Use of cell death-related protein in preparing l-lysine

Abstract

Provided is the use of a cell death-related protein in preparing L-lysine. Further provided is a bacterial strain capable of reducing toxic substances or eliminating foams during a fermentation process, so as to produce L-lysine with a high yield. In order to solve the technical problem, the described use is the use of a protein, or a substance for regulating the expression of a coding gene of the protein, or a substance for regulating the activity or content of the protein, in preparing L-lysine or improving the yield of L-lysine, the protein being specifically one or more of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10 and SEQ ID No. 12. Experiments prove that knocking out the coding gene of the described protein helps to improve the yield of L-lysine in the bacterial strain.

Description

Application of cell death-related proteins in the preparation of L-lysine Technical Field

[0001] The present application relates to the application of cell death-related proteins in the field of biotechnology in the preparation of L-lysine. Background Art

[0002] Microbial fermentation is the main production method for producing lysine at present. The method has low raw material cost, mild reaction conditions and is easy to achieve large-scale production. However, the fermentation performance and conversion rate of lysine strains are still relatively large. In the process of producing lysine by microbial fermentation, as lysine accumulates, a large amount of toxic substances will be produced during the lysine fermentation process, causing the thalline to autolyze, and the fermentation process produces a large amount of foam, which requires increasing the amount of defoamer used, thereby increasing production costs, and a large amount of foam will also reduce the lysine yield of the thalline. Therefore, how to eliminate or alleviate the stress inhibition caused by the aforementioned toxic substances and foam is the key to obtaining efficient lysine production strains.

[0003] SUMMARY OF THE INVENTION

[0004] The technical problem to be solved by this application is to provide a strain that reduces toxic substances or eliminates foam during fermentation, thereby increasing the yield of L-lysine. The technical problem to be solved is not limited to the technical subject matter described above, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above technical problems, this application provides the following technical solutions:

[0006] The present application provides an application, which includes D1), D2), D3) or D4).

[0007] D1) Use of the protein in preparing L-lysine or increasing L-lysine production;

[0008] D2) Use of a substance that regulates the expression of the protein-encoding gene in preparing L-lysine or increasing L-lysine production;

[0009] D3) Use of a substance that regulates the activity of the protein in preparing L-lysine or increasing L-lysine production;

[0010] D4) Use of the substance for regulating protein content in preparing L-lysine or increasing L-lysine yield;

[0011] The above protein comprises G1), G2), G3), G4) or G5).

[0012] G1) includes the protein encoded by the NCgl1706 gene and the protein encoded by the NCgl1707 gene;

[0013] G2) includes the protein encoded by the NCgl2777 gene;

[0014] G3) includes the protein encoded by the NCgl1050 gene and the protein encoded by the NCgl1051 gene;

[0015] G4) includes the protein encoded by the NCgl1706 gene, the protein encoded by the NCgl1707 gene, and the protein encoded by the NCgl2777 gene;

[0016] G5) includes any two or more combinations of G1), G2), G3) or G4);

[0017] G5) The combination includes any of the following:

[0018] P1) includes the protein encoded by the NCgl1706 gene, the protein encoded by the NCgl1707 gene, the protein encoded by the NCgl1050 gene, and the protein encoded by the NCgl1051 gene;

[0019] P2) includes the protein encoded by the NCgl1050 gene, the protein encoded by the NCgl1051 gene, the protein encoded by the NCgl2777 gene, the protein encoded by the NCgl1706 gene, and the protein encoded by the NCgl1707 gene;

[0020] P3) includes the protein encoded by the NCgl1050 gene, the protein encoded by the NCgl1051 gene, and the protein encoded by the NCgl2777 gene;

[0021] The above-mentioned protein includes K1), K2), K3), K4), K5), K6) or K7).

[0022] K1) The protein is composed of two proteins, a protein encoded by the NCgl1706 gene and a protein encoded by the NCgl1707 gene;

[0023] K2) the protein is a protein encoded by the NCgl2777 gene;

[0024] K3) The protein is composed of two proteins: a protein encoded by the NCgl1050 gene and a protein encoded by the NCgl1051 gene;

[0025] K4) The protein is composed of three proteins: a protein encoded by the NCgl2777 gene, a protein encoded by the NCgl1706 gene, and a protein encoded by the NCgl1707 gene;

[0026] K5) The protein is composed of four proteins: a protein encoded by the NCgl1050 gene, a protein encoded by the NCgl1051 gene, a protein encoded by the NCgl1706 gene, and a protein encoded by the NCgl1707 gene;

[0027] K6) The protein consists of five proteins: a protein encoded by the NCgl1050 gene, a protein encoded by the NCgl1051 gene, a protein encoded by the NCgl2777 gene, a protein encoded by the NCgl1706 gene, and a protein encoded by the NCgl1707 gene;

[0028] K7) The protein consists of three proteins: a protein encoded by the NCgl1050 gene, a protein encoded by the NCgl1051 gene, and a protein encoded by the NCgl2777 gene.

[0029] In the above, the protein encoded by the NCgl1050 gene in A1) includes A1-1), A1-2) or A1-3).

[0030] A1-1) a protein having an amino acid sequence containing SEQ ID No. 11;

[0031] A1-2) a protein derived from A1-1) or having 80% or more identity with the protein represented by A1-1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence represented by SEQ ID No. 11, having the same function as the protein encoded by the NCgl1050 gene;

[0032] A1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1-1) or A1-2);

[0033] In the above, the protein encoded by the NCgl1051 gene in A2) includes A2-1), A2-2) or A2-3).

[0034] A2-1) a protein having an amino acid sequence containing SEQ ID No. 12;

[0035] A2-2) a protein derived from A2-1) or having 80% or more identity with the protein represented by A2-1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence represented by SEQ ID No. 12, having the same function as the protein encoded by the NCgl1051 gene;

[0036] A2-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A2-1) or A2-2);

[0037] In the above, the protein encoded by the NCgl2777 gene in A3) includes A3-1), A3-2) or A3-3).

[0038] A3-1) a protein having an amino acid sequence containing SEQ ID No. 7;

[0039] A3-2) a protein derived from A3-1) or having 80% or more identity with the protein represented by A3-1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence represented by SEQ ID No. 7, having the same function as the protein encoded by the NCgl2777 gene;

[0040] A3-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A3-1) or A3-2);

[0041] In the above, the protein encoded by the NCgl1706 gene in A4) includes A4-1), A4-2) or A4-3).

[0042] A4-1) a protein having an amino acid sequence containing SEQ ID No. 2;

[0043] A4-2) a protein derived from A4-1) or having 80% or more identity with the protein represented by A4-1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence represented by SEQ ID No. 2, having the same function as the protein encoded by the NCgl1706 gene;

[0044] A4-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A4-1) or A4-2);

[0045] In the above, the protein encoded by the NCgl1707 gene in A5) includes A5-1), A5-2) or A5-3).

[0046] A5-1) a protein having an amino acid sequence containing SEQ ID No. 4;

[0047] A5-2) a protein derived from A5-1) or having 80% or more identity with the protein represented by A5-1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence represented by SEQ ID No. 4, having the same function as the protein encoded by the NCgl1707 gene;

[0048] A5-3) A fusion protein having the same function as A5-1) or A5-2) obtained by connecting a tag to the N-terminus and / or C-terminus.

[0049] The above-mentioned identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAS T2.1, the identity of a pair of amino acid sequences can be calculated by searching using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0050] The above 80% or more identity may be 80%, 85%, 90% or 95% or more identity. The above 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The above 85% or more identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Said 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Said 95% or more identity can be at least 95%, 96%, 97%, 98% or 99% identity.

[0051] In the present application, the regulation may be upregulating, enhancing or increasing the content and / or activity of the aforementioned protein in the cell, or downregulating, inhibiting or reducing the content and / or activity of the aforementioned protein in the cell.

[0052] In some specific embodiments of the present application, the regulation of the expression of the gene encoding the protein can specifically be inhibition, reduction or down-regulation of the expression of the gene encoding the protein. The inhibition, reduction or down-regulation of the expression of the gene encoding the protein can be achieved by gene knockout or gene silencing.

[0053] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing its DNA sequence.

[0054] The gene silencing refers to the phenomenon of not expressing or underexpressing a gene without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or underexpressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level due to DNA methylation, heterochromatinization and position effects, and the other is post-transcriptional gene silencing, that is, gene inactivation by specifically inhibiting the target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0055] In the present application, the substance is U1), U2), U3), U4), U5) or U6): U1) regulation at the transcription level of the gene; U2) regulation after transcription of the gene (that is, regulation of splicing or processing of the primary transcript of the gene); U3) regulation of RNA transport of the gene (that is, regulation of transport of the mRNA of the gene from the nucleus to the cytoplasm); U4) regulation of translation of the gene; U5) regulation of mRNA degradation of the gene; U6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0056] When the regulation is inhibition, reduction or downregulation, the substance may be an agent for knocking out the protein-encoding gene, such as an agent for knocking out the protein-encoding gene by homologous recombination, or an agent for knocking out the protein-encoding gene by the CRISPR-Cas system.

[0057] The reagents include B1), B2), B3), B4) or B5):

[0058] B1), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or a nucleic acid molecule that inhibits, reduces or down-regulates the activity or content of the protein; the nucleic acid molecule includes a DNA molecule or an RNA molecule;

[0059] B2), expressing the gene encoding the RNA molecule described in B1);

[0060] B3), an expression cassette containing the gene described in B2);

[0061] B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0062] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

[0063] In the above, the DNA molecule in B1) includes a nucleic acid molecule (such as circular DNA (such as a vector) or linear DNA) that knocks out the gene encoding the protein in the cell by homologous recombination.

[0064] The nucleotide sequence of the NCgl1050 gene contains the sequence of SEQ ID No.9, the nucleotide sequence of the NCgl1051 gene contains the sequence of SEQ ID No.11 (540bp), the nucleotide sequence of the NCgl2777 gene contains the sequence of SEQ ID No.6 (1974bp), the nucleotide sequence of the NCgl1706 gene contains the sequence of SEQ ID No.1 (1524bp), and the nucleotide sequence of the NCgl1707 gene contains the sequence of SEQ ID No.3 (672bp).

[0065] The present application also provides a recombinant bacterium, which does not contain or lacks the aforementioned protein.

[0066] Furthermore, the recombinant bacteria does not contain or lacks the coding gene of the aforementioned protein.

[0067] Furthermore, the recombinant bacteria include bacteria. Specifically, the recombinant bacteria can be bacteria.

[0068] The recombinant bacteria can be obtained by knocking out or silencing the gene encoding the protein in a recipient bacterium, and the recipient bacterium contains the gene encoding the protein.

[0069] Furthermore, the recombinant bacteria include Corynebacterium glutamicum. The recombinant bacteria can specifically be Corynebacterium glutamicum.

[0070] The use of biomaterials related to the aforementioned proteins in the preparation or improvement of glutamic acid, lysine, valine, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutaric acid, citric acid, ornithine, and citrulline, preferably in the preparation of L-lysine or in increasing the yield of L-lysine, also falls within the scope of protection of this application.

[0071] The biological material includes:

[0072] B1), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or a nucleic acid molecule that inhibits, reduces or down-regulates the activity or content of the protein; the nucleic acid molecule includes a DNA molecule or an RNA molecule;

[0073] B2), expressing the gene encoding the RNA molecule described in B1);

[0074] B3), an expression cassette containing the gene described in B2);

[0075] B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0076] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

[0077] The expression cassette described in the biological material above refers to DNA capable of expressing the proteins described above in host cells. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing any of the aforementioned proteins. The regulatory sequences are capable of directing the coding sequence to express any of the aforementioned proteins in a suitable host cell under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequence to the coding region of the protein-encoding nucleic acid sequence, the regulatory sequence may be provided with a linker. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. A regulatory sequence may also be a suitable transcriptional terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that is functional in the selected host cell may be used in this application. A regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the selected host cell may be used in this application. A regulatory sequence may also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the encoded protein into the cell's secretory pathway. Any signal peptide coding region that directs the expressed protein into the secretory pathway of the selected host cell may be used in this application. It may also be desirable to add regulatory sequences that can regulate protein expression based on the growth conditions of the host cells. Examples of regulatory systems are those that can turn gene expression on or off in response to chemical or physical stimuli (including in the presence of regulatory compounds). Other examples of regulatory sequences are those that enable gene amplification. In these instances, the protein-encoding nucleic acid sequence should be operably linked to the regulatory sequences.

[0078] The recombinant vector may include a nucleic acid molecule encoding the above-mentioned protein, a promoter, and transcription and translation termination signals. When preparing the recombinant vector, the nucleic acid molecule encoding the above-mentioned protein can be located in the vector so as to be operably linked to an appropriate expression control sequence. The recombinant vector can be any vector (e.g., a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed-loop plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may include any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid can be used, or two or more vectors or plasmids, or transposons, that collectively comprise the entire DNA that will be introduced into the host cell genome can be used. The vector contains one or more selectable markers that are convenient for selecting transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or prototrophy to an auxotroph. Examples of bacterial selectable markers include the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Vectors contain elements that enable the vector to be stably integrated into the host cell genome or to ensure autonomous replication of the vector in the cell, independent of the cellular genome. In the case of autonomous replication, the vector may also contain an origin of replication, enabling the vector to replicate autonomously in the target host cell. The origin of replication may contain a mutation that renders it temperature-sensitive in the host cell (see, for example, Ehrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1433). One or more copies of a nucleic acid molecule encoding any of the above-described proteins may be inserted into the host cell to increase the production of the gene product. The number of copies of the nucleic acid molecule can be increased by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selectable marker along with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and, thereby, the additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selective agent. The procedures used to connect the above-mentioned elements to construct the recombinant expression vectors described herein are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0079] The term "operably linked" is defined herein as a configuration in which a regulatory sequence is appropriately positioned relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein.

[0080] The present application also provides a method for preparing a recombinant microorganism, comprising the following steps: down-regulating, reducing or inhibiting the expression level of a gene in a target microorganism, or / and down-regulating, reducing or inhibiting the expression level or activity of a protein encoded by the gene in the target microorganism, to obtain a recombinant microorganism; the genes include the aforementioned NCgl1706 gene, NCgll707 gene, NCgl2777 gene, NCgl1050 gene and / or NCgll051 gene.

[0081] Specifically, the gene may be the NCgl1706 gene, NCgll707 gene, NCgl2777 gene, NCgl1050 gene and NCgll051 gene; the gene may be the NCgl2777 gene, the NCgl1706 gene and the NCgl1707 gene; the gene may also be the NCgl1050 gene, the NCgl1051 gene and the NCgl2777 gene; the gene may also be the NCgl1050 gene, the NCgl1051 gene, the NCgl1706 gene and the NCgl1707 gene; the gene may be the NCgl1706 gene and the NCgll707 gene; the gene may be the NCgl2777 gene; the gene may be the NCgl1050 gene and the NCgll051 gene.

[0082] Furthermore, the target microorganism in the above method includes Corynebacterium glutamicum. The target microorganism can specifically be Corynebacterium glutamicum. The increasing L-lysine production includes increasing the L-lysine production of bacteria.

[0083] The present application also provides a whole-cell catalyst, comprising the aforementioned recombinant bacteria or the aforementioned biomaterial. The present application also provides a method for preparing L-lysine, comprising fermenting L-lysine using the aforementioned recombinant bacteria, biomaterial or whole-cell catalyst. Beneficial effects

[0084] The present application proves through experiments that compared with the target microorganism Corynebacterium glutamicum YP097158, the recombinant Corynebacterium glutamicum YPL-ΔNCgl1706-NCgl1707-ΔNCgl2777-ΔNCgl1050-NCgl1051 with the NCgl1050 gene, NCgl1051 gene, NCgl2777 gene, NCgl1706 gene and NCgl1707 gene knocked out, and the recombinant Corynebacterium glutamicum YPL-ΔNCgl1050-NCgl1051 with the NCgl1050 gene, NCgl1051 gene, NCgl2777 gene l2777-ΔNCgl1050-NCgll051, recombinant Corynebacterium glutamicum YPL-ΔNCgl1706-NCgll707-ΔNCgl1050-NCgll051 with NCgl1050, NCgl1051, NCgl1706 and NCgl1707 genes knocked out, recombinant Corynebacterium glutamicum YPL-ΔNCgl1050-NCgll051 with NCgl1050 and NCgl1051 genes knocked out, The L-lysine production of the recombinant Corynebacterium glutamicum YPL-ΔNCgl1706-NCgll707-ΔNCgl2777 with the NCgl2777 gene deleted, the NCgl1706 gene and the NCgl1707 gene deleted, and the recombinant Corynebacterium glutamicum YPL-ΔNCgl1706-NCgll707 with the NCgl1706 gene and the NCgl1707 gene deleted was significantly improved.

[0085] Preservation Instructions

[0086] Bacterial species: Corynebacterium glutamicum

[0087] Latin name: Corynebacterium glutamicum

[0088] Strain ID: YP097158

[0089] Depository: General Microbiology Center of China Culture Collection Administration

[0090] Abbreviation of depository institution: CGMCC

[0091] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0092] Deposit date: August 16, 2016

[0093] Deposit number: CGMCC No.12856. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 shows the RT-qPCR detection results of NCgl1706-NCgll707.

[0095] Figure 2 shows the RT-qPCR detection results of NCgl2777.

[0096] Figure 3 shows the RT-qPCR detection results of NCgl1050-NCgll051.

[0097] Figure 4 shows the RT-qPCR detection results of NCgl1706-NCgll707-NCgl2777.

[0098] Figure 5 shows the RT-qPCR detection results of NCgl1706-NCgll707-NCgl1050-NCgll051.

[0099] Figure 6 shows the RT-qPCR detection results of NCgl2777-NCgl1050-NCgll051.

[0100] Figure 7 shows the RT-qPCR detection results of NCgl1706-NCgll707-NCgl2777-NCgl1050-NCgll051. Modes for Carrying Out the Invention

[0101] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0102] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0103] The present application is further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvements by those skilled in the art and do not in any way limit the present application.

[0104] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0105] definition:

[0106] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0107] As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide (polyucleotide), which is composed of a heterocyclic base, a sugar and one or more phosphate groups. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T) and uracil (U)) are typically derivatives of purines or pyrimidines, but it will be understood that natural and non-natural base analogs are also included. Naturally occurring sugars are pentoses (five-carbon sugars), deoxyribose (which forms DNA) or ribose (which forms RNA), but it will be understood that natural and non-natural sugar analogs are also included. Nucleic acids are typically linked by phosphodiester bonds to form nucleic acids or polynucleotides, but may also be linked by other bonds known in the art, such as phosphorothioate bonds.

[0108] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0109] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid (ie, a polynucleotide sequence) to which it has been linked.

[0110] As used herein, the term "fermentation" broadly refers to the conversion of an organic material by host cells into a target substance, for example, by propagating a culture of recombinant host cells in a medium that includes a carbon source so that the recombinant host cells convert the carbon source into lysine.

[0111] As used herein, the term "L-lysine" is L-2,6-diaminohexanoic acid (L-Lysine).

[0112] Wild-type Corynebacterium glutamicum ATCC13032 strain: a product of the American Type Culture Collection (ATCC).

[0113] The Corynebacterium glutamicum strain YP097158 in the following examples is described in the Chinese patent document entitled "A Recombinant Strain for High-yield L-Lysine, Its Construction Method, and Application" with grant number "CN110607313B." The strain number is YP097158 and was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on August 16, 2016, under the deposit number CGMCC No. 12856. Hereinafter, it is referred to as Corynebacterium glutamicum CGMCC No. YP097158.

[0114] The data in the following examples were processed using GraphPad Prism 8 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference.

[0115] The quantitative tests in the following examples were repeated three times and the results were averaged. -ΔΔCt Methods The qRT-PCR results were analyzed and the relative gene expression levels were calculated.

[0116] Example 1: Construction of an engineered strain with NCgl1706-NCgll707 gene deletion in the genome

[0117] Based on the sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, the NCgl1706-NCgll707 genes in the genome of Corynebacterium glutamicum YP097158 (from Corynebacterium glutamicum ATC C13032) were knocked out using the pk18mobsacB plasmid (sequencing confirmed that the complete NCgl1706-NCgll707 genes were retained on the chromosome of the YP097158 strain) to further study the effects of these genes on L-lysine synthesis.

[0118] 1. Construction of knockout plasmid

[0119] Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the fragments at both ends of the NCgl1706-NCgl1707 gene as upstream and downstream homology arm fragments. The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0120] P1:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATCCAAGCGACCACAAG-3' (SEQ ID No. 14);

[0121] P2:5'-CTATTTTCAGGAACATTTACGTGTCGTGAGCGATAAAAAACAC-3' (SEQ ID No. 15);

[0122] P3:5'-GTGTTTTTTATCGCTCACGACACGTAAATGTTCCTGAAAATAG-3' (SEQ ID No. 16);

[0123] P4: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCGTGCGATT-3' (SEQ ID No. 17).

[0124] Among the above primers, the underlined nucleotide sequence is the homologous sequence on pK18, which is used to integrate the DNA fragment (upstream homologous arm-downstream homologous arm) into the pK18mobsacB plasmid (Addgene) by homologous recombination. P2 and P3 are used to connect the upstream homologous arm and the downstream homologous arm by homologous recombination.

[0125] Construction method: Using the genome of Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed with primers P1 / P2, P3 / P4 and high-fidelity DNA polymerase KAPA HiFi HotStart (purchased from Shanghai Huaya Si Chuang Biotechnology Co., Ltd., KK2601), respectively, to obtain a 797bp upstream homology arm fragment (sequence as shown in bases 1-797 in SEQ ID No. 5) and a 693bp downstream homology arm fragment (sequence as shown in bases 798-1490 in SEQ ID No. 5) of the NCgl1706-NCgll707 gene knockout. The amplified products were electrophoresed and purified using a column DNA gel recovery kit. The two recovered DNA fragments were ligated with the pK18mobsacB plasmid (purchased from Addgene) purified after Xbal I / BamH I digestion using NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The monoclonal clone grown after transformation of the ligation product was identified by PCR using primers M13F (5′-TGTAAAA CGACGGCCAGT-3′, SEQ ID No.18) / M13R (5′-CAGGAAACAGCTATGACC-3′, SEQ ID No.19) to obtain the positive knockout vector pK18-ΔNCgl1706-NCgll707, which contains a 1490 bp homologous DNA fragment of the knockout NC gl1706-NCgll707 gene (sequence SEQ ID No.5). The plasmid contains kanamycin resistance as a selection marker, and the plasmid was sent for sequencing.

[0126] PCR amplification system: 5× HiFi with Mg2+ Buffer 10μL, dNTP Mix (10mM) 1.5μL, primers (10pM) 1.6μL each, KAPA HiFi HotStart (1U / μL) 0.5μL, supplemented with ddH2O to a total volume of 50μL. PCR amplification program: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s, followed by overextension at 72°C for 5 min.

[0127] 2. Construction of knockout strains

[0128] The knockout plasmid pK18-ΔNCgl1706-NCgll707, which was sequenced correctly, was electrotransformed into Corynebacterium glutamicum YP 097158 and cultured according to the medium components and culture conditions shown in Table 1. The resulting single colonies were identified by PCR using primers P1 / P4: strains that simultaneously amplified bands of 1490 bp and 3931 bp were positive strains, while strains that only amplified the 3931 bp band were considered the original strains. The positive strains were cultured on 15% sucrose solid medium (shown in Table 1) for 30 h, and single colonies were selected for screening on solid medium plates containing kanamycin and those that did not. Monoclonal strains that grew on medium without kanamycin but did not grow on medium containing kanamycin were further identified by PCR using primers P1 / P4. Strains that amplified a band of 1490 bp (SEQ ID No. 5) were positive strains with the NCgl1706-NCgll707 gene coding region deleted. The gene fragment of the positive strain (NCgl1706-NCgll707 gene coding region knocked out) was amplified again using P1 / P4 primers and sequenced, and the strain with correct sequencing was named YPL-ΔNCgl1706-NCgll707.

[0129] The recombinant bacterium YPL-ΔNCgl1706-NCgll707 is a recombinant Corynebacterium glutamicum obtained by replacing the 1881670 to 1885600 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 5, while keeping other nucleotide sequences unchanged, that is, the NCgl1706 gene coding region (nucleotide sequence is SEQ ID No. 1) in the Corynebacterium glutamicum YP097158 genome, encoding the amino acid sequence shown in SEQ ID No. 2, that is, the 1882467 to 1883990 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) and the NCgl1707 gene coding region (sequence is SEQ ID No. 3), encoding SEQ ID The amino acid sequence shown in No. 4, i.e., bases 1884217 to 1884888 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014), was deleted, while keeping the other nucleotides in the genome of Corynebacterium glutamicum YP097158 unchanged to obtain a recombinant bacterium.

[0130] Table 1 Composition of culture medium and culture conditions

[0131] Note: The culture temperature is 32°C and the culture time is 30 hours.

[0132] 3. RT-qPCR detection of recombinant strains

[0133] The recombinant bacteria YPL-ΔNCgl1706-NCgll707 and Corynebacterium glutamicum YP097158 were cultured for 24 h and then centrifuged at 4°C to collect the cells. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108). The RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription levels of the genes NCgl1706 and NCgll707 were detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). 16S RNA was selected as the internal reference gene, and the primers were designed as follows:

[0134] NCgl1706-F:5'-GACCGTGACCATCTCTACATC-3' (SEQ ID No. 20);

[0135] NCgl1706-R:5'-TTCATTGCGTGACCACAC-3' (SEQ ID No. 21);

[0136] NCgl1707-F:5'-TGTATCCGTAGTAATCCTGGC-3' (SEQ ID No. 22);

[0137] NCgl1707-R: 5'-GGGACGATGAATCCAAATG-3' (SEQ ID No. 23).

[0138] As can be seen from Figure 1, compared with Corynebacterium glutamicum YP097158, the expression levels of NCgl1706 and NCgll707 genes in the recombinant strain YPL-ΔNCgl1706-NCgll707 were significantly decreased, proving that the knockout was successful.

[0139] Example 2: Construction of an engineered strain with a genome-deleted NCgl2777 gene

[0140] Based on the NCBI-published sequence of Corynebacterium glutamicum ATCC13032, the NCgl2777 gene in the genome of L-lysine-producing strain YP097158 (derived from Corynebacterium glutamicum ATCC13032) was deleted using the pk18mobsacB plasmid (sequencing confirmed that the intact NCgl2777 gene was retained on the chromosome of the YP097158 strain). This allowed for a more in-depth study of the effects of these genes on L-lysine synthesis.

[0141] 1. Construction of knockout plasmid

[0142] Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the fragments at both ends of the NCgl2777 gene as upstream and downstream homology arm fragments. The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0143] P5:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGACAGCAATAACGGTGATGG-3' (SEQ ID No. 24);

[0144] P6: 5'-CAAAATGAGAAGGAAAACTTCCAATGAGAAGTTGATCAAC-3' (SEQ ID No. 25);

[0145] P75'-GTTGATCAACTTCTCATTGGAAGTTTTCCTTCTCATTTTG-3' (SEQ ID No. 26);

[0146] P85'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCGGATTTGGAGATGTCG-3' (SEQ ID No. 27).

[0147] Among the above primers, the underlined nucleotide sequence is the homologous sequence on pK18, which is used to integrate the DNA fragment (upstream homologous arm-downstream homologous arm) into the pK18mobsacB plasmid (Addgene) by homologous recombination. P6 and P7 are used to connect the upstream homologous arm and the downstream homologous arm by homologous recombination.

[0148] Construction method: The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR amplification was performed with primers P5 / P6 and P7 / P8 and high-fidelity DNA polymerase KAPA HiFi HotStart (purchased from Shanghai Huaya Si Chuang Biotechnology Co., Ltd., KK2601) to obtain a 788bp upstream homology arm fragment (nucleotide sequence as shown in SEQ ID No. 8, 1-788) and a 692bp downstream homology arm fragment (nucleotide sequence as shown in SEQ ID No. 8, 789-1480). The amplified products were electrophoresed and purified using a column DNA gel recovery kit. The two recovered DNA fragments were ligated with the pK18mobsacB plasmid (purchased from Addgene) purified after digestion with Xbal I / BamH I using NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The monoclonal clone grown after transformation of the ligation product was identified by PCR using primers M13F / M13R to obtain a positive knockout vector pK18-ΔNCgl2777, which contained a 1480 bp homologous DNA fragment of the knockout NCgl2777 gene (SEQ ID No. 8). The plasmid contained kanamycin resistance as a selection marker, and the plasmid was sent for sequencing.

[0149] PCR amplification system: 5×HiFi with Mg 2+ Buffer 10 μL, dNTP Mix (10 mM) 1.5 μL, primers (10 pM) 1.6 μL each, KAPA HiFi HotStart (1 U / μL) 0.5 μL, and ddH₂O to a total volume of 50 μL. PCR amplification program: pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 98°C for 20 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s, followed by over-extension at 72°C for 5 min.

[0150] 2. Construction of knockout strains

[0151] The knockout plasmid pK18-ΔNCgl2777, which had been sequenced correctly, was electroporated into Corynebacterium glutamicum YP097158 and cultured according to the medium composition and culture conditions shown in Table 1. The resulting single colonies were identified by PCR using primers P5 / P8: strains that simultaneously amplified bands of 1480 bp and 3201 bp were considered positive strains, while strains that only amplified the 3201 bp band were considered protozoa. Positive strains were cultured on 15% sucrose solid medium (shown in Table 1) for 30 h, and single colonies were selected for screening on solid medium plates containing kanamycin and those that did not. Monoclonal strains that grew on the medium without kanamycin but did not grow on the medium containing kanamycin were further identified by PCR using primers P5 / P8. Strains that amplified a band of 1480 bp (SEQ ID No. 8) were positive strains with the NCgl2777 gene coding region deleted. The gene fragment of the positive strain (NCgl2777 gene coding region knocked out) was amplified again using P5 / P8 primers and sequenced, and the strain with correct sequencing was named YPL-ΔNCgl2777.

[0152] The recombinant bacterium YPL-ΔNCgl2777 is a recombinant Corynebacterium glutamicum obtained by replacing bases 3071113 to 3074313 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 8, while keeping other nucleotide sequences unchanged, that is, the NCgl2777 gene coding region in the Corynebacterium glutamicum YP097158 genome (sequence shown in SEQ ID No. 6, encoding the amino acid sequence shown in SEQ ID No. 7, i.e., bases 3071901 to 3073621 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) is deleted, and the other nucleotides in the genome of Corynebacterium glutamicum YP097158 are kept unchanged.

[0153] 3. RT-qPCR detection of recombinant strains

[0154] The recombinant bacteria YPL-ΔNCgl2777 and Corynebacterium glutamicum YP097158 were cultured for 24 h, then centrifuged at 4°C to collect the cells. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108). The RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription level of the NC gl2777 gene was detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). 16S RNA was selected as the internal reference gene, and the primers were designed as follows:

[0155] NCgl2777-F:5'-CCTGAAATCTGGGCACTTG-3' (SEQ ID No. 28);

[0156] NCgl2777-R: 5'-TTAGAACGGAAACCCTTGTCC-3' (SEQ ID No. 29).

[0157] As can be seen from Figure 2, compared with Corynebacterium glutamicum YP097158, the expression level of NCgl2777 gene in the recombinant bacteria YPL-ΔNCgl2777 was significantly decreased, proving that the gene NCgl2777 was successfully knocked out.

[0158] Example 3: Construction of an engineered strain with NCgl1050-NCgll051 gene deletion in the genome

[0159] Based on the sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, the NCgl1050-NCgll051 genes in the genome of L-lysine-producing strain YP097158 (from Corynebacterium glutamicum ATCC13032) were knocked out using the pk18mobsacB plasmid (sequencing confirmed that the complete NCgl1050-NCgll051 genes were retained on the chromosome of the YP097158 strain) to further study the effects of these genes on L-lysine synthesis.

[0160] 1. Construction of knockout plasmid

[0161] Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the fragments at both ends of the NCgl1050-NCgl1051 gene as upstream and downstream homology arm fragments. The primers were designed as follows (synthesized by Shanghai Invitrogen):

[0162] P9: 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAACTTGCCTGATTGGGAAG-3' (SEQ ID No. 30);

[0163] P10: 5'-GATGTGAGGTAAGAAAACAGATGCGAAACCGGCGCCAAC-3' (SEQ ID No. 31);

[0164] P11:5'-GTTGGCGCCGGTTCGCATCTGTTTTCTTACCTCACATC-3' (SEQ ID No. 32);

[0165] P12: 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTGAATCTTTAGGGAAACCCAG-3' (SEQ ID No. 33).

[0166] Among the above primers, the underlined nucleotide sequence is the homologous sequence on pK18, which is used to integrate the DNA fragment (upstream homologous arm-downstream homologous arm) into the pK18mobsacB plasmid (Addgene) by homologous recombination. P10 and P11 are used to connect the upstream homologous arm and the downstream homologous arm by homologous recombination.

[0167] Construction method: The genome of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR amplification was performed with primers P9 / P10 and P11 / P12 and high-fidelity DNA polymerase KAPA HiFi HotStart (purchased from Shanghai Huaya Si Chuang Biotechnology Co., Ltd., KK2601) to obtain an upstream homology arm fragment of 834 bp (sequence as shown in 1-834 in SEQ ID No.13) and a downstream homology arm fragment of 882 bp (sequence as shown in 835-1716 in SEQ ID No.13) of the NCgl1050-NCgll051 gene knockout. The amplified products were electrophoresed and purified using a column DNA gel recovery kit. The two recovered DNA fragments were ligated with the pK18mobsacB plasmid (purchased from Addgene) purified after Xbal I / BamH I digestion using NEBuilder enzyme (purchased from NEB) at 50°C for 30 min. The monoclonal clone grown after transformation of the ligation product was identified by PCR using primers M13F / M13R to obtain the positive knockout vector pK18-ΔNCgl1050-NCgll051, which contained a 1716 bp homologous DNA fragment (SEQ ID No. 13) of the knockout NCgl1050-NCgll051 gene. The plasmid contained kanamycin resistance as a screening marker, and the plasmid was sent for sequencing.

[0168] PCR amplification system: 5×HiFi with Mg 2+ Buffer 10μL, dNTP Mixture (10mM) 1.5μL, primers (10pM) 1.6μL each, KAPA HiFi HotStart (1U / μL) 0.5μL, add ddH2O to a total volume of 50μL.

[0169] PCR amplification program: pre-denaturation at 95°C for 5 min, (denaturation at 98°C for 20 s; annealing at 60°C for 15 s; extension at 72°C for 30 s; 30 cycles), over-extension at 72°C for 5 min.

[0170] 2. Construction of knockout strains

[0171] The knockout plasmid pK18-ΔNCgl1050-NCgll051, which was sequenced correctly, was electrotransformed into Corynebacterium glutamicum YP 097158 and cultured according to the medium components and culture conditions shown in Table 1. The resulting single colonies were identified by PCR using primers P9 / P12: strains that simultaneously amplified bands of 1716 bp and 3391 bp were positive strains, while strains that only amplified the 3391 bp band were considered the original strains. After the positive strains were cultured on 15% sucrose solid medium (shown in Table 1) for 30 h, single colonies were selected and screened on solid medium plates containing kanamycin and those that did not. Monoclonal strains that grew on medium containing kanamycin but did not grow on medium containing kanamycin were further identified by PCR using primers P9 / P12. Strains that amplified a band of 1716 bp (SEQ ID No. 13) were positive strains with the NCgl1050-NCgll051 gene coding region deleted. The gene fragment of the positive strain (NCgl1050-NCgll051 gene coding region knocked out) was amplified again using P9 / P12 primers and sequenced, and the strain with correct sequencing was named YPL-ΔNCgl1050-NCgll051.

[0172] The recombinant bacterium YPL-ΔNCgl1050-NCgll051 is a recombinant Corynebacterium glutamicum obtained by replacing bases 1140507 to 1143897 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 13, while keeping other nucleotide sequences unchanged, that is, the NCgl1050 gene coding region (nucleotide sequence of SEQ ID No. 9, encoding the amino acid sequence shown in SEQ ID No. 10, i.e., bases 1141341 to 1142475 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) and the NCgl1051 gene coding region (nucleotide sequence of SEQ ID No. 11, encoding SEQ ID The amino acid sequence shown in No. 12, i.e., positions 1142476 to 1143015 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014), was deleted, while keeping the other nucleotides in the genome of Corynebacterium glutamicum YP097158 unchanged to obtain a recombinant bacterium.

[0173] 3. RT-qPCR detection of recombinant strains

[0174] The recombinant bacteria YPL-ΔNCgl1050-NCgll051 and Corynebacterium glutamicum YP097158 were cultured for 24 h and then centrifuged at 4°C to collect the cells. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108). The RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription levels of the genes NCgl1050 and NCgll051 were detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). 16sRNA was selected as the internal reference gene, and the primers were designed as follows:

[0175] NCgll050-F:5'-ACACGGCACTGGGTATGTTC-3' (SEQ ID No. 34);

[0176] NCgll050-R:5'-CGTTCGGATTCCTTGTAGATG-3' (SEQ ID No. 35);

[0177] NCgll051-F:5'-CAGAACTTGGATTGCGTG-3' (SEQ ID No. 36);

[0178] NCgll051-R: 5'-GGGTGATTCCTTCAACAGC-3' (SEQ ID No. 37).

[0179] As can be seen from Figure 3, compared with Corynebacterium glutamicum YP097158, the expression levels of NCgl1050 and NCgl1051 genes in the recombinant bacteria YPL-ΔNCgl1050-NCgll051 were significantly decreased, proving that the genes NCgl1050 and NCgl1051 were successfully knocked out.

[0180] Example 4: Construction of an engineered strain with NCgl1706-NCgl1707 and NCgl2777 gene deletions

[0181] Based on the sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, the NCgl1706-NCgll707 and NCgl2777 genes in the genome of L-lysine-producing strain YP097158 (from Corynebacterium glutamicum ATCC13032) were knocked out using the pk18mobsacB plasmid (sequencing confirmed that the complete NCgl1706-NCgll707 and NCgl2777 genes were retained on the chromosome of the YP097158 strain). This was done to further investigate the effects of these genes on L-lysine synthesis.

[0182] The knockout plasmid pK18-ΔNCgl2777, constructed correctly in Example 2, was electroporated into the recombinant strain YPL-ΔNCgl1706-NCgl1707 constructed in Example 1. Culture was performed according to the medium composition and culture conditions shown in Table 1. Single colonies resulting from the culture were identified by PCR using primers P5 / P8: strains that simultaneously amplified bands of 1480 bp and 3201 bp were considered positive strains, while strains that only amplified the 3201 bp band were considered primary strains. Positive strains were cultured on 15% sucrose solid medium (shown in Table 1) for 30 h. Single colonies were selected and screened on solid medium plates containing and without kanamycin. Single colonies that grew on the medium without kanamycin but not on the medium containing kanamycin were further identified by PCR using primers P5 / P8. Strains that amplified a band of 1480 bp (SEQ ID No. 8) were positive strains with a knockout of the NCgl2777 gene coding region. The gene fragment of the positive strain was amplified again using P5 / P8 primers and sequenced, and the correctly sequenced strain was named YPL-ΔNCgl1706-NCgll707-ΔNCgl2777.

[0183] The recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl2777 is obtained by replacing the 1881670 to 1885600 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 5, and replacing the 3071113 to 3074313 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 8, while keeping other nucleotide sequences unchanged, that is, the NCgl1706 gene coding region (nucleotide sequence of SEQ ID No. 1, encoding SEQ ID No. 2) in the genome of Corynebacterium glutamicum YP097158 was obtained. The amino acid sequence shown in SEQ ID No.2, i.e., positions 1882467 to 1883990 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) were knocked out; the coding region of the NCgl1707 gene (the nucleotide sequence is SEQ ID No.3, encoding the amino acid sequence shown in SEQ ID No.4, i.e., positions 1884217 to 1884888 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) were knocked out; the coding region of the NCgl2777 gene (the nucleotide sequence is SEQ ID No.6, encoding the amino acid sequence shown in SEQ ID The amino acid sequence shown in No. 7, i.e., positions 3071901 to 3073621 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014), was deleted, while keeping the other nucleotides in the genome of Corynebacterium glutamicum YP097158 unchanged to obtain a recombinant bacterium.

[0184] The recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl2777 and Corynebacterium glutamicum YP097158 were cultured for 24 h and then centrifuged at 4 ° C to collect the bacteria. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108), and the RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription levels of genes NCgl1706, NCgll707 and NCgl2777 were detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). The internal reference gene was 16S RNA, and the primers were used in Examples 1 and 2.

[0185] As can be seen from Figure 4, compared with Corynebacterium glutamicum YP097158, the expression levels of NCgl1706, NCgll707 and NCgl2777 genes in the recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl2777 were significantly decreased, proving that the genes NCgl1706, NCgll707 and NCgl2777 were successfully knocked out.

[0186] Example 5: Construction of an engineered strain with NCgl1706-NCgll707 and NCgl1050-NCgll051 gene deletions in the genome

[0187] Based on the sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, the NCgl1706-NCgll707 genes and NCgl1050-NCgll051 genes in the genome of L-lysine-producing strain YP097158 (from Corynebacterium glutamicum ATCC13032) were knocked out using the pk18mobsacB plasmid (sequencing confirmed that the complete NCgl1706-NCgll707 genes and NCgl1050-NCgll051 genes were retained on the chromosome of the YP097158 strain) to further study the effects of these genes on L-lysine synthesis.

[0188] The knockout plasmid pK18-ΔNCgl1050-NCgll051 constructed correctly in Example 3 was electroporated into the recombinant bacteria YPL-ΔNCgl1706-NCgll707 constructed in Example 1, and cultured according to the medium components and culture conditions shown in Table 1. The single colonies produced by the culture were identified by PCR using primers P9 / P12: the strain that could simultaneously amplify bands of 1716 bp and 3391 bp was a positive strain, and the strain that only amplified the 3391 bp band was the original strain. After culturing the positive strains on 15% sucrose solid medium (shown in Table 1) for 30 hours, single clones were selected and screened on solid medium plates containing and without kanamycin. Single clones that grew on medium without kanamycin but not on medium containing kanamycin were further identified by PCR using primers P9 / P12. Strains that amplified a 1716 bp band (SEQ ID No. 13) were identified as positive strains with knockout of the NCgl1050-NCgl1051 gene coding region. Gene fragments from the positive strains were amplified again using primers P9 / P12 and sequenced. The strain with the correct sequence was named YPL-ΔNCgl1706-NCgl707-ΔNCgl1050-NCgl1051.

[0189] The recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl1050-NCgll051 is a recombinant Corynebacterium glutamicum obtained by replacing the 1881670 to 1885600 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 5, and replacing the 1140507 to 1143897 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 13, while keeping other nucleotide sequences unchanged, that is, the NCgl1706 gene coding region in the Corynebacterium glutamicum YP097158 genome (nucleotide sequence is SEQ ID No. 1, encoding SEQ ID The amino acid sequence shown in SEQ ID No.2, i.e., positions 1882467 to 1883990 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) was knocked out; the coding region of the NCgl1707 gene (the nucleotide sequence is SEQ ID No.3, encoding the amino acid sequence shown in SEQ ID No.4, i.e., positions 1884217 to 1884888 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014)) was knocked out; the coding region of the NCgl1050 gene (the nucleotide sequence is SEQ ID No.9, encoding the amino acid sequence shown in SEQ ID No.10, i.e., positions 1141341 to 1142475 of the ATCC13032 genome (GenBank: NC_03450, 22-DE C-2014)) was knocked out; the coding region of the NCgl1051 gene (the nucleotide sequence is SEQ ID No. As shown in ID No. 11, encoding the amino acid sequence shown in SEQ ID No. 12, that is, positions 1142476 to 1143015 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) were knocked out, while keeping the other nucleotides in the genome of Corynebacterium glutamicum YP097158 unchanged.

[0190] The recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl1050-NCgll051 and Corynebacterium glutamicum YP097158 were cultured for 24 h and then centrifuged at 4 ° C to collect the bacteria. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108), and the RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription levels of genes NCgl1706, NCgll707, NCgl1050 and NCgl1051 were detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). The internal reference gene was 16S RNA, and the primers were used in Examples 1 and 3.

[0191] As can be seen from Figure 5, compared with Corynebacterium glutamicum YP097158, the expression levels of NCgl1706, NCgll707, NCgl1050 and NCgl1051 genes in the recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl1050-NCgll051 were significantly decreased, proving that the genes NCgl1706, NCgll707, NCgl1050 and NCgl1051 were successfully knocked out.

[0192] Example 6: Construction of an engineered strain with NCgl2777 and NCgl1050-NCgll051 gene deletions in the genome

[0193] Based on the sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, the NCgl2777 and NCgl1050-NCgll051 genes in the genome of L-lysine-producing strain YP097158 (from Corynebacterium glutamicum ATCC13032) were knocked out using the pk18mobsacB plasmid (sequencing confirmed that the complete NCgl2777 and NCgl1050-NCgll051 genes were retained on the chromosome of the YP097158 strain). This was done to further investigate the effects of these genes on L-lysine synthesis.

[0194] The knockout plasmid pK18-ΔNCgl1050-NCgll051 constructed correctly in Example 3 was electroporated into the recombinant bacteria YPL-ΔNCgl2777 constructed in Example 2, and cultured according to the medium components and culture conditions shown in Table 1. The single colonies produced by the culture were identified by PCR using primers P9 / P12: the strain that could simultaneously amplify bands of 1716 bp and 3391 bp was a positive strain, and the strain that only amplified the 3391 bp band was the original strain. After culturing the positive strains on 15% sucrose solid medium (shown in Table 1) for 30 hours, single clones were selected and screened on solid medium plates containing and without kanamycin. Single clones that grew on the medium without kanamycin but not on the medium containing kanamycin were further identified by PCR using primers P9 / P12. Strains that amplified a 1716 bp band (shown in SEQ ID No. 13 (1716 bp)) were positive strains with knockout of the NCgl1050-NCgl1051 gene coding region. Gene fragments from the positive strains were amplified again using primers P9 / P12 and sequenced. The strain with the correct sequence was named YPL-ΔNCgl2777-ΔNCgl1050-NCgl1051.

[0195] The recombinant bacterium YPL-ΔNCgl2777-ΔNCgl1050-NCgll051 is a recombinant Corynebacterium glutamicum obtained by replacing bases 3071113 to 3074313 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 8 and bases 1140507 to 1143897 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 13, while keeping other nucleotide sequences unchanged, that is, the NCgl2777 gene coding region in the Corynebacterium glutamicum YP097158 genome (nucleotide sequence is SEQ ID No. 6, encoding the amino acid sequence shown in SEQ ID No. 7, that is, the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) is replaced by SEQ ID No. 13. The 3071901 to 3073621 positions of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) were knocked out; the NCgl1050 gene coding region (the nucleotide sequence is shown in SEQ ID No.9, encoding the amino acid sequence shown in SEQ ID No.10, i.e., positions 1141341 to 1142475 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014)) was knocked out; the NCgl1051 gene coding region (the nucleotide sequence is SEQ ID No.11, encoding the amino acid sequence shown in SEQ ID No.12, i.e., positions 1142476 to 1143015 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014)) was knocked out, and the other nucleotides in the genome of Corynebacterium glutamicum YP097158 were kept unchanged to obtain a recombinant bacterium.

[0196] The recombinant bacteria YPL-ΔNCgl2777-ΔNCgl1050-NCgll051 and Corynebacterium glutamicum YP097158 were cultured for 24 h and then centrifuged at 4 ° C to collect the bacteria. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108), and the RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription levels of genes NCgl2777, NCgl1050 and NCgll051 were detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). The internal reference gene was 16S RNA, and the primers were used in Examples 2 and 3.

[0197] As can be seen from Figure 6, compared with Corynebacterium glutamicum YP097158, the expression levels of NCgl2777, NCgl1050 and NCgl1051 genes in the recombinant bacteria YPL-ΔNCgl2777-ΔNCgl1050-NCgl1051 were significantly decreased, proving that the genes NCgl2777, NCgl1050 and NCgl1051 were successfully knocked out.

[0198] Example 7: Construction of an engineered strain with NCgl1706-NCgl1707, NCgl2777, and NCgl1050-NCgll051 gene deletions in the genome

[0199] Based on the sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, the NCgl1706-NCgll707 genes, NCgl2777 genes, and NCgl1050-NCgll051 genes in the genome of L-lysine-producing strain YP097158 (from Corynebacterium glutamicum ATCC13032) were knocked out using the pk18mobsacB plasmid (sequencing confirmed that the complete NCgl1706-NCgll707 genes, NCgl2777 genes, and NCgl1050-NCgll051 genes were retained on the chromosome of the YP097158 strain) to further study the effects of these genes on L-lysine synthesis.

[0200] The knockout plasmid pK18-ΔNCgl1050-NCgll051 constructed correctly in Example 3 was electroporated into the recombinant bacteria YPL-ΔNCgl1706-NCgll707-ΔNCgl2777 constructed in Example 4, and cultured according to the medium components and culture conditions shown in Table 1. The single colonies produced by the culture were identified by PCR using primers P9 / P12: the strain that could simultaneously amplify bands of 1716 bp and 3391 bp was a positive strain, and the strain that only amplified the 3391 bp band was the original strain. After culturing the positive strains on 15% sucrose solid medium (shown in Table 1) for 30 hours, single clones were selected and screened on solid medium plates containing and without kanamycin. Single clones that grew on the medium without kanamycin but not on the medium containing kanamycin were further identified by PCR using primers P9 / P12. Strains that amplified a 1716 bp band (SEQ ID No. 13) were identified as positive strains with knockout of the NCgl1050-NCgl1051 gene coding region. Gene fragments from the positive strains were amplified again using primers P9 / P12 and sequenced. The strain with the correct sequence was named YPL-ΔNCgl1706-NCgl1707-ΔNCgl2777-ΔNCgl1050-NCgl1051.

[0201] The recombinant strain YPL-ΔNCgl1706-NCgl1707-NCgl2777-NCgl1050-NCgl1051 replaced the 1881670 to 1885600 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 5, and replaced the 3071113 to 3074313 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. 8, and replaced the 1881670 to 1885600 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) with SEQ ID No. No.13 replaced the 1140507 to 1143897 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014), while keeping other nucleotide sequences unchanged to obtain the recombinant Corynebacterium glutamicum, i.e., the NCgl1706 gene coding region (nucleotide sequence is SEQ ID No.1, encoding the amino acid sequence shown in SEQ ID No.2, i.e., the 1882467 to 1883990 bases of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014)) in the Corynebacterium glutamicum YP097158 genome was deleted; the NCgl1707 gene coding region (nucleotide sequence is SEQ ID No.3, encoding the amino acid sequence shown in SEQ ID The amino acid sequence shown in SEQ ID No.4, i.e., positions 1884217 to 1884888 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) were knocked out; the coding region of the NCgl2777 gene (nucleotide sequence of SEQ ID No.6, encoding the amino acid sequence shown in SEQ ID No.7, i.e., positions 3071901 to 3073621 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) were knocked out; the coding region of the NCgl1050 gene (nucleotide sequence of SEQ ID No.9, encoding SEQ ID The amino acid sequence shown in No.10, i.e., positions 1141341 to 1142475 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014) was knocked out; the NCgl1051 gene coding region (nucleotide sequence as SEQ ID No.11, encoding the amino acid sequence shown in SEQ ID No.12, i.e., positions 1142476 to 1143015 of the ATCC13032 genome (GenBank: NC_03450, 22-DEC-2014)) was knocked out, and the other nucleotides in the genome of Corynebacterium glutamicum YP097158 were kept unchanged to obtain a recombinant bacterium.

[0202] The recombinant bacteria YPL-ΔNCgl1706-NCgll707-NCgl2777-NCgl1050-NCgll051 and Corynebacterium glutamicum YP097158 were cultured for 24 h and then centrifuged at 4 ° C to collect the bacteria. RNA was extracted using an RNA extraction kit (purchased from Takara, Code No.: 9108), and the RNA was reverse transcribed into cDNA using a Premix reverse transcription kit (purchased from Takara, Code No.: RR036Q). The transcription levels of genes NCgl1706, NCgll707, NCgl2777, NCgl1050 and NCgll051 were detected using a qPCR kit (purchased from Takara, Code No.: RR42LR). The internal reference gene was 16S RNA, and the primers were used in Examples 1-3.

[0203] As can be seen from Figure 7, compared with Corynebacterium glutamicum YP097158, the expression levels of NCgl1706, NCgll707, NCgl2777, NCgl1050 and NCgl1051 genes in the recombinant bacteria YPL-ΔNCgl1706-NCgll707-NCgl2777-NCgl1050-NCgll051 were significantly decreased, proving that the genes NCgl1706, NCgll707, NCgl2777, NCgl1050 and NCgl1051 were successfully knocked out.

[0204] Example 8, L-lysine fermentation experiment

[0205] The strains constructed in Examples 1-7 and the L-lysine-producing strain YP097158 were fermented in a BLBIO-5GC-4-H fermenter (Shanghai Bailun Biotechnology Co., Ltd.) using the culture medium shown in Table 2 and the control process shown in Table 3. After fermentation, the L-lysine production was detected by the ninhydrin colorimetric method. Each strain was repeated three times, and the results are shown in Table 4.

[0206] Table 2 Fermentation medium formula (the rest is water)

[0207] Table 3 Fermentation control process Note: In the table, “F12h” means fermentation for 12 hours, “F12-F32h” means fermentation for 12-32 hours, and “F32 h” means fermentation for 32 hours.

[0208] Table 4 L-lysine production and significance analysis

[0209] The results are shown in Table 4. Knocking out the NCgl1706-NCgl707 gene coding region, the NCgl2777 gene coding region, or the NCgl1050-NCgl1051 gene coding region in Corynebacterium glutamicum all contribute to the improvement of L-lysine production.

[0210] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experiments, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In short, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including changes that depart from the disclosed scope in the present application and are made using conventional techniques known in the art.

[0211] Cross-reference to related applications:

[0212] This application claims priority to a Chinese patent application filed with the Patent Office of China on November 30, 2023, with application number 202311620424.3 and invention name "Application of cell apoptosis-related proteins in the preparation of L-lysine". The entire contents of the patent application are hereby incorporated by reference into this document. Industrial Applicability

[0213] The present invention can significantly increase the production of L-lysine.

Claims

1. Application, characterized in that, The application includes the application of the protein or a substance regulating the expression of the protein encoding gene or a substance regulating the activity or content of the protein in preparing L-lysine or increasing the yield of L-lysine, wherein the protein is any one of the following: G1) includes the protein encoded by the NCgl1706 gene and the protein encoded by the NCgl1707 gene; G2) includes the protein encoded by the NCgl2777 gene; G3) includes the protein encoded by the NCgl1050 gene and the protein encoded by the NCgl1051 gene; G4) includes the protein encoded by the NCgl1706 gene, the protein encoded by the NCgl1707 gene, and the protein encoded by the NCgl2777 gene; G5) includes any two or more combinations of G1), G2), G3) or G4); Wherein, the protein encoded by the NCgl1050 gene in A1) includes any one of the following: A1-1) a protein having an amino acid sequence of SEQ ID No. 11; A1-2) a protein derived from A1-1) or having 80% or more identity with the protein shown in A1-1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 11, having the same function as the protein encoded by the NCgl1050 gene; A1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1-1) or A1-2); A2) The protein encoded by the NCgl1051 gene includes any of the following: A2-1) a protein having an amino acid sequence of SEQ ID No. 12; A2-2) a protein derived from A2-1) or having 80% or more identity with the protein shown in A2-1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 12, having the same function as the protein encoded by the NCgl1051 gene; A2-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A2-1) or A2-2); A3) The protein encoded by the NCgl2777 gene includes any of the following: A3-1) a protein having an amino acid sequence of SEQ ID No. 7; A3-2) a protein derived from A3-1) or having 80% or more identity with the protein shown in A3-1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 7, having the same function as the protein encoded by the NCgl2777 gene; A3-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A3-1) or A3-2); A4) The protein encoded by the NCgl1706 gene includes any of the following: A4-1) a protein having an amino acid sequence of SEQ ID No. 2; A4-2) a protein derived from A4-1) or having 80% or more identity with the protein shown in A4-1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 2, having the same function as the protein encoded by the NCgl1706 gene; A4-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A4-1) or A4-2); A5) The protein encoded by the NCgl1707 gene includes any of the following: A5-1) a protein having an amino acid sequence of SEQ ID No. 4; A5-2) a protein derived from A5-1) or having 80% or more identity with the protein shown in A5-1) obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 4, having the same function as the protein encoded by the NCgl1707 gene; A5-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A5-1) or A5-2).

2. The use according to claim 1, characterized in that: The G5) includes any of the following: P1) includes the protein encoded by the NCgl1706 gene, the protein encoded by the NCgl1707 gene, the protein encoded by the NCgl1050 gene, and the protein encoded by the NCgl1051 gene; P2) includes the protein encoded by the NCgl1050 gene, the protein encoded by the NCgl1051 gene, the protein encoded by the NCgl2777 gene, the protein encoded by the NCgl1706 gene, and the protein encoded by the NCgl1707 gene; P3) includes the protein encoded by the NCgl1050 gene, the protein encoded by the NCgl1051 gene and the protein encoded by the NCgl2777 gene.

3. The use according to any one of claims 1-2, characterized in that: The regulation is to inhibit, reduce or down-regulate the expression of the gene encoding the protein of claim 1 or the activity or content of the protein.

4. The use according to any one of claims 1 to 3, characterized in that: The substance includes an agent for knocking out the gene encoding the protein.

5. The use according to claim 4, characterized in that: The reagent may be any of the following: B1), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or a nucleic acid molecule that inhibits, reduces or down-regulates the activity or content of the protein; the nucleic acid molecule is a DNA molecule or an RNA molecule; B2), expressing the coding gene of the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

6. The use according to claim 5, characterized in that: B1) The DNA molecule is a nucleic acid molecule that knocks out the gene encoding the protein in the cell by homologous recombination.

7. The use according to claim 5, characterized in that: The nucleotide sequence of the NCgl1050 gene contains SEQ ID No.9, the nucleotide sequence of the NCgl1051 gene contains SEQ ID No.11, the nucleotide sequence of the NCgl2777 gene contains SEQ ID No.6, the nucleotide sequence of the NCgl1706 gene contains SEQ ID No.1, and the nucleotide sequence of the NCgl1707 gene contains SEQ ID No.

3.

8. A recombinant bacterium, characterized in that: The recombinant bacterium does not contain or lacks the protein described in claim 1.

9. The recombinant bacterium according to claim 8, characterized in that The recombinant bacteria does not contain or lacks the gene encoding the protein in claim 1.

10. The recombinant bacterium according to claim 9, characterized in that The recombinant bacteria are obtained by knocking out or silencing the coding gene of the protein in the receptor bacteria, and the receptor bacteria contain the coding gene of the protein.

11. The recombinant bacterium according to claim 10, characterized in that The recombinant bacteria include bacteria.

12. The recombinant bacterium according to claim 11, characterized in that The recombinant bacteria include Corynebacterium glutamicum.

13. Use of a biological material related to the protein of claim 1 in preparing L-lysine or increasing the yield of L-lysine, characterized in that: The biological material is any of the following: B1), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or a nucleic acid molecule that inhibits, reduces or down-regulates the activity or content of the protein; the nucleic acid molecule includes a DNA molecule or an RNA molecule; B2), expressing the coding gene of the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

14. A whole cell catalyst, characterized in that The whole-cell catalyst comprises the recombinant bacteria according to claim 12.

15. A whole cell catalyst, characterized in that The whole cell catalyst comprises the biological material described in claim 13.

16. A method for preparing L-lysine, comprising fermenting L-lysine using the recombinant bacteria according to any one of claims 8 to 12.

17. A method for preparing L-lysine, comprising fermenting the biological material according to claim 13 to prepare L-lysine.

18. A method for preparing L-lysine, comprising fermenting L-lysine using the whole cell catalyst according to claim 14.

19. A method for preparing a recombinant microorganism, characterized in that: The method comprises the following steps: down-regulating, reducing or inhibiting the expression level of a gene in a target microorganism, or / and down-regulating, reducing or inhibiting the expression level or activity of a protein encoded by the gene in the target microorganism to obtain a recombinant microorganism; the gene comprises the NCgl1706 gene, NCgll707 gene, NCgl2777 gene, NCgl1050 gene and / or NCgll051 gene in claim 6.

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