Evolution of enzyme catalysts caged in biomimetic gel-shell beads

Abstract

Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these manmade compartments can be identified readily by fluorescence-activated sorting at rates >107 GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components. Evolution of Catalysts Caged in Biomimetic Gel-Shell Beads AbstractNatural evolution relies on improvement of biological entities by rounds of

Methods for the directed evolution of proteins

“…3E). Using the fluorogenic organophosphate analogues described above, the researchers sorted GSBs based on phosphotriesterase activity to identify parathion hydrolase variants that more rapidly degrade organophosphate pesticides 90 .…”

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

“…The key technical module to make this possible is a microfluidic droplet sorter that has so far relied exclusively on fluorescent readouts (fluorescence-activated droplet sorting, FADS) (10,11). By alternatively transforming microfluidic emulsions to double emulsions (12) or hydrogel beads equipped with polyelectrolyte shells (13), sorting in standard flow cytometers also becomes possible. However, to date, all ultrahigh-throughput screening campaigns implemented in microcompartmentalized formats (including microcapillary arrays) have so far relied on detection of a fluorescent product (13)(14)(15)(16)(17)(18)(19)(20).…”

“…By alternatively transforming microfluidic emulsions to double emulsions (12) or hydrogel beads equipped with polyelectrolyte shells (13), sorting in standard flow cytometers also becomes possible. However, to date, all ultrahigh-throughput screening campaigns implemented in microcompartmentalized formats (including microcapillary arrays) have so far relied on detection of a fluorescent product (13)(14)(15)(16)(17)(18)(19)(20). When assays that lack this type of readout are to be used (e.g., involving instead the widely used absorbance detection), droplet screening is currently impossible: Colony screening assays (relying on precipitation of insoluble product and thus encumbered by poor dynamic range and assay quality) or microwell-plate screening with sophisticated robots (higher quality but expensive in terms of capital expenditure and running costs) remain the only options.…”

Discovery in Droplets

“…Droplet-templated GSBs were critical in the evolution of phophotriesterase (PTE) activity, an enzyme that neutralizes pesticides and nerve gas agents. 20 One round of FACS produced a PTE variant with ~8-fold improved catalytic efficiency. A four-device microfluidic platform has been used to improve the catalysis of the X-motif ribozyme against a trans substrate.…”