Improved subtilisin YaB production in Bacillus subtilis using engineered synthetic expression control sequences.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Alkaline elastase YaB, a subtilisin-type protease from Bacillus YaB, is a valuable meat tenderizer.
• Previous attempts to express YaB in Bacillus subtilis yielded low protein levels compared to the parental strain.

Purpose of the Study:

• To investigate the impact of various expression control sequences on alkaline elastase YaB production in Bacillus subtilis.
• To identify optimal synthetic expression control sequences (SECSs) for enhanced protein yield.

Main Methods:

• Cloning and expression of the alkaline elastase YaB gene (ale) in Bacillus subtilis.
• Utilizing native B. subtilis expression control sequences (cdd, veg), a synthetic expression control sequence (SECS), and engineered SECSs.
• Engineering SECSs via Polymerase Chain Reaction, modifying UP elements and Shine-Dargarno (SD) sequences.

Main Results:

• Engineered SECSs and a standard SECS showed higher expression efficiencies than native control sequences (ale, cdd, veg).
• Substitution of the SD sequence in SECS led to greater YaB expression than UP element substitution.
• Combined substitution of both UP element and SD sequence in engineered SECSs resulted in the highest YaB expression levels.

Conclusions:

• Engineering synthetic expression control sequences is an effective method for improving alkaline elastase YaB production in Bacillus subtilis.
• Optimized engineered SECSs can achieve high-level expression of homologous and heterologous proteins in Bacillus subtilis.