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We developed lytic selection and evolution (LySE), a novel method for bacterial gene cluster evolution. LySE achieves high mutation rates with controlled selection, accelerating directed evolution for biotechnology applications.

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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.