Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization

Background A raw starch-degrading alpha-amylase from Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins. Results AmyZ1 secretory production was achieved in B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter P-spoVG-P-spoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (P-groE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular alpha-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL.h. Conclusions This is the first report of recombinant expression of AmyZ1 in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading alpha-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.