Tuning recombinant protein expression to match secretion capacity

Abstract

BackgroundThe secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity.ResultsHere we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres.ConclusionsThe methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1047-z) contains supplementary material, which is available to authorized users.

Contemporary Tools for Regulating Gene Expression in Bacteria

“…In a recent study it was shown that, by re-engineering the expression platform domain of a translation-mediating riboswitch, it was possible to overcome these limitations [90]. Additionally, riboswitches are highly context sensitive, and several studies have sought to understand and overcome this limitation through the selection of codon-optimised 5' coding linkers and signal peptides [21,107], or by integrating riboswitches into a bicistronic design [108].…”

“…Recent work by Dixon and coworkers demonstrated the use of riboswitch-mediated regulation using a T7 RNAP-based cascade for improving the expression control and permitting expression titration [21]. The tight and tuneable regulation afforded by the RiboTite system resulted in a dynamic range that had increased >10 000-fold upon induction.…”

“…The tight and tuneable regulation afforded by the RiboTite system resulted in a dynamic range that had increased >10 000-fold upon induction. This made it possible to match protein production rate with secretion capacity, reducing overload of the secYEG-dependent pathway, and increasing secretion titres under fed-batch fermentation [21].…”

“…Devices such as these may enable harmonious production while reducing inhibition on biomass accumulation. Decoupling has been achieved using several different mechanisms, including: (C) carbon-dependent production [23]; (D) multi-layered, riboswitch-mediated control of basal expression and orthogonal regulation to decouple growth and expression [21]; and (E) inducible, proteasedependent control of functional protein levels [118].…”

“…robust and predictable genetic circuits it is essential that a balance be struck between circuit function and cell viability, particularly if we hope to apply the given genetic circuitry for large-scale fermentations over extended timescales. Modern tools for regulating gene expression allow construction of better production platforms through fine-tuning of gene expression, dynamic regulation, and autonomous, burden-responsive, feedback [10,[17][18][19][20][21] to help overcome this barrier.…”