CN111635454B - Method for screening arginine high-producing strain by using biosensor - Google Patents

Method for screening arginine high-producing strain by using biosensor Download PDF

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CN111635454B
CN111635454B CN202010501763.XA CN202010501763A CN111635454B CN 111635454 B CN111635454 B CN 111635454B CN 202010501763 A CN202010501763 A CN 202010501763A CN 111635454 B CN111635454 B CN 111635454B
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徐美娟
饶志明
刘平平
杨套伟
张显
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Abstract

The invention discloses a method for screening arginine high-producing strains by using a biosensor, wherein the biosensor contains a gene argR' for coding repressor protein, a gene pargC for coding a promoter and a marker gene sacB.

Description

Method for screening arginine high-producing strain by using biosensor
Technical Field
The invention relates to a method for screening arginine-producing strains by using a biosensor, belonging to the technical field of biology.
Background
L-arginine, an essential amino acid in the metabolic process of the human body, has important physiological and biochemical significance. It is an important intermediate metabolite of the organism urea cycle, and has very wide application in the fields of food, medicine, cosmetics and the like. It can stimulate the immune system, promote wound healing, participate in muscle metabolism, enhance muscle vitality, and convert blood ammonia into urea to be discharged from the body so as to maintain nitrogen balance. Meanwhile, the L-arginine also has the effects of stimulating the generation of insulin, epinephrine and the like in vivo, rapidly reducing the content of blood sugar and fat in vivo and the like. Therefore, the L-arginine has great application and research values.
With the increase of market demand, the research on the production of L-arginine by microbial fermentation is more and more focused. In order to obtain high-yielding strains, genetic engineering breeding methods and mutation breeding methods are two commonly used means. The genetic engineering breeding method can more purposefully carry out directional modification on the strains and improve the yield of the strains. The mutation breeding method has no more limitation, can generate more abundant mutants and obtain more excellent variant strains. However, because the mutation breeding method has a large randomness, in order to obtain a high-yield strain meeting the conditions, screening must be performed from a large number of mutants, and the workload is large, so that a method for screening the arginine high-yield strain with higher efficiency is urgently needed to overcome the defects of the existing screening method.
Disclosure of Invention
In order to solve the technical problem, the invention provides a repressor protein, and the amino acid sequence of the repressor protein is shown as SEQ ID No. 1.
In one embodiment of the invention, the nucleotide sequence of the gene argR' encoding the repressor protein is shown in SEQ ID No. 2.
The invention also provides a biosensor, which is a carrier carrying a gene argR' coding for repressor protein, a gene pargC coding for a promoter and a marker gene sacB.
In one embodiment of the invention, the vector is plasmid pDXW-10.
In one embodiment of the invention, the nucleotide sequence of the gene argR' encoding a repressor protein is shown in SEQ ID No. 2.
In one embodiment of the invention, the nucleotide sequence of the gene PargC encoding the promoter is shown in SEQ ID No. 3.
In one embodiment of the invention, the nucleotide sequence of the marker gene sacB is shown as SEQ ID No. 4.
In one embodiment of the invention, the marker gene sacB is located upstream of the gene PargC encoding the promoter and downstream of the gene argR' encoding the repressor protein.
In one embodiment of the invention, the gene argR' encoding the repressor protein is located downstream of the gene encoding the promoter tac-M carried by plasmid pDXW-10 itself.
The invention also provides a recombinant strain, and the recombinant strain contains the biosensor.
In one embodiment of the present invention, the recombinant strain is a host arginine producing strain.
The invention also provides a method for screening the arginine high-yield strain, which comprises the steps of inoculating the recombinant strain into a liquid culture medium containing cane sugar for culture to obtain a culture solution, and then detecting OD in the culture solution600Value, OD600The mutagenic strain corresponding to the culture solution with relatively high value is the mutagenic strain with relatively strong arginine production capacity; or, the method is to inoculate the recombinant strain to a solid culture medium containing cane sugar and culture the recombinant strain until bacterial colonies grow out, and the mutagenized strain with relatively large bacterial colonies is the mutagenized strain with relatively strong arginine production capacity.
The invention also provides the application of the biosensor or the recombinant strain or the method in screening arginine-producing strains.
[ advantageous effects ]
(1) The invention provides a biosensor with higher efficiency for screening arginine high-producing strains, which comprisesThere are a gene argR' coding for a repressor, a gene PargC coding for a promoter and a marker gene sacB. Wherein, the gene argR' coding the repressor protein changes the 145 th glycine of the starting sequence into the lysine. The marker gene sacB is a sucrose lethal gene, and the expression of the marker gene sacB can make the strain die finally in the presence of sucrose. After the biosensor is introduced into a strain to be screened, if the strain to be screened can produce arginine at a higher level, a repressor ArgR in the biosensor can be activated by arginine and combined with a PargC promoter, so that the expression of a marker gene sacB which is started by the PargC promoter in the biosensor is inhibited, and the strain to be screened can continue to grow and propagate. Moreover, the stronger the arginine producing ability of the strain to be screened, the more normal the growth and reproduction of the strain to be screened tend to be. At this time, it is only necessary to detect OD in the culture solution obtained by culturing each strain to be screened by means of an ultraviolet spectrophotometer600The OD in the culture solution obtained by culturing each strain to be screened is compared600The strains to be screened with relatively strong arginine production capacity can be obtained according to the height of the value. After ArgR protein mutation, the sensitivity of ArgR protein to arginine is reduced, and arginine with higher level can be activated to inhibit the expression of marker gene sacB, only the strain with higher arginine level can grow in a screening plate, and more strains with lower arginine level are eliminated. Therefore, arginine-producing strains can be screened more efficiently using the biosensor of the present invention.
(2) The invention provides a method for improving the screening efficiency of high-yield strains of arginine, which comprises the steps of mutating a gene for coding repressor protein, wherein the gene argR 'for coding the repressor protein is obtained by mutating glycine at the 145 th site of a starting sequence into lysine, so that the sensitivity of the argR protein to arginine is reduced, then, the mutated argR' gene is reconnected to a biosensor plasmid containing a gene PargC for coding a promoter and a marker gene sacB, a mutant biosensor is introduced into a strain to be screened and then the strain to be screened is cultured, and if the arginine produced by the strain to be screened reaches a certain level, the repressor protein in the biosensor can be activated by arginine and is combined with the PargC promoter, so that the expression of the marker gene sacB started by the PargC promoter in the biosensor is inhibited by the repressor protein. The marker gene sacB is a sucrose lethal gene, the inhibition of the marker gene sacB can make the strain to be screened grow and reproduce, and the stronger the arginine producing ability of the strain to be screened is, the more normal the growth and reproduction thereof tend to be. After mutation of ArgR protein, the sensitivity of ArgR protein to arginine is reduced, and higher level of arginine is needed to be activated, so that the expression of marker gene sacB is inhibited. At this time, in the screening process, more mutagenic strains with lower arginine yield can be discarded because the mutagenic strains cannot grow or grow at a lower speed, so that most of the strains which fail in mutagenesis can be screened out quickly and in a large batch by using the method for screening the arginine-producing strains, the probability of obtaining the arginine-producing strains is greatly increased, and meanwhile, the labor and the time for obtaining the arginine-producing strains are reduced.
Drawings
FIG. 1: plasmid map of biosensor pSenArg-sacB.
Detailed Description
The plasmid pDXW-10 referred to in the following examples is described in the patent application publication No. CN 104531747A; the plasmid pK18mobsacB referred to in the examples below was purchased from Youbao organisms; corynebacterium crenatum (Corynebacterium crenatum) SYPA5-5, referred to in the examples below, is described in the patent application publication No. CN1441055A with accession number CGMCC NO.0890 (the strain in the patent application publication is numbered SDNN403, which the inventors renumber to SYPA5-5 during the experiments).
The media involved in the following examples are as follows:
screening the plates: 10g/L of sucrose, 0.2g/L of agar powder, 120-150 g/L of glucose, 30-50 g/L of ammonium sulfate, 10-20 g/L of yeast extract, 0.2-0.6 g/L of magnesium sulfate heptahydrate, 0.5-1.5 g/L of potassium chloride, 1-2 g/L of monopotassium phosphate, 0.01-0.04 g/L of ferrous sulfate heptahydrate and 0.01-0.04 g/L of manganese sulfate monohydrate, and the pH value is 7.0-7.2.
BHI liquid medium: 37.5g/L brain and heart infusion.
BHI solid plate: 37.5g/L of brain-heart infusion and 0.2g/L of agar powder.
Seed culture medium: 40-60 g/L glucose, 20-30 g/L ammonium sulfate, 10-20 g/L yeast extract, 0.3-0.7 g/L magnesium sulfate heptahydrate and 1-2 g/L potassium dihydrogen phosphate, and the pH value is 7.0-7.2.
Fermentation medium: 120-150 g/L glucose, 30-50 g/L ammonium sulfate, 10-20 g/L yeast extract, 0.2-0.6 g/L magnesium sulfate heptahydrate, 0.5-1.5 g/L potassium chloride, 1-2 g/L potassium dihydrogen phosphate, 0.01-0.04 g/L ferrous sulfate heptahydrate and 0.01-0.04 g/L manganese sulfate monohydrate, and the pH value is 7.0-7.2.
The detection methods referred to in the following examples are as follows:
the method for detecting the arginine content comprises the following steps:
1. sakaguchi process
Weighing 5g of 1-naphthol, dissolving in n-propanol, adding 2.5mL of 1% diacetyl, and preparing a color developing agent after the volume is up to 100 mL; adding 1mL of color developing agent, 4mL of NaOH with the concentration of 0.375mol/L and 1mL of arginine standard solution diluted to the gradient concentration into a tube, reacting at 30 ℃ for 30min, and measuring the light absorption value at 520nm to prepare a standard curve; and centrifuging the bacterial liquid obtained by culturing the strain to be detected to obtain supernatant, diluting by 100 times, determining, and substituting the measured light absorption value into a standard function equation for calculation.
2. High performance liquid chromatography
Selecting a C18 chromatographic column; 0.05mol/L acetate buffer (pH 6.4) -50% acetonitrile solution (75:25) as a mobile phase; the detection wavelength is 362 nm; the column temperature was 30 ℃.
Example 1: construction of biosensor for screening arginine high-producing strain
The method comprises the following specific steps:
using genome DNA of Corynebacterium crenatum SYPA5-5 as a template, and argR-F1 (nucleotide sequence is shown in SEQ ID NO. 5) and argR-R1 (nucleotide sequence is shown in SEQ ID NO. 6) as primers, and obtaining gene argR (nucleotide sequence is shown in SEQ ID NO. 7) for coding repressor protein through PCR amplification; and performing tapping and purification on the amplified gene argR for coding the repressor protein, then connecting the gene argR with a shuttle plasmid pDXW-10 subjected to EcoR I and Not I double enzyme digestion to obtain a recombinant plasmid argR-pDXW-10, and performing sequencing verification on the recombinant plasmid argR-pDXW-10 to prove that the connection is successful.
By taking the genome DNA of Corynebacterium crenatum SYPA5-5 and plasmid pK18mobsacB as templates, respectively taking PargC-sacB-F1 (nucleotide sequence is shown in SEQ ID NO. 8) and PargC-sacB-R1 (nucleotide sequence is shown in SEQ ID NO. 8), PargC-sacB-F2 (nucleotide sequence is shown in SEQ ID NO. 9) and PargC-sacB-R2 (nucleotide sequence is shown in SEQ ID NO. 10), PargC-gfp-F2 (nucleotide sequence is shown in SEQ ID NO. 11) and PargC-gfp-R2 (nucleotide sequence is shown in SEQ ID NO. 12) as primers, and obtaining a gene PargC (nucleotide sequence is shown in SEQ ID NO. 3) for coding a promoter and a marker gene PargB (nucleotide sequence is shown in SEQ ID NO. 4) by PCR amplification; chemically synthesizing a gene gfp (the nucleotide sequence is shown as SEQ ID NO. 13) for coding the fluorescent protein; connecting a gene PargC for coding a promoter and a marker gene sacB with a recombinant plasmid argR-pDXW-10 by a fusion PCR technology to obtain a biosensor pSenArg-sacB, connecting the gene PargC for coding the promoter and a gene gfp for coding a fluorescent protein with the recombinant plasmid argR-pDXW-10 by the fusion PCR technology to obtain the biosensor pSenArg-gfp, and performing sequencing verification on the biosensor pSenArg-sacB and the biosensor pSenArg-gfp to prove that the connection is successful; in the biosensor pSenArg-sacB, the marker gene sacB is positioned at the upstream of a gene PargC coding for a promoter and at the downstream of a gene argR coding for a repressor protein, and the gene argR coding for the repressor protein is positioned at the downstream of a gene coding for a promoter tac-M carried by the plasmid pDXW-10; in the biosensor pSenArg-gfp, the gene gfp encoding the fluorescent protein was located upstream of the gene PargC encoding the promoter and downstream of the gene argR encoding the repressor protein, and the gene argR encoding the repressor protein was located downstream of the gene encoding the promoter tac-M carried by the plasmid pDXW-10 itself.
Example 2: preliminary verification of biosensor for screening arginine-producing strains
The method comprises the following specific steps:
respectively electrotransfering the biosensor pSenArg-sacB and the biosensor pSenArg-gfp into Corynebacterium crenatum (Corynebacterium crenatum) SYPA5-5 to obtain recombinant Corynebacterium crenatum/pSenArg-sacB and recombinant Corynebacterium crenatum/pSenArg-gfp; the recombinant Corynebacterium crenatum/pSenArg-sacB and the recombinant Corynebacterium crenatum/pSenArg-gfp are streaked and inoculated on a solid plate without cane sugar, and cultured for 48h at 30 ℃ for activation to obtain a single colony; taking recombinant Corynebacterium crenatum/pDXW-10 containing pDXW-10 empty plasmid as a positive control, picking recombinant Corynebacterium crenatum/pSenArg-sacB single colony, streaking the single colony into solid plates containing 10g/L of sucrose and respectively containing arginine with the concentrations of 0, 10, 20, 40, 60, 80 and 100mM, culturing at 30 ℃ for 48 hours, and observing the growth condition of each colony on the solid plates containing arginine with different concentrations; taking recombinant Corynebacterium crenatum/pDXW-10 containing pDXW-10 empty plasmid as a positive control, selecting recombinant Corynebacterium crenatum/pSenArg-gfp single colonies, inoculating the single colonies into liquid culture media respectively containing arginine with the concentrations of 0, 10, 20, 40, 60, 80 and 100mM, culturing at 30 ℃ for 48 hours, and observing the fluorescence intensity in a fermentation liquid by using a fluorescence microscope; therefore, the sacB gene can respond to different growth conditions of arginine with different concentrations and is more sensitive than the response condition of the gfp gene to arginine with different concentrations.
Example 3: mutation of the arginine repressor ArgR
The method comprises the following specific steps:
(1) two genes constituting the argR' gene are amplified by using genomic DNA of Corynebacterium crenatum SYPA5-5 as a template and primers G145K-F1 (the nucleotide sequence is shown as SEQ ID NO. 14), G145K-R1 (the nucleotide sequence is shown as SEQ ID NO. 15), G145K-F2 (the nucleotide sequence is shown as SEQ ID NO. 16) and G145K-R2 (the nucleotide sequence is shown as SEQ ID NO. 17).
(2) Subsequently, a mutated argR' gene in which the 145 th glycine is mutated into a lysine (G145K) is amplified by a fusion PCR technique using primers G145K-F1 (the nucleotide sequence is shown in SEQ ID NO. 14) and G145K-R2 (the nucleotide sequence is shown in SEQ ID NO. 16).
(3) Then, primers are designed by the same method, glutamine (Q125) at the 125 th position and glycine (G145) at the 145 th position in the amino acid sequence of the repressor protein ArgR are subjected to site saturation mutation respectively, and mutation is carried out to the rest 20 amino acids, and the mutated argR' gene is obtained. And finally, sequencing and verifying the mutated argR' gene.
Example 4: ArgR protein mutation result verification
The method comprises the following specific steps:
the biosensor plasmid of example 1 was double digested with EcoRI and NotI, respectively, followed by agarose gel electrophoresis, and the larger vector fragment was recovered by tapping. Subsequently, each mutated argR' gene was ligated to a vector fragment to obtain a mutated biosensor plasmid. Then each mutated biosensor plasmid is electrotransformed into corynebacterium crenatum SYPA5-5 to obtain a mutant strain.
Each mutant strain was streaked in a BHI solid plate for activation, and then inoculated in a sucrose-containing BHI liquid medium, respectively, and 60mM arginine was added to the medium, respectively. And after culturing for 20-24 h, respectively taking culture solutions of different mutant strains, extracting RNA, carrying out reverse transcription on the RNA into cDNA, and verifying the change condition of the transcription level of the marker gene sacB by using a real-time fluorescent quantitative PCR technology.
As a result, it was found that the transcription level of the marker gene sacB was changed to some extent only when the G145 site was replaced with lysine (G145K) and when the Q125 site was replaced with glutamic acid and leucine (Q125E and Q125L), respectively. Compared with the wild strain, the transcription level of the sacB gene in the mutant strain G145K is improved by 2.26 times, and the transcription levels of the sacB gene in the mutant strains Q125E and Q125L are respectively reduced by 2.38 times and 1.27 times. It can be seen that in the mutant strain G145K, ArgR protein had reduced sensitivity to arginine. The reason for this was analyzed to find that the binding pocket of the mutant G145K became large and that the ArgR protein required more arginine to be activated.
Example 5: high-throughput screening of arginine-producing strains by using mutated biosensor
The method comprises the following specific steps:
1. mutagenesis
And (2) carrying out streak activation on the corynebacterium crenatum containing the mutation biosensor in a BHI solid plate, and culturing at the temperature of 30 ℃ for 24-36 h. Then, a single colony is selected and inoculated in a BHI liquid culture medium, and is subjected to shaking culture at 30 ℃ and 180rpm to the middle and later logarithmic growth stages to obtain a bacterial liquid. OD of the bacterial liquid with sterile physiological saline600Diluting the value to 0.5-0.6, uniformly coating 10 mu L of bacteria liquid on a sterile metal slide, placing the slide in a fixed circular groove above a rotary positioning table by using forceps, rotating the slide to the lower part of a plasma generator, and adjusting the height of the rotary positioning table by using a knob to ensure that the distance between a sample to be processed and a jet outlet of the plasma generator is about 2 mm. And after the sample is placed, closing the door of the operation room and setting parameters. The carrier gas flow rate was selected to be 10L/min, the radio frequency power was 100W, and the mutagenesis time was 30 s. After the mutagenesis of the sample is finished, opening an operation door, putting the slide glass into a centrifuge tube containing 990 mu L of sterile normal saline, then transferring to a super clean bench, diluting the treated bacterial liquid step by using the sterile normal saline, respectively uniformly coating 100 mu L of diluted bacterial liquid on a screening flat plate, and culturing for 24-48 h at 30 ℃. .
2. Preliminary screening
And (3) coating the mutagenized bacterial liquid into a screening plate containing 10g/L of sucrose, and culturing for 30-48 h at the temperature of 30 ℃. Since the strain with higher arginine production in the screening plate grows better, the strain needs higher arginine production to grow in the screening plate after ArgR protein mutation, and the strain with lower arginine production cannot grow or grows small colonies, so the strain is discarded. In the original biosensor, about 1.57% of the mutant strains in the total number of cells can grow on the screening plate, and in the mutated biosensor, only 2.24% of the mutant strains can grow on the screening plate, so that the workload is further reduced, and the screening efficiency is improved.
3. Double sieve
Taking corynebacterium crenatum containing pDXW-10 empty plasmid as a positive control, selecting a strain with a large bacterial colony (the bacterial colony diameter is not less than 1.20mm) from a screening plate, inoculating the strain into a 24-well plate containing a seed culture medium, culturing at 30 ℃ for 24 hours to obtain a culture solution, measuring the yield of arginine in the culture solution by a sakaguchi reagent method, and selecting a mutagenized strain with the yield of the 24-well plate being more than 10% of that of the positive control. The sieve yield of this step was 19.9%; wherein the screening rate is the number of mutant strains with the yield higher than that of a positive control by more than 10 percent/the total number of mutant strains subjected to rescreening.
4. Further rescreening
Inoculating seed liquid corresponding to the mutagenized strain obtained by re-screening into a fermentation culture medium according to the inoculation amount of 5-10%, and performing shaking culture at 30 ℃ and 220rpm for 96 hours to obtain fermentation liquor; the arginine yield in the fermentation broth is determined by high performance liquid chromatography, and the mutagenized strain with the yield 15 percent higher than that of the positive control is selected. The sieve yield of this step was 25.4%; wherein the screening rate is the number of mutant strains with a yield of 15% higher than that of the positive control/the total number of mutant strains subjected to further rescreening.
According to the whole screening process, the mutant strain is subjected to primary screening, secondary screening and further secondary screening by using the mutated biosensor pSenArg-sacB, the screening rates of the secondary screening and the further secondary screening are respectively as high as 19.9 percent and 25.4 percent, and the screening rates are respectively improved by 3.9 percent and 5.4 percent compared with those of the original biosensor. At the same time, more low-yielding strains were discarded during the primary screening. It is shown that the screening of arginine-producing strains using the mutated biosensor pSenArg-sacB greatly increases the probability of obtaining arginine-producing strains, and reduces the labor and time required for obtaining arginine-producing strains.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for screening arginine-producing strain using biosensor
<130> BAA200447A
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 171
<212> PRT
<213> Artificial sequence
<400> 1
Met Ser Leu Gly Ser Thr Pro Ser Thr Pro Glu Asn Leu Asn Pro Val
1 5 10 15
Thr Arg Thr Arg Arg Gln Ala Leu Ile Leu Gln Ile Leu Asp Lys Gln
20 25 30
Lys Val Thr Ser Gln Val Gln Leu Ser Glu Leu Leu Leu Asp Glu Gly
35 40 45
Ile Asp Ile Thr Gln Ala Thr Leu Ser Arg Asp Leu Asp Glu Leu Gly
50 55 60
Ala Arg Lys Val Arg Pro Asp Gly Gly Arg Ala Tyr Tyr Ala Val Gly
65 70 75 80
Pro Val Asp Ser Ile Ala Arg Glu Gly Leu Arg Gly Pro Ser Glu Lys
85 90 95
Leu Arg Arg Met Leu Asp Glu Leu Leu Val Ser Thr Asp His Ser Gly
100 105 110
Asn Ile Ala Met Leu Arg Thr Pro Pro Gly Ala Ala Gln Tyr Leu Ala
115 120 125
Ser Phe Ile Asp Arg Val Gly Leu Lys Glu Val Val Gly Thr Ile Ala
130 135 140
Lys Asp Asp Thr Val Phe Val Leu Ala Arg Asp Pro Leu Thr Gly Lys
145 150 155 160
Glu Leu Gly Glu Leu Leu Ser Gly Arg Thr Thr
165 170
<210> 2
<211> 516
<212> DNA
<213> Artificial sequence
<400> 2
atgtcccttg gctcaacccc gtcaacaccg gaaaacttaa atcccgtgac tcgcactgca 60
cgccaagctc tcattttgca gattttggac aaacaaaaag tcaccagcca ggtacaactg 120
tctgaattgc tgctggatga aggcatcgat atcacccagg ccaccttgtc ccgagatctc 180
gatgaactcg gtgcacgcaa ggttcgcccc gatgggggac gcgcctacta cgcggtcggc 240
ccagtagata gcatcgcccg cgaagatctc cggggtccgt cggagaagct gcgccgcatg 300
cttgatgaac tgctggtttc tacagatcat tccggcaaca tcgcgatgct gcgcaccccg 360
ccgggagctg cccagtacct ggcaagtttc atcgataggg tggggctgaa agaagtcgtt 420
ggcaccatcg ctaatgatga caccgttttc gttctcgccc gtgatccgct cacaggtaaa 480
gaactaggtg aattactcag cgggcgcacc acttaa 516
<210> 3
<211> 145
<212> DNA
<213> Artificial sequence
<400> 3
aaattcatgc ttttacccac ttgcagtttt agctgtaggt gggtttttgc atgtctaacc 60
cgtcttttat gcacaccctc gcaatgaatc aaaaaattat gcatgaataa tttgcatgat 120
catgcataac gtgtatggtg taact 145
<210> 4
<211> 1422
<212> DNA
<213> Artificial sequence
<400> 4
atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 60
gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 120
tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 180
gaaaaatatc aagtttctga atttgattcg tccacaatta aaaatatctc ttctgcaaaa 240
ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 300
cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 360
atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 420
cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 480
caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 540
gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 600
gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 660
gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 720
gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 780
tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 840
gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 900
gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 960
gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1020
attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1080
ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 1140
tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 1200
gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa 1260
ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 1320
tcaacgtttg cgccgagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa 1380
gacagcatcc ttgaacaagg acaattaaca gttaacaaat aa 1422
<210> 5
<211> 50
<212> DNA
<213> Artificial sequence
<400> 5
aacaatttca cacaggaaac agaattcaaa gaggagaaat actagatgtc 50
<210> 6
<211> 44
<212> DNA
<213> Artificial sequence
<400> 6
ggagatctgg taccctcgag gcggccgctt aagtggtgcg cccg 44
<210> 7
<211> 33
<212> DNA
<213> Artificial sequence
<400> 7
cccaagctta aattcatgct tttacccact tgc 33
<210> 8
<211> 36
<212> DNA
<213> Artificial sequence
<400> 8
gcaaactttt tgatgttcat agttacacca tacacg 36
<210> 9
<211> 36
<212> DNA
<213> Artificial sequence
<400> 9
cgtgtatggt gtaactatga acatcaaaaa gtttgc 36
<210> 10
<211> 32
<212> DNA
<213> Artificial sequence
<400> 10
gccttaagtt atttgttaac tgttaattgt cc 32
<210> 11
<211> 44
<212> DNA
<213> Artificial sequence
<400> 11
cgatgcgtcc ggcgtagagg atccaaattc atgcttttac ccac 44
<210> 12
<211> 53
<212> DNA
<213> Artificial sequence
<400> 12
gttcttctcc cttacccatc taatcctcct ttagttacac catacacgtt atg 53
<210> 13
<211> 717
<212> DNA
<213> Artificial sequence
<400> 13
atgggtaagg gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaagcttc ctgttccttg gccaacactt 180
gtcactactc ttacttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg 240
cacgacttct tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc 300
aaggacgacg ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc 360
aacagaatcg agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag 420
ttggaataca actacaactc ccacaacgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540
cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600
ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660
cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717
<210> 14
<211> 32
<212> DNA
<213> Artificial sequence
<400> 14
cgcggatccg aattcatgtc ccttggctca ac 32
<210> 15
<211> 33
<212> DNA
<213> Artificial sequence
<400> 15
gaaaacggtg tcatccttag cgatggtgcc aac 33
<210> 16
<211> 33
<212> DNA
<213> Artificial sequence
<400> 16
gttggcacca tcgctaagga tgacaccgtt ttc 33
<210> 17
<211> 24
<212> DNA
<213> Artificial sequence
<400> 17
gcggccgctt aagtggtgcg cccg 24

Claims (10)

1. A repressor protein, characterized in that its amino acid sequence is shown in SEQ ID No. 1.
2. The repressor protein as set forth in claim 1 wherein the nucleotide sequence of the gene argR' encoding the repressor protein is as set forth in SEQ ID No. 2.
3. A biosensor comprising the repressor protein as set forth in claim 1.
4. A biosensor according to claim 3, wherein the biosensor is a vector carrying the gene argR' encoding the repressor protein according to claim 1, the gene PargC encoding the promoter, and the marker gene sacB.
5. A biosensor as claimed in claim 4 wherein the nucleotide sequence of the gene PargC encoding the promoter is as shown in SEQ ID No. 3.
6. The biosensor of claim 5, wherein the nucleotide sequence of the marker gene sacB is shown in SEQ ID No. 4.
7. A biosensor as claimed in any one of claims 4 to 6 wherein the marker gene sacB is located upstream of the gene pargC encoding the promoter and downstream of the gene argR' encoding the repressor protein.
8. A recombinant strain comprising the biosensor of any one of claims 3-7.
9. A method for screening arginine-producing strains, which comprises inoculating the recombinant strain of claim 8 to a liquid medium containing sucrose, culturing to obtain a culture solution, and detecting OD in the culture solution600Value, OD600The mutagenic strain corresponding to the culture solution with relatively high value is the arginine production capacity phaseFor strong mutagenic strains; or, the method is to inoculate the recombinant strain of claim 8 on a solid culture medium containing cane sugar and culture the recombinant strain until colonies grow out, and the mutagenized strain with relatively large colonies is the mutagenized strain with relatively strong arginine production capacity.
10. Use of the biosensor according to any one of claims 3 to 7 or the recombinant strain according to claim 8 or the method according to claim 9 for screening arginine-producing strains.
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