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Review
. 2016 Jul;100(13):5719-28.
doi: 10.1007/s00253-016-7550-4. Epub 2016 May 12.

Tunable recombinant protein expression in E. coli: enabler for continuous processing?

Lukas Marschall  1 , Patrick Sagmeister  2 , Christoph Herwig  3   4
Affiliations
Review

Tunable recombinant protein expression in E. coli: enabler for continuous processing?

Lukas Marschall et al. Appl Microbiol Biotechnol. 2016 Jul.

Abstract

Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in Escherichia coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death, and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell's metabolic capacities to a maximum. Within this article, we review genetic and process technological aspects of tunable expression systems in E. coli, providing a roadmap for the industrial exploitation of the reviewed technologies. We attempt to differentiate the term "expression tuning" from its inflationary use by providing a concise definition and highlight interesting fields of application for this versatile new technology. Dependent on the type of inducer (metabolizable or non-metabolizable), different process strategies are required in order to achieve tuning. To fully profit from the benefits of tunable systems, an independent control of growth rate and expression rate is indispensable. Being able to tackle problems such as long-term culture stability and constant product quality expression tuning is a promising enabler for continuous processing in biopharmaceutical production.

Keywords: All-or-none induction; Continuous processing; E. coli; Transcription; Tunable.

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Figures

Fig. 1
Fig. 1
By reducing the protein expression level, the cells can be maintained in a productive state for a longer time, which results in higher end product titers (q p specific cellular productivity)
Fig. 2
Fig. 2
In order to maintain a constant expression level under subsaturating inducer concentrations, a constant inducer concentration within the cell is mandatory. Therefore, all routes of transport into (active import, diffusion) and out of the cell (metabolism, inactivation, export) have to be considered. As not all of these rates are accessible, a constant inducer concentration within the cell can only be approximated. When using non-metabolizable inducers, this condition can be approximated by adjusting the extracellular inducer concentration to the biomass concentration. In the case of metabolizable inducers, a mixed-feeding strategy, where the uptake of the main substrate and inducing substrate can be controlled independently, needs to be applied in order to retain the additional degree of freedom of expression tuning
Fig. 3
Fig. 3
For expression tuning, several induction strategies have been used. One-point addition of non-metabolizable inducer (a) is commonly applied, but provides no novel degree of freedom for process control. The inducer concentration is only added at the timepoint of induction and not adjusted throughout the process. It is therefore submitted to change due to catabolism, dilution by growth, and inactivation. By inducer titration, the inducer concentration is continuously added to the culture and concentration changes can therefore be accounted for. When using a metabolizable inducer for inducer titration (b), the specific growth rate and the protein expression cannot be controlled individually as the inducer acts as substrate as well. Independent control of specific growth rate and protein expression rate and therefore a novel degree for process control is gained when performing inducer titration with non-metabolizable inducers (c). In this case, addition of substrate controls the specific growth rate and addition of inducer controls the induction rate. Another way to retain this novel degree of freedom is to use metabolizable inducers in a mixed-feed environment (d). Here, specific growth rate is controlled by addition of main substrate and inducing substrate and induction rate is controlled by the ratio of inducing substrate to main substrate. For reasons of easier comprehensibility, the overall influence of specific growth rate on protein expression is not illustrated in this graphic representation
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