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

Abstract

Tuning of transcription is a promising strategy to overcome challenges associated with a non-suitable expression rate like outgrowth of segregants, inclusion body formation, metabolic burden and inefficient translocation. By adjusting the expression rate-even on line-to purposeful levels higher product titres and more cost-efficient production processes can be achieved by enabling culture long-term stability and constant product quality. Some tunable systems are registered for patents or already commercially available. Within this contribution, we discuss the induction mechanisms of various Escherichia coli inherent promoter systems with respect to their tunability and review studies using these systems for expression tuning. According to the current level of knowledge, some promoter systems were successfully used for expression tuning, and in some cases, analytical evidence on single-cell level is still pending. However, only a few studies using tunable strains apply a suitable process control strategy. So far, expression tuning has only gathered little attention, but we anticipate that expression tuning harbours great potential for enabling and optimizing the production of a broad spectrum of products in E. coli.

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

Fig. 1

Two possible ways of achieving transcription tuning with the p_lac system are described in literature. One reported way to achieve a graded response is to use a lacZ ⁺ and lacY ⁺ strain with lactose as inducer (a). In this case, lactose induces the expression of lacZ (negative feedback) and therefore turns the all-or-none response caused by induction of lacY (positive feedback) into a graded response (Afroz et al. 2014b). Another possibility to achieve a graded response is to use a lacY ⁻ strain with IPTG (b) (Jensen et al. ; Khlebnikov and Keasling 2002). As the gratuitous inducers (IPTG and TMG) are also recognized by LacY, but cannot be metabolized, the positive feedback needs to be interrupted by deleting lacY (Marbach and Bettenbrock 2012). The transacetylase activity of LacA modifies IPTG in a way that it loses its inducing ability and therefore leads to a decrease in single-cell expression levels by decreasing the inducer concentration (Marbach and Bettenbrock 2012). That is why using a lacA ⁻ mutant is recommended (not shown in this graphic). For better clarity, the role of CRP in the regulation of the lac operon is not illustrated in this graphic, but is nicely described elsewhere (Ozbudak et al. 2004)