Autotransporters represent a widespread family of secreted proteins in Gram-negative bacteria.
Their seemingly easy secretion mechanism and modular structure make them interesting
candidates for cell surface display of heterologous proteins. The most widely applied host
organism for this purpose is Escherichia coli. Pseudomonas stutzeri A15 is an interesting
candidate host for environmentally relevant biotechnological applications. With the recently
characterized P. stutzeri A15 EstA autotransporter at hand, all tools for developing a surface
display system for environmental use are available. More general, this system could serve as
a case-study to test the broad applicability of autotransporter based surface display.
Based on the P. stutzeri A15 EstA autotransporter β-domain, a surface display expression
module was constructed for use in P. stutzeri A15. Proof of concept of this module was
presented by successful surface display of the original EstA passenger domain, which retained
its full esterase activity. Almost all of the tested heterologous passenger domains however were not exposed
at the cell surface of P. stutzeri A15, as assessed by whole cell proteinase K treatment. Only for a beta-lactamase protein,
cell surface display in P. stutzeri A15 was comparable to presentation of the original EstA passenger domain.
Development of expression modules based on the full-length EstA autotransporter did not resolve these problems.
Since only one of the tested heterologous passenger proteins could be displayed at the cell
surface of P. stutzeri A15 to a notable extent, our results indicate that the EstA autotransporter cannot be regarded as a broad spectrum cell surface display system in P. stutzeri A15.