[發明專利]高產普魯蘭酶的重組枯草芽孢桿菌的構建方法

在審

• 申請號: 201710410090.5
• 申請日: 2017-06-03
• 公開（公告）號: CN107058367A
• 公開（公告）日: 2017-08-18
• 發明（設計）人: 紀明華;俞峰;劉校函;王輝;李統鋒;丁少明
• 申請（專利權）人: 白銀賽諾生物科技有限公司
• 主分類號: C12N15/75
• 分類號: C12N15/75;C12N15/56;C12R1/125
• 代理公司: 蘭州振華專利代理有限責任公司62102
• 代理人: 張真

1.一種高產普魯蘭酶的重組枯草芽孢桿菌的構建方法，其特征在于：將人工合成的普魯蘭酶基因表達框Tpu1及Tpu2分別原位替代枯草芽孢桿菌的堿性蛋白酶基因aprE以及淀粉酶基因amyE，并將抗性基因ermC和kan敲除后得到所述重組枯草芽孢桿菌，具體步驟如下：

a.設計并基因合成線性DNA片段Tpu1及Tpu2；

b.以枯草芽孢桿菌A164為出發菌株進行分子構建，將Tpu1與枯草芽孢桿菌A164感受態細胞混合，進行轉化，獲得具有紅霉素抗性的重組菌株CNP1；

c.將Tpu2與枯草芽孢桿菌CNP1感受態細胞混合，進行轉化，獲得具有紅霉素/卡那霉素雙抗性的重組菌株CNP2ek；

d.轉入Cre表達質粒，進行紅霉素/卡那霉素雙抗性的消除，得到重組枯草芽孢桿菌CNP2，即重組枯草芽孢桿菌。

2.如權利要求1所述的高產普魯蘭酶的重組枯草芽孢桿菌的構建方法，其特征在于：所述基因表達框Tpu1及Tpu2分別經密碼子優化、基因合成后獲得， Tpu1的核苷酸序列為：

caaatgcgct gaatgcctat gttacaggaa ttggggccaa taaacggatt gtattgtggg60

atacgacgct gaacaaactt gacgattcag aaattctgtt tattatgggc cacgaaatgg120

gccattatgt catgaagcac gtttacatcg gtctggctgg ctatttgctc gtgtcgctcg180

ccggatttta tgtcattgat aagctttaca agcggacggt tcgcctaacc cgcagcatgt240

ttcatttaga agggcggcat gatcttgcgg cacttccgct gttattgctt ttgttttctg300

ttttgagctt tgcggttacg cctttttcta atgctgtctc gcgttatcag gagaataagg360

ctgaccagta tgggatcgag ttgacagaga acagagaagc cgctgttaaa acgtttcagg420

atttggccgt gacggggctg agccaggttg atcctccggt gcttgtgaag attttcagag480

gcagccatcc gtcgatcatg gaacggattc aacatgcgga gaaagaagag aatgcgccgg540

aacatcagga tgctgacaaa taaaaagaag caggtatgga ggaacctgct tctttttact600

attattgtgc agctgcttgc ctgcactgca gatcgattgt ttgagaaaag aagaagacca660

taaaaatacc ttgtctgtca tcagacaggg tattttttat gctgtccaga ctgtccgctg720

tgtaaaaaaa aggaataaag gggggttgac attattttac tgatatgtat aatataattt780

gtataagaaa atggagtcga aacgtaagat gaaaccttag ataaaagtgc tttttttgtt840

gcaattgaag aattattaat gttaagctta attaaagata atatctttga attgtaacgc900

ccctcaaaag taagaactac aaaaaaagaa tacgttatat agaaatatgt ttgaaccttc960

ttcagattac aaatatattc ggacggactc tacctcaaat gcttatctaa ctatagaatg1020

acatacaagc acaaccttga aaatttgaaa atataactac caatgaactt gttcatgtga1080

attatcgctt tatttaattt tctcaattca atatataata tgccaataca ttgttacaag1140

tagaaattaa gacacccttg atagccttac tatacctaac atgatgtagt attaaatgaa1200

tatgtaaata tatttatgat aagaagcgac ttatttataa tcattacata tttttctatt1260

ggaatgatta agattccaat agaatagtgt ataaattatt tatcttgaaa ggagggatgc1320

ctaaaaacga agaacattaa aaacatatat ttgcaccgtc taatggattt atgaaaaatc1380

attttatcag tttgaaaatt atgtattatg cttacattcc atcgtatcat ccgtaaaggc1440

tggatgttcc ttcttgcttt ccttcttaca gcttctcttt tctgccctac aggccaacat1500

gctaaagctg ctgctcaacc tgctgtttct aacgcttacc ttgatgcttc taaccaagtt1560

cttgttaaac tttctcaacc tttcacactt ggcgaaggct cttctggctt cacagttcat1620

gatgatacag ctaacaaaga tatccctgtt acatctgttt ctgatgctaa ccaagttaca1680

gctgttcttg ctggcacatt ccaacatatc ttcggcggct ctgattgggc tcctgataac1740

cataacacac ttcttaaaaa agttaactct aacctttacc aattctctgg caaccttcct1800

gaaggcaact accaatacaa agttgctctt aacgattctt ggaacaaccc ttcttaccct1860

tctgataaca tcaaccttac agttcctgct ggcggcgctc atgttacatt ctcttacatc1920

ccttctacac atgctgttta cgatacaatc aacaacccta acgctgatct tcaagttgat1980

tcttctggcg ttaaaacaga tcttgttgct gttacacttg gcgaaaaccc tgatgtttct2040

catacacttt ctatccaaac agaagattac caagctggcc aagttatccc tcgtaaagtt2100

cttgattctt ctcaatacta ctactctggc gatgatcttg gcaacacata cacaaaaaac2160

gctacaacat tcaaagtttg ggctcctaca tctacacaag ttaacgttct tctttacaac2220

tctgctacag gcgctgttac aaaaacagtt cctatgacag cttctggcca tggcgtttgg2280

gaagctacag ttaaccaaga tcttgaaaac tggtactaca tgtacgaagt tacaggccaa2340

ggctctacac gtacagctgt tgatccttac gctacagcta tcgctcctaa cggcacacgt2400

ggcatgatcg ttgatcttgc taaaacagat cctgctggct gggaatctga taaacatatc2460

acacctaaaa acatcgaaga tgaagttatc tacgaaatgg atgttcgtga tttctctatc2520

gattctaact ctggcatgaa aaacaaaggc aaataccttg ctcttacaga aaaaggcaca2580

aaaggccctg ataacgttaa aacaggcgtt gattctctta aacaacttgg catcacacat2640

gttcaacttc aacctgtttt cgctttcaac tctgttaacg aaaacgatcc tacacaatac2700

aactggggct acgatcctcg taactacaac gttcctgaag gccaatacgc tacaaacgct2760

aacggcacaa cacgtatcaa agaattcaaa gaaatggttc tttctcttca tcaagatcat2820

atcggcgtta acatggatgt tgtttacaac catacattcg ctacacaaat ctctgatttc2880

gataaaatcg ttcctgaata ctactaccgt acagatgatg ctggcaacta cacaaacggc2940

tctggcacag gcaacgaaat cgctgctgaa cgtcctatgg ttcaaaaatt catcatcgat3000

tctcttaaat tctgggttaa cgaataccat gttgatggct tccgtttcga tcttatggct3060

cttcttggca aagatacaat gtctaaagct gctacacaac ttcatgctat cgatcctggc3120

atcgctcttt acggcgaacc ttggacaggc ggcacatctg ctcttcctgc tgatcaactt3180

cttacaaaag gcgctcaaaa aggcatgggc gttgctgttt tcaacgataa ccttcgtaac3240

ggccttgatg gctctgtttt cgattcttct gctcaaggct tcgctacagg cgctacaggc3300

cttacagatg ctatcaaaaa cggcgttgaa ggctctatca acgatttcac agcttctcct3360

ggcgaaacaa tcaactacgt tacatctcat gataactaca cactttggga taaaatcgct3420

caatctaacc ctaacgattc tgaagctgat cgtatcaaaa tggatgaact tgctcaagct3480

atcgttatga catctcaagg catccctttc atgcaaggcg gcgaagaaat gcttcgtaca3540

aaaggcggca acgataactc ttacaacgct ggcgatgttg ttaacgaatt cgattggtct3600

cgtaaagctc aataccctga tgttttcaac tactactctg gccttatcca tcttcgtctt3660

gatcatcctg ctttccgtat gacaacagct aacgaaatca actctcatct tcaattcctt3720

aactctcctg aaaacacagt tgcttacgaa ctttctgatc atgctaacaa agatacatgg3780

ggcaacatcg ttgttatcta caaccctaac aaaacagctg aaacaatcaa ccttccttct3840

ggcaaatggg aaatcaacgc tacatctggc aaagttggcg aatctacact tggccaagct3900

gaaggctctg ttcaagttcc tggcatctct atgatgatcc ttcatcaaga agtttctcct3960

tctgatggca aataaagaag caggttcctc catacctgct tctttttaag gatccaagct4020

tgcggccgct accgttcgta tagcatacat tatacgaagt tatttaaacc gtgtgctcta4080

cgaccaaaac tataaaacct ttaagaactt tcttttttta caagaaaaaa gaaattagat4140

aaatctctca tatcttttat tcaataatcg catccgattg cagtataaat ttaacgatca4200

ctcatcatgt tcatatttat cagagctcgt gctataatta tactaatttt ataaggagga4260

aaaaatatgg gcatttttag tatttttgta atcagcacag ttcattatca accaaacaaa4320

aaataagtgg ttataatgaa tcgttaataa gcaaaattca tataaccaaa ttaaggaggg4380

aaataatgaa cgagaaaaat ataaaacaca gtcaaaactt tattacttca aaacataata4440

tagataaaat aatgacaaat ataagattaa atgaacatga taatatcttt gaaatcggct4500

caggaaaagg ccattttacc cttgaattag taaagaggtg taatttcgta actgccattg4560

aaatagacca taaattatgc aaaactacag aaaataaact tgttgatcac gataatttcc4620

aagttttaaa caaggatata ttgcagttta aatttcctaa aaaccaatcc tataaaatat4680

atggtaatat accttataac ataagtacgg atataatacg caaaattgtt tttgatagta4740

tagctaatga gatttattta atcgtggaat acgggtttgc taaaagatta ttaaatacaa4800

aacgctcatt ggcattactt ttaatggcag aagttgatat ttctatatta agtatggttc4860

caagagaata ttttcatcct aaacctaaag tgaatagctc acttatcaga ttaagtagaa4920

aaaaatcaag aatatcacac aaagataaac aaaagtataa ttatttcgtt atgaaatggg4980

ttaacaaaga atacaagaaa atatttacaa aaaatcaatt taacaattcc ttaaaacatg5040

caggaattga cgatttaaac aatattagct ttgaacaatt cttatctctt ttcaatagct5100

ataaattatt taataagtaa gttaagggat gcataaactg catcccttaa cttgtttttc5160

gtgtgcctat tttttgtgaa ttgattatcg atcttttgcg ccatggataa cttcgtatag5220

catacattat acgaacggta catatgcccg ggggtaccga attccctgct ggtcgccatt5280

ttggctcttt accctctcct tttaaaaaaa ttcagagtag acttacttaa aagactattc5340

tgtgaattta ttgtaataga tggaataata ttttagtaga cccatttttt tgagatgatt5400

ttatctctat ttaggtatat catctctcgc tatttccgta gagactcgaa ataactattt5460

tcatacagaa aagaacgaaa atagacatga gtaaatgttc attatgctga aatgtcatgc5520

ttttttaggt taaatgctcc tgagtcccgg caaattcctg tcgaaaaaat tcgttcaaat5580

gacctgcgtg tgcttccgtg agaacaatgg atattatcgt gatatttttt caaagcatga5640

tgataaaagt attctgaaaa ataaacttta cagaaaaggg atagaatgaa aaaattattg5700

agagggagga agaaataaga tgaacattcg ccaagcaaag acatcagatg cggccgcca 5760

tgcgccgctg tttaaccaat atcgggaatt ttatagacag gcatccgatt tgcaaggggc5820

agaggctttt ttgaaagctc gtttggaaaa tcacgagtct gttattttga tagcagaaga5880

aaatg5885；

Tpu2的核苷酸序列為：

atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg60

tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat120

gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc180

aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca240

tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac300

tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa360

tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc420

atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac480

tggacacatg gaaacacaca aattaaaaac tggtctgatc gcctgcactg catagaatct540

aatattataa ctaaattttc taaaaaaaac attggaatag acatttattt tgtatatgat600

gaaataaagt tagtttattg gataaacaaa ctaactttat taaggtagtt gatggataaa660

cttgttcact taaatcaatc gaaacgtaag atgaaacctt agataaaagt gctttttttg720

ttgcaattga agaattatta atgttaagct taattaaaga taatatcttt gaattgtaac780

gcccctcaaa agtaagaact acaaaaaaag aatacgttat atagaaatat gtttgaacct840

tcttcagatt acaaatatat tcggacggac tctacctcaa atgcttatct aactatagaa900

tgacatacaa gcacaacctt gaaaatttga aaatataact accaatgaac ttgttcatgt960

gaattatcgc tttatttaat tttctcaatt caatatataa tatgccaata cattgttaca1020

agtagaaatt aagacaccct tgatagcctt actataccta acatgatgta gtattaaatg1080

aatatgtaaa tatatttatg ataagaagcg acttatttat aatcattaca tatttttcta1140

ttggaatgat taagattcca atagaatagt gtataaatta tttatcttga aaggagggat1200

gcctaaaaac gaagaacatt aaaaacatat atttgcaccg tctaatggat ttatgaaaaa1260

tcattttatc agtttgaaaa ttatgtatta tgcttatcaa caaatctaaa aaattcttcg1320

ttttctcttt catcttcgtt atgatgcttt ctctttcttt cgttaacggc gaagttgcta1380

aagctgctca acctgctgtt tctaacgctt accttgatgc ttctaaccaa gttcttgtta1440

aactttctca acctttcaca cttggcgaag gctcttctgg cttcacagtt catgatgata1500

cagctaacaa agatatccct gttacatctg tttctgatgc taaccaagtt acagctgttc1560

ttgctggcac attccaacat atcttcggcg gctctgattg ggctcctgat aaccataaca1620

cacttcttaa aaaagttaac tctaaccttt accaattctc tggcaacctt cctgaaggca1680

actaccaata caaagttgct cttaacgatt cttggaacaa cccttcttac ccttctgata1740

acatcaacct tacagttcct gctggcggcg ctcatgttac attctcttac atcccttcta1800

cacatgctgt ttacgataca atcaacaacc ctaacgctga tcttcaagtt gattcttctg1860

gcgttaaaac agatcttgtt gctgttacac ttggcgaaaa ccctgatgtt tctcatacac1920

tttctatcca aacagaagat taccaagctg gccaagttat ccctcgtaaa gttcttgatt1980

cttctcaata ctactactct ggcgatgatc ttggcaacac atacacaaaa aacgctacaa2040

cattcaaagt ttgggctcct acatctacac aagttaacgt tcttctttac aactctgcta2100

caggcgctgt tacaaaaaca gttcctatga cagcttctgg ccatggcgtt tgggaagcta2160

cagttaacca agatcttgaa aactggtact acatgtacga agttacaggc caaggctcta2220

cacgtacagc tgttgatcct tacgctacag ctatcgctcc taacggcaca cgtggcatga2280

tcgttgatct tgctaaaaca gatcctgctg gctgggaatc tgataaacat atcacaccta2340

aaaacatcga agatgaagtt atctacgaaa tggatgttcg tgatttctct atcgattcta2400

actctggcat gaaaaacaaa ggcaaatacc ttgctcttac agaaaaaggc acaaaaggcc2460

ctgataacgt taaaacaggc gttgattctc ttaaacaact tggcatcaca catgttcaac2520

ttcaacctgt tttcgctttc aactctgtta acgaaaacga tcctacacaa tacaactggg2580

gctacgatcc tcgtaactac aacgttcctg aaggccaata cgctacaaac gctaacggca2640

caacacgtat caaagaattc aaagaaatgg ttctttctct tcatcaagat catatcggcg2700

ttaacatgga tgttgtttac aaccatacat tcgctacaca aatctctgat ttcgataaaa2760

tcgttcctga atactactac cgtacagatg atgctggcaa ctacacaaac ggctctggca2820

caggcaacga aatcgctgct gaacgtccta tggttcaaaa attcatcatc gattctctta2880

aattctgggt taacgaatac catgttgatg gcttccgttt cgatcttatg gctcttcttg2940

gcaaagatac aatgtctaaa gctgctacac aacttcatgc tatcgatcct ggcatcgctc3000

tttacggcga accttggaca ggcggcacat ctgctcttcc tgctgatcaa cttcttacaa3060

aaggcgctca aaaaggcatg ggcgttgctg ttttcaacga taaccttcgt aacggccttg3120

atggctctgt tttcgattct tctgctcaag gcttcgctac aggcgctaca ggccttacag3180

atgctatcaa aaacggcgtt gaaggctcta tcaacgattt cacagcttct cctggcgaaa3240

caatcaacta cgttacatct catgataact acacactttg ggataaaatc gctcaatcta3300

accctaacga ttctgaagct gatcgtatca aaatggatga acttgctcaa gctatcgtta3360

tgacatctca aggcatccct ttcatgcaag gcggcgaaga aatgcttcgt acaaaaggcg3420

gcaacgataa ctcttacaac gctggcgatg ttgttaacga attcgattgg tctcgtaaag3480

ctcaataccc tgatgttttc aactactact ctggccttat ccatcttcgt cttgatcatc3540

ctgctttccg tatgacaaca gctaacgaaa tcaactctca tcttcaattc cttaactctc3600

ctgaaaacac agttgcttac gaactttctg atcatgctaa caaagataca tggggcaaca3660

tcgttgttat ctacaaccct aacaaaacag ctgaaacaat caaccttcct tctggcaaat3720

gggaaatcaa cgctacatct ggcaaagttg gcgaatctac acttggccaa gctgaaggct3780

ctgttcaagt tcctggcatc tctatgatga tccttcatca agaagtttct ccttctgatg3840

gcaaataaag aagcaggttc ctccatacct gcttcttttt aagggatcca agcttgcggc3900

cgctaccgtt cgtatagcat acattatacg aagttatcgg gccagtttgt tgaagattag3960

atgctataat tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct4020

gaataagaac ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga4080

aaagggaatg agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat4140

tgttcatgaa attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg4200

tgtttatggc tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg4260

tgtcatgtca acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt4320

ggaagtgaat tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga4380

ttggccgctt acacatggtc aatttttctc tattttgccg atttatgatt caggtggata4440

cttagagaaa gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat4500

ttgtgccctt atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt4560

gcaaggaccg acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat4620

gttgattggt ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc4680

agttaagcaa tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg4740

tcaactttcc gactctgaga aacttctgga atcgctagag aatttctgga atgggattca4800

ggagtggaca gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac4860

gatgacctct aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt4920

agtaattatc atggctgtca tcccgggata acttcgtata gcatacatta tacgaacggt4980

acatatgccc gggggtaccg aattccctgc tggtcgccat tttggctcga catggatgag5040

cgatgatgat atccgtttag gctgggcggt gatagcttct cgttcaggca gtacgcctct5100

tttcttttcc agacctgagg gaggcggaaa tggtgtgagg ttcccgggga aaagccaaat5160

aggcgatcgc gggagtgctt tatttgaaga tcaggctatc actgcggtca atagatttca5220

caatgtgatg gctggacagc ctgaggaact ctcgaacccg aatggaaaca accagatatt5280

tatgaatcag cgcggctcac atggcgttgt gctggcaaat gcaggttcat cctctgtctc5340

tatcaatacg gcaacaaaat tgcctgatgg caggtatgac aataaagctg gagcgggttc5400

atttcaagtg aacgatggta aactgacagg cacgatcaat gccaggtctg tagctgtgct5460

ttatcctgat gatattgcaa aagcgcctca tgttttcctt gagaattaca aaacaggtgt5520

aacacattct ttcaatgatc aactgacgat taccttgcgt gcagatgcga atacaacaaa5580

agccgtttat caaatcaata atggaccaga cgacaggcgt ttaaggatgg agatcaattc5640

acaatcggaa aaggagatcc aatttggcaa aacatacacc atcatgttaa aaggaacgaa5700

cagtgatggt gtaacgagga ccgagaaata cagttttgtt aaaagagatc cagcgtcggc5760

caaaaccatc ggctatcaaa atccgaatca ttggagccag gtaaatgctt atatctataa5820

acatgatggg agccgagtaa ttgaattgac cggatcttgg cctggaaaac caatgactaa5880

aaatgcagac ggaatttaca cgctgacgct gcctgcggac acggatacaa ccaacgcaaa5940

agtgattttt aataatggca gcgcccaagt gcccggtcag aatcagcctg gctttgatta6000

cgtgctaaat ggtttatata atgactcggg cttaagcggt tctcttcccc attga 6055。