CN115125245B - Promoter mutant P alpha-rpsT and application thereof - Google Patents
Promoter mutant P alpha-rpsT and application thereof Download PDFInfo
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Abstract
According to the invention, a promoter mutant with improved strength is obtained by directionally modifying a bacillus-derived promoter-10 region sequence, and the nucleotide sequence is shown as SEQ ID NO: 1. The invention also provides an expression vector and an expression system containing the promoter mutant. The promoter mutant is used for controlling gene expression, is particularly applied to the field of bacillus amyloliquefaciens metabolic engineering, can improve the heterologous expression activity of green fluorescent protein GFP to 178%, and lays a foundation for mediating the expression of heterologous proteins in bacillus amyloliquefaciens expression systems.
Description
Technical Field
The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to a promoter enhanced mutant Palpha-rpsT and application thereof.
Background
The green fluorescent protein (Green fluorescent protein, GFP) is a protein which can be excited by blue-violet light to emit green fluorescence, and can maintain the fluorescence for more than 10min under the laser of 450-490 nm. The luminescence of green fluorescent protein is caused by chromophore, after GFP is folded, amino acid residue at 66 th position is dehydrogenated under aerobic condition, so that the chromophore is cyclized to form p-hydroxy benzyl-5-imidazolone (p-HBI), the process is spontaneously formed under aerobic condition, and green fluorescence can be emitted under the condition of excitation light 488 nm.
GFP as a reporter gene has high sensitivity and has no influence on the structural function of a target gene, and can generate stronger fluorescence at a specific wavelength. The intensity of the expression element can be known from the fluorescence intensity of GFP. High throughput screening of expression elements is achieved. Provides a new method for realizing high-throughput screening of the expression element in the bacillus amyloliquefaciens.
Bacillus amyloliquefaciens is a gram-positive bacterium, is favored as a microbial cell factory because of the excellent extracellular protein secretion capacity and a recognized safety strain, is not only a producer of various enzyme preparations, but also can produce various antibacterial substances, is widely applied to industries such as agriculture, industrial foods, medicines and the like, is an ideal host for expressing and secreting exogenous proteins in a prokaryotic expression system, and becomes an important mode strain in the prokaryotic expression system.
The use of a strong promoter is one of the key factors to achieve efficient expression of foreign proteins, and a promoter (promoter) is a specific DNA sequence that recognizes, binds to and initiates transcription by RNA polymerase (RNA Pol). The bacterial promoter is a target sequence combined with RNA polymerase, is an essential regulatory element for gene expression in bacteria, and determines the intensity and the time of bacterial gene expression. The expression of bacterial genes can be changed through mutation of key areas of the promoter, so that the research on the growth and development of thalli and metabolic regulation is realized. The promoter is the basis for constructing various expression systems and realizing the expression of heterologous genes, so that screening and obtaining a strong promoter are very effective methods for mediating the expression of exogenous protein genes and improving the yield of the exogenous protein genes.
Disclosure of Invention
Aiming at the current industrial demand and the deficiency of the prior art, the invention mainly aims to provide a promoter enhanced mutant and a genetically engineered bacterium expression system for realizing the efficient expression of a target gene.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a promoter mutant, the nucleotide sequence of which is shown in SEQ ID NO: 1.
In a second aspect, the invention provides an expression vector comprising said promoter mutant.
In a third aspect, the invention provides an expression system comprising a promoter mutant or expression vector as described above.
In a fourth aspect, the present invention provides the use of a promoter mutant as described above for controlling gene expression, in particular in the field of bacillus amyloliquefaciens metabolic engineering.
The beneficial effects are that:
according to the invention, the promoter mutant with improved strength is obtained by analyzing and reforming a bacillus-derived promoter, and the promoter mutant is suitable for a bacillus amyloliquefaciens expression system, can efficiently and heterologously express green fluorescent protein GFP, improves GFP expression activity to 178%, mediates the expression of the heterologous protein in the bacillus amyloliquefaciens expression system, and lays a foundation for promoting the efficient expression and industrial production of the green fluorescent protein. The promoter mutant provided by the invention has a good effect when being used for improving the expression of other exogenous protein genes in bacillus amyloliquefaciens.
Drawings
Fig. 1: recovering and verifying the fragments of the recombinant vector; wherein M: marker,1: ply-2,2: pα -rpsT.
Fig. 2: expression activity of GFP gene of recombinant green fluorescent protein.
Detailed Description
The invention is further described below by means of specific embodiments. Unless otherwise indicated, the technical means, materials, etc. to which the following embodiments relate may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
In a first aspect, the invention provides a promoter mutant, the nucleotide sequence of which is shown in SEQ ID NO: 1.
In a second aspect, the invention provides an expression vector comprising said promoter mutant. The backbone of the expression vector may be any expression vector of bacillus subtilis known in the art.
According to a preferred embodiment of the invention, the expression vector is pWB980.
In a third aspect, the invention provides an expression system comprising a promoter mutant or expression vector as described above. The expression system may be any host suitable for the promoter mutants or expression vectors of the invention, such as bacillus amyloliquefaciens.
According to a preferred embodiment of the present invention, the host is a Bacillus amyloliquefaciens genetically engineered bacterium Δ6 Δeps Δpgs Δ3049-3052 (the genetically engineered bacterium is obtained by knocking out six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs and phage-associated genes 3049-3052 starting from CGMCC No.11218, see in particular patent application 202111182462.6).
In a fourth aspect, the present invention provides the use of a promoter mutant as described above for controlling gene expression, in particular in the field of bacillus amyloliquefaciens metabolic engineering.
According to a preferred embodiment of the invention, the promoter mutants are used to control the expression of the Green Fluorescent Protein (GFP) gene in a Bacillus amyloliquefaciens system,
the present invention will be described in more detail with reference to specific examples. In the following examples, unless otherwise specified:
the culture medium used in the invention comprises:
LB medium: 5g/L of yeast powder, 10g/L of peptone and 5g/L of sodium chloride;
preparation of bacillus amyloliquefaciens competent solution:
LBS: 91.085g/L sorbitol; 10g/L of sodium chloride; peptone 10g/L; 5g/L yeast powder;
washing buffer: 91.085g/L sorbitol; mannitol 91.085g/L; 100ml of 10% glycerol;
resuspension buffer: 91.085g/L sorbitol; mannitol 91.085g/L; 100mL of 10% glycerol; 14% PEG 6000 140g/L.
Example 1: novel promoter mutant and construction of plasmid thereof
The promoter ply-2 (nucleotide sequence shown as SEQ ID NO: 2) derived from the a-amylase gene of Bacillus subtilis, which was found to have two-10 regions and two-35 regions by analysis by promoter predictive analysis software iProEP, softberry and the like, was subjected to a multi-point mutation (TGATAGAAT) in the first-10 region sequence (TCTTATATT) to obtain a novel promoter mutant designated as P.alpha. -rpsT, the nucleotide sequence of which is shown as SEQ ID NO: 1.
The nucleotide sequence is shown as SEQ ID NO:1 are synthesized by the biological company with the cleavage sites EcoRI and HindIII.
The digestion of the expression vector and the linkage to the target gene are as follows:
1) Extracting a vector plasmid P alpha-rpsT-PUC 57 containing a mutant promoter and a plasmid of a vector pWB980, and then carrying out double digestion on the plasmids according to required restriction enzymes (EcoRI, hindIII), wherein the digestion conditions are 37 ℃ for 2 hours;
2) Carrying out glue recovery and purification on the vector fragments of the enzyme-digested target fragments Palpha-rpsT and pWB 980;
3) The recovered target fragment P alpha-rpsT and pWB980 carrier fragments are connected, the connection condition is 16 ℃, the connection is carried out for 6 hours or overnight, and the connection system is as follows:
p.alpha. -rpsT fragment 4.5. Mu.L
pWB980 vector fragment 0.5. Mu.L
The ligation products were chemically transformed into Bacillus subtilis WB600 as follows;
1) Selecting a single colony of the newly activated bacillus subtilis WB600, and culturing the single colony in 5mL of LB liquid medium at 37 ℃ for 220r/min overnight;
2) Transfer 100 μl of culture solution into 5mL SPI culture medium, culturing at 37deg.C and 220r/min until OD600 = 1.2 (about 3-4 h) at the end of logarithmic growth;
3) 200 mu L of culture solution grown to the end of a log phase is taken to be placed in 2mL of SPII culture medium, and is cultured for 1.5h at 37 ℃ and 100 r/min;
4) 20 mu L of 10mmol/L EGTA is added into the thallus of the SPII culture medium, and the mixture is cultured for 10min at 37 ℃ and 100 r/min;
5) Adding the connection product into the SPII, and culturing at 37 ℃ for 30min at 100 r/min;
6) The rotation speed is regulated to 220r/min, the culture is continued for 1.5 hours, bacterial liquid is coated on an LB screening plate containing 100 mug/mL kanamycin, the culture is carried out for 12 hours at 37 ℃, and positive transformants are screened for verification (shown in figure 1).
Example 2: green fluorescent protein genetically engineered bacterium
The green fluorescent protein gene (nucleotide sequence is shown as SEQ ID NO:3, genBank: MN 443913.1) is taken as a reporter gene, the reporter gene is synthesized by biological company, the vector is PUC57, and the synthesized gene sequence is provided with HindIII and BamHI enzyme cutting sites.
The green fluorescent protein gene and the P alpha-rpsT-pWB 980 vector containing the mutated promoter are subjected to enzyme digestion connection to construct a recombinant expression vector containing the promoter P alpha-rpsT and GFP gene expression cassette, and transformed into bacillus subtilis WB 600; extracting plasmids, and transferring the recombinant plasmids into a bacillus amyloliquefaciens genetic engineering bacterium delta 6 delta eps delta pgs delta 3049-3052 (the genetic engineering bacterium is obtained by knocking out six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs and phage related genes 3049-3052 from CGMCC No. 11218), thereby obtaining a recombinant strain for heterologous expression of alkaline protease.
Ligation of expression vector containing mutant promoter P.alpha. -rpsT-pWB980 with target gene GFP:
1) Respectively extracting plasmids containing promoter combinations P alpha-rpsT-pWB 980 and GFP, and then carrying out double digestion on the plasmids according to the required restriction enzyme (HindIII, bamHI), wherein the digestion conditions are 37 ℃ for 2 hours; the enzyme digestion system is as follows:
2) Performing gel recovery and purification on the enzyme section;
3) The recovered GFP fragment and the P.alpha. -rpsT-pWB980 fragment were ligated, at 16℃for 6h or overnight, and the ligation system was as follows:
GFP fragment 4.5. Mu.L
Linear P.alpha. -rpsT-pWB980 fragment 0.5. Mu.L
Solution I 5.0μL。
The ligation products were chemically transformed into Bacillus subtilis WB600 as follows;
1) Selecting a single colony of the newly activated bacillus subtilis WB600, and culturing the single colony in 5mL of LB liquid medium at 37 ℃ for 220r/min overnight;
2) Transfer 100 μl of culture solution into 5mL SPI culture medium, culturing at 37deg.C and 220r/min until OD600 = 1.2 (about 3-4 h) at the end of logarithmic growth;
3) 200 mu L of culture solution grown to the end of a log phase is taken to be placed in 2mL of SPII culture medium, and is cultured for 1.5h at 37 ℃ and 100 r/min;
4) 20 mu L of 10mmol/L EGTA is added into the thallus of the SPII culture medium, and the mixture is cultured for 10min at 37 ℃ and 100 r/min;
5) Adding the connection product into the SPII, and culturing at 37 ℃ for 30min at 100 r/min;
6) The rotation speed is regulated to 220r/min, the culture is continued for 1.5 hours, bacterial liquid is coated on an LB screening plate containing 100 mug/mL kanamycin, the culture is carried out for 12 hours at 37 ℃, and positive transformants are screened for verification.
The recombinant plasmid in WB600 was extracted and electrotransformed into Bacillus amyloliquefaciens Δ6 Δeps Δpgs Δ3049-3052 by the following method:
1) Cleaning the electric rotating cup with 75% alcohol, irradiating for more than 20min under ultraviolet, and pre-cooling on ice;
2) 100. Mu.L of competent plasmid DNA was mixed with 10ng of plasmid DNA and added to the electrorotating cup and placed on ice for 2min;
3) 2500V electric shock, the electric shock time is generally 4-6ms;
4) Immediately after the electric shock, 1ml of resuscitation medium was added, and resuscitated for 3 hours at 37 ℃. Plating, culturing at 37 ℃ for 12 hours, and screening positive transformants for verification.
Meanwhile, recombinant bacteria with the same expression system constructed by using the promoter ply-2 are used as control bacteria, and the difference between the recombinant bacteria and the control bacteria is that the promoters of alkaline protease genes are different.
Example 3: expression and analysis of green fluorescent protein gene
Single colonies of recombinant genetically engineered bacteria on fresh plates are respectively inoculated into 50mL of kanamycin-resistant LB seed culture medium, subjected to shaking culture at 37 ℃ and 220rpm for 12 hours, inoculated into LB fermentation culture medium containing kanamycin resistance in the same inoculum size, and subjected to fermentation culture at 37 ℃ and 220 rpm.
Respectively taking fermentation liquids of each recombinant bacterium for fermentation culture for 12h, 24h, 36h, 48h, 60h and 72h, measuring absorbance values of the fermentation liquids by using an enzyme-labeled instrument under the wavelengths of excitation waves 488nm and emission waves 523nm, and drawing a fluorescence curve (shown in figure 2) by taking time as an abscissa and taking OD as an ordinate.
Through measurement, the activity of the green fluorescent protein in each recombinant bacterium fermentation broth reaches the highest at 60 hours. The fluorescence value of the recombinant bacterium expressed green fluorescent protein constructed by the promoter mutant Pa-rpsT is 17654, which is 178% of the fluorescence activity of the recombinant bacterium constructed by the primary promoter ply-2. The invention provides a promoter mutant with obviously improved strength, and the high-efficiency heterologous expression of the green fluorescent protein is realized by using the promoter mutant.
Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.
Sequence listing
<110> university of Tianjin science and technology
<120> a promoter mutant pα -rpsT and use thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 596
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cattatgttt gaatttccgt ttaaagaatg ggctgcaagc cttgtgtttt tgttcatcat 60
tatgatagaa tactgcatca gggctgcggc atccggaatg ctcatgccga gaatagacac 120
caaagaagaa ctgcaaaaac gggtgaagca gcagcgaata gaatcaattg cggtcgcctt 180
tgcggtagtg gtgcttacga tgtacgacag ggggattccc catacattct tcgcttggct 240
gaaaatgatt cttcttttta tcgtctgcgg cggcgttctg tttctgcttc ggtatgtgat 300
tgtgaagctg gcttacagaa gagcggtaaa agaagaaata aaaaagaaat catctttttt 360
gtttggaaag cgagggaagc gttcacagtt tcgggcagct ttttttatag gaacattgat 420
ttgtattcac tctgccaagt tgttttgata gagtgattgt gataatttta aatgtaagcg 480
ttaacaaaat tctccagtct tcacatcggt ttgaaaggag gaagcggaag aatgaagtaa 540
gagggatttt tgactccgaa gtaagtcttc aaaaaatcaa ataaggagtg tcaaga 596
<210> 2
<211> 596
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 2
cattatgttt gaatttccgt ttaaagaatg ggctgcaagc cttgtgtttt tgttcatcat 60
tatcttatat tactgcatca gggctgcggc atccggaatg ctcatgccga gaatagacac 120
caaagaagaa ctgcaaaaac gggtgaagca gcagcgaata gaatcaattg cggtcgcctt 180
tgcggtagtg gtgcttacga tgtacgacag ggggattccc catacattct tcgcttggct 240
gaaaatgatt cttcttttta tcgtctgcgg cggcgttctg tttctgcttc ggtatgtgat 300
tgtgaagctg gcttacagaa gagcggtaaa agaagaaata aaaaagaaat catctttttt 360
gtttggaaag cgagggaagc gttcacagtt tcgggcagct ttttttatag gaacattgat 420
ttgtattcac tctgccaagt tgttttgata gagtgattgt gataatttta aatgtaagcg 480
ttaacaaaat tctccagtct tcacatcggt ttgaaaggag gaagcggaag aatgaagtaa 540
gagggatttt tgactccgaa gtaagtcttc aaaaaatcaa ataaggagtg tcaaga 596
<210> 3
<211> 712
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 60
aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 120
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 180
caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 240
cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 300
cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 360
catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 420
gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 480
ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 540
ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 600
cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 660
gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ag 712
Claims (6)
1. A promoter mutant, which is characterized in that the nucleotide sequence of the promoter mutant is shown in SEQ ID NO: 1.
2. An expression vector comprising the promoter mutant of claim 1.
3. The expression vector of claim 2, wherein the backbone of the expression vector is pWB980.
4. An expression system comprising the promoter mutant of claim 1 or the expression vector of claim 3.
5. The expression system of claim 4, wherein the host of the expression system is bacillus amyloliquefaciens.
6. The expression system of claim 5, further comprising a nucleotide sequence set forth in SEQ ID NO:3, and is controlled by the promoter mutant.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795937A (en) * | 2018-06-14 | 2018-11-13 | 天津科技大学 | The startup sub-portfolio and its genetic engineering bacterium of efficient heterogenous expression alkali protease |
| CN112094781A (en) * | 2020-09-27 | 2020-12-18 | 江南大学 | Bacillus amyloliquefaciens and application thereof |
| CN112522173A (en) * | 2020-12-23 | 2021-03-19 | 天津科技大学 | Engineering bacterium for producing heterologous alkaline protease and construction method thereof |
| CN113151270A (en) * | 2021-04-02 | 2021-07-23 | 天津科技大学 | Promoter for efficiently expressing alkaline protease and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795937A (en) * | 2018-06-14 | 2018-11-13 | 天津科技大学 | The startup sub-portfolio and its genetic engineering bacterium of efficient heterogenous expression alkali protease |
| CN112094781A (en) * | 2020-09-27 | 2020-12-18 | 江南大学 | Bacillus amyloliquefaciens and application thereof |
| CN112522173A (en) * | 2020-12-23 | 2021-03-19 | 天津科技大学 | Engineering bacterium for producing heterologous alkaline protease and construction method thereof |
| CN113151270A (en) * | 2021-04-02 | 2021-07-23 | 天津科技大学 | Promoter for efficiently expressing alkaline protease and application thereof |
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