Cell surface display of enzymes in Pseudomonas stutzeri A15
Research carried out by and Toon Nicolay, coordinated by Jos Vanderleyden and Stijn Spaepen
The expression of proteins on the cell surface of bacteria is interesting for many biotechnological applications. In case an enzyme is displayed on the cell surface of bacteria, the bacteria can be used in analytical assays or they can act as “biofactories”. Therefore, the (laborious) purification of the enzyme of interest is no longer necessary. Apart from this advantage, immobilized proteins have in many cases proven to be more stable than their free counterparts. The cell surface display of a protein also links the displayed protein and the corresponding gene which makes it a tool for the screening of peptide or enzyme libraries. In other applications, the cell surface display of an epitope gives rise to recombinant vaccines and the display of heavy metal fixing proteins can be a means for bioremediation.
In this study we focus on on the characterization of the esterase autotransporter of Pseudomonas stutzeri A15 and its use as possible biotechnological tool. Autotransporters, also known as type Va secretion, are a good tool for bacterial cell surface display because of the inherent simplicity of their protein (and gene) structure. Moving from the N-terminus to the C-terminus they consist of a signal peptide, a passenger domain and a translocator domain which may be involved in the translocation of the passenger domain. Replacement of the DNA sequence of the passenger domain by the gene of another protein results in the cell surface display of the corresponding protein
Overview of autotransporter biogenesis.
Autotransporters consist of an N-terminal signal peptide (SP) for Sec dependent transport across the inner membrane.
The signal peptide is followed by the functional domain, named the passenger domain. The C-terminal part of the protein is called the translocation unit which consists of two domains, a β-barrel for insertion in the outer membrane and an α-helical linker domain that passes through the β-barrel. After Sec mediated transport across the inner membrane and signal peptide cleavage, there is evidence that the passenger domain can undergo already some folding in the periplasm. The extent of this folding and the involvement of periplasmic chaperones are still a matter of debate. Also the exact mechanism of insertion of the β-barrel, the translocation of the passenger domain and the role of the Bam complex during these processes remain unclear.
Pseudomonas stutzeri A15 was isolated from the roots of paddy rice. The species Pseudomonas stutzeri is nutritionally versatile and is widely distributed in the environment. His robustness to environmental stresses and wide distribution makes Pseudomonas stutzeri a potential candidate for several biotechnological applications of cell surface display in the environment.
