Advertisement

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
  • 300 Downloads

Abstract

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.

Keywords

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

Abbreviations

B. amyloliquefaciens

Bacillus amyloliquefaciens

B. clausii

Bacillus clausii

B. subtilis

Bacillus subtilis

E. coli

Escherichia coli

amyQ

Amylase from B. amyloliquefaciens BF. 7658

BaNPR

Neutral protease from B. amyloliquefaciens K11

BcaprE

Alkaline protease from B. clausii

bp

Base pair

DO

Dissolved oxygen

GRAS

Generally recognized as safe

LB

Luria-Bertani

MoDMP

Mimicking-of-DNA-methylation-patterns pipeline

MTase

Methyltransferase

PCR

Polymerase chain reaction

POE-PCR

Prolonged overlap extension-PCR

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SCA

Single-chain antibody

TCA

Trichloroacetic acid

Notes

Acknowledgements

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)

References

  1. 1.
    Cai D, Wei X, Qiu Y, Chen Y, Chen J, Wen Z, Chen S (2016) High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase. J Appl Microbiol 121:704–712CrossRefGoogle Scholar
  2. 2.
    Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut PR, Vater J, Süssmuth R, Liesegang H, Strittmatter A, Gottschalk G, Borriss R (2007) Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol 25:1007–1014CrossRefGoogle Scholar
  3. 3.
    Feng J, Gu Y, Quan Y, Zhang W, Cao M, Gao W, Song C, Yang C, Wang S (2015) Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens. Sci Rep 5:13814CrossRefGoogle Scholar
  4. 4.
    Gu Y, Zheng J, Feng J, Cao M, Gao W, Quan Y, Dang Y, Wang Y, Wang S, Song C (2017) Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements. Appl Microbiol Biotechnol 101:4163–4174CrossRefGoogle Scholar
  5. 5.
    Guan C, Cui W, Cheng J, Liu R, Liu Z, Zhou L, Zhou Z (2016) Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis. Nat Biotechnol 33:372–379Google Scholar
  6. 6.
    Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res Int 2013:329121CrossRefGoogle Scholar
  7. 7.
    Harwood CR, Cranenburgh R (2008) Bacillus protein secretion: an unfolding story. Trends Microbiol 16:73–79CrossRefGoogle Scholar
  8. 8.
    Küppers T, Steffen V, Hellmuth H, O’Connell T, Bongaerts J, Maurer KH, Wiechert W (2014) Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer. Microb Cell Fact 13:46CrossRefGoogle Scholar
  9. 9.
    Liao Y, Huang L, Wang B, Zhou F, Pan L (2015) The global transcriptional landscape of Bacillus amyloliquefaciens XH7 and high-throughput screening of strong promoters based on RNA-seq data. Gene 571:252–262CrossRefGoogle Scholar
  10. 10.
    Liao Y, Wang B, Ye Y, Pan L (2018) Determination and optimization of a strong promoter element from Bacillus amyloliquefaciens by using a promoter probe vector. Biotechnol Lett 40:119–126CrossRefGoogle Scholar
  11. 11.
    Martin G, Simon S, Rebekka B, David F, Yang Y, Dieter J (2007) Bacillus megaterium—an alternative expression system. J Biotechnol 131S:S220Google Scholar
  12. 12.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  13. 13.
    Mizukami M, Hanagata H, Miyauchi A (2010) Brevibacillus expression system: host-vector system for efficient production of secretory proteins. Curr Pharm Biotechnol 11:251–258CrossRefGoogle Scholar
  14. 14.
    Nijland R, Kuipers OP (2008) Optimization of protein secretion by Bacillus subtilis. Recent Pat Biotechnol 2:79–87CrossRefGoogle Scholar
  15. 15.
    Niu DD, Zuo ZR, Shi GY, Wang ZX (2009) High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system. Microb Cell Fact 8:58CrossRefGoogle Scholar
  16. 16.
    Phan TT, Tran LT, Schumann W, Nguyen HD (2015) Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb Cell Fact 14:72CrossRefGoogle Scholar
  17. 17.
    Schallmey MA, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17CrossRefGoogle Scholar
  18. 18.
    Schumann W (2007) Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol 62:137–189CrossRefGoogle Scholar
  19. 19.
    Tanyildizi MS, Ozer D, Elibol M (2007) Production of bacterial α-amylase by B. amyloliquefaciens under solid substrate fermentation. Biochem Eng J 37:294–297CrossRefGoogle Scholar
  20. 20.
    Vehmaanperä J, Steinborn G, Hofemeister J (1991) Genetic manipulation of Bacillus amyloliquefaciens. J Biotechnol 19:221–240CrossRefGoogle Scholar
  21. 21.
    Wang H, Yang L, Ping Y, Bai Y, Luo H, Huang H, Yao B (2016) Engineering of a Bacillus amyloliquefaciens strain with high neutral protease producing capacity and optimization of its fermentation conditions. PLoS ONE 11:e0146373CrossRefGoogle Scholar
  22. 22.
    Wang P, Tian J, Yu X, Chang M, Chu X, Wu N (2016) A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens. Sci Rep 6:22229CrossRefGoogle Scholar
  23. 23.
    Wei X, Zhou Y, Chen J, Cai D, Wang D, Qi G, Chen S (2015) Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization. J Ind Microbiol Biotechnol 42:287–295CrossRefGoogle Scholar
  24. 24.
    Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694:299–310CrossRefGoogle Scholar
  25. 25.
    Wong SL, Ye R, Nathoo S (1994) Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system. Appl Environ Microbiol 60:517–523Google Scholar
  26. 26.
    Wu XC, Lee W, Tran L, Wong SL (1991) Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol 173:4952–4958CrossRefGoogle Scholar
  27. 27.
    Wu XC, Ng SC, Near RI (1993) Efficient production of a functional single-chain antidigoxin antibody via an engineered Bacillus subtilis expression-secretion system. Nat Biotechnol 11:71–76CrossRefGoogle Scholar
  28. 28.
    Xue G-P, Johnson JS, Dalrymple BP (1999) High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J Microbiol Meth 34:183–191CrossRefGoogle Scholar
  29. 29.
    Yang L, Wang H, Lv Y, Bai Y, Luo H, Shi P, Huang H, Yao B (2016) Construction of a rapid feather-degrading bacterium by overexpression of a highly efficient alkaline keratinase in its parent strain Bacillus amyloliquefaciens K11. J Agric Food Chem 64:78–84CrossRefGoogle Scholar
  30. 30.
    You C, Zhang XZ, Zhang YH (2012) Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl Environ Microbiol 78:1593–1595CrossRefGoogle Scholar
  31. 31.
    Zakataeva NP, Nikitina OV, Gronskiy SV, Romanenkov DV, Livshits VA (2010) A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains. Appl Microbiol Biotechnol 85:1201–1209CrossRefGoogle Scholar
  32. 32.
    Zhang G, Wang W, Deng A, Sun Z, Zhang Y, Liang Y, Che Y, Wen T (2012) A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. PLoS Genet 8:e1002987CrossRefGoogle Scholar
  33. 33.
    Zhang GQ, Bao P, Zhang Y, Deng AH, Chen N, Wen TY (2011) Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing. Anal Biochem 409:130–137CrossRefGoogle Scholar
  34. 34.
    Zhang K, Su L, Duan X, Liu L, Wu J (2017) High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system. Microb Cell Fact 16:32CrossRefGoogle Scholar

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

Personalised recommendations