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Flavin-Based Fluorescent Protein EcFbFP Auto-Guided Surface Display of Methyl Parathion Hydrolase in Escherichia coli

  • Lu Bian
  • Zhen Zhang
  • Rong-xing Tang
  • Wei Shen
  • Li-xin MaEmail author
Original paper
  • 28 Downloads

Abstract

Methyl parathion hydrolase (MPH) plays an important role in degrading a range of organophosphorus compounds. In order to display MPH on the cell surface of Escherichia coli strain RosettaBlue™, the Flavin-based fluorescent protein EcFbFP was severed as an auto-anchoring matrix. With net negative charges of EcFbFP supplying the driving forces, fusion protein MPH-EcFbFP through a two-step auto-surface display process was finally verified by (a) inner membrane translocation and (b) anchoring at outer membrane. Cells with surface-displayed MPH obtained activity of 0.12 U/OD600 against substrate methyl parathion. MPH when fused with engineered EcFbFP containing 20 net negative charges exhibited fivefold higher anchoring efficiency and tenfold higher enzymatic catalytic activity of 1.10 U/OD600. The above result showed that MPH was successfully displayed on cell surface and can be used for biodegradation of methyl parathion.

Keywords

Flavin-based fluorescent protein EcFbFP Cell-surface display Net negative charge Methyl parathion hydrolase 

Abbreviations

FMN

Flavin mononucleotide

MPH

Methyl parathion hydrolase

EcFbFP

Escherichia coli codon-optimized FMN-based fluorescent protein

EcFbFP(0)

EcFbFP mutant with 0 net charge

EcFbFP(2)

EcFbFP mutant with 2 net positive charges

EcFbFP(4)

EcFbFP mutant with 4 net positive charges

EcFbFP(-20)

EcFbFP mutant with 20 net negative charges

MPH-EcFbFP

MPH fused with EcFbFP

MPH-EcFbFP(-20)

MPH fused with EcFbFP(-20)

sfGFP

Super folder green fluorescent protein

mCherry

MCherry fluorescent protein

sfGFP-MPH

MPH fused with sfGFP

mCherry-MPH

MPH fused with mCherry

Notes

Acknowledgements

The authors would like to thank professor Li Yi in Hubei University for the critical revision of the manuscript, thank Dr. Mohanty P.B. in University of Oklahoma (U.S.A.) for the analysis of protein structure.

Author Contributions

L.B., Z.Z., and L.M. conceived, designed, and coordinated the study, and wrote the paper. R.T. performed the experiments shown in Figs. 1, 2, 3, 4, and 5. L.B. performed tables, and contributed to paper writing. W.S. performed the experiments shown in Fig. 5.

Funding

This work was supported by Foundation for National Key Basic Research Program of China (No. 2013CB910801 to L.M.), National Science Foundation of Hubei Province (No. 2013CFA133 to L.M.), High and New Technology Industrial Innovative Research Groups of the Wuhan Science and technology Bureau’s department (No. 2014CFA126, to L.M.).

Compliance with Ethical Standards

Conflict of interest

The authors declared that they have no conflicts of interest with the contents of this article.

Supplementary material

12033_2019_204_MOESM1_ESM.docx (840 kb)
Supplementary Material 1 (DOCX 840 kb)

References

  1. 1.
    Theriot, C. M., & Grunden, A. M. (2011). Hydrolysis of organophosphorus compounds by microbial enzymes. Applied Microbiology and Biotechnology, 89, 35–43.CrossRefGoogle Scholar
  2. 2.
    Du, D., Chen, W., Zhang, W., Liu, D., Li, H., & Lin, Y. (2010). Covalent coupling of organophosphorus hydrolase loaded quantum dots to carbon nanotube/Au nanocomposite for enhance detection of methyl parathion. Biosensors & Bioelectronics, 25, 1370–1375.CrossRefGoogle Scholar
  3. 3.
    Liu, F. Y., Hong, M. Z., Liu, D. M., Li, Y. M., Shou, P. S., Yan, H., et al. (2007). Biodegradation of methyl parathion by Acineobacter radioresistens USTB-04. Journal of Environmental Science, 19, 1257–1260.CrossRefGoogle Scholar
  4. 4.
    Fu, G. P., Cui, Z., Huang, T., & Li, S. P. (2004). Expression, purification, and characterization of a novel methyl parathion hydrolase. Protein Expression and Purification, 36, 170–176.CrossRefGoogle Scholar
  5. 5.
    Pakala, S. B., Gorla, P., Pinjari, A. B., Krovidi, R. K., Baru, R., Yanamandra, M., et al. (2007). Biodegradation of methyl parathion and ρ-nitrophenol: Evidence for the presence of a ρ-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001. Applied Microbiology and Biotechnology, 73, 1452–1462.CrossRefGoogle Scholar
  6. 6.
    Shen, Y. J., Lu, P., Mei, H., Yu, H. J., Hong, Q., & Li, S. P. (2009). Isolation of a methyl paration-degrading strain Stenotrophomonas sp. SMSP-1 and cloning of the ophc2 gene. Biodegradation, 21, 785–792.CrossRefGoogle Scholar
  7. 7.
    Yang, C., Zhu, Y., Yang, J., Liu, Z., Qiao, C. L., & Mulchandani, A. (2008). Development of an autofluorescent whole-cell biocatalyst by displaying dual functional moieties of a coculture with organophosphate-mineralizing activity. Applied and Environmental Microbiology, 74, 7737–7739.Google Scholar
  8. 8.
    Kang, D. G., Lim, G. B., & Cha, H. J. (2005). Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli. Journal of Biotechnology, 118, 379–385.CrossRefGoogle Scholar
  9. 9.
    Kang, D. G., Choi, S. S., & Cha, H. J. (2006). Enhanced biodegradation of toxic organophosphate compounds using recombinant Escherichia coli with Sec pathway-driven periplasmic secretion of organophosphorus hydrolase. Biotechnology Progress, 22, 406–410.CrossRefGoogle Scholar
  10. 10.
    Richins, R. D., Kaneva, I., Mulchandani, A., & Chen, W. (1997). Biodegradaiton of organophosphorus pesticides by surface-expressed organophosphorus hydrolase. Nature Biotechnology, 15, 984–987.CrossRefGoogle Scholar
  11. 11.
    Shimazu, M., Mulchandani, A., & Chen, W. (2001). Simultaneous degradation of organophosphorus pesticides and ρ-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase. Biotechnology and Bioengineering, 76, 318–324.CrossRefGoogle Scholar
  12. 12.
    Lei, Y., Mulchandani, A., & Chen, W. (2005). Improved degradation of organophosphorus nerve agents and ρ-nitrophenol by Pseudomonas putida JS444 with surface-expressed organophosphorus hydrolase. Biotechnology Progress, 2005, 678–681.Google Scholar
  13. 13.
    Yang, C., Cai, N., Dong, M., Jiang, H., Li, J., Qiao, C., et al. (2008). Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates. Biotechnology and Bioengineering, 99, 30–37.CrossRefGoogle Scholar
  14. 14.
    Takayama, K., Suye, S., Kuroda, K., Ueda, M., Kitaguchi, T., Tsuchiyama, K., et al. (2006). Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae. Biotechnology Progress, 22, 939–943.CrossRefGoogle Scholar
  15. 15.
    Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in Biotechnology, 21, 45–52.CrossRefGoogle Scholar
  16. 16.
    Samuelson, P., Gunneriusson, E., Nygren, P. A., & Stahl, S. (2002). Display of proteins on bacteria. Jounal of Biotchnology, 96, 129–154.Google Scholar
  17. 17.
    Velaithan, V., Chin, S. C., Yusoff, K., Illias, R., & Rahim, R. A. (2014). Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli. Annals of Microbioloy, 64, 543–550.CrossRefGoogle Scholar
  18. 18.
    Ismail, N. F., Hamdan, S., Mahadi, N. M., Murad, A. M., Rabu, A., Bakar, F. D., et al. (2011). A mutant l-aspraginase II signal peptide improves the secretion of recombinant cyclo detrin glucano transferase and the viability of Escherichia coli. Biotechnology Letters, 33, 999–1005.CrossRefGoogle Scholar
  19. 19.
    Jonet, M. A., Mahadi, N. M., Murad, A. M., Rabu, A., Baker, F. D., Rahim, R. A., et al. (2012). Optimization of a heterologous signal peptide by site-directed mutagensis for improved secretion of recombinant proteins in Escherichia coli. Journal of Molecular Microbiology and Biotechnology, 22, 48–58.CrossRefGoogle Scholar
  20. 20.
    Avila-Perez, M., Vreede, M. J., Tang, Y., Bende, O., Losi, A., Gartner, W., et al. (2009). In vivo mutational analysis of YtvA from Bacillus subtilis: Mechanism of light activation of the general stress response. Journal of Biological Chemistry, 284, 24958–24964.CrossRefGoogle Scholar
  21. 21.
    Jung, H. C., Park, J. H., Park, S. H., Lebeault, J. M., & Pan, J. G. (1998). Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein. Enyzme and Microbial Technology, 22, 348–354.CrossRefGoogle Scholar
  22. 22.
    Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M., & Heyneker, H. L. (1995). Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164, 49–53.CrossRefGoogle Scholar
  23. 23.
    Petersen, T. N., Brunak, S., Heijne, G., & Nielsen, H. (2011). SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nature Methods, 8, 785–786.CrossRefGoogle Scholar
  24. 24.
    Walter, J., Hausmann, S., Drepper, T., Puls, M., Eggert, T., & Dihne, M. (2012). Flavin mononucleotide-based fluorescent proteins function in mammalian cells without oxygen requirement. PLoS ONE, 7, e43921.CrossRefGoogle Scholar
  25. 25.
    Thomas, J. D., Daniel, R. A., Errington, J., & Robinson, C. (2001). Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Molecular Microbiology, 39, 47–53.CrossRefGoogle Scholar
  26. 26.
    Yang, C., Freudl, R., Qiao, C., & Mulchandani, A. (2010). Cotranslocation of methyl parathion hydrolase to the periplasm and of organophosphorus hydrolase to the cell surface of Escherichia coli by the Tat pathway and ice nucleation protein display system. Applied and Environment Microbiology, 76, 434–440.CrossRefGoogle Scholar
  27. 27.
    Moglich, A., & Moffat, K. (2007). Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA. Journal of Molecular Biology, 373, 112–126.CrossRefGoogle Scholar
  28. 28.
    Kudva, R., Denks, K., Kuhn, P., Vogt, P. A., Muller, M., & Koch, H. G. (2013). Protein translocation across the inner membrane of Gram-negative bacteria: The Sec and Tat dependent protein transport pathways. Research in Microbiology, 164, 505–534.CrossRefGoogle Scholar
  29. 29.
    Drepper, T., Gensch, T., & Pohl, M. (2013). Advanced in vivo applications of blue light photoreceptors as alternative fluorescent proteins. Photochemical & Photobiological Sciences, 12, 1125–1134.CrossRefGoogle Scholar
  30. 30.
    Arnold, T., Zeth, K., & Linke, D. (2010). Omp85 from the thermophilic cyanobacterium Thermosynechococcus elongatus differs from proteobacterial Omp85 in structure and domain composition. Journal of Biological Chemistry, 285, 18003–18015.CrossRefGoogle Scholar
  31. 31.
    Knowles, T. J., Scott-Tucker, A., Overduin, M., & Henderson, I. R. (2009). Membrane protein architects: The role of the BAM complex in outer membrane protein assembly. Nature Reviews Microbiology, 7, 206–214.CrossRefGoogle Scholar
  32. 32.
    van Bloois, E., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: Surface display of proteins on Escherichia coli. Trends in Biotechnology, 29, 79–86.CrossRefGoogle Scholar
  33. 33.
    Zhang, Z., Tang, R., Zhu, D., Wang, W., Yi, L., & Ma, L. (2017). Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli. Sci Rep, 7, 6990.CrossRefGoogle Scholar
  34. 34.
    Stahl, S., & Uhlen, M. (1997). Bacterial surface display: Trends and progress. Trends in Biotechnology, 15, 185–192.CrossRefGoogle Scholar
  35. 35.
    Gao, D., Wang, S., Li, H., Yu, H., & Qi, Q. (2015). Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli. Microbial Cell Factories, 14, 49.CrossRefGoogle Scholar
  36. 36.
    Sacks, V., Eshkenazi, I., Neufeld, T., Dosoretz, C., & Rishpon, J. (2000). Immobilized parathion hydrolase: An amperometric sensor for parathion. Analytical Chemistry, 72, 2055–2058.CrossRefGoogle Scholar
  37. 37.
    Singh, B. K., & Walker, A. (2006). Microbial degradation of organophosphorus compounds. FEMS Microbiology Reviews, 30, 428–471.CrossRefGoogle Scholar
  38. 38.
    Strong, L. C., Mctavish, H., Sadowsky, M. J., & Wackett, L. P. (2000). Field-scale remediaiton of atrazine-contaminated soil using recombinant Escherichia coli expressing atrazine chlorohydrolase. Environmental Microbiology, 2, 91–98.CrossRefGoogle Scholar
  39. 39.
    Zhang, R. F., Cui, Z. L., Zhang, X. Z., Jiang, J. D., Gu, J. D., & Li, S. P. (2006). Cloning of the organophosphorus pesticide hydrolase gene clusters of seven degradative bacteria isolated from a methyl parathion contaminated site and evidence of their horizontal gene transfer. Biodegradation, 17, 465–472.CrossRefGoogle Scholar
  40. 40.
    Noureddini, H., Gao, X., & Philkana, R. S. (2005). Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresource Technology, 96, 769–777.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, College of Life SciencesHubei UniversityWuhanChina

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