RU2793368C1 - New version of the transcription regulator and a method for obtaining l-valine with its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: polypeptide is proposed that is involved in the production of L-valine consisting of the amino acid sequence represented by SEQ ID NO: 1, in which glutamic acid, which is the amino acid corresponding to position 247 in SEQ ID NO: 3, is replaced by lysine. Also proposed are a polynucleotide encoding the said polypeptide, a microorganism Corynebacterium glutamicum that produces L-valine and contains the said polypeptide or polynucleotide encoding the said polypeptide, and a method for producing L-valine.

EFFECT: increasing the yield of L-tryptophan.

5 cl, 3 tbl, 2 ex

Description

FIELD OF THE INVENTION

The present disclosure relates to a novel transcription regulator variant, a strain of Corynebacterium glutamicum containing this variant, and to a method for producing L-valine using this strain.

PRIOR ART

Various studies are being conducted to develop highly efficient microorganisms and fermentation process technologies for the production of L-amino acids and other beneficial substances. For example, a target-substance-specific approach is mainly used, which increases the expression of a gene encoding an enzyme involved in the biosynthesis of L-valine, or which removes genes that are not necessary for biosynthesis (US 8465962 B2).

However, research is still needed to effectively increase the L-valine producing capacity as the demand for L-valine increases.

Prior Art Documents

Patent Documents

US 8465962 B2 (published 06/18/2013) Non-patent documents

JOURNAL OF BACTERIOLOGY, Oct. 2011, p.5155-5163 (published on 07/22/2011)

DESCRIPTION OF THE INVENTION

Technical problem

The purpose of this disclosure is to provide a transcription regulator variant consisting of the amino acid sequence represented by SEQ ID NO: 1, in which glutamic acid (Glu, E), which is the amino acid corresponding to position 247 of the amino acid sequence of SEQ ID NO: 3, is replaced by lysine ( Lys, K).

Another object of the present disclosure is to provide a polynucleotide encoding a variant of the present disclosure.

Yet another object of the present disclosure is to provide a strain of Corynebacterium glutamicum that contains a variant of the present disclosure, or a polynucleotide encoding the variant, and has the ability to produce L-valine.

Yet another object of the present disclosure is to provide a method for producing L-valine, which comprises culturing in a medium a strain of Corynebacterium glutamicum that contains a variant of the present disclosure or a polynucleotide encoding the variant and has the ability to produce L-valine.

The best way to implement the invention

The present disclosure will be described in detail as follows. In the meantime, each of the descriptions and embodiments disclosed in this disclosure may apply to other descriptions and embodiments. In other words, all combinations of different elements disclosed in the present disclosure are within the scope of the present disclosure. In addition, the scope of the present disclosure should not be considered to be limited by the specific description provided below. In addition, a number of articles and patent documents are referenced and cited throughout the present specification. The entire content disclosed in the cited articles and patent documents is hereby incorporated by reference in order to more clearly describe the level of the technical field to which the present invention belongs and the content of the present invention.

According to one aspect of the present disclosure, a variant transcription regulator is provided, consisting of the amino acid sequence shown in SEQ ID NO: 1, in which glutamic acid (Glu, E), which is the amino acid corresponding to position 247 of the amino acid sequence of SEQ ID NO: 3, is replaced by lysine (Lys, K).

The variant of the present disclosure may have, contain, or essentially consist of the amino acid sequence represented by SEQ ID NO: 1.

In a variant of the present disclosure, the amino acid corresponding to position 247 based on the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence shown by SEQ ID NO: 1 is lysine, and the variant may contain an amino acid sequence having at least 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% 99.7% or 99.9% or greater homology or identity with the amino acid sequence shown by SEQ ID NO: 1. Obviously, variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included within the scope of this disclosure, so long as the amino acid sequences have such homology or identity and exhibit efficacy consistent with that of the variant of the present disclosure.

Examples thereof include variants having a sequence addition or deletion that does not change the function of the variant of the present disclosure, at the N-terminus, C-terminus of an amino acid sequence and/or within that amino acid sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution.

The term "conservative substitution" means the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such an amino acid substitution can typically exist based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. Typically, a conservative substitution may or may not affect the activity of proteins or polypeptides.

In the present disclosure, the term "variant" refers to a polypeptide that has an amino acid sequence different from the amino acid sequence of the variant prior to modification by conservative substitution and/or modification of one or more amino acids, but retains functions or properties. Such a variant can usually be identified by modifying one or more amino acids of the amino acid sequence of a given polypeptide and evaluating the properties of that modified polypeptide. In other words, the ability of a given variant can be increased, left unchanged, or reduced compared to the ability of the polypeptide before the change. Some variants may include variants in which one or more parts, such as the N-terminal leader sequence or the transmembrane domain, have been deleted. Other variants may include variants in which a portion of the N- and/or C-terminus of the mature protein has been removed. The term "variant" can be used interchangeably with and is not limited to terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent, as long as it is a term used with the meaning of variation. For the purposes of the present disclosure, this variant may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in which glutamic acid (Glu, E), which is the amino acid corresponding to position 247 of the amino acid sequence of SEQ ID NO: 3, has been replaced by lysine (Lys, K).

This variant may contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that co-translationally or post-translationally participates in protein translocation can be conjugated to the N-terminus of this variant. This variant can be conjugated to other sequences or linkers in such a way as to be identified, purified or synthesized.

In the present disclosure, the term "homology" or "identity" means the degree of similarity between two given amino acid or base sequences, and may be expressed as a percentage. The terms "homology" and "identity" can often be used interchangeably.

Homology or sequence identity of a conserved polynucleotide a or polypeptide is determined by standard alignment algorithms and the default gap penalty set by the application program can be shared. Substantially homologous or identical sequences are generally capable of hybridizing to all or part of the sequence under conditions of moderate to high stringency. Obviously, hybridization also includes the hybridization of polynucleotide a with a polynucleotide containing a common type codon or a degenerate codon.

Whether any two polynucleotide or polypeptide sequences share homology, similarity or identity can be determined using known computer algorithms such as the FASTA program, for example using default parameters as in Pearson et at, (1988) Proc. Natl. Acad. sci. USA 85:2444. Alternatively, homology, similarity, or identity can be determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (including GCG software package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al, JMOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994; and CARILLO et al (1988) SIAM J Applied Math 48:1073) For example, National Center for Biotechnology Information BLAST or ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as announced, for example, in Smith and Waterman, A dv. Appl. Math (1981) 2:482. Finally, the result of the GAP program can be defined as the value obtained by dividing the number of similar aligned characters (namely, nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. The default parameters for the GAP program may include (1) a binary comparison matrix (including values of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al, (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL substitution matrix (EMBOSS version NCBI NUC4.4)), as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each character in each gap (or a penalty of 10 for opening a gap, a penalty of 0.5 for extending a gap); and (3) no end gap penalty.

As an example of the present disclosure, a variant of the present disclosure may exhibit transcriptional regulator activity. A variant of the present disclosure may exhibit activity such that it has an increased ability to produce L-valine compared to a wild-type polypeptide.

In the present disclosure, the term "transcriptional regulator - iolR" is a transcription regulator involved in the use of myo-inositol. The term "transcriptional regulator" of the present disclosure may be used interchangeably with IolR. In the present disclosure, the sequence of the transcription regulator can be obtained from GenBank NCBI - a well-known database (eg, WP 003857140.1, etc.). In particular, it may be a polypeptide showing the activity of a transcription regulator encoded by the iolR gene, but is not limited to it.

In the present disclosure, the term "corresponding" refers to amino acid residues at the positions listed in the polypeptide, or to amino acid residues that are similar, identical or homologous to the residues listed in this polypeptide. The identification of an amino acid at the corresponding position may be determined by the specific amino acid in the sequence that refers to the specific sequence. The term "relevant region" as used herein generally refers to a similar or corresponding position in a related protein or reference protein.

For example, an arbitrary amino acid sequence aligns with SEQ ID NO: 3, and based on this, each amino acid residue of that amino acid sequence can be numbered with respect to the amino acid residue of SEQ ID NO: 3 and the numeric position of the corresponding amino acid residue. For example, a sequence alignment algorithm as described herein can determine the position of an amino acid or the position at which a modification occurs, such as a substitution, insertion, or deletion, by comparison with a position in a requested sequence (also referred to as a "reference sequence").

For such alignments, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et at , 2000, Trends Genet. 16:276-277) and the like, but the program and algorithm are not limited thereto, and a sequence alignment program, a pairwise sequence comparison algorithm, and the like known in the art may be suitably used.

Another aspect of the present disclosure is to provide a polynucleotide a encoding a variant of the present disclosure.

In the present disclosure, the term "polynucleotide" is a strand of DNA or RNA, having a certain or greater length, as a polymer of nucleotides, in which nucleotide monomers are joined into a long chain by covalent bonds, and, more specifically, means a polynucleotide fragment a encoding a given variant.

A polynucleotide encoding a variant of the present disclosure may comprise a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present disclosure, a polynucleotide of the present disclosure may have or contain the sequence of SEQ ID NO: 2. A polynucleotide of the present disclosure may consist or essentially consist of the sequence of SEQ ID NO: 2.

In another embodiment, in a polynucleotide of the present disclosure, the base corresponding to position 739 based on the nucleic acid sequence of SEQ ID NO: 4 in the nucleic acid sequence represented by SEQ ID NO: 2 is A, and the polynucleotide may comprise a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%- 99.5%, 99.7% or 99.9% or greater homology or identity with the nucleic acid sequence represented by SEQ ID NO: 2. Obviously, polynucleotides having amino acid sequences in which certain sequences deleted, modified, substituted, conservatively substituted, or added are also included within the scope of this disclosure, provided that those nucleic acid sequences share such homology or identity and encode a polypeptide or protein exhibiting an efficacy corresponding to that of the variant of the present disclosure.

In a polynucleotide of the present disclosure, various modifications can be made in the coding region, provided that the amino acid sequence of the variant of the present disclosure is not altered when considering codon degeneracy or preferred codons in organisms that are destined to express the variant of the present disclosure. In particular, the polynucleotide of the present disclosure has or contains a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95 % or greater, 96% or greater, 97% or greater, 98% or greater, but less than 100% homology or identity with the sequence of SEQ ID NO: 2 or may consist of or essentially consists of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96 % or greater, 97% or greater, 98% or greater, but less than 100% homology or identity with the sequence of SEQ ID NO: 2, but not limited to it. Here, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 247 of SEQ ID NO: 1 may be one of the codons encoding lysine.

The polynucleotide of the present disclosure may comprise a probe that can be derived from a sequence of a known gene, such as a sequence without limitation, provided that it is a sequence that can hybridize to a complementary sequence of all or part of the sequence of the polynucleotide of the present disclosure under stringent conditions. "Stringent conditions" means conditions that allow for specific hybridization between polynucleotides. These conditions are specifically described in documents (see J. Sambrook et al, Molecular Cloning, A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples thereof include conditions under which polynucleotides having higher homology or identity, namely: polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater homology or identity, hybridize with each other , while polynucleotides having less homology or identity do not hybridize with each other, or conditions under which washing is carried out once, in particular two to three times at a salt concentration and temperature equivalent to 60°C, 1×SSC ( citrate and sodium chloride solution), 0.1% SDS (sodium dodecyl sulfate), in particular at 60°C, 0.1×SSC, 0.1% SDS, more specifically at 68°C, 0.1×SSC , 0.1% SDS, which are wash conditions for conventional Southern hybridization.

Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are tolerated, depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing with each other. For example, in relation to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Therefore, a polynucleotide of the present disclosure may also contain substantially similar nucleic acid sequences, as well as isolated nucleic acid fragments that are complementary to the entire sequence.

In particular, a polynucleotide having homology or identity with a polynucleotide of the present disclosure can be detected using hybridization conditions, including a hybridization step at a T _m value of 55°C and the conditions described above. The T _m value may be 60° C., 63° C. or 65° C., but is not limited to, and may be appropriately adjusted by those skilled in the art according to purpose.

The appropriate stringency for hybridization of a polynucleotide depends on the length and degree of complementarity of the given polynucleotide, and the variables are well known in the art (e.g., J. Sambrook et al, Molecular Cloning, A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989).

Another aspect of the present disclosure is the provision of a vector containing the polynucleotide of the present disclosure. This vector may be an expression vector for carrying out the expression of a polynucleotide in a host cell, but is not limited to it.

The vector of the present disclosure may include a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) such that the polypeptide of interest can be expressed in a suitable host. The regulatory region of expression may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA binding site for the ribosome, and a sequence regulating transcription and translation termination. This vector can be transformed into a suitable host cell and then can replicate or function independently of the host genome, or can integrate itself into the genome.

The vector used in the present disclosure is not specifically limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, HXP, ASHII, ARP, t10, t11, Charon4A, Charon21A and the like can be used as the phage vector or cosmid vector, and the pDZ system, pBR system, pUC, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, and the like. In particular, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and pCClBAC vectors, and the like can be used.

For example, a polynucleotide encoding a polypeptide of interest can be inserted into a chromosome via an intracellular chromosomal insertion vector. Insertion of a given polynucleotide into a chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. This vector may further contain a selectable marker to confirm the insertion into the chromosome. This selectable marker serves to select cells transformed with vectors, i.e. to confirm the insertion of a nucleic acid molecule of interest, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides can be used. In the medium treated with the selective agent, only cells expressing the selectable marker survive or exhibit other phenotypic characteristics, and thus transformed cells can be selected.

In the present disclosure, the term "transformation" means that a vector containing a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may be localized by insertion into the chromosome of the host cell, or localized off the chromosome, so long as it can be expressed in the host cell. This polynucleotide contains DNA and/or RNA encoding the polypeptide of interest. This polynucleotide can be inserted in any form, provided that it can be introduced into the host cell and can be expressed. For example, a given polynucleotide can be introduced into a host cell as an expression cassette, which is a genetic construct containing all the elements required for autonomous expression. This expression cassette typically may contain a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. This expression cassette may be in the form of an expression vector capable of autonomous replication. A polynucleotide can be introduced into a host cell by itself and is operably linked to a sequence required for expression in the host cell, but is not limited to.

As used above, "operably linked" means that a polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a variant of interest according to the present disclosure.

Yet another aspect of the present disclosure is the provision of a strain of Corynebacterium glutamicum that contains a variant of the present disclosure or a polynucleotide of the present disclosure.

The strain of the present disclosure may contain a modified polypeptide of the present disclosure, a polynucleotide encoding the polypeptide, or a vector containing the polynucleotide of the present disclosure.

In the present disclosure, a "strain (or microorganism)" includes all wild-type or naturally or artificially genetically modified microorganisms, and may be a microorganism in which a specific mechanism is attenuated or enhanced due to the insertion of an external gene or enhancement of activity, or inactivation of an endogenous a gene, and it may be a microorganism containing a genetic modification to produce a polypeptide, protein or product of interest.

The strain of the present disclosure may be a strain containing any one or more variants of the present disclosure, a polynucleotide of the present disclosure, or a vector containing a polynucleotide of the present disclosure; a strain modified to express a variant of the present disclosure or a polynucleotide of the present disclosure; a strain (eg, a recombinant strain) expressing a variant of the present disclosure or a polynucleotide of the present disclosure; or a strain (eg, a recombinant strain) that exhibits, but is not limited to, activity of the variant of the present disclosure.

The strain of the present disclosure may be a strain having the ability to produce L-valine.

The strain of the present disclosure may be a microorganism having a naturally occurring transcription regulator activity and/or the ability to produce L-valine, or a microorganism in which a variant of the present disclosure or a polynucleotide encoding the variant (or a vector containing the polynucleotide) is administered to a parent strain that does not have transcription regulator activity or L-valine producing ability, and/or in which L-valine producing ability is conferred to, but not limited to, the parent strain.

For example, the strain of the present disclosure is a cell or microorganism that has been transformed with a vector containing a polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure and expresses the variant of the present disclosure. For the purposes of the present disclosure, the strain of the present disclosure may include all microorganisms that contain the variant of the present disclosure and can produce L-valine. For example, the strain of the present disclosure may be a recombinant strain in which a polynucleotide encoding a variant of the present disclosure is introduced into a natural wild-type microorganism or an L-valine producing microorganism, in order to thereby express a transcriptional regulator variant and have increased ability to produce L-valine. The recombinant strain having an increased ability to produce L-valine may be a microorganism having an increased ability to produce L-valine compared to a natural wild-type microorganism or a microorganism not modified with a transcription regulator (namely, a microorganism expressing a wild-type transcription regulator (SEQ ID NO: 3), or a microorganism that does not express the modified (SEQ ID NO: 1) protein, but is not limited to. For example, a strain having an increased ability to produce L-valine according to the present disclosure may be a microorganism having an increased ability to produce L-valine compared to Corynebaterium glutamicum, which contains a polypeptide of SEQ ID NO: 3 or a polynucleotide encoding this polypeptide, but not limited to them. As an example, a microorganism not modified with a transcription regulator, which is a target strain for comparing the increase in the ability to produce L-valine, can be a strain of ATCC14067 and/or a strain of Corynebaterium glutamicum CA08-0072 (KCCM1120IP), but is not limited to.

For example, a recombinant strain having an increased production capacity may have an L-valine production capacity increased by about 1% or more, by about 2.5% or more, by about 5% or more, by about 6% or more , about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5 % or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5%, or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.1% or more, or about 22.2% or more (the upper limit is not specifically limited and may be, for example, , about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, or about 15% or less) as compared to the L-valine-producing ability of the parent strain before modification or with the unmodified microorganism, but the increased amount is not limited thereto, provided that the ability to produce has an increased increased value compared to the ability to produce the parent strain before the change or with an unmodified microorganism. In another example, a recombinant strain having an increased production capacity may have an L-valine production capacity increased by about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more About 1.16 times or more About 1.17 times or more About 1.18 times or more About 1.19 times or more About 1.2 times or more, about 1.21 times or more, about 1.22 times or more (the upper limit is not particularly limited, and may, for example, be about 10 times or less, about 5 times or less, about or less, or about 2 times or less) compared to the L-valine-producing ability of the parental strain before modification or with an unmodified microorganism.

More specifically, a recombinant strain having an increased production capacity may have an L-valine production capacity increased by about 22.2% (or about 1.22 times) compared to the L-valine production ability of the parental strain before the change. or with an unmodified microorganism, but the increase rate is not limited thereto. The term "about" is an interval including all of plus/minus 0.5; plus/minus 0.4; plus/minus 0.3; plus/minus 0.2; plus/minus 0.1 and the like, and includes all values in the range equal to or similar to the value after the term "about", but is not limited to them.

In the present disclosure, "non-modified microorganism" does not exclude strains containing a mutation that may occur in microorganisms in nature, and may be a wild-type strain or a natural strain itself, or may be a strain before a change in trait through genetic variation due to natural or artificial factors. For example, the unmodified microorganism may be a strain that has not or has not yet been introduced the protein variant described herein. The term "unmodified microorganism" can be used interchangeably with the phrases "pre-modification strain", "pre-modification microorganism", "unchanged strain", "unmodified strain", "unchanged microorganism", or "reference microorganism".

В другом примере настоящего раскрытия микроорганизм по настоящему раскрытию может представлять собой Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

In the present disclosure, "attenuation" of the activity of a polypeptide includes both cases where the activity is reduced compared to endogenous activity or absent.

Attenuation may also include the case where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism by changing the polynucleotide encoding the polypeptide and the like, the case where the overall level of activity and/or concentration (expression level) of the polypeptide in the cell is lower than the level of activity or concentration of the natural strain by inhibiting the expression of the gene of the polynucleotide encoding the polypeptide or inhibiting translation into the polypeptide, the case where the polynucleotide is not expressed at all, and/or the case where the activity of the polypeptide is not shown even when expressed polynucleotide. The term "endogenous activity" means the activity of a particular polypeptide, which was originally possessed by the parental strain before changing the trait, or a wild-type microorganism, or an unmodified microorganism when the trait is changed by genetic variation due to natural or artificial factors. The phrase "endogenous activity" can be used interchangeably with the phrase "activity before modification". The fact that the activity of a polypeptide is "inactivated, deficient, reduced, down-regulated, reduced or attenuated" compared to endogenous activity means that the activity of the polypeptide is reduced relative to the activity of the particular polypeptide that the parent strain originally had before the trait change or a wild-type microorganism, or an unmodified microorganism.

Such weakening of the activity of the polypeptide can be carried out by any method known in this field, but this method is not limited to them, and weakening can be achieved using various methods well known in this field (for example, Nakashima N. et al, Bacterial cellular engineering by genome editing and gene silencing Int J Mol Sci 2014; 15(2): 2773-2793, Sambrook et al, Molecular Cloning 2012, et al.).

In particular, the weakening of the activity of the polypeptide in the present disclosure may be:

1) deletion of all or part of the gene encoding the polypeptide;

2) modification of the regulatory region of expression (or regulatory sequence of expression) to reduce the expression of the gene encoding the polypeptide;

3) modifying the amino acid sequence constituting the polypeptide to eliminate or reduce the activity of the polypeptide (eg, deletion/substitution/addition of one or more amino acids in the amino acid sequence);

4) modification of the sequence of the gene encoding the polypeptide to eliminate or weaken the activity of this polypeptide (for example, deletion/substitution/addition of one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene to encode a polypeptide that has been modified to eliminate or weaken activity of the given polypeptide);

5) modification of the initiating codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR (5'-untranslated region) region;

6) introducing an antisense oligonucleotide (eg, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure to which the ribosome cannot attach;

8) adding a promoter to be transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

9) a combination of two or more than two selected from 1)-8), but is not particularly limited to them.

For example:

1) Deletion of part or all of a gene encoding a polypeptide can be the removal of the entire polynucleotide encoding the endogenous polypeptide of interest on the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

2) Modification of the expression regulatory region (or expression regulatory sequence) may be a deletion, insertion, non-conservative or conservative substitution, or the appearance of a change in the expression regulatory region (or expression regulatory sequence) due to their combination, or replacement by a sequence showing weaker activity . The expression regulatory region contains, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination.

3) Modification of the start codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region, may be, for example, replacing the base sequence encoding a different start codon having a lower expression rate of the polypeptide compared to the endogenous start codon, but not limited to her.

4) and 5) A modification of the amino acid sequence or sequence of a polynucleotide may be a deletion, insertion, or a non-conservative or conservative substitution of the amino acid sequence of the polypeptide or of the polynucleotide sequence encoding the polypeptide, or the appearance of a change in the sequence due to their combination, or a substitution by an amino acid sequence or sequence a polynucleotide modified to exhibit weaker activity, or an amino acid sequence, or a polynucleotide sequence modified to be inactive such that the activity of the polypeptide is reduced, but is not limited to. For example, gene expression can be inhibited or attenuated by introducing a change in the sequence of a polynucleotide and the formation of a stop codon, but the modification is not limited thereto.

6) To introduce an antisense oligonucleotide (eg, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide, reference can be made to documents such as Weintraub, H. et al, Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.

7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure to which the ribosome cannot attach, in order to make mRNA translation impossible or to slow down the rate of mRNA translation .

8) The addition of a promoter to be transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the sequence of the gene encoding the polypeptide (reverse transcription engineering, RTE) can serve to attenuate the activity by obtaining an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide .

In the present disclosure, the term "enhancement" of the activity of the polypeptide means that the activity of the polypeptide is increased compared to endogenous activity. The term "enhancement" can be used interchangeably with terms such as activation, upregulation, overexpression and increase. Here, activation, amplification, upregulation, overexpression, and increase can include both showing activity that was not originally present, and showing improved activity compared to endogenous activity or activity before modification. The term "endogenous activity" means the activity of a particular polypeptide, which was originally possessed by the parental strain before changing the trait or unmodified microorganism when the trait is changed through genetic variation due to natural or artificial factors. This can be used interchangeably with "activity before modification". The fact that the activity of a polypeptide is "upregulated", "upregulated", "overexpressed" or "increased" compared to endogenous activity means that the activity of the polypeptide is improved relative to the activity and/or concentration (expression level) of a particular polypeptide, which the parent strain originally possesses before the trait change or unmodified microorganism.

This enhancement can be achieved by introducing a foreign polypeptide or by increasing the endogenous activity and/or concentration (expression level) of the polypeptide. An increase in the activity of a polypeptide can be evidenced by an increase in the degree of activity and level of expression of the polypeptide or the amount of product produced from the polypeptide.

Various methods well known in the art can be used to enhance the activity of a polypeptide, and this method is not limited, provided that the activity of the polypeptide of interest can be enhanced relative to the activity of the microorganism before the modification. In particular, genetic engineering and/or protein engineering well known to those skilled in the art can be used, which are traditional molecular biology techniques, but this technique is not limited to them (e.g., Sitnicka et at, Functional Analysis of Genes. Advances in Cell Biology 2010, Vol. 2. 1-16; Sambrook et at, Molecular Cloning 2012; and the like).

In particular, enhancing the activity of a polypeptide of the present disclosure may be:

1) an increase in the number of intracellular copies of the polynucleotide encoding the polypeptide;

2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence showing strong activity;

3) modification of the initiating codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region;

4) modification of the amino acid sequence of the polypeptide to increase the activity of this polypeptide;

5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of that polypeptide (eg, modifying the polynucleotide sequence of a polypeptide gene to encode a polypeptide that has been modified to enhance the activity of that polypeptide);

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

7) codon optimization of the polynucleotide encoding the polypeptide;

8) analysis of the tertiary structure of the polypeptide to select and modify or chemically modify the exposed site; or

9) a combination of two or more than two selected from (1) - (8), but not specifically limited to them.

More specific:

1) An increase in the intracellular copy number of a polynucleotide encoding a polypeptide can be achieved by introducing into the host cell a vector that can replicate and function independently of the host and to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the increase can be achieved by introducing one copy or two or more than two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out by the introduction of a vector capable of inserting the polynucleotide into the chromosome of the host cell, into the host cell, but is not limited to. This vector is as described above.

2) Replacement of a gene expression regulatory region (or an expression regulatory sequence) on a chromosome encoding a polypeptide with a sequence exhibiting strong activity may be, for example, a deletion, an insertion, a non-conservative or conservative substitution, or the occurrence of a change in sequence due to a combination thereof , or replacement with a sequence showing stronger activity, such that the activity of the regulatory region of expression is further enhanced. The regulatory region of expression is not specifically limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription and translation termination, and the like. For example, the replacement may be, but is not limited to, replacing the original promoter with a strong promoter.

Examples of known strong promoters include CJ1-CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter ( US 10584338 B2), promoter 02 (US 10273491 B2), tkt promoter and ucsA promoter, but are not limited to them.

3) Modification of the initiation codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region, may be, for example, a replacement with a base sequence encoding a different initiation codon having a higher expression rate of the polypeptide compared to the endogenous initiation codon, but not limited to it.

4) and 5) Modification of the amino acid sequence or polynucleotide sequence can be a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding this polypeptide, or the appearance of a change in this sequence due to their combination or replacement by an amino acid sequence or sequence a polynucleotide modified to exhibit stronger activity, or an amino acid sequence or polynucleotide sequence modified to be more active such that the activity of the polypeptide is enhanced, but is not limited to. This replacement can be specifically carried out by inserting a polynucleotide into the chromosome by homologous recombination, but not limited to it. Used here, the vector may additionally contain a selectable marker to confirm insertion into the chromosome. The selectable marker is as described above.

6) The introduction of a foreign polypeptide showing the activity of the polypeptide may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide showing the same or similar activity to this polypeptide. This foreign polynucleotide is not limited in its origin or sequence, provided that it exhibits the same or similar activity to this polypeptide. The introduction can be carried out by a suitable choice of transformation method, known to experts in this field. Since the introduced polynucleotide is expressed in the host cell, the polypeptide can be produced and its activity can be increased.

7) Codon optimization of a polynucleotide encoding a polypeptide can be codon optimization of an endogenous polynucleotide so as to increase transcription or translation in the host cell, or codon optimization of a foreign polynucleotide so as to achieve optimized transcription and translation in the host cell.

8) Analyzing the tertiary structure of a polypeptide for selection and modification or chemically modifying an exposed site can be carried out, for example, to determine a template candidate protein according to the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a repository of information on sequences of known database proteins for confirming the structure based on this and for selecting and modifying or chemically modifying the exposed portion to be modified or chemically modified.

Such an increase in the activity of a polypeptide can be an increase in the activity or concentration or level of expression of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type microbial strain or in the microbial strain before modification, or an increase in the amount of product produced from the polypeptide, but is not limited to. .

In the microorganism of the present disclosure, partial or complete modification (e.g., modification to encode a protein variant as described above) of a polynucleotide can be induced by (a) homologous recombination using a vector to insert into a chromosome in the microorganism, or by editing the genome using a genetically modified nuclease (e.g., CRISPR-Cas9), and/or (b) treatment with light such as ultraviolet rays and radiation, and/or chemical agents, but not limited to. The method for modifying part or all of a gene may include a method using recombinant DNA technology. For example, by introducing into the microorganism a nucleotide sequence or a vector containing a nucleotide sequence homologous to a gene of interest, part or all of the gene can be removed to induce homologous recombination.

The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

In the microorganism of the present disclosure, the variant, polynucleotide, L-valine, and the like are as described in other aspects.

According to still another aspect of the present disclosure, a method for producing an L-amino acid is provided, which comprises culturing in a medium a strain of Corynebacterium glutamicum containing a variant of the present disclosure or a polynucleotide of the present disclosure.

The method for producing an L-amino acid of the present disclosure may include culturing in a medium a strain of Corynebacterium glutamicum containing a variant of the present disclosure or a polynucleotide of the present disclosure or a vector of the present disclosure.

In addition, the L-amino acid of the present disclosure may be L-valine.

In the present disclosure, the term "cultivation" means the cultivation of the Corynebacterium glutamicum strain of the present disclosure under suitably controlled environmental conditions. The culturing method of the present disclosure can be carried out in accordance with a suitable medium and culturing conditions known in the art. Such a culture method can be easily adjusted and used by those skilled in the art according to the selected strain. In particular, the cultivation may be of batch type, continuous type, and/or fed-batch type, but is not limited to them.

In the present disclosure, the term "medium" means a mixed substance containing the nutrients required for cultivating the strain of Corynebacterium glutamicum of the present disclosure as the main component, and this medium supplies nutrients, growth factors and the like, including water, which are indispensable for survival. and development. In particular, as the medium and other culture conditions used for culturing the strain of Corynebacterium glutamicum of the present disclosure, any one can be used without particular limitation, as long as it is a medium used for conventional cultivation of microorganisms. The strain of Corynebacterium glutamicum of the present disclosure can be cultivated in an ordinary medium containing proper carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, and the like, while controlling temperature, pH, and the like under aerobic conditions.

In particular, a culture medium for a strain of the genus Corynebacterium can be found in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington, D.C., USA, 1981).

In the present disclosure, carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid; amino acids such as glutamic acid, methionine and lysine; etc. Natural organic nutrients such as hydrolysed starch, molasses, raw molasses, rice bran, cassava, sugar cane residue and liquid corn extract can be used. In particular, carbohydrates such as glucose and sterilized pre-treated molasses (namely, molasses converted to reducing sugar) can be used, and suitable amounts of other carbon sources can be used in a variety of ways without limitation. These carbon sources can be used alone or in combination with, but not limited to, two or more.

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

Phosphorus sources may include monopotassium phosphate, dipotassium phosphate, or their respective sodium salts. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate and the like can be used. In addition to these, amino acids, vitamins and/or suitable precursors and the like may be contained. These components or precursors may be added to the medium in batches or continuously, but the method of addition is not limited to them.

During cultivation of the Corynebacterium glutamicum strain of the present disclosure, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in the correct manner. During culture, foaming can be suppressed by using an antifoam such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas may be injected into the environment to maintain the aerobic state of the environment, or the gas may not be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected in order to maintain the anaerobic and microaerobic conditions, but the method of maintaining this state is not limited to them.

When cultivating according to the present disclosure, it is possible to maintain the culturing temperature from 20°C to 45°C, in particular, from 25°C to 40°C, and this strain can be cultivated for about 10 to 160 hours, but the culturing conditions are not limited to them. .

The L-amino acid produced by the cultivation of the present disclosure may be secreted into the medium or may remain in the cells.

The method for producing the L-amino acid of the present disclosure may further include the step of obtaining the strain of Corynebacterium glutamicum of the present disclosure, the step of preparing a culture medium for the strain, or a combination thereof (in any order), for example, before the cultivation step.

The method for producing an L-amino acid according to the present disclosure may further include the step of isolating the L-amino acid from a medium according to cultivation (media exposed to culture) or from a strain of Corynebacterium glutamicum. After the culture step, an isolation step may be further included.

The isolation may serve to collect the L-amino acid of interest by a suitable method known in the art according to the microorganism culture method of the present disclosure, for example, a batch, continuous or fed-batch culture method. For example, centrifugation, filtration, precipitant treatment of the crystallized protein (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography can be used, HPLC (high performance liquid chromatography), or a combination of both. The L-amino acid of interest can be isolated from the medium or microorganism by a suitable method known in the art.

The method for producing an L-amino acid according to the present disclosure may further include a purification step. Purification can be carried out by a suitable method known in this field. In one example, when the process for producing an L-amino acid of the present disclosure includes both a isolation step and a purification step, the isolation step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously, or by being combined into one step, but the manner in which these steps are carried out is not limited thereto.

In the method of the present disclosure, the variant, polynucleotide, vector, strain, and the like are as described in other aspects.

Yet another aspect of the present disclosure is to provide a composition for producing an L-amino acid which comprises a strain of Corynebacterium glutamicum containing a variant of the present disclosure, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a polynucleotide of the present disclosure; the medium in which the strain of Corynebacterium glutamicum was cultured; or a combination of two or more than two of them.

The composition of the present disclosure may further comprise any suitable excipients of common use in amino acid production compositions. Such excipients may be, for example, a preservative, humectant, dispersing agent, suspending agent, buffering agent, stabilizer or isotonic agent, but are not limited to .

In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, L-amino acid, and the like are as described in other aspects.

Useful effects

In the case of cultivating a strain of Corynebacterium glutamicum containing the novel version of the transcription regulator of the present disclosure, it is possible to produce L-valine in a higher yield compared to the case of existing microorganisms having unmodified polypeptides.

Method for carrying out the invention

Below, the present disclosure will be described in more detail by way of Examples. However, the following Examples are only preferred embodiments for illustrating the present disclosure, and thus are not intended to limit the scope of the present disclosure. However, technical issues not described in the present description of the invention can be sufficiently understood and easily implemented by experts in the technical field of the present disclosure or in similar technical fields.

Example 1 Construction of a Vector for Expression in a Microorganism of a Transcriptional Regulator Variant

In order to confirm the effect of a variant (E247K; SEQ ID NO: 1) in which glutamic acid (Glu, E) at position 247 of the protein consisting of the amino acid sequence of SEQ ID NO: 3 was replaced by lysine (Lys, K), on the production of L-valine, a vector was constructed to construct a strain expressing this variant using plasmid pDCM2 (Korean Patent Publication No. 10-2020-0136813) to insert and replace a gene in the Corynebacterium genome as follows.

PCR (Polymerase Chain Reaction) was performed using wild-type Corynebacterium glutamicum gDNA ATCC14067 as a template, a primer pair having sequences of SEQ ID NOs: 5 and 6, and a primer pair having sequences of SEQ ID NOs: 7 and 8. PCR was again performed with overlapping primers using a mixture of the two fragments obtained above as a template and a pair of primers having the sequences of SEQ ID NO: 5 and SEQ ID NO: 8 to obtain a fragment. PCR was performed as follows: denaturation at 94°C for 5 minutes; 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute 30 seconds; and 72°C for 5 minutes. The pDCM2 vector was treated with smaI and the PCR product obtained above was cloned into it by fusion. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-iolR(E247K). The primer sequences used in this example are described in the following Table 1:

Example 2 Evaluation of the L-Valine Producing Ability of a Microorganism Expressing a Transcription Regulator Variant

2-1. Construction of a strain expressing a transcriptional regulator variant

The vector constructed in Example 1 was transformed into Corynebacterium glutamicum CA08-0072 (KSCM11201P) (US 8465962).

The strain in which this variant was introduced was selected from strains in which homologous recombination occurred using SEQ ID NOs: 9 and 10. The selected strain was named CA08-0072_iolR_E247K. The primer sequences used in this example are described in the following Table 2:

2-2. Comparison of the ability to produce L-valine of strains expressing a transcriptional regulator variant

The ability to produce L-valine was analyzed by evaluating the flask fermentation titer of each strain constructed in Example 2-1 and the parental control strain.

First, each colony was subcultured in nutrient medium, and then each strain was inoculated into a 250 ml baffled flask containing 25 ml of production medium, and subjected to shaking culture at 200 rpm at 30° C. for 72 hours. Then, the concentration of L-valine was analyzed by HPLC, and the analyzed concentration of L-valine was shown in Table 3 below.

Nutrient medium (pH 7.2)

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

Production medium (pH 7.0)

100 g glucose, 40 g ammonium sulfate, 2.5 g soy protein, 5 g corn extract solids, 3 g urea, 1 g dipotassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 30 g calcium carbonate (based on 1 liter of distilled water).

This experiment was repeated 3 times, and the average values of the results of its analysis are presented in Table 3 below.

As shown in Table 3, strain CA08-0072 iolR E247K showed an increased ability to produce L-valine compared to the ability to produce L-valine of the control group.

From the foregoing description, those skilled in the technical field to which the present disclosure belongs will appreciate that the present disclosure may be embodied in other specific forms without altering its technical nature or essential characteristics. In this regard, it should be understood that the embodiments described above are in all respects illustrative and not restrictive. The scope of the present disclosure should be construed to include all changes or modified forms deriving from the meaning and scope of the claims to be described below, and not from the above detailed description and equivalent ideas.

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<110> CJ CheilJedangCorporation

<120> New transcription regulator variant and method

production of L-valine with its use

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Asp Arg Asn Gly Pro Ile Pro Leu Tyr Phe Gln Val Ala Gln Arg Leu

20 25 30

Glu Asp Gly Ile Arg Ser Gly Val Leu Pro Pro Gly Ala Arg Leu Glu

35 40 45

Asn Glu Ile Ser Val Ala Lys His Leu Asn Val Ser Arg Pro Thr Val

50 55 60

Arg Arg Ala Ile Gln Glu Val Val Asp Lys Gly Leu Leu Val Arg Arg

65 70 75 80

Arg Gly Val Gly Thr Gln Val Val Gln Ser His Val Thr Arg Pro Val

85 90 95

Glu Leu Thr Ser Phe Phe Asn Asp Leu Lys Asn Ala Asn Leu Asp Pro

100 105 110

Lys Thr Arg Val Leu Glu His Arg Leu Leu Ala Ala Ser Ser Ala Ile

115 120 125

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

130 135 140

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

145 150 155 160

Leu Pro Pro Ala Phe Asn Asp Val Ser Leu Asp Glu Leu Glu Glu Lys Gly

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Leu Asp Asn Ser Gly Gln Val Ile Glu Leu Gly Ser His Cys Tyr Arg

225 230 235 240

Pro Asp Met Tyr Asn Phe Lys Thr Thr Leu Val Ala Arg

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ctcccccg gagcacgcct agaaaacgag atctccgtgg cgaaacacct caacgtatcc 180

cgccccaccg tccgccgcgc catccaagaa gtcgtagaca aaggcctcct agttcgtcgc 240

cgcggtgttg gcacccaggt cgtccaaagc cacgtcaccc gcccagtcga actgaccagt 300

ttcttcaacg acctcaaaaa cgccaacctg gaccccaaaa cccgagtcct cgagcaccgc 360

ctccttgctg caagttccgc catcgcagaa aaactcggag tttccgcagg tgacgaagtc 420

ctcctcatcc gccgcctccg ctccaccgga gacatccccg tagcgatcct ggaaaactac 480

ctccccccag cattcaacga cgtctccctc gacgaactgg aaaaaggcgg actctacgat 540

gcactgcgca gccgtggcgt tgtcctaaaa atcgccaacc agaaaatcgg tgcgcgccga 600

gcagtcggtg aagaaagcac cctcctcgac atcgaagacg gcggaccact tctcaccgtc 660

gaacgcgttg cattggataa ttccggccaa gtaatcgagt tgggaagcca ctgctaccgc 720

ccagatatgt acaactttaa aaccactctg gtggccaggt aa 762

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Met Thr Thr Glu Ala Pro Ile Trp Pro Ala Glu Leu Phe Glu Asp Leu

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Glu Asp Gly Ile Arg Ser Gly Val Leu Pro Pro Gly Ala Arg Leu Glu

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Arg Arg Ala Ile Gln Glu Val Val Asp Lys Gly Leu Leu Val Arg Arg

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Arg Gly Val Gly Thr Gln Val Val Gln Ser His Val Thr Arg Pro Val

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Glu Leu Thr Ser Phe Phe Asn Asp Leu Lys Asn Ala Asn Leu Asp Pro

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Lys Thr Arg Val Leu Glu His Arg Leu Leu Ala Ala Ser Ser Ala Ile

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

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

180 185 190

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

195 200 205

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

210 215 220

Leu Asp Asn Ser Gly Gln Val Ile Glu Leu Gly Ser His Cys Tyr Arg

225 230 235 240

Pro Asp Met Tyr Asn Phe Glu Thr Thr Leu Val Ala Arg

245 250

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atgaccaccg aagctcccat ttggccagcc gaactcttcg aagacctcga ccgcaacgga 60

ccaatccccc tctacttcca agtagcccaa cgcctcgaag acggcatccg cagtggagtc 120

ctcccccg gagcacgcct agaaaacgag atctccgtgg cgaaacacct caacgtatcc 180

cgccccaccg tccgccgcgc catccaagaa gtcgtagaca aaggcctcct agttcgtcgc 240

cgcggtgttg gcacccaggt cgtccaaagc cacgtcaccc gcccagtcga actgaccagt 300

ttcttcaacg acctcaaaaa cgccaacctg gaccccaaaa cccgagtcct cgagcaccgc 360

ctccttgctg caagttccgc catcgcagaa aaactcggag tttccgcagg tgacgaagtc 420

ctcctcatcc gccgcctccg ctccaccgga gacatccccg tagcgatcct ggaaaactac 480

ctccccccag cattcaacga cgtctccctc gacgaactgg aaaaaggcgg actctacgat 540

gcactgcgca gccgtggcgt tgtcctaaaa atcgccaacc agaaaatcgg tgcgcgccga 600

gcagtcggtg aagaaagcac cctcctcgac atcgaagacg gcggaccact tctcaccgtc 660

gaacgcgttg cattggataa ttccggccaa gtaatcgagt tgggaagcca ctgctaccgc 720

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agatatgtac aactttaaaa ccactctggt ggc 33

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gtcgactcta gaggatcccc ttgagctaag tatcctatga 40

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ccaagaagtc gtagacaaag 20

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ttgagctaagtatcctatga 20

<---

Claims (5)

1. A polypeptide involved in the production of L-valine, consisting of the amino acid sequence shown by SEQ ID NO: 1, in which glutamic acid, which is the amino acid corresponding to position 247 of SEQ ID NO: 3, is replaced by lysine. 2. A polynucleotide encoding a polypeptide according to claim 1. 3. The microorganism Corynebacterium glutamicum for producing L-valine, containing the polypeptide according to claim 1 or a polynucleotide encoding this polypeptide. 4. The microorganism according to claim 3, which has an increased ability to produce L-valine compared to Corynebacterium glutamicum containing a polypeptide of SEQ ID NO: 3 or a polynucleotide encoding this polypeptide. 5. A method for producing L-valine, comprising cultivating a microorganism Corynebacterium glutamicum containing a polypeptide according to claim 1 or a polynucleotide encoding this polypeptide in a medium.