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Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection
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Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection
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Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection
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Journal Article
Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection
2019
Overview
Background
Our laboratory has constructed a
Bacillus stearothermophilus
α-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
α-amylase in
B. subtilis
is very low.
Results
In this study, the extracellular production level of
B. stearothermophilus
α-amylase in
B. subtilis
was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SP
YojL
) was obtained by screening 173
B. subtilis
signal peptides. Although the extracellular α-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 α-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 SP
YojL
while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL
−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
α-amylase extracellular production levels in
B. subtilis
using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from
B. stearothermophilus
in
B. subtilis
. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in
B. subtilis
.
Publisher
BioMed Central,Springer Nature B.V,BMC
Subject
/ Amylases
/ Bacillus subtilis - genetics
/ Bacillus subtilis - metabolism
/ Bacterial Proteins - metabolism
/ Chemistry and Materials Science
/ E coli
/ Enzymes
/ Geobacillus stearothermophilus - enzymology
/ Geobacillus stearothermophilus - genetics
/ High-cell-density fermentation
/ Microbial Genetics and Genomics
/ Molecular Chaperones - genetics
/ Molecular Chaperones - metabolism
/ Mutant
/ Mutation
/ Peptides
/ Proteins
