Synthetic N-terminal coding sequences for fine-tuning gene expression and metabolic engineering in Bacillus subtilis

N-terminal coding sequences (NCSs) of genes significantly influence gene expression at the translation level and are important for fine-tuning gene expression in bacteria, however, engineering NCSs to fine-tune metabolic pathways is challenging. Here, we developed a statistics-guided native and synthetic NCSs engineering approach to fine-tune gene expression in the industrially important microorganism Bacillus subtilis. This method is based on experimentally characterizing and statistically analyzing 96 rationally selected NCSs from B. subtilis endogenous genes. These NCSs exhibited a magnitude difference of greater than 4 orders in their ability to drive gene expression in 4 different dynamic patterns, including growth-coupled, growth-delayed, consistent expression, and inhibitory patterns. Synthetic and native NCSs were used to fine-tune expression of key enzymes, identified via pathway analysis and kinetic modeling, in the biosynthetic pathway of the useful nutraceutical N-acetylneuraminic acid (NeuAc). We observed a 3.21-fold improvement in NeuAc biosynthesis, indicating that NCSs can provide a synthetic biology toolbox to fine-tune gene expression for metabolic engineering.