Lysine efflux protein and use thereof

Abstract

The application discloses a lysine efflux protein and application thereof. The lysine efflux protein provided by the application is an arginine efflux protein mutant, specifically, a mutant obtained by point mutation of an ArgO arginine efflux protein, and the mutation site contains part or all of the following positions: the 3rd position, the 28th position, the 58th position, the 75th position, the 78th position, the 105th position, the 116th position, the 121st position, the 145th position, the 168th position, the 188th position, the 196th position and the 205th position from the N terminal. The application obtains the arginine efflux protein mutant with improved lysine efflux capacity through a method of directed evolution. Compared with the wild type which almost does not efflux lysine, the mutant has obviously improved lysine efflux capacity. The arginine efflux protein mutant obtained by the application is more conducive to improving the yield of lysine, and lays a foundation for obtaining a new lysine efflux protein.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a lysine efflux protein and its applications. Background Technology

[0002] The history of Corynebacterium glutamicum dates back to the 1950s, when Japanese scientists, through extensive strain screening, obtained the first strain capable of producing lysine and applied it to large-scale production. Subsequently, scientists have continuously studied this bacterium to improve lysine production efficiency. L-lysine, as a basic essential amino acid, is present in very low amounts in food and is easily destroyed during processing; therefore, it is often referred to as the first limiting amino acid. The global annual production of L-lysine is approximately 3 million tons, and market demand continues to increase, making it a product with promising prospects in the international market. Therefore, research on L-lysine production is essential.

[0003] In recent years, amino acid transport systems have received increasing attention. Microbial amino acid transport systems directly participate in amino acid uptake and efflux, playing a crucial role in cellular physiological metabolism and the efficient biosynthesis and transport of amino acids. Three main types of known lysine efflux proteins are known: YbjE from *Escherichia coli*, LysE from *Corynebacterium glutamicum*, and the newly discovered MGLE from *E. coli*. Developing novel lysine transport proteins is of great significance for constructing new lysine fermentation strains. ArgO's natural function is as an efflux protein for arginine; it almost never effluxes lysine. ArgO consists of 211 amino acids and has six transmembrane α-helical segments. Currently, there is limited research and molecular modification of this efflux protein, and no reports have been found on improving its lysine efflux capacity through molecular modification. Therefore, obtaining an efflux protein with strong lysine efflux capacity through molecular modification is of great significance for creating new lysine fermentation strains. Summary of the Invention

[0004] The purpose of this invention is to provide an ArgO mutant with significant lysine efflux capacity, its encoding gene, and related applications.

[0005] In a first aspect, the present invention claims protection for an arginine efflux protein mutant.

[0006] The arginine efflux protein mutant claimed in this invention is obtained by point mutation of ArgO arginine efflux protein, and the mutation site contains (or is) some or all of the following: positions 3, 28, 58, 75, 78, 105, 116, 121, 145, 168, 188, 196 and 205 from the N-terminus.

[0007] Preferably, the amino acid sequence of the arginine efflux protein mutant has more than 95% identity with the sequence containing only the above-mentioned mutation sites.

[0008] The percentage of 95% or more is further preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0009] The arginine efflux protein mutant has the function of effluxing lysine. Preferably, compared with the ArgO arginine efflux protein, the arginine efflux protein mutant has a stronger ability to efflux lysine.

[0010] Furthermore, the arginine efflux protein mutant can be any of the following:

[0011] (A1) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 188th position from the N-terminus (corresponding to mutant M6).

[0012] (A2) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 3rd position from the N-terminus (corresponding to mutant M1);

[0013] (A3) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 105th position from the N-terminus (corresponding to mutant M2).

[0014] (A4) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 121st position from the N-terminus (corresponding to mutant M3).

[0015] (A5) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 168th position from the N-terminus (corresponding to mutant M4).

[0016] (A6) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 28th and 196th positions from the N-terminus (corresponding to mutant M5).

[0017] (A7) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 58th, 75th, 78th and 116th positions from the N-terminus (corresponding to mutant M7).

[0018] (A8) The arginine efflux protein mutant is a protein obtained by point mutation of at least the following sites (or the following sites) of the ArgO arginine efflux protein: the 145th and 205th positions from the N-terminus (corresponding to mutant M8).

[0019] In the arginine efflux protein mutant, the point mutations at the 3rd position from the N-terminus are specifically S3P, at the 28th position is specifically N28D, at the 58th position is specifically G58R, at the 75th position is specifically G75V, at the 78th position is specifically A78V, at the 105th position is specifically K105R, at the 116th position is specifically L116 stop codon, at the 121st position is specifically L121P, at the 145th position is specifically K145R, at the 168th position is specifically A168T, at the 188th position is specifically G188 stop codon, at the 196th position is specifically L196S, and at the 205th position is specifically H205Y.

[0020] For amino acid substitutions, the following nomenclature is used: original amino acid (wild type), position (i.e., position in SEQ ID No. 1), substituted amino acid (including the stop codon). Accordingly, mutants with mutations at different sites in SEQ ID No. 1 are named sequentially.

[0021] In a specific embodiment of the present invention, the arginine efflux protein mutant is any one of the following:

[0022] (a1) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: G188 stop codon (corresponding to mutant M6, the amino acid sequence is shown in SEQ ID No.7).

[0023] That is, only amino acid residues 1-187 of the ArgO arginine efflux protein shown in SEQ ID No.1 are expressed to obtain the arginine efflux protein mutant (M6).

[0024] (a2) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: S3P (corresponding to mutant M1, the amino acid sequence is shown in SEQ ID No.2).

[0025] (a3) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: K105R (corresponding to mutant M2, the amino acid sequence is shown in SEQ ID No.3).

[0026] (a4) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: L121P (corresponding to mutant M3, the amino acid sequence is shown in SEQ ID No.4).

[0027] (a5) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: A168T (corresponding to mutant M4, the amino acid sequence is shown in SEQ ID No.5).

[0028] (a6) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: N28D, L196S (corresponding to mutant M5, the amino acid sequence is shown in SEQ ID No.6).

[0029] (a7) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: G58R, G75V, A78V, L116 stop codon (corresponding to mutant M7, the amino acid sequence is shown in SEQ ID No.8).

[0030] The arginine efflux protein mutant (M7) was obtained by site-directed mutations of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 into G58R, G75V, and A78V, and expressing only amino acid residues 1-115.

[0031] (a8) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: K145R, H205Y (corresponding to mutant M8, the amino acid sequence is shown in SEQ ID No.9).

[0032] Secondly, the present invention claims protection for biological materials related to arginine efflux protein mutants.

[0033] The biomaterials related to arginine efflux protein mutants claimed in this invention may be any of the following:

[0034] (I) The nucleic acid molecule encoding the arginine efflux protein mutant;

[0035] (II) Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.

[0036] The nucleic acid molecule encoding the ArgO arginine efflux protein from Escherichia coli K12 is the DNA molecule shown in SEQ ID No. 10.

[0037] Furthermore, the nucleic acid molecule encoding the arginine efflux protein mutant is specifically any of the following:

[0038] (B1) DNA molecule shown in SEQ ID No. 16 or positions 1-564 of SEQ ID No. 16 (corresponding to mutant M6);

[0039] (B2) DNA molecule shown in SEQ ID No. 11 (corresponding to mutant M1);

[0040] (B3) DNA molecule shown in SEQ ID No. 12 (corresponding to mutant M2);

[0041] (B4) DNA molecule shown in SEQ ID No. 13 (corresponding to mutant M3);

[0042] (B5) DNA molecule shown in SEQ ID No. 14 (corresponding to mutant M4);

[0043] (B6) DNA molecule shown in SEQ ID No. 15 (corresponding to mutant M5);

[0044] (B7) DNA molecule shown in SEQ ID No. 17 or positions 1-348 of SEQ ID No. 17 (corresponding to mutant M7);

[0045] (B8) DNA molecule shown in SEQ ID No. 18 (corresponding to mutant M8).

[0046] In SEQ ID No. 16, bits 562-564 are the stop codon TGA. In SEQ ID No. 17, bits 346-348 are the stop codon TAG.

[0047] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by cloning a nucleic acid molecule encoding the arginine efflux protein mutant into the multiple cloning sites (such as SacI and BamHI) of the pTRCmob vector.

[0048] The recombinant bacteria may be Corynebacterium glutamicum containing the nucleic acid molecule.

[0049] Thirdly, the present invention claims protection for the use of the arginine efflux protein mutant or biological material described above in any of the following:

[0050] (C1) produces lysine;

[0051] (C2) Increases lysine production;

[0052] (C3) Enhances lysine efflux capacity;

[0053] (C4) Preparation of feed additives and / or food fortifiers and / or cosmetic additives;

[0054] (C5) as a nutrient and / or therapeutic agent.

[0055] Fourthly, the present invention claims a method for producing lysine and / or increasing lysine yield and / or increasing lysine efflux capacity.

[0056] The method for producing lysine and / or increasing lysine yield and / or increasing lysine efflux capacity claimed in this invention may include the following steps: expressing the arginine efflux protein mutant described above in a recipient bacterium to obtain a recombinant bacterium; and fermenting the recombinant bacterium to obtain lysine from the fermentation broth.

[0057] Furthermore, the expression of the arginine efflux protein mutant in the recipient bacteria can be achieved by introducing the aforementioned "nucleic acid molecule encoding the arginine efflux protein mutant" into the recipient bacteria.

[0058] Furthermore, the nucleic acid molecule encoding the arginine efflux protein mutant can be introduced into the recipient bacteria in the form of a recombinant vector.

[0059] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by cloning the "nucleic acid molecule encoding the arginine efflux protein mutant" into the multiple cloning sites (such as SacI and BamHI) of the pTRCmob vector.

[0060] Furthermore, the recipient bacterium is Corynebacterium glutamicum.

[0061] Furthermore, the *Corynebacterium glutamicum* can be a recombinant bacterium obtained by mutating position 311 of the lysC gene protein encoded by wild-type *Corynebacterium glutamicum* (e.g., ATCC13032) from T to I, position 59 of the hom gene protein encoded by V to A, position 458 of the pyc gene protein encoded by P to S, and knocking out the LysE gene. That is, the genotype of the *Corynebacterium glutamicum* is ATCC13032 lysC. T311I hom V59A pyc P458S △LysE.

[0062] In the method, the culture medium for fermentation can be CGXII medium, and the culture conditions can be 30°C, 220 rpm for 38 h.

[0063] In a specific embodiment of the present invention, the recombinant bacteria are inoculated into CGXII seed culture medium and cultured at 30°C and 220 rpm for 12 h, and then the initial OD is used. 600 The inoculum was 0.15g in CGXII fermentation medium and cultured at 30℃ and 220rpm for 38h.

[0064] In this invention, the lysine is specifically L-lysine.

[0065] In this invention, the improvement in lysine production and the improvement in lysine efflux capacity refer to the increased lysine production and lysine efflux capacity of the mutant (i.e., the arginine efflux protein mutant mentioned above) compared to the wild type (i.e., the ArgO arginine efflux protein).

[0066] This invention utilizes directed evolution to obtain arginine efflux protein mutants that enhance lysine efflux. Compared to the wild type, which exhibits almost no lysine efflux, the mutants show a significantly increased ability to efflux lysine. The arginine efflux protein mutants obtained in this invention are more conducive to increasing lysine production, laying the foundation for obtaining new efflux proteins that efflux lysine. Attached Figure Description

[0067] Figure 1 This is a comparison of lysine production between the efflux protein mutant and the wild-type lysine produced in 25 mL of CGXII fermentation medium. The control group labeled ArgO represents the wild-type control. The vertical axis, "lysine g / L," indicates the amount of L-lysine produced per L of fermentation broth. ** indicates a significant difference between the corresponding mutant and the wild-type, and *** indicates an extremely significant difference.

[0068] Figure 2 The effect of canavanine on the growth of Corynebacterium glutamicum expressing arginine efflux protein and its efflux mutant is shown in the figure. Canavanine (a structural analog of arginine that affects normal cell growth) can be effluxed extracellularly by arginine efflux protein, reducing its toxicity to cells. Therefore, the efflux capacity of arginine efflux protein to remove canavanine can be detected by measuring cell viability. Detailed Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] pTRCmob vector: provided by Professor Zheng Ping's research group at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and described in the article "Yu Wang, et al. A Novel Corynebacterium glutamicum L-GlutamateExporter. Applied and Environmental Microbiology, Vol.84, No.6". It is available to the public from the applicant and can only be used to repeat the experiments of this invention and may not be used for other purposes.

[0072] Example 1: Gene Cloning of Arginine Efflux Protein

[0073] The ArgO arginine efflux protein target gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and the gene sequence is shown in SEQ ID No. 10. It was then ligated between the SacI and BamHI restriction sites of the pTRCmob vector. Sequencing confirmed the successful construction of the vector and the recombinant plasmid was obtained and named pTRCmob-ArgO.

[0074] The structure of pTRCmob-ArgO is described as follows: a recombinant plasmid obtained by cloning the DNA fragment shown in SEQ ID No. 10 into the SacI and BamHI restriction sites of the pTRCmob vector. SEQ ID No. 10 is the target gene for the wild-type ArgO arginine efflux protein, encoding the wild-type ArgO arginine efflux protein shown in SEQ ID No. 1.

[0075] Example 2: Screening of arginine efflux protein mutants with enhanced lysine efflux capacity

[0076] To enhance the ability of the aforementioned arginine efflux protein to efflux lysine, primers were designed using the recombinant plasmid pTRCmob-ArgO constructed in Example 1 as a template (upstream primer: 5′-ACCATGGAATTCGAGCTCATG-3′; downstream primer: 5′-GGTCGACTCTAGAGGATCCCTA-3′), and Mn was added. 2+ The target gene was randomly mutated using PCR (this random mutation occurred only between positions 4-633 of the ArgO gene, and not at other positions in the vector). The mutated product was electroporated into Corynebacterium glutamicum ATCC13032lysC. T311I hom V59A pyc P458S After obtaining recombinant bacteria by overnight culture at 30°C in LysE competent cells, the resulting single colonies were inoculated into 96-well plates (containing 150 μL LBG medium and 25 mg / mL Kana) using sterile toothpicks. After culturing at 30°C and 800 rpm for 38 h on a shaker, lysine production was detected using a biosensor. Analysis of the results led to the selection of eight mutant strains with increased lysine efflux, named M1, M2, M3, M4, M5, M6, M7, and M8.

[0077] Among them, the Corynebacterium glutamicum ATCC13032 lysC T311I hom V59A pyc P458S △LysE was constructed according to the following steps: in Corynebacterium glutamicum ATCC13032 lysC T311I hom V59A pyc P458SThe strain (which is "AHP-3" in the article "J. Ohnishi·S, et al. A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl Microbiol Biotechnol (2002) 58:217–223", is a recombinant strain obtained by mutating position 311 of the lysC gene encoding protein in the ATCC13032 genome from T to I, mutating position 59 of the hom gene encoding protein from V to A, and mutating position 458 of the pyc gene encoding protein from P to S) was obtained by knocking out the LysE gene according to the method in the article "Tan Yanzhen. Construction of Corynebacterium glutamicum gene knockout system. Jiangnan University, Master's Thesis, 2012". First, the knockout plasmid pK18mobsacB-LRLysE was constructed: using the genome of Corynebacterium glutamicum ATCC13032 as a template, and using LysE-LF(5′-ATCCCGCCACGGGATTAGCTTCA-3′) / LysE-LR(5′-CGTGACCTATGGAAGTACTTAAGTAAAATGATTGG-3′) as primers, the upstream homologous fragment (left homologous arm) L of the LysE gene was amplified. Using LysE-RF(5′-TTTTCGCGGGTTTTGGAATCGGTGGC-3′) / LysE-RR(5′-GCTGCCCGCTTCTGATTCATCAGC-3′) as primers, the downstream homologous fragment (right homologous arm) R of the LysE gene was amplified. After purification, the upstream and downstream homologous fragments were subjected to overlap PCR at a molar ratio of 1:1 to obtain the overlap fragment LR. Using plasmid pK18mobsacB as a template and primers pK18mob-sacB plasmid backbone-F (5′-TGATGAATCAGAAGCGGGCAGCGCACTGGCCGTCGTTTTACAA-3′) / pK18mob-sacB plasmid backbone-R (5′-GAAGCTAATCCCGTGGCGGGATCATGTCATAGCTGTTTCCTGTG-3′), a pK18mob-sacB plasmid backbone with homologous fragments to the overlapping fragment LR was amplified. The overlapping fragment was ligated to the pK18mob-sacB plasmid backbone using homologous recombination; after confirmation by sequencing, the knockout plasmid pK18mobsacB-LRLysE was obtained. Then, plasmid pK18mobsacB-LRLysE was electroporated into Corynebacterium glutamicum ATCC13032 lysC.T311I hom V59A pyc P458S The culture medium was plated onto a solid medium containing kanamycin and incubated at 30°C for 36 hours. Single colonies grew on the plates. Using the single colonies as templates and LysE-LF(5′-ATCCCGCCACGGGATTAGCTTCA-3′) / LysE-RR(5′-GCTGCCCGCTTCTGATTCATCAGC-3′) as primers, colony PCR amplification of upstream and downstream homologous fragments L and R confirmed the successful first round of exchange. Successfully validated single clones were transferred to TSB medium (formula: 5 g / L glucose, 5 g / L yeast extract, 9 g / L soybean peptone, 20 g / L MOPS, 3 g / L urea, 0.5 g / L succinic acid, 0.1 g / L magnesium sulfate heptahydrate, 1 g / L dipotassium hydrogen phosphate trihydrate, 10 μg / L biotin, 0.1 mg / L LVB1) and cultured overnight at 30°C and 220 rpm. Then, 3% inoculum was transferred to TSB medium containing 10% sucrose and cultured for 10 h at 30°C and 220 rpm. After culture, the inoculum was diluted 10 times. -3 10 -4 Spread the culture onto TSB medium containing 10% sucrose; after single colonies grow, colony PCR is performed using LysE-LF(5′-ATCCCGCCACGGGATTAGCTTCA-3′) / LysE-RR(5′-GCTGCCCGCTTCTGATTCATCAGC-3′) as primers. Bacteria with correct bands are transferred and sequenced; those with correct sequencing are identified as the knockout strain *Corynebacterium glutamicum* ATCC13032lysC. T311I hom V59A pyc P458S △lysE.

[0078] Gene sequencing was performed on the above mutant strains. The specific mutated amino acid sites, as well as the specific amino acid sequences and gene sequences after mutation, are detailed in Table 1.

[0079] Table 1. Mutated amino acid sites

[0080]

[0081] Note: The amino acid substitutions in the table are named as follows: original amino acid (wild type), position (i.e., position in SEQ ID No. 1), substituted amino acid.

[0082] Example 3: Expression of arginine efflux protein and its efflux mutant in Corynebacterium glutamicum

[0083] Positive bacteria from the wild type and the eight mutants shown in Table 1 obtained in Example 2 were scraped off with an inoculation loop and inoculated into 10 mL of CGXII seed culture medium. The culture was then incubated at 30°C and 220 rpm in a constant temperature shaker (Shanghai Zhichu Instrument Co., Ltd., Shanghai, China) for 12 h, and then incubated with the initial OD... 600 The inoculum was 0.15g in 25mL CGXII fermentation medium (inoculated into a 500mL Erlenmeyer flask) and cultured at 30℃ and 220rpm on a constant temperature shaker (Shanghai Zhichu Instrument Co., Ltd., Shanghai, China). After 38h of culture, the fermentation broth was collected by centrifugation, and the yield of L-lysine in the fermentation broth was detected using a biosensor (Jinan Yanke Experimental Instrument Co., Ltd., Shandong, China).

[0084] The CGXII seed culture medium was prepared using water as the solvent, and the solutes and their concentrations were as follows: 5 g / L glucose, 20 g / L ammonium sulfate, 5 g / L urea, 1 g / L potassium dihydrogen phosphate, 1.3 g / L dipotassium hydrogen phosphate, 80 g / L MOPS, 0.01 g / L calcium chloride, 0.25 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.01 g / L manganese sulfate, 0.001 g / L zinc sulfate, 0.2 mg / L copper sulfate, 0.02 mg / L nickel chloride, 0.03 g / L dihydroxybenzoic acid, 0.5 μg / L biotin, and 0.1 mg / L thiamine HCl VB1.

[0085] The solvent for the CGXII fermentation medium was water, and the solutes and their concentrations were as follows: 80 g / L glucose, 20 g / L ammonium sulfate, 5 g / L urea, 1 g / L potassium dihydrogen phosphate, 1.3 g / L dipotassium hydrogen phosphate, 80 g / L MOPS, 0.01 g / L calcium chloride, 0.25 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, 0.01 g / L manganese sulfate, 0.001 g / L zinc sulfate, 0.2 mg / L copper sulfate, 0.02 mg / L nickel chloride, 0.03 g / L dihydroxybenzoic acid, 0.5 μg / L biotin, and 0.1 mg / L thiamine HCl VB1.

[0086] Test results as follows Figure 1 , Figure 1 The cultures were grown in 25 mL of CGXII fermentation medium. The results showed that the efflux of lysine from the efflux protein mutant was 1.6-2 times that of wild-type lysine, with mutant M6 showing the best effect.

[0087] Example 4: Effect of canavanine on the growth of Corynebacterium glutamicum expressing arginine efflux protein and its efflux mutant

[0088] Canavanine is a structural analogue of arginine and is toxic to Corynebacterium glutamicum, affecting normal cell growth and reproduction. However, canavanine that enters the cell can be expelled outside the cell by arginine efflux proteins, reducing its toxicity to the cell. Therefore, the efflux capacity of arginine efflux proteins can be detected by cell viability (see “JHSchwartz, et al. Analysis of the inhibition of growth produced by canavanine inescherichia coli. July 1960 Journal of Bacteriology 79(6):794-9”). Wild-type and mutant M2 strains were inoculated separately into 96-well flat-bottomed plates containing 150 μL of CGXII seed culture medium using toothpicks. They were incubated at 30°C and 800 rpm on a shaker (Shanghai Zhichu Instrument Co., Ltd., Shanghai, China) for 12 h. Then, they were inoculated into CGXII fermentation medium containing different concentrations of canavagine (inoculated into 96-well conical-bottomed plates, 150 μL each) using a replicator and incubated at 30°C and 800 rpm on a shaker (Shanghai Zhichu Instrument Co., Ltd., Shanghai, China) for 34 h. The OD values ​​of each strain were detected using a microplate reader (VersaMax continuous wavelength microplate reader, MD, USA). 600 value.

[0089] Test results as follows Figure 2 The results showed that the growth of each strain decreased with increasing canavanine concentration. The half-lethal concentration of the wild-type strain was about 0.5 g / L, while that of the mutant M2 was about 1 g / L. This indicates that the efflux protein mutant M2 has an enhanced ability to efflux the arginine analog canavanine, and also verifies that the efflux ability of mutant M2 is enhanced.

[0090] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A lysine efflux protein and its application <130> GNCLN220351 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 211 <212> PRT <213> Escherichia coli <400> 1 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 2 <211> 211 <212> PRT <213> Artificial sequence <400> 2 Met Phe Pro Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 3 <211> 211 <212> PRT <213> Artificial sequence <400> 3 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Arg Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 4 <211> 211 <212> PRT <213> Artificial sequence <400> 4 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Pro Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 5 <211> 211 <212> PRT <213> Artificial sequence <400> 5 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Thr Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 6 <211> 211 <212> PRT <213> Artificial sequence <400> 6 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asp Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Ser Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 7 <211> 187 <212> PRT <213> Artificial sequence <400> 7 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Lys Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val 180 185 <210> 8 <211> 115 <212> PRT <213> Artificial sequence <400> 8 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Arg Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Val Gly Val Val Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met 115 <210> 9 <211> 211 <212> PRT <213> Artificial sequence <400> 9 Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile 1 5 10 15 Leu Pro Leu Gly Pro Gln Asn Ala Phe Val Met Asn Gln Gly Ile Arg 20 25 30 Arg Gln Tyr His Ile Met Ile Ala Leu Leu Cys Ala Ile Ser Asp Leu 35 40 45 Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln 50 55 60 Ser Pro Trp Leu Leu Ala Leu Val Thr Trp Gly Gly Val Ala Phe Leu 65 70 75 80 Leu Trp Tyr Gly Phe Gly Ala Phe Lys Thr Ala Met Ser Ser Asn Ile 85 90 95 Glu Leu Ala Ser Ala Glu Val Met Lys Gln Gly Arg Trp Lys Ile Ile 100 105 110 Ala Thr Met Leu Ala Val Thr Trp Leu Asn Pro His Val Tyr Leu Asp 115 120 125 Thr Phe Val Val Leu Gly Ser Leu Gly Gly Gln Leu Asp Val Glu Pro 130 135 140 Arg Arg Trp Phe Ala Leu Gly Thr Ile Ser Ala Ser Phe Leu Trp Phe 145 150 155 160 Phe Gly Leu Ala Leu Leu Ala Ala Trp Leu Ala Pro Arg Leu Arg Thr 165 170 175 Ala Lys Ala Gln Arg Ile Ile Asn Leu Val Val Gly Cys Val Met Trp 180 185 190 Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala Tyr Ala Gln Ala 195 200 205 Leo Phe Serum 210 <210> 10 <211> 636 <212> Ms <213> Escherichia coli <400> 10 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agttag 636 <210> 11 <211> 636 <212> DNA <213> Artificial sequence <400> 11 atgtttcctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gctttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agctag 636 <210> 12 <211> 636 <212> DNA <213> Artificial sequence <400> 12 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattaatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaggcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agttag 636 <210> 13 <211> 636 <212> DNA <213> Artificial sequence <400> 13 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg cggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ccgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agttag 636 <210> 14 <211> 636 <212> DNA <213> Artificial sequence <400> 14 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc aacctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agttag 636 <210> 15 <211> 636 <212> DNA <213> Artificial sequence <400> 15 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat ggatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgcctcgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agttag 636 <210> 16 <211> 636 <212> DNA <213> Artificial sequence <400> 16 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gtgatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agttag 636 <210> 17 <211> 636 <212> DNA <213> Artificial sequence <400> 17 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tcgcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct gggtcggcgt agtcttcttg 240 ctgtggtatg gttttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgtaggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caaaacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgct atgtggttta ttgccttgca gctggcgaga 600 gacggtattg ctcatgcaca agccttgttc agctag 636 <210> 18 <211> 636 <212> DNA <213> Artificial sequence <400> 18 atgttttctt attactttca aggtcttgca cttggggcgg ctatgatcct accgctcggt 60 ccacaaaatg cttttgtgat gaatcagggc atacgtcgtc agtaccacat tatgattgcc 120 ttactttgtg ctatcagcga tttggtcctg atttgcgccg ggatttttgg tggcagcgcg 180 ttattgatgc agtcgccgtg gttgctggcg ctggtcacct ggggcggcgt agccttcttg 240 ctgtggtatg gctttggcgc ttttaaaaca gcaatgagca gtaatattga gttagccagc 300 gccgaagtca tgaagcaagg cagatggaaa attatcgcca ccatgttggc agtgacctgg 360 ctgaatccgc atgtttacct ggatactttt gttgtactgg gcagccttgg cgggcaactt 420 gatgtggaac caagacgctg gtttgcactc gggacaatta gcgcctcttt cctgtggttc 480 tttggtctgg ctcttctcgc agcctggctg gcaccgcgtc tgcgcacggc aaaagcacag 540 cgcattatca atctggttgt gggatgtgtt atgtggttta ttgccttgca gctggcgaga 600 gacggtattg cttatgcaca agccttgttc agctag 636

Claims

1. Application of arginine efflux protein mutants in any of the following: (C1) produces lysine; (C2) Increases lysine production; (C3) Enhances lysine efflux capacity; (C4) Preparation of feed additives and / or food fortifiers and / or cosmetic additives; The arginine efflux protein mutant is any one of the following: (a1) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: G188 stop codon; (a2) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: S3P; (a3) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: K105R; (a4) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: L121P; (a5) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: A168T; (a6) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: N28D and L196S. (a7) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: G58R, G75V, A78V and L116 stop codon. (a8) The arginine efflux protein mutant is a protein obtained by point mutation of the amino acid residues of the ArgO arginine efflux protein shown in SEQ ID No. 1 at the following sites: K145R and H205Y.

2. Application of biomaterials related to arginine efflux protein mutants in any of the following: (C1) produces lysine; (C2) Increases lysine production; (C3) Enhances lysine efflux capacity; (C4) Preparation of feed additives and / or food fortifiers and / or cosmetic additives; The biomaterials associated with the arginine efflux protein mutant are any of the following: (I) A nucleic acid molecule encoding the arginine efflux protein mutant as described in claim 1; (II) Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.

3. The application according to claim 2, characterized in that: The nucleic acid molecule is any one of the following: (B1) DNA molecule shown in SEQ ID No. 16 or positions 1-564 of SEQ ID No. 16; (B2) The DNA molecule shown in SEQ ID No. 11; (B3) DNA molecule shown in SEQ ID No. 12; (B4) DNA molecule shown in SEQ ID No. 13; (B5) DNA molecule shown in SEQ ID No. 14; (B6) DNA molecule shown in SEQ ID No. 15; (B7) DNA molecule shown in SEQ ID No. 17 or positions 1-348 of SEQ ID No. 17; (B8) The DNA molecule shown in SEQ ID No. 18; and / or The recombinant bacteria is Corynebacterium glutamicum containing the nucleic acid molecule.

4. A method for producing lysine and / or increasing lysine yield and / or increasing lysine efflux capacity, comprising the following steps: expressing the arginine efflux protein mutant of claim 1 in a recipient bacterium to obtain a recombinant bacterium; fermenting the recombinant bacterium to obtain lysine from the fermentation broth; wherein the recipient bacterium is Corynebacterium glutamicum.

5. The method according to claim 4, characterized in that: The arginine efflux protein mutant is expressed in the recipient bacteria by introducing the nucleic acid molecule of claim 2 or 3 into the recipient bacteria.

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