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Schuller, A; Cserjan-Puschmann, M; Tauer, C; Jarmer, J; Wagenknecht, M; Reinisch, D; Grabherr, R; Striedner, G; .
(2020): Escherichia coli σ
Microb Cell Fact. 2020; 19(1):58 FullText FullText_BOKU

Background 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 sigma(70) 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. Results We showed that, in genome-integrated E. coli expression systems that used sigma(70) 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 systems regulated by a single lacO site and the lac repressor element, lacI(Q), on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.
Authors BOKU Wien:
Cserjan Monika
Grabherr Reingard
Schuller Artur
Striedner Gerald
Tauer Christopher
Find related publications in this database (using NML MeSH Indexing)
DNA-Directed RNA Polymerases/genetics
Escherichia coli/genetics*
Gene Expression Regulation, Bacterial*
Genome, Bacterial*
Green Fluorescent Proteins/genetics
Lac Operon/genetics
Lac Repressors/genetics*
Promoter Regions, Genetic*
Recombinant Proteins
Viral Proteins/genetics

Find related publications in this database (Keywords)
Recombinant protein expression
Escherichia coli
LacI autoregulation
Tunable expression
sigma(70) promoters
Genome-integrated expression systems

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