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

Background: A raw starch-degrading α-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-P 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). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-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 α-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.