Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system
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Bacillus amyloliquefaciens K11 is a hyperproducer of extracellular neutral protease, which can produce recombinant homologous protein steadily and is amenable to scale up to high-cell density fermentation. The present study aims to genetically modify strain K11 as a highly efficient secretory expression system for high-level production of heterologous proteins. Using B. amyloliquefaciens K11 and alkaline protease gene BcaprE as the expression host and model gene, the gene expression levels mediated by combinations of promoters PamyQ, PaprE and Pnpr and signal peptides SPamyQ, SPaprE and SPnpr were assessed on shake flask level. The PamyQ-SPaprE was found to be the best secretory expression cassette, giving the highest enzyme activities of extracellular BcaprE (13,800 ± 308 U/mL). Using the same expression system, the maltogenic α-amylase Gs-MAase and neutral protease BaNPR were successfully produced with the enzyme activities of 19. ± 0.2 U/mL and 17,495 ± 417 U/mL, respectively. After knocking out the endogenous neutral protease-encoding gene Banpr, the enzyme activities of BcaprE and Gs-MAase were further improved by 25.4% and 19.4%, respectively. Moreover, the enzyme activities of BcaprE were further improved to 30,200 ± 312 U/mL in a 15 L fermenter following optimization of the fermentation conditions. In the present study, the genetically engineered B. amyloliquefaciens strain 7-6 containing PamyQ-SPaprE as the secretory expression cassette was developed. This efficient expression system shows general applicability and represents an excellent industrial strain for the production of heterologous proteins.
KeywordsBacillus amyloliquefaciens High-level expression system Secretory expression cassette Gene inactivation General applicability
- B. amyloliquefaciens
- B. clausii
- B. subtilis
- E. coli
Amylase from B. amyloliquefaciens BF. 7658
Neutral protease from B. amyloliquefaciens K11
Alkaline protease from B. clausii
Generally recognized as safe
Polymerase chain reaction
Prolonged overlap extension-PCR
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
This research was supported by the National Key R&D Program of China (2016YFD0501409-02), the National Science Foundation for Young Scientists of China (31702149), and the China Modern Agriculture Research System (CARS-42).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
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