• Title/Summary/Keyword: Bacillus subtilis expression vector

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Expression of Bacillus macerans Cyclodextrin Glucanotransferase in Bacillus subtilis

  • Kim, Chang-Sup;Han, Nam-Soo;Kweon, Dae-Hyuk;Seo, Jin-Ho
    • Journal of Microbiology and Biotechnology
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    • v.9 no.2
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    • pp.230-233
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    • 1999
  • A plasmid vector was constructed for the expression and secretion of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in Bacillus subtilis. The vector, pUBACGT, was composed of the ribosome-binding sequence, signal sequence, and cgt gene from B. macerans under the control of amyR2, the promoter of amyE gene coding for $\alpha$-amylase from B. subtilis var. natto. Bacillus subtilis LKS88, a mutant strain lacking genes for an amylase and two proteases, was used as a host for the transformation of the plasmid vector. The transformants were selected on kanamycin-containing Luria-Bertani plates. The starch hydrolyzing activity was observed on the starch-containing plates by the iodine method and cyclodextrin-forming activity was detected in the culture medium. A SDS-PAGE analysis showed that most of the expressed CGTase in the recombinant B. subtilis was secreted into the medium at a high expression level.

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Cloning of Promoters from Alkali-tolerant Bacillus sp. (알카리 내성 Bacillus속 Promoter의 Cloning)

  • 유주현;구본탁;공인수;정용준;박영서
    • Microbiology and Biotechnology Letters
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    • v.16 no.2
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    • pp.126-130
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    • 1988
  • Promoters of an alkali-tolerant Bacillus sp. isolated from soil have been cloned in Bacillus subtilis using promoter probe vector pPL703. The CAT specific activity of a clone harboring the strongest promoter activity among these transformants was 8.01. This activity was 2.5 times higher than that of Bacillus subtilis harboring expression vector pPL708 and was increased after the end of the logarithmic growth phase. In the 2.8kb of inserted DNA fragment, BamHI and Sal I recognition sites were located.

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High-Level Expression and Secretion of Bacillus pumilus Lipase B26 in Bacillus subtilis Chungkookjang

  • Lee, Mi-Hwa;Song, Jae-Jun;Choi, Yoon-Ho;Hong, Seung-Pyo;Rha, Eu-Gene;Kim, Hyung-Kwoun;Lee, Seung-Goo;Poo, Har-Young;Lee, Sang-Chul;Seu, Young-Bae;Sung, Moon-Hee
    • Journal of Microbiology and Biotechnology
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    • v.13 no.6
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    • pp.892-896
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    • 2003
  • High-level expression of the lipase B26 gene from Bacillus pumilus was achieved using Bacillus subtilis Chungkookjang isolated from the Korean traditional fermented bean paste, Chungkookjang. For the secretory production of recombinant lipase B26 in a Bacillus host system, pLipB26 was constructed by ligating the lipase B26 gene into the recently designed Escherichia coli-Bacillus shuttle vector, pLipSM, and that was then transformed into B. subtilis Chungkookjang. Among the various vector, medium, and host combinations, B. subtilis Chungkookjang harboring the pLipB26 exhibited the highest lipase activity in PY medium, and B. subtilis Chungkookjang secreted two times more enzymes than B. subtilis DB 104 under the same condition. When B. subtilis Chungkookjang harboring the pLipB26 was cultured in a 5-1 jar-fermentor containing 21 of a PY medium, the maximum lipase activity (140 U/ml) and production yield (0.68 g/l) were obtained during the late exponential phase from a cell-free culture broth. Although B. subtilis Chungkookjang also secreted extracellular proteases at the late exponential phase, these results suggested the potential of B. subtilis Chungkookjang as a host for the secretory production of foreign proteins.

Construction of Shuttle Promoter-probe and Expression Vectors for Escherichia coli and Bacillus subtilis, and Expression of B. thuringiensis subsp. kurstaki HD-73 Crystal Protein Gene in the Two Species

  • Park, Seung-Hwan;Koo, Bon-Tag;Shin, Byung-Sik;Kim, Jeong-Il
    • Journal of Microbiology and Biotechnology
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    • v.1 no.1
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    • pp.37-44
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    • 1991
  • A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.

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Properties of Promoters Transferred to the Donor Strain, Alkali-tolerant Bacillus sp. YA-14. (공여 균주인 알카리 내성 Bacillus속에 도입된 Promoter 의 특성)

  • 유주현;구본탁;정용준
    • Microbiology and Biotechnology Letters
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    • v.17 no.3
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    • pp.188-192
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    • 1989
  • The promoters from akali-tolerant Bacillus sp. YA-14 chromosomal DMA cloned in B. subtilis using pPL703 were stably transferred to the donor strain. In alkali-tolerant Bacillus sp., the promoters revealed similiar properties with in B. subtilis but were preyed to be more efficient than in B. subtilis comparing with pPL708. Alkali-tolerant Bacillus sp. harboring the recombinant plasmid, p-l2Bl, was abnormally more inducible with chloramphenicol than B. subtilis haying the plasmid. Therefore the host-vector system using this recombinant plasmid and alkali-tolerant Bacillus sp. was expected to be more available.

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Isolation of a Promoter Element that is Functional in Bacillus subtilis for Heterologous Gene Expression

  • Maeng, Chang-Jae;Kim, Hyung-Kwoun;Park, Sun-Yang;Koo, Bon-Tag;Oh, Tae-Kwang;Lee, Jung-Kee
    • Journal of Microbiology and Biotechnology
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    • v.11 no.1
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    • pp.85-91
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    • 2001
  • To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.

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Construction of a Shuttle Vector for Protein Secretory Expression in Bacillus subtilis and the Application of the Mannanase Functional Heterologous Expression

  • Guo, Su;Tang, Jia-Jie;Wei, Dong-Zhi;Wei, Wei
    • Journal of Microbiology and Biotechnology
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    • v.24 no.4
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    • pp.431-439
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    • 2014
  • We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.

Overexpression and Purification of Bacillus subtilis Glutamyl-tRNA Synthetase in Escherichia coli (대장균에서 Bacillus subtilis glutamyl-tRNA synthetase의 과발현 및 정제)

  • Oh, Jong-Shin;Yoon, Jang-Ho;Hong, Kwang-Won
    • Applied Biological Chemistry
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    • v.45 no.4
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    • pp.190-194
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    • 2002
  • Expression of Bacillus subtilis glutamyl-tRNA synthetase (GluRS) in Escherichia coli is lethal for the host, probably because this enzyme misaminoacylates ${tRNA_l}^{Gln}$ with glutamate in vivo. In order to overexpress B. subtilis GluRS, encoded by the gltX gene, in E. coli, this gene was amplified from B. subtilis 168 chromosomal DNA using PCR method and the entire coding region was cloned into a pET11a expression vector so that it was expressed under the control or the T7 Promoter. The resulting recombinant pEBER plasmid was transformed into E. coli Novablue (DE3) bearing the T7 RNA polymerase gene for expression. After IPTG treatment, the overproduced enzyme was purified using ammonium sulfate fractionation, Source Q anion exchange chromatography, Superdex-200 gel filtration, and Mono Q anion exchange chromatography. The purified enzyme yielded 18-fold increase in specific activity over the crude cell extract and its molecular weight was approximately 55 kDa on SDS-PAGE.

Cloning and Expression of Inulin Fructotransferase Gene of Arthrobacter sp. A-6 in Escherichia coli and Bacillus subtilis

  • Kim, Hwa-Young;Kim, Chan-Wha;Choi, Yong-Jin
    • Journal of Microbiology and Biotechnology
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    • v.10 no.2
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    • pp.275-280
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    • 2000
  • The inulin fructotransferse (depolymerizing) (IFTase, EC 2.4.1.93) gene of Arthrobacter sp. A-6 was cloned and expressed in Escherichia coli and Bacillus subtilis. The IFTase gene consisted of an ORF of 1.311 nucleotides encoding a polypeptide of 436 amino acids containing a signal peptide of 31 amino acids in the N-terminus. The molecular mass of the IFTase based on the nucleotide sequence was calculated to be 46.116 Da. The recombinant E. coli $DH5{\alpha}$ cells expressing the Arthrobacter sp. A-6 IFTase gene produced most of the IFTase intracelularly. In contrast, the recombinant B. subtilis DB 104 carrying the IFTas gene on a B. subtilis-E. Coli expression vector secreted the IFTase into the culture fluid efficiently.

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Expression of a Bacillus subtilis Mannanase Gene in Corynebacterium lactofementum (Corynebacterium lactofermentum에서 Bacillus subtilis의 Mannanase 유전자 발현)

  • Yoon, Ki-Hong
    • Microbiology and Biotechnology Letters
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    • v.37 no.4
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    • pp.405-407
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    • 2009
  • A Bacillus subtilis mannanase gene was subcloned into an Escherichia coli- Corynebacterium lactofermentum shuttle vector pHE83, and the resultant plasmid pHE83M was transferred into an endogenous plasmid-free strain of C. lactofermentum. Mannanase produced by the recombinant C. lactofermentum (pHE83M) was secreted extracellulary at the level of 86%, and the remaining activity of mannanase was detected in the cell-free extract. The maximum mannanase productivity of the recombinant strain reached 8.1 unit/mL in the culture filtrate of LB medium. According to the zymogram of mannanase on SDS-PAGE, it was found that the mannanase produced by the recombinant C. lactofermentum could be maintained stably with a migration identical to the mannanase produced by the parental strain, B. subtilis WL-3.