Browse > Article
http://dx.doi.org/10.5352/JLS.2007.17.9.1248

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

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)
Publication Information
Journal of Life Science / v.17, no.9, 2007 , pp. 1248-1254 More about this Journal
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.
Keywords
Secretion; Clostridium thermocellum; endoglucanase; Saccharomyces cerevisiae; exoinulinase signal sequence;
Citations & Related Records
Times Cited By KSCI : 2  (Citation Analysis)
연도 인용수 순위
1 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   과학기술학회마을
2 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   DOI
3 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   DOI   ScienceOn
4 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   DOI   ScienceOn
5 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
6 Shuster, J. R. 1991. Gene expression in yeast: protein secretion. Curr. Opinion Biotechnol. 2, 685-690   DOI   ScienceOn
7 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   DOI   ScienceOn
8 Tomme, P., R. A. Warren and N. R. Gilkes. 1995. Cellulose hydrolysis by bacteria and fungi. Adv. Microbiol. Physiol. 37, 1-81   DOI
9 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   DOI
10 Miller, G. L., R. Blum, W. E. Glennon and A. L. Burton. 1960. Measurement of carboxymethyl cellulase activity. Anal. Biochem. 2, 127-132
11 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   DOI   ScienceOn
12 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   DOI
13 Hungate, R. E. 1950. The anaerobic insesophilic cellulolytic bacteria. Bacterial. Rev. 14, 1-49
14 Ito, H. Y., Fukuda, K. Murata and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168
15 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   DOI   ScienceOn
16 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
17 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685
18 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
19 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   DOI   ScienceOn
20 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   DOI   ScienceOn
21 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
22 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
23 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   DOI
24 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   DOI   ScienceOn
25 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   DOI