756 NATURE CHEMISTRY | VOL 6 | SEPTEMBER 2014 | www.nature.com/naturechemistry news & views C hemists, biologists and engineers have been developing high-throughput screening (HTS) strategies for synthetic molecules since the invention of combinatorial chemistry by Geysen 1 and Nielsen 2 during the 1980s and early 1990s. All of these approaches have a similar theme: (i) a randomized chemical library, (ii) a molecular ‘bait-and-hook’ selection strategy and (iii) an engineered screening method to select for ‘hits’. As well as a plethora of chemical systems currently available, biological systems, for example, yeast 3 and phage 4 display, have also been co-opted and engineered for HTS selection of protein binding molecules for a diverse array of applications including drug leads, novel enzymes and agents for diagnostic assays. A popular HTS strategy is the in vitro compartmentalization (IVC) method 5 . Tis relies on the formation of emulsion droplets in microfuidic systems that capture molecular substrates so that each droplet is efectively a single reaction capsule. Te droplet formation can be tuned — by adjusting the fow rates of the emulsion components — so that a substrate can be encased with a single molecule of enzyme in a single droplet (the content of droplets follows a Poisson distribution). Screening for enzyme activity can then be performed using modifed fuorescence-activated cell sorting (FACS) that can enable ultra-high-throughput screening (UHTS). One of the attractive benefts of IVC is the potential ability to couple genotype and phenotype — that is, to maintain the connection between the genetic content (DNA sequence) and the observable characteristics (for example, enzyme activity). Tis is, however, a major limitation of emulsion droplet systems because once the emulsion is broken it is difcult to track genotypes. Various labelled DNA, afnity capture or proxy barcoding strategies have been developed to circumvent this limitation 6–9 . Briefy, these strategies are secondary ‘selection’ methods that indirectly report on the genotype of the newly evolved protein. Now, writing in Nature Chemistry, Florian Hollfelder and co-workers have described 10 a convenient and low-cost approach that allows simultaneous purifcation of both an enzyme and the DNA that encodes it. Tey use this system to perform a high-throughput evolution of a phosphotriesterase enzyme that can be used in the detoxifcation of pesticides or nerve agents. Teir “all- in-one” strategy (Fig. 1) relies on the generation of “biomimetic gel-shell beads” for IVC. In a similar fashion to other droplet formation/FACS strategies, their method frst generates emulsion droplets so that each droplet encapsulates a single bacterium carrying a variant of the enzyme (at both the protein and DNA levels), enzyme substrate, low-melting agarose, lysis bufer and an alginate polyanion. Lysis of the trapped bacteria followed by solidifcation of the agarose in the presence of poly(allylamine-hydrochloride) results in the formation of a porous agarose gel- bead containing the released enzyme, the encoding DNA and the enzyme substrate. Tus both the phenotype and encoding genotype (an expression plasmid) are trapped in a convenient bead-like substrate amenable to a standard FACS process. Afer identifying beads that contain active enzymes, the encoding DNA can ENZYME CATALYSIS Evolution made easy Directed evolution is a powerful tool for the development of improved enzyme catalysts. Now, a method that enables an enzyme, its encoding DNA and a fluorescent reaction product to be encapsulated in a gel bead enables the application of directed evolution in an ultra-high-throughput format. Eugene J. H. Wee and Matt Trau 1 2 3 4 5 6 Expression in E. coli Droplet formation, cell lysis Catalysis Selection by FACS Gel-shell bead formation Genotype recovery Plasmid Enzyme Substrate (with tag) Product (with tag) Figure 1 | Directed evolution of enzymes in gel-shell beads 10 . A library of enzymes is created by expression within Escherichia coli carrying the encoding plasmid DNA. The bacteria are encapsulated in droplets so that each droplet contains a single enzyme and the DNA that encodes it. Cell lysis releases the enzyme so that it can be tested as a catalyst with the activity measured using fluorescence. Gel-shell bead formation traps the enzyme with its encoding DNA and the fluorescent reaction product so that active enzymes can be identified in a high-throughput fashion for analysis of the DNA sequence, and potentially for further rounds of enzyme evolution. © 2014 Macmillan Publishers Limited. All rights reserved