van Loo B, Heberlein M, Mair P, Zinchenko A, Schüürmann J, Eenink BDG, Dilkaute C, Jose J, Hollfelder F and Bornberg-Bauer E
High-Throughput, Lysis-free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets
ACS Catalysis, 2018
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. For this purpose we combine the use of microdroplet-based single-variant activity sorting with E. coli autodisplay. This setup allows circumventing complications arising from cell lysis during screening for enzymatic activity. We successfully displayed the moderately efficient (k
M = 4.8×103 s-1 M-1) homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1) on the surface of E. coli. For the first step in a 4-step screening procedure we quantitatively screened >105 SpAS1 variants for improved sulfatase activity using fluorescence activated droplet sorting. The display of the sulfatase variants on living E. coli cells ensured the continuous linkage of genotype and phenotype during droplet sorting. It allowed for direct recovery by simple regrowth of the sorted cells, enriching the percentage of improved variants. When compared to a system involving cell lysis prior to activity measurements during screening, 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 screening procedure allowed us to identify 16 SpAS1-variants with 1.1- to 6.2-fold improved catalytic efficiency compared to wild type, toward the model sulfatase substrate 4-nitrophenyl sulfate. All beneficial mutations occurred in positions that were difficult to predict, i.e. no conserved active site residues were directly affected. The combination of five such mutations as observed in the best variants finally resulted in an SpAS1 mutant with 28-fold improved catalytic efficiency (k
M = 1.35×105 s-1 M-1) compared
to the wild type.