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Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory
I-Son Ng
National Cheng Kung University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1659-5814
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
E. coli W3110, T7RNA polymerase, T7 promoter, recombinant protein, cadaverine
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CURRENT STATUS: Under Review
Version 1
Posted 20 May, 2020
No community comments so far
Editor assigned
On 16 May, 2020
Reviewers invited
Invitations sent on 16 May, 2020
Submission checks complete
On 15 May, 2020
Editor invited
On 15 May, 2020
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On 12 May, 2020
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Subject Areas
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This preprint is under consideration at Microbial Cell Factories. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory
I-Son Ng
National Cheng Kung University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1659-5814
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 20 May, 2020
No community comments so far
Editor assigned
On 16 May, 2020
Reviewers invited
Invitations sent on 16 May, 2020
Submission checks complete
On 15 May, 2020
Editor invited
On 15 May, 2020
First submitted
On 12 May, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Applied & Industrial Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6