CN115125247B - Combined promoter palpha 2-alpha 2 and application thereof - Google Patents
Combined promoter palpha 2-alpha 2 and application thereof Download PDFInfo
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Abstract
According to the invention, a novel combined promoter palpha 2-alpha 2 with improved strength is obtained by analyzing and reforming a promoter from bacillus subtilis. The invention also provides an expression vector and an expression system containing the combined promoter. The combined promoter is used for controlling gene expression, is especially applied to the field of bacillus amyloliquefaciens metabolic engineering, can improve the expression activity of alkaline protease by 144%, lays a foundation for mediating the expression of heterologous alkaline protease genes in bacillus amyloliquefaciens expression systems, and promotes the efficient expression and industrial production of alkaline protease.
Description
Technical Field
The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to a combined promoter pα2- α2 and application thereof.
Background
Alkaline protease (Alkaline protease), an enzyme capable of catalyzing and hydrolyzing peptide bonds, the active center of which contains serine, also known as serine protease, is an enzyme capable of hydrolyzing protein peptide bonds in the pH value alkalescence range, and has the functions of hydrolyzing peptide bonds, hydrolyzing amide bonds, ester bonds, and transesterifying and transpeptiding. Such enzymes are widely found in animal pancreas, bacteria, mold, and the enzyme activity can be specifically inhibited by Diisopropylphosphoryl Fluoride (DFP), phenylmethylsulfonyl fluoride (PMSF), potato Inhibitor (PI), and the like.
Alkaline proteases find wide use in the food, washing, and tanning industries. As the microbial protease is extracellular enzyme, compared with animal and plant source protease, the microbial protease has the advantages of relatively simple downstream technical treatment, low cost, wide sources, easy culture of thalli, high yield and the like, has all the characteristics of the animal and plant protease, has stronger hydrolysis capability and alkali resistance than neutral protease, has higher heat resistance and has certain esterase activity, and is easy to realize industrial production.
Bacillus amyloliquefaciens is taken as a gram positive bacterium, and is an ideal host for expressing and secreting exogenous proteins in a prokaryotic expression system at present because of the characteristics of non-pathogenicity, strong protein secretion capability, and relatively easy transfer of recombinant DNA and good fermentation foundation and production technology, and becomes an important mode strain in the prokaryotic expression system.
One of the key factors for realizing the efficient expression of the foreign protein is the use of a high-strength and easily controllable promoter. The promoter (promoter) is a specific DNA sequence that recognizes, binds to and initiates transcription by an 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 insertion or deletion of the promoter, so that the research on the growth and development of thalli and metabolic regulation is realized. Promoters are also the basis for constructing various expression systems to achieve heterologous gene expression. Therefore, screening for a strong promoter is a very effective method for mediating the expression of protease genes and improving the yield of alkaline proteases.
Disclosure of Invention
Aiming at the current industrial demand and the deficiency of the prior art, the invention mainly aims to provide a promoter and a genetically engineered bacterium expression system for realizing the efficient expression of target genes.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a combined promoter, the nucleotide sequence of which is shown in SEQ ID NO: 2.
In a second aspect, the invention provides an expression vector comprising said combined promoter.
In a third aspect, the invention provides an expression system comprising a combination promoter or expression vector as described above.
In a fourth aspect, the present invention provides the use of a combination promoter 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, a novel combined promoter with improved strength is obtained by analyzing and reforming a bacillus subtilis-derived promoter, the combined promoter is suitable for a bacillus amyloliquefaciens expression system, the alkaline protease expression activity can be improved by 144%, a foundation is laid for mediating the expression of a heterologous alkaline protease gene in the bacillus amyloliquefaciens expression system, and the efficient expression and the industrial production of alkaline protease are promoted.
Drawings
Fig. 1: recovering and verifying each fragment of the recombinant vector in the example 2; wherein M: a Marker;1: promoters pα2- α2;2: control promoter ply-2.
Fig. 2: recombinant strain expressed alkaline protease activity comparison in example 2.
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 combined promoter, the nucleotide sequence of which is shown in SEQ ID NO: 2.
In a second aspect, the invention provides an expression vector comprising said combined promoter. 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 combination promoter or expression vector as described above. The expression system may be any host suitable for the combined promoters or expression vectors of the present invention, such as bacillus amyloliquefaciens.
According to a preferred embodiment of the present invention, the host is a Bacillus amyloliquefaciens genetically engineered bacterium Δ6Δeps ΔpgsΔ3049-3052 (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 combination promoter 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 combined promoter is used to control the expression of the alkaline protease gene aprE in the Bacillus amyloliquefaciens system, preferably the alkaline protease gene has a nucleotide sequence as set forth in SEQ ID NO:3, genBank: FJ940727.1.
The present invention will be described in more detail with reference to specific examples. In the following examples, unless otherwise specified:
the culture medium and the enzyme activity determination method used in the invention are as follows:
seed culture medium: 5g/L yeast powder, 10g/L peptone and 5g/L sodium chloride.
Fermentation medium: 64g/L corn flour, 40g/L bean cake powder, 4g/L disodium hydrogen phosphate, 0.3g/L potassium dihydrogen phosphate and 0.7g/L high temperature amylase.
Bacillus subtilis competent preparation medium:
SP-a salt solution: (NH 4) 2 SO 4 4g/L，K 2 HPO 4 ·3H2O 28g/L，KH 2 PO 4 12g/L, sodium citrate 2g/L;
SP-B salt solution: mgSO (MgSO) 4 ·7H 2 O 0.4g/L；
100 XCAYE solution: 20g/L casein hydrolysate and 100g/L yeast powder;
SPI (200 mL): 98mL of SP-A saline solution, 98mL of SP-B saline solution, 2mL of 50% glucose, 100 XCAYE 2mL;
SPII medium (600 mL): SPI 588mL,50mmol/L CaCl 2 6mL，250mmol/L MgCl 2 6mL；
100 XEGTA solution: 10mmol/L EGTA solution.
Bacillus amyloliquefaciens competent preparation medium:
LBS medium: 5g/L of yeast powder, 10g/L of peptone, 5g/L of sodium chloride and 9.1085g/L of sorbitol;
resuscitating medium: 5g/L of yeast powder, 10g/L of peptone, 5g/L of sodium chloride, 9.1085g/L of sorbitol and 6.92246g/L of mannitol.
The method for measuring the enzyme activity of the alkaline protease used in the invention is carried out according to the GB/T23527-2009 annex B Fu Lin Fen method, namely 1 enzyme activity unit (U/mL) is defined as the amount of enzyme required by 1mL of enzyme solution to react for 1min at 40 ℃ and pH of 10.5 to hydrolyze casein to generate 1 mug of tyrosine.
Example 1: novel combined promoter and construction of plasmid thereof
The promoter ply-2 (nucleotide sequence shown as SEQ ID NO: 1) of the a-amylase gene derived from Bacillus subtilis is analyzed by promoter predictive analysis software iProEP, softberry and the like, and found to have two-10 regions and two-35 regions, the front and rear two fragments comprising the-10 region and the-35 region are respectively named pα1 and pα2, two fragments pα21 and pα22 with homologous arms of pα2 are obtained by PCR amplification by taking ply-2 as a template, and the two fragments are further subjected to tandem combination to obtain a novel combined promoter named pα2- α2, the nucleotide sequence of which is shown as SEQ ID NO: 2. Primers used are shown in the following table (note: capital letters indicate homology arms).
| Primer name | Primer sequences | Annealing temperature |
| Pα2-F1 | TATGGTTTTGGTCGGCACTtgaagctggcttacagaagagc | 52℃ |
| Pα2-R1 | TTCTGTAAGCCAGCTTCAtcttgacactccttatttgatt | 52℃ |
| pWB980-F | TCAAATAAGGAGTGTCAAGAccaggagggctggaa | 52℃ |
| pWB980-R | TCTTCTGTAAGCCAGCTTCAagtgccgaccaaaac | 52℃ |
| Pα2-F2 | AATCAAATAAGGAGTGTCAAGAtgaagctggcttacagaag | 52℃ |
| Pα2-R2 | GCTTCTTCCAGCCCTCCTGGtcttgacactccttatttgattt | 52℃ |
The annealing temperature of the promoter fragment was 52℃and the extension time corresponded to the length of the gene, and the reaction procedure was as follows:
the annealing temperature of the vector was 52℃and the extension time corresponded to the length of the gene, and the reaction procedure was as follows:
the linear pWB980 vector was seamlessly cloned into a promoter by the following method:
1) Extracting a pWB980 plasmid, and carrying out PCR by taking the pWB980 plasmid as a template;
2) Performing glue recovery and purification on the target fragment;
3) And (3) connecting the recovered promoter fragments palpha 21 and palpha 22 with the linear pWB980 carrier fragments, connecting the promoter fragments and the linear pWB980 carrier fragments in a water bath at 50 ℃ for 15min, and then rapidly placing the promoter fragments and the linear pWB980 carrier fragments on ice for standing for a plurality of minutes.
The connection system is as follows:
vector 2uL
Promoter fragment 3uL
Seamless cloning of enzyme 10uL
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.
Example 2: recombinant alkaline protease genetically engineered bacteria
The alkaline protease gene aprE (GenBank: FJ 940727.1) was amplified by PCR using the Bacillus alcalophilus genome as a template.
The reaction system used for amplifying the target gene was 50. Mu.L, as follows:
the annealing temperature of aprE was 58℃and the extension time corresponds to the length of the gene, the reaction procedure was as follows:
respectively carrying out enzyme digestion on alkaline protease gene aprE and palpha 2-alpha 2-pWB980 plasmid containing a combined promoter, connecting, constructing a recombinant expression vector containing the promoters palpha 2-alpha 2 and aprE gene expression cassette, and carrying out transformation and transformation into bacillus subtilis WB 600; extracting plasmids, and transferring the recombinant plasmids into a bacillus amyloliquefaciens genetic engineering bacterium delta 6 deltaeps deltapgs 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 the recombinant strain of the heterologously expressed alkaline protease.
The enzyme digestion system is as follows:
the digestion of the expression vector and the connection with the target gene are as follows:
1) Extracting vector plasmid palpha 2-alpha 2-pWB980 containing the combined promoter, and then carrying out double digestion on the plasmid according to the required restriction enzymes (HindIII and SphI), wherein the digestion conditions are 37 ℃ and 2 hours;
2) Carrying out the same double enzyme digestion on the target gene, and carrying out gel recovery and purification on the fragments;
3) The recovered target gene fragment and the linearized pα2- α2-pWB980 fragment are connected under the following connection conditions at 16 ℃ for 6 hours or overnight:
4.5 mu L of the target Gene fragment
Linear pα2- α2-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 extracted in WB600 was electrotransformed into Bacillus amyloliquefaciens Δ6Δeps ΔpgsΔ3049-3052 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 (fragment recovery verification is shown in figure 1).
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 recombinant alkaline protease genetically engineered bacteria
Single colonies of recombinant genetically engineered bacteria and control bacteria on fresh plates were inoculated into 50mL of kanamycin-resistant seed medium, cultured with shaking at 37℃and 220rpm for 12h, inoculated into fermentation medium containing kanamycin resistance in the same inoculum size, and cultured with fermentation at 37℃and 220 rpm.
According to national standard GB/T23527-2009 annex B Fu Lin Fenfa, measuring the enzyme activity of alkaline protease in recombinant genetically engineered bacterium fermentation supernatant, respectively taking fermentation liquids of recombinant bacterium fermentation culture for 12h, 24h, 36h, 48h and 60h, and measuring that the activity of the recombinant alkaline protease in the recombinant bacterium fermentation supernatant containing the combined promoter reaches the maximum value of 25686U/mL when 48h is measured, wherein the activity is 144% of the expression activity of a control bacterium (shown in figure 2). The invention provides a combined promoter pα2- α2 with higher strength, and the expression activity of alkaline protease in a bacillus subtilis system can be obviously improved by using the promoter.
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
SHANDONG LONCT ENZYMES Co.,Ltd.
<120> a combined promoter pα2- α2 and use thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 596
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 1
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> 2
<211> 588
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tgaagctggc ttacagaaga gcggtaaaag aagaaataaa aaagaaatca tcttttttgt 60
ttggaaagcg agggaagcgt tcacagtttc gggcagcttt ttttatagga acattgattt 120
gtattcactc tgccaagttg ttttgataga gtgattgtga taattttaaa tgtaagcgtt 180
aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 240
gggatttttg actccgaagt aagtcttcaa aaaatcaaat aaggagtgtc aagatgaagc 300
tggcttacag aagagcggta aaagaagaaa taaaaaagaa atcatctttt ttgtttggaa 360
agcgagggaa gcgttcacag tttcgggcag ctttttttat aggaacattg atttgtattc 420
actctgccaa gttgttttga tagagtgatt gtgataattt taaatgtaag cgttaacaaa 480
attctccagt cttcacatcg gtttgaaagg aggaagcgga agaatgaagt aagagggatt 540
tttgactccg aagtaagtct tcaaaaaatc aaataaggag tgtcaaga 588
<210> 3
<211> 1143
<212> DNA
<213> Bacillus alcalophilus (Bacillus alcalophilus)
<400> 3
atgaagaaac cgttggggaa aattgtcgca agcaccgcac tactcatttc tgttgctttt 60
agttcatcga tcgcatcggc tgctgaagaa gcaaaagaaa aatatttaat tggctttaat 120
gagcaggaag ctgtcagtga gtttgtagaa caagtagagg caaatgacga ggtcgccatt 180
ctctctgagg aagaggaagt cgaaattgaa ttgcttcatg aatttgaaac gattcctgtt 240
ttatccgttg agttaagccc agaagatgtg gacgcgcttg aactcgatcc agcgatttct 300
tatattgaag aggatgcaga agtaacgaca atggcgcaat cagtgccatg gggaattagc 360
cgtgtgcaag ccccagctgc ccataaccgt ggattgacag gttctggtgt aaaagttgct 420
gtcctcgata caggtatttc cactcatcca gacttaaata ttcgtggtgg cgctagcttt 480
gtaccagggg aaccatccac tcaagatggg aatgggcatg gcacacatgt ggccgggacg 540
attgctgctt taaacaattc gattggcgtt cttggcgtag cgccgagcgc ggaactatac 600
gctgttaaag tattaggggc gagcggttca ggttcggtca gctcgattgc ccaaggattg 660
gaatgggcag ggaacaatgg catgcacgtt gctaatttga gtttaggaag cccttcgcca 720
agtgccacac ttgagcaagc tgttaatagc gcgacttcta gaggcgttct tgttgtagcg 780
gcatctggga attcaggtgc aggctcaatc agctatccgg cccgttatgc gaacgcaatg 840
gcagtcggag ctactgacca aaacaacaac cgcgccagct tttcacagta tggcgcaggg 900
cttgacattg tcgcaccagg tgtaaacgtg cagagcacat acccaggttc aacgtatgcc 960
agcttaaacg gtacatcgat ggctactcct catgttgcag gtgcagcagc ccttgttaaa 1020
caaaagaacc catcttggtc caatgtacaa atccgcaatc atctaaagaa tacggcaacg 1080
agcttaggaa gcacgaactt gtatggaagc ggacttgtca atgcagaagc ggcaacacgc 1140
taa 1143
<210> 4
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tatggttttg gtcggcactt gaagctggct tacagaagag c 41
<210> 5
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ttctgtaagc cagcttcatc ttgacactcc ttatttgatt 40
<210> 6
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tcaaataagg agtgtcaaga ccaggagggc tggaa 35
<210> 7
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tcttctgtaa gccagcttca agtgccgacc aaaac 35
<210> 8
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
aatcaaataa ggagtgtcaa gatgaagctg gcttacagaa g 41
<210> 9
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gcttcttcca gccctcctgg tcttgacact ccttatttga ttt 43
<210> 10
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
ccaagcttat gaagaaaccg ttggggaaaa ttg 33
<210> 11
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ggcatgctta gcgtgttgcc gcttctgc 28
Claims (6)
1. A combined promoter, characterized in that the nucleotide sequence of the combined promoter is shown in SEQ ID NO: 2.
2. An expression vector comprising the combination promoter 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 combination promoter 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 combined promoter.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210671305.XA CN115125247B (en) | 2022-06-14 | 2022-06-14 | Combined promoter palpha 2-alpha 2 and application thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108004239A (en) * | 2017-10-27 | 2018-05-08 | 天津科技大学 | A kind of Novel promoter of high efficient expression protease |
| 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 |
| CN109852615A (en) * | 2019-01-17 | 2019-06-07 | 天津科技大学 | A kind of bidirectional promoter that can express alkali protease, application, plasmid and genetic engineering bacterium |
| 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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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108004239A (en) * | 2017-10-27 | 2018-05-08 | 天津科技大学 | A kind of Novel promoter of high efficient expression protease |
| 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 |
| CN109852615A (en) * | 2019-01-17 | 2019-06-07 | 天津科技大学 | A kind of bidirectional promoter that can express alkali protease, application, plasmid and genetic engineering bacterium |
| 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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