Comparison of P aprE , P amyE , and P P43 promoter strength for β-galactosidase and staphylokinase expression in Bacillus subtilis

Kim, June-Hyung; Hwang, Bum-Yeol; Roh, Jiwon; Lee, Jong-Ki; Wong, Sui‐Lam; Yun, Hyungdon; Lee, Sun‐Gu; Kim, Byung‐Gee

doi:10.1007/s12257-007-0102-0

Cited by 22 publications

(16 citation statements)

References 28 publications

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“…Due to the substantial importance of the promoter in gene transcription and expression, many studies have focused on promoter modification and substitution (Blazeck and Alper, , ; Darst, ; Ishihama, ). In B. subtilis , various constitutive and inducible promoters have been identified or developed: P43 is the constitutive, strong promoter of the (deoxy) cytidine deaminase gene (Kim et al, ) and widely used for the expression of amylase, β‐lactamase, keratinase and dehydrogenase, etc. (Li et al, ; Liu et al, ; Wang et al, ; Wu et al, ); P spac is an artificial promoter that contains the phage SPO‐1 core promoter and the E. coli lacI ‐ lacO operon (Lecointe et al, ; Yansura and Henner, ); additionally, other promoters, e.g., P amyE , P aprE , P cry3Aa , and P luxS have been reported (Kim et al, ; Liu et al, ; Yang et al, ), and B. licheniformis P luxS increased P43 intensity by eightfold (Yang et al, ).…”

Section: Discussionmentioning

confidence: 99%

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In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters

Song

et al. 2016

Biotech & Bioengineering

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Surfactin-family lipopeptides are green biosurfactants with substantial industrial potential. The major problem prohibiting surfactin use is the low titer of the wild producer, Bacillus subtilis. Using transcriptomic analysis, four strong promoters, PgroE, Pcdd, PrplK, and PsspE, were identified and cloned from the genome of B. subtilis THY-7, a novel surfactin producer that has been identified from soil with a 0.55 g/L surfactin titer. An optimal promoter, PgroE, was selected to replace the native THY-7 surfactin synthase (SrfA) promoter through single-cross homologous recombination; however, the resulting engineered strain containing the PgroE substitution did not synthesize surfactin. The sucrose-inducible promoters PsacB and PsacP were then substituted in place of PsrfA, and the resulting engineered strains produced 1.09 and 0.22 g/L surfactin, respectively. An artificial, sucrose-inducible Pg1 promoter was produced through fusion of the PgroE and PsacB ribonucleic antiterminator (RAT), and the engineered strain containing the Pg1-substitution produced a surfactin titer of 1.44 g/L. An artificial IPTG-inducible promoter, Pg2, was constructed from a PgroE-lacO fusion and then substituted for the chromosomal PsrfA locus, and the surfactin titer of the resulting THY-7/Pg2-srfA increased to 5.98 g/L. The driving capacity of Pg2 was further improved by the inclusion of two point mutations in the -35 and -10 regions to produce the novel promoter Pg3. Pg3 exhibited super-strong activity as measured by lacZ reporter gene overexpression (approximately 3000 U). The Pg3-substitution strain THY-7/Pg3-srfA produced up to 9.74 g/L surfactin in a 5 L fermentor. The maximum productivity was 0.30 g/L/h, and the greatest yield reached 0.14 g surfactin/g sucrose. Biotechnol. Bioeng. 2017;114: 832-842. © 2016 Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

“…β‐Galactosidase reporter activity in recombinant strains was measured as previously described (Kim et al, ; Yang et al, ). Culture samples were taken at various times and the cells were collected by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters

Song

et al. 2016

Biotech & Bioengineering

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“…In a study to compare the expression of beta-galactosidase and staphylokinase enzymes under the promoters of aprE, amyE, and p43, the results indicated the higher power of promoter p43 in expressing these two enzymes. The promoter has the ability to continuously and strongly express its under-control gene and is an overlapping promoter which is expressed in both logarithmic and inertia phases continuously (34). Secretory expression of MPH enzyme in the host strain WB800 has shown that mpd gene is expressed under the promoter in both logarithmic and inertia phases (35).…”

Section: Discussionmentioning

confidence: 99%

Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

Firouzjaei

Abolmaali

Khodi

et al. 2021

J Shahrekord Univ Med Sci

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Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.

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“…The common way is using a strong promoter, including inducible promoters P sacB , P spac [4], P xyl [18], and constitutive promoters, such as P lepA , P amyE , P43 [18]. Other ways, like incorporating a fused signal sequence to improving protein secretion or deleting various host proteases, were also reported as efficient methods [5]. A strong promoter is the essential element to achieve highlevel expression in B. subtilis genetic engineering.…”

Section: Discussionmentioning

confidence: 99%

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

Guo¹,

Tang²,

Wei³

et al. 2014

Journal of Microbiology and Biotechnology

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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.

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Comparison of P aprE , P amyE , and P P43 promoter strength for β-galactosidase and staphylokinase expression in Bacillus subtilis

Abstract: G Comparison of P aprE , P amyE , and P P43 Promoter Strength for β-Galactosidase and Staphylokinase Expression in Bacillus subtilis

Cited by 22 publications

References 28 publications

In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters

In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters

Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

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

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