CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum.

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Metabolic engineering

Background:

• Corynebacterium glutamicum is a key industrial microorganism for producing valuable compounds.
• Gene repression using dCas9/CRISPR interference (CRISPRi) is a powerful tool for identifying metabolic engineering targets.
• Understanding gene function is crucial for optimizing microbial production hosts.

Purpose of the Study:

• To construct and utilize a CRISPRi library targeting 74 genes in C. glutamicum.
• To identify genes influencing the biosynthesis of the carotenoid pigment decaprenoxanthin.
• To validate CRISPRi findings through gene deletion and assess their potential for metabolic engineering.

Main Methods:

• Construction of a CRISPRi library targeting 74 genes in Corynebacterium glutamicum.
• Screening the library to identify genes affecting decaprenoxanthin biosynthesis.
• Confirmation of CRISPRi results via gene deletion experiments.

Main Results:

• CRISPRi-mediated repression of the carotenogenesis repressor gene crtR increased decaprenoxanthin production.
• CRISPRi screening identified 14 genes affecting decaprenoxanthin biosynthesis; 11 decreased and 3 increased production.
• Gene deletions of pgi and gapA significantly improved decaprenoxanthin levels (43-fold and 9-fold, respectively).

Conclusions:

• The developed CRISPRi library is effective for identifying metabolic engineering targets in C. glutamicum.
• Gene repression can be successfully translated into stable gene deletions for enhanced compound production.
• This approach provides valuable insights into optimizing carotenoid biosynthesis pathways.