Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis through signal peptide and translation efficiency optimization

A strategy that combines signal peptide (SP) optimization and translation efficiency improvement was used to enhance the extracellular production of raw starch-degrading α-amylase AmyZ1 from Pontibacillus sp. ZY in Bacillus subtilis. The optimal SPYpuA screened from 173 B. subtilis SPs mediated a 1.28-fold higher AmyZ1 activity than the original SPamyQ. Then, strategies of translation efficiency improvement, including optimizing the Shine-Dalgarno-like (SD-like) sequence, constructing a dual 5′-untranslated region (dual-UTR), optimizing the spacer region of the UTR, and optimizing the 5′-proximal coding sequence, were used to enhance AmyZ1 production. The resulting recombinant strain (BZYACO6) containing SD-like3, dUTR2, and an optimized 5′-proximal coding sequence (O6) produced the highest extracellular α-amylase activity of 2974.0 U/mL in shake flask, 2.07-fold higher than that of the original strain BZd34 (1437.6 U/mL). After fermentation optimization, the BZYACO6 strain produced an extracellular AmyZ1 activity of 3915.2 U/mL in shake flask and 25,070 U/mL in 3-L fermenter, representing the highest level of recombinant raw starch-degrading α-amylase production reported to date. Furthermore, this work provides a strategic reference for enhancing the extracellular production of other recombinant proteins in B. subtilis.