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.

Iterative Machine Learning for Classification and Discovery of Single-Molecule Unfolding Trajectories from Force Spectroscopy Data.

We report the application of machine learning techniques to expedite classification and analysis of protein unfolding trajectories from force spectroscopy data. Using kernel methods, logistic regression, and triplet loss, we developed a workflow called Forced Unfolding and Supervised Iterative Online (FUSION) learning where a user classifies a small number of repeatable unfolding patterns encoded as images, and a machine is tasked with identifying similar images to classify the remaining data. We tested the workflow using two case studies on a multidomain XMod-Dockerin/Cohesin complex, validating the approach first using synthetic data generated with a Monte Carlo algorithm and then deploying the method on experimental atomic force spectroscopy data. FUSION efficiently separated traces that passed quality filters from unusable ones, classified curves with high accuracy, and identified unfolding pathways that were undetected by the user. This study demonstrates the potential of machine learning to accelerate data analysis and generate new insights in protein biophysics.

High-throughput screening, next generation sequencing and machine learning: advanced methods in enzyme engineering.

Enzyme engineering is an important biotechnological process capable of generating tailored biocatalysts for applications in industrial chemical conversion and biopharma. Typical enhancements sought in enzyme engineering and in vitro evolution campaigns include improved folding stability, catalytic activity, and/or substrate specificity. Despite significant progress in recent years in the areas of high-throughput screening and DNA sequencing, our ability to explore the vast space of functional enzyme sequences remains severely limited. Here, we review the currently available suite of modern methods for enzyme engineering, with a focus on novel readout systems based on enzyme cascades, and new approaches to reaction compartmentalization including single-cell hydrogel encapsulation techniques to achieve a genotype-phenotype link. We further summarize systematic scanning mutagenesis approaches and their merger with deep mutational scanning and massively parallel next-generation DNA sequencing technologies to generate mutability landscapes. Finally, we discuss the implementation of machine learning models for computational prediction of enzyme phenotypic fitness from sequence. This broad overview of current state-of-the-art approaches for enzyme engineering and evolution will aid newcomers and experienced researchers alike in identifying the important challenges that should be addressed to move the field forward.