Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Directed evolution is crucial for engineering biomolecules and understanding adaptation.
• Current methods are labor-intensive and low-throughput, limiting complex functional studies.
• Replicate studies of molecular evolution are challenging with existing techniques.

Purpose of the Study:

• To develop a high-throughput, scalable system for directed evolution in vivo.
• To overcome limitations of existing experimental strategies for biomolecular engineering.
• To enable routine, rapid evolution of cellular function.

Main Methods:

• Development of OrthoRep, an orthogonal DNA polymerase-plasmid pair in yeast.
• Achieving mutation rates ~100,000-fold higher than the host genome.
• Utilizing serial passaging for continuous and rapid gene evolution.

Main Results:

• Successfully evolved drug-resistant malarial dihydrofolate reductases (DHFRs) in 90 independent replicates.
• Revealed a complex fitness landscape with common adaptive trajectories and epistatic constraints.
• Identified rare adaptive outcomes and a suboptimal fitness peak impacting evolution.

Conclusions:

• OrthoRep provides a straightforward, scalable, and high-throughput platform for directed evolution.
• Enables routine investigation of complex evolutionary dynamics and fitness landscapes.
• Opens new avenues for engineering biomolecular and cellular functions.