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Optimized Medium Improves Expression and Secretion of Extremely Thermostable Bacterial Xylanase, XynB, in Kluyveromyces lactis

  • Yin, Tie (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Miao, Li-Li (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Guan, Fei-Fei (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Wang, Gui-Li (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Peng, Qing (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Li, Bing-Xue (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Guan, Guo-Hua (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University) ;
  • Li, Ying (State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University)
  • Received : 2010.05.28
  • Accepted : 2010.07.27
  • Published : 2010.11.28

Abstract

An extremely thermostable xylanase gene, xynB, from the hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. The response surface methodology (RSM) was also applied to optimize the medium components for the production of XynB secreted by the recombinant K. lactis. The secretion level (102 mg/l) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/l, 16 U/ml) in the original medium (yeast extract, lactose, and peptone; YLP). The secretory efficiency of mature XynB was also improved when using the YLU medium. When the mRNA levels of 13 characterized secretion-related genes in the K. lactis cultured in YLP and YLU were detected using a semiquantitative RT-PCR method, the unfolded protein response (UPR)-related genes, including ero1, hac1, and kar2, were found to be up-regulated in the K. lactis cultured in YLU. Therefore, the nutrient ingredients, especially the nitrogen source, were shown to have a significant influence on the XynB secretory efficiency of the host K. lactis.

Keywords

References

  1. Bao, W. G. and H. Fukuhara. 2001. Secretion of human proteins from yeast: Stimulation by duplication of polyubiquitin and protein disulfide isomerase genes in Kluyveromyces lactis. Gene 272: 103-110. https://doi.org/10.1016/S0378-1119(01)00564-9
  2. Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondal. 2001. Microbial xylanases and their industrial applications: A review. Appl. Microbiol. Biotechnol. 56: 326-338. https://doi.org/10.1007/s002530100704
  3. Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156-159.
  4. Dorner, A. J. and R. J. Kaufman. 1994. The levels of endoplasmic reticulum proteins and ATP affect folding and secretion of selective proteins. Biologicals 22: 103-112. https://doi.org/10.1006/biol.1994.1016
  5. Dujon, B., D. Sherman, G. Fischer, P. Durrens, S. Casaregola, I. Lafontaine, et al. 2004. Genome evolution in yeasts. Nature 430: 35-44. https://doi.org/10.1038/nature02579
  6. Gasser, B., M. Sauer, M. Maurer, G. Stadlmayr, and D. Mattanovich. 2007. Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. Appl. Environ. Microbiol. 73: 6499-6507. https://doi.org/10.1128/AEM.01196-07
  7. Huang, D., P. R. Gore, and E. V. Shusta. 2008. Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss. Biotechnol. Bioeng. 101: 1264-1275. https://doi.org/10.1002/bit.22019
  8. Kulkarni, N., A. Shendye, and M. Rao. 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23: 411-456. https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680- 685. https://doi.org/10.1038/227680a0
  10. Lodi, T., B. Neglia, and C. Donnini. 2005. Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1. Appl. Environ. Microbiol. 71: 4359-4363. https://doi.org/10.1128/AEM.71.8.4359-4363.2005
  11. Mattanovich, D., B. Gasser, H. Hohenblum, and M. Sauer. 2004. Stress in recombinant protein producing yeasts. J. Biotechnol. 113: 121-135. https://doi.org/10.1016/j.jbiotec.2004.04.035
  12. Nakamura, S., K. Wakabayashi, R. Nakai, R. Aono, and K. Horikoshi. 1993. Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Appl. Environ. Microbiol. 59: 2311-2316.
  13. Nishikawa, S. I., S. W. Fewell, Y. Kato, J. L. Brodsky, and T. Endo. 2001. Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J. Cell Biol. 153: 1061-1070. https://doi.org/10.1083/jcb.153.5.1061
  14. Raimondi, S., E. Zanni, C. Talora, M. Rossi, C. Palleschi, and D. Uccelletti. 2008. SOD1, a new Kluyveromyces lactis helper gene for heterologous protein secretion. Appl. Environ. Microbiol. 74: 7130-7137. https://doi.org/10.1128/AEM.00955-08
  15. Travers, K. J., C. K. Patil, L. Wodicka, D. J. Lockhart, J. S. Weissman, and P. Walter. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101: 249-258. https://doi.org/10.1016/S0092-8674(00)80835-1
  16. Uccelletti, D., S. Anticoli, and C. Palleschi. 2007. The apyrase KlYnd1p of Kluyveromyces lactis affects glycosylation, secretion, and cell wall properties. FEMS Yeast Res. 7: 731-739. https://doi.org/10.1111/j.1567-1364.2007.00229.x
  17. Uccelletti, D., D. Staneva, S. Rufini, P. Venkov, and C. Palleschi. 2005. Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p. FEMS Yeast Res. 5: 735-746. https://doi.org/10.1016/j.femsyr.2005.01.004
  18. van Ooyen, A. J., P. Dekker, M. Huang, M. M. Olsthoorn, D. I. Jacobs, P. A. Colussi, and C. H. Taron. 2006. Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res. 6: 381-392. https://doi.org/10.1111/j.1567-1364.2006.00049.x
  19. W'esolowski-Louvel, M., K. D. Breunig, and H. Fukuhara. 1996. In K. Wolf (ed.). Nonconventional Yeasts in Biotechnology: A Handbook, pp. 139-202. Springer-Verlag, Berlin, New York.
  20. Walsh, D. J. and P. L. Bergquist. 1997. Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis. Appl. Environ. Microbiol. 63: 3297-3300.
  21. Walsh, D. J., M. D. Gibbs, and P. L. Bergquist. 1998. Expression and secretion of a xylanase from the extreme thermophile, Thermotoga strain FjSS3B.1, in Kluyveromyces lactis. Extremophiles 2: 9-14. https://doi.org/10.1007/s007920050037
  22. Wamalwa, B. M., G. Zhao, M. Sakka, P. M. Shiundu, T. Kimura, and K. Sakka. 2007. High-level heterologous expression of Bacillus halodurans putative xylanase Xyn11A (BH0899) in Kluyveromyces lactis. Biosci. Biotechnol. Biochem. 71: 688- 693. https://doi.org/10.1271/bbb.60477
  23. Winterhalter, C. and W. Liebl. 1995. Two extremely thermostable xylanases of the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl. Environ. Microbiol. 61: 1810-1815.
  24. Yang, M. H., Y. Li, G. H. Guan, and Z. Q. Jiang. 2005. Highlevel expression of an extreme-thermostable xylanase B from Thermotoga maritima $MSB_{8}$ in Escherichia coli and Pichia pastoris. Wei Sheng Wu Xue Bao 45: 236-240.
  25. Zhang, J., J. Pan, G. Guan, Y. Li, W. Xue, G. Tang, A. Wang, and H. Wang. 2008. Expression and high-yield production of extremely thermostable bacterial xylanase B in Aspergillus niger. Enzyme Microb. Technol. 43: 513-516. https://doi.org/10.1016/j.enzmictec.2008.07.010
  26. Zhengqiang, J., A. Kobayashi, M. M. Ahsan, L. Lite, M. Kitaoka, and K. Hayashi. 2001. Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside. J. Biosci. Bioeng. 92: 423-428. https://doi.org/10.1263/jbb.92.423

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