CN102174558A - Method for increasing output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes - Google Patents

Method for increasing output of antibacterial peptides of bacillus subtilis through knockout (i)abrB(/i) genes Download PDF

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Publication number
CN102174558A
CN102174558A CN 201110071211 CN201110071211A CN102174558A CN 102174558 A CN102174558 A CN 102174558A CN 201110071211 CN201110071211 CN 201110071211 CN 201110071211 A CN201110071211 A CN 201110071211A CN 102174558 A CN102174558 A CN 102174558A
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abrb
gene
carrier
abrb5
subtilis
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CN102174558B (en
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陆兆新
曹国强
吕凤霞
别小妹
张充
钟蕾
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Nanjing Agricultural University
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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

By knocking out AbrBGene improves the method for antisepsis peptide of wilted hay bacilli output
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 microorganism 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 on agricultural, be used to carry out biological control, 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 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 thei
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 it is deep day by day along with what the antibacterial peptide synthesis secretion was studied, begun to adopt Protocols in Molecular Biology gradually, come original strain is carried out improvement of genes by transforming in its synthesis secretion 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 a starting strain with subtilis ATCC 9943 at first, with escherichia coli cloning carrier pGEM-T is gene knockout carrier pAbrB-Cm of 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 are template pcr amplification part 5 ' end with subtilis ATCC 9943 genomic dnas AbrBGene and upstream gene thereof obtain the 500bp gene fragment; According to subtilis AbrBGene design primer AbrB3 '-F and AbrB3 '-R are template pcr amplification part 3 ' end with subtilis ATCC 9943 genomic dnas AbrBGene and downstream gene thereof obtain the 500bp gene fragment; According to DNA design primer CM-F and the CM-R of pHCMC04, be the chloramphenicol resistance gene expression cassette that the template pcr amplification comprises promotor and terminator with the pHCMC04 plasmid, obtain the 1500bp gene fragment; Three genes that amplification obtains are cloned into the pMD19-T carrier respectively, transformed into escherichia coli ( Escherichia coli) DH5a, after empirical tests, the order-checking correctly, difference called after p AbrB5 '-T, p AbrB3 '-T, p CM-T preserves standby under-20 ℃ of conditions;
Primer Sequence Restriction enzyme site
AbrB5’-F 5’ GAATTCACGCCCTGAAAAAGAATAATTAAAA3’ EcoRI
AbrB5’-R 5’ TCTAGACTACACGTCCTAATTCATCAAC3’ XbaI
AbrB3’-F 5’AAA TCTAGAGGCGGTAAATTGGTTCTTAGT3’ XbaI
AbrB3’-R 5’ACA GCATGCTTGCTGTAACACGTGAACTCACT3’ SphI
Cm-F 5’ TGA TCTAGAGGATTTTTCGCTACGCTCAAATC3’ XbaI
Cm-R 5’ TGA TCTAGAATGCAAGGAGATGGCGCCCAAC 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 handling through identical double digestion, connects with the T4 dna ligase, makes up pGEM- AbrB5 'Carrier, enzyme are cut the correct back of checking and are preserved standby under-20 ℃ of conditions;
p AbrB3 '-T uses XbaI/ SphIObtain behind the double digestion AbrB3 'Fragment is with the pGEM-that handles through identical double digestion AbrB5 'Carrier connects with the T4 dna ligase, makes up pGEM- AbrBCarrier, enzyme are cut the correct back of checking and are preserved standby under-20 ℃ of conditions;
p CM-T uses XbaIEnzyme is cut the back and is obtained 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, transformed into escherichia coli ( Escherichia coli) DH5a, be used for next step conversion;
(2) structure of FMB-29 mutant strain
With subtilis ATCC 9943 is that starting strain prepares competent cell, adopts 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 its broad-spectrum antibacterial activity, biological degradation and toxicity are low, therefore more and more be subject to people's attention, have the potential application prospect at aspects such as Plant diseases biological control, food antiseptic and fodder additivess.Have a strong impact on for food safety at present pesticide residue, chemical food and feed additive especially, production has important practical sense for agricultural product security to explore natural antimicrobial substance.
Carried out big quantity research at the zymotechnique and the isolation technique of antibacterial peptide under the laboratory condition both at home and abroad.Though 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 utilize the pAbrB-Cm carrier that any subtilis is carried out improvement of genes by this method, 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 surfactin and fengycin in the thalline tunning are identified by mass spectrum.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 is template pcr amplification part 3 ' end with subtilis ATCC 9943 genomic dnas 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, DNA is the chloramphenicol resistance gene expression cassette that the template pcr amplification comprises promotor and terminator with pHCMC04 plasmid (available from Bacillus Genetic Stock Center), obtains the 1500bp gene fragment.Three genes that amplification obtains are cloned into pMD19-T carrier (available from Takara company) respectively, transformed into escherichia coli ( 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 preserves standby under-20 ℃ of conditions;
Title Sequence Restriction enzyme site
AbrB5’-F 5’ GAATTCACGCCCTGAAAAAGAATAATTAAAA3’ EcoRI
AbrB5’-R 5’ TCTAGACTACACGTCCTAATTCATCAAC3’ XbaI
AbrB3’-F 5’AAA TCTAGAGGCGGTAAATTGGTTCTTAGT3’ XbaI
AbrB3’-R 5’ACA GCATGCTTGCTGTAACACGTGAACTCACT3’ SphI
Cm-F 5’ TGA TCTAGAGGATTTTTCGCTACGCTCAAATC3’ XbaI
Cm-R 5’ TGA TCTAGAATGCAAGGAGATGGCGCCCAAC 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 handling through identical double digestion, connects with the T4 dna ligase, makes up pGEM- AbrB5 'Carrier, enzyme are cut the correct back of checking and are preserved standby under-20 ℃ of conditions;
p AbrB3 '-T uses XbaI/ SphIObtain behind the double digestion AbrB3 'Fragment is with the pGEM-that handles through identical double digestion AbrB5 'Carrier connects with the T4 dna ligase, makes up pGEM- AbrBCarrier, enzyme are cut the correct back of checking and are preserved standby under-20 ℃ of conditions;
p CM-T uses XbaIEnzyme is cut the back and is obtained 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, transformed into escherichia coli ( 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 takes place 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, the i.e. bacterial strain that we improved.
(3) molecule of proteolytic enzyme deactivated strain checking
Title Sequence
CmT-F 5’GGATTTTTCGCTACGCTCAAATCCTTTAAA 3’
CmT-R 5’ATGCAAGGAGATGGCGCCCAACAGTCCCCC 3’
Whether subtilis FMB 29 mutant bacterias are lacked AbrBGene carries out the molecule checking.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, be that template is carried out pcr amplification with 2 bacillus subtilis FMB, 29 genomic dnas that obtain through resistance screening 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, and at 33 ° of C, 180 rpm cultivate 36 h down, 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 then spending the night, and the 11000g centrifugal collecting precipitation is neutralized to pH 7 with NaOH behind the 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 behind the molecular weight with surfactin C15 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 (4)

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
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; Design primer AbrB3 '-F and AbrB3 '-R is template pcr amplification part 3 ' end with subtilis ATCC 9943 genomic dnas AbrBGene and downstream gene thereof obtain the 500bp gene fragment; Design primer CM-F and CM-R are the chloramphenicol resistance gene expression cassette that the template pcr amplification comprises promotor and terminator with the pHCMC04 plasmid, obtain the 1500bp gene fragment; Three genes that amplification obtains are cloned into the pMD19-T carrier respectively, transformed into escherichia coli ( Escherichia coli) DH5a, after empirical tests, the order-checking correctly, difference called after p AbrB5 '-T, p AbrB3 '-T, p CM-T preserves standby under-20 ℃ of conditions;
p AbrB5 '-T uses EcoRI/ XbaIObtain behind the double digestion AbrB5 'Fragment with the pGEM-T carrier of handling through identical double digestion, connects with the T4 dna ligase, makes up pGEM- AbrB5 'Carrier, enzyme are cut the correct back of checking and are preserved standby under-20 ℃ of conditions;
p AbrB3 '-T uses XbaI/ SphIObtain behind the double digestion AbrB3 'Fragment is with the pGEM-that handles through identical double digestion AbrB5 'Carrier connects with the T4 dna ligase, makes up pGEM- AbrBCarrier, enzyme are cut the correct back of checking and are preserved standby under-20 ℃ of conditions;
p CM-T uses XbaIEnzyme is cut the back and is obtained 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, transformed into escherichia coli DH5a was used for next step conversion;
(2) structure of FMB 29 mutant strains
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 takes place 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.
4. claim 2 is described knocks out AbrBThe antisepsis peptide of wilted hay bacilli product that gene bacterial strain FMB 29 produces.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102978149A (en) * 2012-12-25 2013-03-20 江南大学 Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis
CN103045527A (en) * 2012-12-25 2013-04-17 江南大学 Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof
CN103060252A (en) * 2012-12-25 2013-04-24 江南大学 Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof

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
《Biotechnol Lett》 20110126 Guoqiang Cao et al. A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides 第1050页第3段以及表2 4 第33卷, *
《JOURNAL OF BACTERIOLOGY》 20071231 Mark A. Strauch et al. Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression 全文 1-4 第198卷, 第21期 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102978149A (en) * 2012-12-25 2013-03-20 江南大学 Recombination bacillus subtilis with high yield of acetylglucosamine, and application of recombination bacillus subtilis
CN103045527A (en) * 2012-12-25 2013-04-17 江南大学 Acetyl-glucosamine accumulating recombinant bacillus subtilis and application thereof
CN103060252A (en) * 2012-12-25 2013-04-24 江南大学 Bacillus subtilis engineering bacteria with high yield of acetylglucosamine 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
CN103060252B (en) * 2012-12-25 2015-04-15 江南大学 Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof

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