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Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes.

Jian Wang1, Chenyi Li1, Tian Jiang1

  • 1School of Chemical, Materials and Biomedical Engineering, College of Engineering, The University of Georgia, Athens, GA, 30602, USA.

Metabolic Engineering
|November 14, 2022
PubMed
Summary
This summary is machine-generated.

We developed a biosensor-assisted titratable CRISPR interference (BATCH) screening approach for microbial bioproduction. This method optimizes metabolic engineering and accelerates high-throughput screening, enhancing production of valuable compounds.

Keywords:
BiosensorButyrateMismatch CRISPRiTitratable repressionp-Coumaric acid

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Area of Science:

  • Synthetic Biology
  • Metabolic Engineering
  • Microbial Bioproduction

Background:

  • Rewiring metabolic fluxes and high-throughput screening are key challenges in microbial bioproduction.
  • Advances in synthetic biology offer new tools for metabolic engineering.

Purpose of the Study:

  • To develop a biosensor-assisted titratable CRISPR interference (BATCH) screening approach.
  • To enable optimal rewiring of metabolic fluxes and accelerate high-throughput phenotype screening in Escherichia coli.

Main Methods:

  • Developed a programmable mismatch CRISPR interference system with a one-pot sgRNA pool for tunable gene knockdown.
  • Designed doubly mismatched sgRNA pools targeting 20 genes in E. coli.
  • Optimized PadR-based and HpdR-based biosensors for p-coumaric acid and butyrate production screening, respectively.
  • Utilized eGFP fluorescence for high-production phenotype screening.

Main Results:

  • Achieved a full range of gene knockdown efficacy using mismatch CRISPR interference.
  • Increased p-coumaric acid titer by 40.6% to 1308.6 mg/l by targeting pfkA and ptsI.
  • Increased butyrate titer by 19.0% and 25.2% by targeting sucA and ldhA, respectively.
  • Demonstrated the general applicability of the BATCH screening system for different compounds.

Conclusions:

  • Established a plug-and-play approach for multilevel modulation of metabolic fluxes.
  • Enabled high-throughput screening of high-production microbial strains.
  • The BATCH system significantly enhances metabolic engineering and bioproduction efficiency.