in the O-acetylhomoserine degradation pathway, was obtained by PCR using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a matrix. The nucleotide sequence information for the metY gene (NCBI accession number Ncgl0625, SEQ ID NO: 32) was obtained from the US National Institutes of Health (NIH GenBank). Based on this information, primers (SEQ ID NOs: 33 and 34) including the N-terminal region and linker region of the metY gene, and primers (SEQ ID NOs: 35 and 36) including the C-terminal region and linker region of the metY gene were synthesized. The primer sequences are presented in Table 14 below.

PCR was performed using chromosomal DNA from ATCC13032 as a template with primers SEQ ID NOs: 33 and 34 and SEQ ID NOs: 35 and 36. PfuUltra™ High Confidence DNA Polymerase (Stratagene) was used as the polymerase, and PCR was performed by repeating 30 cycles of denaturation at 96°C for 30 seconds, annealing at 53°C for 30 seconds, and polymerization at 72°C for 1 minute. The result was a 548 bp amplified gene that included the N-terminal region and the linker region of the metY gene, and a 550 bp amplified gene that included the C-terminal region and the linker region of the metY gene. PCR was performed using the two amplified DNA genes obtained above as templates by repeating 10 cycles of denaturation at 96°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 1 minute, and after adding primers with SEQ ID NO: 33 and 34, the polymerization reaction was further repeated 20 times. As a result, an inactivating cassette of 1077 bp was obtained, including the N-terminus-linker-C-terminus of the metY gene. The resulting gene was treated with restriction enzymes XbaI and SalI at the end of the fragment obtained by PCR, and ligated with the vector pDZ (US 9109242 B2), treated with restriction enzymes XbaI and SalI, and the final construct was cloned into the recombinant vector pDZ-ΔmetY, into which The metY inactivation cassette was cloned.

The vector pDZ-ΔmetY thus constructed was transformed into strain ATCC13032ΔmetB by an electrical pulse method, and ATCC13032ΔmetBΔmetY in which the metY gene was inactivated in the chromosome was obtained by a second crossover process. The inactivated metY gene was finally confirmed by comparison with ATCC13032, in which the metY gene was not inactivated after PCR using primers SEQ ID NO: 7 and 10.

To increase the production of O-acetylhomoserine, the vector pDZ-lysC (L377K) constructed in Example 7-2 was transformed into strain ATCC13032 ΔmetB ΔmetY using the electrical pulse method to introduce mutation (L377K) (US 10662450 B2) to induce feedback inhibition of L-lysine and L-threonine of the lysC gene, into the lysC gene (SEQ ID NO: 11) encoding aspartokinase obtained from Corynebacterium glutamicum ATCC13032. Then, Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K), in which a nucleotide mutation was introduced into the lysC gene in the chromosome, was obtained through a secondary crossover process. The gene into which the nucleotide mutation was introduced was finally confirmed by PCR using primers SEQ ID NOs: 15 and 18, followed by sequencing, and comparison of the sequence with the wild type lysC gene.

The vector pDC-gltA (M312I) constructed in Example 3-1 was transformed into strain ATCC13032 ΔmetB ΔmetY lysC (L377K) using the electrical pulse method, and Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K) gltA was obtained through a secondary crossover process (M312I ), in which a nucleotide mutation was introduced into the gltA gene on the chromosome. The introduction of the gltA gene mutation into the finally transformed strain was confirmed by performing PCR using primers SEQ ID NO: 7 and SEQ ID NO: 8, and then analyzing the basic sequence, thereby confirming that the mutation had been introduced into the strain.

To compare the O-acetylhomoserine-producing ability of the Corynebacterium glutamicum ATCC13032 ΔmetB ΔmetY lysC (L377K) and ATCC13032 ΔmetB ΔmetY lysC (L377K) gltA(M312I) strains constructed above, these strains were cultivated in the manner described above, and O-acetylhomoserine in was analyzed in culture medium.

The strains were inoculated into a 250 ml corner baffled flask containing 25 ml of the following medium (inoculation loop) and cultured with shaking at 200 rpm at 37°C for 20 hours. The concentration of O-acetylhomoserine was analyzed using HPLC, and the analyzed concentration is presented in Table 15 below.

L-O-acetylhomoserine production medium (pH 7.2)

Glucose 30 g, KH ₂ PO ₄ 2 g, urea 3 g, (NH ₄ )SO ₄ 40 g, peptone 2.5 g, CSL (Sigma) 5 g (10 ml), MgSO ₄ ⋅7H ₂ O 0.5 g, methionine 400 mg, CaCO ₃ 20 g (based on 1 liter of distilled water)

As can be seen from the results presented in Table 15, it was confirmed that strain ATCC13032 ΔmetB ΔmetY lysC(L377K) gltA(M312I), into which the gltA(M312I) mutation was introduced, had a 31% improved O-acetyl-L-homoserine-producing ability. ability compared to strain ATCC13032 ΔmetB ΔmetY lysC(L377K).

Through the above results, it can be confirmed that the 312th amino acid in the amino acid sequence of gltA, which is citrate synthase, is an important position for the activity of gltA enzyme.

One skilled in the art will appreciate that the teachings of the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics. Accordingly, the described embodiments should in all cases be considered only as illustrative and not limiting. Thus, the scope of the description of the present invention is indicated in the claims rather than in the previous description. All changes that are made within the meaning and range of equivalence of the claims are intended to be covered within the scope of the description of the present invention.

--->

LIST OF SEQUENCES

<110>CJ CheilJedang Corporation

<120> NEW MODIFIED POLYPEPTIDE WITH REDUCED ACTIVITY

CITRATE SYNTHASE AND METHOD FOR OBTAINING L-AMINO ACID FROM ITS

USE

<130>OPA20120

<150> KR 10-2020-0010823

<151> 2020-01-30

<160> 37

<170> KoPatentIn 3.0

<210> 1

<211> 437

<212>PRT

<213>Unknown

<220>

<223> gltA amino acid

<400> 1

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

Ser Asn His Gly Gly Asp Ala Thr Glu 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 Gly 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> 2

<211> 1314

<212> DNA

<213>Unknown

<220>

<223> gltA nucleotide

<400> 2

atgtttgaaa gggatatcgt ggctactgat aacaacaagg ctgtcctgca ctaccccggt

60

ggcgagttcg aaatggacat catcgaggct tctgagggta acaacggtgt tgtcctgggc

120

aagatgctgt ctgagactgg actgatcact tttgacccag gttatgtgag cactggctcc

180

accgagtcga agatcaccta catcgatggc gatgcgggaa tcctgcgtta ccgcggctat

240

gacatcgctg atctggctga gaatgccacc ttcaacgagg tttcttacct acttatcaac

300

ggtgagctac caaccccaga tgagcttcac aagtttaacg acgagattcg ccaccacacc

360

cttctggacg aggacttcaa gtcccagttc aacgtgttcc cacgcgacgc tcacccaatg

420

gcaaccttgg cttcctcggt taacattttg tctacctact accaggacca gctgaaccca

480

ctcgatgagg cacagcttga taaggcaacc gttcgcctca tggcaaaggt tccaatgctg

540

gctgcgtacg cacaccgcgc acgcaagggt gctccttaca tgtacccaga caactccctc

600

aatgcgcgtg agaacttcct gcgcatgatg ttcggttacc caaccgagcc atacgagatc

660

gacccaatca tggtcaaggc tctggacaag ctgctcatcc tgcacgctga ccacgagcag

720

aactgctcca cctccaccgt tcgtatgatc ggttccgcac aggccaacat gtttgtctcc

780

atcgctggtg gcatcaacgc tctgtccggc ccactgcacg gtggcgcaaa ccaggctgtt

840

ctggagatgc tcgaagacat caagagcaac cacggtggcg acgcaaccga gttcatgaac

900

aaggtcaaga acaaggaaga cggcgtccgc ctcatgggct tcggacaccg cgtttacaag

960

aactacgatc cacgtgcagc aatcgtcaag gagaccgcac acgagatcct cgagcacctc

1020

ggtggcgacg atcttctgga tctggcaatc aagctggaag aaattgcact ggctgatgat

1080

tacttcatct cccgcaagct ctacccgaac gtagacttct acaccggcct gatctaccgc

1140

gcaatgggct tcccaactga cttcttcacc gtattgttcg caatcggtcg tctgccagga

1200

tggatcgctc actaccgcga gcagctcggt gcagcaggca acaagatcaa ccgcccacgc

1260

caggtctaca ccggcaacga atcccgcaag ttggttcctc gcgaggagcg ctaa

1314

<210> 3

<211> 437

<212>PRT

<213> Artificial Sequence

<220>

<223> gltA M312I amino acid

<400> 3

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

Ser Asn His Gly Gly Asp Ala Thr Glu 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 Gly 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> 4

<211> 1314

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA M312I nucleotide

<400> 4

atgtttgaaa gggatatcgt ggctactgat aacaacaagg ctgtcctgca ctaccccggt

60

ggcgagttcg aaatggacat catcgaggct tctgagggta acaacggtgt tgtcctgggc

120

aagatgctgt ctgagactgg actgatcact tttgacccag gttatgtgag cactggctcc

180

accgagtcga agatcaccta catcgatggc gatgcgggaa tcctgcgtta ccgcggctat

240

gacatcgctg atctggctga gaatgccacc ttcaacgagg tttcttacct acttatcaac

300

ggtgagctac caaccccaga tgagcttcac aagtttaacg acgagattcg ccaccacacc

360

cttctggacg aggacttcaa gtcccagttc aacgtgttcc cacgcgacgc tcacccaatg

420

gcaaccttgg cttcctcggt taacattttg tctacctact accaggacca gctgaaccca

480

ctcgatgagg cacagcttga taaggcaacc gttcgcctca tggcaaaggt tccaatgctg

540

gctgcgtacg cacaccgcgc acgcaagggt gctccttaca tgtacccaga caactccctc

600

aatgcgcgtg agaacttcct gcgcatgatg ttcggttacc caaccgagcc atacgagatc

660

gacccaatca tggtcaaggc tctggacaag ctgctcatcc tgcacgctga ccacgagcag

720

aactgctcca cctccaccgt tcgtatgatc ggttccgcac aggccaacat gtttgtctcc

780

atcgctggtg gcatcaacgc tctgtccggc ccactgcacg gtggcgcaaa ccaggctgtt

840

ctggagatgc tcgaagacat caagagcaac cacggtggcg acgcaaccga gttcatgaac

900

aaggtcaaga acaaggaaga cggcgtccgc ctcatcggct tcggacaccg cgtttacaag

960

aactacgatc cacgtgcagc aatcgtcaag gagaccgcac acgagatcct cgagcacctc

1020

ggtggcgacg atcttctgga tctggcaatc aagctggaag aaattgcact ggctgatgat

1080

tacttcatct cccgcaagct ctacccgaac gtagacttct acaccggcct gatctaccgc

1140

gcaatgggct tcccaactga cttcttcacc gtattgttcg caatcggtcg tctgccagga

1200

tggatcgctc actaccgcga gcagctcggt gcagcaggca acaagatcaa ccgcccacgc

1260

caggtctaca ccggcaacga atcccgcaag ttggttcctc gcgaggagcg ctaa

1314

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> pCR-gltA F

<400> 5

ctaatctcga ggtcacccat gtttgaaagg

thirty

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> pCR-gltA R

<400> 6

tcgcgaggag cgctaaaacc ggttgat

27

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA F

<400> 7

caatgctggc tgcgtacgc

19

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA R

<400> 8

ctcctcgcga ggaaccaact

20

<210> 9

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA M312I Up F

<400> 9

gtgaattcga gctcggtacc cgcgggaatc ctgcgttacc gc

42

<210> 10

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA M312I Up R

<400> 10

tgtaaacgcg gtgtccgaag ccgatgaggc ggacgccgtc tt

42

<210> 11

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA M312I Down F

<400> 11

aagacggcgt ccgcctcatc ggcttcggac accgcgttta ca

42

<210> 12

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> gltA M312I Down R

<400> 12

ggtcgactct agaggatccc cttagcgctc ctcgcgagga ac

42

<210> 13

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA F

<400> 13

accgaaattg gcttgggtgc cagcccagct gatgcctac

39

<210> 14

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA R

<400> 14

aagcttgcat gcctgcagct taaagtcacc tacgttttgt ac

42

<210> 15

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> lysC up F

<400> 15

tcctctagag ctgcgcagtg ttgaatacg

29

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> lysC up R

<400> 16

tggaaatctt ttcgatgttc acgttgacat

thirty

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> lysC down F

<400> 17

acatcgaaaa gatttccacc tctgagattc

thirty

<210> 18

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> lysC down R

<400> 18

gactctagag ttcacctcag agacgatta

29

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> hom up F

<400> 19

tcctctagac tggtcgcctg atgttctac

29

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> hom up R

<400> 20

cacgatcaga tgtgcatcat cat

23

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> hom down F

<400> 21

atgatgatgc acatctgatc gtg

23

<210> 22

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> hom down R

<400> 22

gactctagat tagtcccttt cgaggcgga

29

<210> 23

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> ilvA V323A up F

<400> 23

acggatccca gactccaaag caaaagcg

28

<210> 24

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> ilvA V323A up R

<400> 24

acaccacggc agaaccaggt gcaaaggaca

thirty

<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> ilvA V323A down F

<400> 25

ctggttctgc cgtggtgtgc atcatctctg

thirty

<210> 26

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> ilvA V323A down R

<400> 26

acggatccaa ccaaacttgc tcacactc

28

<210> 27

<211> 1161

<212> DNA

<213>Unknown

<220>

<223> Corynebacterium glutamicum metB

<400> 27

ttgtcttttg acccaaacac ccagggtttc tccactgcat cgattcacgc tgggtatgag

60

cgacgact actacggttc gattaacacc ccaatctatg cctccaccac cttcgcgcag

120

aacgctccaa acgaactgcg caaaggctac gagtacaccc gtgtgggcaa ccccaccatc

180

gtggcattag agcagaccgt cgcagcactc gaaggcgcaa agtatggccg cgcattctcc

240

tccggcatgg ctgcaaccga catcctgttc cgcatcatcc tcaagccggg cgatcacatc

300

gtcctcggca acgatgctta cggcggaacc taccgcctga tcgacaccgt attcaccgca

360

tggggcgtcg aatacaccgt tgttgatacc tccgtcgtgg aagaggtcaa ggcagcgatc

420

aaggacaaca ccaagctgat ctgggtggaa accccaacca acccagcact tggcatcacc

480

gacatcgaag cagtagcaaa gctcaccgaa ggcaccaacg ccaagctggt tgttgacaac

540

accttcgcat ccccatacct gcagcagcca ctaaaactcg gcgcacacgc agtcctgcac

600

tccaccacca agtacatcgg aggacactcc gacgttgttg gcggccttgt ggttaccaac

660

gaccaggaaa tggacgaaga actgctgttc atgcagggcg gcatcggacc gatcccatca

720

gttttcgatg catacctgac cgcccgtggc ctcaagaccc ttgcagtgcg catggatcgc

780

cactgcgaca acgcagaaaa gatcgcggaa ttcctggact cccgcccaga ggtctccacc

840

gtgctctacc caggtctgaa gaaccaccca ggccacgaag tcgcagcgaa gcagatgaag

900

cgcttcggcg gcatgatctc cgtccgtttc gcaggcggcg aagaagcagc taagaagttc

960

tgtacctcca ccaaactgat ctgtctggcc gagtccctcg gtggcgtgga atccctcctg

1020

gagcacccag caaccatgac ccaccagtca gctgccggct ctcagctcga ggttccccgc

1080

gacctcgtgc gcatctccat tggtattgaa gacattgaag acctgctcgc agatgtcgag

1140

caggccctca ataaccttta g

1161

<210> 28

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> metB_N_del F

<400> 28

tctagacgcc cgcatactgg cttc

24

<210> 29

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> metB_N_del R

<400> 29

cccatccact aaacttaaac agatgtgatc gcccggc

37

<210> 30

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> metB_C_del F

<400> 30

tgtttaagtt tagtggatgg ggaagaacca cccaggcc

38

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> metB_C_del R

<400> 31

gtcgaccaat cgtccagagg gcg

23

<210> 32

<211> 1314

<212> DNA

<213>Unknown

<220>

<223> Corynebacterium glutamicum metY

<400> 32

atgccaaagt acgacaattc caatgctgac cagtggggct ttgaaacccg ctccattcac

60

gcaggccagt cagtagacgc acagaccagc gcacgaaacc ttccgatcta ccaatccacc

120

gctttcgtgt tcgactccgc tgagcacgcc aagcagcgtt tcgcacttga ggatctaggc

180

cctgtttact cccgcctcac caacccaacc gttgaggctt tggaaaaccg catcgcttcc

240

ctcgaaggtg gcgtccacgc tgtagcgttc tcctccggac aggccgcaac caccaacgcc

300

attttgaacc tggcaggagc gggcgaccac atcgtcacct ccccacgcct ctacggtggc

360

accgagactc tattccttat cactcttaac cgcctgggta tcgatgtttc cttcgtggaa

420

aaccccgacg accctgagtc ctggcaggca gccgttcagc caaacaccaa agcattcttc

480

ggcgagactt tcgccaaccc acaggcagac gtcctggata ttcctgcggt ggctgaagtt

540

gcgcaccgca acagcgttcc actgatcatc gacaacacca tcgctaccgc agcgctcgtg

600

cgcccgctcg agctcggcgc agacgttgtc gtcgcttccc tcaccaagtt ctacaccggc

660

aacggctccg gactgggcgg cgtgcttatc gacggcggaa agttcgattg gactgtcgaa

720

aaggatggaa agccagtatt cccctacttc gtcactccag atgctgctta ccacggattg

780

aagtacgcag accttggtgc accagccttc ggcctcaagg ttcgcgttgg ccttctacgc

840

gacaccggct ccaccctctc cgcattcaac gcatgggctg cagtccaggg catcgacacc

900

ctttccctgc gcctggagcg ccacaacgaa aacgccatca aggttgcaga attcctcaac

960

aaccacgaga aggtggaaaa ggttaacttc gcaggcctga aggattcccc ttggtacgca

1020

accaaggaaa agcttggcct gaagtacacc ggctccgttc tcaccttcga gatcaagggc

1080

ggcaaggatg aggcttgggc atttatcgac gccctgaagc tacactccaa ccttgcaaac

1140

atcggcgatg ttcgctccct cgttgttcac ccagcaacca ccacccattc acagtccgac

1200

gaagctggcc tggcacgcgc gggcgttacc cagtccaccg tccgcctgtc cgttggcatc

1260

gagaccattg atgatatcat cgctgacctc gaaggcggct ttgctgcaat ctag

1314

<210> 33

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> metY_N_del F

<400> 33

tctagaccat cctgcaccat ttag

24

<210> 34

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> metY_N_del R

<400> 34

cccatccact aaacttaaac acgctcctgc caggttc

37

<210> 35

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> metY_C_del F

<400> 35

tgtttaagtt tagtggatgg gcttggtacg caaccaagg

39

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> metY_C_del R

<400> 36

gtcgacgatt gctccggctt cgg

23

<210> 37

<211> 1266

<212> DNA

<213>Unknown

<220>

<223> Corynebacterium glutamicum lysC

<400> 37

gtggccctgg tcgtacagaa atatggcggt tcctcgcttg agagtgcgga acgcattaga

60

aacgtcgctg aacggatcgt tgccaccaag aaggctggaa atgatgtcgt ggttgtctgc

120

tccgcaatgg gagacaccac ggatgaactt ctagaacttg cagcggcagt gaatcccgtt

180

ccgccagctc gtgaaatgga tatgctcctg actgctggtg agcgtatttc taacgctctc

240

gtcgccatgg ctattgagtc ccttggcgca gaagcccaat ctttcacggg ctctcaggct

300

ggtgtgctca ccaccgagcg ccacggaaac gcacgcattg ttgatgtcac tccaggtcgt

360

gtgcgtgaag cactcgatga gggcaagatc tgcattgttg ctggtttcca gggtgttaat

420

aaagaaaccc gcgatgtcac cacgttgggt cgtggtggtt ctgacaccac tgcagttgcg

480

ttggcagctg ctttgaacgc tgatgtgtgt gagatttact cggacgttga cggtgtgtat

540

accgctgacc cgcgcatcgt tcctaatgca cagaagctgg aaaagctcag cttcgaagaa

600

atgctggaac ttgctgctgt tggctccaag attttggtgc tgcgcagtgt tgaatacgct

660

cgtgcattca atgtgccact tcgcgtacgc tcgtcttata gtaatgatcc cggcactttg

720

attgccggct ctatggagga tattcctgtg gaagaagcag tccttaccgg tgtcgcaacc

780

gacaagtccg aagccaaagt aaccgttctg ggtatttccg ataagccagg cgaggctgcg

840

aaggttttcc gtgcgttggc tgatgcagaa atcaacattg acatggttct gcagaacgtc

900

tcttctgtag aagacggcac caccgacatc accttcacct gccctcgttc cgacggccgc

960

cgcgcgatgg agatcttgaa gaagcttcag gttcagggca actggaccaa tgtgctttac

1020

gacgaccagg tcggcaaagt ctccctcgtg ggtgctggca tgaagtctca cccaggtgtt

1080

accgcagagt tcatggaagc tctgcgcgat gtcaacgtga acatcgaatt gatttccacc

1140

tctgagattc gtatttccgt gctgatccgt gaagatgatc tggatgctgc tgcacgtgca

1200

ttgcatgagc agttccagct gggcggcgaa gacgaagccg tcgtttatgc aggcaccgga

1260

cgctaa

1266

<---

Claims (15)

1. A modified polypeptide having citrate synthase activity, wherein the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1 is replaced by another amino acid. 2. The modified polypeptide according to claim 1, where the amino acid corresponding to position 312 is replaced by isoleucine. 3. A modified polypeptide having citrate synthase activity and having 80% or more and less than 100% sequence homology with the amino acid sequence of SEQ ID NO: 1, where the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, replaced by another amino acid. 4. The modified polypeptide according to claim 1, which consists of the amino acid sequence SEQ ID NO: 3. 5. A polynucleotide encoding a modified polypeptide according to any one of claims. 1-4. 6. An expression vector containing the polynucleotide according to claim 5. 7. A microorganism producing L-amino acid containing a modified polypeptide according to any one of paragraphs. 1-4, a polynucleotide encoding this modified polypeptide, or a vector containing this polynucleotide. 8. The microorganism according to claim 7, wherein the L-amino acid is any one or more amino acids selected from the group consisting of leucine, lysine, valine, isoleucine and O-acetylhomoserine. 9. The microorganism according to claim 7, which is a microorganism of the genus Corynebacterium . 10. The microorganism according to claim 9, where the microorganism of the genus Corynebacterium is Corynebacterium glutamicum . 11. A method for producing L-amino acids, including: cultivation in the medium of a microorganism containing a modified polypeptide according to any one of claims. 1-4, a polynucleotide encoding this modified polypeptide, or a vector containing this polynucleotide. 12. The method according to claim 11, which further includes isolating the L-amino acid from the culture medium or microorganism. 13. The method of claim 12, wherein the L-amino acid is any one or more amino acids selected from the group consisting of leucine, lysine, valine, isoleucine and O-acetylhomoserine. 14. The use of an L-amino acid producing microorganism to produce an L-amino acid, wherein said microorganism contains a modified polypeptide having citrate synthase activity, in which the amino acid corresponding to position 312 from the N-terminus of the polypeptide consisting of the amino acid sequence SEQ ID NO: 1, replaced by another amino acid; a polynucleotide encoding the modified polypeptide; or a vector containing this polynucleotide.