Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR-AsCpf1 system

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Bacillus subtilis is a versatile microbial chassis for producing valuable chemicals.
• Iterative genome editing is crucial for optimizing microbial strains but can be challenging.
• Existing genome editing tools may lack efficiency or require complex procedures.

Purpose of the Study:

• To develop and validate a novel CRISPR-AsCpf1-based system for efficient and iterative genome editing in Bacillus subtilis.
• To demonstrate the system's utility for genetic modifications related to hyaluronic acid biosynthesis.
• To engineer a B. subtilis strain for heterologous hyaluronic acid production.

Main Methods:

• Construction of CRISPR-AsCpf1 expression plasmids utilizing a temperature-sensitive replicon.
• Application of the system for gene insertion (hasA, tuaD) and large genomic deletion (eps gene cluster).
• Assessment of editing efficiency and plasmid curing for iterative modifications.

Main Results:

• Achieved high genome editing efficiencies (80%-100%) for gene insertion and deletion in B. subtilis 168.
• Successfully implemented iterative genome engineering for hyaluronic acid (HA) biosynthesis pathway.
• Engineered B. subtilis strain produced 1.39 g/L of heterologous hyaluronic acid.

Conclusions:

• The developed CRISPR-AsCpf1 system offers a highly efficient and rapid method for bacterial genome engineering.
• This system provides valuable tools and guidance for optimizing B. subtilis for industrial applications.
• The successful HA production demonstrates the system's potential for metabolic engineering and chemical synthesis.