Ultrahigh‐Throughput Screening to Identify E. coli Cells Expressing Functionally Active Enzymes on their Surface

Abstract

We show here that E. coli bacteria that display esterases or lipases on their cell surface together with horseradish peroxidase (HRP) are capable of hydrolysing carboxylic acid esters of biotin tyramide. The tyramide radicals generated by the coupled lipase-peroxidase reaction were short-lived and therefore became covalently attached to reactive tyrosine residues that were located in close vicinity on the surface of a bacterial cell that displayed lipase activity. Up to 120 000 biotinylated tyramide derivatives could be covalently coupled through HRP activation to the surface of a single living E. coli cell. Differences in cellular esterase activity were found to correlate with the amount of biotin tyramide deposited on the cell surface. Selective biotin tyramide labelling of cells that had lipase activity allowed their isolation by magnetic cell sorting from a 1:10(6) mixture of control cells. This strategy of covalently attaching a biotin label to esterase-proficient bacteria might open new avenues to ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes with enhanced activities or enantioselectivities.

Single-cell Characterization of Autotransporter-mediated Escherichia coli Surface Display of Disulfide Bond-containing Proteins

“…Our work also opens avenues for the engineering of recombinantly displayed proteins mediated by the Ag43 system comprising only the ␣-helix and the ␤-barrel by employing high-throughput flow cytometry (51,52). A special feature of the AT extracellular transport system is that by including the autoproteolysis domain, switching between surface display and secretion of recombinant protein is rather straightforward (40).…”

Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

“…A major bottleneck in the directed evolution of esterases, however, is the high-throughput enzyme activity determination (34). Either substrates containing chromophores have been used, which is a substantial constraint of the strategy, or highly sophisticated equipment, such as gas chromatography, mass spectroscopy, or high-performance liquid chromatography, has been applied, limiting the number of reactions determined within a certain period of time (3,37,41). Recently, we reported on a quantitative screening method for hydrolases by the use of microplates with integrated fluorochrome pH sensors (19,20,29).…”

Directed Evolution of Industrial Biocatalysts

“…Differences in cellular esterase activity were found to correlate well with the amount of biotin tyramide deposited on the cell surface. This selective biotin tyramide labeling of cells with lipase activity allowed their isolation by magnetic cell sorting [65] .…”