Abstract
Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and β-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an α-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and β-glucosidase was able to produce ethanol from β-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.
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Abdeev, R.M., Goldenkova, I.V., Musiychuk, K.A., and Piruzian, E.S. (2001). Exploring the properties of thermostable Clostridium thermocellum cellulase CelE for the purpose of its expression in plants. Biochemistry 66, 808–813.
Arikan, B., Unaldi, M.N., Guvenmez, H., and Coral, G. (2002). Some Properties of Crude Carboxymethyl Cellulase of Aspergillus niger Z10 wild-Type Strain. Turk. J. Biol. 26, 209–213.
Benitez, J., Silva, A., Vazquez, R., Noa, M.D., and Hollenberg, C.P. (1989). Secretion and glycosylation of Clostridium thermocellum endoglucanase A encoded by the celA gene in Saccharomyces cerevisiae. Yeast 5, 299–306.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Cho, K.M., and Yoo, Y.J. (1999). Novel SSF process form ethanol production from microcrystalline cellulose using the δ-integrated recombinant yeast, L2612δGC. J. Microbiol. Biotechnol. 9, 340–345
Den Haan, R., Rose, S.H., Lynd, L.R., and van Zyl, W.H. (2007). Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. Metab. Eng. 9, 87–94.
Fujita, Y., Takahashi, S., Ueda, M., Tanaka, A., Okada, H., Morikawa, Y., Kawaguchi, T., Arai, M., Fukuda, H., and Kondo, A. (2002). Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl. Environ. Microbiol. 68, 5136–5141.
Guedon, E., Desvaux, M., and Petitdemange, H. (2002). Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering. Appl. Environ. Microbiol. 68, 53–58.
Hahn-Hagerdal, B., Wahlbom, C.F., Gardonyi, M., van Zyl, W.H., Cordero Otero, R.R., and Jonsson, L.J. (2001). Metabolic engineering of Saccharomyces cerevisiae for xylose utilization. Adv. Biochem. Eng. Biotechnol. 73, 53–84.
Hall, J., Hazlewood, G.P., Barker, P.J., and Gilbert, H.J. (1988). Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity. Gene 69, 29–38.
Islam, R., Cicek, N., Sparling, R., and Levin, D. (2008). Influence of initial cellulose concentration on the carbon flow distribution during batch fermentation by Clostridium thermocellum ATCC 27405. Appl. Microbiol. Biotechnol. 82, 141–148.
Katahira, S., Mizuike, A., Fukuda, H., and Kondo, A. (2006). Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain. Appl. Microbiol. Biotechnol. 72, 1136–1143.
Kotaka, A., Bando, H., Kaya, M., Kato-Murai, M., Kuroda, K., Sahara, H., Hata, Y., Kondo, A., and Ueda, M. (2008). Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase. J. Biosci. Bioeng. 105, 622–627.
Lee, J.H., Kim, K., Park, E.H., Ahn, K., and Lim, C.J. (2007). Expression, characterization and regulation of a Saccharomyces cerevisiae monothiol glutaredoxin (Grx6) gene in Schizosaccharomyces pombe. Mol. Cells 24, 316–322.
Lynd, L.R., and Grethlein, H.E. (1987). Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell-free broth from Clostridium thermocellum. Biotechnol. Bioeng. 29, 92–100.
Machida, M., Ohtsuki, I., Fukui, S., and Yamashita, I. (1988). Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 54, 3147–3155.
Murai, T., Ueda, M., Kawaguchi, T., Arai, M., and Tanaka, A. (1998). Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing beta-glucosidase and carboxymethylcellulase from Aspergillus aculeatus. Appl. Environ. Microbiol. 64, 4857–4861.
Okada, H., Sekiya, T., Yokoyama, K., Tohda, H., Kumagai, H., and Morikawa, Y. (1998). Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products. Appl. Microbiol. Biotechnol. 49, 301–308.
Patni, N.J., and Alexander, J.K. (1971). Utilization of glucose by Clostridium thermocellum: presence of glucokinase and other glycolytic enzymes in cell extracts. J. Bacteriol. 105, 220–225.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). A Laboratory Manual, 2nd ed., (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory).
Schwarz, W.H. (2001). The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol. Biotechnol. 56, 634–649.
Schwarz, W.H., Bronnenmeier, K., Grabnitz, F., and Staudenbauer, W.L. (1987). Activity staining of cellulases in polyacrylamide gels containing mixed linkage β-glucans. Anal. Biochem. 164, 72–77.
Stagoj, M.N., Comino, A., and Komel, R. (2006). A novel GAL recombinant yeast strain for enhanced protein production. Biomol. Eng. 23, 195–199.
Teeri, T.T. (1997). Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol. 15, 160–167.
Tokatlidis, K., Salamitou, S., Beguin, P., Dhurjati, P., and Aubert, J.P. (1991). Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components. FEBS Lett. 291, 185–188.
Van Maris, A.J., Abbott, D.A., Bellissimi, E., Van den Brink, J., Kuyper, M., Luttik, M.A., Wisselink, H.W., Scheffers, W.A., Van Dijken, J.P., and Pronk, J.T. (2006). Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek 90, 391–418.
Van Rooyen, R., Hahn-Hagerdal, B., La Grange, D.C., and Van Zyl, W.H. (2005). Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains. J. Biotechnol. 120, 284–295.
Wood, B.E., and Ingram, L.O. (1992). Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum. Appl. Environ. Microbiol. 58, 2103–2110.
Wood, T.M., and Bhat, K.M. (1988). Methods for measuring cellulase activities. Methods Enzymol. 160, 87–112.
Zhou, S., and Ingram, L.O. (2001). Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase. Biotechnol. Lett. 23, 1455–1462.
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Jeon, E., Hyeon, Je., Suh, D.J. et al. Production of cellulosic ethanol in Saccharomyces cerevisiae heterologous expressing Clostridium thermocellum endoglucanase and Saccharomycopsis fibuligera β-glucosidase genes. Mol Cells 28, 369–373 (2009). https://doi.org/10.1007/s10059-009-0131-y
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DOI: https://doi.org/10.1007/s10059-009-0131-y