Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Efficient Secretory Expression of Recombinant Endoxylanase from Bacillus sp. HY-20 in Saccharomyces cerevisiae

출아효모(Saccharomyces cerevisiae)에서 Bacillus sp. HY-20균주의 재조합 endoxylanase의 효율적 분비 발현

  • Kim, Min-Ji (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Kim, Bo-Hyun (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Nam, Soo-Wan (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Choi, Eui-Sung (Biochemicals and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Shin, Dong-Ha (Insect Biotech Co. Ltd.) ;
  • Cho, Han-Young (Industrial Biomaterials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Son, Kwang-Hee (Industrial Biomaterials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Park, Ho-Yong (Industrial Biomaterials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Kim, Yeon-Hee (Department of Biotechnology and Bioengineering, Dong-Eui University)
  • 김민지 (동의대학교 생명공학과) ;
  • 김보현 (동의대학교 생명공학과) ;
  • 남수완 (동의대학교 생명공학과) ;
  • 최의성 (한국생명공학연구원 바이오화학연구센터) ;
  • 신동하 ((주)인섹트 바이오텍) ;
  • 조한영 (한국생명공학연구원 산업바이오소재연구센터) ;
  • 손광희 (한국생명공학연구원 산업바이오소재연구센터) ;
  • 박호용 (한국생명공학연구원 산업바이오소재연구센터) ;
  • 김연희 (동의대학교 생명공학과)
  • Received : 2013.05.30
  • Accepted : 2013.07.01
  • Published : 2013.07.30
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Abstract

The XylP gene, which encodes endoxylanase in Bacillus sp. HY-20, was subcloned, and two expression plasmids, pG-xylP and pGMF-xylP were constructed. These plasmids, which contain different signal sequences, XylP s.s and $MF{\alpha}_{opt}$ s.s, respectively, for the secretory expression of endoxylanase, were transformed into Saccharomyces cerevisiae SEY2102 and FY833, respectively. The recombinant endoxylanases were successfully expressed, with a total activity range of 23.7-70.1 unit/ml according to the expression system and host strain. The endoxylanase activity in SEY2102/pGMF-xylP reached a maximum of 88.1 unit/ml in baffled flask culture. Most of the recombinant endoxylanase was efficiently secreted in the extracellular fraction, and the $MF{\alpha}_{opt}$ s.s was more efficient for secreting endoxylanase in yeast than the XylP s.s. Therefore, the expression system developed in this study produces large extracellular amounts of endoxylanase using S. cerevisiae as the host strain, and it could be used in bioethanol production and industrial applications.

Bacillus sp. HY-20균주 유래 endoxylanase를 코드하는 XylP 유전자를 효모에서 발현시키기 위해 두 개의 발현 플라스미드 pG-xylP와 pGMF-xylP를 구축하였다. 이들 플라스미드는 endoxylanase의 분비발현을 위해 각각 다른 분비서열인 XylP 유전자의 자체 분비서열(XylP s.s)과 최적화된 $MF{\alpha}$ 분비서열($MF{\alpha}_{opt}$ s.s)을 가지고 있으며, S. cerevisiae SEY2102와 FY833균주에 형질전환되어 그 분비활성이 비교 조사되었다. 재조합 endoxylanase는 분비발현시스템과 숙주세포에 따라 23.7~70.1 unit/ml의 활성으로 효모 세포에서 성공적으로 발현되었고, 그 중 SEY2102/pGMF-xylP 형질전환주를 이용해 baffled-flask 배양을 실시한 결과 최대 88.1 unit/ml의 endoxylanase 활성을 보임을 확인하였다. 대부분의 재조합 endoxylanase는 세포 외 분획에 효율적으로 분비 생산되었으며, $MF{\alpha}_{opt}$ 분비서열이 XylP 유전자의 자체 분비서열보다 endoxylanase를 더 효율적으로 분비시킴을 확인할 수 있었다. 그러므로 본 연구에서 개발된 발현시스템은 효모를 숙주세포로 하여 많은 양의 세포 외 endoxylanase의 생산을 가능하게 하고, 바이오에탄올 생산 및 산업적 응용에도 유용하게 사용 될 수 있으리라 기대된다.

Keywords

References

  1. Beg, Q. K., Kapoor, M., Mahajan, L. and Hoondal, G. S. 2001. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56, 326-338. https://doi.org/10.1007/s002530100704
  2. Chen, H. G., Yan, X., Liu, X. Y., Wang, M. D., Huang, H. M., Jia, X. C. and Wang, J. A. 2006. Purification and characterization of novel bifunctional xylanase, XynIII, isolated from Aspergillus niger A-25. J Microbiol Biotechnol 16, 1132-1138.
  3. Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162, 156-159.
  4. Emr, S. D., Schekman, R., Flessel, M. C. and Thorner, J. 1983. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci USA 80, 7080-7084. https://doi.org/10.1073/pnas.80.23.7080
  5. Gietz, R. D., Schiestl, R. H., Willems, A. R. and Woods, R. A. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355-360. https://doi.org/10.1002/yea.320110408
  6. Gorgens, J. F., Planas, J., van Zyl, W. H., Knoetze, J. H. and Hahn-Hagerdal, B. 2004. Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium. Yeast 21, 1205-1217. https://doi.org/10.1002/yea.1175
  7. Heo, S. Y., Kim, J. K., Kim, Y. M. and Nam, S. W. 2004. Xylan hydrolysis by treatment with endoxylanase and $\beta$ -xylosidase expressed in yeast. J Microbiol Biotechnol 14, 171-177.
  8. Huy, N. D., Kim, S. W. and Park, S. M. 2011. Heterologous expression of endo-1,4-beta-xylanaseC from Phanerochaete chrysosporium in Pichia pastoris. J Biosci Bioeng 111, 654-657. https://doi.org/10.1016/j.jbiosc.2011.02.010
  9. Khandeparker, R. and Numan, M. T. 2008. Bifunctional xylanases and their potential use in biotechnology. J Ind Microbiol Biotechnol 35, 635-644. https://doi.org/10.1007/s10295-008-0342-9
  10. La Grange, D. C., Pretorius, I. S. and van Zyl, W. H. 1996. Expression of a Trichoderma reesei b-xylanase gene (XYN2) in Saccharomyces cerevisiae. Appl Environ Microbiol 62, 1036-1044.
  11. La Grange, D. C., Pretorius, I. S., Claeyssens, M. and van Zyl, W. H. 2001. Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger $\beta$-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes. Appl Environ Microbiol 67, 5512-5519. https://doi.org/10.1128/AEM.67.12.5512-5519.2001
  12. Latchinian-Sadek, L. and Thomas, D. Y. 1993. Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase. J Biol Chem 268, 534-540.
  13. Lee, J. H., Lim, M. Y., Kim, M. J., Heo, S. Y., Seo, J. H., Kim, Y. H. and Nam, S. W. 2007. Constitutive coexpression of Bacillus endoxylanase and Trichoderma endoglucanase Genes in Saccharomyces cerevisiae. J Microbiol Biotechnol 17, 2076-2080.
  14. Lee, L. H., Kim, D. Y., Han, M. K., Oh, H. W., Ham, S. J., Park, D. S., Bae, K. S., Sok, D. E., Shin, D. H., Son, K. H. and Park, H. Y. 2009. Characterization of an extracellular xylanase from Bacillus sp. HY-20, a bacterium in the gut of Apis mellifera. Korean J Microbiol 45, 332-338.
  15. Liu, M. Q. and Liu, G. F. 2008. Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Protein Expr Purif 57, 101-107. https://doi.org/10.1016/j.pep.2007.10.020
  16. Liu, M. Q., Weng, X. Y. and Sun, J. Y. 2006. Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan. Protein Expr Purif 48, 292-299. https://doi.org/10.1016/j.pep.2006.04.007
  17. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31, 426-428. https://doi.org/10.1021/ac60147a030
  18. Nuyens, F., van Zyl, W. H., Iserentant, D., Verachtert, H. and Michiels, C. 2001. Heterologous expression of the Bacillus pumilus endo-b-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 56, 431-434. https://doi.org/10.1007/s002530100670
  19. Polizeli, M. L. T. M., Rizzatti, A. C. S., Monti, R., Terenzi, H. F., Jorge, J. A. and Amorim, D. S. 2005. Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67, 577-591. https://doi.org/10.1007/s00253-005-1904-7
  20. Subramaniyan, S. and Prema, P. 2002. Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22, 33-64. https://doi.org/10.1080/07388550290789450
  21. Tachaapaikoon, C., Lee, Y. S., Ratanakhanokchai, K., Pinitglang, S., Kyu, K. L., Roh, M. S. and Lee, S. K. 2006. Purification and characterization of two endoxylanases from an alkaliphilic Bacillus halodurans C-1. J Microbiol Biotechnol 16, 613-618.
  22. Walsh, D. J., Gibbs, M. D. and Bergquist, P. L. 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
  23. Wamalwa, B. M., Zhao, G., Sakka, M., Shiundu, P. M., Kimura, T. and Sakka, K. 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
  24. Whang, J., Ahn, J., Chun, C. S., Son, Y. J., Lee, H. and Choi, E. S. 2009. Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae. Proc Biochem 44, 1190-1192. https://doi.org/10.1016/j.procbio.2009.06.009
  25. Winston, F., Dollard, C. and Ricupero-Hovasse, S. L. 1995. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53-55. https://doi.org/10.1002/yea.320110107
  26. Wong, K. K, Tan, L. U. and Saddler, J. N. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganism: function and application. Microbiol Rev 52, 305-317.
  27. Yin, T., Miao, L. L., Guan, F. F., Wang, G. L., Peng, Q., Li, B. X., Guan, G. H. and Li, Y. 2010. Optimized medium improves expression and secretion of extremely thermostable bacterial xylanase, XynB, in Kluyveromyces lactis. J Microbiol Biotechnol 20, 1471-1480. https://doi.org/10.4014/jmb.1005.05041
  28. Yoshida, S., Ono, T., Matsuo, N. and Kusakabe, I. 1994. Structure of hardwood xylan and specificity of Streptomyces b-xylanase toward the xylan. Biosci Biotechnol Biochem 58, 2068-2070. https://doi.org/10.1271/bbb.58.2068
  29. Zhang, G. M., Huang, J., Huang, G. R., Ma, L. X. and Zhang, X. E. 2007. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Appl Microbiol Biotechnol 74, 339-346. https://doi.org/10.1007/s00253-006-0648-3

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