Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

Microbial Cell Factories
Artur SchullerGerald Striedner

Abstract

The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. We showed that, in genome-integrated E. coli expression systems that used σ70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression ...Continue Reading

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Citations

Jan 25, 2021·Journal of Biotechnology·Jens KastenhoferOliver Spadiut
Feb 2, 2021·Microbial Cell Factories·Patrick StargardtJuergen Mairhofer

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Methods Mentioned

BETA
flow cytometry
PCR

Software Mentioned

ImageQuant TL
Kaluza

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