Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing.

Understanding the complex relationships between enzyme sequence, folding stability and catalytic activity is crucial for applications in industry and biomedicine. However, current enzyme assay technologies are limited by an inability to simultaneously resolve both stability and activity phenotypes and to couple these to gene sequences at large scale. Here we present the development of enzyme proximity sequencing, a deep mutational scanning method that leverages peroxidase-mediated radical labeling with single cell fidelity to dissect the effects of thousands of mutations on stability and catalytic activity of oxidoreductase enzymes in a single experiment. We use enzyme proximity sequencing to analyze how 6399 missense mutations influence folding stability and catalytic activity in a D-amino acid oxidase from Rhodotorula gracilis. The resulting datasets demonstrate activity-based constraints that limit folding stability during natural evolution, and identify hotspots distant from the active site as candidates for mutations that improve catalytic activity without sacrificing stability. Enzyme proximity sequencing can be extended to other enzyme classes and provides valuable insights into biophysical principles governing enzyme structure and function.

Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening.

Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of d-Amino acid oxidase from Rhodotorula gracilis (RgDAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between RgDAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled library screening by fluorescence activated cell sorting (FACS). We screened a RgDAAOx variant library containing ∼106 clones while lowering the d-Ala substrate concentration over three sorting rounds in order to isolate variants with low Km. After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower Km than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the RgDAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes.

Shift of spawning season and effects of climate warming on developmental stages of a grayling (Salmonidae).

River-dwelling fish, such as European graylings (Thymallus thymallus), are susceptible to changes in climate because they can often not avoid suboptimal temperatures, especially during early developmental stages. We analyzed data collected in a 62-year-long (1948-2009) population monitoring program. Male and female graylings were sampled about three times/week during the yearly spawning season in order to follow the development of the population. The occurrence of females bearing ripe eggs was used to approximate the timing of each spawning season. In the last years of the study, spawning season was more than 3 weeks earlier than in the first years. This shift was linked to increasing water temperatures as recorded over the last 39 years with a temperature logger at the spawning site. In early spring water temperatures rose more slowly than in later spring. Thus, embryos and larvae were exposed to increasingly colder water at a stage that is critical for sex determination and pathogen resistance in other salmonids. In summer, however, fry were exposed to increasingly warmer temperatures. The changes in water temperatures that we found embryos, larvae, and fry were exposed to could be contributing to the decline in abundance that has occurred over the last 30-40 years.