CN109337855A - A kind of efficient method for reducing fermentation of bacillus liquid viscosity - Google Patents
A kind of efficient method for reducing fermentation of bacillus liquid viscosity Download PDFInfo
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- CN109337855A CN109337855A CN201811383908.XA CN201811383908A CN109337855A CN 109337855 A CN109337855 A CN 109337855A CN 201811383908 A CN201811383908 A CN 201811383908A CN 109337855 A CN109337855 A CN 109337855A
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Abstract
The invention belongs to field of fermentation engineering, more particularly to a kind of method for efficiently reducing fermentation of bacillus liquid viscosity, it is characterized by: knocking out the eps operon of bacterial strain expression system to be rebuilt, the eps operon for specially knocking out bacterial strain expression system to be rebuilt is that the fragment upstream eps-F of eps operon after amplification is inserted between the KpnI and BamHI of pBTS plasmid, segments downstream eps-R is inserted between BamHI and XhoI, new plasmid pBTS-eps is constructed, electrotransformation plasmid is obtained in transformation bacterial strain, culture medium culture and PCR identification.The present invention hinders transposon mutagenesis, and then substantially reduces fermentation broth viscosity, promotes tunning separation, reduces cost, greatly improve efficiency.
Description
Technical field
The invention belongs to field of fermentation engineering, and in particular to a method of efficiently reduce fermentation of bacillus liquid viscosity.
Background technique
What is carried out after the completion of fermentation work is the mask work of tunning, is generally removed by way of being centrifuged, filtering
Tunning, the mode separation and fermentation product of column chromatography is concentrated in thallus by the way of ultrafiltration.But the viscosity of fermentation liquid can shadow
Above-mentioned separation process is rung, working efficiency is reduced, improves cost.
In Chinese patent 201711034848.6, patent name is a kind of method for reducing fermentation of bacillus liquid viscosity
In, by way of knocking out pgs gene, prevent the synthesis of Polyurethane-epoxy resin.Its viscosity that fermentation liquid can be significantly reduced promotees
Into thalli growth, Fermentation Substance Concentration is improved.In tunning separation process, the efficiency of separation is also significantly increased, is reduced
Production cost.But fermentation broth viscosity is still higher, especially in filtering and ultra-filtration process, film clogging is very serious.Filter
The replacement cycle of film is short, and filter efficiency is low.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of method for efficiently reducing fermentation of bacillus liquid viscosity,
In the expression system based on Z12 bacterial strain, by deleting the full gene of eps operon, the formation of biomembrane is hindered, bacterium colony is raw
Long form changes, while significantly reducing fermentation broth viscosity, reduces the filter membrane replacing number during subsequent filter, reduces hair
The cost of ferment product separation.
The efficient method for reducing fermentation of bacillus liquid viscosity of one of present invention of the above technical problem is solved, it is special
Sign is: the eps operon of bacterial strain expression system to be rebuilt knocked out, hinders transposon mutagenesis, and then reduce fermentation broth viscosity,
Promote tunning separation.
The expression system nucleotide sequence is as shown in SEQ ID No.:1.
The eps operon for knocking out bacterial strain expression system to be rebuilt is the fragment upstream of eps operon after expanding
Eps-F is inserted between the KpnI and BamHI of pBTS plasmid, and segments downstream eps-R is inserted between BamHI and XhoI, building
New plasmid pBTS-eps, electrotransformation plasmid are obtained in transformation bacterial strain, culture medium culture and PCR identification.
The nucleotide sequence of eps operon is as shown in SEQ ID No.:2 after the amplification.
The plasmid pBTS-eps nucleotide sequence is as shown in SEQ ID No.:3.
The mother strains Z24 is △ (eps), △ (pgs), △ (aprE), △ (bpr), △ (epr), △ (mpr), △
(nprB),△(nprE),△(vpr),xylAB::PT7-TerT7,wprA::(xylR-PxylABrpoT7)。
The efficient method for reducing fermentation of bacillus liquid viscosity, specifically includes the following steps:
(1) DNA for extracting bacterial strain to be rebuilt, passes through the fragment upstream eps-F and segments downstream of primer amplification eps operon
eps-R；
It is expanded using the KOD-Plus-Neo enzyme of Toyobo, amplification condition program: 94 DEG C, 2min；(94 DEG C, 30s；
60 DEG C, 30s；68 DEG C, 1min) x25；72 DEG C, 2min.
(2) by digestion, eps-F is inserted between the KpnI and BamHI of pBTS plasmid, eps-R is inserted into BamHI
Between XhoI, new plasmid pBTS-eps is constructed.
(3) pBTS-eps is transferred in bacillus to be rebuilt by electrotransformation, obtains positive transformants bacterial strain；
(4) the positive transformants bacterium colony in picking (3), which enters in fluid nutrient medium, is cultivated, and cultivation temperature is 45 DEG C, training
The time is supported greater than for 24 hours；
(5) bacterium solution 100ul -500ul in step (4) is taken, is applied on the LB plate of the kanamycins containing 30mg/L, is cultivated
Temperature is 45 DEG C, is incubated overnight；
(6) bacterium colony in picking step (5), which enters in fluid nutrient medium, is cultivated, and every 8-16h passage is primary, until
Screen nonreactive bacterial strain；
(7) bacterium solution in step (6) is taken to be diluted coated plate, dilution 105-106Times, it is incubated overnight；
(8) bacterium colony in picking step (7) carries out Resistance Identification, until obtaining 5-10 plants of nonreactive bacterial strains；
(9) nonreactive bacterial strain is inoculated into fluid nutrient medium, is incubated overnight, extract bacterial genomes, carry out PCR identification；
Positive strain continues sequencing identification, and sequencing qualification result is the Bacillus strain that positive strain knocks out eps gene to obtain the final product.
Bacillus to be rebuilt is bacillus subtilis 168, Z12 bacterial strain, solution starch gemma in the step (1) and (3)
The bacillus such as bacillus, bacillus pumilus, bacillus licheniformis.
Bacillus to be rebuilt is gene --- the withered grass bud of pgs for having knocked out synthesis γ-PGA in the step (3)
The bacillus such as spore bacillus 168, Z12 bacterial strain, bacillus amyloliquefaciens, bacillus pumilus, bacillus licheniformis.
Primer sequence is as follows in the step (1):
The method of the present invention hinders the formation of biomembrane, and bacterium colony growthform changes, while it is viscous to significantly reduce fermentation liquid
Degree reduces the filter membrane replacing number during subsequent filter, reduces the cost of tunning separation.Reduced rate fermentation broth viscosity rate
Up to 79% or more, improved for 3.5 or more ultrafiltration membrane cleaning frequency, substantially reduce cost, improve efficiency.
Specific embodiment
Invention is further explained With reference to embodiment:
Embodiment 1
Embodiment 1
1, by EcoR I and Apa I restriction endonuclease cut pBAV1K-T5-GFP (http://www.addgene.org/
Vector-database/) plasmid carries out homologous recombination clone with the MCS segment of synthesis, obtains plasmid pBAV1K.
In this experiment and follow-up test:
Restriction endonuclease uses the quick restriction endonuclease of Thermo company, and segment recycling is recycled using the glue of Chengdu Fu Ji biotech firm
Kit (DE-02011), MCS segment are synthesized by Jin Wei intelligence Biotechnology Co., Ltd, and homologous recombination is using Tiangeng
EsayGeno Quick Casting Cloning Kit (VI201-02), coli strain top10, preparation competence use KCM method,
Plasmid extraction uses the universal plasmid Mini Kit (DE-01001) of good fortune border biology.
2, design primer, amplification same sense mutation are deleted the pBAV1K segment of Nde I restriction enzyme site, are cloned using homologous recombination
Mode junction fragment, obtain plasmid be named as pBTS.Primer in this experiment and subsequent experimental is had by golden only intelligence biotechnology
The synthesis of limit company.
Embodiment 2
1, bacterial strain method for transformation is referring to hypertonic conversion method (High osmolarity improves the electro-
transformation efficiency of the gram-positive bacteria Bacillus subtilis and
Bacillus licheniformis, 1999).The new scribing line single colonie for taking bacterial strain to be transformed, is inoculated into about 3ml GM (LB+
0.5M D-sorbite) in culture medium, 37 DEG C, 180rpm is incubated overnight.The next morning is inoculated into 50ml GM culture by 1:100
In base, 37 DEG C, 180rpm culture when bacterium solution grows into OD and reaches between 0.85-0.95, bacterium solution is put into and is pre-chilled on ice
10min；4 DEG C, 5000g, 5min, centrifugation remove supernatant, be pre-chilled in equal volume EM (0.5M D-sorbite+0.5M mannitol+
10% glycerine water solution) thallus is resuspended, 4 DEG C again, 5000g, 5min, supernatant is removed in centrifugation, washes repeatedly 4 times altogether；It is added about
Thallus is resuspended in the EM of 1/40 volume, guarantees bacterial concentration between 1-1.3 × 1010cfu/ml.The above-mentioned bacterium solution of 60ul is taken, is added
1ul plasmid to be transformed, plasmid concentration are greater than 100ng/ul, and piping and druming mixes, and the 1mm electric shock cup of pre-cooling is then added.Electric converter
(Eppendorf Eporator) parameter setting, 2.1kV is practical to shock by electricity the time in 4.0-5.0ms, has been likely to conversion bacterium colony
Growth.After electric shock, immediately plus 1ml RM (LB+0.5M D-sorbite+0.38M mannitol), piping and druming are mixed, and it is sterile to be transferred to 5ml
In centrifuge tube, 37 DEG C, 180rpm cultivates 3h.Bacterium solution is centrifuged, is diluted by a certain percentage after removing supernatant, takes 200ul to be applied to and contains
Have on the LB plate of 30mg/L kanamycins, 37 DEG C are incubated overnight, while being coated with the bacterium solution without electrotransformation and doing negative control.The
Two day morning observed bacterium colony growing state, if reformer plate has bacterium colony growth, negative control does not have, then shows to convert successfully.
Embodiment 3
1, design primer amplification Z12 bacterial strain pas upstream region of gene 558bp segment pgs-F, be inserted into pBTS KpnI and
Between the site BamHI, plasmid pBTS-pgs-F is obtained；The segment aprE-R of downstream 693bp is expanded, insertion plasmid pBTS-pgs-F's
Between BamHI and XhoI, the plasmid pBTS-pgs for knocking out pgs gene is obtained.It is carried out using the KOD-Plus-Neo enzyme of Toyobo
Amplification, amplification condition program: 94 DEG C, 2min；(94 DEG C, 30s；60 DEG C, 30s；68 DEG C, 1min) x25；
72 DEG C, 2min.
Primer sequence:
It is expanded using the KOD-Plus-Neo enzyme of Toyobo, amplification condition program: 94 DEG C, 2min；(94 DEG C, 30s；
60 DEG C, 30s；68 DEG C, 1min) x25；72 DEG C, 2min.
2, using the hypertonic conversion method conversion pBTS-pgs in embodiment 2 to enter Z20 bacterial strain, (Z20 is the derivative bacterium of Z12
Strain, genotype are as follows: △ (aprE), △ (bpr), △ (epr), △ (mpr), △ (nprB), △ (nprE), △ (vpr),
xylAB::PT7-TerT7,wprA::(xylR-PxylABrpoT7)。
Original strain Z12 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, preservation
Number for 12750), Z20-pBTS-pgs bacterial strain is obtained.
3, bacterium Z20-pBTS-pgs is taken to be inoculated into 3ml LB culture medium, 45 DEG C, 180rpm, culture is for 24 hours；Separately meet bacterium Z20-
PBTS is compareed.
4, the bacterium solution 200ul in 3 is taken to be applied on the LB plate containing 30mg/L kanamycins, 45 DEG C are incubated overnight.If
Z20-pBTS does not have bacterium colony growth, and Z20-pBTS-pgs has bacterium colony growth, then the bacterial strain obtained is completed to recombinate for the first time, is named as
Z20-pBTS-pgs-45。
5, it connects in bacterium Z20-pBTS-pgs-45 to 3ml LB culture medium, is incubated overnight, (Chengdu good fortune border is raw for extracting genome
Object Technology Co., Ltd., bacterial genomes DNA extraction kit, DE-05311), PCR amplification pgs segment is verified (Chengdu
Fu Ji Bioisystech Co., Ltd, 2 × fast PCR react premix system, DP-20041).
6, it takes and is verified as positive Z20-pBTS-pgs-45 bacterial strain in 5, be inoculated into 3ml LB base, 37 DEG C, 180rpm training
It supports, it is primary every 8-16h passage, after continuous passage 8 times, bacterium solution is taken to dilute 105Times, it is coated on the LB plate of nonreactive, obtains
Single colonie.
7, the single colonie in 6 is taken, while being crossed on LB and LB plate containing 30mg/L kanamycins, nonreactive is screened
Bacterium colony needs to obtain 5-10 plants of nonreactive bacterium colonies.
8, the nonreactive bacterium colony in 7 is taken, is inoculated into 3ml LB culture medium, 37 DEG C, 180rpm is incubated overnight, and extracts bacterium base
Because of group, homologous recombination result (wild type or knocking out pgs gene) is verified by PCR amplification pgs segment, completes second of weight
Group.Positive segment is taken, Song Jinwei intelligence Biotechnology Co., Ltd further progress sequence verification, obtaining positive bacterial strain is
Knock out bacterial strain --- Z22 (△ (pgs), △ (aprE), △ (bpr), △ (epr), △ (mpr), △ (nprB), △ of pgs gene
(nprE),△(vpr),xylAB::PT7-TerT7,wprA::(xylR-PxylABrpoT7))。
Embodiment 4
1, design primer amplification Z12 bacterial strain eps operon upstream 535bp segment eps-F, be inserted into pBTS KpnI and
Between the site BamHI, plasmid pBTS-eps-F is obtained；The segment eps-R of downstream 536bp is expanded, insertion plasmid pBTS-eps-F's
Between BamHI and XhoI, the plasmid pBTS-eps for knocking out pgs gene is obtained.
It is expanded using the KOD-Plus-Neo enzyme of Toyobo, amplification condition program: 94 DEG C, 2min；(94 DEG C, 30s；
60 DEG C, 30s；68 DEG C, 1min) x25；72 DEG C, 2min.
2, Z22 bacterial strain (bacterial strain in embodiment 3) is entered using the hypertonic conversion method conversion pBTS-eps in embodiment 2,
Obtain Z22-pBTS-eps bacterial strain.
3, bacterium Z22-pBTS-eps is taken to be inoculated into 3ml LB culture medium, 45 DEG C, 180rpm, culture is for 24 hours；Separately meet bacterium Z22-
PBTS is compareed.
4, the bacterium solution 200ul in 3 is taken to be applied on the LB plate containing 30mg/L kanamycins, 45 DEG C are incubated overnight.If
Z22-pBTS does not have bacterium colony growth, and Z22-pBTS-eps has bacterium colony growth, then the bacterial strain obtained is completed to recombinate for the first time, is named as
Z22-pBTS-eps-45。
5, it connects in bacterium Z22-pBTS-eps-45 to 3ml LB culture medium, is incubated overnight, (Chengdu good fortune border is raw for extracting genome
Object Technology Co., Ltd., bacterial genomes DNA extraction kit, DE-05311), PCR amplification eps segment is verified (Chengdu
Fu Ji Bioisystech Co., Ltd, 2 × fast PCR react premix system, DP-20041).
6, it takes and is verified as positive Z22-pBTS-eps-45 bacterial strain in 5, be inoculated into 3ml LB base, 37 DEG C, 180rpm training
It supports, it is primary every 8-16h passage, after continuous passage 8 times, bacterium solution is taken to dilute 106Times, it is coated on the LB plate of nonreactive, obtains
Single colonie.
7, the single colonie in 6 is taken, while being crossed on LB and LB plate containing 30mg/L kanamycins, nonreactive is screened
Bacterium colony needs to obtain 5-10 plants of nonreactive bacterium colonies.
8, the nonreactive bacterium colony in 7 is taken, is inoculated into 3ml LB culture medium, 37 DEG C, 180rpm is incubated overnight, and extracts bacterium base
Because of group, homologous recombination result (wild type or knocking out eps operon) is verified by PCR amplification eps segment, completes second of weight
Group.Positive segment is taken, Song Jinwei intelligence Biotechnology Co., Ltd further progress sequence verification, obtaining positive bacterial strain is
Knock out the bacterial strain of pgs gene --- Z24 (△ (eps), △ (pgs), △ (aprE), △ (bpr), △ (epr),
△(mpr),△(nprB),△(nprE),△(vpr),xylAB::PT7-TerT7,wprA::(xylR-
PxylABrpoT7))。
Test one
1, bacterium Z20, Z22 and Z24 are met to producing in protein culture medium, 30 DEG C, 200rpm cultivates 72h.Protein culture medium is produced to match
Side are as follows:
Basal medium
Yeast extract 1%
Tryptone 2%
Soluble starch 1%
(NH4) 2,SO4 0.5%
Phosphate mother liquor (100 ×)
K2HPO4 210g/L
NaH2PO4 120g/L
(NH4)6Mo7O24 1g/L
Microelement mother liquor (1000 ×)
MgCl2.7H2O 200g/L
FeSO4.7H2O 50g/L
CuSO4.5H2O 10g/L
MnSO4.H2O 10g/L
ZnSO4.7H2O 20g/L
Calcium chloride mother liquor (1000 ×)
CaCl2.2H2O 50g/L
Xylose solution (100 ×)
250g/L
Above-mentioned basal medium, phosphate mother liquor, microelement mother liquor, calcium chloride mother liquor and xylose solution are respectively configured,
121 DEG C, 20min high temperature and humidity sterilization treatment, the mixing of peace ratio, inoculum concentration 1% before being inoculated with.
2, bacterium solution is collected, 5000g is centrifuged 5mins and removes thallus.
3, fermentation broth viscosity is measured using NDJ-5S (power occasion science and technology), wherein the fermentation broth viscosity of Z20 bacterial strain is
162mPa.S (No. 1 rotor, 30rpm), the viscosity of Z22 bacterial strain fermentation liquor are 12.5mPa.S (No. 0 rotor, 30rpm), Z24 bacterial strain
The viscosity of fermentation liquid is only 2.7mPa.S (No. 0 rotor, 60rpm).
4, using Z24 as mother strains, in the protein expression strain of building, fermentation liquid is in ultra-filtration process, filter membrane blocking
Situation ratio Z22 bacterial strain mitigates very much, and the ultrafiltration membrane cleaning frequency is increased to about 700 liters from about 200 liters.Fermentation is significantly improved to produce
The separative efficiency of object.
Basic principles and main features and advantages of the present invention of the invention, above-described embodiment has been shown and described above
It is merely illustrated the principles of the invention with described in specification, without departing from the spirit and scope of the present invention, the present invention
It will also have various changes and improvements, these changes and improvements are fallen in scope of the claimed invention.The present invention claims
The range of protection is defined by the appending claims and its equivalent thereof.
SEQUENCE LISTING
<110>Chengdu Mei Yide Bioisystech Co., Ltd
<120>a kind of method for efficiently reducing fermentation of bacillus liquid viscosity
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 15741
<212> DNA
<213>artificial synthesized
<220>
<223>expression system nucleotide sequence
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gcagaaaattcacggcattggcgtcaataccgagcgtttccggcctgtcagtccgatagagcagcaaagactcaga
gaaaagcacgggttccgtgaagatgattttatattggtttatccggctgagctcaatctgaacaaaaaccagaagc
agttaattgaagccgcagccttgctaaaagaaaaaattccctcactccgccttgtgtttgccggggaaggggcaat
ggaacatacgtatcaaacgttagctgaaaagcttggtgcctccgcccatgtctgtttttacggcttttgcagcgac
atacatgagttgattcagcttgcggatgtatctgtcgcatccagcattagagaaggcctcggtatgaatgtgcttg
agggaatggcggcagaacaaccggcgatcgccacagataatcgcgggcatcgggaaatcatccgcgacggagaaaa
cggttttctgatcaaaatcggtgacagtgctgcttttgcccgccggattgaacagctttaccataagccggagctc
tgccgaaagctgggacaggaaggccgaaaaacagccttgcgcttctcggaggcgcgaacggtggaagaaatggcag
atatttattccgcgtacatggatatggatacaaaggagaaaagcgtatgaactcaggaccgaaagtttctgtcatt
atgggcatttataattgcgaacgcactttggcagaaagcatagaatccatactcagccaatcctataaaaattggg
agctgattttgtgcgatgatgcgtcaacagacggcacgctccgtatcgcgaagcagtatgccgctcattacagcga
ccgcatcaaactgattcaaaacaaaacaaataagcggcttgccgcatcattaaatcattgtctttcgcatgcgaca
ggcgattatatcgcacgtcaggacggagatgacctttcgtttccgcgccgtctggaaaagcaggtcgcgtttttag
aaaagcaccgacactatcaggtggttggcaccggcatgcttgtgtttgatgaatttggcgtaagaggcgcccgcat
tctgccttctgttccggagccgggcatcatggcaaaagggactccattttgccacggcacgattatgatgagagcg
agtgcctaccgcacgctgaaaggctaccggtcggtgcggcggacgagacgaatggaagatattgatttgtggcttc
gcttttttgaagagggcttcaggggctataatcttcaggaagccttgtataaagtgagggaagacagcgatgcatt
caaacggcggtcatttacgtattcaatcgacaatgccattcttgtctatcaggcgtgcagacgcttgaagcttcct
ttatctgattacatatatatcgcaaaaccgttaattcgcgcctttatgcctgcagctgtgatgaatcgctaccata
aaaaaagagtgatgaaccaaaaggaagggcttgtcaagcatgaatagcagccaaaagcgcgtgctccatgttctca
gcggcatgaacaggggcggcgcggaaaccatggtaatgaatttatatcggaagatggacaaaagcaaagtgcaatt
tgattttttaacgtatcgaaatgatccgtgcgcttatgatgaagagattttatctttaggcgggcggcttttttat
gtcccgagcattgggcaaagcaatccccttacatttgtgaggaatgtgagaaacgcgataaaagaaaatgggccgt
tcagcgccgttcatgcgcacacggatttccaaacgggttttatcgcccttgcggcaaggctcgccggagtgccggt
cagggtatgccactcccacaatacgtcttggaagaccggcttcaactggaaggatcgattgcagctgctcgtgttc
aggcggctcattttggcaaatgcgacagcgctgtgtgcctgcggagaggatgcgggcaggtttttatttggacagt
ccaatatggagcgggagcgtgttcaccttcttcctaacgggattgaccttgagttgttcgccccaaatgggcaggc
ggctgatgaagaaaaagcagcacgcggcattgcagccgaccggctcatcattggccatgtggcccggtttcatgaa
gtgaaaaaccacgcgttcctgttgaagcttgccgcacatctcaaggaaagaggcattcgctttcagctcgttctgg
cgggagacgggccgttgtgcggggagatagaggaggaggcgcggcagcagaatttgctatcagacgtcctcttttt
aggcacggaagaacggatccatgaactgatgcgaacattcgatgtatttgtcatgccgtctctgtacgaaggcttg
ccggttgtgcttgtggaagcgcaggcgtcggggcttccatgcatcatttcagacagcattacagaaaaagtcgacg
ccggtctcgggcttgtcacaagattaagtctttctgagccgatcagcgtctgggctgaaaccattgcaagggcggc
cgccgcaggcaggccgaagcgtgagttcatcaaagaaacactcgctcaacttggctacgatgcacagcaaaatgta
ggagcgctgctgaatgtatacaacatcagcacggaaaaggaccataaccgatgattgtatatgccgtcaatatggg
gattgtatttatttggtcttggttcgctaaaatgtgcggcggccgtgatgattcgcttgccacggggtatcggccg
aataagcttttgatctggattccgctcgcttcacttgtgctcgtgtcaggtctccgctatcgagtcggcacggatt
ttcagacgtacacgctgttgtacgaattggcgggcgattatcaaaatgtgtggcagatattcggtttcggcacagc
gaaaacagcgacagatccggggtttaccgcactcctttggctgatgaatttcatcacggaagatcctcaaatcatg
tattttacggtggcggtcgtgacctacagctttattatgaagacactcgccgactatggcaggccgtttgagctga
gtgtctttttatttttgggaacctttcattattacgcatcttttaacggcatcaggcaatacatggtggcagctgt
tttgttttgggcgatccgttatatcattagcgggaactggaagcgatatttcctgattgtgctggtcagctcgctc
tttcattcgtcggcgctgattatgattccagtgtactttattgtcagaagaaaagcctggtcaccggcgatattcg
gcctatccgctttatttctcggcatgacatttttatatcaaaaatttatttctgtgtttgtcgttgtacttgaaaa
cagctcatacagccattatgaaaaatggctcatgacgaacacaaatggaatgaatgtgatcaaaatcgctgttttg
gttctgccgctgttccttgcattttgctataaagaacgactgcggagtctgtggccgcaaattgatattgtcgtca
atttgtgcctgctaggttttttgttcggccttttggccacaaaggacgtgatttttgccagattcaatatttattt
cggtctgtatcaaatgatcctagtcccttatttcgtcaggatatttgatgaaaaatcgaacgctcttatctatatc
gctatcgttgtttgttattttctttacagttatttgcttatgccggtcgattcatcggttctgccttacagaacga
ttttttcccggtaatttatgcacgaggagccggtcattatggaaacacctgcggttagtctgttagtcgctgttta
taacacagaaacatatatcagaacgtgtctcgaatcactgcggaaccagacaatggacaatattgaaatcatcatt
gtcaatgacggttcagctgacgccagcccggatatcgcagaagaatacgccaaaatggacaacaggttcaaggtga
ttcatcaggaaaaccagggactcggtgcggttcggaataaaggcattgaagctgcacgcggcgaatttatcgcgtt
tatcgattcagacgattggattgagcctgattattgcgagcagatgctccggacagcaggcgatgaaactgatctg
gtcatttgcaattacgccgcagagtttgaggacactggcaaaaccatggactctgacattgcccaaacctatcagg
atcagccgaaggagcactatatcaaggcgttattcgaagggaaggtcagagggttttcatggaacaaactgtacag
aagaagcatgattgaagcccatcggctgtcgtttccgctccgaggcgagctggagcatgtcgaggatcagtttttc
agcttcagggctcattttttcgcccgctcagtatcctacgtaaaaacgccgctctatcattatcgaattcaccttt
cctccattgtgcagcgctatcagaaaaaattgtttgaatcaggacttgcgctgtatgagacgaatgcggcgttttt
acaggagaacaacaaactggaggagtatcgcaaggagcttgatacctttatcgttcttcacagcagcatctgtatg
ctgaatgaatggaaaacgagcggcagccgccggctgtttgaaaagcttagaaatgttggcgtgatatgcgcggacc
cggtgtttcaagaaagtctttcaaaaacgggtactgctccttttgacgcaaaacggtcatgcctgcttctgatggc
gaaatacagaatgattccgttcgtcgctatggcatcggctgtgtatcagcgggtgatcgagtacaaaatgagaaac
agagggtgaggacatgtcgttacaatcgttgaaaatcaattttgcagaatggctgctgctaaaggtcaaatacccg
tcccaatattggctgggagcggcagatcaaccggtaaaggccgcagcacatcagaaaaaaatcatactgaccctgc
tgccgtcccatgacaatttgggagatcacgcaattgcttatgccagcaaggcatttcttgagcaagaatacccgga
ctttgacatcgtcgaggtcgatatgaaggacatttacaaatcagcaaaaagcctgatccgctcgcgccatccggag
gatatggtctttatcatcggcggcggaaacatgggggatttataccgttatgaggagtggacgcgccgcttcatca
ttaaaacattccatgactatcgggttgtccagcttccggcaacggctcatttttctgacacgaaaaaagggcgcaa
agagctgaaacgggcacagaaaatttataatgcgcaccccggcctattgctgatggcgcgggatgaaacaacgtat
caatttatgaaacagcattttcaagaaaaaacaattttgaagcagccggacatggtgctgtatttagacagaagca
aggctcccgcagaacgcgaaggggtttatatgtgtttgcgcgaggatcaggaaagcgtgcttcaggaggagcagag
gaaccgggtcaaggctgcgctatgtgaggaattcggcgagatcaaatcctttacgacaacgatcggccgccgggtc
agccgcgatacacgcgaacatgaacttgaagcactgtggtctaagctgcaaagcgcagaagccgtcgtcactgaca
ggcttcatggcatgattttttgcgcgctgacaggaacgccgtgtgttgtcattcgctcctttgaccataaggtgat
ggagggctatcaatggcttaaagacatcccgttcatgaagctgatagaacatccggagccagagcgcgtaacagcc
gcagtcaatgagcttttaacaaaagaaacatcccgtgcaggctttccgagagatgtgtattttaaaggtctgcgtg
acaaaatcagcggtgaagcgcaatgatcccgctcgtcagcattattgtcccgatgtataatgttgaaccatttata
gaagagtgcattgattctttgcttcgtcaaacgctttctgatattgaaatcatcctcgtgaatgacggaacaccgg
atcgttcaggcgaaattgcagaggactatgcaaaacgggatgcgagaatccgggtcattcatcaggcaaacggcgg
gcttagttcagcgcgaaatacgggaataaaggccgcgcggggcacttacatcggctttgtagacggagacgattat
gtatcatccgccatgttccagaggctgactgaagaagcggagcaaaatcagcttgacatcgtcggatgcggttttt
acaagcagtcatcggacaggcggacatatgtgccgccgcagcttgaggcaaaccgcgtgctgacgaaaccagaaat
gactgaacagcttaaacatgctcacgaaacgagatttatctggtatgtatggcgttatctttaccgtcgtgagctt
tttgaaagggcgaatctgctgtttgatgaagacatccgttttgctgaagactctccctttaatttgtccgcttttc
gcgaagcggagcgggtgaaaatgcttgatgaaggattgtacatttatcgtgaaaacccgaacagcctgacagaaat
cccttataagccggcgatggatgaacatcttcaaaaacaatatcaggctaaaatcgcattctacaatcactacggc
ttagcaggcgcatgtaaagaagatttgaatgtgtacatttgcaggcaccagcttccgatgcttttggcaaatgcct
gtgcttctccgaattcgccgaaagacatcaaaaagaagatcagacagattttatcctatgacatggtgcggcaggc
tgtcagacatacaccgtttcagcatgagaaattattaagaggagagcgtttggtattagcactgtgtaaatggcgg
ctcacttttctcatcaagctgtttttcgagcagcgggggacaatgaaaggcagtgcgaagcaggcatgaaattcac
gataaatttcagcgcgaatctcacggcttttctcttgtctgttttcctatcggtttggatgacgccttttattgtc
aaaacgctcggtgtcgaagcgtttggctttgttcacttgacacaaaatgtgattaattacttttcggttattaccg
tggcgctcagctcggttgtcgttcggttcttttctgttgctgcccacaggggagagcgggagaaagcaaatgcgta
tatcagcaattatttagccgcctctgttttgatttccttgctgctcttgctgccgcttgcgggttcggcttttttt
attgaccgcgtcatgaatgtgccgcaagcgcttttggcagatgtgcgtttgtcgattttaatcggcagtgtgctgt
ttattttaacgtttctgatggcgggcttcggcgctgcaccattttatgccaaccgcctttatatcaccagttccat
tcaggcggtgcaaatgcttatacgggtgctgtctgtgctgctcctgtttgcatgctttgcgccgaaaatctggcag
atccagcttgcagctttagctggtgctgttattgcgtctgtgctgtctttctatttcttcaaaaagctgattccgt
ggttttcgtttcgtatgaaggatctttcattccgtacaagcaaggagctgtttcaagcgggcgcatggagctccgt
caatcaaatcggcgtcctgctttttttgcagattgatctgttaaccgccaatttgatgctgggggcgtctgcatcc
gggaaatacgcggcgattatccagtttccgctgcttttgcgcagcttggccggaacggtcgcatccctgtttgcgc
ccatcatgacttcatattattcaaaaggcgatatggaaggattgatgaattacgccaataaggcagtaaggctcaa
tggtcttttgcttgcacttcctgctgccttattgggcggattggcgggaccttttctgacaatctggctcggaccg
tccttttcaacgatagcaccgcttttatttattcatgccggatacttggttgtcagcctcgcctttatgccgctgt
tttatatatggaccgcttttaatcaacaaaaaacaccggcgattgttaccctgctgttaggtgcggtgaatgtggt
gctggcggtcacgctgagcgggccggctcatctcggtctgtacggcataacattggcaggggccatttctcttatt
ttaaaaaatgccatctttacgccgctttacgtatcccgcattacaggctacaaaaagcacgtgttccttaaaggca
taatcgggcctctttcagccgctgtatttgcctggacggtctgtaaggcaattcagttcattgtgaagattgacag
ctggccgtcattgatagcgacgggagtgacagtcagcttttgctacgctgttttcgcttttatgctcgtttgtaca
aaagaggaaagacagctggtattaaaacggtttcgaaaaacgaaaggagctgtgaatctttgatcctgaaacgact
ttttgatctgacggccgccatttttttgttgtgctgtacaagtgtgatcattctgttcaccatcgccgttgtcagg
ctgaaaataggatcacctgtcttctttaagcaagtaaggccgggcctgcacggcaaaccgttcaccctatataagt
tccggacaatgacggatgaacgggacagtaaaggaaatctgctgcctgatgaagtccggctgacgaaaacgggcag
gctgatcagaaagctgagcattgatgagcttccgcagctcctgaatgtcctgaagggcgatctgagccttgtcggg
ccgcggccgcttttgatggactatctgcctctttatacagaaaaacaggcacggcgccatgaggtgaagccgggta
tcacaggctgggcgcaaatcaatggcagaaacgcgatttcctgggaaaagaaatttgaattagatgtttggtacgt
tgacaactggtcattttttctcgatttgaaaattttatgtttgacggtgcgaaaggtccttgtttcagaagggatt
cagcaaaccaatcatgtgaccgcggaacggtttaccggaagcggagatgtgtcctcatgaaaaatgtggccattgt
gggtgacggcggtcacggaaaggtgatcagagagctgataaacgcccgctcagatacgcgcttagccgcggtgctg
gatgataaattcaaaacgttcgaaggcggaaaagaatggtacacaggaccgccgaaagccgttactgaactgcgca
ggctcattcctgatgtgctgtttctgattgctgttgggaataacagtgtcagaaaacagctggcggagcgactggg
actggggaaagatgattttattacattgattcacccgtcagccatcgtcagcaagtcggctgtcattggggaaggg
acagtgattatggcgggcgcgatcattcaggcggatgcgcgcatcggcgcccactgcatcatcaatacgggtgcag
tggcagagcacgacaatcaaatcagcgattacgttcatctgtccccgcgtgccacgctgtcaggagcggtttccgt
tcaggaaggcgctcacgtcggaaccggtgcatccgtcataccgcagatcataatcggggcttggagcattgtcgga
gccggctccgcggtgatccgttccataccggacagggtaacggcggccggtgctccggcacgcatcatttcttcca
ttcaaacatcaaacaaaggatgattctatgcataaaaaaatctacttatctccccctcatatgagcggcagggagc
agcactatatttcagaagcctttcgctcaaactggattgcgccgcttgggccgctcgtgaattcatttgaagaaca
gctggcagaacgagtcggtgtaaaagcagcagctgcggtcggctcaggaacggcggcgattcatctcgcgctgcgt
ttgcttgaggtaaaagaaggtgacagcgtgttttgccagtccttcacatttgtagcaaccgccaatccgattctat
atgaaaaagcggtgcccgtctttattgattctgagcctgatacgtggaatatgtcgccgacagcccttgaaagagc
attggaggaagcgaaaagaaacggaacactgccaaaagcggtaattgccgtcaatctatatgggcagagcgcgaaa
atggatgaaatcgtaagcctgtgtgatgcatacggagttcctgtcattgaggacgcagccgaatctctcggcacag
tctataaagggaagcaaagcggaacattcgggcgcttcggcattttttcatttaacgggaacaaaatcatcaccac
atcagggggagggatgctcgtttcaaatgatgaagccgcaattgaaaaagcaagatttctcgcttcgcaggcacga
gagccggctgttcattatcagcacagtgaaattggacataattacaggctgagcaatatcttagccggcgtcggca
ttgcccagcttgaggtgctggatgagcgagtggagaaaagaaggaccattttcacgagatacaaaaatgcgctcgg
tcacttagacggcgtccgctttatgccagagtacgcagcaggcgtatccaatcgctggctcaccacgctcacactt
gataacgggctcagcccatatgacatagttcaacgtcttgctgaagaaaacattgaagcccgtccgctgtggaagc
cgctccatacccagccgctgttcgatccggctttattttattctcatgaagatacaggaagcgtatgcgaagattt
gttcaagcgaggaatctgtctcccatcggggtccaatatgacagaagatgagcaaggccgggtcattgaagtgcta
ctgcacttattccatactgtcgaggtgaagaaatggacagcaagcattcgatgatcagcctgaaacagaaactgtc
cgggctgctcgacgtcattccgaaacaatcagagattatatatgctgactatcctctatatgggaatgtaggggat
ttatttattatgaaaggaacagaagccttctttaaagaacacgggattcgggtcagaaaacgctggaatccagaca
atttcccaatcgggcgaaagcttgatccgaatctcatcatcgtctgccagggaggcggaaacttcggggatctgta
tccgtattatcaaggctttagagagaaaatcgtccaaacctatccgaaccacaaaattgtgatcctgccgcaatcg
atttattttcaaaacaaagacaacctcaagcggacggcagagatattttctaagcatgcgaaccttcacatcatga
caagggaaaaagcctcctatgctacggcacaggcctattttacaacaaatcacattcagcttctgcctgatatggc
tcatcagctgtttcccgtcattcccacgcagcagccgtccaatcaaaagctgagatttatccgaacagatcatgaa
gcaaaccaggcgcttcaggaacacgcagaagcggaaagctacgactggcgcacggtgctgtcagcttcagaccgcc
ggacgattgcttttctccaaacgctgaacgtcttgaataaaaaagcaggcaaccctttgcccattgcgtatatatg
ggaaaaatactcggattatatcgtccaaaaagcgattcggtttttcagccgttacgaatcggtggaaacatcaagg
ctgcacggccacatcctgtcttctcttcttcaaaaagaaaacacggtcattgataattcctacgggaaaaacgcca
attactttcatacctggatggaaggcgtgccaagcacccgtctcatccagcacgcctcaaagaaggaaaaccttcc
tgctcacatgtga 15741
<210> 2
<211> 1071
<212> DNA
<213>artificial synthesized
<220>
<223>nucleotide sequence of eps operon after expanding
<400> 2
caagacgttcagcgtttggagaaaagcaatcgtccggcggtctgaagctgacagcaccgtgcgccagtcgta
gctttccgcttctgcgtgttcctgaagcgcctggtttgcttcatgatctgttcggataaatctcagcttttgattg
gacggctgctgcgtgggaatgacgggaaacagctgatgagccatatcaggcagaagctgaatgtgatttgttgtaa
aataggcctgtgccgtagcataggaggctttttcccttgtcatgatgtgaaggttcgcatgcttagaaaatatctc
tgccgtccgcttgaggttgtctttgttttgaaaataaatcgattgcggcaggatcacaattttgtggttcggatag
gtttggacgattttctctctaaagccttgataatacggatacagatccccgaagtttccgcctccctggcagacga
tgatgagattcggatcaagctttcgcccgattgggaaattgtctggattccagcgttttctgacccgaatcccgtg
ttcttta cccctttctgttaatgattggattataaaagaaaacgttattatttaaaaattgcaaaataagccaat
aagttctctttagagaacaaaatcatgattttcctctaatttactgcacttcccttattattttaaattttataaa
gaacgaaaaattccttataatgaacgaaataacgacaggaatagaggagaatttcatattatgattggaagaatta
tccgtttgtaccgtaaaagaaaaggctattctattaatcagctggctgttgagtcaggcgtatccaaatcctattt
aagcaagattgaaagaggcgttcacacgaatccgtccgttcaatttttaaaaaaagtttctgccacactggaagtt
gaattaacagaattatttgacgcagaaacaatgatgtatgaaaaaatcagcggcggtgaagaagaatggcgcgtac
atttagtgcaagccgtacaagccgggatggaaaaggaagaattgttcacttttacgaacagactcaagaaagaaca
gcctgaaactgc 1071
<210> 3
<211> 3835
<212> DNA
<213>artificial synthesized
<220>
<223>plasmid pBTS-eps nucleotide sequence
<400> 3
ggtacccaagacgttcagcgtttggagaaaagcaatcgtccggcggtctgaagctgacagcaccgtgcgcca
gtcgtagctttccgcttctgcgtgttcctgaagcgcctggtttgcttcatgatctgttcggataaatctcagcttt
tgattggacggctgctgcgtgggaatgacgggaaacagctgatgagccatatcaggcagaagctgaatgtgatttg
ttgtaaaataggcctgtgccgtagcataggaggctttttcccttgtcatgatgtgaaggttcgcatgcttagaaaa
tatctctgccgtccgcttgaggttgtctttgttttgaaaataaatcgattgcggcaggatcacaattttgtggttc
ggataggtttggacgattttctctctaaagccttgataatacggatacagatccccgaagtttccgcctccctggc
agacgatgatgagattcggatcaagctttcgcccgattgggaaattgtctggattccagcgttttctgacccgaat
cccgtgttctttaggatcccccctttctgttaatgattggattataaaagaaaacgttattatttaaaaattgcaa
aataagccaataagttctctttagagaacaaaatcatgattttcctctaatttactgcacttcccttattatttta
aattttataaagaacgaaaaattccttataatgaacgaaataacgacaggaatagaggagaatttcatattatgat
tggaagaattatccgtttgtaccgtaaaagaaaaggctattctattaatcagctggctgttgagtcaggcgtatcc
aaatcctatttaagcaagattgaaagaggcgttcacacgaatccgtccgttcaatttttaaaaaaagtttctgcca
cactggaagttgaattaacagaattatttgacgcagaaacaatgatgtatgaaaaaatcagcggcggtgaagaaga
atggcgcgtacatttagtgcaagccgtacaagccgggatggaaaaggaagaattgttcacttttacgaacagactc
aagaaagaacagcctgaaactgcctcgagatccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggc
ctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtgtcat
tttatttcccccgtttcagcatcaagaacctttgcataacttgctctatatccacactgataattgccctcaaacc
ataatctaaaggcgctagagtttgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccgctcc
cctaaggcgaataaaagccattaaatcttttgtatttaccaaattatagtcatccactatatctaagagtaaattc
ttcaattctcttttttggctttcatcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaatatctt
ttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatgagtcaaatattcataagaacctttgat
ataatcaagtatctcaacatgagcaactgaactattccccaattttcgcttaatcttgttcctaacgctttctatt
gttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatagtgctttccatttcgtataacatcac
tactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggactgacagccatagatacgcccaaact
ctctaatttttccttccaatcattaggaattgagtcaggatataataaaaatccaaaatttctagctttagtattt
ttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcg
cttattttttttgttctgattcctttcttgcatattcttctatagctaacgccgcaaccgcagattttgaaaaacc
tttttgtttcgccatatctgttaattttttatcttgctcttttgtcagagaaatcataactctttttttcgattct
gaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgtatcactttgtaatcataaaaacaaca
ataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggcgattcgttggcgattgaaaaacccc
ttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggcactcggcacttaatggggggtcgtag
tacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaatttttttcaaaaattttccagcgct
accgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaatttcattccctcatactcccttgagc
ctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcatcggctttcataatctaacagacaac
atcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataacgcctgttttaacgattatgccgata
actaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgttttaataaaaaatcgcctagtgcttgga
ttctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactg
gatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccagtaaaatataatattttattttctcc
caatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatcgacc
ggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactttgcc
atctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtcttt
aaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccagat
cgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcgtatagggacaatccgatatgtcgat
ggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagggctttgttcatcttcatacccttcc
gagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttg
gaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccg
gctgtccgtcatttttaaatataggatttcattttctcccaccagcttatataccttagcaggagacattccttcc
gtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgatattctcattttagccatttattatt
tccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttc
cttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgttttcaaagttggcgtataacatagtatc
gacggagccgattttgaaaccacaattatgatagaattt 3835
Claims (8)
1. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity, it is characterised in that: knock out bacterial strain expression system to be rebuilt
Eps operon, hinder transposon mutagenesis, and then reduce fermentation broth viscosity, promote tunning separation.
2. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to claim 1, it is characterised in that: described
Expression system nucleotide sequence is as shown in SEQ ID No.:1.
3. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to claim 1, it is characterised in that: described
The eps operon for knocking out bacterial strain expression system to be rebuilt is that the fragment upstream eps-F of eps operon after amplification is inserted into pBTS
Between the KpnI and BamHI of plasmid, segments downstream eps-R is inserted between BamHI and XhoI, constructs new plasmid pBTS-
Eps, electrotransformation plasmid are obtained in transformation bacterial strain, culture medium culture and PCR identification.
4. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to claim 3, it is characterised in that: described
The nucleotide sequence of eps operon is as shown in SEQ ID No.:2 after amplification.
5. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to claim 3, it is characterised in that: described
Plasmid pBTS-eps nucleotide sequence is as shown in SEQ ID No.:3.
6. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to any one of claim 1-3, special
Sign is: the bacterial strain to be rebuilt be bacillus subtilis 168, Z12 bacterial strain, bacillus amyloliquefaciens, bacillus pumilus,
The bacillus strain such as clothing bacillus.
7. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to claim 3, it is characterised in that: described
Transformation bacillus is the gene for having knocked out synthesis γ-PGA --- the bacillus subtilis 168 of pgs, Z12 bacterial strain, solution starch
The bacillus such as bacillus, bacillus pumilus, bacillus licheniformis.
8. a kind of method for efficiently reducing fermentation of bacillus liquid viscosity according to claim 3, it is characterised in that: described
The primer sequence for expanding eps operon is as follows:
eps-F-F gaggtacc CAAGACGTTCAGCGTTTGGAGA；
eps-F-R gaggatcc TAAAGAACACGGGATTCGGGTCA；
eps-R-F gaggatcc CCCCTTTCTGTTAATGATTGGAT；
eps-R-R gactcgag GCAGTTTCAGGCTGTTCTTTCTT。
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| CN112239742A (en) * | 2020-08-24 | 2021-01-19 | 天津科技大学 | Bacillus licheniformis for producing alkaline protease and application thereof |
| CN112522173A (en) * | 2020-12-23 | 2021-03-19 | 天津科技大学 | Engineering bacterium for producing heterologous alkaline protease and construction method thereof |
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