Selecting Better Biocatalysts by Complementing Recoded Bacteria

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded orga...

Full description

Saved in:
Bibliographic Details
Published in:Angewandte Chemie International Edition 2023-01, Vol.62 (2), p.e202213942-n/a
Main Authors: Rubini, Rudy, Jansen, Suzanne C., Beekhuis, Houdijn, Rozeboom, Henriëtte J., Mayer, Clemens
Format: Article
Language:English
Subjects:
Amino acids
Amino Acids - chemistry
Bacteria
Biocatalysts
Carbamoylases
Catalysis
Directed Evolution
Enzymatic activity
Enzymes
Evolution
Genetic-Code Expansion
Growth rate
Humans
In Vivo Selection
Precursors
Proenzymes
Synthetic Biology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non‐canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild‐type enzyme for ncAA‐precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts. An in vivo selection strategy is presented, in which bacteria addicted to non‐canonical amino acids (ncAAs) are complemented by enzymes that can yield these building blocks from synthetic precursors. As growth rates under selective conditions correlate with enzyme activities, serial passaging elicited better biocatalysts from populations harboring enzyme libraries. The platform was used to improve the activity of carbamoylases for ncAA‐precursors.
ISSN:1433-7851
1521-3773
1521-3773
DOI:10.1002/anie.202213942