One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.

Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1 000 000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at -80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers

Proteome-wide landscape of solubility limits in a bacterial cell

Proteins are prone to aggregate when expressed above their solubility limits. Aggregation may occur rapidly, potentially as early as proteins emerge from the ribosome, or slowly, following synthesis. However, in vivo data on aggregation rates are scarce. Here, we classified the Escherichia coli proteome into rapidly and slowly aggregating proteins using an in vivo image-based screen coupled with machine learning. We find that the majority (70%) of cytosolic proteins that become insoluble upon overexpression have relatively low rates of aggregation and are unlikely to aggregate co-translationally. Remarkably, such proteins exhibit higher folding rates compared to rapidly aggregating proteins, potentially implying that they aggregate after reaching their folded states. Furthermore, we find that a substantial fraction (similar to 35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our study provides a global view of aggregation rates and hence solubility limits of proteins in a bacterial cell.Peer reviewe

Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics

This is the final version of the article. It was first available from NPG via http://dx.doi.org/10.1038/ncomms10008Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.This research was funded by the Engineering and Physical Sciences Research Council (EPSRC) and the Biological and Biotechnological Research Council (BBSRC). FH is an ERC Starting Investigator. PYC holds a fellowship of the EU ITN PhosChemRec, CM of the ITN ProSA and ENEFP. BK and MM were supported by postdoctoral Marie-Curie fellowships. The authors thank Sean Devenish and Nobuhiko Tokuriki for useful comments on the manuscript and Gabrielle Potocki-Veronese and Stephane Emond for help with the design of the library strategy. We thank Raindance for the gift of EA surfactant

Promiscuous enzymes from functional metagenomics

Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.This research was funded by the Engineering and Physical Sciences Research Council (EPSRC) and the Biological and Biotechnological Research Council (BBSRC). FH is an ERC Starting Investigator. PYC holds a fellowship of the EU ITN PhosChemRec, CM of the ITN ProSA and ENEFP. BK and MM were supported by postdoctoral Marie-Curie fellowships. The authors thank Sean Devenish and Nobuhiko Tokuriki for useful comments on the manuscript and Gabrielle Potocki-Veronese and Stephane Emond for help with the design of the library strategy. We thank Raindance for the gift of EA surfactant.This is the final version of the article. It was first available from NPG via http://dx.doi.org/10.1038/ncomms1000

Rational design of balanced dual-targeting antibiotics with limited resistance

Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins. ULD1 and ULD2 are excellently potent against a broad range of gram-positive bacteria. Notably, the efficacy of these compounds was tested against a broad panel of multidrug-resistant Staphylococcus aureus clinical strains. Antibiotics with clinical relevance against staphylococcal infections fail to inhibit a significant fraction of these isolates, whereas both ULD1 and ULD2 inhibit all of them (minimum inhibitory concentration [MIC] ≤1 μg/mL). Resistance mutations against these compounds are rare, have limited impact on compound susceptibility, and substantially reduce bacterial growth. Based on their efficacy and lack of toxicity demonstrated in murine infection models, these compounds could translate into new therapies against multidrug-resistant bacterial infections

Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution

We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 107 monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viabl