Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein

Abstract

BackgroundMany valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the ‘hijack’ of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant.ResultsWe report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5′UTR and first 47 amino acidsof FliC from E. coli (but no 3′UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L−1, in low cell density culture.ConclusionsWe have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1058-4) contains supplementary material, which is available to authorized users.

Comparison of Enzyme Secretion and Ferulic Acid Production by Escherichia coli Expressing Different Lactobacillus Feruloyl Esterases

“…Nevertheless, the understanding of the atypical protein secretion pathway provides a new solution for the secretory expression of foreign proteins. Recombinant proteins had been successfully secreted by these pathways in E. coli ( Green et al, 2019 ). Therefore, the secretion mechanism of feruloyl esterase can broaden the means of protein secretion in E. coli .…”

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“…The understanding of the atypical protein secretion pathway provides a new solution for the secretory expression of foreign proteins. Recombinant proteins had been successfully secreted by these pathways in E. coli [35]. Therefore, the secretion mechanism of feruloyl esterase can broaden the means of protein secretion in E. coli.…”

Functional divergence of flagellar type III secretion system: A case study in a non-flagellated, predatory bacterium

“…Wild-type OH11 and its derivative strains were grown at 28 °C in 1/10 TSB until OD 600 reached 1.5. Total secreted proteins from bacterial cultures were obtained as described previously [21] with a slight modification. Briefly, 40 mL of each culture sample was subjected to centrifugation (6000 rpm, 4 °C, and 30 min) to separate supernatants from whole cells.…”