Selecting Better Biocatalysts by Complementing Recoded Bacteria

• 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
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202213942

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.