RU2840267C2 - Lyse variant and method for producing l-arginine using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a polypeptide involved in the production of l-arginine, having 80 % or more sequence identity with SEQ ID NO: 1, and wherein any one or more amino acids from amino acids corresponding to positions 50, 153 and 215 in SEQ ID NOs: 1, is replaced with another amino acid, where the amino acid corresponding to position 50 is replaced with valine, leucine or isoleucine, the amino acid corresponding to position 153 is replaced with serine, threonine or glutamine, the amino acid corresponding to position 215 is substituted with serine, threonine or glutamine. Also disclosed are a polynucleotide encoding said polypeptide, a Corynebacterium microorganism for producing L-arginine, containing said polypeptide or polynucleotide encoding said polypeptide, and method of producing L-arginine.

EFFECT: invention increases output of L-arginine.

8 cl, 5 tbl, 3 ex

Description

AREA OF TECHNOLOGY

The present disclosure relates to a LysE variant, a microorganism of the genus Corynebacterium containing the variant, and a method for producing L-arginine using the same.

PRIOR ART

L-arginine is used for medical purposes, such as liver function stimulants, brain function stimulants, complex amino acid preparations, etc., and it is a substance that has recently attracted attention as a food, such as fish pie additives, health drink additives, salt replacement for high blood pressure patients, etc.

Research is ongoing on the use of microorganisms for producing high concentrations of industrially applicable L-arginine, and there have been reports on a method of using variant strains derived from microorganisms of the genus Brevibacterium or Corynebacterium, which are glutamate-producing strains, a method of using an amino acid-producing strain whose growth is improved by cell fusion, etc.

However, although it has been reported that lysE from the genus Corynebacterium, which is a lysine exporter, also exports L-arginine (Bellmann A, et al, Microbiology, 147:1765-1774, 2001), further investigation is required to increase L-arginine production.

DESCRIPTION OF THE INVENTION

Technical problem

According to the present disclosure, a variant of LysE is provided.

Technical solution

The purpose of the present disclosure is to provide a variant of LysE in which any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 in SEQ ID NO: 1 are replaced with another amino acid.

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

Yet another object of the present disclosure is to provide a microorganism comprising a LysE variant of the present disclosure, or a polynucleotide encoding the variant.

Yet another object of the present disclosure is to provide a method for producing L-arginine comprising the step of culturing a microorganism according to the present disclosure.

Beneficial effects

The yield of L-arginine production by microorganisms can be increased using the embodiment of the present disclosure.

Detailed Description of Preferred Embodiments

The present disclosure will be described in detail as follows. Meanwhile, each description and embodiment disclosed in this disclosure can also be applied to other descriptions and embodiments. That is, all combinations of different elements disclosed in this disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited to the specific description described below. In addition, throughout this specification of the invention, reference is made to and cited a number of articles and patent documents. The disclosures of the cited articles and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which the present disclosure pertains.

According to one aspect of the present disclosure, there is provided a variant of LysE in which any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 in SEQ ID NO: 1 are replaced with another amino acid.

The term "LysE" as used here refers to the lysine export protein encoded by the LysE gene.

In particular, LysE may be a naturally occurring polypeptide or a wild-type polypeptide, may be a mature polypeptide thereof, or may include a variant or a functional fragment thereof, but may include any without limitation, provided that it may be a parent variant of a LysE variant of the present disclosure.

The sequence of LysE of the present disclosure can be accessed from GenBank of NCBI (National Institute of Biotechnology Information), etc., which is a well-known database.

In one embodiment, LysE of the present disclosure may be from the genus Corynebacterium and may, for example, be from Corynebacterium glutamicum, Corynebacterium suranareeae, Corynebacterium callunae, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium pacaense, but is not limited to.

In one embodiment, LysE of the present disclosure may be a polypeptide of SEQ ID NO: 1. In one embodiment, LysE of the present disclosure may be a polypeptide having about 60% or greater sequence identity with the polypeptide of SEQ ID NO: 1, and any polypeptide without limitation may be included in the LysE protein, so long as it has an activity identical to or corresponding to the activity of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

For example, LysE can be a protein derived from the genus Corynebacterium having at least about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, or about 99.5% or greater identity to SEQ ID NO: 1. Examples thereof may include proteins with NCBI accession no.

In any embodiment of the above embodiments, LysE of the present disclosure may have or include the amino acid sequence of SEQ ID NO: 1 or may essentially consist of this amino acid sequence, but it may include polypeptides/proteins having the same activity without limitation.

Furthermore, it is obvious that any polypeptide/protein having an amino acid sequence having a deletion, modification, substitution or addition in a portion of this sequence may also be included within the scope of the polypeptide/protein to be mutated according to the present disclosure, provided that it has such homology or identity and exhibits L-lysine export activity.

The term "homology" or "identity" as used herein refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms "homology" and "identity" can often be used interchangeably.

Homology or sequence identity of conserved polynucleotides or polypeptides can be determined by a standard alignment algorithm, and gap penalties set by default by the program to be used can be used together. Substantially homologous or identical sequences can generally hybridize to each other in whole or in part under moderately stringent or very stringent conditions. It is obvious that hybridization also includes hybridization to a polynucleotide containing conventional codons or codons taking into account the degeneracy of the codons in the polynucleotide.

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

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

The term "variant" as used herein refers to a polypeptide that has a different amino acid sequence from the sequence of the variant before modification by conservative substitution and/or modification of one or more amino acids, but retains the functions or properties. Such a variant can generally be identified by making a modification of one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of the variant may be increased, unchanged, or decreased compared to the ability of the polypeptide before the change. In addition, 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 has been deleted from the mature protein. The term "variant" may be used interchangeably with, and is not limited to, terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent, provided that it is a term used with the meaning of change.

In addition, the variant may include 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 may be conjugated to the N-terminus of the variant. The variant may be conjugated to other sequences or linkers in a manner that can be identified, purified, or synthesized.

A LysE variant provided in the present disclosure may be a polypeptide comprising an amino acid sequence in which any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 in SEQ ID NO: 1 are replaced with another amino acid.

For example, a LysE variant of the present disclosure may be a variant comprising substitution of any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 from the N-terminus of SEQ ID NO: 1 in any polypeptide or protein having lysine export activity with another amino acid.

The term "another amino acid" is not limited, as long as it is an amino acid different from the amino acid before the substitution. Meanwhile, in the present disclosure, when expressing "a specific amino acid is replaced", it is obvious that the amino acid is replaced with an amino acid different from the amino acid before the substitution, unless it is otherwise described as being replaced with another amino acid.

In one embodiment, with respect to the variant of the present disclosure, in the amino acid sequence of SEQ ID NO: 1, which is a reference protein, the amino acid corresponding to position 50 may be an amino acid other than phenylalanine, and/or the amino acid corresponding to position 153 may be an amino acid other than asparagine, and/or the amino acid corresponding to position 215 may be an amino acid other than asparagine.

In any embodiment of the above embodiments with respect to the variant, the amino acid corresponding to position 50 in SEQ ID NO: 1 may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, histidine and arginine. For example, the amino acid corresponding to position 50 in SEQ ID NO: 1 of the variant may be a non-polar amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan and proline. For example, the amino acid corresponding to position 50 in SEQ ID NO: 1 of the variant may be a branched amino acid selected from, but not limited to, valine, leucine, and isoleucine.

In any embodiment of the above embodiments with respect to the variant, the amino acid corresponding to position 153 in SEQ ID NO: 1 can be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, histidine and arginine. For example, the amino acid corresponding to position 153 in SEQ ID NO: 1 of the variant can be a polar amino acid selected from serine, threonine, cysteine, tyrosine and glutamine. For example, the amino acid corresponding to position 153 in SEQ ID NO: 1 of the variant can be serine, threonine or glutamine, but is not limited to them.

In any embodiment of the above embodiments with respect to the variant, the amino acid corresponding to position 215 in SEQ ID NO: 1 can be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, histidine and arginine. For example, the amino acid corresponding to position 215 in SEQ ID NO: 1 of the variant can be a polar amino acid selected from serine, threonine, cysteine, tyrosine and glutamine. For example, the amino acid corresponding to position 215 in SEQ ID NO: 1 of the variant can be serine, threonine or glutamine, but is not limited to them.

In any embodiment of the above-described embodiments, a variant of the present disclosure may have any one or more amino acids of the amino acid residues at positions 50, 153 and 215 in the amino acid sequence of SEQ ID NO: 1 replaced with another amino acid. In any embodiment of the above-described embodiments, a variant of the present disclosure may have any amino acid of the amino acid residues at positions 50, 153 and 215 in the amino acid sequence of SEQ ID NO: 1 replaced with another amino acid. The amino acids after the replacement of the amino acid residue at position 50, 153 and 215 in the amino acid sequence of SEQ ID NO: 1 may be the same as the amino acid after the replacement of the amino acid residue corresponding to position 50, 153 or 215 in the amino acid sequence of SEQ ID NO: 1.

In any embodiment of the above embodiments, a variant of the present disclosure may have a substitution of any one or more amino acids of the amino acid residues at positions 50, 153 and 215 in the amino acid sequence of SEQ ID NO: 1 with another amino acid and may have at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, or less than 100% identity to SEQ ID NO: 1.

In any embodiment of the above embodiments, a variant of the present disclosure may consist of an amino acid sequence selected from SEQ ID NO: 27-35, or may have or include an amino acid sequence having at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.8% or more, or 100% homology or identity thereto, or may consist essentially of, but is not limited to, a given amino acid sequence.

It is also apparent that a variant having an amino acid sequence having a deletion, modification, substitution, conservative replacement or addition of some sequence also falls within the scope of the present disclosure, provided that the amino acid sequence has such homology or identity and exhibits efficacy corresponding to that of the variant of the present disclosure.

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

The term "conservative substitution" refers to the replacement of one amino acid with another amino acid that has similar structural and/or chemical properties. Such amino acid substitution may typically occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. Typically, a conservative substitution may have little or no effect on the activity of proteins or polypeptides.

The term "corresponding" as used herein refers to amino acid residues at positions listed in a polypeptide, or to amino acid residues that are similar, identical, or homologous to residues listed in a polypeptide. Identification of an amino acid at a corresponding position may identify a particular amino acid in a sequence that relates to a particular sequence. The term "corresponding region" as used herein generally refers to an analogous or corresponding position in a related protein or reference protein.

For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 1 and, based on this, each amino acid residue of this amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, a sequence alignment algorithm as described in the present disclosure can determine the position of an amino acid or the position at which a modification such as a substitution, insertion or deletion occurs by comparison with an amino acid in a query 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 al., 2000), Trends Genet. 16: 276-277), etc., but not limited to them, can be used, and a sequence alignment program, a pairwise sequence comparison algorithm, etc., which are known in the art, can be appropriately used.

According to another aspect of the present disclosure, there is provided a polynucleotide encoding a variant of the present disclosure.

The term "polynucleotide" as used herein is a strand of DNA or RNA of a certain or greater length, in the form of a polymer of nucleotides in which the nucleotide monomers are linked into a long chain by covalent bonds.

In the polynucleotide of the present disclosure, various modifications can be made in the coding region, provided that the amino acid sequence is not changed, taking into account codon degeneracy or preferred codons in organisms that are intended to express the protein.

In any embodiment of the above embodiments, the polynucleotide of the present disclosure may be available from the lysE sequence in the NCBI GenBank, etc., which is a known database.

In particular, a polynucleotide encoding a variant of the LysE protein of the present disclosure may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or may consist of or consist essentially of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 2, but is not limited thereto. In this regard, in the sequence having homology or identity, the codon encoding the amino acid corresponding to position 50 in SEQ ID NO: 1 may be one of the codons encoding amino acids other than phenylalanine, and/or the codon encoding the amino acid corresponding to position 153 may be one of the codons encoding amino acids other than asparagine, and/or the codon encoding the amino acid corresponding to position 215 may be one of the codons (e.g., SEQ ID NOs: 36-38) encoding amino acids other than asparagine, but is not limited thereto.

In addition, a polynucleotide of the present disclosure may include a probe that is obtainable from a known gene sequence, such as, but not limited to, a sequence that is capable of hybridizing to a complementary sequence, to all or part of a polynucleotide sequence of the present disclosure, under stringent conditions. The term "stringent conditions" means conditions that ensure specific hybridization between polynucleotides. These conditions are specifically described in the documents (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples thereof include conditions under which polynucleotides having higher homology or identity, i.e., polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, while polynucleotides having less homology or identity do not hybridize with each other, or conditions in which washing is carried out once, in particular two to three times, at a salt concentration and temperature equivalent to 60°C, lx SSC (sodium chloride citrate solution), 0.1% SDS (sodium dodecyl sulfate), in particular at 60°C, 0.1x SSC, 0.1% SDS, more in particular at 68°C, 0.lx SSC, 0.1% SDS, which represent the washing conditions for conventional Southern hybridization.

Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are allowed, depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, a polynucleotide of the present disclosure may also include 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 the polynucleotide of the present disclosure can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and the above-described conditions. In addition, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for hybridization of a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and these variables are well known in the art (e.g., J. Sambrook et al., supra).

According to yet another aspect of the present disclosure, there is provided a vector comprising a polynucleotide encoding a LysE variant. The LysE variant and the polynucleotide encoding it are the same as described above.

In one embodiment, the vector may be, but is not limited to, an expression vector for expressing a polynucleotide in a host cell.

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

The vector used in the present disclosure is not particularly 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, IXII, ASHII, ARP, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or a cosmid vector, and pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as a plasmid vector. In particular, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCClBAC, etc. vector can be used.

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

The term "transformation" as used herein means that a vector comprising a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism in such a way that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be located, regardless of position, either inserted into the chromosome of the host cell or located outside the chromosome, so long as it can be expressed in the host cell. The polynucleotide includes DNA and/or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form, so long as it can be introduced into the host cell and can be expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements required for autonomous expression. The expression cassette may typically 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 autonomous replication. In addition, the polynucleotide may be introduced into the host cell in its own form and may be operably linked to, but not limited to, a sequence required for expression in the host cell.

Additionally, the term "operably linked" means that the polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the variant of the present disclosure.

According to yet another aspect of the present disclosure, there is provided a microorganism comprising a LysE variant or a polynucleotide encoding the variant. The LysE variant and the polynucleotide encoding it are as described above.

A microorganism of the present disclosure may include any one or more LysE variants of the present disclosure, a polynucleotide encoding the same, and a vector comprising the polynucleotide.

The term "microorganism (or strain)" as used herein includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it may be a microorganism in which a specific mechanism is weakened or strengthened due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and it may be a microorganism incorporating genetic modification to produce a polypeptide, protein or product of interest.

A microorganism of the present disclosure may be, but is not limited to, a microorganism comprising any one or more variants of the present disclosure, a polynucleotide encoding a variant of the present disclosure, and a vector comprising a polynucleotide encoding a variant of the present disclosure; a microorganism modified to express a variant of the present disclosure or a polynucleotide encoding a variant of the present disclosure; a microorganism (e.g., a recombinant strain) expressing a variant of the present disclosure or a polynucleotide encoding a variant of the present disclosure; or a microorganism (e.g., a recombinant strain) having the activity of a variant of the present disclosure.

The microorganism of the present disclosure may have the ability to export and/or produce L-arginine.

The microorganism of the present disclosure may be, but is not limited to, a microorganism that naturally expresses the LysE protein or has the ability to export and/or produce arginine, or a microorganism in which a variant of the present disclosure or a polynucleotide encoding the variant (or a vector comprising the polynucleotide) is introduced into a parent strain that does not have the LysE protein or the ability to export and/or produce arginine, and/or in which the parent strain is given the ability to export and/or produce arginine. For example, the microorganism of the present disclosure may be, but is not limited to, a microorganism in which a LysE variant of the present disclosure is introduced, and thereby the ability to export and/or produce L-arginine is increased.

For example, a strain of the present disclosure is a cell or microorganism that has been transformed with a polynucleotide of the present disclosure or a vector comprising a polynucleotide encoding a variant of the present disclosure to express the variant of the present disclosure. With respect to the aspects of the present disclosure, a strain of the present disclosure may include any microorganism that comprises a variant of the present disclosure and is capable of exporting or producing L-arginine. For example, a 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 naturally occurring wild-type microorganism or into a microorganism producing L-arginine to express a LysE variant and to have an increased ability to export and/or produce L-arginine. A recombinant strain having an increased ability to export and/or produce L-arginine may be a microorganism having an increased ability to export and/or produce L-arginine compared to a natural wild-type microorganism or a microorganism in which LysE is not modified (i.e., a microorganism expressing wild-type LysE (SEQ ID NO: 1) or a microorganism that does not express a modified LysE protein), but is not limited to them.

The term "unmodified microorganism" as used herein does not exclude strains comprising a mutation that may occur naturally in microorganisms, and may be the wild-type strain itself or a natural strain before the trait has been altered by genetic change due to natural or artificial factors. For example, an unmodified microorganism may be a strain in which the LysE variant described herein is not introduced or has not yet been introduced. The term "unmodified microorganism" may be used interchangeably with the terms "strain before modification", "microorganism before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

In one embodiment, the microorganism of the present disclosure may be a microorganism of the genus Corynebacterium. For example, the microorganism of the present disclosure may be Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotoleram, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, in particular, but not limited to, Corynebacterium glutamicum.

The microorganism exporting and/or producing L-arginine according to the present disclosure may be characterized in that it comprises the LysE variant disclosed in this specification, thereby having an enhanced ability to perform the production and/or export of L-arginine, as desired.

In any embodiment of the above-described embodiments, the microorganism producing and/or exporting L-arginine, or the microorganism having the ability to perform the production and/or export of L-arginine, in the present disclosure may be a microorganism in which some of the genes involved in the L-arginine biosynthesis pathway are enhanced or weakened, or some of the genes involved in the L-arginine degradation pathway are enhanced or weakened.

In any embodiment of the above embodiments, the microorganism may be a microorganism in which some of the genes involved in the L-arginine biosynthetic pathway are enhanced or reduced. Examples of L-arginine biosynthetic enzymes include N-acetylglutamyl phosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyltransferase (argF), argininosuccinic acid synthase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthase (carAB). The arginine biosynthetic enzymes present on the Arg operon (argCJBDFRGH) can be regulated by the arginine repressor encoded by argR (J Bacteriol. 2002Dec;184(23):6602-14.). Therefore, the ability to produce L-arginine can be provided or enhanced by deleting or reducing the activity of the arginine repressor (argR) (US2002-0045223) and/or by expressing one or more genes from the genes associated with biosynthesis. For example, the arginine repressor can be a polypeptide represented by the amino acid sequence of SEQ ID NO: 39, but is not limited thereto.

The term "reduction" of the activity of a polypeptide as used herein is intended to encompass all of the reduction in activity or lack of activity compared to endogenous activity. The term "reduction" may be used interchangeably with inactivation, deficiency, down-regulation, reduction, decrease, attenuation, or the like.

The reduction may also include the case where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation, etc. of the polynucleotide encoding the polypeptide, the case where the total activity and/or concentration (expression level) of the polypeptides in the cell is lower compared to the natural strain due to inhibition of expression of the gene of the polynucleotide encoding the polypeptide or inhibition of translation into the polypeptide, the case where expression of the polynucleotide does not occur and/or the case where the polypeptide has no activity despite expression of the polynucleotide. The term "endogenous activity" refers to the activity of a particular polypeptide that was originally possessed by the parent strain before modification or by a wild-type or unmodified microorganism when transformed by a genetic mutation caused by natural or artificial factors. This term may be used interchangeably with the term "activity before modification". "Inactivation, deficiency, reduction, down-regulation, diminution, or attenuation" of the activity of a polypeptide compared to its endogenous activity means that the activity of the polypeptide is reduced compared to the activity of the particular polypeptide originally possessed by the parent strain prior to transformation or by the unmodified microorganism.

The reduction in activity of the polypeptide may be accomplished by any method known in the art, but is not limited thereto, and may be achieved using various methods well known in the art (e.g., 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, etc.).

In particular, the reduction in activity of the polypeptide of the present disclosure can be achieved by the following:

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

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

3) modification of the amino acid sequence constituting the polypeptide to eliminate or reduce the activity of the polypeptide (e.g. deletion/replacement/addition of one or more amino acids in the amino acid sequence);

4) modification of the sequence of a gene encoding a polypeptide to eliminate or reduce the activity of the polypeptide (e.g. deletion/replacement/addition of one or more nucleotides in the nucleotide sequence of a polypeptide gene encoding a polypeptide that is modified to eliminate or reduce the activity of the polypeptide);

5) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region (5'-untranslated region) of the transcript of the gene encoding the given polypeptide;

6) introduction of an antisense oligonucleotide (e.g. antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide above the Shine-Dalgarno sequence to form a secondary structure that prevents ribosome attachment;

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

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

For example:

1) Deletion of part or all of a gene encoding a polypeptide may be the elimination of the entire polynucleotide encoding the endogenous target protein in the chromosome, replacement with a polynucleotide having a deletion of some nucleotides, or replacement with a marker gene.

In addition, 2) a modification of the expression control region (or expression control sequence) may be a mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having less activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling termination of transcription and translation.

In addition, 3) and 4) the modification of the amino acid sequence or the polynucleotide sequence may be a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or a replacement with an amino acid sequence or a polynucleotide sequence modified to have a weaker activity, or an amino acid sequence or a polynucleotide sequence improved to have no activity, so as to reduce the activity of the polypeptide, but not limited to them. For example, gene expression can be inhibited or weakened by introducing a mutation into the polynucleotide sequence and forming a stop codon, but not limited to it.

In addition, 5) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the transcript of the gene encoding the polypeptide may be, for example, a replacement with a nucleotide sequence encoding not the endogenous initiation codon, but another initiation codon that has a lower rate of expression of the polypeptide, but is not limited to it.

6) To introduce an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to the transcript of a gene encoding a polypeptide, one can refer, for example, to the literature [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 of the gene encoding the polypeptide upstream of the Shine-Dalgarno sequence in such a way as to form a secondary structure that prevents ribosome attachment, which may prevent or reduce the rate of translation of the mRNA.

8) Addition of a promoter that is transcribed in the opposite direction relative to the 3' end of the open reading frame (ORF) sequence of the gene encoding the polypeptide (reverse transcription engineering, RTE) can create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, reducing the activity of the polypeptide.

As used herein, the term "enhancement" of the activity of a polypeptide means that the activity of the polypeptide is increased compared to the endogenous activity. The term "enhancement" may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase may include both demonstrating an activity that was not originally possessed and demonstrating an improved activity compared to the endogenous activity or the activity before modification. The term "endogenous activity" means the activity of a particular polypeptide that was originally possessed by the parent strain before the change in the trait or by the unmodified microorganism when the trait is changed by genetic change due to natural or artificial factors. This term may be used interchangeably with the term "activity before modification". The fact that the activity of a polypeptide is "enhanced," "up-regulated," "overexpressed," or "increased" compared to the endogenous activity means that the activity of the polypeptide is improved compared to the activity and/or concentration (expression level) of the particular polypeptide originally possessed by the parent strain before the trait change or by the unmodified microorganism.

The enhancement may be achieved by introducing a foreign polypeptide or enhancing the endogenous activity and/or concentration (expression level) of the polypeptide. The enhancement of the activity of the polypeptide may be confirmed by increasing the activity level and expression level of the corresponding polypeptide or the amount of product produced from the corresponding polypeptide.

To enhance the activity of a polypeptide, various methods well known in the art can be used, and the method is not limited, as long as the activity of the polypeptide of interest can be enhanced compared to the activity of the microorganism before modification. In particular, genetic engineering and/or protein engineering well known to those skilled in the art can be used, which are traditional methods of molecular biology, but the method is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

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

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

2) replacement of the regulatory region of gene expression on the chromosome encoding the polypeptide with a sequence demonstrating strong activity;

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

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

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

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

7) optimization of codons 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) to (8), but not specifically limited to them.

More specifically:

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

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

Examples of known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (US Patent No. US 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 1,0584,338 B2), the O2 promoter (US Patent No. US 1,0273,491 B2), the tkt promoter, the yccA promoter, etc.

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

4) and 5) The modification of the amino acid sequence or the polynucleotide sequence may be, but is not limited to, the occurrence of a change in the sequence due to a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or a combination thereof, or a replacement with an amino acid sequence or polynucleotide sequence modified to exhibit a stronger activity, or an amino acid sequence or polynucleotide sequence modified to be more active, such that the activity of the polypeptide is enhanced. This substitution can be specifically carried out by, but is not limited to, inserting the polynucleotide into the chromosome by homologous recombination. The vector used herein may further comprise a selectable marker for confirming the insertion into the chromosome. The selectable marker is as described above.

6) The introduction of a foreign polynucleotide exhibiting the activity of a polypeptide may be the introduction into a host cell of a foreign polynucleotide encoding a polypeptide exhibiting an activity identical or similar to this polypeptide. There is no limitation on its origin or sequence, provided that this foreign polynucleotide exhibits an activity identical or similar to this polypeptide. The method used in the introduction can be carried out by appropriately selecting a transformation method known to those skilled in the art. Since the introduced polynucleotide is expressed in the host cell, the polypeptide can be produced and its activity can be increased.

7) Optimization of codons of a polynucleotide encoding a polypeptide may be optimization of codons of an endogenous polynucleotide in such a way as to increase transcription or translation in the host cell, or optimization of codons of a foreign polynucleotide in such a way as to carry out optimized transcription and translation in the host cell.

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

Such enhancement of the activity of the polypeptide may be, but is not limited to, an increase in the activity or concentration or expression level 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 corresponding polypeptide.

In the microorganism of the present disclosure, partial or complete modification of a polynucleotide may be induced by (a) homologous recombination using a vector for insertion into a chromosome in the microorganism or genome editing 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 a part or all of a gene may include a method using recombinant DNA technology. For example, by introducing a nucleotide sequence or a vector containing a nucleotide sequence homologous to a gene of interest into a microorganism to cause homologous recombination, a part or all of the gene may be deleted. The nucleotide sequence or vector to be introduced may include, but is not limited to, a dominant selectable marker.

According to yet another aspect of the present disclosure, there is provided a method for producing L-arginine comprising the step of culturing a microorganism comprising a LysE variant or a polynucleotide encoding it.

The LysE variant encoding its polynucleotide, including its microorganism and L-arginine are as described above.

The method for producing L-arginine according to the present disclosure may include the step of culturing in a medium a microorganism containing the LysE variant described herein or a polynucleotide encoding it, or a vector comprising this polynucleotide.

The term "cultivating" as used herein means growing the microorganism of the present disclosure under appropriately controlled environmental conditions. The culturing method of the present disclosure can be carried out in accordance with an appropriate environment and culturing conditions known in the art. Such a culturing method can be easily adjusted and used by those skilled in the art according to the selected strain. In particular, the culturing may be of a batch type, a continuous type, and/or a fed-batch type, but is not limited to them.

The term "medium" as used herein means a mixed substance containing nutrients required for culturing the microorganism of the present disclosure as a main component, and the medium supplies nutrients, growth factors, etc., including water, which are essential for survival and development. In particular, any of the medium and other culture conditions used for culturing the microorganism of the present disclosure may be used without particular limitation, as long as it is a medium used for conventional culturing of microorganisms. The microorganism of the present disclosure can be cultured in a conventional medium containing proper carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, etc., while controlling temperature, pH, etc. under aerobic conditions.

For example, a culture medium for a strain of the genus Corynebacterium can be found in the document ["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, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, raw molasses, rice bran, cassava, sugar cane residue and liquid corn steep liquor can be used. In particular, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugar) can be used, and suitable amounts of other carbon sources can be used in various ways without limitation. These carbon sources can be used singly or in combination of two or more than two, but are not limited to them.

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

Phosphorus sources may include monopotassium phosphate, dipotassium phosphate or their corresponding sodium salts. Inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and/or suitable precursors, etc. may be included. These components or precursors may be added to the medium in portions or continuously, but the method of addition is not limited to them.

In addition, during the cultivation of the microorganism according to the present disclosure, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in a proper manner. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. In addition, oxygen or an oxygen-containing gas can be injected into the medium to maintain an aerobic state of the medium, or the gas may not be injected, or nitrogen gas, hydrogen gas, or carbon dioxide gas can be injected to maintain an anaerobic and microaerobic state, but the method for maintaining the state is not limited to these.

In the culture according to the present disclosure, the culture temperature can be maintained at 20°C to 45°C, particularly 25°C to 40°C, and the strain can be cultured for about 10 to 160 hours, but the culture conditions are not limited thereto.

L-arginine produced by the culturing according to the present disclosure may be secreted into the medium or may remain in the cells.

The method for producing L-arginine according to the present disclosure may further comprise the steps of obtaining a microorganism according to the present disclosure, preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

The method for producing L-arginine according to the present disclosure may further comprise a step of isolating L-arginine from a medium according to the culturing (a medium exposed to the culture) or from a microorganism. After the culturing step, a step of isolating may further be included.

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

The method for producing L-arginine according to the present disclosure may further comprise a purification step. The purification may be carried out by a suitable method known in the art. In one example, when the method for producing L-arginine according to the present disclosure comprises both an isolation step and a purification step, these isolation steps and purification steps may be carried out continuously or discontinuously, regardless of the order, or may be carried out simultaneously, or by combining into one step, but are not limited to them.

In the method of the present disclosure, the LysE variant, polynucleotide, vector, microorganism/strain, etc. are as described in other aspects.

According to yet another aspect of the present disclosure, there is provided a composition for producing L-arginine comprising a LysE variant of the present disclosure, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present disclosure; a culture medium thereof, or a combination thereof.

The composition of the present disclosure may further comprise arbitrary suitable excipients that are commonly used in compositions for producing L-arginine. Such excipients may be, for example, a preservative, a humectant, a dispersing agent, a suspending agent, a buffering agent, a stabilizer or an isotonic agent, etc., but are not limited to them.

With respect to the composition of the present disclosure, the LysE variant, polynucleotide, vector, microorganism/strain, medium, etc. are as described in other aspects.

According to yet another aspect of the present disclosure, there is provided a method for increasing the ability of a microorganism to export and/or produce L-arginine or a method for providing a microorganism with the ability to export and/or produce L-arginine, comprising the step of modifying the microorganism to express a LysE variant of the present disclosure.

With respect to the method for increasing the ability of the microorganism of the present disclosure to produce L-arginine, the variant, polynucleotide, vector, microorganism/strain, etc. are the same as those described in other aspects.

The method for modifying a microorganism may include the step of introducing a LysE variant, a polynucleotide encoding it, or a recombinant vector containing this polynucleotide into the microorganism. However, this modification method is not limited thereto, and methods known in the art, including the method disclosed in this document, can be used without limitation.

According to yet another aspect of the present disclosure, there is provided the use of a LysE variant of the present disclosure in effecting L-arginine export.

According to yet another aspect of the present disclosure, there is provided the use of a microorganism comprising one or more LysE variants of the present disclosure, a polynucleotide encoding the variant, and a vector comprising the polynucleotide, in producing L-arginine.

The LysE variant, polynucleotide, vector, microorganism are as described in other aspects.

Method of carrying out the invention

Below, the present disclosure will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples serve only to illustrate the present disclosure, and the scope of the present disclosure is not intended to be limited by these Examples and Experimental Examples.

Example 1. Obtaining different variants with substitution of amino acid residues at positions 50, 153 and 215 LysE

The present inventors determined that amino acid residues at positions 50, 153 and 215 in SEQ ID NO: 1 are important positions for LysE activity and generated variants in which the corresponding amino acid residues were replaced with another amino acid. In particular, the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template for PCR (polymerase chain reaction) using the primer pairs from Table 1 (SEQ ID NOs: 3 and 5, SEQ ID NOs: 4 and 6, SEQ ID NOs: 3 and 7, SEQ ID NOs: 4 and 8, SEQ ID NOs: 3 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 3 and 11, SEQ ID NOs: 4 and 12, SEQ ID NOs: 3 and 13, SEQ ID NOs: 4 and 14, SEQ ID NOs: 3 and 15, SEQ ID NOs: 4 and 16, SEQ ID NOs: 3 and 17, SEQ ID NOs: 4 and 18, SEQ ID NOs: 3 and 19, SEQ ID NOs: 4 and 20, SEQ ID NOs: 3 and 21, SEQ ID NOs: 4 and 22) and overlapping primer PCR using the primer pair of SEQ ID NOs: 3 and 4, thereby obtaining homologous recombination fragments each having a mutant sequence of LysE-F50L, F50I, F50V, N153S, N153T, N153Q, N215T, N215S, or N215Q. At this time, the PCR reaction was carried out by 30 cycles consisting of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and extension at 72°C for 1 minute. Then, the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813), which does not replicate in Corynebacterium glutamicum, and each PCR-amplified fragment were treated with BamHI and XbaI, which are restriction enzymes for insertion into the chromosome, and then ligated using DNA ligase. Then, each product was transformed into E. coli DH5α and plated on LB solid medium containing kanamycin (25 mg/L).

Colonies transformed with the plasmid into which the target gene was inserted were selected by PCR, and then each plasmid was obtained using the plasmid isolation method and named pDCM2-lysE(F50L), pDCM2-lysE(F50I), pDCM2-lysE(F50V), _P DCM2-lysE(N153S), _P DCM2-lysE(N153T), _P DCM2-lysE(N153Q), pDCM2-lysE(N215T), _P DCM2-lysE(N215S), _P DCM2-lysE(N215Q), respectively.

Example 2. Obtaining a strain with an introduced LysE variant originating from Corynebacterium glutamicum KSSM10741P and assessing the ability to produce L-arginine

In order to introduce the LysE mutation into Corynebacterium glutamicum KCSM10741P (US 8034602 B2), which is an L-arginine producing strain, transformation was performed using the obtained recombinant plasmid (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Then, secondary recombination was carried out on a solid medium plate containing 4% sucrose, and PCR was performed on the transformant in which secondary recombination was completed using a pair of primers (SEQ ID NOs: 3 and 4) to identify the introduction of each mutation into the LysE gene on the chromosome. At this time, PCR reaction was carried out through 30 cycles consisting of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds and extension at 72°C for 1 minute. The strains with the introduced LysE variant selected from it were named KCCM10741P-lysE(F50L), KCCM10741P-lysE(F50I), KCCM10741P-lysE(F50V), KCCM10741P-lysE(N153S), KCCM10741P-lysE(N153T), KCCM10741P-lysE(N153Q), KCCM10741P-lysE(N215T), KCCM10741P-lysE(N215S), KCCM10741P-lysE(N215Q), respectively.

To analyze the ability of strains with the introduced LysE variant to produce L-arginine, they were cultured with the parent strain, Corynebacterium glutamicum strain KCCM10741P, in the following manner, respectively.

The parent strain Corynebacterium glutamicum KCCM10741P and the above-obtained strains with the LysE variant were inoculated into a 250-mL baffled flask containing 25 mL of the seed medium described below, respectively, and cultured at 30°C with shaking at 200 rpm for 20 hours. Then, 1 mL of the seed culture was inoculated into a 250-mL baffled flask containing 24 mL of the production medium and cultured at 30°C with shaking at 200 rpm for 72 hours. The compositions of the seed medium and the production medium are as follows.

Seed medium (pH 7.2)

20 g glucose, 45 g ammonium sulfate, 2 g magnesium sulfate heptahydrate, 2 g monobasic potassium phosphate, 10 g ammonium chloride, 0.01 mg biotin, 0.1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 10 mg iron (II) sulfate, 0.02 mg zinc sulfate, 0.5 mg copper sulfate (based on 1 liter of distilled water).

Production medium (pH 7.2)

60 g glucose, 45 g ammonium sulfate, 2 g magnesium sulfate heptahydrate, 2 g monobasic potassium phosphate, 10 g ammonium chloride, 0.01 mg biotin, 0.1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 10 mg ferrous sulfate, 10 mg manganese sulfate, 0.02 mg zinc sulfate, 0.5 mg copper sulfate, 30 g calcium carbonate (based on 1 liter of distilled water).

After completion of the culture, the L-arginine producing capacity was measured by HPLC (Waters 2478) (Table 2).

As a result, it was confirmed that all strains with the introduced LysE variant demonstrated a 20% increase in the ability to produce L-arginine compared to the parent strain.

Example 3. Obtaining a strain with an introduced LysE variant originating from Corynebacterium glutamicum CJ1R, having the ability to produce L-arginine, and evaluating the ability to produce L-arginine

In the following manner it was confirmed whether or not other L-arginine-producing strains of Corynebacterium glutamicum also had the same effect as in Example 2.

A Corynebacterium glutamicum strain having the ability to produce L-arginine was obtained by introducing one kind of mutation (AargR) into the wild-type strain (ATCC13869). In detail, a recombinant vector for deleting the argR gene (SEQ ID NO: 40) was constructed in the same manner as in Example 1, and the primers used for constructing this vector are shown in Table 3.

The plasmid into which the desired fragment was inserted was selected by PCR, and this plasmid was named pDCM2-AargR. A wild-type Corynebacterium glutamicum strain was transformed using pDCM2-AargR in the same manner as in Example 2, and the transformant was subjected to PCR using a pair of primers (SEQ ID NOs: 23 and 26) to identify a strain with a deletion of argR on the chromosome, which was named CJ1R.

The strain variants were prepared by introducing each of the nine LysE variants into CJ1R in the same manner as in Example 2, and each was named CJ1R-lysE(F50L), CJ1R-lysE(FSOI), CJ1R-lysE(F50V), CJ1R-lysE(N153S), CJ1R-lysE(N153T), CJ1R-lysE(N153Q), CJ1R-lysE(N215T), CJ1R-lysE(N215S), CJ1R-lysE(N215Q).

To analyze the L-arginine producing ability of the CJ1R-derived strains with the introduced LysE variant, these strains were cultured in the same manner as in Example 2, and after completion of the culture, the L-arginine producing ability was measured by HPLC (Waters 2478) (Table 4).

Table 4

As a result, it was confirmed that all strains with the introduced LysE variant demonstrated a 19% increase in the ability to produce L-arginine compared to the parent strain.

Based on the above description, it will be understood by those skilled in the art that the present disclosure may be embodied in another specific form without changing its technical essence or essential characteristics. In this regard, it should be understood that the above embodiment is not limiting, but illustrative in all respects. The scope of the present disclosure is defined by the appended claims rather than by the preceding description, and therefore, it is intended that all changes and modifications that fall within the scope of the claims or the equivalents of such scope be embraced by the claims.

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ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

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<INSDQualifier>

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<INSDSeq_sequence>aaggtgccggcgatgatcaaaaagacgtcagaaat</INSDSeq_sequ

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<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q29">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N153Q-BF</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N153Q-BF</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q30">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggttgcaaccgaatgcgtatttggacgcgtttg</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q31">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N215T-AR</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N215T-AR</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q32">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgacgacggtgatccagcgccacaccttggggctg</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q33">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N215T-BF</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N215T-BF</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q34">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggatcaccgtcgtcgtggcagttgtgatgacc</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="19">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q35">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N215S-AR</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N215S-AR</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q36">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgacgacgctgatccagcgccacaccttggggctg</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="20">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q37">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N215S-BF</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N215S-BF</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q38">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggatcagcgtcgtcgtggcagttgtgatgacc</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="21">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q39">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N215Q-AR</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N215Q-AR</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q40">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cgacgacttggatccagcgccacaccttggggctg</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="22">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q41">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_lysE-N215Q-BF</INSDQualifier_value>

<NonEnglishQualifier_value>primer_lysE-N215Q-BF</NonEnglishQua

lifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q42">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggatccaagtcgtcgtggcagttgtgatgacc</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="23">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q43">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_argR-AF</INSDQualifier_value>

<NonEnglishQualifier_value>primer_argR-AF</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q44">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcggtacccggggatccagcaggccttaagggtaag</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="24">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q45">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_argR-AR</INSDQualifier_value>

<NonEnglishQualifier_value>primer_argR-AR</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q46">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aggggcgctgtcttacctcggctggtgggccagc</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="25">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q47">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_argR-BF</INSDQualifier_value>

<NonEnglishQualifier_value>primer_argR-BF</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q48">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggtaagacagcgcccctagttcaaggcttgttaatc</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="26">

<INSDSeq>

<INSDSeq_length>37</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..37</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q49">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer_argR-BR</INSDQualifier_value>

<NonEnglishQualifier_value>primer_argR-BR</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..37</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q50">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgcaggtcgactctagaaccgttgaactgcttgccag</INSDSeq_se

quence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="27">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q51">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE F50L</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q52">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

LIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="28">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q53">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N153S</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q54">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

FIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLSPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="29">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q55">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N215T</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q56">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

FIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWITVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="30">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q57">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE F50I</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q58">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

IIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="31">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q59">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE F50V</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q60">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

VIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="32">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q61">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N153T</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q62">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

FIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLTPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="33">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q63">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N153Q</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q64">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

FIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLQPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="34">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q65">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N215S</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q66">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

FIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWISVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="35">

<INSDSeq>

<INSDSeq_length>233</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q67">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N215Q</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..233</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q68">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFL

FIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLGGSAV

ATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLI

WFPLVGFGAAALSRPLSSPKVWRWIQVVVAVVMTALAIKLMLMG</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="36">

<INSDSeq>

<INSDSeq_length>702</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..702</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q69">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE F50L nt</INSDQualifier_value>

<NonEnglishQualifier_value>LysE F50L

nt</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..702</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q70">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggaaatcttcattacaggtctgcttttgggggccagtcttttgctgt

ccatcggaccgcagaatgtactggtgattaaacaaggaattaagcgcgaaggactcattgcggttcttct

cgtgtgtttaatttctgacgtctttttgctcatcgccggcaccttgggcgttgatcttttgtccaatgcc

gcgccgatcgtgctcgatattatgcgctggggtggcatcgcttacctgttatggtttgccgtcatggcag

cgaaagacgccatgacaaacaaggtggaagcgccacagatcattgaagaaacagaaccaaccgtgcccga

tgacacgcctttgggcggttcggcggtggccactgacacgcgcaaccgggtgcgggtggaggtgagcgtc

gataagcagcgggtttgggtgaagcccatgttgatggcaatcgtgctgacctggttgaacccgaatgcgt

atttggacgcgtttgtgtttatcggcggcgtcggcgcgcaatacggcgacaccggacggtggattttcgc

cgctggcgcgttcgcggcaagcctgatctggttcccgctggtgggtttcggcgcagcagcattgtcacgc

ccgctgtccagccccaaggtgtggcgctggatcaacgtcgtcgtggcagttgtgatgaccgcattggcca

tcaaactgatgttgatgggttag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="37">

<INSDSeq>

<INSDSeq_length>702</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..702</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q71">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N153S nt</INSDQualifier_value>

<NonEnglishQualifier_value>LysE N153S

nt</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..702</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q72">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggaaatcttcattacaggtctgcttttgggggccagtcttttgctgt

ccatcggaccgcagaatgtactggtgattaaacaaggaattaagcgcgaaggactcattgcggttcttct

cgtgtgtttaatttctgacgtctttttgttcatcgccggcaccttgggcgttgatcttttgtccaatgcc

gcgccgatcgtgctcgatattatgcgctggggtggcatcgcttacctgttatggtttgccgtcatggcag

cgaaagacgccatgacaaacaaggtggaagcgccacagatcattgaagaaacagaaccaaccgtgcccga

tgacacgcctttgggcggttcggcggtggccactgacacgcgcaaccgggtgcgggtggaggtgagcgtc

gataagcagcgggtttgggtgaagcccatgttgatggcaatcgtgctgacctggttgagcccgaatgcgt

atttggacgcgtttgtgtttatcggcggcgtcggcgcgcaatacggcgacaccggacggtggattttcgc

cgctggcgcgttcgcggcaagcctgatctggttcccgctggtgggtttcggcgcagcagcattgtcacgc

ccgctgtccagccccaaggtgtggcgctggatcaacgtcgtcgtggcagttgtgatgaccgcattggcca

tcaaactgatgttgatgggttag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="38">

<INSDSeq>

<INSDSeq_length>702</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..702</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q73">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>LysE N215T nt</INSDQualifier_value>

<NonEnglishQualifier_value>LysE N215T

nt</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..702</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q74">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggaaatcttcattacaggtctgcttttgggggccagtcttttgctgt

ccatcggaccgcagaatgtactggtgattaaacaaggaattaagcgcgaaggactcattgcggttcttct

cgtgtgtttaatttctgacgtctttttgttcatcgccggcaccttgggcgttgatcttttgtccaatgcc

gcgccgatcgtgctcgatattatgcgctggggtggcatcgcttacctgttatggtttgccgtcatggcag

cgaaagacgccatgacaaacaaggtggaagcgccacagatcattgaagaaacagaaccaaccgtgcccga

tgacacgcctttgggcggttcggcggtggccactgacacgcgcaaccgggtgcgggtggaggtgagcgtc

gataagcagcgggtttgggtgaagcccatgttgatggcaatcgtgctgacctggttgaacccgaatgcgt

atttggacgcgtttgtgtttatcggcggcgtcggcgcgcaatacggcgacaccggacggtggattttcgc

cgctggcgcgttcgcggcaagcctgatctggttcccgctggtgggtttcggcgcagcagcattgtcacgc

ccgctgtccagccccaaggtgtggcgctggatcaccgtcgtcgtggcagttgtgatgaccgcattggcca

tcaaactgatgttgatgggttag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="39">

<INSDSeq>

<INSDSeq_length>171</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..171</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q75">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Corynebacterium sp.</INSDQualifier_value>

<NonEnglishQualifier_value>Species of the genus

Corynebacterium</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MSLGSTPSTPENLNPVTRTARQALILQILDKQKVTSQVQLSELLLDEGI

DITQATLSRDLDELGARKVRPDGGRAYYAVGPVDSIAREDLRGPSEKLRRMLDELLVSTDHSGNIAMLRT

PPGAAQYLASFIDRVGLKEVVGTIAGDDTVFVLARDPLTGKELGELLSGRTT</INSDSeq_sequence

>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="40">

<INSDSeq>

<INSDSeq_length>516</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..516</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>genomic DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q76">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Corynebacterium sp.</INSDQualifier_value>

<NonEnglishQualifier_value>Species of the genus

Corynebacterium</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgtcccttggctcaaccccgtcaacaccggaaaacttaaatcccgtga

ctcgcactgcacgccaagctctcattttgcagattttggacaaacaaaaagtcaccagccaggtacaact

gtctgaattgctgctggatgaaggcatcgatatcacccaggccaccttgtcccgagatctcgatgaactc

ggtgcacgcaaggttcgccccgatgggggacgcgcctactacgcggtcggcccagtagatagcatcgccc

gcgaagatctccggggtccgtcggagaagctgcgccgcatgcttgatgaactgctggtttctacagatca

ttccggcaacatcgcgatgctgcgcaccccgccgggagctgcccagtacctggcaagtttcatcgatagg

gtggggctgaaagaagtcgttggcaccatcgctggcgatgacaccgttttcgttctcgcccgtgatccgc

tcacaggtaaagaactaggtgaattactcagcgggcgcaccacttaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (22)

1. A polypeptide involved in the production of L-arginine, having 80% or more sequence identity to SEQ ID NO: 1, and in which any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 in SEQ ID NO: 1 are replaced by another amino acid, where the amino acid corresponding to position 50 is replaced by valine, leucine or isoleucine, the amino acid corresponding to position 153 is replaced by serine, threonine or glutamine, the amino acid corresponding to position 215 is replaced by serine, threonine, or glutamine. 2. The polypeptide of claim 1, wherein said polypeptide is from the genus Corynebacterium . 3. The polypeptide of claim 1, wherein any one amino acid from the amino acid residues at positions 50, 153 and 215 in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid, where the amino acid corresponding to position 50 is replaced by valine, leucine or isoleucine, the amino acid corresponding to position 153 is replaced by serine, threonine or glutamine, the amino acid corresponding to position 215 is replaced by serine, threonine, or glutamine. 4. The polypeptide of claim 1, involved in the production of L-arginine, having 90% or more sequence identity with SEQ ID NO: 1. 5. A polynucleotide encoding a polypeptide according to any one of claims 1-4. 6. A microorganism of the genus Corynebacterium for producing L-arginine, comprising a polypeptide involved in the production of L-arginine, in which any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 in SEQ ID NO: 1 are replaced by another amino acid, where the amino acid corresponding to position 50 is replaced by valine, leucine or isoleucine, the amino acid corresponding to position 153 is replaced by serine, threonine or glutamine, the amino acid corresponding to position 215 is replaced by serine, threonine, or glutamine; or a polynucleotide encoding a polypeptide involved in the production of L-arginine. 7. The microorganism according to claim 6, which is Corynebacterium glutamicum . 8. A method for producing L-arginine, comprising the step of culturing a microorganism of the genus Corynebacterium containing a polypeptide involved in the production of L-arginine, in which any one or more amino acids from the amino acids corresponding to positions 50, 153 and 215 in SEQ ID NO: 1 are replaced by another amino acid, where the amino acid corresponding to position 50 is replaced by valine, leucine or isoleucine, the amino acid corresponding to position 153 is replaced by serine, threonine or glutamine, the amino acid corresponding to position 215 is replaced by serine, threonine, or glutamine; or a polynucleotide encoding a polypeptide involved in the production of L-arginine.