Systematic engineering of transport and transcription to boost alkaline α-amylase production in Bacillus subtilis.

In the present work, we used systematic engineering at transport and transcription levels to significantly enhance alkaline α-amylase production in Bacillus subtilis 168M. Signal peptide YwbN' proved to be optimal. Alkaline α-amylase production was elevated by deleting a putative peptide segment of YwbN'. Insertion of arginine (R) between residues 5 and 6 of YwbN'∆p further increased the protein yield. Enhancing positive charges at sites 4 and 10 and decreasing the hydrophobicity of the H-region of YwbN'∆p were critical for improving alkaline α-amylase production in B. subtilis 168M. PHpaII was the optimal promoter, and deleting - 27T or - 31A from PHpaII enhanced the transcription of the target gene. Using a single-pulse feeding-based fed-batch system, alkaline α-amylase activity of B. subtilis 168M P∆-27T was increased by 250.6-fold, compared with B. subtilis 168M A1.