Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints

Marschall, Lukas; Sagmeister, Patrick; Herwig, Christoph

doi:10.1007/s00253-016-8045-z

Cited by 49 publications

(32 citation statements)

References 113 publications

(208 reference statements)

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“…The regulation of transcription in E. coli has recently received considerable attention, because it is the first step in the process of recombinant protein production [35][36][37][38]. Transcription control of the GOI allows a cell to divide up its resources between cellular and recombinant proteins in a physiologically balanced manner.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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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 σ 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 σ 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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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“…Note that, given the characteristics of some of these bacterial species, they could potentially fulfil more than one application. synthetic promoters, libraries of ribosome binding sites and other regulatory elements, plasmidial origins of replication, resistance markers, affinity tags for protein purification and efficient transcriptional terminators (Terpe, 2006;Rosano and Ceccarelli, 2014;Dvo r ak et al, 2015;Marschall et al, 2017;Segall-Shapiro et al, 2018). Following the very spirit of synthetic biology, some of these tools have adopted specific standards and protocols followed suit, such as the Registry of Standard Biological Parts (Peccoud et al, 2008) or the BioBricks repository (Røkke et al, 2014).…”

Section: Escherichia Colimentioning

confidence: 99%

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

Calero

Nikel

2018

Microbial Biotechnology

243

184

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The last few years have witnessed an unprecedented increase in the number of novel bacterial species that hold potential to be used for metabolic engineering. Historically, however, only a handful of bacteria have attained the acceptance and widespread use that are needed to fulfil the needs of industrial bioproduction - and only for the synthesis of very few, structurally simple compounds. One of the reasons for this unfortunate circumstance has been the dearth of tools for targeted genome engineering of bacterial chassis, and, nowadays, synthetic biology is significantly helping to bridge such knowledge gap. Against this background, in this review, we discuss the state of the art in the rational design and construction of robust bacterial chassis for metabolic engineering, presenting key examples of bacterial species that have secured a place in industrial bioproduction. The emergence of novel bacterial chassis is also considered at the light of the unique properties of their physiology and metabolism, and the practical applications in which they are expected to outperform other microbial platforms. Emerging opportunities, essential strategies to enable successful development of industrial phenotypes, and major challenges in the field of bacterial chassis development are also discussed, outlining the solutions that contemporary synthetic biology-guided metabolic engineering offers to tackle these issues.

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“…The use of different promoters in recombinant protein expression systems, in general, has been reviewed extensively [96][97][98][99]. The promoters used in heterologous expression systems vary significantly in their nature, activity, location, and mode of action.…”

Section: Promoters Signal Peptides and Targeted Expressionmentioning

confidence: 99%

Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update

Satheeshkumar

2020

Mol Biotechnol

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Single chain variable fragments (scFvs) are generated by joining together the variable heavy and light chain of a monoclonal antibody (mAb) via a peptide linker. They offer some advantages over the parental mAb such as low molecular weight, heterologous production, multimeric form, and multivalency. The scFvs were produced against more than 50 antigens till date using 10 different plant species as the expression system. There were considerable improvements in the expression and purification strategies of scFv in the last 24 years. With the growing demand of scFv in therapeutic and diagnostic fields, its biosynthesis needs to be increased. The easiness in development, maintenance, and multiplication of transgenic plants make them an attractive expression platform for scFv production. The review intends to provide comprehensive information about the use of plant expression system to produce scFv. The developments, advantages, pitfalls, and possible prospects of improvement for the exploitation of plants in the industrial level are discussed.

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Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints

Cited by 49 publications

References 113 publications

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

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

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update

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