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

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 that 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.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

“…Design of a selection for deoxycytidine deamination PACE has enabled the rapid laboratory evolution of diverse protein functions, including protein-protein interactions 35 , tRNA synthetases 36 , DNA-binding proteins [37][38][39] , proteases 40,41 , polymerases 42 , metabolic enzymes [43][44][45] and base editors 7,12 . During PACE, the evolving protein is encoded on the selection phage (SP), which infect Escherichia coli host cells 46 .…”

Molecular Dynamics-Derived Descriptor Informs the Impact of Mutation on the Catalytic Turnover Number in Lactonase Across Substrates

“…The natural occurrence of beneficial mutations has been reported to be below 1%, and therefore, extensive iterations and experimental screening of each is often required. Consequently, the a priori identification of beneficial enzyme variants that catalyze non-native substrates remains a major obstacle. − …”

A Closed Form Model for Molecular Ratchet-Type Chemically Induced Dimerization Modules

“…The N-terminal half (N-T7) encodes a portion of the polymerase essential for converting T7 RNAP from the initiation to an elongation complex; in the absence of N-T7, C-T7 can bind at T7 DNA promoters and initiate transcription but produces only abortive transcripts . The Dickinson lab has developed this split T7 system coupled to molecular ratchets for use as general purpose biosensors, , and other laboratories have used similar architectures for sense and response applications . These bacterial sensors have observed responsiveness in the micromolar range, even though the same sensors in other configurations can have picomolar limits of detection.…”