Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Secretory Overexpression of Clostridium Endoglucanase A in Saccharomyces cerevisiae Using GAL10 Promoter and Exoinulinase Signal Sequeice.

Saccharomyces cerevisiae에서 GAL 10 promoter와 exoinulinase 분비 서열을 이용한 Clostridium endoglucanase A의 과발현·분비

  • Lim, Myung-Ye (Department of Biomaterial Control, Dong-Eui University) ;
  • Lee, Jin-Woo (Department of Biotechnology & Bioengineering, Dong-Eui University) ;
  • Lee, Jae-Hyung (Department of Biomaterial Control, Dong-Eui University) ;
  • Kim, Yeon-Hee (Department of Biotechnology & Bioengineering, Dong-Eui University) ;
  • Seo, Jin-Ho (Department of Agricultural Biotechnology, Seoul National University) ;
  • Nam, Soo-Wan (Department of Biomaterial Control, Department of Biotechnology & Bioengineering, Dong-Eui University)
  • 임명예 (동의대학교 바이오물질제어학과) ;
  • 이진우 (동의대학교 생명공학과) ;
  • 이재형 (동의대학교 바이오물질제어학과) ;
  • 김연희 (동의대학교 생명공학과) ;
  • 서진호 (서울대학교 농생명공학부) ;
  • 남수완 (동의대학교 바이오물질제어학과, 생명공학과)
  • Published : 2007.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

The secretory overexpression of Clostridium thermocellum endoglucanase A gene (celA) was examined in Saccharomyces cerevisiae using Kluyveromyces marxianus exoinulinase (INU1) signal sequence and GAL10 promoter. The two plasmids, pYEG-CT1 with its own signal sequence, and pYInu-CT1 with INU1 signal sequence were introduced to S. cerevisiae SEY2102 and S. cerevisiae 2805 host strains, respectively, and then each transformant was selected on the synthetic defined media lacking uracil. The expression level and secretion efficiency of endoglucanase A was increased by $18{\sim}22%$ and 11%, respectively, by INU1 signal sequence over celA signal sequence. By considering the high level of expression (361 unit/I), plasmid stability (89%), and secretion efficiency (70%), S. cerevisiae 2805 harboring plasmid pYInu-CT1 was selected as the opti-mal host vector system for the production of cellulose-degrading enzyme and recombinant yeast probiotic. The total expression and secretion efficiency of endoglucanase A was 418 unit/l and 73%, respectively, in the batch fermentation of S. cerevisiae 2805/pYlnu-CT1 on galactose medium. The mo-lecular weight of secreted endoglucanase A was found to be greater than 100 kDa, presumably due to the N-linked glycosylation.

Kluyveromyces marxianus 유래의 exoinulinase (INU1) 분비 서열과 GAL10 promoter를 이용하여 Clostridium thermocellum endoglucanase A gene (celA)의 과발현과 분비 성능 검증연구를 수행하였다. 자체 분비 서열을 가지는 pYEG- CT1 과 INU1 분비 서열을 가지는 pYInu-CT1, 두개의 plasmid는 S. cer-evisiae SEY2102와 S. cerevisiae 2805에 각각 형질전환시켜 ur-acil이 결핍된 배지에서 선별하였다. celA gene 자체 분비 서열보다 INU1 분비 서열을 사용했을 때 발현량과 분비효율은 각각 약 $18{\sim}22%$ 와 11% 향상되었다. 이 중 361 unit/l의 발현율과 89%의 plasmid 안정성, 그리고 70%의 분비효율을 보인 S. cerevisiae 2805/pYinu-CT1 효모 형질전환체를 cellolose를 효과적으로 분해하는 재조합 효모 생균체로 선별하였다. Galactose 배지내에서 S. cerevisiae 2805/ pYInu-CT1의 발효조 회분배양 결과, 총발현량과 분비효율은 각각 418 unit/l 와 73% 로 나타났다. 또한 분비된 endoglucanase A는 분자량 100kDa 이상에서 활성 밴드를 보여, N-linked 당쇄부가에 의해 상당한 비율의 당쇄가 부가됨을 추측할 수 있었다.

Keywords

References

  1. Beguin, P., P. Cornet and J. P. Aubert. 1985. Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellulm. J. Bacteriol. 162, 102-105
  2. Broker, M., O. Bauml, A. Gottig, J. Ochs, M. Bodenbenner and E. Amann, 1991. Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: Dependence of the expression levels from host-vector system and medium condition. Appl. Microbiol. Biotechnol. 34, 756-764
  3. Choi, J. Y., J. O. Aim, S. I. Kim and H. J. Shin. 2006. Expression of thermostable $\alpha$-glucosidase from Thermus caldophilus GK24 in recombinant Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 16, 2000-2003
  4. Chung, B. H. S. W. Nam, B. M. Kim and Y. H. Park. 1996. Highly-dfficient secretion of heterologous protein from Saccharomyces cerevisiae using inulinase signal peptide. Biotechnol. Bioeng. 49, 473-479 https://doi.org/10.1002/(SICI)1097-0290(19960220)49:4<473::AID-BIT15>3.0.CO;2-B
  5. Chung, D. K., D. H. Shin, B. W. Kim, J. K. Nam, I. S. Han and S. W. Nam. 1997. Expression and secretion of Clostridium thermocullum endoglucanase A gene (celA) in different Saccharomyces cerevisiae strains. Biotechnol. Lett. 19, 503-506 https://doi.org/10.1023/A:1018320916579
  6. Emr, S. D., R. Schekman, M. C. Flessel and J. Thorner. 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
  7. Halliwell, G. and M. P. Bryant. 1963. The cellulolytic activity of pure strains of bacteria from the rumen of cattle. J. Gen. Microbiol. 32, 441-448 https://doi.org/10.1099/00221287-32-3-441
  8. Henrissat, B., H. Driguez, C. Viet and M. Schulein. 1985. Synergism of cellulases from Trichoderma reesei in the degradation if cellulose. Bio/Technol. 3, 722-726 https://doi.org/10.1038/nbt0885-722
  9. Hungate, R. E. 1950. The anaerobic insesophilic cellulolytic bacteria. Bacterial. Rev. 14, 1-49
  10. Ito, H. Y., Fukuda, K. Murata and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168
  11. Kang, H. A., S. W. Nam, K. S. Kwon, B. H. Chung and M. H. Yu. 1996. High-level secretion of human $\alpha_{1}$-antitrypsin form Saccharomyces cerevisiae using inulinase signal sequence. J. Biotechnol. 48, 15-24 https://doi.org/10.1016/0168-1656(96)01391-0
  12. Knowles, J., M. PenttiIa, T. Teeri, H. Nevalainen, I. Salovuori and P. Lehtovaara-Helenius. 1985. Yeast strains producing cellulolytic enzymes and methods and means for constructing them. International Patent Publication No. WO 85-04672
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685
  14. Laloux O., J. P. Cassart, J. Delcour, J. V. Beeumen and J. Vandenhaute. 1991. Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424. FEBS J. 289, 64-68 https://doi.org/10.1016/0014-5793(91)80909-M
  15. Lim C. K., Y. K. Kim, K. H. Kim, C. H. Kim, S. K. Rhee and S. W. Nam. 2004. Expression and secretion of Zymononas mobilis levansucrase in Saccahromyces cerevisiae. Kor. J. Life Sci. 14, 429-434 https://doi.org/10.5352/JLS.2004.14.3.429
  16. Miller, G. L., R. Blum, W. E. Glennon and A. L. Burton. 1960. Measurement of carboxymethyl cellulase activity. Anal. Biochem. 2, 127-132
  17. Russell, J. B. and D. B. Dombrowski. 1980. Effect of pH on the efficiency of growth by pure culture of rumen bacteria in continuous culture. Appl. Environ. Microbiol. 39, 604-610
  18. Schultz, L., H. Z. Markus, K. J. Hofmann, D. L. Montgomery, C. T. Dunwiddie, P. J. Kniskern, R. B. Freedman, R. W. Ellis and M. F. Tuite. 1994. Using molecular genetics to improve the production of recombinant proteins by the yeast Saccharomyces cerevisiae. Ann. NY Acad. Sci. 721, 148-157 https://doi.org/10.1111/j.1749-6632.1994.tb47387.x
  19. Scotti, P. A., R. Chambert and M. F. Petit-Glatron. 1994. Extracellular levansucrase of Bacillus subtilis produced in yeast remains in the cell under its precursor form. Yeast 10, 29-38 https://doi.org/10.1002/yea.320100104
  20. Scotti, P. A., M. Praestegaard, R. Chambert and M. F. Petit-Glatron, 1996. The targeting of Bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part. Yeast 12, 953-963 https://doi.org/10.1002/(SICI)1097-0061(199608)12:10<953::AID-YEA998>3.0.CO;2-#
  21. Shoemaker, S. P., D. H. Gelfand, M. A. Innis, S. Y. Kwok, M. B. Landner and V. Schweickart. 1984. Recombinant fungal cellobiohydrolases. European Patent Application No. 84110305.4
  22. Shuster, J. R. 1991. Gene expression in yeast: protein secretion. Curr. Opinion Biotechnol. 2, 685-690 https://doi.org/10.1016/0958-1669(91)90035-4
  23. Skiper, N., M. Sutherland, R. W. Davies, D. Kilburn, R. C. Miller, Jr., A. Warren and R. Womg. 1985. Secretion of a bacterial cellulase by yeast. Science 230, 958-960 https://doi.org/10.1126/science.230.4728.958
  24. Tomme, P., R. A. Warren and N. R. Gilkes. 1995. Cellulose hydrolysis by bacteria and fungi. Adv. Microbiol. Physiol. 37, 1-81 https://doi.org/10.1016/S0065-2911(08)60143-5
  25. Woodward, J., M. K. Hayes and N. E. Lee. 1988. Hydrolysis of cellulose by saturating and non-saturating concentration of cellulase: Implications for synergism. Bio/Technol. 6, 301-304 https://doi.org/10.1038/nbt0388-301

Cited by

  1. Construction of an expression system for the secretory production of recombinant α-agarase in yeast vol.34, pp.6, 2012, https://doi.org/10.1007/s10529-012-0864-0
  2. Optimization for Production of Exo-β-1,3-glucanase (Laminarinase) from Aspergillus oryzae in Saccharomyces cerevisiae vol.26, pp.5, 2011, https://doi.org/10.7841/ksbbj.2011.26.5.427