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.

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.

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In today's clinical practice, monoclonal antibodies have become a well-established therapy option for a range of indications, such as cancer and autoimmune diseases [1]. To develop various specific antibodies, huge antibody libraries have to be screened. For this purpose phage display has been used with great success in the last 25 years. Nevertheless, this method is associated with some drawbacks as the possible discrimination of the most potent binders during the biopanning process, the incompatibility with flow cytometry or the size limitation of the protein displayed on the surface [2]. To circumvent these disadvantages, we developed a screening tool using E. coli cells presenting a full-length antibody on their surface. The presentation of antibodies and in particular their libraries enables the screening for new variants against pre-given epitopes using flow-cytometry without losing the highly potent binders. In this work, the autodisplay technique [3,4] was utilized to present a functional full-length antibody on the surface. As a proof of principle, the display of the antibody T84.66 which is directed against carcinoembryonic antigen (CEA) was investigated. Based on this antibody a library was generated. Therefore, restriction sites were introduced in front of and behind the complementarity determining region 3 (CDR3). This enables the exchange of the CDR3 through a randomized fragment. After ligation, this construct was used to transform E. coli UT5600 (DE3) via electroporation. The resulting library consists of up to 10 5 clones which can be analysed and sorted via flow cytometry after incubation with a fluorescently labelled target protein. To examine the optimal conditions for the screening, two different autotransporters in combination with two promoters were investigated: the AIDA-1 autotransporter [3] under control of a T7-promoter and the EhaA-autotransporter [4] controlled by an araBAD promoter. Experiments with the T84.66 antibody as a passenger revealed that the EhaA-autotransporter under araBAD control suited better with regard to surface presentation and cell survival after sorting via flow cytometry. These results indicate that it is possible to generate a full-length antibody library on the surface of E. coli which afterwards can be screened with the advantageous high-throughput screening system of flow cytometry. Further investigations should be performed to identify an antibody variant out of the constructed library which binds a pre-given epitope of therapeutical interest.