Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene...

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Growing demand for sustainable chemical and fuel production necessitates advanced biological fermentation processes.
• Thermophilic microorganisms offer advantages for industrial bioprocesses but require enhanced genome editing tools for effective utilization.
• Existing CRISPR/Cas9 gene editing in *Parageobacillus thermoglucosidasius* is hampered by constitutive Cas9 expression, leading to low efficiency and off-target mutations.

Purpose of the Study:

• To develop a controllable and efficient CRISPR/Cas9 gene editing system for *Parageobacillus thermoglucosidasius*.
• To mitigate the toxicity and off-target effects associated with Cas9 expression in this thermophilic bacterium.
• To enhance the efficiency of mutant generation for industrial applications.

Main Methods:

• Engineered a synthetic riboswitch responsive to theophylline to control Cas9 expression.
• Developed an inducible CRISPR/Cas9 system named RiboCas93.
• Assessed the dose-dependent control of gene expression by the riboswitch.
• Evaluated transformation efficiency, editing efficiency, and off-target mutations (SNPs).

Main Results:

• Demonstrated dose-dependent control of Cas9 expression using the theophylline-inducible riboswitch.
• Achieved 100% efficiency in mutant generation.
• Significantly increased transformation efficiency of editing vectors.
• Reduced Cas9 toxicity, evidenced by a decrease in observed single nucleotide polymorphisms (SNPs).

Conclusions:

• RiboCas93 provides an efficient and controllable method for genome editing in *Parageobacillus thermoglucosidasius*.
• The inducible system overcomes limitations of constitutive Cas9 expression, enhancing industrial biomanufacturing potential.
• This advancement facilitates rapid and precise mutant generation in thermophilic bacteria.