CN102174558B - Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes - Google Patents

Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes Download PDF

Info

Publication number
CN102174558B
CN102174558B CN 201110071211 CN201110071211A CN102174558B CN 102174558 B CN102174558 B CN 102174558B CN 201110071211 CN201110071211 CN 201110071211 CN 201110071211 A CN201110071211 A CN 201110071211A CN 102174558 B CN102174558 B CN 102174558B
Authority
CN
China
Prior art keywords
abrb
gene
abrb5
carrier
abrb3
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.)
Expired - Fee Related
Application number
CN 201110071211
Other languages
Chinese (zh)
Other versions
CN102174558A (en
Inventor
陆兆新
曹国强
吕凤霞
别小妹
张充
钟蕾
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.)
Nanjing Agricultural University
Original Assignee
Nanjing Agricultural University
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 Nanjing Agricultural University filed Critical Nanjing Agricultural University
Priority to CN 201110071211 priority Critical patent/CN102174558B/en
Publication of CN102174558A publication Critical patent/CN102174558A/en
Application granted granted Critical
Publication of CN102174558B publication Critical patent/CN102174558B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a method for increasing the output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes, and belongs to the field of biotechnology. The method comprises the following steps of: firstly establishing a gene knockout vector pAbrB-Cm by taking bacillus subtilis ATCC9943 as a starting strain and taking a cloning vector pGEM-T of escherichia coli as a skeleton; and establishing a high-yield antibacterial peptide strain FMB29 through a knockout method of genes of a genome abrB of the bacillus subtilis ATCC9943 by applying a double exchange process based on a homologous recombination principle. Through high performance liquid chromatograph, the capacity of producing surfactins and fengycins of improved strains is greatly improved, and the existence of the surfactins and fengycins in fermentation products of improved strains FMB29 is identified through mass spectrum.

Description

Improve the method for antisepsis peptide of wilted hay bacilli output by knocking out the abrB gene
Technical field
The present invention relates to by knocking out AbrBGene improves the method for antisepsis peptide of wilted hay bacilli output, belongs to biological technical field.
Background technology
Subtilis ( Bacillus subtilis) be one of safe microorganisms of generally acknowledging at present; its antimicrobial metabolite is rich and varied; especially lipopeptide antibiotic substance; has a broad antifungal spectrum but also have the function of bio-surfactant not only; therefore not only can be used for carrying out biological control in agricultural, and in industry and environment protection, also have a wide range of applications.
Subtilis ( Bacillus subtilis) ATCC 9943 a kind ofly can secrete surfactin, the subtilis of the antibacterial peptide materials such as fengycin (Val é rie Leclere, Romain Marti, Max B é chet. The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties. Arch Microbiol. (2006) 186:475-483).Wherein, surfactin is as a kind of bio-surfactant; compare with chemical surfactant; it has the biodegradability of having; low toxicity, the advantage that structure is abundant, the raising that requires along with the environmental compatibility to industrial raw material; it is applied to environment protection, and oil production aspect potentiality will be increasing.But in the reality, it is widespread use in industry and environment protection not, traces it to its cause, and is because its production cost is huge, just becomes the key problem that reduces its production cost so improve its output.
At present, in the world wide, the method that improves its output mainly is the screening strain excellent, and optimization of fermentation conditions, and the method that seldom adopts Protocols in Molecular Biology that bacterial classification is improved improves the output that it produces antimicrobial substance.But day by day deep along with what the antibacterial peptide synthesis secretion was studied, begun gradually to adopt Protocols in Molecular Biology, come original strain is carried out improvement of genes by transforming in its synthesis secretion the method such as signal path, thereby improve himself synthesis secretion antibacterial peptide ability.
AbrBGene (Gene ID:937009), as a kind of modulin in the subtilis, play an important role in the various physiological metabolism processes of subtilis, correlative study shows, AbrB albumen has certain inhibition for antimicrobial substance synthetic.
Present method is at first take subtilis ATCC 9943 as starting strain, gene knockout carrier pAbrB-Cm take escherichia coli cloning carrier pGEM-T as framework construction, utilize the homologous recombination principle, knock out in subtilis ATCC 9943 genomes by the double exchange process AbrBThe method of gene has made up a high yield antibacterial peptide bacterial strain FMB 29.
Summary of the invention
Technical problem
The objective of the invention is to utilize Protocols in Molecular Biology, make up AbrBGene knockout carrier transforms by homologous recombination in subtilis ATCC 9943 genomes through electricity AbrBGene knockout, thus make up a kind of bacillus subtilis strain FMB 29 of high yield antibacterial peptide.
Technical scheme
1, by knocking out AbrBGene improves the method for antisepsis peptide of wilted hay bacilli output, it is characterized in that:
(1) AbrBThe structure of gene knockout carrier pAbrB-Cm
According to subtilis AbrBGene design primer AbrB5 '-F and AbrB5 '-R, the 5 ' end take subtilis ATCC 9943 genomic dnas as template pcr amplification part AbrBGene and upstream gene thereof obtain the 500bp gene fragment; According to subtilis AbrBGene design primer AbrB3 '-F and AbrB3 '-R, the 3 ' end take subtilis ATCC 9943 genomic dnas as template pcr amplification part AbrBGene and downstream gene thereof obtain the 500bp gene fragment; According to DNA design primer CM-F and the CM-R of pHCMC04, comprise the chloramphenicol resistance gene expression cassette of promotor and terminator take the pHCMC04 plasmid as the template pcr amplification, obtain the 1500bp gene fragment; Three genes that amplification obtains are cloned into respectively the pMD19-T carrier, the conversion intestinal bacteria ( Escherichia coli) DH5a, after empirical tests, the order-checking correctly, difference called after p AbrB5 '-T, p AbrB3 '-T, p CM-T saves backup under-20 ℃ of conditions;
Primer Sequence Restriction enzyme site
AbrB5’-F 5’ GAATTCACGCCCTGAAAAAGAATAATTAAAA 3’ EcoRI
AbrB5’-R 5’ TCTAGACTACACGTCCTAATTCATCAAC 3’ XbaI
AbrB3’-F 5’AAA TCTAGAGGCGGTAAATTGGTTCTTAGT 3’ XbaI
AbrB3’-R 5’ACA GCATGCTTGCTGTAACACGTGAACTCACT 3’ SphI
Cm-F 5’ TGA TCTAGAGGATTTTTCGCTACGCTCAAATC 3’ XbaI
Cm-R 5’ TGA TCTAGA ATGCAAGGAGATGGCGCCCAAC 3’ XbaI
The pcr amplification system is as follows:
10x Pfu PCR buffer 10μl
10 μ Μ upstream primers, 10 μ l
10 μ M downstream primers, 10 μ l
2.5mM dNTPs 8μl
The subtilis genomic dna
Perhaps pHCMC04 plasmid DNA 1 μ l
Pfu archaeal dna polymerase 1 μ l
ddH 2O 60μl
The PCR program is 94 ℃ of 2min; 34 circulations: 94 ℃ of 45s, 58 ℃ of 50s, 72 ℃ of 4min; 72 ℃ of 10min;
p AbrB5 '-T uses EcoRI/ XbaIObtain behind the double digestion AbrB5 'Fragment with the pGEM-T carrier of processing through identical double digestion, connects with the T4 dna ligase, makes up pGEM- AbrB5 'Carrier, enzyme save backup under-20 ℃ of conditions after cutting checking correctly;
p AbrB3 '-T uses XbaI/ SphIObtain behind the double digestion AbrB3 'Fragment is with the pGEM-that processes through identical double digestion AbrB5 'Carrier connects with the T4 dna ligase, makes up pGEM- AbrBCarrier, enzyme save backup under-20 ℃ of conditions after cutting checking correctly;
p CM-T uses XbaIEnzyme is cut rear acquisition CMFragment is cut the pGEM-of processing with the process same enzyme AbrBCarrier connects with the T4 dna ligase, makes up AbrBAfter gene knockout carrier pAbrB-Cm, enzyme cut checking correctly, the conversion intestinal bacteria ( Escherichia coli) DH5a, be used for next step conversion;
(2) structure of FMB-29 mutant strain
Prepare competent cell take subtilis ATCC 9943 as starting strain, adopt electric method for transformation, with what make up AbrBGene knockout carrier pAbrB-Cm is transformed into subtilis ATCC 9943, at genome AbrBGene locus generation double exchange, screening obtains chlorampenicol resistant bacterial strain, called after FMB 29 on the CM resistant panel; This bacterial strain AbrBGene is replaced by chloramphenicol resistance gene, becomes to have lacked AbrBThe mutant strain of gene is resulting bacterial strain.
Described knocking out AbrBGene bacillus subtilis strain FMB 29 can use in producing the Bacillus subtilus antibacterial peptide, produces the antisepsis peptide of wilted hay bacilli product.
Beneficial effect
Subtilis ( Bacillus subtilis) in the growth metabolism process, can produce different types of antimicrobial substance, wherein antibacterial peptide is occupied an leading position.Because anti-microbial activity, biological degradation and the toxicity of its wide spectrum are low, therefore more and more be subject to people's attention, have potential application prospect at aspects such as Plant diseases biological control, food antiseptic and fodder additivess.Have a strong impact on for food safety for present pesticide residue, chemical food and feed additive especially, exploring natural antimicrobial substance has important realistic meaning for crop production safety.
Carried out large quantity research for zymotechnique and the isolation technique of antibacterial peptide under the laboratory condition both at home and abroad.Although the optimization by zymotechnique can improve the antibacterial peptide production level, fermentation yield still is difficult to large-scale industrial production.Therefore from the molecular biology level, utilize genetic engineering technique that bacterial classification is carried out the means that orderly improvement will become a kind of new raising yield of antibacterial peptides.
The present invention successfully makes up a plant height and produces antibacterial peptide bacterial strain FMB 29 by Protocols in Molecular Biology.Compare with original strain ATCC 9943, the bacterial strain FMB 29 synthesis secretion antibacterial peptide abilities of process strain improvement improve greatly, and wherein surfactin output is brought up to 1091 mg/L from 777 mg/L, has improved 1.4 times; The output of fengycin is brought up to 2435mg/L from 646 mg/L, has improved 3.77 times.In addition, this invention has also disclosed the have resistance inhibitor action of AbrB albumen to antibiotic peptide material synthesis secretion, can by this method, utilize the pAbrB-Cm carrier that any subtilis is carried out improvement of genes, to obtain to improve accordingly bacterial classification, reach the purpose that improves yield of antibacterial peptides.
Description of drawings
Fig. 1. technical scheme;
Fig. 2. AbrBThe structure of gene knockout carrier;
Fig. 3. AbrBThe structure of genetically deficient bacterial strain;
Fig. 4. AbrBGene knock-out bacterial strain PCR checking;
Fig. 5. AbrBThe electrospray ionization mass spectrum of gene knock-out bacterial strain fermentation lipopeptid extract.
Embodiment
The present invention at first makes up AbrBGene knockout carrier pAbrB-Cm, through electricity transform with the pAbrB-Cm carrier be transformed into subtilis ( Bacillus subtilis) ATCC 9943(is available from The Global Bioresource Center), through double exchange homologous recombination process with in its genome AbrBGene replaces with chloramphenicol resistance gene, reaches to knock out AbrBThe purpose of gene, thus make up the bacillus subtilis strain FMB 29 that a plant height produces antibacterial peptide.By PCR method mutant strain is identified; Utilize high performance liquid chromatography (HPLC) to analyze the output of improved strain surfactin and fengycin, and by mass spectrum surfactin and fengycin in the thalline tunning are identified.Embodiment is as follows:
(1) AbrBThe structure of gene knockout carrier pAbrB-Cm
According to subtilis AbrBGene (Gene ID:937009) design primer AbrB5 '-F and AbrB5 '-R, with subtilis ( Bacillus subtilis) ATCC 9943 genomic dnas are template pcr amplification part 5 ' end AbrBGene and upstream gene thereof obtain the 500bp gene fragment; According to subtilis AbrBGene (Gene ID:937009) design primer AbrB3 '-F and AbrB3 '-R, the 3 ' end take subtilis ATCC 9943 genomic dnas as template pcr amplification part AbrBGene and downstream gene thereof obtain the 500bp gene fragment; DNA(http according to pHCMC04: //www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC04. htm) design primer CM-F and CM-R, the chloramphenicol resistance gene expression cassette that comprises promotor and terminator take pHCMC04 plasmid (available from Bacillus Genetic Stock Center) DNA as the template pcr amplification obtains the 1500bp gene fragment.Three genes that amplification obtains are cloned into respectively pMD19-T carrier (available from Takara company), the conversion intestinal bacteria ( Escherichia coli) DH5a (available from precious biotechnology company limited), after empirical tests, order-checking are correct, called after p respectively AbrB5 '-T, p AbrB3 '-T, p CM-T saves backup under-20 ℃ of conditions;
Title Sequence Restriction enzyme site
AbrB5’-F 5’ GAATTCACGCCCTGAAAAAGAATAATTAAAA 3’ EcoRI
AbrB5’-R 5’ TCTAGACTACACGTCCTAATTCATCAAC 3’ XbaI
AbrB3’-F 5’AAA TCTAGAGGCGGTAAATTGGTTCTTAGT 3’ XbaI
AbrB3’-R 5’ACA GCATGCTTGCTGTAACACGTGAACTCACT 3’ SphI
Cm-F 5’ TGA TCTAGAGGATTTTTCGCTACGCTCAAATC 3’ XbaI
Cm-R 5’ TGA TCTAGA ATGCAAGGAGATGGCGCCCAAC 3’ XbaI
The pcr amplification system is as follows:
10x Pfu PCR buffer 10μl
10 μ Μ upstream primers, 10 μ l
10 μ M downstream primers, 10 μ l
2.5mM dNTPs 8μl
The subtilis genomic dna
Perhaps pHCMC04 plasmid DNA 1 μ l
Pfu archaeal dna polymerase 1 μ l
ddH 2O 60μl
The PCR program is 94 ℃ of 2min; 34 * (94 ℃ of 45s; 58 ℃ of 50s; 72 ℃ of 4min; 72 ℃ of 10min.
p AbrB5 '-T uses EcoRI/ XbaIObtain behind the double digestion AbrB5 'Fragment with the pGEM-T carrier of processing through identical double digestion, connects with the T4 dna ligase, makes up pGEM- AbrB5 'Carrier, enzyme save backup under-20 ℃ of conditions after cutting checking correctly;
p AbrB3 '-T uses XbaI/ SphIObtain behind the double digestion AbrB3 'Fragment is with the pGEM-that processes through identical double digestion AbrB5 'Carrier connects with the T4 dna ligase, makes up pGEM- AbrBCarrier, enzyme save backup under-20 ℃ of conditions after cutting checking correctly;
p CM-T uses XbaIEnzyme is cut rear acquisition CMFragment is cut the pGEM-of processing with the process same enzyme AbrBCarrier connects with the T4 dna ligase, makes up AbrBAfter gene knockout carrier pAbrB-Cm, enzyme cut checking correctly, the conversion intestinal bacteria ( Escherichia coli) DH5a (available from precious biotechnology company limited), be used for next step conversion;
(2) structure of FMB 29 mutant strains
With subtilis ATCC 9943(available from The Global Bioresource Center) the preparation competent cell, adopt electric method for transformation, with what obtain AbrBGene knockout carrier pAbrB-Cm is transformed into subtilis ATCC 9943, at genome AbrBDouble exchange occurs in the site, and screening obtains chlorampenicol resistant bacterial strain, called after FMB 29 on the CM resistant panel.This bacterial strain AbrBGene is replaced by chloramphenicol resistance gene, becomes to have lacked AbrBThe mutant strain of gene, i.e. our bacterial strain of improveing.
(3) Molecular of proteolytic enzyme deactivated strain
Title Sequence
CmT-F 5’ GGATTTTTCGCTACGCTCAAATCCTTTAAA 3’
CmT-R 5’ ATGCAAGGAGATGGCGCCCAACAGTCCCCC 3’
Whether subtilis FMB 29 mutant bacterias are lacked AbrBGene carries out Molecular.Because in the bacterial strain AbrBGene is replaced by chloramphenicol resistance gene, so we are according to chloramphenicol resistance gene among the pHCMC04 (http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC04. htm) design primer CmT-F/CmT-R, carry out pcr amplification take 2 bacillus subtilis FMB, 29 genomic dnas that obtain through resistance screening as template respectively, amplify and expected results product (Fig. 4 of the same size, the 1st, 2 swimming lane).Through the molecular biology checking, we have obtained disappearance really AbrBThe improvement bacillus subtilis strain FMB 29 of gene.
(4)Improvement bacillus subtilis strain FMB 29 yield of antibacterial peptides HPLC analyze
Subtilis FMB 29 seed liquor are inoculated in the Landy fermention medium with 5% concentration, at 33 ° of C, cultivate 36 h under 180 rpm, get the antimicrobial substance fermented liquid.Fermented liquid 11000g is centrifugal, and 15 min remove thalline, with 6 M HCl supernatant liquor are adjusted to pH 2, static spending the night then, and the 11000g centrifugal collecting precipitation is neutralized to pH 7 with NaOH after adding methyl alcohol, obtains the antibacterial peptide crude extract.
Through the analysis of HPLC, ATCC9943 compares with original strain, and surfactin output is brought up to 1091 mg/L from 777 mg/L among the improved strain FMB29, has improved 1.4 times; The output of fengycin is brought up to 2435mg/L from 646 mg/L, has improved 3.77 times.
Figure 87856DEST_PATH_IMAGE001
(5)Improved strain subtilis FMB 29 fermentation lipopeptid mass spectroscopy
In order further to have surfactin and fengycin in checking improved strain subtilis FMB 29 tunnings, we carry out ESI-MS/CID with 36 hours lipopeptid extract of subtilis FMB 29 fermentations and analyze.As can be seen from Figure 5, [M+H] +Be the signal of m/z 1058.62, deduction Na +Identical with surfactin C15 molecular weight behind the molecular weight; [M+H]+be the signal of m/z 1526.40, deduction Na +Molecular weight with fengicin C17 behind the molecular weight is identical, really have surfactin and fengycin(Lijun Sun. Zhaoxin Lu. Xiaomei Bie. Isolation and characterization of a co-producer of fengycins and surfactins in proof improved strain subtilis FMB 29 tunnings, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi. World J Microbiol Biotechnol, 2006,22:1259-1266).
SEQUENCE LISTING
＜110〉Agricultural University Of Nanjing
＜120〉by knocking out the method for abrB gene raising Bacillus subtilus yield of antibacterial peptides
＜130〉specification sheets
<160> 8
<170> PatentIn version 3.1
<210> 1
<211> 31
<212> DNA
＜213〉synthetic
<220>
<221> AbrB5'-F
<222> (1)..(31)
<223>
<400> 1
gaattcacgc cctgaaaaag aataattaaa a 31
<210> 2
<211> 28
<212> DNA
＜213〉synthetic
<220>
<221> AbrB5'-R
<222> (1)..(28)
<223>
<400> 2
tctagactac acgtcctaat tcatcaac 28
<210> 3
<211> 30
<212> DNA
＜213〉synthetic
<220>
<221> AbrB3'-F
<222> (1)..(30)
<223>
<400> 3
aaatctagag gcggtaaatt ggttcttagt 30
<210> 4
<211> 32
<212> DNA
＜213〉synthetic
<220>
<221> AbrB3'-R
<222> (1)..(32)
<223>
<400> 4
acagcatgct tgctgtaaca cgtgaactca ct 32
<210> 5
<211> 32
<212> DNA
＜213〉synthetic
<220>
<221> Cm-F
<222> (1)..(32)
<223>
<400> 5
tgatctagag gatttttcgc tacgctcaaa tc 32
<210> 6
<211> 31
<212> DNA
＜213〉synthetic
<220>
<221> Cm-R
<222> (1)..(31)
<223>
<400> 6
tgatctagaa tgcaaggaga tggcgcccaa c 31
<210> 7
<211> 30
<212> DNA
＜213〉synthetic
<220>
<221> CmT-F
<222> (1)..(30)
<223>
<400> 7
ggatttttcg ctacgctcaa atcctttaaa 30
<210> 8
<211> 30
<212> DNA
＜213〉synthetic
<220>
<221> CmT-R
<222> (1)..(30)
<223>
<400> 8
atgcaaggag atggcgccca acagtccccc 30

Claims (3)

1. one kind by knocking out AbrBGene obtains the method for the bacillus subtilis strain of antibacterial peptide surfactin or fengicin output increased, it is characterized in that:
(1) AbrBThe structure of gene knockout carrier pAbrB-Cm
Design primer AbrB5 '-F and AbrB5 '-R:
AbrB5’-F： 5’ GAATTCACGCCCTGAAAAAGAATAATTAAAA 3’ EcoRI
AbrB5’-R：5’ TCTAGACTACACGTCCTAATTCATCAAC 3’ XbaI
With subtilis ( Bacillus subtilis) ATCC 9943 genomic dnas are template pcr amplification part 5 ' end AbrBGene and upstream gene thereof obtain the 500bp gene fragment;
Design primer AbrB3 '-F and AbrB3 '-R:
AbrB3’-F： 5’AAA TCTAGAGGCGGTAAATTGGTTCTTAGT 3’ XbaI
AbrB3’-R：5’ACA GCATGCTTGCTGTAACACGTGAACTCACT 3’ SphI
3 ' the end take subtilis ATCC 9943 genomic dnas as template pcr amplification part AbrBGene and downstream gene thereof obtain the 500bp gene fragment;
Design primer CM-F and CM-R:
Cm-F：5’ TGA TCTAGAGGATTTTTCGCTACGCTCAAATC 3’ XbaI
Cm-R：5’ TGA TCTAGA ATGCAAGGAGATGGCGCCCAAC 3’ XbaI
The chloramphenicol resistance gene expression cassette that comprises promotor and terminator take the pHCMC04 plasmid as the template pcr amplification obtains the 1500bp gene fragment; Three genes that amplification obtains are cloned into respectively the pMD19-T carrier, the conversion intestinal bacteria ( Escherichia coli) DH5a, after empirical tests, the order-checking correctly, difference called after p AbrB5 '-T, p AbrB3 '-T, p CM-T saves backup under-20 ℃ of conditions;
p AbrB5 '-T uses EcoRI/ XbaIObtain behind the double digestion AbrB5 'Fragment with the pGEM-T carrier of processing through identical double digestion, connects with the T4 dna ligase, makes up pGEM- AbrB5 'Carrier, enzyme save backup under-20 ℃ of conditions after cutting checking correctly;
p AbrB3 '-T uses XbaI/ SphIObtain behind the double digestion AbrB3 'Fragment is with the pGEM-that processes through identical double digestion AbrB5 'Carrier connects with the T4 dna ligase, makes up pGEM- AbrBCarrier, enzyme save backup under-20 ℃ of conditions after cutting checking correctly;
p CM-T uses XbaIEnzyme is cut rear acquisition CMFragment is cut the pGEM-of processing with the process same enzyme AbrBCarrier connects with the T4 dna ligase, makes up AbrBAfter gene knockout carrier pAbrB-Cm, enzyme cut checking correctly, transform escherichia coli DH5a, be used for next step conversion;
(2) structure of FMB29 mutant strain
With subtilis ATCC 9943 preparation competent cells, adopt electric method for transformation, with what obtain AbrBGene knockout carrier pAbrB-Cm is transformed into subtilis ATCC 9943, at genome AbrBDouble exchange occurs in the site, and screening obtains chlorampenicol resistant bacterial strain, called after FMB 29 on the CM resistant panel; This bacterial strain AbrBGene is replaced by chloramphenicol resistance gene, becomes to have lacked AbrBThe mutant strain of gene is resulting bacterial strain.
2. what the described method of claim 1 obtained knocks out AbrBGene bacterial strain FMB 29.
3. claim 2 is described knocks out AbrBThe application of gene bacterial strain FMB 29 in producing antisepsis peptide of wilted hay bacilli.
CN 201110071211 2011-03-24 2011-03-24 Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes Expired - Fee Related CN102174558B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201110071211 CN102174558B (en) 2011-03-24 2011-03-24 Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110071211 CN102174558B (en) 2011-03-24 2011-03-24 Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes

Publications (2)

Publication Number Publication Date
CN102174558A CN102174558A (en) 2011-09-07
CN102174558B true CN102174558B (en) 2013-04-10

Family

ID=44517801

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110071211 Expired - Fee Related CN102174558B (en) 2011-03-24 2011-03-24 Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes

Country Status (1)

Country Link
CN (1) CN102174558B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103060252B (en) * 2012-12-25 2015-04-15 江南大学 Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof
CN103045527A (en) * 2012-12-25 2013-04-17 江南大学 Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof
CN102978149B (en) * 2012-12-25 2014-01-29 江南大学 Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2133416A1 (en) * 2007-02-22 2009-12-16 Kao Corporation Recombinant microorganism

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2133416A1 (en) * 2007-02-22 2009-12-16 Kao Corporation Recombinant microorganism

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Guoqiang Cao et al..A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides.《Biotechnol Lett》.2011,第33卷第1050页第3段以及表2. *
Mark A. Strauch et al..Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression.《JOURNAL OF BACTERIOLOGY》.2007,第198卷(第21期),全文. *

Also Published As

Publication number Publication date
CN102174558A (en) 2011-09-07

Similar Documents

Publication Publication Date Title
Grujić et al. Spent mushroom compost as substrate for the production of industrially important hydrolytic enzymes by fungi Trichoderma spp. and Aspergillus niger in solid state fermentation
CN106459885B (en) Avermectin is expressed similar to the recombinant microorganism and application thereof of object
CN101402959B (en) Promotor replacement method for improving volume of production of bacillus subtilis surfactin
CN102399737A (en) Biological engineering bacterial strain for producing novel microorganism source fungicide and application of biological engineering bacterial strain
CN105087455A (en) Genetic engineering strain for producing phenazine-1-carboxylic acid and application of genetic engineering strain
Rhee et al. Complete genome sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1
CN101705265A (en) Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz
CN102174558B (en) Method for increasing output of antibacterial peptides of bacillus subtilis through knockout abrB genes
CN105368866A (en) Application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel
CN102220367B (en) Method for increasing yield of antibacterial peptide of bacillus subtilis through overexpression of yerP gene
CN102220366B (en) Method for increasing yield of Bacillus subtilis antimicrobial peptide by overexpression comA gene
CN102242142B (en) Method for increasing output of bacillus subtilis antibacterial peptide through knock-out of phrC gene
CN105802952B (en) A method of mushroom strain protection is carried out using uracil auxotrophy
JP2016531589A (en) Isolated Streptomyces bacteria
CN101475944B (en) Promoter replacement method for improving Bacillus amyloliquefaciens yield
CN103409341A (en) Application of relA gene in increase of moenomycin yield of streptomyces bambergiensis and strain
Khalid-Bin-Ferdaus et al. Commercial production of alpha amylase enzyme for potential use in the textile industries in Bangladesh
CN102807958A (en) Bacterial strain capable of secreting cellulase as well as cellulase extraction method and application thereof
CN105255936A (en) Method for establishing salinispora arenlocola CNS-205 gene genetic manipulation system
CN109022474A (en) The method for improving anti-fungus peptide bacillomycin D yield by being overexpressed spo0A gene
Naumov et al. Taxonomic genetics of methylotrophic yeast genus Komagataella: new biological species K. kurtzmanii
CN101348775B (en) Enterobacteria recombinant strain and use thereof
CN102162003B (en) Method for improving yield of avermectins and producing strain thereof
Buddiwong et al. Screening of thermotolerant yeast isolated from sugarcane plantations in Northeastern part of Thailand
CN103013873A (en) Strain generating heat-stable Beta-glucanase and application of strain

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C17 Cessation of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20130410

Termination date: 20140324