[A CRISPR/dCpf1-based transcriptional repression system for Gluconobacter oxydans]

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Gluconobacter oxydans is industrially significant for carbohydrate oxidation but limited by gene manipulation tools.
• Efficient genome editing and transcriptional regulation are crucial for optimizing G. oxydans industrial applications.

Purpose of the Study:

• To develop a novel CRISPR/dCpf1-mediated transcriptional repression system for Gluconobacter oxydans.
• To overcome limitations in current gene regulation methods for G. oxydans metabolic engineering.

Main Methods:

• Constructed a CRISPR/dCpf1 system expressing nuclease-inactivated Cpf1 protein (dCpf1) in G. oxydans.
• Utilized a 19 nt direct repeat sequence for effective gene transcriptional repression.
• Applied the system for single and multiplex gene repression, and metabolic pathway regulation.

Main Results:

• Achieved effective repression of gene transcription, with a relative repression level up to 97.9% for single gene repression.
• Demonstrated successful multiplex gene repression capabilities.
• Successfully applied the system to regulate the L-sorbose metabolic pathway and respiratory chain.

Conclusions:

• The developed CRISPR/dCpf1 system provides an efficient gene manipulation tool for G. oxydans.
• This system addresses the shortage of effective gene regulation methods, facilitating metabolic engineering.
• The tool is suitable for both single and multiplex gene repression in industrial G. oxydans strains.