Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system

  • Hui Wang
  • Xin Zhang
  • Jin Qiu
  • Kaikai Wang
  • Kun Meng
  • Huiying Luo
  • Xiaoyun Su
  • Rui Ma
  • Huoqing HuangEmail author
  • Bin YaoEmail author
Genetics and Molecular Biology of Industrial Organisms - Original Paper


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.


Bacillus amyloliquefaciens High-level expression system Secretory expression cassette Gene inactivation General applicability 


B. amyloliquefaciens

Bacillus amyloliquefaciens

B. clausii

Bacillus clausii

B. subtilis

Bacillus subtilis

E. coli

Escherichia coli


Amylase from B. amyloliquefaciens BF. 7658


Neutral protease from B. amyloliquefaciens K11


Alkaline protease from B. clausii


Base pair


Dissolved oxygen


Generally recognized as safe




Mimicking-of-DNA-methylation-patterns pipeline




Polymerase chain reaction


Prolonged overlap extension-PCR


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Single-chain antibody


Trichloroacetic acid



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.

Supplementary material

10295_2018_2089_MOESM1_ESM.doc (3.4 mb)
Supplementary material 1 (DOC 3443 kb)


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • Hui Wang
    • 1
  • Xin Zhang
    • 1
  • Jin Qiu
    • 1
  • Kaikai Wang
    • 1
  • Kun Meng
    • 1
  • Huiying Luo
    • 1
  • Xiaoyun Su
    • 1
  • Rui Ma
    • 1
  • Huoqing Huang
    • 1
    Email author
  • Bin Yao
    • 1
    Email author
  1. 1.Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research InstituteChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China

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