Catalysis

Phage-Assisted Continuous Evolution and Selection of Enzymes for Chemical Synthesis

Authors

Abstract

Ligand-dependent biosensors are valuable tools for coupling the intracellular concentrations of small molecules to easily detectable readouts such as absorbance, fluorescence, or cell growth. While ligand-dependent biosensors are widely used for monitoring the production of small molecules in engineered cells and for controlling or optimizing biosynthetic pathways, their application to directed evolution for biocatalysts remains underexplored. As a consequence, emerging continuous evolution technologies are rarely applied to biocatalyst evolution. Here, we develop a panel of ligand-dependent biosensors that can detect a range of small molecules. We demonstrate that these biosensors can link enzymatic activity to the production of an essential phage protein to enable biocatalyst-dependent phage assisted continuous evolution (PACE) and phage-assisted continuous selection (PACS). By combining these phage-based evolution and library selection technologies, we demonstrate we can evolve enzyme variants with improved and expanded catalytic properties. Finally, we show that the genetic diversity resulting from a highly mutated PACS library is enriched for active enzyme variants with altered substrate scope. These results lay the foundation for using phage-based continuous evolution and selection technologies to engineer biocatalysts with novel substrate scope and reactivity.

Content

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Supplementary material

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SUPPORTING INFORMATION FOR: Phage-Assisted Continuous Evolution and Selection of Enzymes for Chemical Synthesis
Materials and instrumentation, general methods, PACE and PACS methods, biocatalysis methods, organic synthesis, supplementary tables and figures, NMR spectra, and references.