Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in Bacillus subtilis

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Synonymous mutations in N-terminal coding sequences (NCS) are utilized to control gene expression.
• Predicting the impact of NCS on protein expression levels is crucial for optimizing biotechnological applications.

Purpose of the Study:

• To develop a statistical model for predicting the effect of NCS on protein expression in Bacillus subtilis WB600.
• To utilize the model for enhancing extracellular enzyme production.

Main Methods:

• Generated a library of 172 NCS synonymous mutants of superfolder green fluorescent protein.
• Developed a prediction model using multiple regression analysis with G/C frequency and minimum free energy as variables.
• Designed novel NCS for 10 signal peptides based on the developed model.

Main Results:

• The prediction model accurately correlated NCS sequence parameters with fluorescence intensity.
• Designing NCS de novo using the model resulted in significant up-regulation (up to 515%) or down-regulation (up to 79%) of extracellular pullulanase yield in B. subtilis.
• Demonstrated successful fine-tuning of gene expression and enzyme production.

Conclusions:

• A predictive statistical model for NCS effects on protein expression in B. subtilis was successfully developed.
• The model serves as a valuable tool for optimizing gene expression and enhancing enzyme production in B. subtilis.
• This approach offers a candidate strategy for precise control of protein yields in microbial systems.