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Journal of microbiology and biotechnology v.20 no.3, 2010년, pp.467 - 473   SCIE 피인용횟수: 2
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High-Level Expression of an Aspergillus niger Endo-$\beta$-1,4-Glucanase in Pichia pastoris Through Gene Codon Optimization and Synthesis

Zhao, Shumiao    (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University   ); Huang, Jun    (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University   ); Zhang, Changyi    (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University   ); Deng, Ling    (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University   ); Hu, Nan    (College of Life Science and Pharmacy, Nanjing University of Technology   ); Liang, Yunxiang    (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University  );
  • 초록

    To improve the expression efficiency of recombinant endo- $\beta$ -1,4-glucanase in P. pastoris, the endo- $\beta$ -1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo- $\beta$ -1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$ -glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$ , and the optimal pH was 5.0 when CMC was used as the substrate.


  • 주제어

    Aspergillus niger .   codon optimization .   endo- $\beta$-1,4-glucanase .   expression .   Pichia pastoris.  

  • 참고문헌 (36)

    1. Cazemier, A. E., J. C. Verdoes, H. J. M. Op den Camp, J. H. P. Hackstein, and A. J. J. van Ooyen. 1999. A $\beta$-1,4-endoglucanaseencoding gene from Cellulomonas pachnodae. Appl. Microbiol.Biotechnol. 52: 232-239. 
    2. David, M. H. and L. Jacek. 2002. DNAWorks: An automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res. 30: e43 
    3. Dienes, D., A. Egyhazi, and K. Reczey. 2004. Treatment of recycled fiber with Trichoderma cellulases. Ind. Crop. Prod. 20: 11-21. 
    4. Duan, X. Y., S. Y. Liu, W. C. Zhang, Q. X. Zhang, and P. J. Gao. 2004. Volumetric productivity improvement for endoglucanase of Trichoderma pseudokoingii S-38. J. Appl. Microbiol. 96: 772-776. 
    5. Huang, Y., G. Krauss, S. Cottaz, H. Driguez, and G. Lipps. 2005. A highly acid-stable and thermostable endo-$\beta$-glucanase from the thermoacidophilic archaeon Sulfolobus solfataricus. Biochem. J. 385: 581-588. 
    6. Lynd, L. R., J. Paul, P. J. Weimer, W. H. van Zyl, and I. S. Pretorius. 2002. Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66: 506-577. 
    7. Nakamura, Y., T. Gojobori, and T. Ikemura. 1999. Codon usage tabulated from the international DNA sequence database; its status 1999. Nucleic Acids Res. 27: 292. 
    8. Palomer, X., E. Dominguez-Puigjaner, M. Vendrell, and I. Llop-Tous. 2004. Study of the strawberry Cel1 endo-(1,4)-glucanase protein accumulation and characterization of its in vitro activity by heterologous expression in Pichia pastoris. Plant Sci. 167: 509-518. 
    9. Peilong, Y., P. Shi, Y. Wang, Y. Bai, K. Meng, H. Luo, T. Yuan, and B. Yao. 2007. Cloning and overexpression of a Paenibacillus $\beta$-glucanase in Pichia pastoris: Purification and characterization of the recombinant enzyme. J. Microbiol. Biotechnol. 17: 58-66.     
    10. Rose, S. H. and W. H. van Zyl. 2002. Constitutive expression of the Trichoderma reesei $\beta$-1,4-xylanase gene (xyn2) and the $\beta$-1,4-endoglucanase gene (egI) in Aspergillus niger in molasses and defined glucose media. Appl. Microbiol. Biotechnol. 58: 461-468. 
    11. Saloheimo, A., B. Henrissat, A. M. Hoffren, O. Teleman, and M. Penttila. 1994. A novel small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast. Mol. Microbiol. 13: 219-228. 
    12. Vervoort, E. B., A. Van Ravestein, N. N. van Peij, J. C. Heikoop, P. J. van Hasstert, G. F. Verheijden, and M. H. Linskens. 2000. Optimizing heterologous expression in Dictyostelium: Importance of 5' codon adaptation. Nucleic Acids Res. 28: 2069-2074. 
    13. Bauer, M. W., L. E. Driskill, W. Callen, M. A. Snead, E. J. Mathur, and R. M. Kelly. 1999. An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes $\beta$-1,4 bonds in mixed-linkage (1→3),(1→4)-TEX>$\beta$-D-glucans and cellulose. J. Bacteriol. 181: 284-290. 
    14. Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316. 
    15. Invitrogen: Pichia Expression Kit: A manual of methods for expression of recombinant proteins in Pichia pastoris. Version M. Invitrogen, San Diego, CA, 2002 
    16. Sheppard, P. O., F. J. Grant, P. J. Oort, C. A. Sprecher, D. C. Foster, F. S. Hagen, A. U. Upshall, G. L. McKnight, and P. J. OHara. 1994. The use of conserved cellulase family-specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum. Gene 150: 163-167. 
    17. Sinclair, G. and F. Y. M. Choy. 2002. Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 26: 96-105. 
    18. Bhat, M. K. 2004. Cellulases and related enzymes in biotechnology. Biotechnol. Adv. 18: 355-383. 
    19. Hong, J., T. Hisanori, A. Shunichi, K. Yamamtot, and H. Kumagai. 2001. Cloning of a gene encoding a highly stable endo-$\beta$-1,4-glucanase from Aspergillus niger and its expression in yeast. J. Biosci. Bioeng. 92: 434-439. 
    20. Teather, R. M. and P. J. Wood. 1982. Use of Congo redpolysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 8: 777-780. 
    21. Ajithkumar, A., R. Andersson, M. Siika-aho, M. Tenkanen, and P. Aman. 2006. Isolation of cellotriosyl blocks from barley $\beta$-glucan with endo-1,4-$\beta$-glucanase from Trichoderma reesei. Carbohyd. Polym. 64: 233-238. 
    22. Hu, S., L. Li, J. Qiao, Y. Guo, L. Cheng, and J. Liu. 2006. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expr. Purif. 47: 249-257. 
    23. Huang, H., P. Yang, H. Luo, H. Tang, N. Shao, T. Yuan, Y. Wang, Y. Bai, and B. Yao. 2008. High-level expression of a truncated 1,3-1,4-$\beta$-D-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation. Appl. Microbiol. Biotechnol. 78: 95-103. 
    24. Kitamoto, N., M. Go, T. Shibayama, T. Kimura, Y. Kito, K. Ohmiya, and N. Tsukagoshi. 1996. Molecular cloning, purification and characterization of two endo-1,4-$\beta$-glucanases from Aspergillus oryzae KBN616. Appl. Microbiol. Biotechnol. 46: 538-544. 
    25. Chang, S. W., G. C. Lee, and J. F. Shaw. 2006. Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J. Agric. Food Chem. 3: 815-822. 
    26. Ooi, T., A. Shinmyo, H. Okada, S. Murao, T. Kawaguchi, and M. Arai. 1990. Complete nucleotide sequence of a gene coding for Aspergillus aculeatus cellulase (F1-CMCase). Nucleic Acids Res. 18: 5884. 
    27. Sakamoto, S., G. Tamura, K. Ito, T. Ishikawa, K. Iwano, and N. Nishiya. 1995. Cloning and sequencing of cellulase cDNA from Aspergillus kawachii and its expression in Saccharomyces cerevisiae. Curr. Genet. 27: 435-439. 
    28. Beguin, P. and J. P. Aubert. 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13: 25-58. 
    29. Shuyan, L., D. Xinyuan, L. Xuemei, and G. Peiji. 2006. A novel thermophilic endoglucanase from a mesophilic fungus Fusarium oxysporum. Chinese Sci. Bull. 51: 191-197. 
    30. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428. 
    31. Teng, D., Y. Fan, Y. L.Yang, Z. G. Tian, L. Luo, and J. H. Wang. 2007. Codon optimization of Bacillus licheniformis $\beta$-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Appl. Microbiol. Biotechnol. 74: 1074-1083. 
    32. Feng, Y., C. J. Duan, H. Pang, X. C. Mo, C. F. Wu, Y. Yu, et al. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl. Microbiol. Biotechnol. 75: 319-328. 
    33. Hale, R. S. and G. Thompson. 1998. Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli. Protein Expr. Purif. 12: 185-188. 
    34. Macauley-Patrick, S., M. L. Fazenda, B. McNeil, and L.M. Harvey. 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast 22: 249-270. 
    35. Nikolay, S. O., J. S. Willem, and A. J. Maarten. 2002. Optimization of the expression of equistatin in Pichia pastoris. Protein Expr. Purif. 24: 18-24. 
    36. Xiong, A. S., Q. H. Yao, and R. H. Peng. 2004. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res. 32: e98. 
  • 이 논문을 인용한 문헌 (2)

    1. 2011. "" Journal of microbiology and biotechnology, 21(10): 1012~1020     
    2. 2011. "" Journal of microbiology and biotechnology, 21(12): 1264~1269     

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