Selecting Better Biocatalysts by Complementing Recoded Bacteria

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• In vivo selections are crucial for directed enzyme evolution but challenging for non-metabolic enzymes.
• Linking enzymatic activity to cellular survival is a key limitation.

Purpose of the Study:

• To develop a versatile in vivo selection strategy for evolving biocatalysts.
• To engineer enzymes providing non-canonical amino acids (ncAAs) from synthetic precursors.

Main Methods:

• Utilized recoded organisms dependent on ncAAs.
• Employed serial passaging under selective conditions.
• Engineered carbamoylases for ncAA precursor conversion.

Main Results:

• Achieved catalytic efficiencies over five orders of magnitude higher than wild-type enzymes.
• Demonstrated correlation between bacterial growth rates and enzymatic activity.
• Successfully evolved improved enzyme variants with minimal intervention.

Conclusions:

• The developed platform enables efficient in vivo directed evolution of biocatalysts.
• The strategy is versatile, requiring minimal human intervention and no specialized equipment.
• This method facilitates the evolution of enzymes for producing essential building blocks like ncAAs.