RU2793433C1 - New variant of cell division membrane protein and method for obtaining l-lysine with its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: polypeptide is proposed that is involved in the production of L-lysine, consisting of the amino acid sequence represented by SEQ ID NO: 1, in which isoleucine being the amino acid corresponding to position 307 of SEQ ID NO: 3 is replaced by valine. Also proposed are the following: a polynucleotide encoding the said polypeptide, a microorganism Corynebacterium glutamicum producing L-lysine and containing the said polypeptide or polynucleotide encoding the said polypeptide, and a method for producing of L-lysine.

EFFECT: increasing the yield of L-lysine.

5 cl, 3 tbl, 2 ex

Description

FIELD OF THE INVENTION

The present disclosure relates to a novel cell division membrane protein variant, a strain of Corynebacterium glutamicum containing the variant, and to a method for producing L-lysine using the strain.

PRIOR ART

Conduct various research to develop highly efficient microorganisms and fermentation process technologies for the production of L-amino acids and other beneficial substances. For example, a target-substance-specific approach is generally used in which the expression of a gene encoding an enzyme involved in L-lysine biosynthesis is increased or in which genes not required for biosynthesis are removed (WO 2008-082181 A1).

However, research is still needed to effectively increase the ability to produce L-lysine as the demand for L-lysine increases.

DESCRIPTION OF THE INVENTION

Technical problem

It is an object of the present disclosure to provide a cell division membrane protein variant consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein isoleucine (Ile, or I), which is the amino acid corresponding to position 307 of the amino acid sequence of SEQ ID NO: 3, has been replaced valine (Val, or V).

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

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

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

Technical solution

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

According to one aspect of the present disclosure, there is provided a variant consisting of the amino acid sequence represented by SEQ ID NO: 1, in which isoleucine (Ile, or I), which is the amino acid corresponding to position 307 of the amino acid sequence of SEQ ID NO: 3, is replaced by valine (Val , or V).

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

In a variant of the present disclosure, the amino acid corresponding to position 307 based on the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence shown by SEQ ID NO: 1 is valine, and the variant may contain an amino acid sequence having at least 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% 99.7% or 99.9% or greater homology or identity with the amino acid sequence shown by SEQ ID NO: 1. Obviously, variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of this disclosure, so long as the amino acid sequences have such homology or identity and exhibit an efficacy corresponding to that of the variant of the present disclosure.

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

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

In the present disclosure, the term "variant" refers to a polypeptide that has an amino acid sequence different from the amino acid sequence of the variant prior to modification by conservative substitution and/or modification of one or more amino acids, but retains functions or properties. Such a variant can usually be identified by modifying one or more amino acids of the amino acid sequence of a given polypeptide and evaluating the properties of that modified polypeptide. In other words, the ability of a given variant can be increased, left unchanged, or reduced compared to the ability of the polypeptide before the change. Some variants may include variants in which one or more than one portion, such as the N-terminal leader sequence or the transmembrane domain, has been deleted. Other variants may include variants in which a portion of the N- and/or C-terminus of the mature protein has been removed. The term "variant" can be used interchangeably with and is not limited to terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent, as long as it is a term used with the meaning of variation. For the purposes of this disclosure, this variant may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein isoleucine (Ile, or I), which is the amino acid corresponding to position 307 of the amino acid sequence of SEQ ID NO: 3, has been replaced valine (Val, or V).

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

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

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

Whether any two polynucleotide or polypeptide sequences share homology, similarity or identity can be determined using known computer algorithms such as the FASTA program, for example using default parameters as in Pearson et al., (1988) Proc. Natl. Acad. sci. USA 85:2444. Alternatively, homology, similarity, or identity can be determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (including GCG software package (Devereux, J., et al., Nucleic Acids Research 12 :387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., 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, National Center for Biotechnology Information BLAST or ClustalW can be used to determine homology, similarity, or identity.

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

As an example of the present disclosure, a variant of the present disclosure may exhibit cell division membrane protein activity.

In the present disclosure, the term "cell division membrane protein" is an enzyme involved in cell division. Specifically, "cell division membrane protein" in the present disclosure may be used interchangeably with the terms "RodA protein" and "RodA". In the present disclosure, the cell division membrane protein sequence can be obtained from GenBank NCBI, a well-known database such as GenBank accession number WP 011013343.1. In particular, the cell division membrane protein may be a polypeptide showing the activity of the cell division membrane protein encoded by the NCgl0043 gene (or the rodA gene), but is not limited thereto.

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

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

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

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

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

A polynucleotide encoding a variant of the present disclosure may comprise a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present disclosure, a polynucleotide of the present disclosure may have or contain the sequence of SEQ ID NO: 2. A polynucleotide of the present disclosure may consist or essentially consist of the sequence of SEQ ID NO: 2. In another embodiment, in the polynucleotide of the present disclosure, the base corresponding to position 919 based on the nucleic acid sequence of SEQ ID NO: 4 in the nucleic acid sequence represented by SEQ ID NO: 2 is G, and this polynucleotide may contain a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or greater homology or identity to the nucleic acid sequence represented by SEQ ID NO: 2. Obviously, polynucleotides having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of this disclosure, provided that these nucleic acid sequences have such homology or identity and encode a polypeptide or protein , demonstrating the effectiveness corresponding to the effectiveness of the variant of the present disclosure.

In a polynucleotide of the present disclosure, various modifications can be made in the coding region, provided that the amino acid sequence of the variant of the present disclosure is not changed when considering the degeneracy of the preferred codons in the organisms that are intended to express the variant of the present disclosure. In particular, the polynucleotide of the present disclosure has or contains a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95 % or greater, 96% or greater, 97% or greater, 98% or greater, but less than 100% homology or identity with the sequence of SEQ ID NO: 2 or may consist of or essentially consists of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% - or greater, 97% or greater, 98% or greater, but less than 100% homology or identity with the sequence of SEQ ID NO: 2, but not limited to it. Here, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 307 in SEQ ID NO: 1 may be one of the codons encoding valine.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The strain of the present disclosure may be a microorganism having naturally occurring cell division membrane protein activity and/or the ability to produce L-lysine, or a microorganism in which a variant of the present disclosure or a polynucleotide encoding the variant (or a vector containing the polynucleotide) , is administered to a parent strain that does not have cell division membrane protein activity or L-lysine production ability, and/or in which L-lysine production ability is imparted to, but is not limited to, the parent strain.

For example, the strain of the present disclosure is a cell or microorganism that has been transformed with a vector containing a polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure and expresses the variant of the present disclosure. For the purposes of the present disclosure, the strain of the present disclosure may include all microorganisms that contain the variant of the present disclosure and can produce L-lysine. For example, the strain of the present disclosure may be a recombinant strain in which a polynucleotide encoding a variant of the present disclosure is introduced into a natural wild type microorganism or an L-lysine producing microorganism to thereby express a cell division membrane protein variant and have increased ability to produce L-lysine. The recombinant strain having an increased ability to produce L-lysine may be a microorganism having an increased ability to produce L-lysine compared to a natural wild-type microorganism or a microorganism not modified with a cell division membrane protein (namely, a microorganism expressing the membrane protein wild-type cell division), but is not limited thereto. As an example, a microorganism not modified with cell division membrane protein, which is a target strain for comparing the increase in the ability to produce L-lysine, can be a strain of ATCC13032 and/or a strain of Corynebaterium glutamicum CJ3P (US 9556463 B2; a full description of which is included in this description by reference), but is not limited to it.

For example, a recombinant strain having an increased production capacity may have an L-lysine production capacity (or productivity) increased by about 1% or more, by about 2.5% or more, by about 5% or more, by about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more About 12% or more About 12.5% or more About 13% or more About 13.5% or more About 14% or more About 14% .5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% % or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5%, or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more (the upper limit is not specifically limited and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or about 35% or less) compared to the ability to produce L -lysine of the parental strain before modification or with an unmodified microorganism. In another example, a recombinant strain having an increased production capacity may have an L-lysine production capacity increased by about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more About 1.16 times or more About 1.17 times or more About 1.18 times or more About 1.19 times or more About 1.2 times or more, about 1.21 times or more, about 1.25 times or more, or about 1.3 times or more (the upper limit is not particularly limited and may, for example, be about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the L-lysine-producing ability of the parental strain before modification or with an unmodified microorganism. More specifically, a recombinant strain having increased production capacity may have L-lysine production capacity (or productivity) increased by about 16.9% (or about 1.17 times) compared to L-lysine production capacity. the parent strain before modification or with an unmodified microorganism, but the increase rate is not limited thereto. The term "about" is an interval including all of plus/minus 0.5; plus/minus 0.4; plus/minus 0.3; plus/minus 0.2; plus/minus 0.1 and the like, and includes all values in the range equal to or similar to the value after the term "about", but is not limited to them.

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

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

In the present disclosure, "weakening" of the activity of the polypeptide includes both cases where the activity is reduced compared to endogenous activity or absent. The term "attenuation" can be used interchangeably with terms such as inactivation, insufficiency, down regulation, reduction, reduction and attenuation.

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

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

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

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

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

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

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

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

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

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

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

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

For example:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

7) codon optimization of the polynucleotide encoding the polypeptide;

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

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

More specific:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

When cultivating according to the present disclosure, the culturing temperature can be maintained at 20°C to 45°C, in particular 25°C to 40°C, and the strain can be cultivated for about 10 to 160 hours, but the culturing conditions are not limited thereto.

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

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

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

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

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

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

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

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

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

Beneficial effects

In the case of cultivating a strain of Corynebacterium glutamicum containing a new variant of the cell division membrane protein of the present disclosure, it is possible to produce L-lysine in a higher yield compared to the case of existing microorganisms having unmodified polypeptides.

Method for carrying out the invention

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

Example 1: Construction of a vector for expression of a cell division membrane protein variant in a microorganism

In order to confirm the effect of a variant (I307V; SEQ ID NO: 1) in which isoleucine (Ile) at position 307 of SEQ ID NO: 3 of the cell division membrane protein was replaced by valine (Val) on L-lysine production, a vector to construct a strain expressing this variant, was constructed using plasmid pDCM2 (Korean Patent Publication No. 10-2020-0136813) to insert and replace a gene in the Corynebacterium genome as follows.

PCR (Polymerase Chain Reaction) was performed using wild-type Corynebacterium glutamicum gDNA ATCC13032 as a template, a primer pair having the sequences of SEQ ID NOs: 5 and 6, and a primer pair having the sequences of SEQ ID NOs: 7 and 8. PCR was again performed with overlapping primers using a mixture of the two fragments obtained above as a template and a pair of primers having the sequences of SEQ ID NO: 5 and SEQ ID NO: 8 to obtain a fragment.

PCR was performed as follows: denaturation at 94°C for 5 minutes; 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute 30 seconds; and 72°C for 5 minutes. Plasmid pDCM2 was treated with smaI, and the PCR product obtained above was cloned into it by fusion. Fusion cloning was performed using the In-Fusion® HD Cloning Kit (Clontech). The resulting vector was designated pDCM2-NCgl0043(I307V). The primer sequences used in this example are described in the following Table 1:

Example 2 Evaluation of the L-Lysine Producing Ability of a Microorganism Expressing a Cell Division Membrane Protein Variant

2-1. Construction of a strain expressing a cell division membrane protein variant

The vector constructed in Example 1 was transformed into Corynebacterium glutamicum CJ3P (US 9556463 B2).

The strain in which this variant was introduced was selected from strains in which homologous recombination occurred using SEQ ID NOs: 9 and 10. The selected strain was designated CJ3P_NCg10043_I307V. The primer sequences used in this example are described in the following Table 2:

2-2. Comparison of the ability to produce L-lysine of strains expressing a cell division membrane protein variant

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

First, each strain was inoculated into a 250 ml baffled flask containing 25 ml of seed medium and subjected to shaking culture at 200 rpm at 30° C. for 20 hours. Then, the seed culture medium was inoculated into a 250 ml baffled flask containing 24 ml of production medium and subjected to shaking culture at 200 rpm at 30° C. for 72 hours. After completion of cultivation, L-lysine production was analyzed by HPLC. The concentration of L-lysine, as well as the increase in the concentration level in the culture medium for each tested strain is shown in Table 3 below.

Seed medium (pH 7.0):

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH ₂ PO ₄ , 8 g K ₂ HPO ₄ , 0.5 g MgSO ₄ -7H ₂ O, 0.1 mg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 2 mg nicotinamide (per 1 liter of distilled water).

Production medium (pH 7.0):

100 g glucose, 40 g (NH ₄ ) ₂ SO ₄ , 2.5 g soy protein, 5 g corn extract solids, 3 g urea, 1 g KH ₂ PO ₄ , 0.5 g MgSO ₄ ⋅7H ₂ O, 100 micrograms of biotin, 1000 micrograms of thiamine chloride, 2000 micrograms of calcium pantothenate, 3000 micrograms of nicotinamide, 30 g of CaCO ₃ (per 1 liter of distilled water).

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

As shown in Table 3, the CJ3P_NCg10043_I307V strain showed an increased ability to produce L-lysine compared to that of the control group.

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

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SEQUENCE LIST

<110> CJ CheilJedang Corporation

<120> NEW MEMBRANE PROTEIN FOR CELL DIVISION AND METHOD OF OBTAINING

L-LYSINE WITH ITS USE

<130>OPP20212331KR

<150> KR 10-2021-0047417

<151> 2021-04-12

<160> 10

<170> KoPatentIn 3.0

<210> 1

<211> 441

<212> PRT

<213> Artificial Sequence

<220>

<223> (I307V)_cell division membrane protein

<400> 1

Met Asn Thr Leu Glu Arg Leu Lys Leu Arg Arg Thr Glu Met Trp Leu

1 5 10 15

Leu Ile Leu Ala Thr Leu Val Val Ser Ile Met Phe Ile Ser Leu Glu

20 25 30

Leu Ala Met Gly Asn Glu Leu Gly Thr His Ile Leu Met Leu Met Gly

35 40 45

Gly Tyr Ile Gly Ile Phe Ile Val Ala His Leu Ala Met Ala Trp Val

50 55 60

Ala Pro Phe Ala Asp Gln Ile Met Leu Pro Val Val Ala Val Leu Asn

65 70 75 80

Gly Ile Gly Leu Val Met Ile Tyr Arg Leu Asp Glu Ala Thr Gly Tyr

85 90 95

Ser Thr Val Asn Ser Gln Leu Met Trp Thr Val Val Gly Val Thr Leu

100 105 110

Met Val Ala Val Leu Leu Leu Leu Arg Asp Tyr Lys Ser Leu Ser Arg

115 120 125

Tyr Ser Tyr Leu Leu Gly Val Val Gly Ile Val Leu Leu Ala Leu Pro

130 135 140

Leu Val Trp Pro Gln Pro Gly Gly Val Glu Ala Arg Ile Trp Ile Trp

145 150 155 160

Leu Gly Pro Phe Ser Ile Gln Pro Gly Glu Phe Ser Lys Ile Leu Leu

165 170 175

Leu Leu Phe Phe Ala Gln Leu Leu Ala Thr Lys Arg Ala Leu Phe Thr

180 185 190

Val Ala Gly Tyr Arg Phe Leu Gly Met Asp Phe Pro Arg Leu Arg Asp

195 200 205

Leu Ala Pro Ile Leu Val Val Trp Ala Leu Ala Ile Leu Ile Met Ala

210 215 220

Gly Ala Asn Asp Phe Gly Pro Ala Leu Leu Leu Phe Thr Thr Val Leu

225 230 235 240

Ala Met Val Tyr Leu Ala Thr Gly Arg Gly Ser Trp Leu Leu Ile Gly

245 250 255

Ala Val Leu Val Ala Val Gly Ala Phe Ala Val Tyr Gln Val Ser Ser

260 265 270

Lys Ile Gln Glu Arg Val Gln Asn Phe Val Asp Pro Val Ala His Tyr

275 280 285

Asp Thr Thr Gly Tyr Gln Leu Ser Gln Ser Leu Phe Gly Met Ser Trp

290 295 300

Gly Gly Val Thr Gly Thr Gly Ile Gly Gln Gly Tyr Pro Asn Met Ile

305 310 315 320

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

325 330 335

Gly Leu Ile Gly Leu Ala Ala Ile Ile Val Leu Phe Gly Val Phe Val

340 345 350

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

355 360 365

Leu Val Ala Ser Gly Leu Ser Met Thr Ile Met Ile Gln Ile Phe Val

370 375 380

Val Val Ala Gly Ile Ser Ser Leu Met Pro Met Thr Gly Leu Thr Thr

385 390 395 400

Pro Phe Met Ser Gln Gly Gly Ser Ser Leu Met Ala Asn Tyr Ile Leu

405 410 415

Met Ala Ile Ile Leu Arg Ile Ser Asp Ser Ala Arg Arg Pro Val Met

420 425 430

Ser Lys Gln Ala Ser Glu Val Ala Ala

435 440

<210> 2

<211> 1326

<212> DNA

<213> Artificial Sequence

<220>

<223> (I307V)_NCgl0043

<400> 2

atgaacacgc ttgaacgatt aaagcttcgt cgcacggaaa tgtggctgct gatacttgcc 60

acactcgttg tgtcgatcat gttcatcagc ctcgagctgg ccatgggcaa tgagttgggt 120

acccatattt tgatgctgat gggcggatat atcggtatct tcatcgtcgc gcacctagcc 180

atggcatggg tggcgccgtt tgctgatcaa atcatgctgc ctgtggtggc ggtgctcaat 240

ggcattggtt tggtgatgat ttatcgcctt gatgaggcca cgggctacag cacggtcaat 300

agccaattga tgtggacggt tgttggcgtc acgctgatgg tggctgtgtt gttgctgttg 360

cgtgattaca agtcgctttc gcgttattcc tacctcctcg gtgtggtggg catcgtgctg 420

ctggcgctgc ctctcgtgtg gccgcagcca ggcggcgtgg aagcccgcat ctggatttgg 480

cttggacctt tctccatcca gccaggtgag ttctccaaga ttttgctgct gctgttcttt 540

gctcagctgc tagccaccaa gcgtgctttg tttactgttg cgggctaccg tttcctcggc 600

atggatttcc ctcgtttgcg tgacctcgcg ccgattcttg tggtgtgggc gttggctatt 660

ttgatcatgg ctggcgccaa cgacttcggt cctgcactgc tgcttttcac taccgttttg 720

gccatggtgt acctggctac cggccgtggt tcctggctgt tgattggtgc tgtgttggtg 780

gctgtcggcg cgttcgcggt gtaccaagtt tcaagcaaga ttcaggaacg cgtgcaaaac 840

ttcgtggatc ctgtggccca ctatgacacc accggttacc agctgtccca gtccttgttt 900

ggcatgagtt ggggcggagt caccggcacc ggcattggtc agggttaccc caacatgatc 960

cctgtcgtgc actcggactt cattctcgca gccattggtg aggagcttgg tctgattggc 1020

ctggcggcca tcatcgtgct gtttggtgtg tttgtcaccc gcggtatgcg caccgctacc 1080

ctggctcgtg acagctacgg aaagctcgtg gcatctggtc tgtcgatgac catcatgatc 1140

cagattttcg tcgtcgtggc aggtatttct tcactgatgc ccatgacagg tttgaccact 1200

ccgtttatgt cccagggtgg ttcatccctg atggctaact acattctgat ggccatcatc 1260

ttgcgtattt ctgacagtgc ccgccgacct gtcatgtcca agcaagcatc ggaggtggct 1320

gcgtga 1326

<210> 3

<211> 441

<212> PRT

<213> Artificial Sequence

<220>

<223> (WT)_cell division membrane protein

<400> 3

Met Asn Thr Leu Glu Arg Leu Lys Leu Arg Arg Thr Glu Met Trp Leu

1 5 10 15

Leu Ile Leu Ala Thr Leu Val Val Ser Ile Met Phe Ile Ser Leu Glu

20 25 30

Leu Ala Met Gly Asn Glu Leu Gly Thr His Ile Leu Met Leu Met Gly

35 40 45

Gly Tyr Ile Gly Ile Phe Ile Val Ala His Leu Ala Met Ala Trp Val

50 55 60

Ala Pro Phe Ala Asp Gln Ile Met Leu Pro Val Val Ala Val Leu Asn

65 70 75 80

Gly Ile Gly Leu Val Met Ile Tyr Arg Leu Asp Glu Ala Thr Gly Tyr

85 90 95

Ser Thr Val Asn Ser Gln Leu Met Trp Thr Val Val Gly Val Thr Leu

100 105 110

Met Val Ala Val Leu Leu Leu Leu Arg Asp Tyr Lys Ser Leu Ser Arg

115 120 125

Tyr Ser Tyr Leu Leu Gly Val Val Gly Ile Val Leu Leu Ala Leu Pro

130 135 140

Leu Val Trp Pro Gln Pro Gly Gly Val Glu Ala Arg Ile Trp Ile Trp

145 150 155 160

Leu Gly Pro Phe Ser Ile Gln Pro Gly Glu Phe Ser Lys Ile Leu Leu

165 170 175

Leu Leu Phe Phe Ala Gln Leu Leu Ala Thr Lys Arg Ala Leu Phe Thr

180 185 190

Val Ala Gly Tyr Arg Phe Leu Gly Met Asp Phe Pro Arg Leu Arg Asp

195 200 205

Leu Ala Pro Ile Leu Val Val Trp Ala Leu Ala Ile Leu Ile Met Ala

210 215 220

Gly Ala Asn Asp Phe Gly Pro Ala Leu Leu Leu Phe Thr Thr Val Leu

225 230 235 240

Ala Met Val Tyr Leu Ala Thr Gly Arg Gly Ser Trp Leu Leu Ile Gly

245 250 255

Ala Val Leu Val Ala Val Gly Ala Phe Ala Val Tyr Gln Val Ser Ser

260 265 270

Lys Ile Gln Glu Arg Val Gln Asn Phe Val Asp Pro Val Ala His Tyr

275 280 285

Asp Thr Thr Gly Tyr Gln Leu Ser Gln Ser Leu Phe Gly Met Ser Trp

290 295 300

Gly Gly Ile Thr Gly Thr Gly Ile Gly Gln Gly Tyr Pro Asn Met Ile

305 310 315 320

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

325 330 335

Gly Leu Ile Gly Leu Ala Ala Ile Ile Val Leu Phe Gly Val Phe Val

340 345 350

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

355 360 365

Leu Val Ala Ser Gly Leu Ser Met Thr Ile Met Ile Gln Ile Phe Val

370 375 380

Val Val Ala Gly Ile Ser Ser Leu Met Pro Met Thr Gly Leu Thr Thr

385 390 395 400

Pro Phe Met Ser Gln Gly Gly Ser Ser Leu Met Ala Asn Tyr Ile Leu

405 410 415

Met Ala Ile Ile Leu Arg Ile Ser Asp Ser Ala Arg Arg Pro Val Met

420 425 430

Ser Lys Gln Ala Ser Glu Val Ala Ala

435 440

<210> 4

<211> 1326

<212> DNA

<213> Artificial Sequence

<220>

<223> (WT)_NCgl0043

<400> 4

atgaacacgc ttgaacgatt aaagcttcgt cgcacggaaa tgtggctgct gatacttgcc 60

acactcgttg tgtcgatcat gttcatcagc ctcgagctgg ccatgggcaa tgagttgggt 120

acccatattt tgatgctgat gggcggatat atcggtatct tcatcgtcgc gcacctagcc 180

atggcatggg tggcgccgtt tgctgatcaa atcatgctgc ctgtggtggc ggtgctcaat 240

ggcattggtt tggtgatgat ttatcgcctt gatgaggcca cgggctacag cacggtcaat 300

agccaattga tgtggacggt tgttggcgtc acgctgatgg tggctgtgtt gttgctgttg 360

cgtgattaca agtcgctttc gcgttattcc tacctcctcg gtgtggtggg catcgtgctg 420

ctggcgctgc ctctcgtgtg gccgcagcca ggcggcgtgg aagcccgcat ctggatttgg 480

cttggacctt tctccatcca gccaggtgag ttctccaaga ttttgctgct gctgttcttt 540

gctcagctgc tagccaccaa gcgtgctttg tttactgttg cgggctaccg tttcctcggc 600

atggatttcc ctcgtttgcg tgacctcgcg ccgattcttg tggtgtgggc gttggctatt 660

ttgatcatgg ctggcgccaa cgacttcggt cctgcactgc tgcttttcac taccgttttg 720

gccatggtgt acctggctac cggccgtggt tcctggctgt tgattggtgc tgtgttggtg 780

gctgtcggcg cgttcgcggt gtaccaagtt tcaagcaaga ttcaggaacg cgtgcaaaac 840

ttcgtggatc ctgtggccca ctatgacacc accggttacc agctgtccca gtccttgttt 900

ggcatgagtt ggggcggaat caccggcacc ggcattggtc agggttaccc caacatgatc 960

cctgtcgtgc actcggactt cattctcgca gccattggtg aggagcttgg tctgattggc 1020

ctggcggcca tcatcgtgct gtttggtgtg tttgtcaccc gcggtatgcg caccgctacc 1080

ctggctcgtg acagctacgg aaagctcgtg gcatctggtc tgtcgatgac catcatgatc 1140

1200

ccgtttatgt cccagggtgg ttcatccctg atggctaact acattctgat ggccatcatc 1260

ttgcgtattt ctgacagtgc ccgccgacct gtcatgtcca agcaagcatc ggaggtggct 1320

gcgtga 1326

<210> 5

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> NCgl0043_1F

<400> 5

tgaattcgag ctcggtaccc ggtgggcatc gtgctgc 37

<210> 6

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> NCgl0043_2R

<400> 6

atgccggtgc cggtgactcc gccccaactc atg 33

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> NCgl0043_3F

<400> 7

catgagttgg ggcggagtca ccggcaccgg cat 33

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> NCgl0043_4R

<400> 8

gtcgactcta gaggatcccc ggaaaccacg tgcgttc 37

<210> 9

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> NCgl0043_5F

<400> 9

cgggctacag cacggtc 17

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<223> NCgl0043_6R

<400> 10

gcgagtactt ggccacc 17

<---

Claims (5)

1. A polypeptide involved in the production of L-lysine, consisting of the amino acid sequence shown by SEQ ID NO: 1, in which isoleucine, which is the amino acid corresponding to position 307 of SEQ ID NO: 3, is replaced by valine. 2. A polynucleotide encoding a polypeptide according to claim 1. 3. The microorganism Corynebacterium glutamicum for producing L-lysine, containing the polypeptide according to claim 1 or a polynucleotide encoding this polypeptide. 4. The microorganism according to claim 3, which has an increased ability to produce L-lysine compared to Corynebacterium glutamicum containing a polypeptide of SEQ ID NO: 3 or a polynucleotide encoding this polypeptide. 5. Method for producing L-lysine, including culturing a microorganismCorynebacterium glutamicumcontaining a polypeptide according to claim 1 or a polynucleotide encoding this polypeptide in the medium.