Bridging continuous and discrete evolution through a controllable, hypermutagenic phage-bacteria system.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Directed evolution methods often present a trade-off between control and throughput.
• Continuous evolution systems offer high throughput but limited control over discrete genetic elements.

Purpose of the Study:

• To engineer a novel method, lytic selection and evolution (LySE), for near-continuous evolution of bacterial gene clusters.
• To balance the speed of continuous systems with the control of discrete approaches in directed evolution.

Main Methods:

• Developed a hypermutagenic T7 DNA polymerase variant fused to a dual adenine-cytosine deaminase for high-frequency transition mutations.
• Implemented a biocontainment strategy using a T7 DNA polymerase-lacking phagemid and an accessory plasmid.
• Utilized alternating cycles of lysis and transduction for selective gene replication and mutagenesis.

Main Results:

• Achieved mutation rates of 3.82 × 10^-5 substitutions per base.
• Evolved a 25-fold increase in tigecycline resistance in 5 cycles.
• Increased endpoint biomass by 50.9% in a bacterial strain utilizing ethylene glycol as a sole carbon source.

Conclusions:

• LySE effectively balances speed and control for directed bacterial evolution.
• The method enables rapid optimization of bacterial gene clusters for desired traits.
• LySE has potential applications in accelerating strain development for industrial biotechnology.