Biocatalysis has historically lagged behind other areas of biotechnology (such as drug discovery) in terms of the speed and throughput at which experiments can be carried out, because of the requirement that reactions must be spatially segregated due to the accumulation of product molecules in bulk solution rather than on the surface of a cell. As a result, enzyme engineering screens are traditionally carried out at throughputs of only thousands of variants per generation, using expensive liquid handling robots operating on 96- or 384-well microtiter plates. To address this unmet need for scalable systems to optimize the catalytic properties of enzymes, we present a new multi-functional protein engineering platform -- which we designate uSCALE (micropore single cell analysis and laser extraction for protein engineering) -- capable of directly imaging massive arrays of spatially segregated single cells and retrieving desired cells using a simple laser-based technique. This micropore-based platform sorts cells based on time-resolved kinetic analysis, enabling the screening of libraries of over 107 protein variants for any phenotype of interest that can be coupled to a fluorescent readout. Cells expressing improved variants of a protein can then be extracted via optical cavitation driven by a targeted laser pulse, establishing a robust genotype-to-phenotype linkage in the pores and enabling iterative directed evolution. In order to validate the uSCALE platform in the context of enzyme engineering, we performed a series of proof-of-concept experiments using yeast-displayed alkaline phosphatase (AP) as a model system. In mock library experiments, uSCALE screens provided quantitative kinetic discrimination between WT AP and an R166S mutant with reduced catalytic activity. As a further rigorous test of the platform, we screened an error-prone PCR library of 107 AP mutants for improved activity on an artificial fluorogenic phosphate ester in the presence of inhibitory concentrations of inorganic phosphate. Three unique clones isolated from these screens exhibit substantially enhanced substrate turnover both in the presence and absence of phosphate, with apparent kcat/Km values up to 16-fold higher and KI values up to 10-fold higher than that of the wild-type enzyme. These results establish the uSCALE platform as an efficient strategy for isolating rare variants with enhanced catalytic properties from large enzyme libraries.

Laser extraction from micropore arrays enables high-throughput enzyme engineering

249th National Meeting (2015)

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