CN116514933A - Lysine efflux protein and application thereof - Google Patents

Lysine efflux protein and application thereof Download PDF

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CN116514933A
CN116514933A CN202210065236.8A CN202210065236A CN116514933A CN 116514933 A CN116514933 A CN 116514933A CN 202210065236 A CN202210065236 A CN 202210065236A CN 116514933 A CN116514933 A CN 116514933A
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arginine
efflux protein
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朱蕾蕾
聂志华
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention discloses lysine efflux protein and application thereof. The lysine efflux protein provided by the invention is an arginine efflux protein mutant, specifically, the arginine efflux protein ArgO is obtained by performing point mutation, and the mutation site contains part or all of the following: from the N-terminus, bits 3, 28, 58, 75, 78, 105, 116, 121, 145, 168, 188, 196, and 205. According to the invention, the arginine efflux protein mutant for improving lysine efflux is obtained by a directed evolution method. Compared with wild type, the mutant has little lysine excretion, and the ability of the mutant to excrete lysine is obviously improved. The arginine efflux protein mutant obtained by the invention is more beneficial to improving the yield of lysine, and lays a foundation for obtaining new efflux protein of the efflux lysine.

Description

Lysine efflux protein and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to lysine efflux protein and application thereof.
Background
The history of Corynebacterium glutamicum began in the 50 s of the 20 th century, when Japanese scientists obtained lysine-producing strains from a large number of strain screens, and applied them to mass production. Subsequently, the bacterium is continuously studied by scientists to improve the productivity of lysine. L-lysine is a basic essential amino acid, is very low in food and is easily destroyed during processing, and is often referred to as the first limiting amino acid. The total annual global production amount of L-lysine is about 300 ten thousand tons, and the market consumption demand is still increasing, so that the L-lysine is a product with good development prospect in the international market. Therefore, it is necessary to investigate the production of L-lysine.
In recent years, amino acid transport systems have received increasing attention. The amino acid transport system of the microorganism directly participates in the intake and the discharge of amino acid, and is important for the physiological metabolism of cells and the efficient biosynthesis and transportation of the amino acid. There are three main types of known lysine efflux proteins: ybjE from escherichia coli, lysE from corynebacterium glutamicum and newly discovered MGLE from escherichia coli. The development of new lysine transporters is of great importance for the construction of new lysine fermentation strains. ArgO, which is a naturally occurring efflux protein of arginine with little efflux of lysine, consists of 211 amino acids and has a6 transmembrane α -helical fragment structure. At present, the research and the molecular modification of the efflux protein are less, and no report for improving the capability of the efflux lysine through the molecular modification is seen. Therefore, the efflux protein with strong lysine efflux capability is obtained by carrying out molecular transformation on the efflux protein, and has important significance for creating new lysine fermentation strains.
Disclosure of Invention
The invention aims to provide an ArgO mutant with remarkable lysine excretion capacity, and a coding gene and related application thereof.
In a first aspect, the invention claims an arginine efflux protein mutant.
The arginine efflux protein mutant disclosed by the invention is obtained by performing point mutation on ArgO arginine efflux protein, and the mutation site contains (or is) part or all of the following: from the N-terminus, bits 3, 28, 58, 75, 78, 105, 116, 121, 145, 168, 188, 196, and 205.
Preferably, the amino acid sequence of the arginine efflux protein mutant has at least 95% identity to a sequence comprising only each of the above-described mutation sites.
The content of the above-mentioned components is more preferably 96% or more, 97% or more, 98% or more or 99% or more.
The arginine efflux protein mutant has the function of efflux lysine. Preferably, the arginine efflux protein mutant has a greater ability to efflux lysine than the ArgO arginine efflux protein.
Further, the arginine efflux protein mutant may be any of the following:
(A1) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: 188 th from the N-terminus (corresponding mutant M6);
(A2) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: 3 rd position from the N-terminus (corresponding mutant M1);
(A3) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: position 105 from the N-terminus (corresponding mutant M2);
(A4) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: position 121 from the N-terminus (corresponding mutant M3);
(A5) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: 168 from the N-terminus (corresponding mutant M4);
(A6) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: from the N-terminus, at positions 28 and 196 (corresponding mutant M5);
(A7) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: positions 58, 75, 78 and 116 (corresponding to mutant M7) from the N-terminus;
(A8) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites (or the following sites) of the ArgO arginine efflux protein: from the N-terminus, at positions 145 and 205 (corresponding to mutant M8).
In the arginine efflux protein mutant, the 3 rd point mutation from the N end is specifically S3P, the 28 th point mutation is specifically N28D, the 58 th point mutation is specifically G58R, the 75 th point mutation is specifically G75V, the 78 th point mutation is specifically A78V, the 105 th point mutation is specifically K105R, the 116 th point mutation is specifically L116 stop code, the 121 th point mutation is specifically L121P, the 145 th point mutation is specifically K145R, the 168 th point mutation is specifically A168T, the 188 th point mutation is specifically G188 stop code, the 196 th point mutation is specifically L196S, and the 205 th point mutation is specifically H205Y.
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 termination code). Accordingly, mutants mutated at different positions of SEQ ID No.1 are named sequentially.
In a specific embodiment of the present invention, the arginine efflux protein mutant is specifically any of the following:
(a1) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: g188 termination code (corresponding mutant M6, amino acid sequence shown in SEQ ID No. 7).
That is, only amino acid residues 1-187 of ArgO arginine efflux protein shown in SEQ ID No.1 are expressed to obtain the arginine efflux protein mutant (M6).
(a2) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: S3P (corresponding mutant M1, the amino acid sequence is shown as SEQ ID No. 2).
(a3) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: K105R (corresponding mutant M2, the amino acid sequence of which is shown as SEQ ID No. 3).
(a4) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: L121P (corresponding mutant M3, the amino acid sequence of which is shown in SEQ ID No. 4).
(a5) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: A168T (corresponding mutant M4, the amino acid sequence of which is shown in SEQ ID No. 5).
(a6) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: N28D, L196S (corresponding mutant M5, amino acid sequence shown in SEQ ID No. 6).
(a7) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: G58R, G75V, A V, L termination code (corresponding to mutant M7, amino acid sequence shown in SEQ ID No. 8).
Namely, the arginine efflux protein mutant (M7) which is obtained by carrying out G58R, G, 75, V, A and 78V site-directed mutagenesis on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 and expressing only amino acid residues 1-115.
(a8) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: K145R, H Y (corresponding mutant M8, amino acid sequence shown in SEQ ID No. 9).
In a second aspect, the invention claims arginine efflux protein mutant related biomaterials.
The biological material related to the arginine efflux protein mutant claimed by the invention can be any of the following materials:
(I) Nucleic acid molecules encoding the arginine efflux protein mutants;
(II) expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.
Wherein, the nucleic acid molecule for encoding the ArgO arginine efflux protein from Escherichia coli K12 is the DNA molecule shown in SEQ ID No. 10.
Further, the nucleic acid molecule encoding the arginine efflux protein mutant is specifically any of the following:
(B1) The DNA molecule shown in SEQ ID No.16 or SEQ ID No.16 at positions 1-564 (corresponding mutant M6);
(B2) A DNA molecule shown in SEQ ID No.11 (corresponding mutant M1);
(B3) A DNA molecule shown in SEQ ID No.12 (corresponding mutant M2);
(B4) The DNA molecule shown in SEQ ID No.13 (corresponding mutant M3);
(B5) A DNA molecule shown in SEQ ID No.14 (corresponding mutant M4);
(B6) The DNA molecule shown in SEQ ID No.15 (corresponding mutant M5);
(B7) A DNA molecule shown in SEQ ID No.17 or SEQ ID No.17 at positions 1-348 (corresponding mutant M7);
(B8) The DNA molecule shown in SEQ ID No.18 (corresponding mutant M8).
Wherein, the 562 th to 564 th bits of SEQ ID No.16 are the stop codon TGA. The 346-348 th bit of SEQ ID No.17 is the stop codon TAG.
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 between multiple cloning sites (e.g., sacI and BamHI) of the pTRCmob vector.
The recombinant bacterium may be Corynebacterium glutamicum containing the nucleic acid molecule.
In a third aspect, the invention claims the use of an arginine efflux protein mutant or biological material as described hereinbefore in any of the following:
(C1) Producing lysine;
(C2) The lysine yield is improved;
(C3) Improving the lysine excretion capacity;
(C4) Preparing feed additives and/or food enhancers and/or cosmetic additives;
(C5) As nutritional and/or therapeutic agents.
In a fourth aspect, the invention claims a method for producing lysine and/or increasing lysine yield and/or increasing lysine efflux capacity.
The method for producing lysine and/or improving the yield and/or the excretion capacity of lysine as claimed in the invention can comprise the following steps: expressing the arginine efflux protein mutant in a recipient bacterium to obtain a recombinant bacterium; and (3) fermenting and culturing the recombinant bacteria, and obtaining lysine from fermentation broth.
Further, expression of the arginine efflux protein mutant in the recipient bacterium may be accomplished by introducing into the recipient bacterium a "nucleic acid molecule encoding the arginine efflux protein mutant" as described above.
Further, the "nucleic acid molecule encoding the arginine efflux protein mutant" may be introduced into the recipient bacterium in the form of a recombinant vector.
In a specific embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by cloning the "nucleic acid molecule encoding the arginine efflux protein mutant" between multiple cloning sites (e.g., sacI and BamHI) of the pTRCmob vector.
Further, the recipient bacterium is corynebacterium glutamicum.
Still further, the corynebacterium glutamicum may be a recombinant bacterium obtained by mutating 311 th position of a lysC gene-encoding protein in the genome of a wild-type corynebacterium glutamicum (e.g., ATCC 13032) from T to I, mutating 59 th position of a hom gene-encoding protein from V to A, mutating 458 th position of a pyc gene-encoding protein from P to S, and knocking out the lysE gene. Namely, the genotype of the corynebacterium glutamicum is ATCC13032lysC T311I hom V59A pyc P458S △LysE。
In the method, the culture medium of the fermentation culture may be CGXII culture medium, and the culture condition may be 30℃and 220rpm for 38 hours.
In a specific embodiment of the present invention, the recombinant bacteria are inoculated into CGXII seed medium, cultured at 30℃and 220rpm for 12 hours, and then at the starting OD 600 An inoculum size of 0.15 was inoculated into CGXII fermentation medium and cultured at 30℃and 220rpm for 38 hours.
In the present invention, the lysine is specifically L-lysine.
In the present invention, the increased lysine productivity and the increased lysine efflux ability are both increased in lysine productivity and lysine efflux ability of a mutant (i.e., the arginine efflux protein mutant described above) as compared to the wild type (i.e., the ArgO arginine efflux protein).
According to the invention, the arginine efflux protein mutant for improving lysine efflux is obtained by a directed evolution method. Compared with wild type, the mutant has little lysine excretion, and the ability of the mutant to excrete lysine is obviously improved. The arginine efflux protein mutant obtained by the invention is more beneficial to improving the yield of lysine, and lays a foundation for obtaining new efflux protein of the efflux lysine.
Drawings
FIG. 1 is a graph showing comparison of the yield of efflux protein mutants with wild-type lysine cultured in 25mL CGXII fermentation medium. ArgO is labeled as wild-type control, and the ordinate "lysine g/L" refers to the amount of L-lysine produced per L of fermentation broth. * Indicating significant differences between the corresponding mutant and wild type, and indicating very significant differences between the corresponding mutant and wild type.
FIG. 2 shows the effect of canavanine on the growth of Corynebacterium glutamicum expressing arginine efflux protein and its efflux mutants. In the figure, canavanine (a structural analogue of arginine, which affects the normal growth of cells) can be discharged outside cells by arginine efflux proteins, so that the toxicity to the cells is reduced. Thus, the efflux ability of arginine efflux protein to canavanine can be detected by the viability of the cells.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
pTRCmob vector: the subject group of Tianjin institute of biotechnology, national academy of sciences Zheng Ping, described in Yu Wang, et al A Novel Corynebacterium glutamicum L-glamate outlet.applied and Environmental Microbiology, vol.84, no.6, is available to the public from the applicant and can only be used for repeated experiments of the present invention, but not for other uses.
Example 1 Gene cloning of arginine efflux protein
The target gene of ArgO arginine efflux protein is synthesized by the complete gene of Suzhou Jin Weizhi biotechnology company, the gene sequence is shown as SEQ ID No.10, then the gene is connected between the enzyme cutting sites SacI and BamHI of the pTRCmob vector, and the recombinant plasmid is obtained after sequencing to confirm that the vector is constructed successfully, and is named as pTRCmob-ArgO.
The structure of pTRCmob-ArgO is described as: the recombinant plasmid obtained by cloning the DNA fragment shown in SEQ ID No.10 between the cleavage sites SacI and BamHI of pTRCmob vector. SEQ ID No.10 is a target gene of wild ArgO arginine efflux protein, and encodes the wild ArgO arginine efflux protein shown in SEQ ID No. 1.
EXAMPLE 2 screening of arginine efflux protein mutants with improved ability to efflux lysine
In order to enhance the ability of the arginine efflux protein to export lysine, primers (upstream primer: 5'-ACCATGGAATTCGAGCTCATG-3'; downstream primer: 5'-GGTCGACTCTAGAGGATCCCTA-3') were designed using the recombinant plasmid pTRCmob-ArgO constructed in example 1 as a template, mn was added 2+ The target gene was randomly mutated by PCR (the random mutation occurred only between positions 4-633 of the ArgO gene and not at other positions of the vector). Electrotransformation of the mutant product into Corynebacterium glutamicum ATCC13032lysC T311I hom V59A pyc P458S After overnight incubation at 30℃to obtain recombinant bacteria in delta LysE competent cells, the grown monoclonal cells were inoculated into 96-well plates (containing 150. Mu.L of LBG medium, 25mg/mL Kana) with sterile toothpicks, respectively, and after shaking culture at 30℃for 38 hours at 800rpm, lysine production was detected with a biosensor. And (3) analyzing the detection result, and finally screening 8 mutant strains with increased lysine efflux, which are named as M1, M2, M3, M4, M5, M6, M7 and M8 respectively.
Wherein the corynebacterium glutamicum ATCC13032lysC T311I hom V59A pyc P458S The DeltaLysE is constructed according to the following steps: in Corynebacterium glutamicum ATCC13032lysC T311I hom V59A pyc P458S Strain (the strain is "AHP-3" in "J.Ohnishi.S, et al A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing variant.Appl Microbiol Biotechnol (2002) 58:217-223", 31 st protein encoded by lysC gene in ATCC13032 genome)Mutation of the gene from T to I at position 1 and mutation of the gene encoding protein from V to A at position 59 and mutation of the gene encoding protein from P to S at position 458) were carried out by the method described in "E.glutamicum gene knockout system was constructed" Tan Yanzhen ", university of Jiangnan, academic paper (Shus), 2012". The knockout plasmid pK18mobsacB-LRLysE was first constructed: the corynebacterium glutamicum ATCC13032 genome is used as a template, lysE-L-F (5'-ATCCCGCCACGGGATTAGCTTCA-3')/LysE-L-R (5'-CGTGACCTATGGAAGTACTT AAGTAAAATGATTGG-3') is used as a primer, an upstream homologous fragment (left homologous arm) L of the LysE gene is obtained by amplification, lysE-R-F (5'-TTTTCGCGGGTTTTGGAATCGGTGGC-3')/LysE-R-R (5'-GCTGCCCGCTTCTGATTCATCAGC-3') is used as a primer, a downstream homologous fragment (right homologous arm) R of the LysE gene is obtained by amplification, and after purification, the upstream and downstream homologous fragments are purified according to a molar ratio of 1:1, overlap PCR was performed to obtain overlap fragment LR. The plasmid pK18mobsacB is used as a template, the pK18mob-sacB plasmid skeleton-F (5'-TGATGAATCAGAAGCGGGCAGCGCACTGGCCGTCGTTTTACAA-3')/pK 18mob-sacB plasmid skeleton-R (5'-GAAGCTAATCCCGTGGCGGGATCATGTCATAGCTGTTTCCTGTG-3') is used as a primer, and the pK18mob-sacB plasmid skeleton of the fragment homologous to the overlapping fragment LR is obtained by amplification. Ligating the overlapping fragments to the pK18mob-sacB plasmid backbone by means of homologous recombination; and obtaining the knocked-out plasmid pK18mobsacB-LRLysE after sequencing and verification. Then the plasmid pK18mobsacB-LRLysE was electrotransferred to Corynebacterium glutamicum ATCC13032lysC T311I hom V59A pyc P458S The colony PCR amplified upstream and downstream homologous fragments L and R confirmed successful first round of exchange by applying the colony PCR to a solid medium containing kanamycin, culturing at 30℃for 36 hours, growing a monoclonal on a plate, and using the monoclonal as a template and LysE-L-F (5'-ATCCCGCCACGGGATTAGCTTCA-3')/LysE-R-R (5'-GCTGCCCGCTTCTGATTCATCAGC-3') as primers. The monoclonal cells which were successfully verified were transferred to TSB medium (formulation: 5g/L glucose, 5g/L yeast powder, 9g/L soybean peptone, 20g/L MOPS,3g/L urea, 0.5g/L succinic acid, 0.1g/L magnesium sulfate heptahydrate, 1g/L dipotassium phosphate trihydrate, 10. Mu.g/L biotin, 0.1mg/L VB 1) and cultured overnight at 30℃and 220 rpm; transfer to 10% sucrose at 3% inoculum sizeCulturing in TSB culture medium at 30deg.C and 220rpm for 10 hr, and diluting 10 after culturing -3 、10 -4 Spread into TSB medium containing 10% sucrose; after single bacterial colony grows out, lysE-L-F (5'-ATCCCGCCACGGGATTAGCTTCA-3')/LysE-R-R (5'-GCTGCCCGCTTCTGATTCATCAGC-3') is used as a primer to carry out colony PCR verification, and bacteria with correct strips are transferred and sequenced; the correct sequencing results in the knock-out strain Corynebacterium glutamicum ATCC13032lysC T311I hom V59A pyc P458S △lysE。
Each mutant strain is subjected to gene sequencing, and specific mutated amino acid sites, and specific amino acid sequences and gene sequences after mutation are shown in Table 1.
TABLE 1 mutant amino acid sites
Note that: amino acid substitutions in the tables, using the following nomenclature: original amino acid (wild type), position (i.e. position in SEQ ID No. 1), substituted amino acid.
EXAMPLE 3 arginine efflux protein and expression of its efflux mutant in Corynebacterium glutamicum
The positive bacteria of the wild type and 8 mutants shown in Table 1 obtained in example 2 were inoculated into 10mL CGXII seed medium and cultured at 30℃and 220rpm in a constant temperature culture shaker (Shanghai know Chu instruments Co., shanghai, china) for 12 hours, followed by an initial OD 600 An inoculum size of 0.15 was inoculated into 25mL CGXII fermentation medium (into 500mL Erlenmeyer flask) and cultured at 30℃and 220rpm on a constant temperature incubator shaker (Shanghai Kochia Instrument Co., shanghai, china). After culturing for 38 hours, the fermentation broth was collected by centrifugation, and the yield of L-lysine in the fermentation broth was measured by a biosensor (Jinan Shandong, china, experimental apparatus Co., ltd.).
Wherein, the solvent of the CGXII seed culture medium is water, and the solute and the concentration are as follows: 5g/L glucose, 20g/L ammonium sulfate, 5g/L urea, 1g/L monopotassium phosphate, 1.3g/L dipotassium phosphate, 80g/L MOPS,0.01g/L calcium chloride, 0.25g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.01g/L manganese sulfate, 0.001g/L zinc sulfate, 0.2mg/L copper sulfate, 0.02mg/L nickel chloride, 0.03g/L dihydroxybenzoic acid, 0.5 μg/L biotin, 0.1mg/L Thiamine HCL VB1.
The solvent of the CGXII fermentation medium is water, and the solute and the concentration are as follows: 80g/L glucose, 20g/L ammonium sulfate, 5g/L urea, 1g/L monopotassium phosphate, 1.3g/L dipotassium phosphate, 80g/L MOPS,0.01g/L calcium chloride, 0.25g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.01g/L manganese sulfate, 0.001g/L zinc sulfate, 0.2mg/L copper sulfate, 0.02mg/L nickel chloride, 0.03g/L dihydroxybenzoic acid, 0.5 μg/L biotin, 0.1mg/L Thiamine HCL VB1.
The detection results are shown in FIG. 1, and FIG. 1 is cultured in 25mL CGXII fermentation medium, and the results show that the efflux of the efflux protein mutant lysine is 1.6-2 times that of the wild-type lysine, and particularly the mutant M6 has the best effect.
EXAMPLE 4 Effect of canavanine on the growth of Corynebacterium glutamicum expressing arginine efflux protein and its efflux mutants
Canavanine is a structural analogue of arginine, which has deleterious effects on corynebacterium glutamicum, affecting normal growth and reproduction of cells; and canavanine entering cells can be discharged outside the cells by arginine efflux protein, so that the toxicity to the cells is reduced. Thus, the efflux ability of arginine efflux proteins can be detected by cell viability (see "JH Schwartz, et al analysis of the inhibition of growth produced by canavanine in escherichia coll. July 1960Journal of Bacteriology 79 (6): 794-9", supra). The wild type strain M2 and the mutant strain M2 were inoculated with toothpick into a 96-well flat bottom plate containing 150. Mu.L of CGXII seed medium, cultured at 30℃and 800rpm in a constant temperature culture shaker (Shanghai Kochia instruments Co., ltd., shanghai, china) for 12 hours, then inoculated with replicators into CGXII fermentation media containing canavanine at different concentrations, respectively, inoculated into a 96 Kong Jian bottom plate, 150. Mu.L of liquid-filled volume, cultured at 30℃and 800rpm in a constant temperature culture shaker (Shanghai Kochia instruments Co., shanghai, china) for 34 hours, and O of each strain was detected with an enzyme-labeled instrument (American MD continuous wavelength enzyme-labeled instrument Versamax)D 600 Values.
The results of the test are shown in FIG. 2, which shows that the growth of each strain decreases with increasing concentration of canavanine, the semi-lethal concentration of the wild-type strain is about 0.5g/L, and the semi-lethal concentration of the mutant M2 is about 1g/L, indicating that the efflux protein mutant M2 has an increased efflux capacity for arginine analogue canavanine, and at the same time, the efflux capacity of the mutant M2 is verified to be increased.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> a lysine efflux protein and use thereof
<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
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Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala
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Leu Phe Ser
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Met Phe Pro Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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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
Leu Phe Ser
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Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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Leu Phe Ser
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Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln
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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
Leu Phe Ser
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Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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Phe Ile Ala Leu Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala
195 200 205
Leu Phe Ser
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Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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Val Leu Ile Cys Ala Gly Ile Phe Gly Gly Ser Ala Leu Leu Met Gln
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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
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Phe Ile Ala Ser Gln Leu Ala Arg Asp Gly Ile Ala His Ala Gln Ala
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Leu Phe Ser
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<210> 7
<211> 187
<212> PRT
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Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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Met Phe Ser Tyr Tyr Phe Gln Gly Leu Ala Leu Gly Ala Ala Met Ile
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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
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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
Leu Phe Ser
210
<210> 10
<211> 636
<212> DNA
<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 (10)

1. The arginine efflux protein mutant is obtained by performing point mutation on ArgO arginine efflux protein, and the mutation site contains part or all of the following: from the N-terminus, bits 3, 28, 58, 75, 78, 105, 116, 121, 145, 168, 188, 196, and 205.
2. The arginine efflux protein mutant of claim 1, wherein: the arginine efflux protein mutant may be any of the following:
(A1) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: 188 th bit from N end;
(A2) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: 3 rd bit from N end;
(A3) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: 105 th bit from the N-terminal;
(A4) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: 121 th bit from N-terminal;
(A5) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: 168 th bit from N end;
(A6) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: from the N-terminus, 28 th and 196 th;
(A7) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: 58 th, 75 th, 78 th and 116 th bits from the N-terminal;
(A8) The arginine efflux protein mutant is a protein obtained by performing point mutation on amino acid residues of at least the following sites of the ArgO arginine efflux protein: bits 145 and 205 from the N-terminal.
3. The arginine efflux protein mutant of claim 1 or 2, wherein: the ArgO arginine efflux protein is ArgO arginine efflux protein from Escherichia coli K12.
4. The arginine efflux protein mutant of claim 3, wherein: the amino acid sequence of the ArgO arginine efflux protein from Escherichia coli K12 is shown as SEQ ID No. 1.
5. The arginine efflux protein mutant of any one of claims 1-4, wherein: in the arginine efflux protein mutant, the 3 rd point mutation is S3P, the 28 th point mutation is N28D, the 58 th point mutation is G58R, the 75 th point mutation is G75V, the 78 th point mutation is A78V, the 105 th point mutation is K105R, the 116 th point mutation is L116 stop code, the 121 th point mutation is L121P, the 145 th point mutation is K145R, the 168 th point mutation is A168T, the 188 th point mutation is G188 stop code, the 196 th point mutation is L196S and the 205 th point mutation are H205Y.
6. The arginine efflux protein mutant of any one of claims 1-5, wherein: the arginine efflux protein mutant is any one of the following:
(a1) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: g188 stop password;
(a2) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of 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 carrying out point mutation on the amino acid residues of 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 carrying out point mutation on the amino acid residues of 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 carrying out point mutation on the amino acid residues of 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 carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: N28D, L196S;
(a7) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: G58R, G75V, A V, L116 stop code;
(a8) The arginine efflux protein mutant is a protein obtained by carrying out point mutation on the amino acid residues of ArgO arginine efflux protein shown in SEQ ID No.1 at the following sites: K145R, H Y.
7. The biological material related to the arginine efflux protein mutant is any of the following:
(I) A nucleic acid molecule encoding the arginine efflux protein mutant of any of claims 1-6;
(II) expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.
8. The biomaterial according to claim 7, wherein: the nucleic acid molecule for encoding the ArgO arginine efflux protein from Escherichia coli K12 is a DNA molecule shown in SEQ ID No. 10;
further, the nucleic acid molecule encoding the arginine efflux protein mutant is any of the following:
(B1) DNA molecules shown in SEQ ID No.16 or SEQ ID No.16 at positions 1-564;
(B2) A DNA molecule shown in SEQ ID No. 11;
(B3) A DNA molecule shown in SEQ ID No. 12;
(B4) A DNA molecule shown in SEQ ID No. 13;
(B5) A DNA molecule shown in SEQ ID No. 14;
(B6) A DNA molecule shown in SEQ ID No. 15;
(B7) A DNA molecule shown in SEQ ID No.17 or SEQ ID No.17 at positions 1-348;
(B8) A DNA molecule shown as SEQ ID No. 18;
and/or
The recombinant bacterium is corynebacterium glutamicum containing the nucleic acid molecule.
9. Use of an arginine efflux protein mutant of any of claims 1-6 or a biological material of claim 7 or 8 in any of the following:
(C1) Producing lysine;
(C2) The lysine yield is improved;
(C3) Improving the lysine excretion capacity;
(C4) Preparing feed additives and/or food enhancers and/or cosmetic additives;
(C5) As nutritional and/or therapeutic agents.
10. A method for producing lysine and/or increasing lysine production and/or increasing lysine efflux capacity comprising the steps of: expressing the arginine efflux protein mutant of any of claims 1-6 in a recipient bacterium to obtain a recombinant bacterium; fermenting and culturing the recombinant bacteria to obtain lysine from fermentation broth;
further, expressing the arginine efflux protein mutant in the recipient bacterium by introducing into the recipient bacterium the nucleic acid molecule encoding the arginine efflux protein mutant of claim 7 or 8;
and/or
Further, the recipient bacterium is corynebacterium glutamicum.
CN202210065236.8A 2022-01-20 2022-01-20 Lysine efflux protein and application thereof Pending CN116514933A (en)

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