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Cited by 94Bacterial Genetics and BiotechnologyMicrobial Metabolic Engineering and BioproductionFungal and yeast genetics research

Novel, Versatile, and Tightly Regulated Expression System for E scherichia coli Strains

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Choi, Young J., Morel, Lyne, Le François, Teffanie, Bourque, Denis, Bourget, Lucie, Groleau, Denis, Massie, Bernard, Míguez, Carlos B.·2010·Applied and Environmental Microbiology
DOI 10.1128/aem.00413-10PMC PMC2916507
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Abstract

A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p -isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T 5 phage promoter combined with the Pseudomonas -based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-β- d -thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTG-induced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.

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  • In the presence of cumate, the pNEW vector increases production yield of recombinant proteins by two to three-fold compared to pET-based IPTG-inducible systems.

    “In the presence of cumate, the pNEW vector is able to increase the production yield of recombinant proteins by two to three-fold compared to pET-based IPTG-inducible systems.”

    From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [72]

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Authors
Choi, Y. J., Morel, L., Le François, T., Bourque, D., Bourget, L., Groleau, D., Massie, B., & Míguez, C. B.
Affiliations
  • McGill University (CA)
  • Microbial and Enzymatic Technology Group
  • Biotechnology Research Institute (CA)
  • Genomics and Gene Therapy Vectors Group, Bioprocess Centre, Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada
Corresponding author
Míguez, Carlos B.
Venue
Applied and Environmental Microbiology
Publisher
American Society for Microbiology
Published
2010-08
Volume
76
Issue
15
Pages
5058-5066
ISSN
  • 0099-2240 (ISSN-L)
  • 1098-5336
DOI
10.1128/aem.00413-10
PMC
PMC2916507
OpenAlex
W2134174762
Cited by
94
References
4
Citations by year
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FWCI
1.62
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Applied and Environmental Microbiology (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • NPARC (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords
transcriptional regulation, inducible expression system, recombinant protein expression, fed-batch fermentation, Escherichia coli
MeSH terms
Benzoates, Cloning, Molecular, Escherichia coli, Genetic Engineering, Genetic Vectors, Pseudomonas, Recombinant Proteins, Transcriptional Activation, Gene Expression, Gene Expression Regulation, Bacterial, Pseudomonas putida

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