Method for improving amino acid production capacity of bacteria

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for improving the amino acid production capacity of bacteria. The invention provides any one of the following applications of an NCgl1815 gene or a homologous gene thereof, an inhibiting factor of the NCgl1815 gene or the homologous gene thereof or an encoding protein of the NCgl1815 gene or the homologous gene thereof: (1) an application in constructing a microorganism for producing lysine or a derivative thereof; (2) application in production of lysine or derivatives thereof by microbial fermentation; and (3) application in improving the yield of lysine or derivatives thereof produced by microorganisms. The reduction of the expression quantity and / or enzyme activity of encoding protein of the NCgl1815 gene or the homologous gene thereof can obviously improve the lysine yield of the strain, and can be used for constructing a lysine production strain, so that the lysine production capacity of the strain is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for improving the amino acid production capacity of bacteria. Background Art

[0002] Lysine has important application value in the fields of feed, medicine, food, chemical industry, etc., so it is of great significance to improve the ability of bacteria to produce lysine. Corynebacterium glutamicum is currently an important lysine-producing bacterium.

[0003] In order to improve the lysine production capacity, the transformation strategies for the production strains include rational transformation based on metabolic pathways, irrational mutagenesis and evolutionary screening. At present, the transformation of lysine strains is mainly focused on the central carbon metabolic pathway and the lysine terminal metabolic pathway. In order to further improve the production efficiency of lysine, it is still necessary to explore targets that are conducive to bacterial synthesis and accumulation of lysine. Summary of the invention

[0004] The present invention provides a method for improving the amino acid production capacity of bacteria.

[0005] The present invention aims to develop a strain with high lysine production. During the research and development process, it was found that knocking out the NCgl1815 gene can significantly promote the accumulation of lysine and improve the lysine production capacity of the strain.

[0006] Based on the above findings, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides any of the following uses of the NCgl1815 gene or its homologous gene, an inhibitor of the NCgl1815 gene or its homologous gene, or a protein encoded by the NCgl1815 gene or its homologous gene:

[0008] (1) Application in constructing a microorganism for producing lysine or its derivatives;

[0009] (2) Application in the production of lysine or its derivatives by microbial fermentation;

[0010] (3) Application in increasing the yield of lysine or its derivatives produced by microorganisms.

[0011] The NCgl1815 gene is a gene encoding a hypothetical protein in Corynebacterium glutamicum, and its encoded protein is a protein containing repeated LGFP (LGFP repeat-containing protein), and its function is currently unknown. The nucleotide sequence of the NCgl1815 gene is shown in SEQ ID NO.2, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.1. Based on the sequence of the NCgl1815 gene and its encoded protein, those skilled in the art can determine the homologous genes of the gene in other Corynebacterium bacteria or other bacteria other than Corynebacterium by homologous sequence retrieval.

[0012] Specifically, homologous genes of NCgl1815 refer to genes that exist in bacteria of other strains, species or genera that have similar functions to NCgl1815 and have high homology. The homologous genes of NCgl1815 preferably have a similarity of at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% to the protein encoded by the NCgl1815 gene.

[0013] In the present invention, the gene inhibitor refers to a substance that can weaken gene expression, and these substances include nucleic acids (such as sgRNA, interfering RNA, etc.), proteins (such as repressor proteins, etc.), compounds, etc.

[0014] Preferably, the use is achieved by reducing the expression level and / or enzyme activity of the protein shown in SEQ ID NO.1 or its homologous protein.

[0015] In the present invention, reducing the expression level and / or enzyme activity can be achieved by any one or more of the following methods (1) to (3):

[0016] (1) mutating the gene to reduce its expression level;

[0017] (2) using an element with lower transcriptional or translational activity to regulate the transcription or translation of the gene, thereby reducing its expression level;

[0018] (3) Mutating the gene so that the sequence of the protein it encodes is changed, thereby reducing the enzyme activity of the protein it encodes.

[0019] The reduction can be weakening or inactivation. The reduction in expression level and / or enzyme activity is at least 30%, at least 50%, at least 70%, at least 90% or completely lost compared to before modification.

[0020] In the above (1), the mutation may be to mutate the start codon of the gene to another start codon other than ATG (for example, GTG or TTG), or to delete, insert or replace one or more bases in the coding region of the gene, so that the expression level of the gene is reduced or even no longer expressed.

[0021] In the above (2), "having lower activity" means having lower activity than the original transcription or translation element of the gene.

[0022] Among them, transcription elements include promoters, enhancers, etc.; translation elements include ribosome binding sites, 5'-UTR, etc.

[0023] Preferably, the above application is achieved by inactivating the coding gene of the protein shown in SEQ ID NO. 1 or its homologous protein in the microorganism.

[0024] In the above application, the microorganism is a bacterium of the genus Corynebacterium, Escherichia or Bacillus.

[0025] Preferably, the microorganism is a bacterium of the genus Corynebacterium, more preferably Corynebacterium glutamicum.

[0026] The present invention has been verified through experiments and has shown that the lysine production of Corynebacterium glutamicum is significantly improved by inactivating the NCgl1815 gene in Corynebacterium glutamicum.

[0027] In addition, the present invention also found that the modification of the protein shown in the above SEQ ID NO.1 can cooperate with the modification of reducing the expression level and / or enzyme activity of one or more enzymes among quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase, so as to further significantly increase the lysine production and produce a superimposed unexpected effect.

[0028] Corynebacterium glutamicum is an aerobic bacterium. When the oxygen in the environment is insufficient, part of the carbon source will be metabolized to produce organic acids, such as acetic acid or lactic acid, to balance the redox state in the cell. Lactic acid metabolism and acetic acid metabolism in Corynebacterium glutamicum are both involved in multiple enzymes, and are regulated by transcription factors, and the metabolic reactions are relatively complex. Lactate dehydrogenase is mainly involved in the synthesis and catabolism of lactic acid in organisms. There are three forms of lactate dehydrogenase in Corynebacterium glutamicum, among which the quinone-dependent L-lactate dehydrogenase (lldD), encoded by NCgl2817, can catalyze L-lactic acid to produce pyruvic acid. There are also multiple enzymatic reactions involving the synthesis and metabolism of acetic acid in Corynebacterium glutamicum, among which acetate kinase (ack) is encoded by the NCgl2656 gene, catalyzing acetate to produce acetyl phosphate, and the reaction is bidirectional; phosphotransacetylase (pta) is encoded by the NCgl2657 gene, catalyzing acetyl phosphate to produce acetyl-CoA, and the reaction is also bidirectional.

[0029] Preferably, the use further comprises: reducing the expression level and / or enzyme activity of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase in the microorganism.

[0030] In some embodiments of the present invention, the use further comprises reducing the expression level and / or enzyme activity of quinone-dependent L-lactate dehydrogenase in the microorganism.

[0031] In some embodiments of the present invention, the use further comprises reducing the expression level and / or enzyme activity of quinone-dependent L-lactate dehydrogenase, acetate kinase and phosphotransacetylase in the microorganism.

[0032] Preferably, the reduction of the expression level and / or enzyme activity is achieved by inactivating the gene encoding the enzyme.

[0033] For Corynebacterium glutamicum, quinone-dependent L-lactate dehydrogenase, acetate kinase and phosphotransacetylase are encoded by NCgl2817, NCgl2656 and NCgl2657 genes, respectively, and their nucleotide sequences are shown in SEQ ID NO. 3, 4 and 5, respectively.

[0034] In a second aspect, the present invention provides a modified Corynebacterium microorganism, wherein the expression level and / or enzyme activity of the protein represented by SEQ ID NO.1 or its homologous protein in the modified Corynebacterium microorganism is reduced.

[0035] Preferably, the modified Corynebacterium microorganism further comprises the following modification: the expression level and / or enzyme activity of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase are reduced.

[0036] The reduction of expression level and / or enzyme activity described above can be achieved by any one or more of the following methods (1) to (3):

[0037] (1) mutating the gene to reduce its expression level;

[0038] (2) using an element with lower transcriptional or translational activity to regulate the transcription or translation of the gene, thereby reducing its expression level;

[0039] (3) Mutating the gene so that the sequence of the protein it encodes is changed, thereby reducing the enzyme activity of the protein it encodes.

[0040] The above reduction may be weakening or inactivation.

[0041] In the above (1), the mutation may be to mutate the start codon of the gene to another start codon other than ATG (for example, GTG or TTG), or to delete, insert or replace one or more bases in the coding region of the gene, so that the expression level of the gene is reduced or even no longer expressed.

[0042] In the above (2), "having lower activity" means having lower activity than the original transcription or translation element of the gene.

[0043] Among them, transcription elements include promoters, enhancers, etc.; translation elements include ribosome binding sites, 5'-UTR, etc.

[0044] In some embodiments of the present invention, a modified Corynebacterium microorganism is provided, wherein the gene encoding the protein shown in SEQ ID NO. 1 or a homologous protein thereof is inactivated.

[0045] Compared with the modification of only inactivating the coding gene of the protein shown in SEQ ID NO.1 or its homologous protein, the modification of inactivating the coding gene of the protein shown in SEQ ID NO.1 or its homologous protein is combined with the modification of inactivating the coding gene of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase, and the yield of lysine is further significantly improved.

[0046] In some embodiments of the present invention, a modified Corynebacterium microorganism is provided, wherein the gene encoding the protein shown in SEQ ID NO. 1 or a homologous protein thereof is inactivated, and at the same time, the gene encoding the quinone-dependent L-lactate dehydrogenase is inactivated.

[0047] In some embodiments of the present invention, a modified Corynebacterium microorganism is provided, wherein the gene encoding the protein shown in SEQ ID NO.1 or its homologous protein is inactivated, and at the same time, the genes encoding quinone-dependent L-lactate dehydrogenase, acetate kinase and phosphotransacetylase are inactivated.

[0048] In the present invention, the inactivation can be achieved by conventional techniques in the art, such as replacing, deleting or inserting one or more bases in the gene so that the gene loses its function.

[0049] In some embodiments of the present invention, inactivation of the gene is achieved by deleting its coding frame.

[0050] Preferably, the modified Corynebacterium microorganism has enhanced lysine-producing ability compared to the microorganism before modification.

[0051] The enhanced lysine productivity may be manifested as an increase in the yield and / or conversion rate of lysine.

[0052] Preferably, the Corynebacterium microorganism is Corynebacterium glutamicum.

[0053] The starting strain of the modified Corynebacterium microorganism (the microorganism before modification) can be any strain capable of synthesizing and accumulating lysine, including but not limited to wild-type Corynebacterium or modified lysine-producing bacteria.

[0054] In some embodiments of the present invention, the starting strain is wild-type Corynebacterium glutamicum ATCC13032 (model bacteria). Corynebacterium glutamicum ATCC13032 can be purchased from public channels. The genome sequence of the related strain has been made public, and the relevant information can be queried on the NCBI website (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_003450.3).

[0055] In some embodiments of the present invention, the starting strain is Corynebacterium glutamicum CGMCC No.11942, which is a lysine-producing bacterium and was deposited in the General Microbiological Center of China Culture Collection Committee on December 25, 2015, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The classification name is: Corynebacterium glutamicum, which has been disclosed in patent CN105734004B.

[0056] In some embodiments of the present invention, the starting strain is Corynebacterium glutamicum CGMCC No.13407, which is a lysine-producing bacterium and was deposited in the General Microbiological Center of China Culture Collection Committee on November 30, 2016, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named as Corynebacterium glutamicum. The strain has been disclosed in patent application CN106635944A.

[0057] The present invention is to carry out the above-mentioned transformation with Corynebacterium glutamicum (wild-type strain, lysine production bacteria through genetic engineering) of different genetic backgrounds as the starting strain, and the transformed strain obtained is carried out fermentation experiment.After verification, in these starting strains, only inactivation NCgl1815 gene or its homologous gene is inactivated, or the transformation of inactivation NCgl1815 gene or its homologous gene is combined with the transformation of the coding gene of one or more enzymes selected from L-lactate dehydrogenase, acetate kinase, phosphotransacetylase that quinone depends on, and the lysine production of strain is all significantly improved.It can be seen from this that the lifting effect of the above-mentioned transformation for the lysine production capacity of strain does not depend on other genetic transformations contained by the above-mentioned strain, and these genetic transformations are only in order to make strain have certain lysine production capacity.Therefore, the lifting effect of the above-mentioned transformation for the lysine production capacity of Corynebacterium glutamicum has universality for the starting strain that can synthesize and accumulate lysine.Thus, it will be appreciated by those skilled in the art that the above-mentioned transformation is introduced into any Corynebacterium glutamicum and can effectively promote the production of lysine.

[0058] In a third aspect, the present invention provides a method for constructing the modified Corynebacterium microorganism described above, the method comprising: modifying the Corynebacterium microorganism so that the expression level and / or enzyme activity of the protein shown in SEQ ID NO.1 or its homologous protein is reduced.

[0059] Preferably, the reduction in the expression level and / or enzyme activity of the protein shown in SEQ ID NO.1 or its homologous protein is achieved by inactivating the gene encoding the protein shown in SEQ ID NO.1 or its homologous protein.

[0060] Preferably, the construction method further comprises: modifying the Corynebacterium microorganism so that the expression amount and / or enzyme activity of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase are reduced.

[0061] In a fourth aspect, the present invention provides a method for producing lysine or its derivatives by fermentation, the method comprising the following steps:

[0062] (1) culturing the modified Corynebacterium microorganism described above to obtain a culture of the microorganism;

[0063] (2) Collecting the produced lysine or its derivatives from the culture obtained in step (1).

[0064] In a fifth aspect, the present invention provides a method for increasing the production of lysine or its derivatives in a Corynebacterium microorganism, the method comprising: modifying the Corynebacterium microorganism so that the expression level of the NCgl1815 gene or its homologous gene and / or the enzyme activity of its encoded protein is reduced.

[0065] Preferably, the method further comprises: modifying the Corynebacterium microorganism so that the expression amount and / or enzyme activity of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase are reduced.

[0066] The beneficial effects of the present invention include at least: the present invention significantly promotes the synthesis and accumulation of lysine by inactivating the NCgl1815 gene or its homologous gene in Corynebacterium, effectively improves the lysine production capacity of Corynebacterium, and significantly improves the lysine yield of Corynebacterium. The above transformation can be used for the construction of lysine production strains, effectively improves the lysine production capacity of strains, and is of great significance for improving the production efficiency of lysine and reducing production costs. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] In the following examples, the amino acid sequence of the protein encoded by the NCgl1815 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the NCgl1815 gene is shown as SEQ ID NO.2.

[0069] The NCgl2817 gene encodes quinone-dependent L-lactate dehydrogenase, and its nucleotide sequence is shown in SEQ ID NO. 3. The NCgl2656 gene encodes acetate kinase, and its nucleotide sequence is shown in SEQ ID NO. 4. The NCgl2657 gene encodes phosphotransacetylase, and its nucleotide sequence is shown in SEQ ID NO. 5.

[0070] The primer sequence information used in the following examples is shown in Table 1.

[0071] Table 1 Primer sequence information

[0072]

[0073]

[0074] If no specific techniques or conditions are specified in the following examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0075] Example 1: Inactivation of the NCgl1815 gene of Corynebacterium glutamicum ATCC 13032 strain

[0076] There are many options for gene inactivation, such as deletion of the coding region, deletion of all or part of the coding region, or introduction of nonsense or missense mutations in the coding region. The above operations may lead to transcription failure or translation failure, and ultimately fail to produce functional proteins.

[0077] In this example, the NCgl1815 gene of ATCC 13032 is inactivated by deleting the entire coding region. The gene inactivation method can adopt the classic genetic manipulation method in Corynebacterium, that is, the secondary exchange recombination strategy based on the pK18mobsacB suicide plasmid. The specific operation method is as follows:

[0078] Step 1: Using the ATCC 13032 genome as a template, PCR amplification was performed with the primer pairs 1815-1f / 1815-1r and 1815-2f / 1815-2r, respectively, to obtain the upstream homology arm and downstream homology arm of the NCgl1815 gene deletion modification. Afterwards, a mixture of the upstream homology arm and the downstream homology arm was used as a template, and amplification was performed with the primer pair 1815-1f / 1815-2r to obtain the full-length fragment after the fusion of the upstream and downstream homology arms. The full-length fragment was double-digested with XbaI and HindIII, and the product was recovered using a gel recovery kit.

[0079] Step 2: The plasmid vector was pK18mobsacB (GenBank: FJ437239.1), which was double-digested with the same restriction endonuclease and subjected to gel recovery.

[0080] Step 3: The products obtained in steps 1 and 2 above were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells. The target transformants were screened on 50 mg / L kanamycin resistance plates, and the plasmids were extracted and sequenced for the obtained transformants. The correctly sequenced plasmid was named pK18mobsacB-ΔNCgl1815.

[0081] Step 4: Prepare competent cells of ATCC 13032 and perform gene recombination according to the method in the C. glutamicum Handbook (Charpter 23).

[0082] The recombinant plasmid pK18mobsacB-ΔNCgl1815 was transformed into ATCC 13032 competent cells by electroporation, and transformants were selected on BHI selection medium containing 15 mg / L kanamycin. The selected transformants were cultured overnight in ordinary BHI liquid medium at 33°C and shaken at 220 rpm on a rotary shaker. During this culture process, the transformants underwent a second recombination, the vector sequence was removed from the genome by gene exchange, and the target deletion mutation was introduced at the same time. The culture was diluted continuously (10 -2 Serial dilution up to 10 -4 ), the dilution was spread on a common BHI solid medium containing 10% sucrose and cultured at 33°C for 48 hours. The grown transformants should carry the target deletion mutation and not carry the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing confirmed that the NCgl1815 gene was successfully deleted. The target transformed strain finally obtained was named 13032-ΔNCgl1815.

[0083] Example 2: Inactivation of the NCgl2817 gene of the 13032-ΔNCgl1815 strain

[0084] There are many options for gene inactivation, such as deletion of the coding region, deletion of all or part of the coding region, or introduction of nonsense or missense mutations in the coding region. The above operations may lead to transcription failure or translation failure, and ultimately fail to produce functional proteins.

[0085] In this example, the NCgl2817 gene of 13032-ΔNCgl1815 was inactivated by deleting the entire coding region. The gene inactivation method can adopt the classic genetic manipulation method in Corynebacterium, that is, the secondary exchange recombination strategy based on the pK18mobsacB suicide plasmid. The specific operation method is as follows:

[0086] Step 1: Using the ATCC 13032 genome as a template, PCR amplification was performed with the 2817-1f / 2817-1r primer pair and the 2817-2f / 2817-2r primer pair to obtain the upstream homology arm and downstream homology arm of the NCgl2817 gene deletion modification. Afterwards, a mixture of the upstream homology arm and the downstream homology arm was used as a template and amplified with the 2817-1f / 2817-2r primer pair to obtain the full-length fragment after the fusion of the upstream and downstream homology arms. The full-length fragment was double-digested with XbaI and HindIII, and the product was recovered using a gel recovery kit.

[0087] Step 2: The plasmid vector was pK18mobsacB (GenBank: FJ437239.1), which was double-digested with the same restriction endonuclease and subjected to gel recovery.

[0088] Step 3: The products obtained in steps 1 and 2 above were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells. The target transformants were screened on 50 mg / L kanamycin resistance plates, and the plasmids were extracted and sequenced for the obtained transformants. The correctly sequenced plasmid was named pK18mobsacB-ΔNCgl2817.

[0089] Step 4: Prepare competent cells of 13032-ΔNCgl1815 and perform gene recombination according to the method in the C. glutamicum Handbook (Charpter 23).

[0090] The recombinant plasmid pK18mobsacB-ΔNCgl2817 was transformed into 13032-ΔNCgl1815 competent cells by electroporation, and the transformants were selected on BHI selection medium containing 15 mg / L kanamycin. The selected transformants were cultured overnight in ordinary BHI liquid medium at 33°C and shaken at 220 rpm on a rotary shaker. During this culture process, the transformants underwent a second recombination, the vector sequence was removed from the genome by gene exchange, and the target deletion mutation was introduced at the same time. The culture was diluted continuously (10 -2 Serial dilution up to 10 -4 ), the dilution was spread on a common BHI solid medium containing 10% sucrose and cultured at 33°C for 48 hours. The grown transformants should carry the target deletion mutation and not carry the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing confirmed that the NCgl2817 gene was successfully deleted. The target transformed strain finally obtained was named 13032-ΔNCgl1815ΔNCgl2817.

[0091] Example 3: Inactivation of NCgl2656 and NCgl2657 genes of 13032-ΔNCgl1815ΔNCgl2817 strain

[0092] NCgl2656 and NCgl2657 encode acetate kinase and phosphotransacetylase, respectively. NCgl2656 and NCgl2657 are adjacent to each other on the ATCC 13032 genome and form an operon. Considering the relevance of their functions, these two genes were inactivated together. There are many options for inactivation, such as deletion of the coding region, deletion of the entire coding region or deletion of part of the coding region, or introduction of nonsense mutations or missense mutations in the coding region. The above operations may lead to transcription failure or translation failure, and ultimately fail to produce functional proteins.

[0093] In this example, the deletion of all coding regions is used as an example to inactivate the NCgl2656 and NCgl2657 genes of the 13032-ΔNCgl1815ΔNCgl2817 strain. The gene inactivation method can adopt the classic genetic manipulation method in Corynebacterium, that is, the secondary exchange recombination strategy based on the pK18mobsacB suicide plasmid. The specific operation method is as follows:

[0094] Step 1: Using the ATCC 13032 genome as a template, PCR amplification was performed with the primer pairs 2657-1f / 2657-1r and 2656-2f / 2656-2r, respectively, to obtain the upstream homology arms and downstream homology arms of the NCgl2656 and NCgl2657 gene deletion modifications. Afterwards, a mixture of the upstream homology arms and the downstream homology arms was used as a template, and amplification was performed with the primer pair 2657-1f / 2656-2r to obtain the full-length fragment after the fusion of the upstream and downstream homology arms. The full-length fragment was double-digested with XbaI and HindIII, and the product was recovered using a gel recovery kit.

[0095] Step 2: The plasmid vector was pK18mobsacB (GenBank: FJ437239.1), which was double-digested with the same restriction endonuclease and subjected to gel recovery.

[0096] Step 3: The products obtained in steps 1 and 2 above were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells. The target transformants were screened on 50 mg / L kanamycin resistance plates, and the plasmids were extracted and sequenced for the obtained transformants. The correctly sequenced plasmid was named pK18mobsacB-ΔNCgl2657-2656.

[0097] Step 4: Prepare competent cells of 13032-ΔNCgl1815ΔNCgl2817 and perform gene recombination according to the method in the C. glutamicum Handbook (Charpter 23).

[0098] The recombinant plasmid pK18mobsacB-ΔNCgl2657-2656 was transformed into 13032-ΔNCgl1815ΔNCgl2817 competent cells by electroporation, and the transformants were selected on BHI selection medium containing 15 mg / L kanamycin. The selected transformants were cultured overnight in ordinary BHI liquid medium at 33°C and shaken at 220 rpm on a rotary shaker. During the culture process, the transformants underwent a second recombination, the vector sequence was removed from the genome by gene exchange, and the target deletion mutation was introduced at the same time. The culture was diluted continuously (10 -2 Serial dilution up to 10 -4 ), the dilution was spread on a common BHI solid medium containing 10% sucrose and cultured at 33°C for 48h. The grown transformants should carry the target deletion mutation and not carry the inserted vector sequence. The target sequence was amplified by PCR, and nucleotide sequencing confirmed that the NCgl2657 and NCgl2656 genes were successfully deleted. The target transformed strain finally obtained was named 13032-ΔNCgl1815ΔNCgl2817ΔNCgl2657-2656.

[0099] Example 4: Inactivation of the NCgl1815 gene of the CGMCC No.11942 strain

[0100] CGMCC No.11942 is a high-lysine-producing strain derived from ATCC 13032 through genetic engineering. The tool plasmid used in ATCC 13032 can also be used in CGMCC No.11942. Therefore, the method for inactivating the NCgl1815 gene of the CGMCC No.11942 strain was referred to the method of Example 1, and the finally obtained modified strain was named 11942-ΔNCgl1815.

[0101] Example 5: Inactivation of the NCgl2817 gene of the 11942-ΔNCgl1815 strain

[0102] The transformation method was similar to that in Example 2, and the transformed strain finally obtained was named 11942-ΔNCgl1815ΔNCgl2817.

[0103] Example 6: Inactivation of NCgl2656 and NCgl2657 genes in strain 11942-NCgl1815ΔNCgl2817

[0104] The transformation method was referred to Example 3, and the finally obtained transformed strain was named 11942-ΔNCgl1815ΔNCgl2817ΔNCgl2657-2656.

[0105] Example 7: Inactivation of the NCgl1815 gene of CGMCC No.13407 strain

[0106] CGMCC No.13407 is a high-lysine-producing strain derived from ATCC 13032 through genetic engineering. The tool plasmid used in ATCC 13032 can also be used in CGMCC No.13407. Therefore, the NCgl1815 gene of CGMCC No.13407 was inactivated according to the method of Example 1, and the resulting modified strain was named 13407-ΔNCgl1815.

[0107] Example 8: Inactivation of the NCgl2817 gene of the 13407-ΔNCgl1815 strain

[0108] The transformation method was similar to that in Example 2, and the transformed strain finally obtained was named 13407-ΔNCgl1815ΔNCgl2817.

[0109] Example 9: Inactivation of NCgl2656 and NCgl2657 genes in strain 13407-ΔNCgl1815ΔNCgl2817

[0110] The transformation method was similar to that in Example 3, and the transformed strain finally obtained was named 13407-ΔNCgl1815ΔNCgl2817ΔNCgl2657-2656.

[0111] Example 10: Lysine production capacity test of modified strains

[0112] The 9 modified strains obtained in Examples 1 to 9 were subjected to fermentation tests to verify whether their amino acid production capacity was enhanced. The corresponding starting strains of the same batch were used as controls.

[0113] The culture medium used in the fermentation experiments was as follows:

[0114] 1. Plate activation medium: BHI 37g / L, agar powder 18g / L.

[0115] 2. Seed culture medium: sucrose 20g / L, yeast powder 5g / L, peptone 10g / L, urea 5g / L, magnesium sulfate heptahydrate 0.4g / L, adjust pH to 7.0.

[0116] 3. Fermentation medium: glucose 60g / L, ammonium sulfate 25g / L, potassium dihydrogen phosphate 2.0g / L, magnesium sulfate heptahydrate 1.0g / L, soybean meal hydrolyzate 10g / L, calcium carbonate 30g / L, adjust pH to 7.0.

[0117] The fermentation method is as follows:

[0118] 1. Seed activation: Take the strain to be verified from the cryopreserved tube, streak it on the seed activation medium, and culture it at 33℃ for 24h;

[0119] 2. Seed culture: Pick the activated seeds 1 from the plate and transfer them to a 500mL Erlenmeyer flask containing 30mL of seed culture medium. Shake and culture at 33℃ and 220r / min for 6h to obtain seed solution.

[0120] 3. Fermentation culture: inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture at 33°C and 220 rpm for 14-15 h. Make 3 parallel cultures for each strain.

[0121] 4.OD 562 Determination: The fermentation broth was diluted 100 times with 0.01M hydrochloric acid, and the absorbance was detected at a wavelength of 562nm using a spectrophotometer. Three parallel tests were performed for each strain, and the average value was calculated. The test results are shown in Table 2, and the data in the table are the average values ​​of the three parallel tests.

[0122] 5. Determination of amino acid concentration: 2 mL of fermentation broth was centrifuged (12000 rpm, 2 min), the supernatant was collected, and the test was performed using Agilent high performance liquid chromatography (HPLC). The test bacteria and the control bacteria were each tested in parallel for 3 times, and the average value was calculated. The test results are shown in Table 2.

[0123] Table 2 Acid production phenotype test of modified strains

[0124]

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Any of the following uses of NCgl1815 gene or its homologous gene, inhibitor of NCgl1815 gene or its homologous gene, or protein encoded by NCgl1815 gene or its homologous gene: (1) Application in constructing a microorganism for producing lysine or its derivatives; (2) Application in the production of lysine or its derivatives by microbial fermentation; (3) Application in increasing the yield of lysine or its derivatives produced by microorganisms.

2. The use according to claim 1, characterized in that: The application is achieved by reducing the expression level and / or enzyme activity of the protein shown in SEQ ID NO.1 or its homologous protein.

3. The use according to claim 1 or 2, characterized in that: The microorganism is a bacterium of the genus Corynebacterium, Escherichia or Bacillus.

4. The use according to any one of claims 1 to 3, characterized in that: The application also includes: reducing the expression amount and / or enzyme activity of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase in the microorganism.

5. A modified Corynebacterium microorganism, characterized in that The expression level and / or enzyme activity of the protein shown in SEQ ID NO.1 or its homologous protein in the modified Corynebacterium microorganism is reduced.

6. The modified Corynebacterium microorganism according to claim 5, characterized in that In the modified Corynebacterium microorganism, the expression level and / or enzyme activity of one or more enzymes selected from quinone-dependent L-lactate dehydrogenase, acetate kinase, and phosphotransacetylase are reduced.

7. The modified Corynebacterium microorganism according to claim 5 or 6, characterized in that The modified Corynebacterium microorganism has enhanced lysine-producing ability compared to the microorganism before modification.

8. The method for constructing a modified Corynebacterium microorganism according to any one of claims 5 to 7, characterized in that: The method comprises: modifying a microorganism of the genus Corynebacterium so that the expression amount and / or enzyme activity of the protein shown in SEQ ID NO.1 or a homologous protein thereof is reduced.

9. The method according to claim 8, characterized in that The reduction of the expression level and / or enzyme activity of the protein shown in SEQ ID NO.1 or its homologous protein is achieved by inactivating the coding gene thereof.

10. A method for producing lysine or its derivatives by fermentation, characterized in that: The method comprises the following steps: (1) culturing the modified Corynebacterium microorganism according to any one of claims 5 to 7 to obtain a culture of the microorganism; (2) Collecting the produced lysine or its derivatives from the culture obtained in step (1).

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