Directed evolution on Ag43 β-domain to enhance protein display efficiency

Autotransporters play a key role in bacterial virulence by facilitating transport of essential virulence factors across the outer membrane, thereby contributing to overall bacterial pathogenicity. Antigen 43 (Ag43), composed of an ɑ- and β- subunit, is an autotransporter in Escherichia coli implicated in bacterial pathogenicity, as evidenced by its role in adhesion, autoaggregation, and biofilm formation. While previous studies have focused on investigating how the functionality of the ɑ-subunit may be improved, the β-subunit remains understudied. Improvements made to the β-subunit have broader implications for the Ag43 autotransporter system as a whole. Optimizing the Ag43 system offers potential for improved heterologous protein production across a range of biotechnological and medical applications. Furthermore, continued refinement could expand its versatility and increase the yield of recombinant proteins for industrial and therapeutic purposes. Using a combination of error-prone PCR, autoaggregation, and colony morphology assays, this study investigated whether sequence changes to the Ag43 β-domain could improve its functionality and transport efficiency. Existing literature have shown that fimbrial protrusions in the extracellular space can hinder Ag43-mediated autoaggregations, however, our pilot assays showed that both DH5ɑ and ΔfimA E. coli autoaggregate similarly in all induction conditions. Error-prone PCR success varied between runs despite constant conditions, suggesting that Taq polymerase exhibits inherent variability. Ag43 β-domain mutant transformants were selected via directed evolution based on their sedimentation rate relative to wild type Ag43, which indicates increased Ag43 transport efficiency. Further selection used colony morphology to compare ruffled edges in Ag43 β-domain mutants versus wild type. Sequencing of selected variants with extreme phenotypes revealed no detectable mutations, suggesting that a more comprehensive sequencing effort, or direct mutation detection methods such as Illumina sequencing may be necessary to detect epPCR-induced diversity.