Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein
Microbial Cell Factories
Many 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 puriication, 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 lagellar type III secretion system (FT3SS). Ordinarily lagellar subunits are exported through the centre
... hrough the centre of the growing lagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain lagellar components (e.g. cap proteins), monomeric lagellar proteins are secreted into the supernatant. Results: We report the creation and iterative improvement of an E. coli strain, by means of a modiied FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the lagellin 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 lagellar 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 irst 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 (CH 2 )], achieving secreted yields of up to 0.29 mg L −1 , in low cell density culture. Conclusions: We 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.