T7 RNA polymerase drives transcription of a reporter gene from T7 promoter, but engenders post-transcriptional silencing of expression

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Results Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At first, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Conclusion Inducer and lacI/lacO regulators on chromosome and plasmid have been investigated in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory in the future.

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CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

10.21203/rs.3.rs-339217/v1 ◽

2021 ◽

Author(s):

Changchuan Ye ◽

Xi Chen ◽

Mengjie Yang ◽

Xiangfang Zeng ◽

Shiyan Qiao

Keyword(s):

Rna Polymerase ◽

Mutant Strain ◽

Aminolevulinic Acid ◽

Expression System ◽

T7 Rna Polymerase ◽

Bacterial Strains ◽

T7 Promoter ◽

E Coli ◽

T7 Expression System ◽

High Level

Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

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A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator

Gene ◽

10.1016/0378-1119(94)90355-7 ◽

1994 ◽

Vol 142 (1) ◽

pp. 61-66 ◽

Cited By ~ 5

Author(s):

Marina I. Lebedeva ◽

Ekaterina V. Rogozhkina ◽

Nikolay A. Tsyba ◽

Sergey V. Mashko

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Recombinant Plasmid ◽

Expression System ◽

T7 Rna Polymerase ◽

T7 Promoter ◽

Lac Operator

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Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences

Biochemistry ◽

10.1021/bi00086a016 ◽

1993 ◽

Vol 32 (35) ◽

pp. 9115-9124 ◽

Cited By ~ 7

Author(s):

Richard A. Ikeda ◽

Lisa L. Chang ◽

G. Sakuntala Warshamana

Keyword(s):

Rna Polymerase ◽

T7 Rna Polymerase ◽

T7 Promoter

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CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

Journal of Biological Engineering ◽

10.1186/s13036-021-00270-9 ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Changchuan Ye ◽

Xi Chen ◽

Mengjie Yang ◽

Xiangfang Zeng ◽

Shiyan Qiao

Keyword(s):

Rna Polymerase ◽

Mutant Strain ◽

Aminolevulinic Acid ◽

Expression System ◽

T7 Rna Polymerase ◽

Bacterial Strains ◽

T7 Promoter ◽

E Coli ◽

T7 Expression System ◽

High Level

AbstractT7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

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Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.353-360.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 353-360

Author(s):

B M Benton ◽

W K Eng ◽

J J Dunn ◽

F W Studier ◽

R Sternglanz ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Target Genes ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Yeast Cells ◽

T7 Rna Polymerase ◽

Bacteriophage T7 ◽

T7 Promoter

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

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Development of an inducible T7 expression system in Bacillus subtilis ATCC 6051a for production of α-L-arabinofuranosidase

10.21203/rs.2.23253/v1 ◽

2020 ◽

Author(s):

Minghua Ji ◽

Sijie Li ◽

Yunhui Liu ◽

Haiying Wu ◽

Qiao Chen ◽

...

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Target Genes ◽

Genetic Manipulation ◽

Fermentation Broth ◽

Expression System ◽

T7 Rna Polymerase ◽

T7 Promoter ◽

Subtilis Atcc ◽

Bacillus Subtilis Atcc

Abstract Background: Xylan is the second most abundant polysaccharide biomass on the earth, the polymers have a backbone of β-1,4-linked xylose residues with various side-chain substitutions, such as arabinose, acetic acid, glucuronic acid, and other esterified groups, thus, the removal of arabinose side groups by α-L-arabinofuranosidases is helpful in various industrial processes involving xylan treatment. Bacillus subtilis ATCC 6051a is known for its excellent capacity of secretory production of recombinant peptides, however, poor experience in genetic manipulation and lack of universal expression elements impede this strain for wider application.Results: Xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, and the transformation efficiency of the engineered strain B. subtilis 164S was increased by more than 1000 folds. B. subtilis 164S was further modified to generate B. subtilis 164T7P which incorporates a D-xylose inducible T7 RNA polymerase. The recombinant GFP expressed by 164T7P is more than thirteen times that achieved by P43 promoter, representing the most efficient expression system that had been ever reported in B. subtilis . Subsequently, abfA1 , encoding a glycoside hydrolase (GH) family 51 enzyme was cloned and overexpressed in 164T7P. The activity of recombinant α-L-arabinofuranosidase (AbfA1) reached 90.6±2.0 U mL -1 in the fermentation broth. Using p NPA as a substrate, kinetic parameters of the crude enzyme were Km of 1.4±0.1 mM and kcat of 139.4 s −1 . The optimum temperature and pH of the recombinant AbfA1 towards p NPA were observed to be 45 °C and pH 6.5, respectively.Conclusion: With enhanced cellular competence and introduction of T7 RNA polymerase, B. subtilis ATCC 6051a was engineered as a versatile cell tool for recombinant production of heterologous peptides employing T7 promoter. The novel expression kit demonstrated a very low level of leaky expression of target genes. The efficiency and applicability of the system were demonstrated by high-level production of a bacterial type α-L-arabinofuranosidase.

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