Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor.

Tryptophan synthase catalyzes the synthesis of a wide array of noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (up to 107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic readouts, which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a sensor to quantitatively report on tryptophan production in droplets. The utility of the sensor was validated by identifying five-fold improved tryptophan synthases from ∼100,000 protein variants. More generally, this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

Enhancing prime editor activity by directed protein evolution in yeast.

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations - the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT - results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.

Effects of algae and fungicides on the fate of a sulfonylurea herbicide in a water-sediment system.

The impact of pesticide mixtures on various soil parameters has been extensively studied, whereas research on effects in the aquatic environment is scarce. Furthermore, investigations on the consequences of chemical mixtures on the biodegradation kinetics of parent compounds remain deficient. Our research intended to evaluate potential effects by combined application of an agriculturally employed tank mixture to aquatic sediment systems under controlled laboratory conditions. The mixture contained two fungicides and one radiolabeled herbicide of which the route and rate of degradation was followed. One set of aquatic sediment vessels was incubated in the dark. A second set of vessels was controlled under identical conditions, except for being continuously irradiated to promote algal growth. In addition, the algal biomass in irradiated aquatic sediment was monitored to determine its effects and a potential role in the biodegradation of iodosulfuron-methyl-sodium. The study results showed that the herbicide, although hydro- and photolytically stable throughout the study, metabolized faster (DT50 1.1-1.2-fold and DT90 2.8-4.5-fold) when continuously irradiated in comparison to dark aquatic sediment. Both fungicides had a significant prolonging effect on the biodegradation rate of the herbicide. In the presence of fungicides, DT90 values increased 1.5-fold in the irradiated, and 2.5-fold in the dark systems. Additionally, algae may have influenced the metabolization of the herbicide in the irradiated systems, where shorter DT90 values were evaluated. Even so, the algal influence was concluded to be indirect.