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