Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection

Background: Our laboratory has constructed a Bacillus stearothermophilus alpha-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus alpha-amylase in B. subtilis is very low. Results: In this study, the extracellular production level of B. stearothermophilus alpha-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and alpha-amylase mutant selection. The alpha-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular alpha-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased alpha-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in alpha-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92h, the alpha-amylase activity of the culture supernatant was 9201.1UmL(-1), which is the highest level that has been reported to date. Conclusions: This is the first report that describes an improvement of B. stearothermophilus alpha-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for alpha-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of alpha-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.