Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

Background: Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed.

High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics.

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities.

Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms.

Discovery of microorganisms producing enzymes that can efficiently hydrolyze cellulosic biomass is of great importance for biofuel production. To date, however, only a miniscule fraction of natural biodiversity has been tested because of the relatively low throughput of screening systems and their limitation to screening only culturable microorganisms. Here, we describe an ultra-high-throughput droplet-based microfluidic system that allowed the screening of over 100,000 cells in less than 20 min.

New glycosidase substrates for droplet-based microfluidic screening.

Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates.

Disulfide bond substitution by directed evolution in an engineered binding protein.

Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways.

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.