Overexpression of a Thermostable α-Amylase through Genome Integration in <i>Bacillus subtilis</i>

A carbohydrate binding module 68 (CBM68) of pullulanase from <i>Anoxybacillus</i> sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in <i>Bacillus subtilis</i>, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain <i>B. subtilis</i> 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of <i>B. subtilis</i> 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by <i>B. subtilis</i> 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of <i>B. subtilis</i> 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702.