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Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems
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Adaptive cargo deletion as an evolutionary dead-end during phage-based directed evolution.

Zidan Li1, Ibrahim Al'Abri1, Nathan Crook2

  • 1Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695, USA.

BMC Research Notes
|June 19, 2026
PubMed
Summary
This summary is machine-generated.

Directed evolution using P1 phage improved pH tolerance in Escherichia coli Nissle by mutating sigma factors. However, plasmid deletion led to an evolutionary dead end, highlighting challenges in continuous directed evolution.

Keywords:
E. coli Nissle 1917Acid toleranceEvolutionary escapeGlobal transcriptional machinery engineering (gTME)Inducible directed evolution (IDE)

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Published on: September 1, 2018

Area of Science:

  • Microbiology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Improving microbial pH tolerance is crucial for industrial applications.
  • Global transcriptional machinery engineering (gTME) offers a pathway to enhance microbial traits.
  • Previous gTME methods focused on single sigma factor evolution.

Purpose of the Study:

  • To evaluate the P1 phage-based inducible directed evolution (IDE) method for gTME.
  • To enhance pH tolerance in Escherichia coli Nissle by simultaneously mutating key sigma factors.
  • To assess the reliability of IDE for microbial strain improvement.

Main Methods:

  • Simultaneous mutagenesis of three sigma factors (rpoD, rpoN, rpoH) in E. coli Nissle.
  • Five rounds of mutagenesis and growth-based selection at pH 4.2 using P1 phage.
  • Transcriptome analysis to understand fitness mechanisms.

Main Results:

  • Early mutations in rpoD (V98E, R603C) reduced lag phase under acidic conditions.
  • Library improvement plateaued after three cycles, with subsequent plasmid deletion dominating.
  • Plasmid deletion enhanced low-pH fitness but created an evolutionary dead end, halting further adaptation.

Conclusions:

  • The P1 phage-based IDE method can improve E. coli pH tolerance.
  • Simultaneous sigma factor evolution and subsequent plasmid deletion present evolutionary limitations.
  • Continuous directed evolution strategies must account for potential evolutionary dead ends caused by substrate changes.