CN107779465A - A kind of method for reducing fermentation of bacillus liquid viscosity - Google Patents

A kind of method for reducing fermentation of bacillus liquid viscosity Download PDF

Info

Publication number
CN107779465A
CN107779465A CN201711034848.6A CN201711034848A CN107779465A CN 107779465 A CN107779465 A CN 107779465A CN 201711034848 A CN201711034848 A CN 201711034848A CN 107779465 A CN107779465 A CN 107779465A
Authority
CN
China
Prior art keywords
pgs
bacillus
fermentation
pbts
bacterial strain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201711034848.6A
Other languages
Chinese (zh)
Other versions
CN107779465B (en
Inventor
任钧
张韬
唐旭
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CHENGDU MYTECH BIOTECHNOLOGY Co.,Ltd.
Sichuan Agricultural University
Original Assignee
Chengdu Mei Yide Bioisystech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chengdu Mei Yide Bioisystech Co Ltd filed Critical Chengdu Mei Yide Bioisystech Co Ltd
Priority to CN201711034848.6A priority Critical patent/CN107779465B/en
Publication of CN107779465A publication Critical patent/CN107779465A/en
Application granted granted Critical
Publication of CN107779465B publication Critical patent/CN107779465B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes

Landscapes

  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention belongs to field of fermentation engineering, more particularly to a kind of method for reducing fermentation of bacillus liquid viscosity, γ PGA gene --- pgs is synthesized by directly knocking out bacillus subtilis, including pgs F are inserted between the KpnI and BamHI of pBTS plasmids, pgs R are inserted between BamHI and XhoI, build new plasmid pBTS pgs, conversion plasmid, which enters, treats knock-out bacterial strain, carry out first time restructuring, and carry out the steps such as second of restructuring, obtain the bacterial strain of the knockout pgs genes without resistance marker, hinder bacillus synthesis γ PGA, and then reduce the viscosity of zymotic fluid, promote thalli growth, improve Fermentation Substance Concentration.

Description

A kind of method for reducing fermentation of bacillus liquid viscosity
Technical field
The invention belongs to field of fermentation engineering, and in particular to a kind of method for reducing fermentation of bacillus liquid viscosity.
Background technology
In the growth course of some bacillus, Polyurethane-epoxy resin（γ-PGA）It is secreted to be formed from cell membrane The pod membrane structure of thalline, also may be dissolved in zymotic fluid.Zymotic fluid is distributed to and necessarily faced when gas, solid content and gas, solid particulate During dividing value, the mutation of gentle, the solid dispersed phase of continuous mobile phase just occurs, shows the change of zymotic fluid non-Newtonianism, shows Viscosity rises.
γ-PGA belong to high molecular polymer, and the degree of polymerization is about between 1,000-15,000.Due to γ-PGA Glutamic Acids Hydrophilic radical on residue is present, and can increase molecular volume by a large amount of water of Hydrogenbond, and then cause zymotic fluid to become abnormal It is sticky.Viscosity increase, ventilation can be influenceed first and is supported, increase stirring resistance, influence thalli growth；Also surface tension can be improved, Bubble collapse is influenceed, and then produces uncontrollable foam during the fermentation, causes fermentation failure or destination protein expression Amount declines.
Method typically by reducing γ-PGA secretions to medium optimization or ferment control, such as patent CN201410174064.3, it is published in specification and adds mass-volume concentration fermentation initial stage in liquid state fermentation culture medium and be 0.5-30g/L KCl, on the basis of HMW high yield weight gamma-polyglutamic acid is kept, viscosity is reduced to original 10- 80%, although these methods achieve some effects, but can only reduce γ-PGA secretory volume, can not fully erased γ- PGA, and repeatability is bad, also without versatility.
Patent CN200810117844.9, disclose in specification in having encapsulated citric acid category bacterial strain, pass through Terminator is integrated after ORF3 partial sequence after promoter, blocks Klebsiella pod membrane to be formed, reduces its fermentation broth viscosity, PDO product separation and Extraction difficulty is reduced, while reduces the pathogenic of thalline.But method can there is do not knock out in this patent Resistance marker, it is impossible to continue to transform bacterial strain or be transferred to plasmid technology problem, repeatability and versatility be not high.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of method for reducing fermentation of bacillus liquid viscosity, knock out Pgs gene is synthesized, hinders γ-PGA synthesis, and then reduces fermentation broth viscosity, there is very high repeatability and versatility.
The method for solving a kind of reduction fermentation of bacillus liquid viscosity in the present invention of above technical problem, its feature exist In：γ-PGA gene --- pgs is synthesized by directly knocking out bacillus subtilis, bacillus synthesis γ-PGA is hindered, enters And the viscosity of zymotic fluid is reduced, promote thalli growth, improve Fermentation Substance Concentration.
The method for reducing fermentation of bacillus liquid viscosity, comprises the following steps：
（1）The DNA of bacterial strain to be rebuilt is extracted, the fragment upstream pgs-F and segments downstream pgs-R of pgs genes are expanded by primer；
Expanded using Toyobo KOD-Plus- Neo enzymes, amplification condition program： 94℃,2min;(94 DEG C, 30s； 60 DEG C, 30s；68 DEG C, 1min) x25；72 DEG C, 2min.
（2）By digestion, pgs-F is inserted between the KpnI and BamHI of pBTS plasmids, pgs-R is inserted into BamHI Between XhoI, pgs genes are knocked out, build new plasmid pBTS-pgs；
(3) pBTS-pgs is transferred in bacillus to be rebuilt by electricity conversion, obtains positive transformants bacterial strain；
(4) picking（3）In positive transformants bacterium colony enter in fluid nutrient medium and cultivated, cultivation temperature is 45 DEG C, during culture Between be more than 24h；
(5) step is taken（4）Middle bacterium solution 100ul -500ul, it is applied on the LB flat boards of the kanamycins containing 30mg/L, cultivation temperature For 45 DEG C, it is incubated overnight；
（6）Picking step（5）In bacterium colony enter in fluid nutrient medium and cultivated, every 8-16h passage once, until screening To nonreactive bacterial strain；
（7）Take step（6）In bacterium solution be diluted coated plate, dilution 105-106Times, it is incubated overnight；
（8）Picking step（7）In bacterium colony carry out Resistance Identification, until obtain 5-10 strain nonreactive bacterial strains；
(9) nonreactive bacterial strain is inoculated into fluid nutrient medium, be incubated overnight, extract bacterial genomes, enter performing PCR identification；It is positive Bacterial strain continues sequencing identification, and sequencing identification result is that positive strain produces the Bacillus strain for knocking out pgs genes.It is described Step（1）Middle knockout pgs genes, while knock out resistance marker.
The step（1）With（3）In bacillus to be rebuilt be bacillus subtilis 168, Z12 bacterial strains, solution starch gemma The bacillus such as bacillus, bacillus pumilus or bacillus licheniformis.
The primer sequence is：
pgs-F-F gaggtaccAGCTGTTCCGATTTTATACTGGTC；
pgs-F-R gaggatccGTTTGTTGCCGTAGCCATCGTG；
pgs-R-F gaggatccCTAATCGACTAAGCCAAACTTCTC；
pgs-R-R gactcgagTAGCCAAAACAGCTCCTCCTTG。
The present invention directly knocks out synthesis γ-PGA gene, bacillus is had very high from synthesizing γ-PGA Repeatability and versatility.
The present invention synthesizes γ-PGA gene --- pgs by directly knocking out bacillus subtilis, including pgs-F is inserted To between the KpnI and BamHI of pBTS plasmids, pgs-R is inserted between BamHI and XhoI, builds new plasmid pBTS- Pgs, conversion plasmid, which enters, treats knock-out bacterial strain, carries out first time restructuring, and carries out the steps such as second of restructuring, is not had The strain step of the knockout pgs genes of resistance marker, bacillus synthesis γ-PGA are hindered, and then reduce the viscosity of zymotic fluid, Promote thalli growth, improve Fermentation Substance Concentration.Pgs genes have been knocked out in the inventive method, and without residual resistance marker base Cause, it can continue to knock out other genes, convenient use, versatility height later.
Embodiment
With reference to specific embodiment, the present invention is described in detail：
Embodiment 1
Embodiment 1
1st, EcoR I and Apa I inscribe cleavages pBAV1K-T5-GFP is passed through（http://www.addgene.org/ vector-database/）Plasmid, homologous recombination clone is carried out with the MCS fragments of synthesis, obtains plasmid pBAV1K.
In this experiment and follow-up test：
Restriction endonuclease use Thermo companies quick restriction endonuclease, fragment recovery using Chengdu Fu Ji biotech firms glue reclaim try Agent box（DE-02011）, MCS fragments synthesize by Jin Wei intelligence bio tech ltd, and homologous recombination uses the EsayGeno of Tiangeng Quick Casting Cloning Kit（VI201-02）, coli strain top10, prepare competence and use KCM methods, plasmid extraction Using the universal plasmid Mini Kit of good fortune border biology（DE-01001）.
2nd, primer is designed, amplification same sense mutation is deleted the pBAV1K fragments of Nde I restriction enzyme sites, 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 Limit company synthesizes.
Embodiment 2
1st, 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 line single bacterium colony of bacterial strain to be transformed is taken, is inoculated into about 3ml GM（LB+ 0.5M D-sorbites）In culture medium, 37 DEG C, 180rpm is incubated overnight.The next morning presses 1:100 are inoculated into 50ml GM cultures In base, 37 DEG C, 180rpm cultures, when bacterium solution grows into OD and reached between 0.85-0.95, bacterium solution is put into precooling on ice 10min；4 DEG C, 5000g, 5min, supernatant is removed in centrifugation, with the EM of isometric precooling（0.5M D-sorbite+0.5M mannitols+ 10% glycerine water solution）Thalline is resuspended, 4 DEG C again, 5000g, 5min, supernatant is removed in centrifugation, repeated washing 4 times altogether；Add about 1/ Thalline is resuspended in the EM of 40 volumes, ensures bacterial concentration between 1-1.3 × 1010cfu/ml.The above-mentioned bacterium solutions of 60ul are taken, add 1ul Plasmid to be transformed, plasmid concentration are more than 100ng/ul, and piping and druming mixes, and then add the 1mm electric shock cups of precooling.Electric conversion instrument （Eppendorf Eporator）Parameter setting, 2.1kV is actual to shock by electricity the time in 4.0-5.0ms, has been only possible to conversion bacterium colony Growth.After electric shock, add 1ml RM immediately（LB+0.5M D-sorbite+0.38M mannitols）, piping and druming mixing, it is sterile to be transferred to 5ml In centrifuge tube, 37 DEG C, 180rpm cultures 3h.Bacterium solution is centrifuged, diluted by a certain percentage after removing supernatant, takes 200ul to be applied to and contains Have on the LB flat boards of 30mg/L kanamycins, 37 DEG C are incubated overnight, while are coated with the bacterium solution without electricity conversion and do negative control.The Two day morning observed colony growth situation, if reformer plate has colony growth, negative control does not have, then shows to convert successfully.
Embodiment 3
1st, the pas upstream region of gene 558bp of primer amplification Z12 bacterial strains fragment pgs-F is designed, inserts pBTS KpnI and BamHI Between site, plasmid pBTS-pgs-F is obtained；Downstream 693bp fragment aprE-R is expanded, insertion plasmid pBTS-pgs-F's Between BamHI and XhoI, obtain knocking out the plasmid pBTS-pgs of pgs genes.
Expanded using Toyobo KOD-Plus- Neo enzymes, amplification condition program： 94℃,2min;(94 DEG C, 30s；60 DEG C, 30s；68 DEG C, 1min) x25；72 DEG C, 2min.
Primer sequence：
pgs-F-F gaggtaccAGCTGTTCCGATTTTATACTGGTC；
pgs-F-R gaggatccGTTTGTTGCCGTAGCCATCGTG；
pgs-R-F gaggatccCTAATCGACTAAGCCAAACTTCTC；
pgs-R-R gactcgagTAGCCAAAACAGCTCCTCCTTG。
2nd, Z12 bacterial strains are entered using the hypertonic conversion method conversion pBTS-pgs in embodiment 2（It is preserved in China Microbiological bacterium Kind preservation administration committee common micro-organisms center CGMCC, preserving number 12750）, obtain Z12-pBTS-pgs bacterial strains.
3rd, take bacterium Z12-pBTS-pgs to be inoculated into 3ml LB culture mediums, 45 DEG C, 180rpm, cultivate 24h；Separately meet bacterium Z12- PBTS is compareed.
4th, the bacterium solution 200ul in 3 is taken to be applied on the LB flat boards containing 30mg/L kanamycins, 45 DEG C are incubated overnight.If Z12-pBTS does not have colony growth, and Z12-pBTS-pgs has colony growth, then the bacterial strain obtained is completed to recombinate for the first time, is named as Z12-pBTS-pgs-45。
5th, connect in bacterium Z12-pBTS-pgs-45 to 3ml LB culture mediums, be incubated overnight, extract genome（Give birth on Chengdu good fortune border Thing Technology Co., Ltd., bacterial genomes DNA extraction kit, DE-05311）, PCR amplification pgs fragments verified（Chengdu Fu Ji Bioisystech Co., Ltd, 2 × fast PCR reaction premix system, DP-20041）.
6th, the Z12-pBTS-pgs-45 bacterial strains that the positive is verified as in 5 are taken, are inoculated into 3ml LB bases, 37 DEG C, 180rpm trainings Support, every 8-16h passages once, after continuous passage 8 times, take bacterium solution to dilute 106Times, it is coated on the LB flat boards of nonreactive, obtains Single bacterium colony.
7th, the single bacterium colony in 6 is taken, while is scoring on LB and LB flat boards containing 30mg/L kanamycins, screens nonreactive Bacterium colony is, it is necessary to obtain 5-10 strain nonreactive bacterium colonies.
8th, the nonreactive bacterium colony in 7 is taken, is inoculated into 3ml LB culture mediums, 37 DEG C, 180rpm is incubated overnight, and extracts bacterium base Because of group, pgs fragments are expanded by PCR and verify homologous recombination result（Wild type knocks out pgs genes）, complete second of weight Group.The fragment of the positive is taken, Song Jinwei intelligence bio tech ltd further carries out sequence verification, and the bacterial strain for obtaining the positive is Knock out bacterial strain --- the Z21 of pgs genes（△(pgs)）.
Embodiment 4：
1st, bacterium Z12 and Z21 are met into zymotic fluid, 37 DEG C, 200rpm cultures 48h.Fermentation liquor formulation is：Bean cake powder 30g/L, bran Skin 10g/L, the g/L of cornstarch 10, the g/L of dusty yeast 5, the g/L of ammonium sulfate 2, the g/L of sodium chloride 10, the g/L of dipotassium hydrogen phosphate 4, The g/L of potassium dihydrogen phosphate 2, the g/L of calcium chloride 0.4, the g/L of magnesium sulfate 0.05, the g/L of ferrous sulfate 0.01, prepared using running water Afterwards, 121 DEG C, 20mins sterilization treatments.
2nd, bacterium solution is collected, 12000g centrifugations 10mins removes the particle in thalline and zymotic fluid.
3rd, using NDJ-5S（Power occasion science and technology）Fermentation broth viscosity is determined, the wherein fermentation broth viscosity of Z12 bacterial strains is 1.52Pa The viscosity of s, Z21 bacterial strain fermentation liquor is only 98mPa s.It can be seen that after knocking out pgs genes, the viscosity of zymotic fluid can be significantly reduced.
Sequence table
SEQUENCE LISTING
<110>Chengdu Mei Yide Bioisystech Co., Ltd
<120>A kind of method for reducing fermentation of bacillus liquid viscosity
<160>4
<170>PatentIn version 3.3
<210> 1
<211> 32
<212> DNA
<213>It is artificial synthesized
<220>
<223>Pgs-F-F sequences
<400> 1
gaggtaccAGCTGTTCCGATTTTATACTGGTC 32
<210> 2
<211> 30
<212> DNA
<213>It is artificial synthesized
<220>
<223>Pgs-F-R sequences
<400> 2
gaggatccGTTTGTTGCCGTAGCCATCGTG 30
<210> 3
<211>
<212> DNA
<213>It is artificial synthesized
<220>
<223>Pgs-R-F sequences
<400> 3
gaggatccCTAATCGACTAAGCCAAACTTCTC 32
<210> 4
<211>
<212> DNA
<213>It is artificial synthesized
<220>
<223>Pgs-R-R sequences
<400> 4
gactcgagTAGCCAAAACAGCTCCTCCTTG 30

Claims (5)

  1. A kind of 1. method for reducing fermentation of bacillus liquid viscosity, it is characterised in that：Closed by directly knocking out bacillus subtilis Into γ-PGA gene --- pgs, bacillus synthesis γ-PGA are hindered, and then reduce the viscosity of zymotic fluid, promote thalline life It is long, improve Fermentation Substance Concentration.
  2. A kind of 2. method for reducing fermentation of bacillus liquid viscosity according to claim 1, it is characterised in that：Including following Step：
    （1）The DNA of bacterial strain to be rebuilt is extracted, the fragment upstream pgs-F and segments downstream pgs-R of pgs genes are expanded by primer；
    （2）By digestion, pgs-F is inserted between the KpnI and BamHI of pBTS plasmids, by pgs-R be inserted into BamHI and Between XhoI, pgs genes are knocked out, build new plasmid pBTS-pgs；
    (3) pBTS-pgs is transferred in bacterium to be rebuilt by electricity conversion, obtains positive transformants bacterial strain；
    (4) picking（3）In positive transformants bacterium colony enter in fluid nutrient medium and cultivated, cultivation temperature is 45 DEG C, during culture Between>24h；
    (5) step is taken（4）Middle bacterium solution 100ul -500ul, it is applied on the LB flat boards of the kanamycins containing 30mg/L, cultivation temperature For 45 DEG C, it is incubated overnight；
    （6）Picking step（5）In bacterium colony enter in fluid nutrient medium and cultivated, every 8-16h passage once, until screening To nonreactive bacterial strain；
    （7）Take step（6）In bacterium solution be diluted coated plate, dilution 105-106Times, it is incubated overnight；
    （8）Picking step（7）In bacterium colony carry out Resistance Identification, until obtain 5-10 strain nonreactive bacterial strains；
    (9) nonreactive bacterial strain is inoculated into fluid nutrient medium, be incubated overnight, extract bacterial genomes, enter performing PCR identification；It is positive Bacterial strain continues sequencing identification, and sequencing identification result is that positive strain produces the Bacillus strain for knocking out pgs genes.
  3. A kind of 3. method for reducing fermentation of bacillus liquid viscosity according to claim 2, it is characterised in that：The step （1）Middle knockout pgs genes, while knock out resistance marker.
  4. A kind of 4. method for reducing fermentation of bacillus liquid viscosity according to claim 2, it is characterised in that：The step （1）With（3）In bacillus to be rebuilt be bacillus subtilis 168, Z12 bacterial strains, bacillus amyloliquefaciens, bacillus pumilus Or the bacillus such as bacillus licheniformis.
  5. 5. a kind of method of reduction fermentation of bacillus liquid viscosity according to any one of claim 1-4, its feature exist In：The primer sequence is：
    pgs-F-F gaggtaccAGCTGTTCCGATTTTATACTGGTC；
    pgs-F-R gaggatccGTTTGTTGCCGTAGCCATCGTG；
    pgs-R-F gaggatccCTAATCGACTAAGCCAAACTTCTC；
    pgs-R-R gactcgagTAGCCAAAACAGCTCCTCCTTG。
CN201711034848.6A 2017-10-30 2017-10-30 Method for reducing viscosity of bacillus fermentation liquid Active CN107779465B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711034848.6A CN107779465B (en) 2017-10-30 2017-10-30 Method for reducing viscosity of bacillus fermentation liquid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711034848.6A CN107779465B (en) 2017-10-30 2017-10-30 Method for reducing viscosity of bacillus fermentation liquid

Publications (2)

Publication Number Publication Date
CN107779465A true CN107779465A (en) 2018-03-09
CN107779465B CN107779465B (en) 2021-02-26

Family

ID=61432435

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711034848.6A Active CN107779465B (en) 2017-10-30 2017-10-30 Method for reducing viscosity of bacillus fermentation liquid

Country Status (1)

Country Link
CN (1) CN107779465B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109337855A (en) * 2018-11-20 2019-02-15 成都美溢德生物技术有限公司 A kind of efficient method for reducing fermentation of bacillus liquid viscosity
CN112239742A (en) * 2020-08-24 2021-01-19 天津科技大学 Bacillus licheniformis for producing alkaline protease and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104087628A (en) * 2014-04-28 2014-10-08 广西大学 Method for reducing viscosity of gamma-polyglutamic acid fermentation liquid
CN106755046A (en) * 2016-11-30 2017-05-31 成都美溢德生物技术有限公司 A kind of method for transforming bacillus gene group

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104087628A (en) * 2014-04-28 2014-10-08 广西大学 Method for reducing viscosity of gamma-polyglutamic acid fermentation liquid
CN106755046A (en) * 2016-11-30 2017-05-31 成都美溢德生物技术有限公司 A kind of method for transforming bacillus gene group

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FENG J等: "Construction of a Bacillus amyloliquefaciens strain for high purity levan production", 《FEMS MICROBIOLOGY LETTERS》 *
LUO Z等: "Microbial Synthesis of Poly-γ-Glutamic Acid: Current Progress, Challenges, and Future Perspectives", 《BIOTECHNOL BIOFUELS》 *
王风青等: "新型食品添加剂γ-聚谷氨酸合成酶基因的克隆及表达", 《食品工业科技》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109337855A (en) * 2018-11-20 2019-02-15 成都美溢德生物技术有限公司 A kind of efficient method for reducing fermentation of bacillus liquid viscosity
CN112239742A (en) * 2020-08-24 2021-01-19 天津科技大学 Bacillus licheniformis for producing alkaline protease and application thereof
CN112239742B (en) * 2020-08-24 2022-10-18 天津科技大学 Bacillus licheniformis for producing alkaline protease and application thereof

Also Published As

Publication number Publication date
CN107779465B (en) 2021-02-26

Similar Documents

Publication Publication Date Title
Zhou et al. Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing
WO2018129980A1 (en) Method for efficiently expressing pullulanase in bacillus subtilis and recombinant bacillus subtilis
CN112522173B (en) Engineering bacterium for producing heterologous alkaline protease and construction method thereof
CN108753636A (en) A kind of yeast and construction method producing tyrosol and hydroxytyrosol
US11447760B2 (en) Special enzyme for galactooligosaccharide production as well as preparation and application thereof
CN110055204B (en) Method for improving fermentation enzyme production of bacillus licheniformis by knocking out spo II Q and pcf genes and application
CN107142225A (en) A kind of pichia yeast recombinant bacterium of overexpression Streptomyces sp. FA1 sources zytase
CN113151270A (en) Promoter for efficiently expressing alkaline protease and application thereof
CN107881140B (en) Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof
CN107779465A (en) A kind of method for reducing fermentation of bacillus liquid viscosity
CN110904174B (en) Application of bacillus licheniformis with deletion of leucine dehydrogenase gene in production of heterologous protein
EP4273250A1 (en) Recombinant microorganism expressing fucosyltransferase, and method of producing 2&#39;-fucosyllactose using same
CN109234299B (en) Method for expressing and preparing lactobiose phosphorylase
CN109295087B (en) Method for expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase
CN113913357A (en) Chassis strain for producing alkaline protease and construction method and application thereof
CN106434730A (en) Nonreactive expression system based on bacillus subtilis and construction method
CN106755046B (en) A method of transformation bacillus gene group
CN114107146A (en) Construction method and application of resistance-marker-free auxotrophic bacillus subtilis
CN114058606B (en) Application of bacillus licheniformis with xpt gene deleted in heterologous protein production
CN110878293B (en) Application of bacillus licheniformis with deletion of yceD gene in production of heterologous protein
CN112175981B (en) Method for site-directed gene knockout of vibrio harveyi based on stimulation of absolute ethyl alcohol or sodium dodecyl sulfonate
CN109593696B (en) Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof
CN113881618A (en) Recombinant bacillus subtilis for secreting lactocasein, and construction method and application thereof
CN114507684B (en) Method for inhibiting target gene expression in Mediterranean salt-rich bacteria
CN115960869B (en) Construction and application of xylanase and secretory expression strain thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Ren Jun

Inventor after: Zhou Xiaoqiu

Inventor after: Zhang Tao

Inventor after: Tang Xu

Inventor before: Ren Jun

Inventor before: Zhang Tao

Inventor before: Tang Xu

CB03 Change of inventor or designer information
TA01 Transfer of patent application right

Effective date of registration: 20200325

Address after: Zhen Hong Yang Cun 610000 Sichuan city of Chengdu province Tianfu Ji Tian Group wusheng Road 1

Applicant after: CHENGDU MYTECH BIOTECHNOLOGY Co.,Ltd.

Applicant after: SICHUAN AGRICULTURAL University

Address before: 610222 Sichuan city of Chengdu province Shuangliu County jitianzhen Wu Sheng Industrial Park Road No. 1

Applicant before: CHENGDU MYTECH BIOTECHNOLOGY Co.,Ltd.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant