CN106676125A - Maltose promoter-containing carrier and maltose promoter mutant - Google Patents
Maltose promoter-containing carrier and maltose promoter mutant Download PDFInfo
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- CN106676125A CN106676125A CN201510753590.XA CN201510753590A CN106676125A CN 106676125 A CN106676125 A CN 106676125A CN 201510753590 A CN201510753590 A CN 201510753590A CN 106676125 A CN106676125 A CN 106676125A
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Abstract
The invention discloses a maltose promoter-containing carrier and a maltose promoter mutant. The invention establishes a novel carrier which can be expressed in a prokaryotic host, a nucleic acid sequence containing an inducible promoter of a maltose operon in bacillus subtilis, and mutant sequences of the nucleic acid sequence, wherein the carrier and the nucleic acid sequence are respectively applied to transformation of host cells so as to express exogenic nucleic acid sequences of encoded polypeptides.
Description
Technical field
The present invention relates in prokaryotic host cell effable carrier, which includes maltose inducible promoter, the promoter be used for encode such as nucleic acid of polypeptide, recombiant protein heterogenous expression.Specifically, the present invention relates to be used to carry out the new carrier of heterogenous expression in host, which includes the promoter region of the maltose operon for being operably connected to transcriptional units, the transcriptional units are comprising being heterologous nucleotide sequence for the host, and the expression of the nucleotide sequence is controlled by the promoter region of the maltose operon.Additionally, the present invention relates to the use of these carriers is used for heterologous coding schedule up to the purposes of such as nucleic acid of polypeptide, recombiant protein.
Background technology
The heterogenous expression production of polypeptide and recombiant protein has great importance in protein engineering, wherein the large-scale production for carrying out polypeptide and recombiant protein using prokaryote has the advantages such as expressing quantity is high, fermentation density is big, fermentation costs are cheap.
The expression vector and expression system set up in prokaryotic cell at present is a lot, wherein abduction delivering is carried out using inducer be used to set up corresponding inducible expression because of the advantage such as its expression intensity is high, expression time is controllable, such as T7, arabinose (Arabinose) inducible expression in gram negative bacteria escherichia coli, the xylose inducible expression in gram positive bacteria bacillus cereuss etc..
By inducible promoter sequence is connected to target protein nucleotide sequence in these systems, build the inducible expression vector containing target protein nucleotide sequence, and the culture under different condition is carried out by this inducible expression vector is converted Host Strains, under the condition of culture for being not added with inducer, repressor is combined and is prevented the transcription of genes of interest with promoter, or the transcription that son cannot be incorporated into activating genes of interest in promoter due to lacking inducer is activated in the case of another kind；Under the condition of culture for adding appropriate inducer, it is initial so as to transcribe that repressor is induced thing inactivation, or inducer combines rear activation promoter with activation in the case of another kind, so as to genes of interest transcribe it is initial.
At present typical inducer can be the substrate needed for prokaryotic host cell metabolism, such as different types of sugar.But some inducers such as gala sugar analogue IPTG, tetracycline etc. have cytotoxicity, some inducers such as price such as Lactose, arabinose, xylose costly, limits application of the corresponding inducible expression in large-scale polypeptide and recombiant protein commercial production.
The content of the invention
The present invention is by providing a kind of new expression vector in prokaryotic hosts, promoter region of the described new support comprising the maltose operon for being operatively attached to transcriptional units or its complementary series or mutant sequence, the transcriptional units are comprising being heterologous nucleotide sequence for the host, and the expression of the nucleotide sequence is controlled by the promoter region of the maltose operon.
Present invention also offers various host cells that heterogenous expression is carried out for the new expression vector, described host cell is transformed through molecular genetics, including the loop-stem structure sequence, alpha-glucosidase gene MalL, the term single gene deletion mycopremna of maltose phosphorylase gene yvdK or the combination gene deletion mycopremna that have lacked 6- phosphoric acid malto-hydrolase gene M alA, 6- phosphoric acid Maltose hydrolysis enzyme gene and its 3 ' end rears；Include the bacterial strain of maltose activating transcription factor gene M alR, Fructus Hordei Germinatus Sugar phosphorylation transporter gene MalP term single genes Enhanced expressing or combination Enhanced expressing simultaneously.
The present invention also provides heterogenous expression purposes of the nucleotide sequence in prokaryotic hosts by new expression vector, the nucleotide sequence for isolating and purifying expressed will be needed to be connected in described new expression vector, which includes the promoter region of maltose operon, the expression vector of the nucleotide sequence that the carrying for building is isolated and purified is converted to prokaryotic host cell, by changing the method that condition of culture produces polypeptide and recombiant protein in host cell.
Description of the drawings
Fig. 1：It is derived from the nucleotide sequence and structure of the MalA promoter regions of the maltose operon of bacillus subtilises (B.subtilis), wherein transcriptional start site is represented with asterisk, -35 regions and -10 regions of presumption are with box indicating, the cre sequences of presumption are represented by dotted lines, ribosome binding sequence represents that with underscore MalA gene-starts are depicted with arrows.
Fig. 2：It is the plasmid map for detecting the carrier pDG of maltose promoter Intensity of Transcription of Endothelial.
Fig. 3：It is the plasmid map of the expression vector pMATE-rep and pMATE-int of the present invention.
Fig. 4：It is green fluorescent protein fluorescence signal intensity (RFU) mapping comprising the bacillus subtilises 1A751 for carrying different maltose promoter mutant plasmid pDG.
Fig. 5：It is green fluorescent protein fluorescence signal intensity (RFU) mapping comprising the bacillus subtilises 1A751 genetic modification bacterial strains for carrying different maltose promoter mutant plasmid pDG.
Fig. 6：It is the structure flow chart of plasmid pDG.
Fig. 7：It is the structure flow chart of plasmid pMATE-rep and pMATE-int.
Fig. 8：It is the SDS-PAGE figures of the bacillus subtilises 1A751 of the pMATE-int plasmids for carrying expressing green fluorescent protein gene GFP genes.
Fig. 9：It is the SDS-PAGE figures of the bacillus subtilises 1A751 of the pMATE-rep plasmids for carrying expressing green fluorescent protein gene GFP genes.
Specific embodiment
If not having other to illustrate, following material and method are used：
Bacterial isolateses and growth conditionss：
Bacillus coli DH 5 alpha and bacillus subtilis 1A751 (BGSC, USA) as main host, for cloning and expressing.Escherichia coli are grown on LB fluid mediums (Luria S.E. et al., Virology 12,1960,348-390) and LB agar plates in 37 DEG C, wherein supplementing 100 μ g/ml ampicillin.Bacillus subtilises are grown on LB fluid mediums and SR fermentation medium in 37 DEG C.Fluid medium and agar plate supplement 20 μ g/ml kanamycin or 5 μ g/ml chloromycetin respectively.In order to induce maltose promoter, add aseptic filtration or autoclaved D-Maltose to 1% (w/v) ultimate density.
Material：
All of chemicals are all available from Sigma-Aldrich or Merck.DNA widow's core former times acid of synthesis is purchased from gold only intelligence Genewiz.Restriction Enzyme and DNA modification enzyme are purchased from New England Biolabs..With the high-fidelity DNA polymerase from Fermentas in the enterprising performing PCR of thermal cycler from Eppendorf.
The preparation and conversion of DNA：
Using the DNA reagent preparation boxes of Tiangen or Omega, according to the description of manufacturer, DNA is separated with bacillus subtilis or from agarose gel from escherichia coli.In all embodiments using the molecular engineering of standard.Using such as Chung C.T. et al., the plasmid DNA transformation escherichia coli of the descriptions of Proc.Natl.Acad.Sci.USA 86,1989,21722175.According to improved " Paris methods " (HarwoodC.R.Molecular Biological Methods for Bacillus, 1990, John Wiley＆Sons Ltd., England), using plasmid DNA or DNA fragmentation conversion bacillus subtilis.
The measure of green fluorescent protein fluorescence intensity RFU：
Culture to OD600 is about into the test strains of 0.6-0.8 in 4 DEG C, 4000rpm is centrifuged 10 minutes collects thallines, with the PBS solution washing thalline 2 times of pre-cooling, shift 150 holes of μ L to 96 black bottom permease target (Corning, USA in), it is positioned over microwell plate SpectraMax M2 microplate reader (Molecular Devices, USA) under room temperature condition, 483nm/525nm is excited and is detected fluorescent emission light intensity RFU, cell concentration is determined in 600nm, fluorescence intensity is calculated and calculated according to RFU/OD600.
Rite-directed mutagenesises or random mutation：
Using the Qucikchange II plasmid DNA mutagenesis kits of Strategene, according to the description of manufacturer, rite-directed mutagenesises or the random mutation based on degenerate primer are carried out to plasmid DNA.The up/down trip primer of mutant primer is reverse complementary sequence, in the design of the end of mutational site 5 ' no less than 8 nucleotide matched with template DNA, in the design of the end of mutational site 3 ' no less than 12 nucleotide matched with template DNA.The program of mutation PCR is similar to Standard PCR but slightly changes, reaction cycle number foundation mutational site number be adjusted, mutational site≤3, period are 12；4≤mutational site≤6, period are 16；Mutational site >=6, period are 20.PCR primer after amplification converts escherichia coli Jing after DpnI digestion with restriction enzyme, and the transformant for obtaining extracts plasmid DNA retention Jing after sequence verification.
Bacillus subtilis genes traceless knockout：
According to improved " the Bacillus subtilis genes knockout technique based on AraR " (Liu S, Endo K, et al.Microbiology.2008；154(Pt 9):2562-2570.) carry out gene traceless knockout.Spectinomycin resistance gene fragment " the P regulated and controled by Arabinose promoter of homology arm will be carried first with homologous recombination principleara- spc " is proceeded in bacillus subtilises 1A751 bacterial strains by method for transformation, the AraR genes in replacement gene group, builds original gene knockout original bacteria, and which carries Spectinomycin resistance.When needing to knock out gene X, upstream 1Kb fragments (being named as UP), downstream 1Kb fragments (being named as DN), gene X fragments to be knocked out (being named as G) and the screening fragment (being named as CR) with AraR genes and chloramphenicol resistance gene cat of X gene are cloned respectively, above-mentioned 4 fragments are permeated fragment with the order of " UP-DN-CR-G " using overlap-PCR methods, and proceeded to by method for transformation and carry out in bacillus subtilises 1A751 homologous recombination, transformant is screened with chlorampenicol resistant.The transformant for obtaining is verified on Spectinomycin resistance LB flat boards, be chosen on chloromycetin growth, and the transformant that do not grow on spectinomycin as positive transformant.By positive transformant in nonresistant LB culture medium with 37 DEG C, 200rpm is cultivated 16 hours, is taken 10uL bacterium solutions and is coated and screened on Spectinomycin resistance flat board, the transformant for obtaining is verified by bacterium colony PCR, determines whether gene knocks out.Bacterium colony PCR is verified into that correct bacterial strain is preserved as the bacterial strain for knocking out X gene.
Used oligonucleotide：
Table 1
The structure of the pDG carriers of 1 deletion promoters sequence of embodiment Carrying Green Fluorescent Protein GFP.
With in bacillus subtilises commonly use integrative plasmid pDL (BGSC, USA) for skeleton, using CPEC PCR cloning methods (Nature Protocols, 6,242-251,2011, doi:The bgaB genes on pDL plasmids are replaced with into GFP genes 10.1038/nprot.2010.181), concrete grammar is to separately design primer pDG-vector.for/pDG-vector.rev Inverse PCR amplification pDL plasmids in the bgaB gene upstream and downstream of pDL plasmids, primer gfp-insert.for/gfp-insert.rev is designed simultaneously from pSG1729 plasmid (BGSC, USA PCR amplifications gfp genes on), 5 ' ends of wherein gfp-insert.for/gfp-insert.rev primers are respectively provided with the homologous sequence of pDL plasmid purpose insertion point both sides 20bp.The gfp Insert Fragments and pDL carrier segments that amplification is obtained is cut after glue reclaim, according to mol ratio 1:1 ratio enters performing PCR reaction in being added to PCR system, the PCR primer of gained directly converts escherichia coli, positive transformant is screened using amicillin resistance, extracting plasmid Hou Songjinwei intelligence company carries out sequencing identification, correct plasmid is sequenced and is named as pDG, profiling results are shown in Fig. 2, structure flow processs of the Fig. 6 for pDG plasmids.
Separation and identification of the embodiment 2 from the MalA promoteres of the maltose operon of bacillus subtilises.
(1) from bacillus subtilises maltose operon MalA promoteres separation.
Using the chromosomal DNA of bacterial chromosome extracts kit (Beijing, China) the separation and Extraction bacillus subtilises 1A751 of Tiangen.Obtain the DNA fragmentation of about 340bp between yfiA and MalA genes using primer Pglv.for/Pglv.rev from the chromosomal DNA of bacillus subtilises 1A751 by PCR amplifications.This fragment is cloned in into the upstream of described pDG plasmid green fluorescent protein GFP genes by BanHI/KpnI sites, the pDG plasmids for carrying the maltose operon MalA promoteres from bacillus subtilises is obtained, is named as pDG-G1.
(2) Structural Identification of maltose operon MalA promoteres.
According to the MalA promoter sequence SEQ ID NO of sequencing gained：1, using online promoter in prokaryote forecast analysis software BPROM (V.Solovyev, A Salamov, 2011, Nova Science Publishers, p.61-78) MalA promoteres are analyzed, -35th area, -10th area, ribosome binding site RBS and cre element in promoter sequence are marked out respectively, is as a result shown in Fig. 1.
(3) in maltose operon MalA promoteres activating transcription factor MalR binding site sequences identification.
Exist while inducer maltose and activating transcription factor MalR are depended on from the transcription initiation of the maltose operon MalA promoteres of bacillus subtilises, activating transcription factor MalR activates the transcriptional activity of promoter after combining promoter DNA sequence, if binding sequence of the promoter deletion with activating transcription factor MalR, promoter transcription activity cannot be activated.In order to position the binding site sequence of activating transcription factor in bacillus subtilises maltose operon MalA promoteres, according to the analysis result of MalA promoter sequence structures in experiment 2, on the basis of pDG-G1 plasmids, before from promoter sequence section start to -35 region sequences, lack this interregional promoter sequence in units of 18bp respectively, build promoter deletion mutant, conversion bacillus subtilises 1A751 and by detection cultivate after thalline fluorescence intensity, judge impact of the deleting DNA sequences to MalA promoter transcription activation capabilities, if thalline unstressed configuration signal after disappearance, binding site of this fragment comprising activating transcription factor is illustrated then, so that it is determined that binding site sequences of the activating transcription factor MalR in MalA promoteres.The final activating transcription factor MalR for obtaining is shown in SEQ ID NO in the binding site sequence of MalA promoteres：2.
The structure of 3 integrated expression vector pMATE-int of embodiment.
Construct containing the maltose operon MalA promoteres or its mutant from bacillus subtilises, the integrated expression vector of Bacillus subtilis genes group amyE gene locis can be incorporated into and pMATE-int is named as.Construction method is：Based on pDL plasmids (BGSC, USA), so that KpnI and BamHI cuttings are from the maltose operon MalA promoteres or its mutant DNA fragments of bacillus subtilises and are connected into pDL plasmids.Plasmid backbone and Insert Fragment are expanded respectively using oligonucleotide pMATE-int-vector.for/pMATE-int-vector.rev and superMCS-insert.for/superMCS-insert.rev, the bgaB genes on plasmid are replaced with the multiple clone site superMCS fragment from pSE420 plasmids in the CPEC PCR cloning methods in embodiment 1, construct integrated expression vector pMATE-int, plasmid map is shown in Fig. 3, plasmid construction process is shown in Fig. 6, and the oligonucleotide for being used is as shown in table 1.
The structure of 4 science expression vector pMATE-rep of embodiment.
Construct containing the maltose operon MalA promoteres or its mutant from bacillus subtilises, the shuttle expression carrier that can be replicated in bacillus subtilises and escherichia coli is simultaneously named as pMATE-rep.Construction method is：With the pMA5 plasmids with pUB110 replicons as skeleton, plasmid backbone is expanded respectively and from bacillus subtilises maltose operon MalA promoteres or the Insert Fragment of its mutant using oligonucleotide pMATE-rep-vector.for/pMATE-rep-vector.rev and PmalA-insert.for/PmalA-insert.rev, MalA promoteres are inserted in pMA5 carriers in the CPEC PCR cloning methods in embodiment 1.Simultaneously according to the method in embodiment 3, after the multiple clone site superMCS fragment insertion MalA promoteres or its mutant from pSE420 plasmids, construct science expression vector pMATE-rep, plasmid map is shown in Fig. 3, plasmid construction process is shown in Fig. 6, and the oligonucleotide for being used is as shown in table 1.
Screening mutant of the embodiment 5 from the maltose operon MalA promoteres of bacillus subtilises.
Based on the pDG carriers in embodiment 1, the mutant library of the maltose operon MalA promoteres from bacillus subtilises is built by rite-directed mutagenesises or random mutation, by detecting the green fluorescence protein gene gfp of promoter abduction delivering evaluating the height of MalA promoter mutation body transcriptional activities after converting bacillus subtilises.Concrete grammar is, using site-directed mutagenesis technique (Strategene, USA rite-directed mutagenesis primer or degenerate primer are designed), -35th area, -15th area, -10th area for MalA promoteres, RBS areas, cre areas, activating transcription factor binding site (operator) region carry out rite-directed mutagenesises or degenerate primer random mutation PCR, build with different plasmid libraries, used oligonucleotide is as shown in table 2.
By the rite-directed mutagenesises plasmid for obtaining so that bacillus subtilises 1A751 is converted after PstI digestion with restriction enzyme linearisations, screened using the LB solid medium flat boards with chlorampenicol resistant, the transformant for being obtained is preserved after PCR checkings are correct.After scribed line culture activation, picking monoclonal is inoculated in the 96 hole depth orifice plates containing 0.5mL culture medium and carries out overnight fermentation.Next day, draw 5 μ L overnight cultures be inoculated in respectively without/containing 1% (w/w) maltose inducer fresh LB in fermented, then the GFP expression strains of unmutated MalA promoteres are carried as control, carry out the measure of fluorescence intensity, measurement result is shown in Fig. 4 A.
The degenerate primer random mutation plasmid library of acquisition is converted into bacillus coli DH 5 alpha and resistant panel is coated, the bacterium colony on flat board is scraped and is resuspended in fresh LB using cell sleaker after culture and cultivated, pDG mixing plasmids with difference MalA promoter mutation bodies are extracted then, bacillus subtilises 1A751 is converted after PstI digestion with restriction enzyme linearisations, screened using the LB solid medium flat boards with chlorampenicol resistant, obtain single copy and be integrated in the GFP expression strains induced by different MalA promoter mutations bodies in Bacillus subtilis genes group.LB of this GFP expression strain Jing 1% (w/w) maltose inducer is contained is cultivated after activation, the fresh bacterium solutions of 1mL are taken in 4 DEG C, 12000rpm is centrifuged 2 minutes collects thallines, with the resuspended washing thalline of phosphate buffered solution PBS 3 times, with 50 times of the resuspended rear dilution of equal-volume PBS, the unicellular green fluorescence sortings of FACS are carried out using flow cytometer (Beckman, USA), 1% cell of RFU fluorescent values highest is chosen and is collected.The cell collected is repeated into above-mentioned FACS assorting rooms 2 times to obtain the stable GFP expression strains induced by different MalA promoter mutations bodies.By this unicellular inoculation in the 96 orifice plate (Corning containing 150 μ L fresh LBs, USA incubated overnight in), next day, draw 5 μ L overnight cultures be inoculated in respectively without/containing 1% (w/w) maltose inducer fresh LB in fermented, then the GFP expression strains of unmutated MalA promoteres are carried as control, carry out the measure of fluorescence intensity.15 plants of bacterial strains that fluorescence intensity substantially increases are chosen according to measurement result, genome amplification MalA promoter fragment sequences is extracted and is sequenced, to obtain the site information of mutant nucleotide sequence, this 15 MalA promoteres are named asmut01PmalA-mut015PmalA.Fluorescent strength determining result is shown in Fig. 4 B, and the sequence of MalA promoter mutation bodies is shown in SEQ ID NO：3-SEQ ID NO：17.
Table 2
Intensity of Transcription of Endothelial optimization of the embodiment 6 from the maltose operon MalA promoteres of bacillus subtilises.
According to document (Yamamoto, H., et al.Journal of bacteriology 183 (17):5110-5121.2001) report, " CG " by the 6th and the 7th nucleotide (i.e. 315 and 316 of bacillus subtilises MalA promoter sequences) of the MalA promoter cre elements of Mutant Bacillus subtilis maltose operon is " AT ", can with past release in the presence of glucose to maltose MalA promoter transcriptions activity depression effect.In addition, according in embodiment 5 for -35th area of MalA promoteres, -15th area, -10th area, RBS regions point mutation experimental result, promoter with -10 area A303T mutation improves Intensity of Transcription of Endothelial while preciseness is kept, hence with point mutation technology, above-mentioned C315A, G316T and A303T mutation is introduced into pDG plasmids, pDG2 Baseline mutation plasmids are constructed.According to flow cytometer FACS the selection results, wherein there are 15 plants of bacterial strains for carrying the sub- pDG plasmids of bacillus subtilises maltose operon MalA promoter mutations that there is the fluorescence intensity for substantially increasing, its maltose operon MalA promoter mutation site informations are obtained after sequencing, and (sequence is shown in SEQ ID NO：3-SEQ ID NO：17).As a result show, mutational site occur mainly in promoter 209,210,212,216,217,219-225 positions, 227 and 228, particularly, after G209T, A210T, A212T, C216T, C217T, G219M, C220K, C222T and A224T site is sported, the Intensity of Transcription of Endothelial of muton is remarkably reinforced.Mutation and fluorescence intensity screening are combined on the basis of pDG2 plasmids to above-mentioned mutational site, the MalA promoter mutation bodies with higher Intensity of Transcription of Endothelial are obtained, sequence is shown in SEQ ID NO：18-SEQ ID NO：32.
The genetic modification of 7 bacillus subtilises expressive host bacterial strain of embodiment and structure.
Respectively the MalA genes in bacillus subtilises 1A751 genomic DNAs, MalA genes and its 3 ' loop-stem structures (MalA-term) of one section of the end similar to terminator, Yvdk-MalL genes are knocked out using previously described genome traceless knockout method, build Bacillus subtilis genes knockout mutations body MATE02-MATE06, its objective is to strengthen sensitivity of the thalline to maltose inducer, extend maltose induction time and reduce maltose inducer consumption.Simultaneously, based on the conformability expression vector pMATE-int built by embodiment 3, locate to insert the MalR genes and MalR-MalP genes by bacillus subtilises MalA promoter regulations respectively behind multiple clone site, and imported in the hay spore for having built expression 1A751 Host Strains by converting, screened using the LB solid medium flat boards with chlorampenicol resistant, the transformant for being obtained is preserved after PCR checkings are correct, obtains genetic modification bacterial strain MATE07-MATE018.Synchronous, based on science expression vector pMA5, locate to insert the MalR genes and MalR-MalP genes by composing type HpaII promoter regulations respectively behind multiple clone site, build pMA5R and pMA5RP and imported in the hay spore expressive host bacterium 1A751 for having built by converting, screened using the LB solid medium flat boards with kalamycin resistance, the transformant for being obtained is preserved after PCR checkings are correct, obtains genetic modification bacterial strain MATE019-MATE024.Then by the pDG plasmid integrations of the carrying GFP genes built in embodiment 1 into above-mentioned genetic modification bacterial strain, transcriptional efficiency impact of the heterogeneic genetic modification on MalA promoteres is evaluated by measuring fluorescence intensity.As shown in table 3, as shown in table 4, the fluorescence intensity comparison diagram of different strains is as shown in Figure 5 for the oligonucleotide for being used for the bacillus cereuss genetic modification bacterial strain that structure is obtained.
Embodiment 8 carries out the fermentation expression of green fluorescent protein using the new expression vector and expressive host.
Based on the 15 bacillus subtilises MalA promoter mutation bodies obtained by embodiment 5, according to the method for embodiment 3, build the pMATE-int integrative plasmids with above mutant promoters and green fluorescent protein GFP genes, bacillus subtilises 1A751 is imported by converting, screened using the LB solid medium flat boards with chlorampenicol resistant, the GFP expression strains induced by different MalA promoter mutations bodies that single copy is integrated in Bacillus subtilis genes group are obtained, the transformant for being obtained after PCR checkings are correct carries out shake flask fermentation and is compared.After bacterial strain scribed line culture activation, picking monoclonal is inoculated in containing after culture 12h in 5mL LB fluid mediums, it is inoculated in fermentation medium of the 25mL without maltose inducer with 1% inoculum concentration and is fermented, adds when OD600 reaches 0.8 1% (w/w) maltose inducer to carry out abduction delivering in the fermentation medium.SDS-PAGE results are as shown in Figure 8.The preferable bacterial strain of 4 plants of expression effects of selection builds the pMATE-rep replication form plasmids with mutant promoters and green fluorescent protein GFP genes according to the method for embodiment 4 on this basis, bacillus subtilises 1A751 is imported by converting, screened using the LB solid medium flat boards with kalamycin resistance, the transformant for being obtained carries out shake flask fermentation comparison after PCR checkings are correct, and sweat is as described above.Whole sweat carries out SDS-PAGE measure at interval of 24h samplings to carry the GFP expression strains of wild type MalA promoteres as control, and SDS-PAGE is as shown in figure 9, the oligonucleotide for being used is as shown in table 4.
Table 3
Table 4
SEQUENCE LISTING
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gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttctt tcttggggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 9
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 9
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt attttttcgt tctttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 10
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 10
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgagt attttttcat tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 11
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 11
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa ggtaaaaaaa
180
ccaaatctct cagacataag gcaaatgagt atttttccgt tctttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 12
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 12
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt atttttccat tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 13
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 13
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaat actttctcgt tatttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 14
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 14
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgata attttttcat tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 15
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 15
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa ttggaagggc
120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttccg tgagcggtaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 16
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 16
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttcgg attaggtgaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc tcatggtata
300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 17
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 17
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga actttttcat tatttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
aatggaattg taaacgttat caaggaggtc
gtcat
335
<210> 18
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 18
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt aatttttcac tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 19
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 19
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt attttttcgt tctttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 20
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 20
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt attttttcga tctttgggaa aaaacactaa
240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 21
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 21
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttcca attctgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 22
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 22
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaat attttttcgt tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 23
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 23
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttctt tcttggggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 24
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 24
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt attttttcgt tctttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 25
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 25
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgagt attttttcat tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 26
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 26
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgagt atttttccgt tctttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 27
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 27
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa ttggaagggc
120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgatt atttttccat tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 28
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 28
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaat actttctcgt tatttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc tcatggtata
300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 29
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 29
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat aaatgattat
60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgata attttttcat tttttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 30
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 30
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttccg tgagcggtaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 31
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 31
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat aaatgattat
60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga aatttttcgg attaggtgaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
<210> 32
<211> 335
<212> DNA
<213> Bacillus subtilis
<400> 32
cttttgtccc ctgccttttc taaattcacg cacaattgga tgttttatat
aaatgattat 60
aaataattcg gcatgtatcc gaatcgtaca aaagaacctt ttcataagaa
ttggaagggc 120
gtatattcac ttaaaattca cagttggtga gactttaaga ttacaaaaaa
ggtaaaaaaa 180
ccaaatctct cagacataag gcaaatgaga actttttcat tatttgggaa
aaaacactaa 240
agttgatcaa atgacctaag tgcgccaaac gtgttacggg acgagctatc
tcatggtata 300
attggaattg taaaatttat caaggaggtc
gtcat
335
Claims (14)
1. one kind effable carrier in prokaryotic host cell, wherein the maltose inducible promoters of the maltose operon comprising the bacillus subtilises for being operatively attached to transcriptional units, the transcriptional units include the heterologous nucleic acid sequence of coded polypeptide.
2. carrier according to claim 1, wherein the promoter is MalA promoteres.
3. the carrier according to any one of claim 1-2, wherein the MalA promoteres are included selected from SEQ ID
NO:1 sequence, its complementary series or its variant.
4. the carrier of aforementioned any one of claim, wherein the carrier is the reproducible shuttle vector in escherichia coli and bacillus subtilis.
5. the carrier of aforementioned any one of claim, wherein the carrier includes the replication origin of the replication origin of plasmid pUC18 and/or pBS72 replicons or rep genes and plasmid pUB110.
6. the carrier of aforementioned any one of claim, wherein the heterologous nucleic acid sequence coded polypeptide or its fragment.
7. the separation in maltose inducible promoters area of the maltose operon of bacillus subtilis and the nucleotide sequence and its complementary seriess of purification or its variant.
8. the nucleotide sequence of the separation of claim 7 and purification, wherein the sequence includes SEQ ID NO:2 Arbitrary Term, its complementary series or its variant.
9. the prokaryotic host cell for converting with the carrier of any one of claim 1-6 or with the nucleotide sequence of claim 7 or 8 Arbitrary Terms.
10. prokaryotic host cell according to claim 9, wherein described prokaryotic host cell includes wild-type cell and lacked loop-stem structure sequence, alpha-glucosidase gene MalL or its homologous genes, maltose phosphorylase gene yvdK or its homogenic term single gene deletion mycopremna or the combination gene deletion mycopremna at 6- phosphoric acid malto-hydrolase gene Ms alA or its homologous genes, 6- phosphoric acid Maltose hydrolysis enzyme genes or its homologous genes and its 3 ' end rears；Include the bacterial strain of maltose activating transcription factor gene M alR or its homologous genes, Fructus Hordei Germinatus Sugar phosphorylation transporter gene MalP or its homogenic term single gene Enhanced expressing or combination Enhanced expressing simultaneously.
11. prokaryotic host cells according to claim 9 or 10, wherein the prokaryotic host cell is gram-positive.
12. according to claim 10 or the prokaryotic host cell of 11 Arbitrary Terms, wherein the host cell belongs to Firmacutes (Firmicutes）.
13. in prokaryotic host cell produce polypeptide method, comprise the following steps：Build the carrier of any one of claim 1-6；Prokaryotic host cell is converted with the carrier；Grown under appropriate culture medium and condition of culture by the host cell after conversion and express the polypeptide.
14. methods according to claim 13, wherein the host cell is grown first in the case where presence does not have inducer, then grow in the case where there is inducer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904223A (en) * | 2017-12-26 | 2018-04-13 | 中国科学院天津工业生物技术研究所 | A kind of algin catenase, the host cell for secreting algin catenase and its application |
| CN110592131A (en) * | 2018-12-14 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | Mutant library construction screening and application of maltose transcriptional activator MalR |
| CN110592080A (en) * | 2018-12-17 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | Optimized maltose promoter mutant and application thereof |
| CN113061608A (en) * | 2021-03-23 | 2021-07-02 | 江南大学 | Evolution method of inducible promoter and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1261581C (en) * | 1998-04-14 | 2006-06-28 | 皮埃尔法博赫药品公司 | Novel constructs for controlled expression of recombinant proteins in prokaryotic cells |
| CN100445391C (en) * | 2002-12-02 | 2008-12-24 | 巴斯福股份公司 | L-rhamnose-inducible expression systems |
| CN101952435A (en) * | 2008-02-01 | 2011-01-19 | 塞瑞斯公司 | Promoter, promoter control elements, and combinations, and use thereof |
| CN101090969B (en) * | 2004-12-07 | 2013-01-16 | 隆萨股份公司 | Rhamnose promoter expression system and uses thereof |
| CN104630229A (en) * | 2015-02-11 | 2015-05-20 | 华南理工大学 | DNA fragment with promoter function and application |
| CN103194477B (en) * | 2013-04-08 | 2015-05-20 | 武汉华美生物工程有限公司 | Fused type prokaryotic expression vector and construction method and application thereof |
-
2015
- 2015-11-05 CN CN201510753590.XA patent/CN106676125B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1261581C (en) * | 1998-04-14 | 2006-06-28 | 皮埃尔法博赫药品公司 | Novel constructs for controlled expression of recombinant proteins in prokaryotic cells |
| CN100445391C (en) * | 2002-12-02 | 2008-12-24 | 巴斯福股份公司 | L-rhamnose-inducible expression systems |
| CN101090969B (en) * | 2004-12-07 | 2013-01-16 | 隆萨股份公司 | Rhamnose promoter expression system and uses thereof |
| CN101952435A (en) * | 2008-02-01 | 2011-01-19 | 塞瑞斯公司 | Promoter, promoter control elements, and combinations, and use thereof |
| CN103194477B (en) * | 2013-04-08 | 2015-05-20 | 武汉华美生物工程有限公司 | Fused type prokaryotic expression vector and construction method and application thereof |
| CN104630229A (en) * | 2015-02-11 | 2015-05-20 | 华南理工大学 | DNA fragment with promoter function and application |
Non-Patent Citations (4)
| Title |
|---|
| YAMAMOTO,H. 等: ""Bacillus subtilis DNA for 76-degree region, complete cds"", 《GENBANK DATABASE》 * |
| YANG M MING 等: ""Development of a Bacillus subtilis expression system using the improved Pglv promoter"", 《MICROBIAL CELL FACTORIES》 * |
| 余小霞 等: "枯草芽孢杆菌表达系统及其启动子研究进展", 《生物技术通报》 * |
| 潘皎 等: "枯草芽孢杆菌基因启动子的分离与鉴定", 《微生物学报》 * |
Cited By (7)
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| CN110592131A (en) * | 2018-12-14 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | Mutant library construction screening and application of maltose transcriptional activator MalR |
| CN110592131B (en) * | 2018-12-14 | 2021-10-22 | 中国科学院天津工业生物技术研究所 | Mutant library construction screening and application of maltose transcriptional activator MalR |
| CN110592080A (en) * | 2018-12-17 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | Optimized maltose promoter mutant and application thereof |
| CN110592080B (en) * | 2018-12-17 | 2021-11-23 | 中国科学院天津工业生物技术研究所 | Optimized maltose promoter mutant and application thereof |
| CN113061608A (en) * | 2021-03-23 | 2021-07-02 | 江南大学 | Evolution method of inducible promoter and application thereof |
| CN113061608B (en) * | 2021-03-23 | 2022-12-13 | 江南大学 | Evolution method of inducible promoter and application thereof |
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Application publication date: 20170517 Assignee: Tianjin Chunyao Biotechnology Co.,Ltd. Assignor: TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, CHINESE ACADEMY OF SCIENCES Contract record no.: X2023980036854 Denomination of invention: A vector containing Maltose promoter and a Maltose promoter mutant Granted publication date: 20200814 License type: Common License Record date: 20230620 |