In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection

Area of Science:

• Biotechnology
• Enzyme Engineering
• Directed Evolution

Background:

• Growth-based screening is crucial for identifying improved biocatalysts in directed evolution.
• High-throughput measurement of subtle growth differences is challenging due to metabolic complexity.

Purpose of the Study:

• To miniaturize and enhance a growth-based directed enzyme evolution process for high-throughput screening.
• To develop a method for accurately assessing enzyme performance through linked growth behavior.

Main Methods:

• Utilized optically clear, nanoliter-volume microcarriers for clone cultivation.
• Employed fluorescence-assisted particle sorting to quantify growth behavior of microcarrier-embedded clones.
• Incorporated an antimetabolite to modulate selection stringency.

Main Results:

• Demonstrated successful miniaturization of growth-based directed evolution.
• Established a correlation between observed growth behavior and enzyme performance improvements.
• Successfully identified moderately improved enzyme variants using the developed assay.

Conclusions:

• Miniaturized growth-based screening in microcarriers enables high-throughput directed evolution.
• This approach allows for precise discrimination of enzyme variants based on performance-linked growth.
• The method is applicable for optimizing enzymes, such as amino acid racemases for synthesis applications.