Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in Bacillus subtilis

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Bacterial protein production is hindered by low expression and poor induction control.
• The Bacillus subtilis ComQXPA quorum sensing system typically activates the P_srfA promoter.

Purpose of the Study:

• To engineer flexible autoinduction expression systems in Bacillus subtilis.
• To enhance protein yield and control induction timing for recombinant protein production.

Main Methods:

• Modified the P_srfA promoter's -35 region with conserved σA-dependent sequences to create P₁.
• Applied semi-rational design to the P₁ promoter's spacer region to develop the P_S1E promoter.
• Optimized ComX and ComA promoter combinations to establish autoinduction systems with varied induction initiation times.

Main Results:

• The P₁ promoter showed an 85% increase in strength compared to P_srfA.
• The engineered P_S1E promoter exhibited an 8.22-fold higher expression level than P_srfA.
• Developed three autoinduction systems with induction initiation ranging from 1.5 to 9.5 hours.
• Achieved 80.2 U/mL of Bacillus deramificans pullulanase, a 36% improvement over the P₅₆₆ constitutive promoter.

Conclusions:

• Engineered flexible autoinduction expression systems in Bacillus subtilis WB600.
• Demonstrated enhanced protein expression and controllable induction dynamics.
• Highlighted the potential of these systems for improving protein and metabolite production in bacteria.