SUMMARY Among the pathways used by gram-negative bacteria for protein secretion, the autotransporter pathway represents a solution of impressive simplicity. Proteins are transported, independent of their nature as recombinant or native passengers, as long as the coding nucleotide sequence is inserted in frame between those of an N-terminal signal peptide and a C-terminal domain, referred to as the β-barrel of the outer membrane translocation unit. The immunoglobulin A1 (IgA1) protease from Neisseria gonorrhoeae was the first identified member of the autotransporter family of secreted proteins. The IgA1 protease was employed in initial experiments investigating autotransporter-mediated surface display of recombinant proteins and to investigate structural and functional requirements. Various other autotransporter proteins have since been described, and the autodisplay system was developed on the basis of the natural Escherichia coli autotransporter protein AIDA-I (adhesin involved in diffuse adherence). Autodisplay has been used for the surface display of random peptide libraries to successfully screen for novel enzyme inhibitors. The autodisplay system was also used for the surface display of functional enzymes, including esterases, oxidoreductases, and electron transfer proteins. Whole E. coli cells displaying enzymes have been utilized to efficiently synthesize industrially important rare organic compounds with specific chirality. Autodisplay of epitopes on the surface of attenuated Salmonella carriers has also provided a novel way to induce immune protection after oral vaccination. This review summarizes the structural and functional features of the autodisplay system, illustrating its discovery and most recent applications. Autodisplay facilitates the export of more than 100,000 recombinant molecules per single cell and permits the oligomerization of subunits on the cell surface as well as the incorporation of inorganic prosthetic groups after transport of apoproteins onto the bacterial surface without disturbing bacterial integrity or viability. We discuss future biotechnical and biomedical applications in the light of these achievements.

Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 10(5) recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.

No abstract

No abstract

No abstract

Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of a high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with E. coli autodisplay. For the first step in a 4-step screening procedure we quantitatively screened >10 5 variants of the homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Display of the sulfatase variants on living E. coli cells ensured the continuous linkage of genotype and phenotype during droplet sorting and allowed for direct recovery by simple regrowth of the sorted cells. The use of autodisplay on living cells simplified and reduced the degree of liquid handling during all steps in the screening procedure to the single event of simply mixing substrate and cells. The percentage of apparent improved variants was enriched >10-fold as a result of droplet sorting. We ultimately identified 25 SpAS1-variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In SpAS1 variants with improved performance toward the bulky fluorescein disulfate, many of the beneficial mutations occur in residues that form hydrogen bonds between α-helices in the C-terminal oligomerization region, suggesting a non-trivial, previously unknown role for the dimer interface in shaping the substrate binding site of SpAS1.

Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N5-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives.