Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Abstract

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering.Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

A step forward to the optimized HlyA type 1 secretion system through directed evolution

“…Oligonucleotides used in this work are listed in Tables S1 and S2 . Plasmid pK184_ EF (Best)- lipA - hlyA1 - Ter - hlyBD (Pourhassan et al 2022a ) was used as the backbone vector for the library generation. Note that the gene encoding the TolC protein is endogenous, and the genes encoding HlyB and HlyD are plasmid-based.…”

“…The screening of the libraries with the para -nitrophenyl butyrate ( p NPB) assay was performed as previously reported in 96-well MTPs (Pourhassan et al 2022a ). Screening of the recombinant library via lipase colorimetric assay proved to be a challenging task as the amount of lipase in the supernatant of clones was high and reached the saturation level of the assay.…”

“…To enhance the secretion efficiency of different classes of secretion systems, different approaches have been applied. For instance, these approaches include modification of secretion signal (Freudl 2018 ), transferring the secretion system to a more efficient secretion host (Eom et al 2014 ), and modification of genetical elements such as enhancer fragment (Pourhassan et al 2022a ). In addition to these approaches, protein engineering has proved to be a powerful tool for tailoring protein properties (Wong et al 2006 ).…”

“…To optimize the secretion efficiency of the system, we recently worked on the HlyA enhancer fragment and have achieved great success (Pourhassan et al 2022a ), which promoted us to a further improvement of the system. In the current study, we report a two-round based KnowVolution campaigns on the inner membrane complex of HlyA T1SS (HlyB and HlyD) to achieve further improved HlyA T1SS secretion.…”

Investigations on the substrate binding sites of hemolysin B, an ABC transporter, of a type 1 secretion system

“…The HlyA secretion system is a paradigm of the T1SS, first identified in an uropathogenic Escherichia coli strain ( Noegel et al, 1979 ). The secretion machinery of this system consists of three membrane components, as follows: the ABC transporter HlyB, the membrane fusion protein HlyD, and the outer membrane TolC ( Holland et al, 2016 ; Pourhassan et al, 2022 ). Only very recently, structural information derived from single particle cryo-EM was reported and revealed an unusual six stoichiometry of the inner membrane components of the HlyA T1SS, HlyB, and HlyD, which form the so-called inner membrane complex ( Zhao et al, 2022 ).…”