Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Optimization of the Functional Expression of Coprinus cinereus Peroxidase in Pichia pastoris by Varying the Host and Promoter

  • Kim, Su-Jin (Korea Research Institute of Chemical Technology) ;
  • Lee, Jeong-Ah (Korea Research Institute of Chemical Technology) ;
  • Kim, Yong-Hwan (Department of Chemical Engineering, Kwangwoon University) ;
  • Song, Bong-Keun (Korea Research Institute of Chemical Technology)
  • Published : 2009.09.30
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Abstract

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the $Mut^s $ Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

Keywords

References

  1. Abdelmoula, S., L. Rekik, A. Gargouri, and R. Mokdad. 2007. High-level expression of human tumor suppressor P53 in the methylotrophic yeast: Pichia pastoris. Protein Expr. Purif. 54: 283-288 https://doi.org/10.1016/j.pep.2007.03.015
  2. Arnold, R. R., J. E. Russell, W. J. Champion, M. Brewer, and J. J. Gauthier. 1982. Bactericidal activity of human lactoferrin: Differentiation from the stasis of iron deprivation. Infect. Immun. 35: 792-799
  3. Arnold, R. R., M. Brewer, and J. J. Gauthier. 1980. Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms. Infect. Immun. 28: 893-898
  4. Bibila, T. and M. C. Flickinger. 1991. A structured model for monoclonal antibody synthesis in exponentially growing and stationary phase hybridoma cells. Biotechnol. Bioeng. 37: 210- 226 https://doi.org/10.1002/bit.260370304
  5. Boer, H., T. T. Teeri, and A. Koivula. 2000. Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. Biotechnol. Bioeng. 69: 486-494 https://doi.org/10.1002/1097-0290(20000905)69:5<486::AID-BIT3>3.0.CO;2-N
  6. Conesa, A., P. J. Punt, and C. A. van den Hondel. 2002. Fungal peroxidases: Molecular aspects and applications. J Biotechnol. 93: 143-158 https://doi.org/10.1016/S0168-1656(01)00394-7
  7. Cregg, J. M., J. L. Cereghino, J. Shi, and D. R. Higgings. 2000. Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16: 23-52 https://doi.org/10.1385/MB:16:1:23
  8. Doring, G., T. Pfestrof, K. Botzenhart, and M. A. Abdallah. 1988. Iron-chelating substances and inflammation. Scand. J. Gastroenterol. 23: 68-69
  9. Ellison III, R. T., and T. J. Giehl. 1991. Killing of Gram-negative bacteria by lactoferrin and lysozyme. J. Clin. Invest. 88: 1080- 1091 https://doi.org/10.1172/JCI115407
  10. Gasser, B., M. Maurer, J. Gach, R. Kunert, and D. Mattanovich. 2006. Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol. Bioeng. 94: 353-361 https://doi.org/10.1002/bit.20851
  11. Hohenblum, H., B. Gasser, M. Maurer, N. Borth, and D. Mattanovich. 2004. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol. Bioeng. 85: 367-375 https://doi.org/10.1002/bit.10904
  12. Joo, H., H. J. Chae, J. S. Yeo, and Y. J. Yoo. 1998. Depolymerization of phenolic polymers using horseradish peroxidase in organic solvent. Process Biochem. 32: 291-296 https://doi.org/10.1016/S0032-9592(96)00092-1
  13. Kim, S. J., J. A. Lee, Y. H. Kim, and B. K. Song. 2009. Functional expression of Coprinus cinereus peroxidase in Pichia pastoris. Process Biochemistry 44: 731-735 https://doi.org/10.1016/j.procbio.2009.03.004
  14. Lee, K., S. Lim, and D. Kim. 2006. Effect of various additives on the production of recombinant HBsAg during methanol induction in Pichia pastoris. Biotechnol. Bioeng. 21: 260-266
  15. Lokman, B. C., V. Joosten, J. Hovenkamp, R. J. Gouka, C. T. Verrips, and C. A. van den Hondel. 2003. Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori. J Biotechnol. 103: 183-190 https://doi.org/10.1016/S0168-1656(03)00109-3
  16. Nakayama, T. and T. Amachi. 1999. Fungal peroxidase: Its structure, function, and application. J. of Molec. Catal. B Enzym. 6: 185-198 https://doi.org/10.1016/S1381-1177(98)00119-2
  17. Petersen, J. F., J. W. Tams, J. Vind, A. Svensson, H. Dalboge, K. G. Welinder, and S. Larsen. 1993. Crystallization and X-ray diffraction analysis of recombinant Coprinus cinereus peroxidase. J. Mol. Biol. 232: 989-991 https://doi.org/10.1006/jmbi.1993.1445
  18. Romanos, M. A., C. A. Scorer, and J. J. Clare. 1992. Foreign gene expression in yeast: A review Yeast 8: 423-488 https://doi.org/10.1002/yea.320080602
  19. Ryan, B. J., N. Carolan, and C. O'Fagain. 2006. Horseradish and soybean peroxidases: Comparable tools for alternative niches? Trends Biotechnol. 24: 355-363 https://doi.org/10.1016/j.tibtech.2006.06.007
  20. Ryu, K., J. P. McEldoon, and J. S. 1995. Kinetic characterization of a fungal peroxidase from Coprinus cinereus in aqueous and organic media. Biocatal. Biotransform. 13: 53-63 https://doi.org/10.3109/10242429509040105
  21. Sawai-Hatanaka, H., T. Ashikari, Y. Tanaka, Y. Asada, T. Nakayama, H. Minakata, et al. 1995. Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase. Biosci. Biotechnol. Biochem. 59: 1221-1228 https://doi.org/10.1271/bbb.59.1221
  22. Schorer, C. A., J. J. Clare, W. R. McCombie, M. A. Romanos, and K. Sreekrishna. 1994. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology 12: 181-184 https://doi.org/10.1038/nbt0294-181
  23. Sears, I. B., J. O'Connor, O. W. Rossanese, and B. S. Glick. 1998. Versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast 14: 787-790
  24. Sreekrishna, K., J. F. Tschopp, and M. Fuke. 1987. Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation Pichia pastoris. Gene 59: 115-125 https://doi.org/10.1016/0378-1119(87)90272-1
  25. Stratton, J., V. Chiruvolu, and M. M. Meagher. 1998. High celldensity fermentation. Methods Molec. Biol. 103: 107-120
  26. Tschopp, J. F., G. Dverlow, R. Kosson, W. Craig, and L. Grinna. 1987. High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris. Biotechnology 5: 1305- 1308 https://doi.org/10.1038/nbt1287-1305
  27. Woo, S., J. Cho, B. Lee, and E. Kim. 2004. Decolorization of melanin by lignin peroxidase from Phanerochaete chrysosporium. Biotechnol. Bioprocess Eng. 9: 256-260 https://doi.org/10.1007/BF02942340