Handal-Marquez, P. | Search bioRxiv

bioRxiv2024

Navigating directed evolution efficiently: optimizing selection conditions and selection output analysis

Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acids (XNAs) synthetases and reverse transcriptases (RTs) capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

synthetic biology↗

bioRxiv2022

Directed evolution of mesophilic HNA polymerases providing insight into DNA polymerase mechanisms

Detailed biochemical characterization of natural and mutant enzymes provides essential clues to understand their mechanisms. There are, however, limits to the throughput of such approaches and they are not without errors. DNA polymerases have benefited from over 50 years of detailed study and remain not fully understood. As such, methods that allow high-throughput interrogation of variants, and viable analysis pipelines to identify relevant variants, become an important tool to accelerate research. Using the DNA polymerase from B. subtilis Phi29 bacteriophage as a model, we demonstrate how coupling focused libraries, selection and deep sequencing can be combined to identify variants of interest for characterization. As selection parameters can be controlled, different areas of an enzymes mechanism can be explored. Focusing selection on faster HNA (1,5-anhydrohexitol nucleic acid) synthesis, we identified P562del as a variant of interest, enriching significantly between rounds. Characterization confirmed its faster HNA synthesis initiation but lower processivity and fidelity. P562 is a non-conserved residue, unlikely to be selected by more traditional approaches, but its deletion recapitulates knowledge on how Phi29 exonuclease, thumb and TPR2 subdomains regulate polymerase function. Our data further support the hypothesis that Phi29 shows a two-state binding to its template: a fast non-replicative complex that transitions to a replication-competent state.

biochemistry↗