CN107058367A - 高产普鲁兰酶的重组枯草芽孢杆菌的构建方法 - Google Patents

Abstract

本发明公开了一种高产普鲁兰酶的重组枯草芽孢杆菌的构建方法。将耐酸热普鲁兰酶基因经密码子优化后，分别和两种不同的分泌肽及启动子进行人工拼接，组成人工操纵子，并整合到枯草芽孢杆菌A164，整合位点分别为碱性蛋白酶（aprE）及淀粉酶基因（amyE）所在处；此外，还通过Cre重组酶的质粒表达，将整合到A164上的抗性基因进行了敲除；所获得的普鲁兰酶重组生产菌株CNP2的普鲁兰酶发酵酶活超过1000 U/mL。

Description

高产普鲁兰酶的重组枯草芽孢杆菌的构建方法

技术领域

本发明属于制糖工业用酶安全生产菌株的构建技术领域，涉及一种高产普鲁兰酶的重组枯草芽孢杆菌的构建方法。

背景技术

普鲁兰酶是食品行业制糖生产技术中不可或缺的一类酶，主要由原核微生物分泌表达的糖链解枝酶。生产应用中多使用这类酶中的I型普鲁兰酶，其能够专一性切开淀粉原料中由α-1,6糖苷键构成的支链分支，因此普鲁兰酶与其它淀粉水解酶共同作用，可以让淀粉得以完全酶解，得到单分子葡萄糖分子，其在淀粉加工等食品行业中有着广阔的市场前景。近年来，国内外对普鲁兰酶生产菌株的研发投入大量人力物力，许多工业酶生产企业先后开发出多种不同类型的重组生产菌株或诱变菌。在适合工业生产应用的多种普鲁兰酶蛋白中，来自长野芽胞杆菌 (Bacillus naganoensis) 的普鲁兰酶（Bn PulB），其酶学特征，包括最适反应温度及pH等均非常适合淀粉工业加工的需求，但该酶的原产菌的表达水平不高，因此其基因常被用于构建于重组表达生产，在适合食品工业用酶的宿主细胞中。枯草芽孢杆菌(Bacillus subtilis)因为其不表达致热源和毒素因子，且其分泌途径强大，因此被列为安全生产菌株（GRAS）之一，常被用于许多胞外酶发酵生产的首选宿主细胞。枯草芽孢杆菌有许多种，其各自的转化性能、分泌能力等相关很大，其中A164以其营养需求不高，发酵密度高，蛋白分泌水平高等原因被视为蛋白生产的理想宿主细胞。

为了提高生产菌株的遗传稳定性，外源表达通常被整合到宿主细胞的基因组中，而抗生素基因常用作筛选标记来使用，但是在食品工业中，携带抗生素基因的重组菌株会带来生物安全性的问题，因此，有必要在完成外源基因的整合后再通过二次重组或者特异性重组的方式，将抗生素基因去除，得到无抗的生产菌株。

发明内容

本发明的目的是提供一种高产普鲁兰酶的重组枯草芽孢杆菌CNP2的构建方法—通过两次基因操作，将两份拷贝普鲁兰酶基因整合到枯草芽孢杆菌的基因组中，并通过特异性重组***，将抗生素基因去除，从而获得了一株安全的普鲁兰酶高产菌株。

为了实现上述目的，本发明所采用的技术方案具体如下：

将人工合成的普鲁兰酶基因表达框Tpu1及Tpu2分别原位替代枯草芽孢杆菌的碱性蛋白酶基因aprE以及淀粉酶基因amyE，并将抗性基因ermC和kan敲除后得到所述重组枯草芽孢杆菌，具体步骤如下：

a.设计并基因合成线性DNA片段Tpu1及Tpu2；

b.以枯草芽孢杆菌A164为出发菌株进行分子构建，将Tpu1与枯草芽孢杆菌A164感受态细胞混合，进行转化，获得具有红霉素抗性的重组菌株CNP1；

所述枯草芽孢杆菌A164，最开始分离自德国马尔堡，该菌种可以通过美国国家菌种保藏中心购买得到，菌种保藏号为ATCC 6051a；

c.将Tpu2与枯草芽孢杆菌CNP1感受态细胞混合，进行转化，获得具有红霉素/卡那霉素双抗性的重组菌株CNP2ek；

d.转入Cre表达质粒，进行红霉素/卡那霉素双抗性的消除，得到重组枯草芽孢杆菌CNP2，即重组枯草芽孢杆菌。

所述基因表达框Tpu1及Tpu2分别经密码子优化、基因合成后获得， Tpu1的核苷酸序列为：

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的核苷酸序列为：

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。

所术基因表达框Tpu1和Tpu2分别包含一份拷贝的普鲁兰酶编码基因pulB，所述pulB基因的核苷酸序列如下：

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。

所述基因表达框Tpu1含有用于敲除aprE基因的同源片断，其核苷酸序列如下：

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；

所述基因表达框Tpu2含有用于敲除amyE基因的同源片断，其核苷酸序列如下：

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。

所述基因表达框Tpu1及Tpu2均含有普鲁兰酶分泌信号肽编码序列以及用于重组的同源臂序列，Tpu1中所述信号肽的核苷酸序列如下：

atgcttacat tccatcgtat catccgtaaa ggctggatgt tccttcttgc tttccttctt 60

acagcttctc ttttctgccc tacaggccaa catgctaaag ctgctgct 108；

Tpu2中所述信号肽的核苷酸序列如下：

atgcttatca acaaatctaa aaaattcttc gttttctctt tcatcttcgt tatgatgctt 60

tctctttctt tcgttaacgg cgaagttgct aaagctgct 99。

所述基因表达框Tpu1中含有启动子Pamy-cryIII及淀粉酶分泌的信号肽序列SPamyL，核苷酸序列如下：

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；

所述基因表达框Tpu2中含有启动子PxylA-cryIII及来自几丁质酶分泌的信号肽序列SPchit，核苷酸序列如下：

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。

本发明的有益效果是：与现有的菌株诱变或者分子生物学构建技术相比，本发明综合利用枯草芽孢杆菌表达***的特点和优势，以基因设计结合基因合成的方式获取线性的人工DNA片断；DNA片断中含有分子生物学操作后期所需的抗性基因及重组序列，利用不同抗性基因的筛选，获得双拷贝的表达基因，再利用特异性重组，一次性地将抗性基因敲除；设计的表达元件中充分利用了RNA稳定因子、高效分泌肽等元素，将重组枯草芽孢杆菌高分泌的优势尽量发挥出来；最终得到产普鲁兰酶的、无抗的重组枯草芽孢杆菌CNP2，该重组菌株经生物反应器发酵证实可以高产普鲁兰酶，发酵32h的酶活大于1000 U/mL。

附图说明

图1为重组枯草芽孢杆菌的构建示意图；

图2为重组枯草芽孢杆菌CNP2的发酵酶活随发酵时间变化的示意图。

具体实施方式

下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明，均为商业化产品。

实施例1：重组枯草芽孢杆菌CNP2的构建

1.整合型DNA的获得

普鲁兰酶表达的操纵子有两个，第一个pulB1包括人工串联启动子Pamy-cryIII、密码子优化的淀粉酶分泌的信号肽序列SPamyL以及密码子优化的普鲁兰酶编码基因pulB，pulB序列见SEQ ID NO. 3，pulB1序列见SEQ ID NO. 10；第二个pulB2包括人工串联启动子PxylA-cryIII、密码子优化的几丁质酶分泌的信号肽序列SPchit以及密码子优化的普鲁兰酶编码基因pulB，pulB2序列见SEQ ID NO. 11。

用于aprE敲除的重组臂及红霉素抗性片段的基因表达框apr-erm-apr如SEQ IDNO. 8所示，用于amyE敲除的重组臂及卡那霉素抗性片段的基因表达框amy-kan-amy如SEQID NO. 9所示。

把pulB1***apr-erm-apr的Pst I和BamH I位点，通过人工合成的方式获得基因表达框Tpu1，把pulB2***amy-kan-amy的Pst I和BamH I位点，通过人工合成的方式获得基因表达框Tpu2。Tpu1和Tpu2的序列分别见SEQ ID NO. 4和SEQ ID NO. 5。

2.重组枯草芽孢杆菌的构建

首先挑取 A164单菌落于含有3 mL液体LB培养基的试管中，37℃、200 r/min过夜培养；然后取3 mL过夜培养菌液转接到新鲜的40 mL富集培养基中37℃、200 r/min培养至 OD600=0.85-0.95，富集培养基的配方为LB+ 90 g/L山梨醇；再将菌液冰水浴10 min，然后5000g，5 min，4℃离心后收集菌体；接着用30 mL预冷的电转培养基，重新悬浮菌体，5000 g离心5 min，4℃离心去上清，如此操作3次，电转培养基的配方为10 g/L葡萄糖+ 92.5 g/L甘露醇+90 g/L山梨醇；洗三次后，利用未完全除去的电转培养基将菌体重悬后，然后分装到1.5mL无菌离心管中，每管分装100 μL，冰上操作；最后在100 μL感受态细胞中加入2μg 线性DNA片段Tpu1，冰上孵育 2 min，加入预冷的电转杯 (1mm) 中，设置电压2.5 kV，电击一次；电击完毕取出杯子并立即加入500 μL复苏培养基，37℃、200 r/min培养，复苏培养基的组分为LB+90 g/L山梨醇+70 g/L甘露醇；复苏 3 h 后，分别涂红霉素抗性板， 37 ℃恒温箱过夜培养，其中，红霉素(Erm)浓度为10 μg/mL。

对筛选得到的转化子，采用菌落PCR的方式进行验证，所用的引物序列见下。

上游引物1P-F：cacccttgatagccttactatacctaac

下游引物1P-R：gatgttatcagaagggtaagaagg

反应体系如下：

PCR程序如下：

1)98℃，2min

2)98℃，30sec

3)52℃，30sec

4)72℃，30sec

5)循环从2)到4)，30次

6)72℃，5min

7)4℃，保存。

经验证为阳性的转化子命名为CNP1，然后采取上述相同的转化方法，用线性片段Tpu2转化CNP1，红霉素和卡那霉素双抗性板筛选，其中，卡那霉素(Kan)的浓度为10 μg/mL。

对筛选得到的转化子，采用菌落PCR的方式进行验证，所用的引物序列见下。

上游引物2P-F：cggacggactctacctcaaatgc

下游引物2P-R：gcgttagaaacagcaggttgagc

反应体系如下：

反应程序和CNP1的验证方法一致。

经验证为阳性的转化子命名为CNP2ek，然后采用上述相同的方法，用Cre酶表达质粒pMK4-cre转化CNP2ek，氯霉素抗性板筛选，其中，氯霉素(Cm)的浓度为5 μg/mL。

对于筛选得到的转化子，采用传代的方式丢失质粒pMK4-cre，取10 μL菌液接种到新鲜的3 mL液体LB培养基中，37 ℃、200 r/min培养，每隔8 h转接1次，总共转接5次，然后取培养液梯度稀释至10⁶倍，涂LB平板，置于恒温培养箱37 ℃、200 r/min培养；待长出新鲜菌落，分别***至氯霉素、红霉素和卡那霉素平板进行抗性反筛。在3种抗性平板上均不能生长的菌，其在无抗 LB 平板上对应的菌落为消除质粒的菌株，为目的菌株。再对其进行菌落PCR验证，第一对引物为1P-F/1P-R，第二对引物为2P-F/2P-R。经验证均为阳性的菌株，命名为CNP2，为带有两份拷贝pulB的无抗的普鲁兰酶生产菌株。

实施例2：重组枯草芽孢杆菌CNP2的发酵产酶

以重组枯草芽孢杆菌CNP2为生产菌种，做摇瓶发酵：用接种环挑取重组枯草芽孢杆菌CNP2接种到装有25 mL液体培养基的200 mL挡板瓶中，37℃、200 r/min进行发酵培养，其中，培养基的主要组分为玉米浆、麦芽糖浆、葡萄糖、酵母粉和无机盐离子等。发酵20 h取样测定酶活，普鲁兰酶酶活测定方法为：取1.0 mL的pH=4.5的醋酸-醋酸钠缓冲液配制酶样或稀释样品（对照组为沸水浴灭活的样品），将其加到1.0 mL 0.5%的普鲁兰多糖溶液中，于60℃水浴锅中水浴30 min，再加入3,5-二硝基水杨酸溶液（DNS）3.0 mL，沸水浴10 min，取出后流水冷却，于550 nm测吸光值。一个酶活力单位定义为：在上述反应条件下，每分钟分解普鲁兰多糖释放出1 μmol葡萄糖所需的酶量。测得的摇瓶发酵20 h的酶活力约为225 U/mL。

采用上罐补料的方式进行发酵，在20 L罐中，以流加麦芽糖浆和氨水控制发酵罐的pH在5.80左右，通风搅拌，溶氧控制为30%左右，还原糖控制为10 g/L左右。取样并测定酶活，普鲁兰酶的活力随发酵时间变化曲线的结果如图例2所示。其中，在32 h左右，酶活力超过1000 U/mL。

上述仅为本发明的部分优选实施例，本发明并不仅限于实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内可以有各种变化和更改，所作的任何变化和更改，均在本发明保护范围之内。

SEQUENCE LISTING

<110> 白银赛诺生物科技有限公司

<120> 高产普鲁兰酶的重组枯草芽孢杆菌的构建方法

<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> 敲除aprE基因的同源片段

<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> 敲除amyE基因的同源片段

<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)

1.一种高产普鲁兰酶的重组枯草芽孢杆菌的构建方法，其特征在于：将人工合成的普鲁兰酶基因表达框Tpu1及Tpu2分别原位替代枯草芽孢杆菌的碱性蛋白酶基因aprE以及淀粉酶基因amyE，并将抗性基因ermC和kan敲除后得到所述重组枯草芽孢杆菌，具体步骤如下：

a.设计并基因合成线性DNA片段Tpu1及Tpu2；

b.以枯草芽孢杆菌A164为出发菌株进行分子构建，将Tpu1与枯草芽孢杆菌A164感受态细胞混合，进行转化，获得具有红霉素抗性的重组菌株CNP1；

c.将Tpu2与枯草芽孢杆菌CNP1感受态细胞混合，进行转化，获得具有红霉素/卡那霉素双抗性的重组菌株CNP2ek；

d.转入Cre表达质粒，进行红霉素/卡那霉素双抗性的消除，得到重组枯草芽孢杆菌CNP2，即重组枯草芽孢杆菌。

2.如权利要求1所述的高产普鲁兰酶的重组枯草芽孢杆菌的构建方法，其特征在于：所述基因表达框Tpu1及Tpu2分别经密码子优化、基因合成后获得， Tpu1的核苷酸序列为：

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的核苷酸序列为：

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.如权利要求1所述的高产普鲁兰酶的重组枯草芽孢杆菌的构建方法，其特征在于：基因表达框Tpu1和Tpu2分别包含一份拷贝的普鲁兰酶编码基因pulB，所述pulB基因的核苷酸序列如下：

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. 如权利要求1所述的高产普鲁兰酶的重组枯草芽孢杆菌的构建方法，其特征在于：所述基因表达框Tpu1含有用于敲除aprE基因的同源片断，其核苷酸序列如下：

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；

所述基因表达框Tpu2含有用于敲除amyE基因的同源片断，其核苷酸序列如下：

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.如权利要求1所述的高产普鲁兰酶的重组枯草芽孢杆菌的构建方法，其特征在于：所述基因表达框Tpu1及Tpu2均含有普鲁兰酶分泌信号肽编码序列以及用于重组的同源臂序列，Tpu1中所述信号肽的核苷酸序列如下：

atgcttacat tccatcgtat catccgtaaa ggctggatgt tccttcttgc tttccttctt 60

acagcttctc ttttctgccc tacaggccaa catgctaaag ctgctgct 108；

Tpu2中所述信号肽的核苷酸序列如下：

atgcttatca acaaatctaa aaaattcttc gttttctctt tcatcttcgt tatgatgctt 60

tctctttctt tcgttaacgg cgaagttgct aaagctgct 99。

6.如权利要求1所述的高产普鲁兰酶的重组枯草芽孢杆菌的构建方法，其特征在于：所述基因表达框Tpu1中含有启动子Pamy-cryIII及淀粉酶分泌的信号肽序列SPamyL，核苷酸序列如下：

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；

所述基因表达框Tpu2中含有启动子PxylA-cryIII及来自几丁质酶分泌的信号肽序列SPchit，核苷酸序列如下：

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。