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Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi).

Sara Cleto1,2, Jaide Vk Jensen1,2,3, Volker F Wendisch3

  • 1Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

ACS Synthetic Biology
|February 2, 2016
PubMed
Summary

CRISPR interference (CRISPRi) technology efficiently represses genes in Corynebacterium glutamicum, enhancing amino acid production. This rapid metabolic engineering method offers an alternative to traditional gene deletion techniques.

Keywords:
C. glutamicumCRISPRiamino acidmetabolic engineeringsgRNA/dCas9

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Area of Science:

  • Microbial biotechnology
  • Metabolic engineering
  • Synthetic biology

Background:

  • Corynebacterium glutamicum is a key industrial microorganism for amino acid production.
  • Conventional metabolic engineering relies on homologous recombination, which is often inefficient due to rare double-crossover events.

Purpose of the Study:

  • To apply CRISPR interference (CRISPRi) technology for efficient gene repression in C. glutamicum.
  • To assess the impact of CRISPRi-mediated gene silencing on amino acid production titers.
  • To establish a rapid and effective metabolic engineering strategy for C. glutamicum.

Main Methods:

  • Utilized CRISPR interference (CRISPRi) with deactivated Cas9 (dCas9) to repress target genes (pgi, pck, pyk) in C. glutamicum.
  • Quantified gene expression changes and determined the effects on l-lysine and l-glutamate production.
  • Compared CRISPRi results with traditional gene deletion methods.

Main Results:

  • CRISPRi successfully repressed pgi and pck gene expression by up to 98% and pyk by up to 97%.
  • Achieved l-lysine and l-glutamate titer enhancement ratios comparable to those obtained via gene deletion.
  • Demonstrated a 3-day workflow for metabolic pathway remodeling.

Conclusions:

  • CRISPRi technology provides a rapid and efficient method for metabolic engineering in C. glutamicum.
  • This approach enables effective pathway remodeling without the need for gene deletions or mutations.
  • CRISPRi facilitates the mapping of gene expression to metabolic outputs, accelerating strain development.