Rational Design Of The N-Terminal Coding Sequence For Regulating Enzyme Expression In Bacillus Subtilis

Synonymous mutation of the N-terminal coding sequence (NCS) has been used to regulate gene expression. We here developed a statistical model to predict the effect of the NCSs on protein expression in Bacillus subtilis WB600. First, a synonymous mutation was performed within the first 10 residues of a superfolder green fluorescent protein to generate a library of 172 NCS synonymous mutants with different expression levels. A prediction model was then developed, which adopted G/C frequency at the third position of each codon and minimum free energy of mRNA as the independent variables, using multiple regression analysis between the 11 sequence parameters of the NCS and their fluorescence intensities. By designing the NCS of the 10 signal peptides de novo according to the model, the extraceliular yield of B. subtilis pullulanase fused to each signal peptide was up-regulated by up to 515% or down-regulated by at most 79%. This work provided a candidate tool for fine-tuning gene expression or enzyme production in B. subtilis.