Specification
Title of the invention: Novel mutant polypeptide with weakened citrate synthase activity and L-amino acid production method using same
technical field
[One]
The present application relates to a novel mutant polypeptide in which the activity of citrate synthase is weakened, a microorganism comprising the mutant polypeptide, and a method for producing L-amino acids using the microorganism.
[2]
background
[3]
Corynebacterium genus (the genus Corynebacterium ) Microorganisms, particularly Corynebacterium glutamicum ( Corynebacterium glutamicum ) is a gram-positive microorganism that is widely used for the production of L-amino acids and other useful substances. In order to produce the L-amino acid and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (US 8048650 B2).
[4]
On the other hand, among L-amino acids, L-lysine, L-threonine, L-methionine, L-isoleucine, and L-glycine are amino acids derived from aspartate, and the synthesis level of oxaloacetate, a precursor of aspartate, is the above. It may affect the level of synthesis of L-amino acids.
[5]
Citrate synthase (CS) is an enzyme that produces citrate by polymerizing acetyl-CoA and oxaloacetate produced in the glycolysis process of microorganisms, and is also an important enzyme that determines carbon influx into the TCA pathway.
[6]
Changes in the phenotype of the L-lysine-producing strain due to the deletion of the gltA gene encoding citrate synthase have been reported in the prior literature (Ooyen et al., Biotechnol. Bioeng., 109(8): 2070-2081, 2012). However, in the case of the gltA gene-deficient strain, the growth of the strain is inhibited, and the sugar consumption rate is greatly reduced, so that the lysine production per unit time is low. Therefore, there is still a need for research that considers the effective increase in the production capacity of L-amino acids and the growth of the strain.
[7]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[8]
The present inventors have completed the present invention by confirming that the production of L-amino acids is increased when a novel mutant polypeptide in which citrate synthase activity is attenuated to a specific level is used.
[9]
means of solving the problem
[10]
The present application provides a variant polypeptide having citrate synthase activity in which the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.
[11]
The present application provides a polynucleotide encoding the variant polypeptide.
[12]
The present application provides a vector comprising the polynucleotide.
[13]
The present application provides a microorganism for producing L-amino acids, comprising the variant polypeptide, a polynucleotide encoding the variant polypeptide, or a vector including the polynucleotide.
[14]
The present application provides a method for producing L-amino acids, comprising culturing a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector including the polynucleotide in a medium.
[15]
Effects of the Invention
[16]
In the case of culturing a microorganism of the genus Corynebacterium that produces L-amino acids in which the activity of the citrate synthase of the present application on the substrate is changed, it is possible to produce L-amino acids in high yield compared to microorganisms having existing unmodified polypeptides do.
[17]
Best mode for carrying out the invention
[18]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
[19]
[20]
One aspect of the present application is a variant polypeptide having citrate synthase activity, in which the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid may be to provide
[21]
In the present application, the sequence of SEQ ID NO: 1 may be an amino acid sequence having citrate synthase activity. Specifically, the sequence of SEQ ID NO: 1 may be 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 a known database, GenBank of NCBI. For example, Corynebacterium glutamicun), but is not limited thereto, and may be included without limitation as long as it is an amino acid sequence of a protein having the same activity as a protein comprising the amino acid sequence. In addition, although the protein having citrate synthase activity in the present application may be a protein including the amino acid sequence of SEQ ID NO: 1, a meaningless sequence added before and after the amino acid sequence of SEQ ID NO: 1 or a naturally occurring mutation, or It does not exclude its latent mutation, and if it has the same or corresponding activity as the protein comprising the amino acid sequence of SEQ ID NO: 1, it corresponds to the protein having the citrate synthase activity of the present application. self-evident to As a specific example, the protein having citrate synthase activity of the present application is the amino acid sequence of SEQ ID NO: 1 or 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more thereof. It may be a protein composed of an amino acid sequence having homology or identity. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein, a protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added is also included within the scope of the protein subject to mutation of the present application. is self-evident
[22]
[23]
As used herein, the term "citrate synthase" is an enzyme that generates citrate by polymerizing acetyl-CoA and oxaloacetate produced in the glycolysis process of microorganisms, and is an enzyme that determines carbon influx into the TCA pathway. It is an important enzyme. Specifically, as a citric acid synthase, it may play a rate-regulating role in the first step of the TCA cycle. The enzyme is also capable of catalyzing the condensation reaction of acetyl coei with a 2-carbon acetate moiety from a molecule of 4-carbon oxaloacetate to form 6-carbon acetate. In the present application, the citrate synthase may be used in combination with citrate synthase, CS, GltA protein, or GltA.
[24]
[25]
As used herein, the term "variant" means that one or more amino acids differ from the recited sequence in conservative substitution and/or modification, but the function of the protein Refers to a polypeptide in which functions or properties are maintained. A variant polypeptide differs from the identified sequence by several amino acid substitutions, deletions or additions. Such variants can generally be identified by modifying one of the polypeptide sequences and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. Such variants can generally be identified by modifying one of the polypeptide sequences and evaluating the reactivity of the modified polypeptide. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, are removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
[26]
As used herein, the term "conservative substitution" means substituting one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. 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, conservative substitutions have little or no effect on the activity of the resulting polypeptide.
[27]
In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated with a signal (or leader) sequence at the N-terminus of the protein that is involved in the transfer of the protein either co-translationally or post-translationally. The polypeptide may also be conjugated with other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.
[28]
[29]
본 출원에서 용어, "시트레이트 신타아제 활성을 가지는 변이형 폴리펩티드"는 시트레이트 신타아제 활성을 가지는 폴리펩티드의 아미노산 서열 일부가 치환됨으로써 야생형에 비해 약화된 시트레이트 신타아제 활성을 가지는 폴리펩티드를 의미한다. 본 출원에서는 시트레이트 신타아제 활성을 가지는 폴리펩티드의 아미노산 서열상에서 하나 이상의 아미노산이 변이되어 그 활성이 야생형과 비교하여 약화되어 탄소흐름이 효율적으로 균형을 이루게 하는 변이형 폴리펩티드를 의미할 수 있다.
[30]
구체적으로 상기 변이형 폴리펩티드는 상기의 시트레이트 신타아제 활성을 갖는 다양한 단백질에서, 서열번호 1의 아미노산 서열의 312번째 위치에 상응하는 아미노산이 다른 아미노산으로 치환된 변이형 폴리펩티드일 수 있다. 상기 '다른 아미노산'은 치환 전과는 다른 아미노산을 의미하며, 치환 전의 아미노산을 제외한 아미노산이면 제한되지 않는다.
[31]
More specifically, the variant polypeptide may be a variant polypeptide in which methionine corresponding to position 312 of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid in the various proteins having the above-mentioned citrate synthase activity. The methionine is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine ), isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (valine) may be substituted with any one or more amino acids selected from the group consisting of, and more specifically, may be a variant sequence substituted with isoleucine, but is not limited thereto.
[32]
In addition, the substituted amino acid residue may include not only natural amino acids but also non-natural amino acids. The non-natural amino acid can be, for example, a D-amino acid, a homo-amino acid, a beta-homo-amino acid, an N-methyl amino acid, an alpha-methyl amino acid, an unconventional amino acid (such as citrulline or naph thylalanine, etc.), but is not limited thereto. On the other hand, in the present application, when it is expressed that 'a specific amino acid is substituted', it is obvious that the amino acid is substituted with an amino acid different from the amino acid before the substitution, even if it is not separately indicated that it is substituted with another amino acid.
[33]
[34]
As used herein, the term “corresponding to” refers to an amino acid residue at a position listed in a protein or peptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a protein or peptide. As used herein, "corresponding region" generally refers to a similar position in a related protein or reference protein.
[35]
In the present application, specific numbering may be used for amino acid residue positions in the polypeptides used in this application. For example, by aligning the polypeptide sequence of the present application with the target polypeptide to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the polypeptide of the present application.
[36]
In the mutant polypeptide having citrate synthase activity provided in the present application, amino acids at specific positions in the aforementioned citrate synthase are substituted, so that the ability to produce L-amino acids can be increased compared to the polypeptide before the mutation.
[37]
[38]
The variant polypeptide may have 80% or more and less than 100% sequence homology to the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. Specifically, the variant polypeptide of the present application 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 obvious that proteins having an amino acid sequence in which some sequences are deleted, modified, substituted or added in addition to the amino acid sequence at position 312 are included within the scope of the present application as long as the amino acid sequence has homology and exhibits efficacy corresponding to the protein.
[39]
[40]
In addition, even if it is described as 'a protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO:' in the present application, if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO: some sequences are deleted, It is apparent that proteins having modified, substituted or added amino acid sequences can also be used in the present application. For example, if it has the same or corresponding activity as the mutant polypeptide, in addition to the specific 312 mutation that imparts a specific activity, a meaningless sequence before and after the amino acid sequence of the corresponding SEQ ID NO. This does not exclude latent mutations, and it is apparent that such sequence additions or mutations fall within the scope of the present application.
[41]
[42]
The mutant polypeptide in which the amino acid corresponding to the 312th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid may include the amino acid sequence of SEQ ID NO: 3. More specifically, the mutant polypeptide in which methionine corresponding to position 312 in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine may consist of SEQ ID NO: 3. In addition, the variant polypeptide may include the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 80% or more and less than 100% homology thereto, but is not limited thereto. Specifically, the variant polypeptide of the present application may include SEQ ID NO: 3 and a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 3 have. In addition, if it is an amino acid sequence having such homology and exhibiting efficacy corresponding to the protein, in addition to the amino acid sequence at position 312, a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application. can be self-evident
[43]
For the purpose of the present application, in the case of a microorganism comprising a mutant polypeptide having weakened citrate synthase activity, the yield of L-amino acid is increased, but the sugar consumption rate is similar to that of the control. This suggests that L-amino acid production can be increased through an appropriate balance between the carbon flow into the TCA pathway and the supply of oxaloacetate used as a precursor of L-amino acid biosynthesis through regulation of citrate synthase activity.
[44]
[45]
The term "homology" refers to the percent identity between two polynucleotide or polypeptide moieties. It refers to the degree of correspondence with a given amino acid sequence or base sequence and may be expressed as a percentage. In the present specification, a homologous sequence having the same or similar activity to a given amino acid sequence or base sequence may be expressed as "% homology". Homology between sequences from one moiety to another can be determined by known art. For example, using standard software that calculates parameters such as score, identity, and similarity, specifically BLAST 2.0, or by Southern hybridization experiments under defined stringent conditions. Appropriate hybridization conditions defined by comparing , Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
[46]
[47]
Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity A polynucleotide encoding a polypeptide may be provided.
[48]
The amino acid sequence of SEQ ID NO: 1, citrate synthase and variant polypeptides are as described above.
[49]
As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.
[50]
The polynucleotide of the present application may be included without limitation as long as it is a polynucleotide sequence encoding a variant polypeptide having citrate synthase activity of the present application. In the present application, the gene encoding the amino acid sequence of citrate synthase is a gltA gene, and the gene may be derived from Corynebacterium glutamicum, but is not limited thereto. In addition, the gene may be a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, more specifically, may be a sequence including the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
[51]
Specifically, the polynucleotide of the present application may contain a variety of coding regions within a range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or considering codons preferred in the organism to express the polypeptide. Deformation can be made. Specifically, any polynucleotide sequence encoding a variant polypeptide in which the amino acid corresponding to position 312 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid may be included without limitation. For example, the variant polypeptide of the present application may be a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. More specifically, it may be composed of the polynucleotide sequence set forth in SEQ ID NO: 4, but is not limited thereto.
[52]
In addition, by hybridizing under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to all or part of the nucleotide sequence, the 312th amino acid in the amino acid sequence of SEQ ID NO: 1 is a different amino acid Any sequence encoding a protein having a substituted citrate synthase activity may be included without limitation. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., supra). For example, 40% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more homology between genes with high homology or identity, or Genes with the same identity are hybridized, and genes with less homology or identity do not hybridize, or wash conditions of normal Southern hybridization at 60°C, 1ΥSSC, 0.1% SDS, specifically At a salt concentration and temperature equivalent to 60°C, 0.1ΥSSC, 0.1% SDS, more specifically 68°C, 0.1ΥSSC, 0.1% SDS, conditions for washing once, specifically 2 to 3 times can be listed. have.
[53]
Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include isolated nucleic acid fragments that are complementary to substantially similar nucleic acid sequences as well as the entire sequence.
[54]
Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above. In addition, the Tm value may be 60° C., 63° C. or 65° C., but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
[55]
The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[56]
[57]
Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity Vectors comprising a polynucleotide encoding a polypeptide can be provided.
[58]
The amino acid sequence of SEQ ID NO: 1, citrate synthase, variant polypeptides and polynucleotides are as described above.
[59]
As used herein, the term "vector" refers to a DNA preparation containing a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable regulatory sequence so that the target polypeptide can be expressed in a suitable host. Such regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
[60]
The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. Specifically, pCR2.1, pDC, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
[61]
The vector usable in the present application is not particularly limited, and a known expression vector may be used. In addition, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. In addition, it may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule has been inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
[62]
As used herein, the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell.
[63]
In addition, the polynucleotide may include DNA and RNA encoding a target protein.
[64]
The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto. The transformation method includes any method of introducing a polynucleotide into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca(H 2 PO 4 ) 2 , CaHPO 4 , or Ca 3 (PO 4 ) 2 ) precipitation, calcium chloride (CaCl 2 )) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method, but is not limited thereto.
[65]
In addition, the term “operably linked” as used herein may mean that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present application and the polynucleotide sequence are functionally linked. An operable linkage may be prepared using a genetic recombination technique known in the art, and site-specific DNA cleavage and ligation may be made using a cleavage and ligation enzyme in the art, but is not limited thereto.
[66]
[67]
Another aspect of the present application is that the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a variant having citrate synthase activity It is possible to provide a microorganism producing L-amino acids, including a polypeptide, a polynucleotide encoding the variant polypeptide, or a vector including the polynucleotide.
[68]
The amino acid sequence of SEQ ID NO: 1, citrate synthase, variant polypeptides, polynucleotides and vectors are as described above.
[69]
The microorganism may include a polynucleotide encoding the variant polypeptide or provide a microorganism transformed with a vector containing a polynucleotide encoding the variant polypeptide, but is not limited thereto.
[70]
In addition, the microorganism can improve the production capacity of L-amino acids without inhibiting the growth or sugar consumption rate of the microorganism compared to the microorganism containing the wild-type polypeptide, and L-amino acids can be obtained from these microorganisms in high yield. have.
[71]
In the present application, the term "microorganism comprising a mutant polypeptide" refers to a microorganism in which L-amino acid-producing ability is conferred to a microorganism having a naturally weak L-amino acid-producing ability or a parent strain having no L-amino acid-producing ability. . Specifically, the microorganism is a microorganism expressing a variant polypeptide comprising one or more amino acid mutations in a polypeptide having citrate synthase activity, wherein the amino acid mutation is at position 312 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 It may include substitution of the corresponding amino acid with another amino acid. The variant polypeptide having a citrate synthase activity expressed from the microorganism may have a weakened activity, but is not limited thereto.
[72]
The microorganism is a cell or microorganism capable of expressing a variant polypeptide by being transformed with a vector containing a polynucleotide encoding a variant polypeptide or a polynucleotide encoding a variant polypeptide, and the object of the present application The host cell or microorganism may be any microorganism capable of producing L-amino acids including the variant polypeptide.
[73]
In the present application, the term "microorganism that produces L-amino acids" includes all microorganisms in which genetic modification has occurred, either naturally or artificially, As a microorganism whose mechanism is strengthened or weakened, it may be a microorganism in which a genetic mutation has occurred or its activity is weakened for the production of a desired L-amino acid. For the purpose of the present application, the microorganism producing the L-amino acid refers to a microorganism capable of producing the desired L-amino acid from a carbon source in the medium, including the mutant polypeptide, in excess compared to the wild-type or unmodified microorganism. can In the present application, the "microorganism producing L-amino acid" may be used interchangeably with "a microorganism having an ability to produce L-amino acid" or "a microorganism producing L-amino acid".

Claims

[Claim 1]
A variant polypeptide having citrate synthase activity in which the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.
[Claim 2]
The variant polypeptide according to claim 1, wherein the amino acid corresponding to position 312 is substituted with isoleucine.
[Claim 3]
The variant polypeptide according to claim 1, wherein the variant polypeptide has 80% or more and less than 100% sequence homology to the amino acid sequence of SEQ ID NO: 1.
[Claim 4]
The variant polypeptide according to claim 1, wherein the variant polypeptide consists of the amino acid sequence of SEQ ID NO: 3.
[Claim 5]
A polynucleotide encoding the variant polypeptide of any one of claims 1 to 4.
[Claim 6]
A vector comprising the polynucleotide of claim 5 .
[Claim 7]
A microorganism producing L-amino acids, comprising the mutant polypeptide of any one of claims 1 to 4, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide.
[Claim 8]
The microorganism according to claim 7, wherein the L-amino acid is at least one selected from the group consisting of leucine, lysine, valine, isoleucine and O-acetyl homoserine.
[Claim 9]
According to claim 7, wherein the microorganism is Corynebacterium genus ( Corynebacterium sp.), L- amino acid producing microorganism.
[Claim 10]
The microorganism of claim 9, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum .
[Claim 11]
The production of L-amino acids, comprising the step of culturing a microorganism comprising the mutant polypeptide of any one of claims 1 to 4, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide in a medium Way.
[Claim 12]
The method of claim 11, wherein the method further comprises recovering L-amino acids from the culture medium or microorganisms.
[Claim 13]
The method of claim 12, wherein the L-amino acid is at least one selected from the group consisting of leucine, lysine, valine, isoleucine and O-acetyl homoserine.
[Claim 14]
A variant polypeptide having citrate synthase activity in which the amino acid corresponding to the 312th position from the N-terminus of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, a polynucleotide encoding the variant polypeptide, or A use for the production of L-amino acids in microorganisms producing L-amino acids, comprising a vector comprising the polynucleotide.