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Redirecting Metabolic Flux in E. Coli via Combinatorial CRISPRi-Mediated Repression for Isopentenol Production
Published: January 25, 2019
Posted: August 02, 2021

Objective

Use CRISPR interference (CRISPRi) to knock down endogenous genes in competing pathways to redirect metabolic flux toward target metabolite.

Approach

CRISPRi-mediated multiplex repression system to silence transcription of several endogenous genes to increase precursor availability in a heterologous isopentenol biosynthesis pathway.

Results/Impacts

  • Designed a single-gRNA library with 15 individual knockdown targets, where 3 gRNA cassettes targeting genes asnA, prpE, and gldA increased isopentenol titer by 18% to 24%.
  • Combined the three single-gRNA cassettes into a two- or three-gRNA array and observed up to 98% enhancement in production by fine-tuning the repression level through titrating dCas9 expression.
  • Strategy shows that multiplex combinatorial knockdown of competing genes using CRISPRi can increase production of the target metabolite.

Tian, T. et al. “Redirecting metabolic flux via combinatorial multiplex CRISPRi-mediated repression for isopentenol production in Escherichia coli.” ACS Synthetic Biology 8(2), 391–402 (2019). [DOI:10.1021/acssynbio.8b00429]

Topic Areas:

  • Research Area: Genomic Analysis and Systems Biology
  • Research Area: Microbes and Communities
  • Research Area: Sustainable Biofuels and Bioproducts
  • Research Area: DOE Bioenergy Research Centers (BRC)

Division: SC-33.2 Biological Systems Science Division, BER

 

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