CN107058367A - The construction method of the recombined bacillus subtilis of high yield Pullulanase - Google Patents

The construction method of the recombined bacillus subtilis of high yield Pullulanase Download PDF

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CN107058367A
CN107058367A CN201710410090.5A CN201710410090A CN107058367A CN 107058367 A CN107058367 A CN 107058367A CN 201710410090 A CN201710410090 A CN 201710410090A CN 107058367 A CN107058367 A CN 107058367A
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bacillus subtilis
gene
pullulanase
tpu2
tpu1
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纪明华
俞峰
刘校函
王辉
李统锋
丁少明
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BAIYIN SAINUO BIOTECHNOLOGY Co Ltd
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BAIYIN SAINUO BIOTECHNOLOGY Co Ltd
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

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Abstract

The invention discloses a kind of construction method of the recombined bacillus subtilis of high yield Pullulanase.By the Pullulanase of resistance to acid heat gene after codon optimization, the secretion peptide different with two kinds and promoter are manually spliced respectively, constitute artificial operons, and are incorporated into bacillus subtilis A 16 4, and integration site is respectively alkali protease（aprE）And amylase gene（amyE）At place；In addition, also by the plasmid expression of Cre recombinases, the resistant gene that would be integrated on A164 is knocked out；The Pullulanase recombinant production bacterial strain CNP2 obtained Pullulanase ferments enzyme activity more than 1000 U/mL.

Description

The construction method of the recombined bacillus subtilis of high yield Pullulanase
Technical field
The invention belongs to constructing technology field of the sugar industry with enzyme safety in production bacterial strain, it is related to a kind of high yield Pullulanase Recombined bacillus subtilis construction method.
Background technology
Pullulanase is indispensable class of enzymes in food service industry Closed Circulation in Sugar Production technology, is mainly secreted by prokaryotic micro-organisms The sugar chain solution branch enzyme of expression.Many I type Pullulanases using in this fermentoid in production application, it being capable of selectivity incision starch original The branch chain component being made up of in material α -1,6 glycosidic bonds, therefore Pullulanase and other amylolytic enzyme collective effects, can allow shallow lake Powder is digested completely, obtains single molecules of glucose molecule, before it has wide market in the food service industrys such as starch processing Scape.In recent years, a large amount of manpower and materials of Innovation Input both at home and abroad to pullulanase production strain, many industrial enzyme manufacturing enterprises are first After develop a variety of different types of recombinant production bacterial strains or mutagenic bacteria.It is being adapted to a variety of Pullulanase eggs of industrial production application Bai Zhong, from Nagano bacillus (Bacillus naganoensis) Pullulanase（Bn PulB）, its zymetology feature, Including optimal reactive temperature and pH etc. be especially suitable for starch industry processing demand, but the original bacterium of the enzyme expression not Height, therefore its gene is commonly used for being implemented in recombination expression production, in the host cell for being adapted to food industry enzyme.Withered grass bud Spore bacillus (Bacillus subtilis) because it does not express pyrogen and the toxin factor, and its secretory pathway is powerful, therefore quilt It is classified as safety in production bacterial strain（GRAS）One of, it is commonly used for the preferred host cell of many ectoenzyme fermenting and producings.Withered grass gemma Bacillus has many kinds, and its respective conversion performance, secretion capacity etc. are related very big, and wherein A164 is not high with its nutritional need, hair Ferment density is high, and the reason such as protein secretion level height is considered as the desired host cell of protein production.
In order to improve the genetic stability of production bacterial strain, heterogenous expression is typically integrated into the genome of host cell, And antibiotic resistance gene is commonly used for selection markers to use, but in the food industry, the recombinant bacterial strain meeting of antibiotic resistance gene is carried The problem of bringing biological safety, therefore, it is necessary to pass through secondary restructuring or special again after the integration of foreign gene is completed Property restructuring mode, antibiotic resistance gene is removed, the production bacterial strain of nonreactive is obtained.
The content of the invention
It is an object of the invention to provide a kind of recombined bacillus subtilis CNP2 of high yield Pullulanase construction method- By genetic manipulation twice, by two parts of copy Pullulanase gene integrations into the genome of bacillus subtilis, and pass through spy Specific recombination systems, antibiotic resistance gene is removed, so as to obtain the Pullulanase superior strain of one plant of safety.
To achieve these goals, the technical solution adopted in the present invention is specific as follows：
By artificial synthesized Pullulanase gene expression frame Tpu1 and Tpu2 the difference alkaline egg in situ for substituting bacillus subtilis White enzyme gene aprE and amylase gene amyE, and the recombinant bacillus bud will be obtained after resistant gene ermC and kan knockout Spore bacillus, comprises the following steps that：
A. design and gene chemical synthesis linear DNA fragment Tpu1 and Tpu2；
B. it is that starting strain carries out molecule construction with bacillus subtilis A 16 4, by Tpu1 and the competence of bacillus subtilis A 16 4 Mixing with cells, is converted, and obtains the recombinant bacterial strain CNP1 with Erythromycinresistant；
The bacillus subtilis A 16 4, is most initially separated from Marburg, Germany, and the strain can be protected by American National strain Tibetan center is commercially available, and culture presevation number is ATCC 6051a；
C. Tpu2 is mixed with bacillus subtilis CNP1 competent cells, converted, obtaining has erythromycin/kanamycins Double recombinant bacterial strain CNP2ek；
D. Cre expression plasmids are transferred to, the Double elimination of erythromycin/kanamycins is carried out, obtains recombined bacillus subtilis CNP2, i.e. recombined bacillus subtilis.
The gene expression frame Tpu1 and Tpu2 is obtained after codon optimization, gene chemical synthesis respectively, Tpu1 nucleotides Sequence is：
caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg 60
atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg 120
gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg 180
ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt 240
ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg 300
ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg 360
ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg 420
atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag 480
gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg 540
aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact 600
attattgtgc agctgcttgc ctgcactgca gatcgattgt ttgagaaaag aagaagacca 660
taaaaatacc ttgtctgtca tcagacaggg tattttttat gctgtccaga ctgtccgctg 720
tgtaaaaaaa aggaataaag gggggttgac attattttac tgatatgtat aatataattt 780
gtataagaaa atggagtcga aacgtaagat gaaaccttag ataaaagtgc tttttttgtt 840
gcaattgaag aattattaat gttaagctta attaaagata atatctttga attgtaacgc 900
ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt ttgaaccttc 960
ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa ctatagaatg 1020
acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt gttcatgtga 1080
attatcgctt tatttaattt tctcaattca atatataata tgccaataca ttgttacaag 1140
tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt attaaatgaa 1200
tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata tttttctatt 1260
ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa ggagggatgc 1320
ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt atgaaaaatc 1380
attttatcag tttgaaaatt atgtattatg cttacattcc atcgtatcat ccgtaaaggc 1440
tggatgttcc ttcttgcttt ccttcttaca gcttctcttt tctgccctac aggccaacat 1500
gctaaagctg ctgctcaacc tgctgtttct aacgcttacc ttgatgcttc taaccaagtt 1560
cttgttaaac tttctcaacc tttcacactt ggcgaaggct cttctggctt cacagttcat 1620
gatgatacag ctaacaaaga tatccctgtt acatctgttt ctgatgctaa ccaagttaca 1680
gctgttcttg ctggcacatt ccaacatatc ttcggcggct ctgattgggc tcctgataac 1740
cataacacac ttcttaaaaa agttaactct aacctttacc aattctctgg caaccttcct 1800
gaaggcaact accaatacaa agttgctctt aacgattctt ggaacaaccc ttcttaccct 1860
tctgataaca tcaaccttac agttcctgct ggcggcgctc atgttacatt ctcttacatc 1920
ccttctacac atgctgttta cgatacaatc aacaacccta acgctgatct tcaagttgat 1980
tcttctggcg ttaaaacaga tcttgttgct gttacacttg gcgaaaaccc tgatgtttct 2040
catacacttt ctatccaaac agaagattac caagctggcc aagttatccc tcgtaaagtt 2100
cttgattctt ctcaatacta ctactctggc gatgatcttg gcaacacata cacaaaaaac 2160
gctacaacat tcaaagtttg ggctcctaca tctacacaag ttaacgttct tctttacaac 2220
tctgctacag gcgctgttac aaaaacagtt cctatgacag cttctggcca tggcgtttgg 2280
gaagctacag ttaaccaaga tcttgaaaac tggtactaca tgtacgaagt tacaggccaa 2340
ggctctacac gtacagctgt tgatccttac gctacagcta tcgctcctaa cggcacacgt 2400
ggcatgatcg ttgatcttgc taaaacagat cctgctggct gggaatctga taaacatatc 2460
acacctaaaa acatcgaaga tgaagttatc tacgaaatgg atgttcgtga tttctctatc 2520
gattctaact ctggcatgaa aaacaaaggc aaataccttg ctcttacaga aaaaggcaca 2580
aaaggccctg ataacgttaa aacaggcgtt gattctctta aacaacttgg catcacacat 2640
gttcaacttc aacctgtttt cgctttcaac tctgttaacg aaaacgatcc tacacaatac 2700
aactggggct acgatcctcg taactacaac gttcctgaag gccaatacgc tacaaacgct 2760
aacggcacaa cacgtatcaa agaattcaaa gaaatggttc tttctcttca tcaagatcat 2820
atcggcgtta acatggatgt tgtttacaac catacattcg ctacacaaat ctctgatttc 2880
gataaaatcg ttcctgaata ctactaccgt acagatgatg ctggcaacta cacaaacggc 2940
tctggcacag gcaacgaaat cgctgctgaa cgtcctatgg ttcaaaaatt catcatcgat 3000
tctcttaaat tctgggttaa cgaataccat gttgatggct tccgtttcga tcttatggct 3060
cttcttggca aagatacaat gtctaaagct gctacacaac ttcatgctat cgatcctggc 3120
atcgctcttt acggcgaacc ttggacaggc ggcacatctg ctcttcctgc tgatcaactt 3180
cttacaaaag gcgctcaaaa aggcatgggc gttgctgttt tcaacgataa ccttcgtaac 3240
ggccttgatg gctctgtttt cgattcttct gctcaaggct tcgctacagg cgctacaggc 3300
cttacagatg ctatcaaaaa cggcgttgaa ggctctatca acgatttcac agcttctcct 3360
ggcgaaacaa tcaactacgt tacatctcat gataactaca cactttggga taaaatcgct 3420
caatctaacc ctaacgattc tgaagctgat cgtatcaaaa tggatgaact tgctcaagct 3480
atcgttatga catctcaagg catccctttc atgcaaggcg gcgaagaaat gcttcgtaca 3540
aaaggcggca acgataactc ttacaacgct ggcgatgttg ttaacgaatt cgattggtct 3600
cgtaaagctc aataccctga tgttttcaac tactactctg gccttatcca tcttcgtctt 3660
gatcatcctg ctttccgtat gacaacagct aacgaaatca actctcatct tcaattcctt 3720
aactctcctg aaaacacagt tgcttacgaa ctttctgatc atgctaacaa agatacatgg 3780
ggcaacatcg ttgttatcta caaccctaac aaaacagctg aaacaatcaa ccttccttct 3840
ggcaaatggg aaatcaacgc tacatctggc aaagttggcg aatctacact tggccaagct 3900
gaaggctctg ttcaagttcc tggcatctct atgatgatcc ttcatcaaga agtttctcct 3960
tctgatggca aataaagaag caggttcctc catacctgct tctttttaag gatccaagct 4020
tgcggccgct accgttcgta tagcatacat tatacgaagt tatttaaacc gtgtgctcta 4080
cgaccaaaac tataaaacct ttaagaactt tcttttttta caagaaaaaa gaaattagat 4140
aaatctctca tatcttttat tcaataatcg catccgattg cagtataaat ttaacgatca 4200
ctcatcatgt tcatatttat cagagctcgt gctataatta tactaatttt ataaggagga 4260
aaaaatatgg gcatttttag tatttttgta atcagcacag ttcattatca accaaacaaa 4320
aaataagtgg ttataatgaa tcgttaataa gcaaaattca tataaccaaa ttaaggaggg 4380
aaataatgaa cgagaaaaat ataaaacaca gtcaaaactt tattacttca aaacataata 4440
tagataaaat aatgacaaat ataagattaa atgaacatga taatatcttt gaaatcggct 4500
caggaaaagg ccattttacc cttgaattag taaagaggtg taatttcgta actgccattg 4560
aaatagacca taaattatgc aaaactacag aaaataaact tgttgatcac gataatttcc 4620
aagttttaaa caaggatata ttgcagttta aatttcctaa aaaccaatcc tataaaatat 4680
atggtaatat accttataac ataagtacgg atataatacg caaaattgtt tttgatagta 4740
tagctaatga gatttattta atcgtggaat acgggtttgc taaaagatta ttaaatacaa 4800
aacgctcatt ggcattactt ttaatggcag aagttgatat ttctatatta agtatggttc 4860
caagagaata ttttcatcct aaacctaaag tgaatagctc acttatcaga ttaagtagaa 4920
aaaaatcaag aatatcacac aaagataaac aaaagtataa ttatttcgtt atgaaatggg 4980
ttaacaaaga atacaagaaa atatttacaa aaaatcaatt taacaattcc ttaaaacatg 5040
caggaattga cgatttaaac aatattagct ttgaacaatt cttatctctt ttcaatagct 5100
ataaattatt taataagtaa gttaagggat gcataaactg catcccttaa cttgtttttc 5160
gtgtgcctat tttttgtgaa ttgattatcg atcttttgcg ccatggataa cttcgtatag 5220
catacattat acgaacggta catatgcccg ggggtaccga attccctgct ggtcgccatt 5280
ttggctcttt accctctcct tttaaaaaaa ttcagagtag acttacttaa aagactattc 5340
tgtgaattta ttgtaataga tggaataata ttttagtaga cccatttttt tgagatgatt 5400
ttatctctat ttaggtatat catctctcgc tatttccgta gagactcgaa ataactattt 5460
tcatacagaa aagaacgaaa atagacatga gtaaatgttc attatgctga aatgtcatgc 5520
ttttttaggt taaatgctcc tgagtcccgg caaattcctg tcgaaaaaat tcgttcaaat 5580
gacctgcgtg tgcttccgtg agaacaatgg atattatcgt gatatttttt caaagcatga 5640
tgataaaagt attctgaaaa ataaacttta cagaaaaggg atagaatgaa aaaattattg 5700
agagggagga agaaataaga tgaacattcg ccaagcaaag acatcagatg cggccgcca 5760
tgcgccgctg tttaaccaat atcgggaatt ttatagacag gcatccgatt tgcaaggggc 5820
agaggctttt ttgaaagctc gtttggaaaa tcacgagtct gttattttga tagcagaaga 5880
aaatg 5885；
Tpu2 nucleotides sequence is classified as：
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg catagaatct 540
aatattataa ctaaattttc taaaaaaaac attggaatag acatttattt tgtatatgat 600
gaaataaagt tagtttattg gataaacaaa ctaactttat taaggtagtt gatggataaa 660
cttgttcact taaatcaatc gaaacgtaag atgaaacctt agataaaagt gctttttttg 720
ttgcaattga agaattatta atgttaagct taattaaaga taatatcttt gaattgtaac 780
gcccctcaaa agtaagaact acaaaaaaag aatacgttat atagaaatat gtttgaacct 840
tcttcagatt acaaatatat tcggacggac tctacctcaa atgcttatct aactatagaa 900
tgacatacaa gcacaacctt gaaaatttga aaatataact accaatgaac ttgttcatgt 960
gaattatcgc tttatttaat tttctcaatt caatatataa tatgccaata cattgttaca 1020
agtagaaatt aagacaccct tgatagcctt actataccta acatgatgta gtattaaatg 1080
aatatgtaaa tatatttatg ataagaagcg acttatttat aatcattaca tatttttcta 1140
ttggaatgat taagattcca atagaatagt gtataaatta tttatcttga aaggagggat 1200
gcctaaaaac gaagaacatt aaaaacatat atttgcaccg tctaatggat ttatgaaaaa 1260
tcattttatc agtttgaaaa ttatgtatta tgcttatcaa caaatctaaa aaattcttcg 1320
ttttctcttt catcttcgtt atgatgcttt ctctttcttt cgttaacggc gaagttgcta 1380
aagctgctca acctgctgtt tctaacgctt accttgatgc ttctaaccaa gttcttgtta 1440
aactttctca acctttcaca cttggcgaag gctcttctgg cttcacagtt catgatgata 1500
cagctaacaa agatatccct gttacatctg tttctgatgc taaccaagtt acagctgttc 1560
ttgctggcac attccaacat atcttcggcg gctctgattg ggctcctgat aaccataaca 1620
cacttcttaa aaaagttaac tctaaccttt accaattctc tggcaacctt cctgaaggca 1680
actaccaata caaagttgct cttaacgatt cttggaacaa cccttcttac ccttctgata 1740
acatcaacct tacagttcct gctggcggcg ctcatgttac attctcttac atcccttcta 1800
cacatgctgt ttacgataca atcaacaacc ctaacgctga tcttcaagtt gattcttctg 1860
gcgttaaaac agatcttgtt gctgttacac ttggcgaaaa ccctgatgtt tctcatacac 1920
tttctatcca aacagaagat taccaagctg gccaagttat ccctcgtaaa gttcttgatt 1980
cttctcaata ctactactct ggcgatgatc ttggcaacac atacacaaaa aacgctacaa 2040
cattcaaagt ttgggctcct acatctacac aagttaacgt tcttctttac aactctgcta 2100
caggcgctgt tacaaaaaca gttcctatga cagcttctgg ccatggcgtt tgggaagcta 2160
cagttaacca agatcttgaa aactggtact acatgtacga agttacaggc caaggctcta 2220
cacgtacagc tgttgatcct tacgctacag ctatcgctcc taacggcaca cgtggcatga 2280
tcgttgatct tgctaaaaca gatcctgctg gctgggaatc tgataaacat atcacaccta 2340
aaaacatcga agatgaagtt atctacgaaa tggatgttcg tgatttctct atcgattcta 2400
actctggcat gaaaaacaaa ggcaaatacc ttgctcttac agaaaaaggc acaaaaggcc 2460
ctgataacgt taaaacaggc gttgattctc ttaaacaact tggcatcaca catgttcaac 2520
ttcaacctgt tttcgctttc aactctgtta acgaaaacga tcctacacaa tacaactggg 2580
gctacgatcc tcgtaactac aacgttcctg aaggccaata cgctacaaac gctaacggca 2640
caacacgtat caaagaattc aaagaaatgg ttctttctct tcatcaagat catatcggcg 2700
ttaacatgga tgttgtttac aaccatacat tcgctacaca aatctctgat ttcgataaaa 2760
tcgttcctga atactactac cgtacagatg atgctggcaa ctacacaaac ggctctggca 2820
caggcaacga aatcgctgct gaacgtccta tggttcaaaa attcatcatc gattctctta 2880
aattctgggt taacgaatac catgttgatg gcttccgttt cgatcttatg gctcttcttg 2940
gcaaagatac aatgtctaaa gctgctacac aacttcatgc tatcgatcct ggcatcgctc 3000
tttacggcga accttggaca ggcggcacat ctgctcttcc tgctgatcaa cttcttacaa 3060
aaggcgctca aaaaggcatg ggcgttgctg ttttcaacga taaccttcgt aacggccttg 3120
atggctctgt tttcgattct tctgctcaag gcttcgctac aggcgctaca ggccttacag 3180
atgctatcaa aaacggcgtt gaaggctcta tcaacgattt cacagcttct cctggcgaaa 3240
caatcaacta cgttacatct catgataact acacactttg ggataaaatc gctcaatcta 3300
accctaacga ttctgaagct gatcgtatca aaatggatga acttgctcaa gctatcgtta 3360
tgacatctca aggcatccct ttcatgcaag gcggcgaaga aatgcttcgt acaaaaggcg 3420
gcaacgataa ctcttacaac gctggcgatg ttgttaacga attcgattgg tctcgtaaag 3480
ctcaataccc tgatgttttc aactactact ctggccttat ccatcttcgt cttgatcatc 3540
ctgctttccg tatgacaaca gctaacgaaa tcaactctca tcttcaattc cttaactctc 3600
ctgaaaacac agttgcttac gaactttctg atcatgctaa caaagataca tggggcaaca 3660
tcgttgttat ctacaaccct aacaaaacag ctgaaacaat caaccttcct tctggcaaat 3720
gggaaatcaa cgctacatct ggcaaagttg gcgaatctac acttggccaa gctgaaggct 3780
ctgttcaagt tcctggcatc tctatgatga tccttcatca agaagtttct ccttctgatg 3840
gcaaataaag aagcaggttc ctccatacct gcttcttttt aagggatcca agcttgcggc 3900
cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt tgaagattag 3960
atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct 4020
gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga 4080
aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat 4140
tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg 4200
tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg 4260
tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt 4320
ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga 4380
ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt caggtggata 4440
cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat 4500
ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt 4560
gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat 4620
gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc 4680
agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg 4740
tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga atgggattca 4800
ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac 4860
gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt 4920
agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta tacgaacggt 4980
acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga catggatgag 5040
cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca gtacgcctct 5100
tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga aaagccaaat 5160
aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca atagatttca 5220
caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca accagatatt 5280
tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat cctctgtctc 5340
tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg gagcgggttc 5400
atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg tagctgtgct 5460
ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca aaacaggtgt 5520
aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga atacaacaaa 5580
agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg agatcaattc 5640
acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa aaggaacgaa 5700
cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc cagcgtcggc 5760
caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt atatctataa 5820
acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac caatgactaa 5880
aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa ccaacgcaaa 5940
agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg gctttgatta 6000
cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc attga 6055。
Institute art gene expression frame Tpu1 and Tpu2 include the Pullulanase encoding gene of copy respectivelypulB, it is describedpulBThe nucleotide sequence of gene is as follows：
caacctgctg tttctaacgc ttaccttgat gcttctaacc aagttcttgt taaactttct 60
caacctttca cacttggcga aggctcttct ggcttcacag ttcatgatga tacagctaac 120
aaagatatcc ctgttacatc tgtttctgat gctaaccaag ttacagctgt tcttgctggc 180
acattccaac atatcttcgg cggctctgat tgggctcctg ataaccataa cacacttctt 240
aaaaaagtta actctaacct ttaccaattc tctggcaacc ttcctgaagg caactaccaa 300
tacaaagttg ctcttaacga ttcttggaac aacccttctt acccttctga taacatcaac 360
cttacagttc ctgctggcgg cgctcatgtt acattctctt acatcccttc tacacatgct 420
gtttacgata caatcaacaa ccctaacgct gatcttcaag ttgattcttc tggcgttaaa 480
acagatcttg ttgctgttac acttggcgaa aaccctgatg tttctcatac actttctatc 540
caaacagaag attaccaagc tggccaagtt atccctcgta aagttcttga ttcttctcaa 600
tactactact ctggcgatga tcttggcaac acatacacaa aaaacgctac aacattcaaa 660
gtttgggctc ctacatctac acaagttaac gttcttcttt acaactctgc tacaggcgct 720
gttacaaaaa cagttcctat gacagcttct ggccatggcg tttgggaagc tacagttaac 780
caagatcttg aaaactggta ctacatgtac gaagttacag gccaaggctc tacacgtaca 840
gctgttgatc cttacgctac agctatcgct cctaacggca cacgtggcat gatcgttgat 900
cttgctaaaa cagatcctgc tggctgggaa tctgataaac atatcacacc taaaaacatc 960
gaagatgaag ttatctacga aatggatgtt cgtgatttct ctatcgattc taactctggc 1020
atgaaaaaca aaggcaaata ccttgctctt acagaaaaag gcacaaaagg ccctgataac 1080
gttaaaacag gcgttgattc tcttaaacaa cttggcatca cacatgttca acttcaacct 1140
gttttcgctt tcaactctgt taacgaaaac gatcctacac aatacaactg gggctacgat 1200
cctcgtaact acaacgttcc tgaaggccaa tacgctacaa acgctaacgg cacaacacgt 1260
atcaaagaat tcaaagaaat ggttctttct cttcatcaag atcatatcgg cgttaacatg 1320
gatgttgttt acaaccatac attcgctaca caaatctctg atttcgataa aatcgttcct 1380
gaatactact accgtacaga tgatgctggc aactacacaa acggctctgg cacaggcaac 1440
gaaatcgctg ctgaacgtcc tatggttcaa aaattcatca tcgattctct taaattctgg 1500
gttaacgaat accatgttga tggcttccgt ttcgatctta tggctcttct tggcaaagat 1560
acaatgtcta aagctgctac acaacttcat gctatcgatc ctggcatcgc tctttacggc 1620
gaaccttgga caggcggcac atctgctctt cctgctgatc aacttcttac aaaaggcgct 1680
caaaaaggca tgggcgttgc tgttttcaac gataaccttc gtaacggcct tgatggctct 1740
gttttcgatt cttctgctca aggcttcgct acaggcgcta caggccttac agatgctatc 1800
aaaaacggcg ttgaaggctc tatcaacgat ttcacagctt ctcctggcga aacaatcaac 1860
tacgttacat ctcatgataa ctacacactt tgggataaaa tcgctcaatc taaccctaac 1920
gattctgaag ctgatcgtat caaaatggat gaacttgctc aagctatcgt tatgacatct 1980
caaggcatcc ctttcatgca aggcggcgaa gaaatgcttc gtacaaaagg cggcaacgat 2040
aactcttaca acgctggcga tgttgttaac gaattcgatt ggtctcgtaa agctcaatac 2100
cctgatgttt tcaactacta ctctggcctt atccatcttc gtcttgatca tcctgctttc 2160
cgtatgacaa cagctaacga aatcaactct catcttcaat tccttaactc tcctgaaaac 2220
acagttgctt acgaactttc tgatcatgct aacaaagata catggggcaa catcgttgtt 2280
atctacaacc ctaacaaaac agctgaaaca atcaaccttc cttctggcaa atgggaaatc 2340
aacgctacat ctggcaaagt tggcgaatct acacttggcc aagctgaagg ctctgttcaa 2400
gttcctggca tctctatgat gatccttcat caagaagttt ctccttctga tggcaaataa 2460。
The gene expression frame Tpu1 contains for knocking outaprEThe homologous segment of gene, its nucleotide sequence is as follows：
caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg 60
atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg 120
gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg 180
ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt 240
ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg 300
ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg 360
ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg 420
atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag 480
gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg 540
aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact 600
attattgtgc agctgcttgc ctgcactgca gggatccaag cttgcggccg ctaccgttcg 660
tatagcatac attatacgaa gttatttaaa ccgtgtgctc tacgaccaaa actataaaac 720
ctttaagaac tttctttttt tacaagaaaa aagaaattag ataaatctct catatctttt 780
attcaataat cgcatccgat tgcagtataa atttaacgat cactcatcat gttcatattt 840
atcagagctc gtgctataat tatactaatt ttataaggag gaaaaaatat gggcattttt 900
agtatttttg taatcagcac agttcattat caaccaaaca aaaaataagt ggttataatg 960
aatcgttaat aagcaaaatt catataacca aattaaggag ggaaataatg aacgagaaaa 1020
atataaaaca cagtcaaaac tttattactt caaaacataa tatagataaa ataatgacaa 1080
atataagatt aaatgaacat gataatatct ttgaaatcgg ctcaggaaaa ggccatttta 1140
cccttgaatt agtaaagagg tgtaatttcg taactgccat tgaaatagac cataaattat 1200
gcaaaactac agaaaataaa cttgttgatc acgataattt ccaagtttta aacaaggata 1260
tattgcagtt taaatttcct aaaaaccaat cctataaaat atatggtaat ataccttata 1320
acataagtac ggatataata cgcaaaattg tttttgatag tatagctaat gagatttatt 1380
taatcgtgga atacgggttt gctaaaagat tattaaatac aaaacgctca ttggcattac 1440
ttttaatggc agaagttgat atttctatat taagtatggt tccaagagaa tattttcatc 1500
ctaaacctaa agtgaatagc tcacttatca gattaagtag aaaaaaatca agaatatcac 1560
acaaagataa acaaaagtat aattatttcg ttatgaaatg ggttaacaaa gaatacaaga 1620
aaatatttac aaaaaatcaa tttaacaatt ccttaaaaca tgcaggaatt gacgatttaa 1680
acaatattag ctttgaacaa ttcttatctc ttttcaatag ctataaatta tttaataagt 1740
aagttaaggg atgcataaac tgcatccctt aacttgtttt tcgtgtgcct attttttgtg 1800
aattgattat cgatcttttg cgccatggat aacttcgtat agcatacatt atacgaacgg 1860
tacatatgcc cgggggtacc gaattccctg ctggtcgcca ttttggctct ttaccctctc 1920
cttttaaaaa aattcagagt agacttactt aaaagactat tctgtgaatt tattgtaata 1980
gatggaataa tattttagta gacccatttt tttgagatga ttttatctct atttaggtat 2040
atcatctctc gctatttccg tagagactcg aaataactat tttcatacag aaaagaacga 2100
aaatagacat gagtaaatgt tcattatgct gaaatgtcat gcttttttag gttaaatgct 2160
cctgagtccc ggcaaattcc tgtcgaaaaa attcgttcaa atgacctgcg tgtgcttccg 2220
tgagaacaat ggatattatc gtgatatttt ttcaaagcat gatgataaaa gtattctgaa 2280
aaataaactt tacagaaaag ggatagaatg aaaaaattat tgagagggag gaagaaataa 2340
gatgaacatt cgccaagcaa agacatcaga tgcggccgcc attgcgccgc tgtttaacca 2400
atatcgggaa ttttatagac aggcatccga tttgcaaggg gcagaggctt ttttgaaagc 2460
tcgtttggaa aatcacgagt ctgttatttt gatagcagaa gaaaatg 2507；
The gene expression frame Tpu2 contains for knocking outamyEThe homologous segment of gene, its nucleotide sequence is as follows：
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg cagggatcca 540
agcttgcggc cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt 600
tgaagattag atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt 660
attaatagct gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa 720
aattatctga aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa 780
gaatgaagat tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta 840
aggctattgg tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg 900
agatgatgtg tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg 960
agtggaaggt ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg 1020
tggaatcaga ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt 1080
caggtggata cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc 1140
acgatgcgat ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta 1200
atattcgtgt gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg 1260
caggtgccat gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct 1320
taactgaagc agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg 1380
taatgtctgg tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga 1440
atgggattca ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac 1500
cattttgaac gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc 1560
tcctagtatt agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta 1620
tacgaacggt acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga 1680
catggatgag cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca 1740
gtacgcctct tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga 1800
aaagccaaat aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca 1860
atagatttca caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca 1920
accagatatt tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat 1980
cctctgtctc tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg 2040
gagcgggttc atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg 2100
tagctgtgct ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca 2160
aaacaggtgt aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga 2220
atacaacaaa agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg 2280
agatcaattc acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa 2340
aaggaacgaa cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc 2400
cagcgtcggc caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt 2460
atatctataa acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac 2520
caatgactaa aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa 2580
ccaacgcaaa agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg 2640
gctttgatta cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc 2700
attga 2705。
The gene expression frame Tpu1 and Tpu2 is containing Pullulanase secretion signal peptide-coding sequence and for recombinating Homology arm sequence, the nucleotide sequence of signal peptide described in Tpu1 is as follows：
atgcttacat tccatcgtat catccgtaaa ggctggatgt tccttcttgc tttccttctt 60
acagcttctc ttttctgccc tacaggccaa catgctaaag ctgctgct 108；
The nucleotide sequence of signal peptide described in Tpu2 is as follows：
atgcttatca acaaatctaa aaaattcttc gttttctctt tcatcttcgt tatgatgctt 60
tctctttctt tcgttaacgg cgaagttgct aaagctgct 99。
Signal peptide sequence containing promoter Pamy-cryIII and amylase secretion in the gene expression frame Tpu1 SPamyL, nucleotide sequence is as follows：
atcgattgtt tgagaaaaga agaagaccat aaaaatacct tgtctgtcat cagacagggt 60
attttttatg ctgtccagac tgtccgctgt gtaaaaaaaa ggaataaagg ggggttgaca 120
ttattttact gatatgtata atataatttg tataagaaaa tggagtcgaa acgtaagatg 180
aaaccttaga taaaagtgct ttttttgttg caattgaaga attattaatg ttaagcttaa 240
ttaaagataa tatctttgaa ttgtaacgcc cctcaaaagt aagaactaca aaaaaagaat 300
acgttatata gaaatatgtt tgaaccttct tcagattaca aatatattcg gacggactct 360
acctcaaatg cttatctaac tatagaatga catacaagca caaccttgaa aatttgaaaa 420
tataactacc aatgaacttg ttcatgtgaa ttatcgcttt atttaatttt ctcaattcaa 480
tatataatat gccaatacat tgttacaagt agaaattaag acacccttga tagccttact 540
atacctaaca tgatgtagta ttaaatgaat atgtaaatat atttatgata agaagcgact 600
tatttataat cattacatat ttttctattg gaatgattaa gattccaata gaatagtgta 660
taaattattt atcttgaaag gagggatgcc taaaaacgaa gaacattaaa aacatatatt 720
tgcaccgtct aatggattta tgaaaaatca ttttatcagt ttgaaaatta tgtattatgc 780
ttacattcca tcgtatcatc cgtaaaggct ggatgttcct tcttgctttc cttcttacag 840
cttctctttt ctgccctaca ggccaacatg ctaaagctgc tgct 884；
The signal peptide sequence secreted in the gene expression frame Tpu2 containing promoter PxylA-cryIII and from chitinase SPchit, nucleotide sequence is as follows：
tagaatctaa tattataact aaattttcta aaaaaaacat tggaatagac atttattttg 60
tatatgatga aataaagtta gtttattgga taaacaaact aactttatta aggtagttga 120
tggataaact tgttcactta aatcaatcga aacgtaagat gaaaccttag ataaaagtgc 180
tttttttgtt gcaattgaag aattattaat gttaagctta attaaagata atatctttga 240
attgtaacgc ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt 300
ttgaaccttc ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa 360
ctatagaatg acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt 420
gttcatgtga attatcgctt tatttaattt tctcaattca atatataata tgccaataca 480
ttgttacaag tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt 540
attaaatgaa tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata 600
tttttctatt ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa 660
ggagggatgc ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt 720
atgaaaaatc attttatcag tttgaaaatt atgtattatg cttatcaaca aatctaaaaa 780
attcttcgtt ttctctttca tcttcgttat gatgctttct ctttctttcg ttaacggcga 840
agttgctaaa gctgct 856。
The beneficial effects of the invention are as follows：Compared with existing strain mutagenesis or molecular biology constructing technology, the present invention The characteristics of comprehensively utilizing bacillus subtilis expression system and advantage, obtain linear in the way of gene design combines gene chemical synthesis Artificial DNA segment；Containing the resistant gene and recombination sequence needed for the molecular biology manipulations later stage in DNA segment, using not With the screening of resistant gene, the expressing gene of double copies is obtained, specificity restructuring is recycled, disposably strikes resistant gene Remove；The elements such as RNA stable factors, efficient secretion peptide are taken full advantage of in the Expression element of design, by recombined bacillus subtilis The advantage of hypersecretion, which is tried one's best, to be brought into play；Production Pullulanase, nonreactive recombined bacillus subtilis CNP2 is finally given, this is heavy Group bacterial strain ferments confirmation through bioreactor can be with high yield Pullulanase, and the enzyme activity for the 32h that ferments is more than 1000 U/mL.
Brief description of the drawings
Fig. 1 is the structure schematic diagram of recombined bacillus subtilis；
The schematic diagram that Fig. 2 changes for recombined bacillus subtilis CNP2 fermentation enzyme activity with fermentation time.
Embodiment
Technical scheme is described in detail with reference to embodiment.The reagent and biomaterial used below If not otherwise specified, it is commercially produced product.
Embodiment 1：Recombined bacillus subtilis CNP2 structure
1. integrated DNA acquisition
The operator of Pullulanase expression has two, firstpulB1Including artificial Gene expression Pamy-cryIII, password The signal peptide sequence SPamyL and the Pullulanase encoding gene of codon optimization of the amylase secretion of son optimizationpulB,pulB Sequence is shown in SEQ ID NO. 3,pulB1Sequence is shown in SEQ ID NO. 10；SecondpulB2Including artificial Gene expression PxylA-cryIII, the signal peptide sequence SPchit and the Propiram of codon optimization of the chitinase secretion of codon optimization Enzyme coding genepulB,pulB2Sequence is shown in SEQ ID NO. 11.
ForaprEThe restructuring arm and the gene expression frame of Erythromycinresistant fragment of knockoutapr-erm-aprSuch as SEQ ID NO. shown in 8, it is used foramyEThe restructuring arm and the gene expression frame of kalamycin resistance fragment of knockoutamy-kan-amySuch as SEQ Shown in ID NO. 9.
pulB1Apr-erm-apr Pst I and BamH I sites are inserted, gene is obtained by artificial synthesized mode Expression cassette Tpu1,pulB2Amy-kan-amy Pst I and BamH I sites are inserted, base is obtained by artificial synthesized mode Because of expression cassette Tpu2.Tpu1 and Tpu2 sequence is shown in SEQ ID NO. 4 and SEQ ID NO. 5 respectively.
2. the structure of recombined bacillus subtilis
Picking A164 single bacteriums are fallen within the test tube containing 3 mL LB liquid mediums first, 37 DEG C, 200 r/min incubated overnights； Then take that 3 mL incubated overnight bacterium solutions are transferred in 40 fresh mL enriched mediums 37 DEG C, 200 r/min are cultivated to OD600 =0.85-0.95, the formula of enriched medium is the g/L sorbierites of LB+ 90；Again by the min of bacterium solution ice-water bath 10, then 5000 G, 5 min, 4 DEG C of collected after centrifugation thalline；Then culture medium is turned with the electricity of 30 mL precoolings, again suspension thalline, 5000 g centrifugations Supernatant is removed in 5 min, 4 DEG C of centrifugations, so operation 3 times, and the formula that electricity turns culture medium is the g/L sweet dews of 10 g/L glucose+92.5 The g/L sorbierites of alcohol+90；Wash after three times, turn after thalline is resuspended culture medium, to be then dispensed into 1.5 using the electricity not removed completely In mL sterile centrifugation tubes, often pipe dispenses 100 μ L, operates on ice；It is linear finally 2 μ g to be added in 100 μ L competent cells DNA fragmentation Tpu1, is incubated 2 min on ice, in the electric revolving cup (1mm) for adding precooling, sets the kV of voltage 2.5, electric shock is once； Electric shock finishes taking-up cup and adds 500 μ L recovery mediums immediately, 37 DEG C, 200 r/min cultures, the group of recovery medium It is divided into the g/L mannitol of LB+90 g/L sorbierites+70；Recover after 3 h, Erythromycinresistant plate, 37 DEG C of insulating boxs are applied respectively Incubated overnight, wherein, erythromycin (Erm) concentration is 10 μ g/mL.
The transformant obtained to screening, is verified, primer sequence used is seen below by the way of bacterium colony PCR.
Sense primer 1P-F：cacccttgatagccttactatacctaac
Anti-sense primer 1P-R：gatgttatcagaagggtaagaagg
Reaction system is as follows：
PCR programs are as follows：
1) 98 DEG C, 2min
2) 98 DEG C, 30sec
3) 52 DEG C, 30sec
4) 72 DEG C, 30sec
5) circulation is from 2) to 4), 30 times
6) 72 DEG C, 5min
7) 4 DEG C, preserve.
Empirical tests are named as CNP1 for positive transformant, then take above-mentioned identical method for transformation, use linear fragment Tpu2 converts CNP1, erythromycin and the Double screen choosing of kanamycins, wherein, the concentration of kanamycins (Kan) is 10 μ g/mL.
The transformant obtained to screening, is verified, primer sequence used is seen below by the way of bacterium colony PCR.
Sense primer 2P-F：cggacggactctacctcaaatgc
Anti-sense primer 2P-R：gcgttagaaacagcaggttgagc
Reaction system is as follows：
Response procedures are consistent with CNP1 verification method.
Empirical tests are named as CNP2ek for positive transformant, then using above-mentioned identical method, with Cre expression of enzymes matter Grain pMK4-cre conversion CNP2ek, the choosing of chlorampenicol resistant screen, wherein, the concentration of chloramphenicol (Cm) is 5 μ g/mL.
The transformant obtained for screening, plasmid pMK4-cre is lost by the way of passage, takes 10 μ L bacterium solutions to be inoculated into In 3 fresh mL LB liquid mediums, 37 DEG C, 200 r/min cultures are transferred 1 time every 8 h, transferred 5 times altogether, then Nutrient solution gradient dilution is taken to 106Times, LB flat boards are applied, 37 DEG C of constant incubator, 200 r/min cultures is placed in；Wait to grow fresh Bacterium colony, difference seal to chloramphenicol, erythromycin and kanamycins flat board carries out resistance counter-selection.Can not in 3 kinds of resistant panels The bacterium of growth, its corresponding bacterium colony on nonreactive LB flat boards, to eliminate the bacterial strain of plasmid, is purpose bacterial strain.Bacterium is carried out to it again Fall PCR checkings, pair of primers is 1P-F/1P-R, and second pair of primer is 2P-F/2P-R.Empirical tests are positive bacterial strain, life Entitled CNP2, for two parts of copiespulBNonreactive pullulanase production strain.
Embodiment 2：Recombined bacillus subtilis CNP2 enzymatic production
Using recombined bacillus subtilis CNP2 as production strain, shake flask fermentation is done：With oese picking recombined bacillus subtilis CNP2 is inoculated into the 200 mL baffle plates bottles equipped with 25 mL fluid nutrient mediums, 37 DEG C, 200 r/min progress fermented and cultureds, its In, the key component of culture medium is corn steep liquor, malt syrup, glucose, dusty yeast and inorganic ion etc..20 h that ferment are sampled Enzyme activity is determined, Pullulanase enzyme activity determination method is：Take 1.0 mL pH=4.5 Acetic acid-sodium acetate buffer enzyme sample or Dilute sample（Control group is the sample that boiling water bath is inactivated）, in the pulullan polysaccharide solution for being added into 1.0 mL 0.5%, in 60 The min of water-bath 30 in DEG C water-bath, adds 3,5- dinitrosalicylic acid solutions（DNS）3.0 mL, the min of boiling water bath 10, take out Flowing water is cooled down afterwards, and light absorption value is surveyed in 550 nm.One enzyme activity unit is defined as：Under the above-described reaction conditions, it is per minute to decompose Pulullan polysaccharide discharges the enzyme amount needed for 1 μm of ol glucose.The h of shake flask fermentation 20 measured enzyme activity is about 225 U/ mL。
Fermented by the way of upper tank feed supplement, in 20 L tanks, add malt syrup and ammoniacal liquor control fermentation tank to flow PH 5.80 or so, ventilation stirring, dissolved oxygen control is 30% or so, and reduced sugar control is 10 g/L or so.Sample and determine enzyme Living, the vigor of Pullulanase is with the result of fermentation time change curve as shown in legend 2.Wherein, in 32 h or so, enzyme activity surpasses Cross 1000 U/mL.
The part preferred embodiment of the present invention is above are only, the present invention is not limited in the content of embodiment.For ability For technical staff in domain, can there are various change and change in the concept of technical solution of the present invention, that is made appoints What changes and changed, within the scope of the present invention.
SEQUENCE LISTING
<110>Baiyin Sino Biotechnology Co., Ltd.
<120>The construction method of the recombined bacillus subtilis of high yield Pullulanase
<130>
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 108
<212> DNA
<213> Artificial
<220>
<223> SPchit
<400> 1
atgcttacat tccatcgtat catccgtaaa ggctggatgt tccttcttgc tttccttctt 60
acagcttctc ttttctgccc tacaggccaa catgctaaag ctgctgct 108
<210> 2
<211> 99
<212> DNA
<213> Artificial
<220>
<223> SPamyL
<400> 2
atgcttatca acaaatctaa aaaattcttc gttttctctt tcatcttcgt tatgatgctt 60
tctctttctt tcgttaacgg cgaagttgct aaagctgct 99
<210> 3
<211> 2460
<212> DNA
<213> Artificial
<220>
<223> pulB
<400> 3
caacctgctg tttctaacgc ttaccttgat gcttctaacc aagttcttgt taaactttct 60
caacctttca cacttggcga aggctcttct ggcttcacag ttcatgatga tacagctaac 120
aaagatatcc ctgttacatc tgtttctgat gctaaccaag ttacagctgt tcttgctggc 180
acattccaac atatcttcgg cggctctgat tgggctcctg ataaccataa cacacttctt 240
aaaaaagtta actctaacct ttaccaattc tctggcaacc ttcctgaagg caactaccaa 300
tacaaagttg ctcttaacga ttcttggaac aacccttctt acccttctga taacatcaac 360
cttacagttc ctgctggcgg cgctcatgtt acattctctt acatcccttc tacacatgct 420
gtttacgata caatcaacaa ccctaacgct gatcttcaag ttgattcttc tggcgttaaa 480
acagatcttg ttgctgttac acttggcgaa aaccctgatg tttctcatac actttctatc 540
caaacagaag attaccaagc tggccaagtt atccctcgta aagttcttga ttcttctcaa 600
tactactact ctggcgatga tcttggcaac acatacacaa aaaacgctac aacattcaaa 660
gtttgggctc ctacatctac acaagttaac gttcttcttt acaactctgc tacaggcgct 720
gttacaaaaa cagttcctat gacagcttct ggccatggcg tttgggaagc tacagttaac 780
caagatcttg aaaactggta ctacatgtac gaagttacag gccaaggctc tacacgtaca 840
gctgttgatc cttacgctac agctatcgct cctaacggca cacgtggcat gatcgttgat 900
cttgctaaaa cagatcctgc tggctgggaa tctgataaac atatcacacc taaaaacatc 960
gaagatgaag ttatctacga aatggatgtt cgtgatttct ctatcgattc taactctggc 1020
atgaaaaaca aaggcaaata ccttgctctt acagaaaaag gcacaaaagg ccctgataac 1080
gttaaaacag gcgttgattc tcttaaacaa cttggcatca cacatgttca acttcaacct 1140
gttttcgctt tcaactctgt taacgaaaac gatcctacac aatacaactg gggctacgat 1200
cctcgtaact acaacgttcc tgaaggccaa tacgctacaa acgctaacgg cacaacacgt 1260
atcaaagaat tcaaagaaat ggttctttct cttcatcaag atcatatcgg cgttaacatg 1320
gatgttgttt acaaccatac attcgctaca caaatctctg atttcgataa aatcgttcct 1380
gaatactact accgtacaga tgatgctggc aactacacaa acggctctgg cacaggcaac 1440
gaaatcgctg ctgaacgtcc tatggttcaa aaattcatca tcgattctct taaattctgg 1500
gttaacgaat accatgttga tggcttccgt ttcgatctta tggctcttct tggcaaagat 1560
acaatgtcta aagctgctac acaacttcat gctatcgatc ctggcatcgc tctttacggc 1620
gaaccttgga caggcggcac atctgctctt cctgctgatc aacttcttac aaaaggcgct 1680
caaaaaggca tgggcgttgc tgttttcaac gataaccttc gtaacggcct tgatggctct 1740
gttttcgatt cttctgctca aggcttcgct acaggcgcta caggccttac agatgctatc 1800
aaaaacggcg ttgaaggctc tatcaacgat ttcacagctt ctcctggcga aacaatcaac 1860
tacgttacat ctcatgataa ctacacactt tgggataaaa tcgctcaatc taaccctaac 1920
gattctgaag ctgatcgtat caaaatggat gaacttgctc aagctatcgt tatgacatct 1980
caaggcatcc ctttcatgca aggcggcgaa gaaatgcttc gtacaaaagg cggcaacgat 2040
aactcttaca acgctggcga tgttgttaac gaattcgatt ggtctcgtaa agctcaatac 2100
cctgatgttt tcaactacta ctctggcctt atccatcttc gtcttgatca tcctgctttc 2160
cgtatgacaa cagctaacga aatcaactct catcttcaat tccttaactc tcctgaaaac 2220
acagttgctt acgaactttc tgatcatgct aacaaagata catggggcaa catcgttgtt 2280
atctacaacc ctaacaaaac agctgaaaca atcaaccttc cttctggcaa atgggaaatc 2340
aacgctacat ctggcaaagt tggcgaatct acacttggcc aagctgaagg ctctgttcaa 2400
gttcctggca tctctatgat gatccttcat caagaagttt ctccttctga tggcaaataa 2460
<210> 4
<211> 5885
<212> DNA
<213> Artificial
<220>
<223> Tpu1
<400> 4
caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg 60
atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg 120
gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg 180
ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt 240
ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg 300
ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg 360
ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg 420
atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag 480
gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg 540
aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact 600
attattgtgc agctgcttgc ctgcactgca gatcgattgt ttgagaaaag aagaagacca 660
taaaaatacc ttgtctgtca tcagacaggg tattttttat gctgtccaga ctgtccgctg 720
tgtaaaaaaa aggaataaag gggggttgac attattttac tgatatgtat aatataattt 780
gtataagaaa atggagtcga aacgtaagat gaaaccttag ataaaagtgc tttttttgtt 840
gcaattgaag aattattaat gttaagctta attaaagata atatctttga attgtaacgc 900
ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt ttgaaccttc 960
ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa ctatagaatg 1020
acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt gttcatgtga 1080
attatcgctt tatttaattt tctcaattca atatataata tgccaataca ttgttacaag 1140
tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt attaaatgaa 1200
tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata tttttctatt 1260
ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa ggagggatgc 1320
ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt atgaaaaatc 1380
attttatcag tttgaaaatt atgtattatg cttacattcc atcgtatcat ccgtaaaggc 1440
tggatgttcc ttcttgcttt ccttcttaca gcttctcttt tctgccctac aggccaacat 1500
gctaaagctg ctgctcaacc tgctgtttct aacgcttacc ttgatgcttc taaccaagtt 1560
cttgttaaac tttctcaacc tttcacactt ggcgaaggct cttctggctt cacagttcat 1620
gatgatacag ctaacaaaga tatccctgtt acatctgttt ctgatgctaa ccaagttaca 1680
gctgttcttg ctggcacatt ccaacatatc ttcggcggct ctgattgggc tcctgataac 1740
cataacacac ttcttaaaaa agttaactct aacctttacc aattctctgg caaccttcct 1800
gaaggcaact accaatacaa agttgctctt aacgattctt ggaacaaccc ttcttaccct 1860
tctgataaca tcaaccttac agttcctgct ggcggcgctc atgttacatt ctcttacatc 1920
ccttctacac atgctgttta cgatacaatc aacaacccta acgctgatct tcaagttgat 1980
tcttctggcg ttaaaacaga tcttgttgct gttacacttg gcgaaaaccc tgatgtttct 2040
catacacttt ctatccaaac agaagattac caagctggcc aagttatccc tcgtaaagtt 2100
cttgattctt ctcaatacta ctactctggc gatgatcttg gcaacacata cacaaaaaac 2160
gctacaacat tcaaagtttg ggctcctaca tctacacaag ttaacgttct tctttacaac 2220
tctgctacag gcgctgttac aaaaacagtt cctatgacag cttctggcca tggcgtttgg 2280
gaagctacag ttaaccaaga tcttgaaaac tggtactaca tgtacgaagt tacaggccaa 2340
ggctctacac gtacagctgt tgatccttac gctacagcta tcgctcctaa cggcacacgt 2400
ggcatgatcg ttgatcttgc taaaacagat cctgctggct gggaatctga taaacatatc 2460
acacctaaaa acatcgaaga tgaagttatc tacgaaatgg atgttcgtga tttctctatc 2520
gattctaact ctggcatgaa aaacaaaggc aaataccttg ctcttacaga aaaaggcaca 2580
aaaggccctg ataacgttaa aacaggcgtt gattctctta aacaacttgg catcacacat 2640
gttcaacttc aacctgtttt cgctttcaac tctgttaacg aaaacgatcc tacacaatac 2700
aactggggct acgatcctcg taactacaac gttcctgaag gccaatacgc tacaaacgct 2760
aacggcacaa cacgtatcaa agaattcaaa gaaatggttc tttctcttca tcaagatcat 2820
atcggcgtta acatggatgt tgtttacaac catacattcg ctacacaaat ctctgatttc 2880
gataaaatcg ttcctgaata ctactaccgt acagatgatg ctggcaacta cacaaacggc 2940
tctggcacag gcaacgaaat cgctgctgaa cgtcctatgg ttcaaaaatt catcatcgat 3000
tctcttaaat tctgggttaa cgaataccat gttgatggct tccgtttcga tcttatggct 3060
cttcttggca aagatacaat gtctaaagct gctacacaac ttcatgctat cgatcctggc 3120
atcgctcttt acggcgaacc ttggacaggc ggcacatctg ctcttcctgc tgatcaactt 3180
cttacaaaag gcgctcaaaa aggcatgggc gttgctgttt tcaacgataa ccttcgtaac 3240
ggccttgatg gctctgtttt cgattcttct gctcaaggct tcgctacagg cgctacaggc 3300
cttacagatg ctatcaaaaa cggcgttgaa ggctctatca acgatttcac agcttctcct 3360
ggcgaaacaa tcaactacgt tacatctcat gataactaca cactttggga taaaatcgct 3420
caatctaacc ctaacgattc tgaagctgat cgtatcaaaa tggatgaact tgctcaagct 3480
atcgttatga catctcaagg catccctttc atgcaaggcg gcgaagaaat gcttcgtaca 3540
aaaggcggca acgataactc ttacaacgct ggcgatgttg ttaacgaatt cgattggtct 3600
cgtaaagctc aataccctga tgttttcaac tactactctg gccttatcca tcttcgtctt 3660
gatcatcctg ctttccgtat gacaacagct aacgaaatca actctcatct tcaattcctt 3720
aactctcctg aaaacacagt tgcttacgaa ctttctgatc atgctaacaa agatacatgg 3780
ggcaacatcg ttgttatcta caaccctaac aaaacagctg aaacaatcaa ccttccttct 3840
ggcaaatggg aaatcaacgc tacatctggc aaagttggcg aatctacact tggccaagct 3900
gaaggctctg ttcaagttcc tggcatctct atgatgatcc ttcatcaaga agtttctcct 3960
tctgatggca aataaagaag caggttcctc catacctgct tctttttaag gatccaagct 4020
tgcggccgct accgttcgta tagcatacat tatacgaagt tatttaaacc gtgtgctcta 4080
cgaccaaaac tataaaacct ttaagaactt tcttttttta caagaaaaaa gaaattagat 4140
aaatctctca tatcttttat tcaataatcg catccgattg cagtataaat ttaacgatca 4200
ctcatcatgt tcatatttat cagagctcgt gctataatta tactaatttt ataaggagga 4260
aaaaatatgg gcatttttag tatttttgta atcagcacag ttcattatca accaaacaaa 4320
aaataagtgg ttataatgaa tcgttaataa gcaaaattca tataaccaaa ttaaggaggg 4380
aaataatgaa cgagaaaaat ataaaacaca gtcaaaactt tattacttca aaacataata 4440
tagataaaat aatgacaaat ataagattaa atgaacatga taatatcttt gaaatcggct 4500
caggaaaagg ccattttacc cttgaattag taaagaggtg taatttcgta actgccattg 4560
aaatagacca taaattatgc aaaactacag aaaataaact tgttgatcac gataatttcc 4620
aagttttaaa caaggatata ttgcagttta aatttcctaa aaaccaatcc tataaaatat 4680
atggtaatat accttataac ataagtacgg atataatacg caaaattgtt tttgatagta 4740
tagctaatga gatttattta atcgtggaat acgggtttgc taaaagatta ttaaatacaa 4800
aacgctcatt ggcattactt ttaatggcag aagttgatat ttctatatta agtatggttc 4860
caagagaata ttttcatcct aaacctaaag tgaatagctc acttatcaga ttaagtagaa 4920
aaaaatcaag aatatcacac aaagataaac aaaagtataa ttatttcgtt atgaaatggg 4980
ttaacaaaga atacaagaaa atatttacaa aaaatcaatt taacaattcc ttaaaacatg 5040
caggaattga cgatttaaac aatattagct ttgaacaatt cttatctctt ttcaatagct 5100
ataaattatt taataagtaa gttaagggat gcataaactg catcccttaa cttgtttttc 5160
gtgtgcctat tttttgtgaa ttgattatcg atcttttgcg ccatggataa cttcgtatag 5220
catacattat acgaacggta catatgcccg ggggtaccga attccctgct ggtcgccatt 5280
ttggctcttt accctctcct tttaaaaaaa ttcagagtag acttacttaa aagactattc 5340
tgtgaattta ttgtaataga tggaataata ttttagtaga cccatttttt tgagatgatt 5400
ttatctctat ttaggtatat catctctcgc tatttccgta gagactcgaa ataactattt 5460
tcatacagaa aagaacgaaa atagacatga gtaaatgttc attatgctga aatgtcatgc 5520
ttttttaggt taaatgctcc tgagtcccgg caaattcctg tcgaaaaaat tcgttcaaat 5580
gacctgcgtg tgcttccgtg agaacaatgg atattatcgt gatatttttt caaagcatga 5640
tgataaaagt attctgaaaa ataaacttta cagaaaaggg atagaatgaa aaaattattg 5700
agagggagga agaaataaga tgaacattcg ccaagcaaag acatcagatg cggccgccat 5760
tgcgccgctg tttaaccaat atcgggaatt ttatagacag gcatccgatt tgcaaggggc 5820
agaggctttt ttgaaagctc gtttggaaaa tcacgagtct gttattttga tagcagaaga 5880
aaatg 5885
<210> 5
<211> 6055
<212> DNA
<213> Artificial
<220>
<223> Tpu2
<400> 5
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg catagaatct 540
aatattataa ctaaattttc taaaaaaaac attggaatag acatttattt tgtatatgat 600
gaaataaagt tagtttattg gataaacaaa ctaactttat taaggtagtt gatggataaa 660
cttgttcact taaatcaatc gaaacgtaag atgaaacctt agataaaagt gctttttttg 720
ttgcaattga agaattatta atgttaagct taattaaaga taatatcttt gaattgtaac 780
gcccctcaaa agtaagaact acaaaaaaag aatacgttat atagaaatat gtttgaacct 840
tcttcagatt acaaatatat tcggacggac tctacctcaa atgcttatct aactatagaa 900
tgacatacaa gcacaacctt gaaaatttga aaatataact accaatgaac ttgttcatgt 960
gaattatcgc tttatttaat tttctcaatt caatatataa tatgccaata cattgttaca 1020
agtagaaatt aagacaccct tgatagcctt actataccta acatgatgta gtattaaatg 1080
aatatgtaaa tatatttatg ataagaagcg acttatttat aatcattaca tatttttcta 1140
ttggaatgat taagattcca atagaatagt gtataaatta tttatcttga aaggagggat 1200
gcctaaaaac gaagaacatt aaaaacatat atttgcaccg tctaatggat ttatgaaaaa 1260
tcattttatc agtttgaaaa ttatgtatta tgcttatcaa caaatctaaa aaattcttcg 1320
ttttctcttt catcttcgtt atgatgcttt ctctttcttt cgttaacggc gaagttgcta 1380
aagctgctca acctgctgtt tctaacgctt accttgatgc ttctaaccaa gttcttgtta 1440
aactttctca acctttcaca cttggcgaag gctcttctgg cttcacagtt catgatgata 1500
cagctaacaa agatatccct gttacatctg tttctgatgc taaccaagtt acagctgttc 1560
ttgctggcac attccaacat atcttcggcg gctctgattg ggctcctgat aaccataaca 1620
cacttcttaa aaaagttaac tctaaccttt accaattctc tggcaacctt cctgaaggca 1680
actaccaata caaagttgct cttaacgatt cttggaacaa cccttcttac ccttctgata 1740
acatcaacct tacagttcct gctggcggcg ctcatgttac attctcttac atcccttcta 1800
cacatgctgt ttacgataca atcaacaacc ctaacgctga tcttcaagtt gattcttctg 1860
gcgttaaaac agatcttgtt gctgttacac ttggcgaaaa ccctgatgtt tctcatacac 1920
tttctatcca aacagaagat taccaagctg gccaagttat ccctcgtaaa gttcttgatt 1980
cttctcaata ctactactct ggcgatgatc ttggcaacac atacacaaaa aacgctacaa 2040
cattcaaagt ttgggctcct acatctacac aagttaacgt tcttctttac aactctgcta 2100
caggcgctgt tacaaaaaca gttcctatga cagcttctgg ccatggcgtt tgggaagcta 2160
cagttaacca agatcttgaa aactggtact acatgtacga agttacaggc caaggctcta 2220
cacgtacagc tgttgatcct tacgctacag ctatcgctcc taacggcaca cgtggcatga 2280
tcgttgatct tgctaaaaca gatcctgctg gctgggaatc tgataaacat atcacaccta 2340
aaaacatcga agatgaagtt atctacgaaa tggatgttcg tgatttctct atcgattcta 2400
actctggcat gaaaaacaaa ggcaaatacc ttgctcttac agaaaaaggc acaaaaggcc 2460
ctgataacgt taaaacaggc gttgattctc ttaaacaact tggcatcaca catgttcaac 2520
ttcaacctgt tttcgctttc aactctgtta acgaaaacga tcctacacaa tacaactggg 2580
gctacgatcc tcgtaactac aacgttcctg aaggccaata cgctacaaac gctaacggca 2640
caacacgtat caaagaattc aaagaaatgg ttctttctct tcatcaagat catatcggcg 2700
ttaacatgga tgttgtttac aaccatacat tcgctacaca aatctctgat ttcgataaaa 2760
tcgttcctga atactactac cgtacagatg atgctggcaa ctacacaaac ggctctggca 2820
caggcaacga aatcgctgct gaacgtccta tggttcaaaa attcatcatc gattctctta 2880
aattctgggt taacgaatac catgttgatg gcttccgttt cgatcttatg gctcttcttg 2940
gcaaagatac aatgtctaaa gctgctacac aacttcatgc tatcgatcct ggcatcgctc 3000
tttacggcga accttggaca ggcggcacat ctgctcttcc tgctgatcaa cttcttacaa 3060
aaggcgctca aaaaggcatg ggcgttgctg ttttcaacga taaccttcgt aacggccttg 3120
atggctctgt tttcgattct tctgctcaag gcttcgctac aggcgctaca ggccttacag 3180
atgctatcaa aaacggcgtt gaaggctcta tcaacgattt cacagcttct cctggcgaaa 3240
caatcaacta cgttacatct catgataact acacactttg ggataaaatc gctcaatcta 3300
accctaacga ttctgaagct gatcgtatca aaatggatga acttgctcaa gctatcgtta 3360
tgacatctca aggcatccct ttcatgcaag gcggcgaaga aatgcttcgt acaaaaggcg 3420
gcaacgataa ctcttacaac gctggcgatg ttgttaacga attcgattgg tctcgtaaag 3480
ctcaataccc tgatgttttc aactactact ctggccttat ccatcttcgt cttgatcatc 3540
ctgctttccg tatgacaaca gctaacgaaa tcaactctca tcttcaattc cttaactctc 3600
ctgaaaacac agttgcttac gaactttctg atcatgctaa caaagataca tggggcaaca 3660
tcgttgttat ctacaaccct aacaaaacag ctgaaacaat caaccttcct tctggcaaat 3720
gggaaatcaa cgctacatct ggcaaagttg gcgaatctac acttggccaa gctgaaggct 3780
ctgttcaagt tcctggcatc tctatgatga tccttcatca agaagtttct ccttctgatg 3840
gcaaataaag aagcaggttc ctccatacct gcttcttttt aagggatcca agcttgcggc 3900
cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt tgaagattag 3960
atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct 4020
gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga 4080
aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat 4140
tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg 4200
tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg 4260
tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt 4320
ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga 4380
ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt caggtggata 4440
cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat 4500
ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt 4560
gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat 4620
gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc 4680
agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg 4740
tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga atgggattca 4800
ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac 4860
gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt 4920
agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta tacgaacggt 4980
acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga catggatgag 5040
cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca gtacgcctct 5100
tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga aaagccaaat 5160
aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca atagatttca 5220
caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca accagatatt 5280
tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat cctctgtctc 5340
tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg gagcgggttc 5400
atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg tagctgtgct 5460
ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca aaacaggtgt 5520
aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga atacaacaaa 5580
agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg agatcaattc 5640
acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa aaggaacgaa 5700
cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc cagcgtcggc 5760
caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt atatctataa 5820
acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac caatgactaa 5880
aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa ccaacgcaaa 5940
agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg gctttgatta 6000
cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc attga 6055
<210> 6
<211> 884
<212> DNA
<213> Artificial
<220>
<223> Pamy-cryIII-SPamyL
<400> 6
atcgattgtt tgagaaaaga agaagaccat aaaaatacct tgtctgtcat cagacagggt 60
attttttatg ctgtccagac tgtccgctgt gtaaaaaaaa ggaataaagg ggggttgaca 120
ttattttact gatatgtata atataatttg tataagaaaa tggagtcgaa acgtaagatg 180
aaaccttaga taaaagtgct ttttttgttg caattgaaga attattaatg ttaagcttaa 240
ttaaagataa tatctttgaa ttgtaacgcc cctcaaaagt aagaactaca aaaaaagaat 300
acgttatata gaaatatgtt tgaaccttct tcagattaca aatatattcg gacggactct 360
acctcaaatg cttatctaac tatagaatga catacaagca caaccttgaa aatttgaaaa 420
tataactacc aatgaacttg ttcatgtgaa ttatcgcttt atttaatttt ctcaattcaa 480
tatataatat gccaatacat tgttacaagt agaaattaag acacccttga tagccttact 540
atacctaaca tgatgtagta ttaaatgaat atgtaaatat atttatgata agaagcgact 600
tatttataat cattacatat ttttctattg gaatgattaa gattccaata gaatagtgta 660
taaattattt atcttgaaag gagggatgcc taaaaacgaa gaacattaaa aacatatatt 720
tgcaccgtct aatggattta tgaaaaatca ttttatcagt ttgaaaatta tgtattatgc 780
ttacattcca tcgtatcatc cgtaaaggct ggatgttcct tcttgctttc cttcttacag 840
cttctctttt ctgccctaca ggccaacatg ctaaagctgc tgct 884
<210> 7
<211> 856
<212> DNA
<213> Artificial
<220>
<223> PxylA-cryIII-SPchit
<400> 7
tagaatctaa tattataact aaattttcta aaaaaaacat tggaatagac atttattttg 60
tatatgatga aataaagtta gtttattgga taaacaaact aactttatta aggtagttga 120
tggataaact tgttcactta aatcaatcga aacgtaagat gaaaccttag ataaaagtgc 180
tttttttgtt gcaattgaag aattattaat gttaagctta attaaagata atatctttga 240
attgtaacgc ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt 300
ttgaaccttc ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa 360
ctatagaatg acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt 420
gttcatgtga attatcgctt tatttaattt tctcaattca atatataata tgccaataca 480
ttgttacaag tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt 540
attaaatgaa tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata 600
tttttctatt ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa 660
ggagggatgc ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt 720
atgaaaaatc attttatcag tttgaaaatt atgtattatg cttatcaaca aatctaaaaa 780
attcttcgtt ttctctttca tcttcgttat gatgctttct ctttctttcg ttaacggcga 840
agttgctaaa gctgct 856
<210> 8
<211> 2507
<212> DNA
<213>Knock out the homologous fragment of aprE genes
<400> 8
caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg 60
atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg 120
gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg 180
ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt 240
ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg 300
ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg 360
ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg 420
atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag 480
gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg 540
aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact 600
attattgtgc agctgcttgc ctgcactgca gggatccaag cttgcggccg ctaccgttcg 660
tatagcatac attatacgaa gttatttaaa ccgtgtgctc tacgaccaaa actataaaac 720
ctttaagaac tttctttttt tacaagaaaa aagaaattag ataaatctct catatctttt 780
attcaataat cgcatccgat tgcagtataa atttaacgat cactcatcat gttcatattt 840
atcagagctc gtgctataat tatactaatt ttataaggag gaaaaaatat gggcattttt 900
agtatttttg taatcagcac agttcattat caaccaaaca aaaaataagt ggttataatg 960
aatcgttaat aagcaaaatt catataacca aattaaggag ggaaataatg aacgagaaaa 1020
atataaaaca cagtcaaaac tttattactt caaaacataa tatagataaa ataatgacaa 1080
atataagatt aaatgaacat gataatatct ttgaaatcgg ctcaggaaaa ggccatttta 1140
cccttgaatt agtaaagagg tgtaatttcg taactgccat tgaaatagac cataaattat 1200
gcaaaactac agaaaataaa cttgttgatc acgataattt ccaagtttta aacaaggata 1260
tattgcagtt taaatttcct aaaaaccaat cctataaaat atatggtaat ataccttata 1320
acataagtac ggatataata cgcaaaattg tttttgatag tatagctaat gagatttatt 1380
taatcgtgga atacgggttt gctaaaagat tattaaatac aaaacgctca ttggcattac 1440
ttttaatggc agaagttgat atttctatat taagtatggt tccaagagaa tattttcatc 1500
ctaaacctaa agtgaatagc tcacttatca gattaagtag aaaaaaatca agaatatcac 1560
acaaagataa acaaaagtat aattatttcg ttatgaaatg ggttaacaaa gaatacaaga 1620
aaatatttac aaaaaatcaa tttaacaatt ccttaaaaca tgcaggaatt gacgatttaa 1680
acaatattag ctttgaacaa ttcttatctc ttttcaatag ctataaatta tttaataagt 1740
aagttaaggg atgcataaac tgcatccctt aacttgtttt tcgtgtgcct attttttgtg 1800
aattgattat cgatcttttg cgccatggat aacttcgtat agcatacatt atacgaacgg 1860
tacatatgcc cgggggtacc gaattccctg ctggtcgcca ttttggctct ttaccctctc 1920
cttttaaaaa aattcagagt agacttactt aaaagactat tctgtgaatt tattgtaata 1980
gatggaataa tattttagta gacccatttt tttgagatga ttttatctct atttaggtat 2040
atcatctctc gctatttccg tagagactcg aaataactat tttcatacag aaaagaacga 2100
aaatagacat gagtaaatgt tcattatgct gaaatgtcat gcttttttag gttaaatgct 2160
cctgagtccc ggcaaattcc tgtcgaaaaa attcgttcaa atgacctgcg tgtgcttccg 2220
tgagaacaat ggatattatc gtgatatttt ttcaaagcat gatgataaaa gtattctgaa 2280
aaataaactt tacagaaaag ggatagaatg aaaaaattat tgagagggag gaagaaataa 2340
gatgaacatt cgccaagcaa agacatcaga tgcggccgcc attgcgccgc tgtttaacca 2400
atatcgggaa ttttatagac aggcatccga tttgcaaggg gcagaggctt ttttgaaagc 2460
tcgtttggaa aatcacgagt ctgttatttt gatagcagaa gaaaatg 2507
<210> 9
<211> 2705
<212> DNA
<213>Knock out the homologous fragment of amyE genes
<400> 9
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg cagggatcca 540
agcttgcggc cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt 600
tgaagattag atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt 660
attaatagct gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa 720
aattatctga aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa 780
gaatgaagat tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta 840
aggctattgg tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg 900
agatgatgtg tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg 960
agtggaaggt ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg 1020
tggaatcaga ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt 1080
caggtggata cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc 1140
acgatgcgat ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta 1200
atattcgtgt gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg 1260
caggtgccat gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct 1320
taactgaagc agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg 1380
taatgtctgg tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga 1440
atgggattca ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac 1500
cattttgaac gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc 1560
tcctagtatt agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta 1620
tacgaacggt acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga 1680
catggatgag cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca 1740
gtacgcctct tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga 1800
aaagccaaat aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca 1860
atagatttca caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca 1920
accagatatt tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat 1980
cctctgtctc tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg 2040
gagcgggttc atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg 2100
tagctgtgct ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca 2160
aaacaggtgt aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga 2220
atacaacaaa agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg 2280
agatcaattc acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa 2340
aaggaacgaa cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc 2400
cagcgtcggc caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt 2460
atatctataa acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac 2520
caatgactaa aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa 2580
ccaacgcaaa agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg 2640
gctttgatta cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc 2700
attga 2705
<210> 10
<211> 3378
<212> DNA
<213> Artificial
<220>
<223> pulB1
<400> 10
atcgattgtt tgagaaaaga agaagaccat aaaaatacct tgtctgtcat cagacagggt 60
attttttatg ctgtccagac tgtccgctgt gtaaaaaaaa ggaataaagg ggggttgaca 120
ttattttact gatatgtata atataatttg tataagaaaa tggagtcgaa acgtaagatg 180
aaaccttaga taaaagtgct ttttttgttg caattgaaga attattaatg ttaagcttaa 240
ttaaagataa tatctttgaa ttgtaacgcc cctcaaaagt aagaactaca aaaaaagaat 300
acgttatata gaaatatgtt tgaaccttct tcagattaca aatatattcg gacggactct 360
acctcaaatg cttatctaac tatagaatga catacaagca caaccttgaa aatttgaaaa 420
tataactacc aatgaacttg ttcatgtgaa ttatcgcttt atttaatttt ctcaattcaa 480
tatataatat gccaatacat tgttacaagt agaaattaag acacccttga tagccttact 540
atacctaaca tgatgtagta ttaaatgaat atgtaaatat atttatgata agaagcgact 600
tatttataat cattacatat ttttctattg gaatgattaa gattccaata gaatagtgta 660
taaattattt atcttgaaag gagggatgcc taaaaacgaa gaacattaaa aacatatatt 720
tgcaccgtct aatggattta tgaaaaatca ttttatcagt ttgaaaatta tgtattatgc 780
ttacattcca tcgtatcatc cgtaaaggct ggatgttcct tcttgctttc cttcttacag 840
cttctctttt ctgccctaca ggccaacatg ctaaagctgc tgctcaacct gctgtttcta 900
acgcttacct tgatgcttct aaccaagttc ttgttaaact ttctcaacct ttcacacttg 960
gcgaaggctc ttctggcttc acagttcatg atgatacagc taacaaagat atccctgtta 1020
catctgtttc tgatgctaac caagttacag ctgttcttgc tggcacattc caacatatct 1080
tcggcggctc tgattgggct cctgataacc ataacacact tcttaaaaaa gttaactcta 1140
acctttacca attctctggc aaccttcctg aaggcaacta ccaatacaaa gttgctctta 1200
acgattcttg gaacaaccct tcttaccctt ctgataacat caaccttaca gttcctgctg 1260
gcggcgctca tgttacattc tcttacatcc cttctacaca tgctgtttac gatacaatca 1320
acaaccctaa cgctgatctt caagttgatt cttctggcgt taaaacagat cttgttgctg 1380
ttacacttgg cgaaaaccct gatgtttctc atacactttc tatccaaaca gaagattacc 1440
aagctggcca agttatccct cgtaaagttc ttgattcttc tcaatactac tactctggcg 1500
atgatcttgg caacacatac acaaaaaacg ctacaacatt caaagtttgg gctcctacat 1560
ctacacaagt taacgttctt ctttacaact ctgctacagg cgctgttaca aaaacagttc 1620
ctatgacagc ttctggccat ggcgtttggg aagctacagt taaccaagat cttgaaaact 1680
ggtactacat gtacgaagtt acaggccaag gctctacacg tacagctgtt gatccttacg 1740
ctacagctat cgctcctaac ggcacacgtg gcatgatcgt tgatcttgct aaaacagatc 1800
ctgctggctg ggaatctgat aaacatatca cacctaaaaa catcgaagat gaagttatct 1860
acgaaatgga tgttcgtgat ttctctatcg attctaactc tggcatgaaa aacaaaggca 1920
aataccttgc tcttacagaa aaaggcacaa aaggccctga taacgttaaa acaggcgttg 1980
attctcttaa acaacttggc atcacacatg ttcaacttca acctgttttc gctttcaact 2040
ctgttaacga aaacgatcct acacaataca actggggcta cgatcctcgt aactacaacg 2100
ttcctgaagg ccaatacgct acaaacgcta acggcacaac acgtatcaaa gaattcaaag 2160
aaatggttct ttctcttcat caagatcata tcggcgttaa catggatgtt gtttacaacc 2220
atacattcgc tacacaaatc tctgatttcg ataaaatcgt tcctgaatac tactaccgta 2280
cagatgatgc tggcaactac acaaacggct ctggcacagg caacgaaatc gctgctgaac 2340
gtcctatggt tcaaaaattc atcatcgatt ctcttaaatt ctgggttaac gaataccatg 2400
ttgatggctt ccgtttcgat cttatggctc ttcttggcaa agatacaatg tctaaagctg 2460
ctacacaact tcatgctatc gatcctggca tcgctcttta cggcgaacct tggacaggcg 2520
gcacatctgc tcttcctgct gatcaacttc ttacaaaagg cgctcaaaaa ggcatgggcg 2580
ttgctgtttt caacgataac cttcgtaacg gccttgatgg ctctgttttc gattcttctg 2640
ctcaaggctt cgctacaggc gctacaggcc ttacagatgc tatcaaaaac ggcgttgaag 2700
gctctatcaa cgatttcaca gcttctcctg gcgaaacaat caactacgtt acatctcatg 2760
ataactacac actttgggat aaaatcgctc aatctaaccc taacgattct gaagctgatc 2820
gtatcaaaat ggatgaactt gctcaagcta tcgttatgac atctcaaggc atccctttca 2880
tgcaaggcgg cgaagaaatg cttcgtacaa aaggcggcaa cgataactct tacaacgctg 2940
gcgatgttgt taacgaattc gattggtctc gtaaagctca ataccctgat gttttcaact 3000
actactctgg ccttatccat cttcgtcttg atcatcctgc tttccgtatg acaacagcta 3060
acgaaatcaa ctctcatctt caattcctta actctcctga aaacacagtt gcttacgaac 3120
tttctgatca tgctaacaaa gatacatggg gcaacatcgt tgttatctac aaccctaaca 3180
aaacagctga aacaatcaac cttccttctg gcaaatggga aatcaacgct acatctggca 3240
aagttggcga atctacactt ggccaagctg aaggctctgt tcaagttcct ggcatctcta 3300
tgatgatcct tcatcaagaa gtttctcctt ctgatggcaa ataaagaagc aggttcctcc 3360
atacctgctt ctttttaa 3378
<210> 11
<211> 3350
<212> DNA
<213> Artificial
<220>
<223> pulB2
<400> 11
tagaatctaa tattataact aaattttcta aaaaaaacat tggaatagac atttattttg 60
tatatgatga aataaagtta gtttattgga taaacaaact aactttatta aggtagttga 120
tggataaact tgttcactta aatcaatcga aacgtaagat gaaaccttag ataaaagtgc 180
tttttttgtt gcaattgaag aattattaat gttaagctta attaaagata atatctttga 240
attgtaacgc ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt 300
ttgaaccttc ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa 360
ctatagaatg acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt 420
gttcatgtga attatcgctt tatttaattt tctcaattca atatataata tgccaataca 480
ttgttacaag tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt 540
attaaatgaa tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata 600
tttttctatt ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa 660
ggagggatgc ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt 720
atgaaaaatc attttatcag tttgaaaatt atgtattatg cttatcaaca aatctaaaaa 780
attcttcgtt ttctctttca tcttcgttat gatgctttct ctttctttcg ttaacggcga 840
agttgctaaa gctgctcaac ctgctgtttc taacgcttac cttgatgctt ctaaccaagt 900
tcttgttaaa ctttctcaac ctttcacact tggcgaaggc tcttctggct tcacagttca 960
tgatgataca gctaacaaag atatccctgt tacatctgtt tctgatgcta accaagttac 1020
agctgttctt gctggcacat tccaacatat cttcggcggc tctgattggg ctcctgataa 1080
ccataacaca cttcttaaaa aagttaactc taacctttac caattctctg gcaaccttcc 1140
tgaaggcaac taccaataca aagttgctct taacgattct tggaacaacc cttcttaccc 1200
ttctgataac atcaacctta cagttcctgc tggcggcgct catgttacat tctcttacat 1260
cccttctaca catgctgttt acgatacaat caacaaccct aacgctgatc ttcaagttga 1320
ttcttctggc gttaaaacag atcttgttgc tgttacactt ggcgaaaacc ctgatgtttc 1380
tcatacactt tctatccaaa cagaagatta ccaagctggc caagttatcc ctcgtaaagt 1440
tcttgattct tctcaatact actactctgg cgatgatctt ggcaacacat acacaaaaaa 1500
cgctacaaca ttcaaagttt gggctcctac atctacacaa gttaacgttc ttctttacaa 1560
ctctgctaca ggcgctgtta caaaaacagt tcctatgaca gcttctggcc atggcgtttg 1620
ggaagctaca gttaaccaag atcttgaaaa ctggtactac atgtacgaag ttacaggcca 1680
aggctctaca cgtacagctg ttgatcctta cgctacagct atcgctccta acggcacacg 1740
tggcatgatc gttgatcttg ctaaaacaga tcctgctggc tgggaatctg ataaacatat 1800
cacacctaaa aacatcgaag atgaagttat ctacgaaatg gatgttcgtg atttctctat 1860
cgattctaac tctggcatga aaaacaaagg caaatacctt gctcttacag aaaaaggcac 1920
aaaaggccct gataacgtta aaacaggcgt tgattctctt aaacaacttg gcatcacaca 1980
tgttcaactt caacctgttt tcgctttcaa ctctgttaac gaaaacgatc ctacacaata 2040
caactggggc tacgatcctc gtaactacaa cgttcctgaa ggccaatacg ctacaaacgc 2100
taacggcaca acacgtatca aagaattcaa agaaatggtt ctttctcttc atcaagatca 2160
tatcggcgtt aacatggatg ttgtttacaa ccatacattc gctacacaaa tctctgattt 2220
cgataaaatc gttcctgaat actactaccg tacagatgat gctggcaact acacaaacgg 2280
ctctggcaca ggcaacgaaa tcgctgctga acgtcctatg gttcaaaaat tcatcatcga 2340
ttctcttaaa ttctgggtta acgaatacca tgttgatggc ttccgtttcg atcttatggc 2400
tcttcttggc aaagatacaa tgtctaaagc tgctacacaa cttcatgcta tcgatcctgg 2460
catcgctctt tacggcgaac cttggacagg cggcacatct gctcttcctg ctgatcaact 2520
tcttacaaaa ggcgctcaaa aaggcatggg cgttgctgtt ttcaacgata accttcgtaa 2580
cggccttgat ggctctgttt tcgattcttc tgctcaaggc ttcgctacag gcgctacagg 2640
ccttacagat gctatcaaaa acggcgttga aggctctatc aacgatttca cagcttctcc 2700
tggcgaaaca atcaactacg ttacatctca tgataactac acactttggg ataaaatcgc 2760
tcaatctaac cctaacgatt ctgaagctga tcgtatcaaa atggatgaac ttgctcaagc 2820
tatcgttatg acatctcaag gcatcccttt catgcaaggc ggcgaagaaa tgcttcgtac 2880
aaaaggcggc aacgataact cttacaacgc tggcgatgtt gttaacgaat tcgattggtc 2940
tcgtaaagct caataccctg atgttttcaa ctactactct ggccttatcc atcttcgtct 3000
tgatcatcct gctttccgta tgacaacagc taacgaaatc aactctcatc ttcaattcct 3060
taactctcct gaaaacacag ttgcttacga actttctgat catgctaaca aagatacatg 3120
gggcaacatc gttgttatct acaaccctaa caaaacagct gaaacaatca accttccttc 3180
tggcaaatgg gaaatcaacg ctacatctgg caaagttggc gaatctacac ttggccaagc 3240
tgaaggctct gttcaagttc ctggcatctc tatgatgatc cttcatcaag aagtttctcc 3300
ttctgatggc aaataaagaa gcaggttcct ccatacctgc ttctttttaa 3350
<210> 12
<211> 6932
<212> DNA
<213> Artificial
<220>
<223> pMK4-cre
<400> 12
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgg 240
ctgcaggtcg acggatcccc gggaattcta cacagcccag tccagactat tcggcactga 300
aattatgggt gaagtggtca agacctcact aggcacctta aaaatagcgc accctgaaga 360
agatttattt gaggtagccc ttgcctacct agcttccaag aaagatatcc taacagcaca 420
agagcggaaa gatgttttgt tctacatcca gaacaacctc tgctaaaatt cctgaaaaat 480
tttgcaaaaa gttgttgact ttatctacaa ggtgtggcat aatgtgtgga attgtgagcg 540
gataacaatt aagcttaagg aggaagcagg tatgtccaat ttactgaccg tacaccaaaa 600
tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat 660
gttcagggat cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg 720
gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga 780
tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca 840
acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag 900
caatgctgtt tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg 960
tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga 1020
aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac 1080
cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg 1140
tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga 1200
gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt 1260
agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc 1320
atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg 1380
attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag 1440
tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat 1500
gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag 1560
tgaaacaggg gcaatggtgc gcctgctgga agatggcgat taggcatgcg aattcactgg 1620
ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 1680
cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 1740
cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc 1800
atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg 1860
catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 1920
tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 1980
ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 2040
tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 2100
atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 2160
tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 2220
aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 2280
acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 2340
acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 2400
ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 2460
ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 2520
caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 2580
ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 2640
aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 2700
aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 2760
tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 2820
aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 2880
cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 2940
ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 3000
gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 3060
agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 3120
atttttaatt taaaaggatc taggtgaaga tccatatcct tctttttctg aaccgacttc 3180
tcctttttcg cttctttatt ccaattgctt tattgacgtt gagcctcgga acccttaaca 3240
atcccaaaac ttgtcgaatg gtcggcttaa tagctcacgc tatgccgaca ttcgtctgca 3300
agtttagtta agggttcttc tcaacgcaca ataaattttc tcggcataaa tgcgtggtct 3360
aatttttatt tttaataacc ttgatagcaa aaaatgccat tccaatacaa aaccacatac 3420
ctataatcga taaccacata acagtcataa aaccactcct ttttaacaaa ctttatcaca 3480
agaaatattt aaattttaaa tgcctttatt ttgaatttta aggggcattt taaagattta 3540
ggggtaaatc atatagtttt atgcctaaaa acctacagaa gcttttaaaa agcaaatatg 3600
agccaaataa atatattcta attctacaaa caaaaatttg agcaaattca gtgtcgattt 3660
tttaagacac tgcccagtta catgcaaatt aaaattttca tgatttttta tagttcctaa 3720
cagggttaaa atttgtataa cgaaagtata atgtttatat aacgttagta taataaagca 3780
ttttaacatt atacttttga taatcgttta tcgtcgtcat cacaataact tttaaaatac 3840
tcgtgcataa ttcaacagct gacctcccaa taactacatg gtgttatcgg gaggtcagct 3900
gttagcactt atattttgtt attgttcttc ctcgatttcg tctatcattt tgtgattaat 3960
ttctcttttt tcttgttctg ttaagtcata aagttcacta gctaaatact ctttttgttt 4020
ccaaatataa aaaatttgat agatatattc ggttggatca atttctttta agtaatctaa 4080
atccccattt tttaatttct ttttagcctc tttaaataat cctgaataaa ctaatacctg 4140
tttaccttta agtgatttat aaaatgcatc aaagactttt tgatttatta aataatcact 4200
atctttacca gaatacttag ccatttcata taattcttta ttattatttt gtcttatttt 4260
ttgaacttga acttgtgtta tttctgaaat gcccgttaca tcacgccata aatctaacca 4320
ttcttgttgg ctaatataat atcttttatc tgtgaaatac gatttattta ctgcaattaa 4380
cacatgaaaa tgaggattat aatcatctct ttttttatta tatgtaatct ctaacttacg 4440
aacatatccc tttataacac tacctacttt ttttctcttt ataagttttc taaaagaatt 4500
attataacgt tttatttcat tttctaattc atcactcatt acattaggtg tagtcaaagt 4560
taaaaagata aactcctttt tctcttgctg cttaatatat tgcatcatca aagataaacc 4620
caatgcatct tttctagctt ttctccaagc acagacagga caaaatcgat ttttacaaga 4680
attagcttta tataatttct gtttttctaa agttttatca gctacaaaag acagaaatgt 4740
attgcaatct tcaactaaat ccatttgatt ctctccaata tgacgtttaa taaatttctg 4800
aaatacttga tttctttgtt ttttctcagt atacttttcc atgttataac acataaaaac 4860
aacttagttt tcacaaacta tgacaataaa aaaagttgct ttttcccctt tctatgtatg 4920
ttttttacta gtcatttaaa acgatacatt aataggtacg aaaaagcaac tttttttgcg 4980
cttaaaacca gtcataccaa taacttaagg gtaactagcc tcgccggcaa tagttaccct 5040
tattatcaag ataagaaaga aaaggatttt tcgctacgct caaatccttt aaaaaaacac 5100
aaaagaccac attttttaat gtggtctttt attcttcaac taaagcaccc attagttcaa 5160
caaacgaaaa ttggataaag tgggatattt ttaaaatata tatttatgtt acagtaatat 5220
tgacttttaa aaaaggattg attctaatga agaaagcaga caagtaagcc tcctaaattc 5280
actttagata aaaatttagg aggcatatca aatgaacttt aataaaattg atttagacaa 5340
ttggaagaga aaagagatat ttaatcatta tttgaaccaa caaacgactt ttagtataac 5400
cacagaaatt gatattagtg ttttataccg aaacataaaa caagaaggat ataaatttta 5460
ccctgcattt attttcttag tgacaagggt gataaactca aatacagctt ttagaactgg 5520
ttacaatagc gacggagagt taggttattg ggataagtta gagccacttt atacaatttt 5580
tgatggtgta tctaaaacat tctctggtat ttggactcct gtaaagaatg acttcaaaga 5640
gttttatgat ttataccttt ctgatgtaga gaaatataat ggttcgggga aattgtttcc 5700
caaaacacct atacctgaaa atgctttttc tctttctatt attccatgga cttcatttac 5760
tgggtttaac ttaaatatca ataataatag taattacctt ctacccatta ttacagcagg 5820
aaaattcatt aataaaggta attcaatata tttaccgcta tctttacagg tacatcattc 5880
tgtttgtgat ggttatcatg caggattgtt tatgaactct attcaggaat tgtcagatag 5940
gcctaatgac tggcttttat aatatgagat aatgccgact gtacttttta cagtcggttt 6000
tctaatgtca ctaacctgcc ccgttagttg aagaaggttt ttatattaca gctccagatc 6060
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 6120
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 6180
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 6240
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 6300
aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 6360
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 6420
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 6480
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 6540
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 6600
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 6660
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 6720
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 6780
ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 6840
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 6900
cgcagcgagt cagtgagcga ggaagcggaa ga 6932

Claims (6)

1. a kind of construction method of the recombined bacillus subtilis of high yield Pullulanase, it is characterised in that：Will be artificial synthesized general The Shandong orchid enzyme gene expression frame Tpu1 and Tpu2 difference alkaline protease gene in situ for substituting bacillus subtilisaprEAnd form sediment Powder enzyme geneamyE, and by resistant geneermCWithkanThe recombined bacillus subtilis is obtained after knockout, specific steps are such as Under：
A. design and gene chemical synthesis linear DNA fragment Tpu1 and Tpu2；
B. it is that starting strain carries out molecule construction with bacillus subtilis A 16 4, by Tpu1 and the competence of bacillus subtilis A 16 4 Mixing with cells, is converted, and obtains the recombinant bacterial strain CNP1 with Erythromycinresistant；
C. Tpu2 is mixed with bacillus subtilis CNP1 competent cells, converted, obtaining has erythromycin/kanamycins Double recombinant bacterial strain CNP2ek；
D. Cre expression plasmids are transferred to, the Double elimination of erythromycin/kanamycins is carried out, obtains recombined bacillus subtilis CNP2, i.e. recombined bacillus subtilis.
2. the construction method of the recombined bacillus subtilis of high yield Pullulanase as claimed in claim 1, it is characterised in that：Institute State gene expression frame Tpu1 and Tpu2 to obtain after codon optimization, gene chemical synthesis respectively, Tpu1 nucleotides sequence is classified as：
caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg 60
atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg 120
gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg 180
ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt 240
ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg 300
ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg 360
ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg 420
atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag 480
gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg 540
aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact 600
attattgtgc agctgcttgc ctgcactgca gatcgattgt ttgagaaaag aagaagacca 660
taaaaatacc ttgtctgtca tcagacaggg tattttttat gctgtccaga ctgtccgctg 720
tgtaaaaaaa aggaataaag gggggttgac attattttac tgatatgtat aatataattt 780
gtataagaaa atggagtcga aacgtaagat gaaaccttag ataaaagtgc tttttttgtt 840
gcaattgaag aattattaat gttaagctta attaaagata atatctttga attgtaacgc 900
ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt ttgaaccttc 960
ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa ctatagaatg 1020
acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt gttcatgtga 1080
attatcgctt tatttaattt tctcaattca atatataata tgccaataca ttgttacaag 1140
tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt attaaatgaa 1200
tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata tttttctatt 1260
ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa ggagggatgc 1320
ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt atgaaaaatc 1380
attttatcag tttgaaaatt atgtattatg cttacattcc atcgtatcat ccgtaaaggc 1440
tggatgttcc ttcttgcttt ccttcttaca gcttctcttt tctgccctac aggccaacat 1500
gctaaagctg ctgctcaacc tgctgtttct aacgcttacc ttgatgcttc taaccaagtt 1560
cttgttaaac tttctcaacc tttcacactt ggcgaaggct cttctggctt cacagttcat 1620
gatgatacag ctaacaaaga tatccctgtt acatctgttt ctgatgctaa ccaagttaca 1680
gctgttcttg ctggcacatt ccaacatatc ttcggcggct ctgattgggc tcctgataac 1740
cataacacac ttcttaaaaa agttaactct aacctttacc aattctctgg caaccttcct 1800
gaaggcaact accaatacaa agttgctctt aacgattctt ggaacaaccc ttcttaccct 1860
tctgataaca tcaaccttac agttcctgct ggcggcgctc atgttacatt ctcttacatc 1920
ccttctacac atgctgttta cgatacaatc aacaacccta acgctgatct tcaagttgat 1980
tcttctggcg ttaaaacaga tcttgttgct gttacacttg gcgaaaaccc tgatgtttct 2040
catacacttt ctatccaaac agaagattac caagctggcc aagttatccc tcgtaaagtt 2100
cttgattctt ctcaatacta ctactctggc gatgatcttg gcaacacata cacaaaaaac 2160
gctacaacat tcaaagtttg ggctcctaca tctacacaag ttaacgttct tctttacaac 2220
tctgctacag gcgctgttac aaaaacagtt cctatgacag cttctggcca tggcgtttgg 2280
gaagctacag ttaaccaaga tcttgaaaac tggtactaca tgtacgaagt tacaggccaa 2340
ggctctacac gtacagctgt tgatccttac gctacagcta tcgctcctaa cggcacacgt 2400
ggcatgatcg ttgatcttgc taaaacagat cctgctggct gggaatctga taaacatatc 2460
acacctaaaa acatcgaaga tgaagttatc tacgaaatgg atgttcgtga tttctctatc 2520
gattctaact ctggcatgaa aaacaaaggc aaataccttg ctcttacaga aaaaggcaca 2580
aaaggccctg ataacgttaa aacaggcgtt gattctctta aacaacttgg catcacacat 2640
gttcaacttc aacctgtttt cgctttcaac tctgttaacg aaaacgatcc tacacaatac 2700
aactggggct acgatcctcg taactacaac gttcctgaag gccaatacgc tacaaacgct 2760
aacggcacaa cacgtatcaa agaattcaaa gaaatggttc tttctcttca tcaagatcat 2820
atcggcgtta acatggatgt tgtttacaac catacattcg ctacacaaat ctctgatttc 2880
gataaaatcg ttcctgaata ctactaccgt acagatgatg ctggcaacta cacaaacggc 2940
tctggcacag gcaacgaaat cgctgctgaa cgtcctatgg ttcaaaaatt catcatcgat 3000
tctcttaaat tctgggttaa cgaataccat gttgatggct tccgtttcga tcttatggct 3060
cttcttggca aagatacaat gtctaaagct gctacacaac ttcatgctat cgatcctggc 3120
atcgctcttt acggcgaacc ttggacaggc ggcacatctg ctcttcctgc tgatcaactt 3180
cttacaaaag gcgctcaaaa aggcatgggc gttgctgttt tcaacgataa ccttcgtaac 3240
ggccttgatg gctctgtttt cgattcttct gctcaaggct tcgctacagg cgctacaggc 3300
cttacagatg ctatcaaaaa cggcgttgaa ggctctatca acgatttcac agcttctcct 3360
ggcgaaacaa tcaactacgt tacatctcat gataactaca cactttggga taaaatcgct 3420
caatctaacc ctaacgattc tgaagctgat cgtatcaaaa tggatgaact tgctcaagct 3480
atcgttatga catctcaagg catccctttc atgcaaggcg gcgaagaaat gcttcgtaca 3540
aaaggcggca acgataactc ttacaacgct ggcgatgttg ttaacgaatt cgattggtct 3600
cgtaaagctc aataccctga tgttttcaac tactactctg gccttatcca tcttcgtctt 3660
gatcatcctg ctttccgtat gacaacagct aacgaaatca actctcatct tcaattcctt 3720
aactctcctg aaaacacagt tgcttacgaa ctttctgatc atgctaacaa agatacatgg 3780
ggcaacatcg ttgttatcta caaccctaac aaaacagctg aaacaatcaa ccttccttct 3840
ggcaaatggg aaatcaacgc tacatctggc aaagttggcg aatctacact tggccaagct 3900
gaaggctctg ttcaagttcc tggcatctct atgatgatcc ttcatcaaga agtttctcct 3960
tctgatggca aataaagaag caggttcctc catacctgct tctttttaag gatccaagct 4020
tgcggccgct accgttcgta tagcatacat tatacgaagt tatttaaacc gtgtgctcta 4080
cgaccaaaac tataaaacct ttaagaactt tcttttttta caagaaaaaa gaaattagat 4140
aaatctctca tatcttttat tcaataatcg catccgattg cagtataaat ttaacgatca 4200
ctcatcatgt tcatatttat cagagctcgt gctataatta tactaatttt ataaggagga 4260
aaaaatatgg gcatttttag tatttttgta atcagcacag ttcattatca accaaacaaa 4320
aaataagtgg ttataatgaa tcgttaataa gcaaaattca tataaccaaa ttaaggaggg 4380
aaataatgaa cgagaaaaat ataaaacaca gtcaaaactt tattacttca aaacataata 4440
tagataaaat aatgacaaat ataagattaa atgaacatga taatatcttt gaaatcggct 4500
caggaaaagg ccattttacc cttgaattag taaagaggtg taatttcgta actgccattg 4560
aaatagacca taaattatgc aaaactacag aaaataaact tgttgatcac gataatttcc 4620
aagttttaaa caaggatata ttgcagttta aatttcctaa aaaccaatcc tataaaatat 4680
atggtaatat accttataac ataagtacgg atataatacg caaaattgtt tttgatagta 4740
tagctaatga gatttattta atcgtggaat acgggtttgc taaaagatta ttaaatacaa 4800
aacgctcatt ggcattactt ttaatggcag aagttgatat ttctatatta agtatggttc 4860
caagagaata ttttcatcct aaacctaaag tgaatagctc acttatcaga ttaagtagaa 4920
aaaaatcaag aatatcacac aaagataaac aaaagtataa ttatttcgtt atgaaatggg 4980
ttaacaaaga atacaagaaa atatttacaa aaaatcaatt taacaattcc ttaaaacatg 5040
caggaattga cgatttaaac aatattagct ttgaacaatt cttatctctt ttcaatagct 5100
ataaattatt taataagtaa gttaagggat gcataaactg catcccttaa cttgtttttc 5160
gtgtgcctat tttttgtgaa ttgattatcg atcttttgcg ccatggataa cttcgtatag 5220
catacattat acgaacggta catatgcccg ggggtaccga attccctgct ggtcgccatt 5280
ttggctcttt accctctcct tttaaaaaaa ttcagagtag acttacttaa aagactattc 5340
tgtgaattta ttgtaataga tggaataata ttttagtaga cccatttttt tgagatgatt 5400
ttatctctat ttaggtatat catctctcgc tatttccgta gagactcgaa ataactattt 5460
tcatacagaa aagaacgaaa atagacatga gtaaatgttc attatgctga aatgtcatgc 5520
ttttttaggt taaatgctcc tgagtcccgg caaattcctg tcgaaaaaat tcgttcaaat 5580
gacctgcgtg tgcttccgtg agaacaatgg atattatcgt gatatttttt caaagcatga 5640
tgataaaagt attctgaaaa ataaacttta cagaaaaggg atagaatgaa aaaattattg 5700
agagggagga agaaataaga tgaacattcg ccaagcaaag acatcagatg cggccgcca 5760
tgcgccgctg tttaaccaat atcgggaatt ttatagacag gcatccgatt tgcaaggggc 5820
agaggctttt ttgaaagctc gtttggaaaa tcacgagtct gttattttga tagcagaaga 5880
aaatg 5885；
Tpu2 nucleotides sequence is classified as：
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg catagaatct 540
aatattataa ctaaattttc taaaaaaaac attggaatag acatttattt tgtatatgat 600
gaaataaagt tagtttattg gataaacaaa ctaactttat taaggtagtt gatggataaa 660
cttgttcact taaatcaatc gaaacgtaag atgaaacctt agataaaagt gctttttttg 720
ttgcaattga agaattatta atgttaagct taattaaaga taatatcttt gaattgtaac 780
gcccctcaaa agtaagaact acaaaaaaag aatacgttat atagaaatat gtttgaacct 840
tcttcagatt acaaatatat tcggacggac tctacctcaa atgcttatct aactatagaa 900
tgacatacaa gcacaacctt gaaaatttga aaatataact accaatgaac ttgttcatgt 960
gaattatcgc tttatttaat tttctcaatt caatatataa tatgccaata cattgttaca 1020
agtagaaatt aagacaccct tgatagcctt actataccta acatgatgta gtattaaatg 1080
aatatgtaaa tatatttatg ataagaagcg acttatttat aatcattaca tatttttcta 1140
ttggaatgat taagattcca atagaatagt gtataaatta tttatcttga aaggagggat 1200
gcctaaaaac gaagaacatt aaaaacatat atttgcaccg tctaatggat ttatgaaaaa 1260
tcattttatc agtttgaaaa ttatgtatta tgcttatcaa caaatctaaa aaattcttcg 1320
ttttctcttt catcttcgtt atgatgcttt ctctttcttt cgttaacggc gaagttgcta 1380
aagctgctca acctgctgtt tctaacgctt accttgatgc ttctaaccaa gttcttgtta 1440
aactttctca acctttcaca cttggcgaag gctcttctgg cttcacagtt catgatgata 1500
cagctaacaa agatatccct gttacatctg tttctgatgc taaccaagtt acagctgttc 1560
ttgctggcac attccaacat atcttcggcg gctctgattg ggctcctgat aaccataaca 1620
cacttcttaa aaaagttaac tctaaccttt accaattctc tggcaacctt cctgaaggca 1680
actaccaata caaagttgct cttaacgatt cttggaacaa cccttcttac ccttctgata 1740
acatcaacct tacagttcct gctggcggcg ctcatgttac attctcttac atcccttcta 1800
cacatgctgt ttacgataca atcaacaacc ctaacgctga tcttcaagtt gattcttctg 1860
gcgttaaaac agatcttgtt gctgttacac ttggcgaaaa ccctgatgtt tctcatacac 1920
tttctatcca aacagaagat taccaagctg gccaagttat ccctcgtaaa gttcttgatt 1980
cttctcaata ctactactct ggcgatgatc ttggcaacac atacacaaaa aacgctacaa 2040
cattcaaagt ttgggctcct acatctacac aagttaacgt tcttctttac aactctgcta 2100
caggcgctgt tacaaaaaca gttcctatga cagcttctgg ccatggcgtt tgggaagcta 2160
cagttaacca agatcttgaa aactggtact acatgtacga agttacaggc caaggctcta 2220
cacgtacagc tgttgatcct tacgctacag ctatcgctcc taacggcaca cgtggcatga 2280
tcgttgatct tgctaaaaca gatcctgctg gctgggaatc tgataaacat atcacaccta 2340
aaaacatcga agatgaagtt atctacgaaa tggatgttcg tgatttctct atcgattcta 2400
actctggcat gaaaaacaaa ggcaaatacc ttgctcttac agaaaaaggc acaaaaggcc 2460
ctgataacgt taaaacaggc gttgattctc ttaaacaact tggcatcaca catgttcaac 2520
ttcaacctgt tttcgctttc aactctgtta acgaaaacga tcctacacaa tacaactggg 2580
gctacgatcc tcgtaactac aacgttcctg aaggccaata cgctacaaac gctaacggca 2640
caacacgtat caaagaattc aaagaaatgg ttctttctct tcatcaagat catatcggcg 2700
ttaacatgga tgttgtttac aaccatacat tcgctacaca aatctctgat ttcgataaaa 2760
tcgttcctga atactactac cgtacagatg atgctggcaa ctacacaaac ggctctggca 2820
caggcaacga aatcgctgct gaacgtccta tggttcaaaa attcatcatc gattctctta 2880
aattctgggt taacgaatac catgttgatg gcttccgttt cgatcttatg gctcttcttg 2940
gcaaagatac aatgtctaaa gctgctacac aacttcatgc tatcgatcct ggcatcgctc 3000
tttacggcga accttggaca ggcggcacat ctgctcttcc tgctgatcaa cttcttacaa 3060
aaggcgctca aaaaggcatg ggcgttgctg ttttcaacga taaccttcgt aacggccttg 3120
atggctctgt tttcgattct tctgctcaag gcttcgctac aggcgctaca ggccttacag 3180
atgctatcaa aaacggcgtt gaaggctcta tcaacgattt cacagcttct cctggcgaaa 3240
caatcaacta cgttacatct catgataact acacactttg ggataaaatc gctcaatcta 3300
accctaacga ttctgaagct gatcgtatca aaatggatga acttgctcaa gctatcgtta 3360
tgacatctca aggcatccct ttcatgcaag gcggcgaaga aatgcttcgt acaaaaggcg 3420
gcaacgataa ctcttacaac gctggcgatg ttgttaacga attcgattgg tctcgtaaag 3480
ctcaataccc tgatgttttc aactactact ctggccttat ccatcttcgt cttgatcatc 3540
ctgctttccg tatgacaaca gctaacgaaa tcaactctca tcttcaattc cttaactctc 3600
ctgaaaacac agttgcttac gaactttctg atcatgctaa caaagataca tggggcaaca 3660
tcgttgttat ctacaaccct aacaaaacag ctgaaacaat caaccttcct tctggcaaat 3720
gggaaatcaa cgctacatct ggcaaagttg gcgaatctac acttggccaa gctgaaggct 3780
ctgttcaagt tcctggcatc tctatgatga tccttcatca agaagtttct ccttctgatg 3840
gcaaataaag aagcaggttc ctccatacct gcttcttttt aagggatcca agcttgcggc 3900
cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt tgaagattag 3960
atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct 4020
gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga 4080
aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat 4140
tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg 4200
tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg 4260
tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt 4320
ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga 4380
ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt caggtggata 4440
cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat 4500
ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt 4560
gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat 4620
gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc 4680
agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg 4740
tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga atgggattca 4800
ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac 4860
gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt 4920
agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta tacgaacggt 4980
acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga catggatgag 5040
cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca gtacgcctct 5100
tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga aaagccaaat 5160
aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca atagatttca 5220
caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca accagatatt 5280
tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat cctctgtctc 5340
tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg gagcgggttc 5400
atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg tagctgtgct 5460
ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca aaacaggtgt 5520
aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga atacaacaaa 5580
agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg agatcaattc 5640
acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa aaggaacgaa 5700
cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc cagcgtcggc 5760
caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt atatctataa 5820
acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac caatgactaa 5880
aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa ccaacgcaaa 5940
agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg gctttgatta 6000
cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc attga 6055。
3. the construction method of the recombined bacillus subtilis of high yield Pullulanase as claimed in claim 1, it is characterised in that：Base Because expression cassette Tpu1 and Tpu2 include the Pullulanase encoding gene of copy respectivelypulB, it is describedpulBThe nucleotides of gene Sequence is as follows：
caacctgctg tttctaacgc ttaccttgat gcttctaacc aagttcttgt taaactttct 60
caacctttca cacttggcga aggctcttct ggcttcacag ttcatgatga tacagctaac 120
aaagatatcc ctgttacatc tgtttctgat gctaaccaag ttacagctgt tcttgctggc 180
acattccaac atatcttcgg cggctctgat tgggctcctg ataaccataa cacacttctt 240
aaaaaagtta actctaacct ttaccaattc tctggcaacc ttcctgaagg caactaccaa 300
tacaaagttg ctcttaacga ttcttggaac aacccttctt acccttctga taacatcaac 360
cttacagttc ctgctggcgg cgctcatgtt acattctctt acatcccttc tacacatgct 420
gtttacgata caatcaacaa ccctaacgct gatcttcaag ttgattcttc tggcgttaaa 480
acagatcttg ttgctgttac acttggcgaa aaccctgatg tttctcatac actttctatc 540
caaacagaag attaccaagc tggccaagtt atccctcgta aagttcttga ttcttctcaa 600
tactactact ctggcgatga tcttggcaac acatacacaa aaaacgctac aacattcaaa 660
gtttgggctc ctacatctac acaagttaac gttcttcttt acaactctgc tacaggcgct 720
gttacaaaaa cagttcctat gacagcttct ggccatggcg tttgggaagc tacagttaac 780
caagatcttg aaaactggta ctacatgtac gaagttacag gccaaggctc tacacgtaca 840
gctgttgatc cttacgctac agctatcgct cctaacggca cacgtggcat gatcgttgat 900
cttgctaaaa cagatcctgc tggctgggaa tctgataaac atatcacacc taaaaacatc 960
gaagatgaag ttatctacga aatggatgtt cgtgatttct ctatcgattc taactctggc 1020
atgaaaaaca aaggcaaata ccttgctctt acagaaaaag gcacaaaagg ccctgataac 1080
gttaaaacag gcgttgattc tcttaaacaa cttggcatca cacatgttca acttcaacct 1140
gttttcgctt tcaactctgt taacgaaaac gatcctacac aatacaactg gggctacgat 1200
cctcgtaact acaacgttcc tgaaggccaa tacgctacaa acgctaacgg cacaacacgt 1260
atcaaagaat tcaaagaaat ggttctttct cttcatcaag atcatatcgg cgttaacatg 1320
gatgttgttt acaaccatac attcgctaca caaatctctg atttcgataa aatcgttcct 1380
gaatactact accgtacaga tgatgctggc aactacacaa acggctctgg cacaggcaac 1440
gaaatcgctg ctgaacgtcc tatggttcaa aaattcatca tcgattctct taaattctgg 1500
gttaacgaat accatgttga tggcttccgt ttcgatctta tggctcttct tggcaaagat 1560
acaatgtcta aagctgctac acaacttcat gctatcgatc ctggcatcgc tctttacggc 1620
gaaccttgga caggcggcac atctgctctt cctgctgatc aacttcttac aaaaggcgct 1680
caaaaaggca tgggcgttgc tgttttcaac gataaccttc gtaacggcct tgatggctct 1740
gttttcgatt cttctgctca aggcttcgct acaggcgcta caggccttac agatgctatc 1800
aaaaacggcg ttgaaggctc tatcaacgat ttcacagctt ctcctggcga aacaatcaac 1860
tacgttacat ctcatgataa ctacacactt tgggataaaa tcgctcaatc taaccctaac 1920
gattctgaag ctgatcgtat caaaatggat gaacttgctc aagctatcgt tatgacatct 1980
caaggcatcc ctttcatgca aggcggcgaa gaaatgcttc gtacaaaagg cggcaacgat 2040
aactcttaca acgctggcga tgttgttaac gaattcgatt ggtctcgtaa agctcaatac 2100
cctgatgttt tcaactacta ctctggcctt atccatcttc gtcttgatca tcctgctttc 2160
cgtatgacaa cagctaacga aatcaactct catcttcaat tccttaactc tcctgaaaac 2220
acagttgctt acgaactttc tgatcatgct aacaaagata catggggcaa catcgttgtt 2280
atctacaacc ctaacaaaac agctgaaaca atcaaccttc cttctggcaa atgggaaatc 2340
aacgctacat ctggcaaagt tggcgaatct acacttggcc aagctgaagg ctctgttcaa 2400
gttcctggca tctctatgat gatccttcat caagaagttt ctccttctga tggcaaataa 2460。
4. the construction method of the recombined bacillus subtilis of high yield Pullulanase as claimed in claim 1, it is characterised in that： The gene expression frame Tpu1 contains for knocking outaprEThe homologous segment of gene, its nucleotide sequence is as follows：
caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg 60
atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg 120
gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg 180
ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt 240
ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg 300
ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg 360
ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg 420
atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag 480
gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg 540
aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact 600
attattgtgc agctgcttgc ctgcactgca gggatccaag cttgcggccg ctaccgttcg 660
tatagcatac attatacgaa gttatttaaa ccgtgtgctc tacgaccaaa actataaaac 720
ctttaagaac tttctttttt tacaagaaaa aagaaattag ataaatctct catatctttt 780
attcaataat cgcatccgat tgcagtataa atttaacgat cactcatcat gttcatattt 840
atcagagctc gtgctataat tatactaatt ttataaggag gaaaaaatat gggcattttt 900
agtatttttg taatcagcac agttcattat caaccaaaca aaaaataagt ggttataatg 960
aatcgttaat aagcaaaatt catataacca aattaaggag ggaaataatg aacgagaaaa 1020
atataaaaca cagtcaaaac tttattactt caaaacataa tatagataaa ataatgacaa 1080
atataagatt aaatgaacat gataatatct ttgaaatcgg ctcaggaaaa ggccatttta 1140
cccttgaatt agtaaagagg tgtaatttcg taactgccat tgaaatagac cataaattat 1200
gcaaaactac agaaaataaa cttgttgatc acgataattt ccaagtttta aacaaggata 1260
tattgcagtt taaatttcct aaaaaccaat cctataaaat atatggtaat ataccttata 1320
acataagtac ggatataata cgcaaaattg tttttgatag tatagctaat gagatttatt 1380
taatcgtgga atacgggttt gctaaaagat tattaaatac aaaacgctca ttggcattac 1440
ttttaatggc agaagttgat atttctatat taagtatggt tccaagagaa tattttcatc 1500
ctaaacctaa agtgaatagc tcacttatca gattaagtag aaaaaaatca agaatatcac 1560
acaaagataa acaaaagtat aattatttcg ttatgaaatg ggttaacaaa gaatacaaga 1620
aaatatttac aaaaaatcaa tttaacaatt ccttaaaaca tgcaggaatt gacgatttaa 1680
acaatattag ctttgaacaa ttcttatctc ttttcaatag ctataaatta tttaataagt 1740
aagttaaggg atgcataaac tgcatccctt aacttgtttt tcgtgtgcct attttttgtg 1800
aattgattat cgatcttttg cgccatggat aacttcgtat agcatacatt atacgaacgg 1860
tacatatgcc cgggggtacc gaattccctg ctggtcgcca ttttggctct ttaccctctc 1920
cttttaaaaa aattcagagt agacttactt aaaagactat tctgtgaatt tattgtaata 1980
gatggaataa tattttagta gacccatttt tttgagatga ttttatctct atttaggtat 2040
atcatctctc gctatttccg tagagactcg aaataactat tttcatacag aaaagaacga 2100
aaatagacat gagtaaatgt tcattatgct gaaatgtcat gcttttttag gttaaatgct 2160
cctgagtccc ggcaaattcc tgtcgaaaaa attcgttcaa atgacctgcg tgtgcttccg 2220
tgagaacaat ggatattatc gtgatatttt ttcaaagcat gatgataaaa gtattctgaa 2280
aaataaactt tacagaaaag ggatagaatg aaaaaattat tgagagggag gaagaaataa 2340
gatgaacatt cgccaagcaa agacatcaga tgcggccgcc attgcgccgc tgtttaacca 2400
atatcgggaa ttttatagac aggcatccga tttgcaaggg gcagaggctt ttttgaaagc 2460
tcgtttggaa aatcacgagt ctgttatttt gatagcagaa gaaaatg 2507；
The gene expression frame Tpu2 contains for knocking outamyEThe homologous segment of gene, its nucleotide sequence is as follows：
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg cagggatcca 540
agcttgcggc cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt 600
tgaagattag atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt 660
attaatagct gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa 720
aattatctga aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa 780
gaatgaagat tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta 840
aggctattgg tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg 900
agatgatgtg tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg 960
agtggaaggt ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg 1020
tggaatcaga ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt 1080
caggtggata cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc 1140
acgatgcgat ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta 1200
atattcgtgt gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg 1260
caggtgccat gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct 1320
taactgaagc agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg 1380
taatgtctgg tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga 1440
atgggattca ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac 1500
cattttgaac gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc 1560
tcctagtatt agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta 1620
tacgaacggt acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga 1680
catggatgag cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca 1740
gtacgcctct tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga 1800
aaagccaaat aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca 1860
atagatttca caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca 1920
accagatatt tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat 1980
cctctgtctc tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg 2040
gagcgggttc atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg 2100
tagctgtgct ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca 2160
aaacaggtgt aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga 2220
atacaacaaa agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg 2280
agatcaattc acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa 2340
aaggaacgaa cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc 2400
cagcgtcggc caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt 2460
atatctataa acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac 2520
caatgactaa aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa 2580
ccaacgcaaa agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg 2640
gctttgatta cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc 2700
attga 2705。
5. the construction method of the recombined bacillus subtilis of high yield Pullulanase as claimed in claim 1, it is characterised in that：Institute State the homology arm sequence of gene expression frame Tpu1 and Tpu2 containing Pullulanase secretion signal peptide-coding sequence and for restructuring Row, the nucleotide sequence of signal peptide described in Tpu1 is as follows：
atgcttacat tccatcgtat catccgtaaa ggctggatgt tccttcttgc tttccttctt 60
acagcttctc ttttctgccc tacaggccaa catgctaaag ctgctgct 108；
The nucleotide sequence of signal peptide described in Tpu2 is as follows：
atgcttatca acaaatctaa aaaattcttc gttttctctt tcatcttcgt tatgatgctt 60
tctctttctt tcgttaacgg cgaagttgct aaagctgct 99。
6. the construction method of the recombined bacillus subtilis of high yield Pullulanase as claimed in claim 1, it is characterised in that：Institute State the signal peptide sequence SPamyL containing promoter Pamy-cryIII and amylase secretion, nucleotides in gene expression frame Tpu1 Sequence is as follows：
atcgattgtt tgagaaaaga agaagaccat aaaaatacct tgtctgtcat cagacagggt 60
attttttatg ctgtccagac tgtccgctgt gtaaaaaaaa ggaataaagg ggggttgaca 120
ttattttact gatatgtata atataatttg tataagaaaa tggagtcgaa acgtaagatg 180
aaaccttaga taaaagtgct ttttttgttg caattgaaga attattaatg ttaagcttaa 240
ttaaagataa tatctttgaa ttgtaacgcc cctcaaaagt aagaactaca aaaaaagaat 300
acgttatata gaaatatgtt tgaaccttct tcagattaca aatatattcg gacggactct 360
acctcaaatg cttatctaac tatagaatga catacaagca caaccttgaa aatttgaaaa 420
tataactacc aatgaacttg ttcatgtgaa ttatcgcttt atttaatttt ctcaattcaa 480
tatataatat gccaatacat tgttacaagt agaaattaag acacccttga tagccttact 540
atacctaaca tgatgtagta ttaaatgaat atgtaaatat atttatgata agaagcgact 600
tatttataat cattacatat ttttctattg gaatgattaa gattccaata gaatagtgta 660
taaattattt atcttgaaag gagggatgcc taaaaacgaa gaacattaaa aacatatatt 720
tgcaccgtct aatggattta tgaaaaatca ttttatcagt ttgaaaatta tgtattatgc 780
ttacattcca tcgtatcatc cgtaaaggct ggatgttcct tcttgctttc cttcttacag 840
cttctctttt ctgccctaca ggccaacatg ctaaagctgc tgct 884；
The signal peptide sequence secreted in the gene expression frame Tpu2 containing promoter PxylA-cryIII and from chitinase SPchit, nucleotide sequence is as follows：
tagaatctaa tattataact aaattttcta aaaaaaacat tggaatagac atttattttg 60
tatatgatga aataaagtta gtttattgga taaacaaact aactttatta aggtagttga 120
tggataaact tgttcactta aatcaatcga aacgtaagat gaaaccttag ataaaagtgc 180
tttttttgtt gcaattgaag aattattaat gttaagctta attaaagata atatctttga 240
attgtaacgc ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt 300
ttgaaccttc ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa 360
ctatagaatg acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt 420
gttcatgtga attatcgctt tatttaattt tctcaattca atatataata tgccaataca 480
ttgttacaag tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt 540
attaaatgaa tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata 600
tttttctatt ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa 660
ggagggatgc ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt 720
atgaaaaatc attttatcag tttgaaaatt atgtattatg cttatcaaca aatctaaaaa 780
attcttcgtt ttctctttca tcttcgttat gatgctttct ctttctttcg ttaacggcga 840
agttgctaaa gctgct 856。
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Publication number Priority date Publication date Assignee Title
CN108998403A (en) * 2018-09-19 2018-12-14 长春大学 A kind of bacillus subtilis recombinant bacterium and its application
CN108998403B (en) * 2018-09-19 2021-12-07 长春大学 Bacillus subtilis recombinant strain and application thereof
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CN112226451A (en) * 2020-10-23 2021-01-15 中国科学院上海高等研究院 Bacillus subtilis expression system and method for producing alpha-L-AFs by using same
CN118147036A (en) * 2024-03-18 2024-06-07 中国科学院天津工业生物技术研究所 Bacillus subtilis capable of stably expressing target product and application thereof
CN118147036B (en) * 2024-03-18 2024-07-05 中国科学院天津工业生物技术研究所 Bacillus subtilis capable of stably expressing target product and application thereof

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