Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Area of Science:

• Metabolic engineering
• Synthetic biology
• Microbial bioproduction

Background:

• Metabolic gene expression regulation is key for optimizing bioproduction yields.
• CRISPR interference (CRISPRi) offers precise gene repression for strain engineering.
• Isoprenol is a valuable bioproduct and precursor for sustainable aviation fuel.

Purpose of the Study:

• To apply CRISPRi for downregulating genes involved in isoprenol biosynthesis pathways in E. coli.
• To enhance isoprenol production titers through multiplexed gene downregulation.
• To assess the scalability of CRISPRi-based metabolic engineering for industrial applications.

Main Methods:

• Utilized CRISPR interference (CRISPRi) with deactivated Cas9 (dCas9) and guide RNA (gRNA) arrays.
• Downregulated 32 essential and non-essential genes in E. coli strains engineered for isoprenol production.
• Constructed a multiplexed gRNA library and performed fed-batch cultivation at 2-L scale.

Main Results:

• Identified specific gRNAs that significantly improved isoprenol titers.
• Achieved a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) in minimal medium.
• Demonstrated scalable titer improvements, reaching 12.4 ± 1.3 g/L isoprenol in fed-batch cultivation.

Conclusions:

• CRISPRi is an effective tool for tuning metabolic flux in microbial hosts.
• Multiplexed gene repression via CRISPRi enhances bioproduct titers.
• The developed strategy is scalable and holds potential for industrial bioproduction.