An RNA-directed recombination reaction can result in a network of interacting RNA species. It is now becoming increasingly apparent that such networks could have been an important feature of the RNA world during the nascent evolution of life on the Earth. However, the means by which such small RNA networks assimilate other available genotypes in the environment to grow and evolve into the more complex networks that are thought to have existed in the prebiotic milieu are not known. Here, we used the ability of fragments of the Azoarcus group I intron ribozyme to covalently self-assemble via genotype-selfish and genotype-cooperative interactions into full-length ribozymes to investigate the dynamics of small (three- and four-membered) networks. We focused on the influence of a three-membered core network on the incorporation of additional nodes, and on the degree and direction of connectivity as single new nodes are added to this core. We confirmed experimentally the predictions that additional links to a core should enhance overall network growth rates, but that the directionality of the link (a "giver" or a "receiver") impacts the growth of the core itself. Additionally, we used a simple mathematical model based on the first-order effects of lower-level interactions to predict the growth of more complex networks, and find that such a model can, to a first approximation, predict the ordinal rankings of nodes once a steady-state distribution has been reached.

Here we report the conception, synthesis and evaluation of new hydrophilic rhodamine-based enzymatic substrates for detection of peptidase activity compatible with high-throughput screening using droplet-based microfluidics.

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. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.
RESULTS:
Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).
CONCLUSIONS:
Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Sign epistasis is a central evolutionary constraint, but its causal factors remain difficult to predict. Here we use the notion of parameterised optima to explain epistasis within a signalling cascade, and test these predictions in Escherichia coli. We show that sign epistasis arises from the benefit of tuning phenotypic parameters of cascade genes with respect to each other, rather than from their complex and incompletely known genetic bases. Specifically, sign epistasis requires only that the optimal phenotypic parameters of one gene depend on the phenotypic parameters of another, independent of other details, such as activating or repressing nature, position within the cascade, intra-genic pleiotropy or genotype. Mutational effects change sign more readily in downstream genes, indicating that optimising downstream genes is more constrained. The findings show that sign epistasis results from the inherent upstream-downstream hierarchy between signalling cascade genes, and can be addressed without exhaustive genotypic mapping.

We report on the impact of surface hydrophobization on the structure of aqueous silica dispersions and how this structure resists drying stress. Hydrophilic silica particles were hydrophobized directly in water using a range of organosilane precursors, with a precise control of the grafting density. The resulting nanostructure was precisely analyzed by a combination of small-angle X-ray scattering (SAXS) and cryo-microscopy (cryo-TEM). Then, the dispersion was progressively concentrated by drying, and the evolution of the nanostructures as a function of the grafting density was followed by SAXS. At the fundamental level, because the hydrophobic character of the silica surfaces could be varied continuously through a precise control of the grafting density, we were able to observe how the hydrophobic interactions change particles interactions and aggregates structures. Practically, this opened a new route to tailor the final structure, the residual porosity, and the damp-proof properties of the fully dried silica. For example, regardless of the nature of the hydrophobic precursor, a grafting density of 1 grafter per nm2 optimized the interparticle interactions in solution in view to maximize the residual porosity in the dried material (0.9 cm3/g) and reduced the water uptake to less than 4% in weight compared to the typical value of 13% for hydrophilic particles (at T = 25 °C and relative humidity = 80%).

We report on a measurement of forces between particles adsorbed at a water–oil interface in the presence of an oil-soluble polymer. The cationic polymer interacts electrostatically with the negatively charged particles, thereby modulating the particle contact angle and the magnitude of capillary attraction between the particles. However, polymer adsorption to the interface also generates an increase in the apparent interfacial viscosity over several orders of magnitude in a time span of a few hours. We have designed an experiment in which repeated motion trajectories are measured on pairs of particles. The experiment gives an independent quantification of the interfacial drag coefficient (10–7–10–4 Ns/m) and of the interparticle capillary forces (0.1–10 pN). We observed that the attractive capillary force depends on the amount of polymer in the oil phase and on the particle pair. However, the attraction appears to be independent of the surface rheology, with changes over a wide range of apparent viscosity values due to aging. Given the direction (attraction), the range (∼μm), and the distance dependence (∼1/S5) of the observed interparticle force, we interpret the force as being caused by quadrupolar deformations of the fluid–fluid interface induced by particle surface roughness. The results suggest that capillary forces are equilibrated in the early stages of interface aging and thereafter do not change anymore, even though strong changes in surface rheology still occur. The described experimental approach is powerful for studying dissipative as well as conservative forces of micro- and nanoparticles at fluid–fluid interfaces for systems out of equilibrium.

Liquid core capsules having a hydrogel membrane are becoming a versatile tool for three-dimensional culture of micro-organisms and mammalian cells. Making sub-millimeter capsules at a high rate, via the breakup of a compound jet in air, opens the way to high-throughput screening applications. However, control of the capsule size monodispersity, especially required for quantitative bioassays, was still lacking. Here, we report how the understanding of the underlying hydrodynamic instabilities that occur during the process can lead to calibrated core–shell bioreactors. The requirements are: i) damping the shear layer instability that develops inside the injector arising from the co-annular flow configuration of liquid phases having contrasting viscoelastic properties; ii) controlling the capillary instability of the compound jet by superposing a harmonic perturbation onto the shell flow; iii) avoiding coalescence of drops during jet fragmentation as well as during drop flight towards the gelling bath; iv) ensuring proper engulfment of the compound drops into the gelling bath for building a closed hydrogel shell. We end up with the creation of numerous identical compartments in which cells are able to form multicellular aggregates, namely spheroids. In addition, we implement an intermediate composite hydrogel layer, composed of alginate and collagen, allowing cell adhesion and thus the formation of epithelia or monolayers of cells.

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. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 10(4) clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost,

De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution. In this work, we show that microfluidic-based screening using fluorescence-activated droplet sorting (FADS) is ideally suited for efficient optimization of designed enzymes with low starting activity, essentially straight out of the computer. We chose the designed retro-aldolase RA95.0, which had been previously evolved by conventional microtiter plate screening, as an example and reoptimized it using the microfluidic-based assay. Our results show that FADS is sufficiently sensitive to detect enzyme activities as low as kcat/Km = 0.5 M(-1)s(-1) The ultra-high throughput of this system makes screening of large mutant libraries possible in which clusters of up to five residues are randomized simultaneously. Thus, combinations of beneficial mutations can be identified directly, leading to large jumps in catalytic activity of up to 80-fold within a single round of evolution. By exploring several evolutionary trajectories in parallel, we identify alternative active site arrangements that exhibit comparably enhanced efficiency but opposite enantioselectivity.

We obtained osmotic pressure data of lysozyme solutions, describing their physical states over a wide concentration range, using osmotic stress for pressures between 0.05 bar and about 40 bar and volume fractions between 0.01 and 0.61. The osmotic pressure vs. volume fraction data consist of a dilute, gas-phase regime, a transition regime with a high-compressibility plateau, and a concentrated regime where the system is nearly incompressible. The first two regimes are shifted towards a higher protein volume fraction upon decreasing the strength or the range of electrostatic interactions. We describe this shift and the overall shape of the experimental data in these two regimes through a model accounting for a steric repulsion, a short-range van der Waals attraction and a screened electrostatic repulsion. The transition is caused by crystallization, as shown by small-angle X-ray scattering. We verified that our data points correspond to thermodynamic equilibria, and thus that they consist of the reference experimental counterpart of a thermodynamic equation of state.