2019/11/18 by Bert van Loo, Magdalena Heberlein, Philip Mair +8 · 1 citation
Biochemistry, Genetics and Molecular Biology · Engineering · #Enzyme Catalysis and Immobilization #Innovative Microfluidic and Catalytic Techniques Innovation #Microbial Metabolic Engineering and Bioproduction
paper · doi:10.1021/acssynbio.9b00274
openalex publication_date 2019/11/18 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/30
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 an ultra 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 ( Sp AS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Compartmentalization of the fluorescent reaction product with living E. coli cells autodisplaying the sulfatase variants 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 Sp AS1 variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In Sp AS1 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 previously unknown role for the dimer interface in shaping the substrate binding site of Sp AS1.