DOI QR코드

DOI QR Code

Enhancement of Excretory Production of an Exoglucanase from Escherichia coli with Phage Shock Protein A (PspA) Overexpression

  • Wang, Y.Y. (Division of Life Science, The Hong Kong University of Science and Technology) ;
  • Fu, Z.B. (Division of Life Science, The Hong Kong University of Science and Technology) ;
  • Ng, K.L. (Division of Life Science, The Hong Kong University of Science and Technology) ;
  • Lam, C.C. (Division of Life Science, The Hong Kong University of Science and Technology) ;
  • Chan, A.K.N. (Division of Life Science, The Hong Kong University of Science and Technology) ;
  • Sze, K.F. (Division of Life Science, The Hong Kong University of Science and Technology) ;
  • Wong, W.K.R. (Division of Life Science, The Hong Kong University of Science and Technology)
  • Received : 2011.01.24
  • Accepted : 2011.03.22
  • Published : 2011.06.28

Abstract

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.

Keywords

References

  1. Abramoff, M. D., P. J. Magelhaes, and S. J. Ram. 2004. Image processing with ImageJ. Biophotonics Ind. 11: 36-42.
  2. DeLisa, M. P., P. Lee, T. Palmer, and G. Georgiou. 2004. Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway. J. Bacteriol. 186: 366- 373. https://doi.org/10.1128/JB.186.2.366-373.2004
  3. Ferrer-Miralles, N., J. Domingo-Espín, J. L. Corchero, E. Vázquez, and A. Villaverde. 2009. Microbial factories for recombinant pharmaceuticals. Microb. Cell Fact. 8: 17. https://doi.org/10.1186/1475-2859-8-17
  4. Fu, Z. B., K. L. Ng, T. L. Lam, and W. K. R. Wong. 2005. Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli. Protein Expr. Purif. 42: 67-77. https://doi.org/10.1016/j.pep.2005.03.029
  5. Fu, Z. B., K. L. Ng, C. Lam, K. Leung, W. Yip, and W. K. R. Wong. 2006. A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli. Protein Expr. Purif. 48: 205-214. https://doi.org/10.1016/j.pep.2006.01.013
  6. Gant, V. A., G. Warnes, I. Phillips, and G. F. Savidge. 1993. The application of flow cytometry to the study of bacterial responses to antibiotics. J. Med. Microbiol. 39:147-154. https://doi.org/10.1099/00222615-39-2-147
  7. Jovanovic, G., L. J. Lloyd, M. P. H. Stumpf, A. J. Mayhew, and M. Buck. 2006. Induction and function of the phage shock protein extra-cytoplasmic stress response in Escherichia coli. J. Biol. Chem. 281: 21147-21161. https://doi.org/10.1074/jbc.M602323200
  8. Kleerebezem, M. and J. Tommassen. 1993. Expression of the pspA gene stimulates efficient protein export in Escherichia coli. Mol. Microbiol. 7: 947-956. https://doi.org/10.1111/j.1365-2958.1993.tb01186.x
  9. Kleerebezem, M., W. Crielaard, and J. Tommassen. 1996. Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the proton motive force under stress conditions. EMBO J. 15: 162-171.
  10. Kobayashi, R., T. Suzuki, and M. Yoshida. 2007. Escherichia coli phage-shock protein A (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged membranes. Mol. Microbiol. 66: 100-109. https://doi.org/10.1111/j.1365-2958.2007.05893.x
  11. Lam, T., S. C. R. Wong, and W. K. R. Wong. 1997. Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength. Enzyme Microb. Technol. 20: 482-488. https://doi.org/10.1016/S0141-0229(96)00203-7
  12. Mason, D. J., R. Lopéz-Amorós, R. Allman, J. M. Stark, and D. Lloyd. 1995. The ability of membrane potential dyes and calcafluor white to distinguish between viable and non-viable bacteria. J. Appl. Bacteriol. 78: 309-315. https://doi.org/10.1111/j.1365-2672.1995.tb05031.x
  13. Mergulhão, F. and G. Monteiro. 2004. Secretion capacity limitations of the Sec pathway in Escherichia coli. J. Microbiol. Biotechnol. 14: 128-133.
  14. Model, P., G. Jovanovic, and J. Dworkin. 1997. The Escherichia coli phage shock protein (psp) operon. Mol. Microbiol. 24: 255-261. https://doi.org/10.1046/j.1365-2958.1997.3481712.x
  15. Papanikou, E., S. Karamanou, and A. Economou. 2007. Bacterial protein secretion through the translocase nanomachine. Nat. Rev. Microbiol. 5: 839-851. https://doi.org/10.1038/nrmicro1771
  16. Sambrook, J. and D. Russell. 2001. Molecular Cloning: A Laboratory Manual; 3rd Ed. Cold Spring Harbor Laboratory Press, New York
  17. Schagger, H. and G. von Jagow. 1987. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379. https://doi.org/10.1016/0003-2697(87)90587-2
  18. Vrancken, K., S. De Keersmaeker, N. Geukens, E. Lammertyn, J. Anne, and L. Van Mellaert. 2006. pspA Over-expression in Streptomyces lividans improves both Sec- and Tat-dependent protein secretion. Appl. Microbiol. Biotechnol. 73: 1150-1157. https://doi.org/10.1007/s00253-006-0571-7
  19. Walsh, G. 2006. Biopharmaceutical benchmarks. 2006. Nat. Biotechnol. 24: 769-776. https://doi.org/10.1038/nbt0706-769
  20. Wang, Y. Y., K. L. Ng, C. C. Lam, K. N. Chan, K. F. Sze, Z. B. Fu, and W. K. R. Wong. 2010. Efficient Bacillus subtilis promoters for graded expression of heterologous genes in Escherichia coli. Res. J. Biotechnol. 5: 5-14. https://doi.org/10.1002/biot.201090003
  21. Wong, D. K., K. H. E. Lam, C. K. P. Chan, Y. C. V. Wong, W. K. R. Wong, and J. Hackett. 1998. Extracellular expression of human epidermal growth factor encoded by an Escherichia coli K-12 plasmid stabilized by the ytl2-incR system of Salmonella Typhimurium. J. Ind. Microbiol. Biotechnol. 21: 31-36. https://doi.org/10.1038/sj.jim.2900557
  22. Wong, W. K. R., E. Lam, R. C. Huang, S. C. Wong, C. Morris, and J. Hackett. 2001. Applications and efficient large-scale production of recombinant human epidermal growth factor. Biotechnol. Genet. Eng. Rev. 18: 51-71. https://doi.org/10.1080/02648725.2001.10648008

Cited by

  1. Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea vol.493, pp.1, 2011, https://doi.org/10.1016/j.gene.2011.11.027
  2. A revolutionary approach facilitating co-expression of authentic human epidermal growth factor and basic fibroblast growth factor in both cytoplasm and culture medium of Escherichia coli vol.97, pp.20, 2013, https://doi.org/10.1007/s00253-013-5090-8
  3. Subcellular localization, interactions and dynamics of the phage‐shock protein‐like Lia response in Bacillus subtilis vol.92, pp.4, 2011, https://doi.org/10.1111/mmi.12586
  4. The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli vol.8, pp.12, 2011, https://doi.org/10.5812/jjm.17790