Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis

Area of Science:

• Synthetic Biology
• Microbial Biotechnology
• Genomics

Background:

• Multiplex genome engineering is crucial for synthetic biology and biotechnology.
• Existing methods for microbial genome engineering are often complex and require multiple transformation cycles.
• Natural transformation offers a potential avenue for simpler genetic manipulation.

Purpose of the Study:

• To develop a simple, rapid, and efficient method for simultaneous multiplex genome engineering (SMGE) in Bacillus subtilis.
• To leverage natural transformation for one-step bacterial genome design.
• To create a platform for accelerated evolution and large-scale genetic engineering.

Main Methods:

• Adapted transformed DNA, competency factors, and recombinases for enhanced co-editing frequencies.
• Utilized all-in-one vectors with multi-gene cassettes for simultaneous variant generation.
• Applied high-throughput screening to optimize the tyrosine biosynthesis pathway for resveratrol production.

Main Results:

• Achieved over 27-fold improvement in co-editing frequencies.
• Successfully generated single to octuplet variants with high genetic diversity in one step.
• Demonstrated the application of SMGE in optimizing a metabolic pathway for commercial compound production.

Conclusions:

• Simultaneous multiplex genome engineering (SMGE) provides a streamlined approach for bacterial genome modification.
• This method accelerates the generation of diverse genetic variants for synthetic biology and biotechnology.
• SMGE in Bacillus subtilis facilitates rapid strain development and optimization for industrial applications.