CN101487008B - Bacillus licheniformis integration expression box and use thereof - Google Patents
Bacillus licheniformis integration expression box and use thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus licheniformis integrated expression cassette and application thereof. The expression cassette orderly contains LacA gene 5'-arm, hemoglobin gene, selection marker gene and LacA gene 3'-arm; the LacA gene 5'-arm is a DNA segment that has the length of at least 600bp and is selected from Beta-galactosidase acetyltransferase gene 5'-end, and the LacA gene 3'-arm is a DNA segment that has the length of at least 600bp and is selected from Beta-galactosidase acetyltransferase gene 3'-end. A foreign gene expression cassette is arranged between the hemoglobin gene and the selection marker gene of the Bacillus licheniformis expression cassette, and comprises an Alpha-amylase gene promoter, a fructan synthase gene promoter, a multiple cloning site and a terminator orderly from upstream to downstream. The Bacillus licheniformis expression cassette of the invention can produce high-temperature starch after being induced into the Bacillus licheniformis with high-temperature amylase generation and can reach the enzyme activity of 9100U in each milliliter of a fermented fluid.
Description
Technical field
The present invention relates to bacillus licheniformis integration expression box and application thereof.
Background technology
The genetically engineered field expression system of sophisticated foreign protein is intestinal bacteria and yeast expression system.What extensively sell on the market also mainly is intestinal bacteria and yeast expression system.
The Bacillus licheniformis expression system has the following advantages:
(1) Bacillus licheniformis has than the more effective protein excretion mechanism of subtilis, product can be secreted into the extracellular, needs the loaded down with trivial details step of smudge cells when having avoided purified product, thereby improves the product rate of recovery, simplifies purification step.
(2) Bacillus licheniformis itself is the no pathogenicity microorganism to the person poultry harmless, does not have colibacillary intracellular toxin, is the microorganism of food grade safety.
(3) Bacillus licheniformis has been used for industrial production plurality of enzymes preparation, zymotechnique maturation for many years.
Summary of the invention
The purpose of this invention is to provide a kind of bacillus licheniformis integration expression box and application thereof.
Expression cassette provided by the present invention comprises LacA gene 5 ' arm, hemoglobin gene, selection markers gene and LacA gene 3 ' arm successively; Described LacA gene 5 ' arm is to be selected from the beta galactose glycosides acetyltransferase gene 5 ' end dna fragmentation of 600bp at least; Described LacA gene 3 ' arm is selected from beta galactose glycosides acetyltransferase gene 3 ' and holds the dna fragmentation of 600bp at least.
Wherein, also can contain the exogenous gene expression box between described hemoglobin gene and the described selection markers gene, described exogenous gene expression box comprises alpha-amylase gene promotor, Polylevulosan synthase gene promoter and signal peptide sequence, multiple clone site and transcription termination sequence successively to the downstream from the upstream.
The hemoglobin gene of described hemoglobin-based because Vitreoscilla comprises promotor, signal peptide sequence and the encoding gene of hemoglobin gene.The nucleotide sequence of described hemoglobin gene is specially the sequence 6 in the sequence table.Described selection markers gene can be erythromycin resistance gene; The nucleotide sequence of described erythromycin resistance gene is specially the sequence 8 in the sequence table.
The nucleotide sequence of described LacA gene 5 ' arm specifically can be the sequence 1 in the sequence table, and the nucleotide sequence of described LacA gene 3 ' arm specifically can be the sequence 5 in the sequence table.The nucleotide sequence of described alpha-amylase gene promotor specifically can be the sequence 2 in the sequence table; The nucleotide sequence of described Polylevulosan synthase gene promoter and signal peptide sequence specifically can be the sequence 3 in the sequence table.The nucleotide sequence of described multiple clone site and transcription termination sequence specifically can be the sequence 7 in the sequence table.
The recombinant expression vector that contains described expression cassette also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of method of producing alpha-amylase.
The method of production alpha-amylase provided by the present invention is that described expression cassette is imported in the Bacillus licheniformis, obtains the reorganization bacterium, and the described reorganization bacterium of fermenting produces alpha-amylase.
Bacillus licheniformis expression cassette of the present invention has LacA gene 5 ' and the 3 ' arm of Bacillus licheniformis, can the double exchange mode be incorporated in the Bacillus licheniformis genome, improves integration efficiency; This expression cassette also has hemoglobin gene, Bacillus licheniformis is sought after the supply of energy and oxygen in the logarithmic phase growth, Tremellineae haemoglobin can be in conjunction with oxygen, when Bacillus licheniformis needs oxygen, can discharge oxygen, and hemoglobin gene 5 ' end has signal peptide sequence, helps oxyphorase and is secreted into the extracellular, is beneficial to fermentation industry production.Bacillus licheniformis expression vector of the present invention is imported in the Bacillus licheniformis of produced high-temperature amylase, produce alpha-amylase with this bacterium, enzyme work can reach the 9100U/ml fermented liquid, improves 2.1 times than wild bacterium.
Description of drawings
Fig. 1 is a Bacillus licheniformis expression vector collection of illustrative plates.
Embodiment
Bacillus licheniformis expression vector of the present invention is made up of A, B, C, D, E, F, G and eight gene fragments of H.A, C, D, F and H gene fragment (are purchased in Chinese industrial microbial strains preservation administrative center from Bacillus licheniformis CICC10181, preserving number is CICC10181) genomic dna .B fragment from the genomic dna of Vitreoscilla vitreoscilla (USS biological collecting center, deposit number is ATCC 15551).E fragment synthetic.(Ohio State Univ-Columbus USA's bacterial classification is preserved center (BacillusGenetic Stock Center (BGSC) to the G fragment from carrier pAXOI
The Ohio State UniversityBuy) adopt conventional fusion PCR Protocols in Molecular Biology that eight gene fragments are connected together.5 ' the end and the 3 ' end that merge the primer sequence of PCR respectively comprise one section template sequence to be merged, and are equivalent to adapter, and two adjacent dna sequence dnas are connected together.The nucleotide sequence of primer is as follows:
Primer PaF:5 '
GaatccATTCAATCTGTGACCAATCGTTG 3 ';
Primer PaR:5 '
CTTAAAACTTAATCCACATATACGACCCAAGCAAGATAC3 '.
Primer PbF:5 ' GTATCTTGCTTGGGTCGTAT
TGTGGATTAAGTTTTAAG3 ';
Primer PbR:5 '
GCGCTTTCTACTCATTTATTTATTCAACCTCTTGAGCG3 '.
Primer PcF:5 ' CGCTCAAGAGGTTGA
ATAAATAAATGAGTAGAAAGCGC3 ';
Primer PcR:5 ' GGATTTGCAGACTACGGG
GATTCTCCTCCCCTTTC3 '.
Primer PdF:5 '
GAAAGGGGAGGAGAATCCCCGTAGTCTGCAAATCC3 ';
Primer PdR:
5’GAATTCGAGCTCGGTACCCCCGGGGGATCCTCTAGAGTCGACCTGCAG
CGTTCATGTCTCC3’。
Primer PeF:
5’
GGAGACATGAACGCTGCAGGTCGACTCTAGAGGATCCCCCGGGGGTACCGAGCTCGAATTC3’；
Primer PeR:
5’
GAATTCGAGCTCGGTACCCCCGGGGGATCCTCTAGAGTCGACCTGCAGCGTTCATGTCTCC3’。
Primer PfF:5 '
CGAGCTCGAATTCGCTTAATTAATTAAGTAACCTGTATTAAAA3 ';
Primer PfR:5 '
TATTTGGTTGAGTACTTTTTCCTTCTGTTGTTTGGGATAG3 '.
Primer PgF:5 '
CTATCCCAAACAACAGAAGGAAAAAGTACTCAACCAAATA3 ';
Primer PgR:5 '
GGTGAATAATGAAAGCGTTAGTAATGGTACTTAAATTGTTTAC3 '.
Primer PhF:5 '
GTAAACAATTTAAGTACCATTACTAACGCTTTCATTATTCACC3 ';
Primer PhR:5 '
GAATCCAGGCATTTGCTGCCAACGGG3 '.
The structure of embodiment 1, Bacillus licheniformis expression vector
1) structure of Bacillus licheniformis expression cassette
Extract the genomic dna of Bacillus licheniformis CICC10181 (purchasing) in Chinese industrial microbial strains preservation administrative center.(extracting method is with reference to Bron, S (1990) .Plasmids.In Molecular BiologicalMethods for Bacillus, pp 75-174.Edited by C.R.Harwood ﹠amp; S.M.cutting.Chichester:Wiley)
Bacillus licheniformis CICC10181 genomic dna with extraction is a template, carries out pcr amplification with different primers respectively.The A gene fragment is used primer PaF and primer PaR, and the C gene fragment is used primer PcF primer PcR, and D gene fragment primer PdF primer PdR, F gene fragment are used primer PfF primer PfR, and the H gene fragment is used primer PhF primer PhR.
PCR reaction system: 10 * PCR Buffer, 5 μ L, each 1 μ L of primer, dna profiling 1 μ L, MgCl
2(25mM) 3 μ L, dNTPs (each 2.5mM) 3 μ L, Pfu-Taq (5U/ μ L) 0.5 μ L, last moisturizing to 50 μ L.
Reaction conditions: 94 ℃ of thermally denature 5min; 94 ℃ of sex change 45s, 52 ℃ of annealing 45s, 72 ℃ are extended 6min, totally 30 circulations; 72 ℃ of last 10min that extend.
The PCR product reclaims test kit with DNA and reclaims, and method is with reference to its specification sheets.
The PCR product checks order, and sequencing result shows that the segmental nucleotide sequence of A is shown in sequence in the sequence table 1; The segmental nucleotide sequence of C is shown in sequence in the sequence table 2; The segmental nucleotide sequence of D is shown in sequence in the sequence table 3; The segmental nucleotide sequence of F is shown in sequence in the sequence table 4; The segmental nucleotide sequence of H is shown in sequence in the sequence table 5.
With reference to Bron, S (1990) .Plasmids.In Molecular Biological Methods forBacillus, pp75-174.Edited by C.R.Harwood ﹠amp; S.M.cutting.Chichester:Wiley extracts Vitreoscilla vitreoscilla ATCC 15551 genomic dnas. and with the genomic dna template primer PbF and primer PbR pcr amplification B fragment.
PCR reaction system: 10 * PCR Buffer, 5 μ L, each 1 μ L of primer, dna profiling 1 μ L, MgCl
2(25mM) 3 μ L, dNTPs (each 2.5mM) 3 μ L, Pfu-Taq (5U/ μ L) 0.5 μ L, last moisturizing to 50 μ L.
Reaction conditions: 94 ℃ of thermally denature 5min; 94 ℃ of sex change 45s, 58 ℃ of annealing 45s, 72 ℃ are extended 6min, totally 30 circulations; 72 ℃ of last 10min that extend.
The PCR product reclaims test kit with DNA and reclaims, and method is with reference to its specification sheets.
The PCR product checks order, and sequencing result shows that the segmental nucleotide sequence of B is shown in sequence in the sequence table 6.
Carry out pcr amplification E fragment with primer PeF and PeR.
PCR reaction system: 10 * PCR Buffer, 5 μ L, each 1 μ L of primer, MgCl
2(25mM) 3 μ L, dNTPs (each 2.5mM) 3 μ L, Pfu-Taq (5U/ μ L) 0.5 μ L, last moisturizing to 50 μ L
Reaction conditions: 94 ℃ of thermally denature 5min; 94 ℃ of sex change 45s, 60 ℃ of annealing 45s, 72 ℃ are extended 6min, totally 30 circulations; 72 ℃ of last 10min that extend.
The PCR product reclaims test kit with DNA and reclaims, and method is with reference to its specification sheets.
The PCR product checks order, and sequencing result shows that the segmental nucleotide sequence of E is shown in sequence in the sequence table 7.
With carrier pAXOI is template, carries out pcr amplification G fragment with primer PgF and primer PgR.
PCR reaction system: 10 * PCR Buffer, 5 μ L, each 1 μ L of primer, MgCl
2(25mM) 3 μ L, dNTPs (each 2.5mM) 3 μ L, Pfu-Taq (5U/ μ L) 0.5 μ L, last moisturizing to 50 μ L.
Reaction conditions: 94 ℃ of thermally denature 5min; 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 6min, totally 30 circulations; 72 ℃ of last 10min that extend.
The PCR product reclaims test kit with DNA and reclaims, and method is with reference to its specification sheets.
The PCR product checks order, and sequencing result shows that the segmental nucleotide sequence of G is shown in sequence in the sequence table 8.
(Huang Liuyu compiles, and PCR state-of-the-art technology principle, method and application p220-225) are assembled A, B, C, D, E, F, G and H gene fragment together to adopt molecular biology to merge round pcr.
Concrete grammar is as follows:
A, B, C, D, E, F, G and the H gene fragment of purifying are pressed a mole molecular ratio: 1: 1: 1: 1: 1: 1: be mixed as template at 1: 1, merge PCR with primer PaF and primer PhR.
The PCR reaction system: template is 100ng, 10 * PCR Buffer, 5 μ L, MgCl
2(25mM) 3 μ L, dNTPs (each 2.5mM) 3 μ L, last moisturizing to 50 μ L after 94 ℃ of this reaction system heating, adds Pfu-Taq (5U/ μ L) 1 μ L; 94 ℃ of thermally denature 5min then; 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 6min, totally 5 circulations; Reaction system is heated to 94 ℃ again, other add 50 μ L reaction solutions (comprise 10 * PCR Buffer, 5 μ L,, MgCl
2(25mM) 5 μ L, dNTPs (each 2.5mM) 3 μ L, Pfu-Taq (5U/ μ L) 1 μ L, each 1 μ L of primer PaF (having the PstI restriction enzyme site) and primer PhR (having the EcoRI restriction enzyme site), last moisturizing to 50 μ L; Carry out following reaction at last again: 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 3.5min, totally 25 circulations; 72 ℃ are extended 10min.
The PCR product reclaims test kit with DNA and reclaims, and method is with reference to its specification sheets.
PCR product length is 3553bp, called after W expression cassette.
2) contain the structure of the Bacillus licheniformis expression vector of W expression cassette
With restriction enzyme PstI and EcoRI double digestion carrier pUC19 (available from the precious biotech firm in Dalian), be connected with W expression cassette with same enzyme double digestion.To connect product conversion Top10 competent escherichia coli cell (available from sky, Beijing root biochemical corp) method for transformation and see the test kit specification sheets.The competent escherichia coli cell shop LB screening dull and stereotyped (containing amp 100ug/ml) that transforms, 37 ℃ of overnight incubation, second day from flat board picking clone insert 37 ℃ of incubated overnight the LB substratum, identify positive colony with primer PaF and primer PhR PCR, amplify the positive clone of segmental clone about 3.5kb, the recombinant plasmid called after pWY (Fig. 1) that positive colony contains.
Embodiment 2, production alpha-amylase
PWY2 is added electroporation with protoplastis transform the Bacillus licheniformis CICC10181 (Chinese industrial microbial strains preservation administrative center) that produces high-temperature starch, concrete grammar is as follows:
Reference literature Romero D, Journal of Microbiology Methods, the method for 2006 Vol, 66 p556-559 obtains Bacillus licheniformis CICC10181 protoplastis.
The electric shock cup of 2ug pWY and 0.2CM with protoplastis and pWY mixing, is placed 10min in precooling on ice on ice, opens electroporation then, transfers voltage to 600V; Protoplastis and pWY mixed solution are moved in the electric shock cup of precooling and shock by electricity, add 1ml cell recovery damping fluid (20g/L BSA and 1M sucrose) then, behind the re-suspended cell, 37 ℃, the 100rpm shaking table was cultivated 12 hours, got 150ul bacterium liquid and coated the DM3 solid medium that contains 10ug/ml erythromycin and (contain 8g/L agar, the 5g/L caseinhydrolysate, the 5g/L yeast powder, 1.5g/L KH
2PO
4, 3.5g/L K
2HPO
4, the substratum of 45.5g/L sorbyl alcohol and 10g/L starch) on, 37 ℃ of incubators were cultivated 72 hours.Containing the clone who grows on the 10ug/ml erythromycin flat board, called after Bacillus licheniformis (Bacillus Licheniformis) WY.
Bacillus licheniformis WY or Bacillus licheniformis (B.Licheniformis-181) CICC10181 (wild bacterium) is respectively according to 0.01g thalline/ml substratum (soybean cake powder 26.1g/L, cottonseed meal 21.1g/L, calcium chloride 0.46g/L, ammonium sulfate 5g/L, Trisodium Citrate 2g/L, dipotassium hydrogen phosphate 14g/L, potassium primary phosphate 6g/L, corn steep liquor 21.1g/L, lactose 105g/L (independent sterilization), the pH value is 6.7) amount be inoculated in the dress 50ml substratum, 37 ℃ of shaking tables are cultivated, and shaking speed is 200rpm, when cultivating 48h, in shaking bottle, add the sucrose induction that final concentration is 20g/L, cultivate 96h and stop cultivating.Three repetitions are established in experiment.
In the fermenting process, get fermented supernatant fluid and carry out enzyme biopsy survey.
The alpha-amylase biopsy is surveyed and is adopted man of People's Republic of China (PRC) standard law to measure (QB/T2306-1997); Enzyme is lived and is defined as at 70 ℃, and under the pH6.0 condition, 1 milligram of Zulkovsky starch of liquefaction in 1 minute becomes the required enzyme amount of dextrin, is an enzyme activity unit, represents with U/ml or U/g.
The result shows, fermentation culture 96 hours, the enzyme work of reorganization Bacillus licheniformis WY reaches the 9100U/ml fermented liquid, biomass reaches 20g bacterium/L fermented liquid, and the work of wild bacterium Bacillus licheniformis (B.Licheniformis-181) CICC10181 enzyme is 4200U/ml, and biomass is the 12g/L fermented liquid.Bacillus licheniformis WY enzyme is lived (9100U/ml fermented liquid) than 2.1 times of wild bacterium raisings.
Sequence table
＜110〉Beijing ZhongTian-Noah Sports Science Co., Ltd.
＜120〉bacillus licheniformis integration expression box and application thereof
<130>CGGNARW92038
<160>8
<210>1
<211>600
<212>DNA
＜213〉bacillus Bacillus licheniformis (Bacillus Licheniformis)
<400>1
attcaatctg?tgaccaatcg?ttgaaggtgt?ggctccaaaa?aggcgtccac?catgcgtcat?60
tcaaagattt?aatatcatgg?ttgtacttct?ttttcagcca?gtctctgaag?gcgtgctgac?120
attgatcaca?atggcattct?cctccgtatt?cgttggaaac?atgccacatc?aaaagggcat?180
gctggctgcc?gtacctttca?gccagcatgc?ggttgatctc?tttcgttttt?tcacggtata?240
cgtatgaagt?gaagcaatga?ttgtgccttc?cgccgtgcag?ctgtttgacg?cgttcagcat?300
tcacgcgcag?cacttccgga?tacttttgcg?acagccaagc?aggacgggcg?ccgctcggtg?360
tcgccaatat?aatccggccg?ccgttccggt?gaatgctctc?aaaaatatca?tccagccatt?420
caaacgtata?gacgccttcc?tccggctcaa?gggcgctcca?tgaaaaaatc?cccactgaaa?480
atgtattcgt?atgggcaagc?ttcatcagct?tgatgtcatc?tgcaagaata?tccggacggt?540
caagccactg?atccggattg?tagtctcccc?cgtgaagcat?gtatcttgct?tgggtcgtat?600
<210>2
<211>128
<212>DNA
＜213〉bacillus Bacillus licheniformis (Bacillus Licheniformis)
<400>2
ataaatgagt?agaaagcgcc?atatcggcgc?ttttcttttg?gaagaaaata?tagggaaaat?60
ggtatttgtt?aaaaattctg?aatatttata?caatatcata?tgtttcacat?tgaaagggga?120
ggagaatc 128
<210>3
<211>313
<212>DNA
＜213〉bacillus Bacillus licheniformis (Bacillus Licheniformis)
<400>3
cccgtagtct?gcaaatcctt?cccgtagtct?gcaaatcctt?ttatgatttt?ctatcaaaca 60
aaagaggaaa?atagaccagt?tgcaatccaa?acgagagtct?aatagaatga?ggtcgaaaag 120
taaatcgcgc?gggtttgtta?ctgataaagc?aggcaagacc?taaaatgtgt?aaagggcaaa 180
gtgtatactt?tggcgtcacc?ccttacatat?tttaggtctt?tttttattgt?gcgtaactaa 240
cttgccatct?tcaaacagga?gggctggaag?aagcagaccg?ctaacacagt?acataaaaaa 300
ggagacatga?acg 313
<210>4
<211>313
<212>DNA
＜213〉bacillus Bacillus licheniformis (Bacillus Licheniformis)
<400>4
taacctgtat?taaaaacacg?gtcagtttca?actgaaccgt?gtttttttct?tctatcccaa 60
acaacagaag 70
<210>5
<211>660
<212>DNA
＜213〉bacillus Bacillus licheniformis (Bacillus Licheniformis)
<400>5
aacgctttca?ttattcaccc?ggagaaacat?ccaccttaaa?gcggatgtcc?tcccaggcct 60
attcttttgc?ttttaccgct?attctggcct?catatttttc?caatgtgact?tcgccggcca 120
gcgtttctcc?ggtcagcata?tctttaactg?ccgacgccaa?cacgacgggc?tgacgttttt 180
ccgtgaaatt?catgatgaaa?atatagtcat?tttcctcatc?ctgtctgact?tgaacggaga 240
cgccaggctg?atgtttcacg?tcaagagccg?gttgaatcgc?tagctcttta?atcaatgttc 300
cataaaaatc?ccgatgaaat?tggctgctta?aacgcgcacc?gatataatac?gttttcccct 360
gtttgtatgg?atggctcgtc?acagccgtcg?tgtccgcata?aaagtcgtcc?tcatagaatc 420
cctcgggatc?tgccgtactg?agcttcagca?cggtcgcgta?atctttcagc?tcatacgacc 480
ggttttgata?acggaccgca?tttttatcgc?ccggatagag?tgtatcggtt?tcaacaggct 540
ccatgccaaa?catctcttgc?agatcttttg?gccatccgcc?taaataggtc?agatcagatt 600
cgttgacgat?tccgctaata?taggtcatca?ccaatgtgcc?cccgttggca?gcaaatgcct 660
<210>6
<211>499
<212>DNA
＜213〉Vitreoscilla (Vitreoscilla sp)
<400>6
tgtggattaa?gttttaagag?gcaataaaga?ttataataag?tgctgctaca?ccatactgat 60
gttagaccag?caaaccatta?acatcatcaa?agccactgtt?cctgtattga?aggagcatgg 120
cgttaccatt?accacgactt?tttataaaaa?cttgtttgcc?aaacaccctg?aagtacgtcc 180
tttgtttgat?atgggtcgcc?aagaatcttt?ggagcagcct?aaggctttgg?cgatgacggt 240
attggcggca?gcgcaaaaca?ttgaaaattt?gccagctatt?ttgcctgcgg?tcaaaaaaat 300
tgcagtcaaa?cattgtcaag?caggcgtggc?agcagcgcat?tatccgattg?tcggtcaaga 360
attgttgggt?gcgattaaag?aagtattggg?cgatgccgca?accgatgaca?ttttggacgc 420
gtggggcaag?gcttatggcg?tgattgcaga?tgtgtttatt?caagtggaag?cagatttgta 480
cgctcaagag?gttgaataa 499
<210>7
<211>63
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>7
ctgcaggtcg?actctagagg?atcccccggg?ggtaccgagc?tcgaattcgc?ttaattaatt 60
aagtaacctg?tattaaaaac?acggtcagtt?tcaactgaac?cgtgtttttt?tcttctatcc 120
caaacaacag?aag 133
<210>8
<211>1278
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>8
gaaaaagtac?tcaaccaaat?aataaaacaa?ttgaatttaa?aagaaaccga?taccgtttac 60
gaaattggaa?caggtaaagg?gcatttaacg?acgaaactgg?ctaaaataag?taaacaggta 120
acgtctattg?aattagacag?tcatctattc?aacttatcgt?cagaaaaatt?aaaactgaat 180
actcgtgtca?ctttaattca?ccaagatatt?ctacagtttc?aattccctaa?caaacagagg 240
tataaaattg?ttgggagtat?tccttacaat?ttaagcacac?aaattattaa?aaaagtggtt 300
tttgaaagcc?atgcgtctga?catctatctg?attgttgaag?aaggattcta?caagcgtacc 360
ttggatattc?accgaacact?agggttgctc?ttgcacactc?aagtctcgat?tcagcaattg 420
cttaagctgc?cagcggaatg?ctttcatcct?aaaccaaaag?taaacagtgt?cttaataaaa 480
cttacccgcc?ataccacaga?tgttccagat?aaatattgga?agctatatac?gtactttgtt 540
tcaaaatggg?tcaagcgaga?atatcgtcaa?ctgtttacta?aaaatcagtt?tcatcaagca 600
gaaaaagtac?tcaaccaaat?aataaaacaa?ttgaatttaa?aagaaaccga?taccgtttac 660
gaaattggaa?caggtaaagg?gcatttaacg?acgaaactgg?ctaaaataag?taaacaggta 720
acgtctattg?aattagacag?tcatctattc?aacttatcgt?cagaaaaatt?aaaactgaat 780
actcgtgtca?ctttaattca?ccaagatatt?ctacagtttc?aattccctaa?caaacagagg 840
tataaaattg?ttgggagtat?tccttacaat?ttaagcacac?aaattattaa?aaaagtggtt 900
tttgaaagcc?atgcgtctga?catctatctg?attgttgaag?aaggattcta?caagcgtacc 960
ttggatattc?accgaacact?agggttgctc?ttgcacactc?aagtctcgat?tcagcaattg 1020
cttaagctgc?cagcggaatg?ctttcatcct?aaaccaaaag?taaacagtgt?cttaataaaa 1080
cttacccgcc?ataccacaga?tgttccagat?aaatattgga?agctatatac?gtactttgtt 1140
tcaaaatggg?tcaagcgaga?atatcgtcaa?ctgtttacta?aaaatcagtt?tcatcaagca 1200
atgaaacacg?ccaaagtaaa?caatttaagt?accattacta?tgaaacacgc?caaagtaaac 1260
aatttaagta?ccattact 1278
Claims (3)
1. an expression cassette comprises LacA gene 5 ' arm, hemoglobin gene, selection markers gene and LacA gene 3 ' arm successively; Also contain the exogenous gene expression box between described hemoglobin gene and the described selection markers gene, described exogenous gene expression box comprises alpha-amylase gene promotor, Polylevulosan synthase gene promoter and signal peptide sequence, multiple clone site and transcription termination sequence successively to the downstream from the upstream;
The nucleotides sequence of described LacA gene 5 ' arm is classified the SEQ ID NO:1 in the sequence table as, the nucleotides sequence of described LacA gene 3 ' arm is classified the SEQ ID NO:5 in the sequence table as, and the nucleotides sequence of described hemoglobin gene is classified the SEQ ID NO:6 in the sequence table as; Described selection markers gene is an erythromycin resistance gene; The nucleotides sequence of described erythromycin resistance gene is classified the SEQ ID NO:8 in the sequence table as;
The nucleotides sequence of described alpha-amylase gene promotor is classified the SEQ ID NO:2 in the sequence table as; The nucleotides sequence of described Polylevulosan synthase gene promoter and signal peptide sequence is classified the SEQ ID NO:3 in the sequence table as; The nucleotides sequence of described multiple clone site and transcription termination sequence is classified the SEQ ID NO:7 in the sequence table as.
2. the recombinant expression vector that contains the described expression cassette of claim 1.
3. a method of producing alpha-amylase is that the described expression cassette of claim 1 is imported in the Bacillus licheniformis of produced high-temperature amylase, obtains the reorganization bacterium, and the described reorganization bacterium of fermenting produces alpha-amylase.
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