Agricultural waste biomass has already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR) and the protein engineered xylitol dehydrogenase (XDH) (with NADP) of Pichia stipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomyces cerevisiae as judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARS strain, in which NADP+-dependent XDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH.
Jeffries, T.W., and Jin, Y.S. (2004). “Metabolic Engineering for Improved Fermentation of Pentoses by Yeasts.” Appl Microbiol Biotechnol. 63: pp 495-509.
Jeffries, T. W. (1983). “Utilization of Xylose by Bacteria, Yeasts, and Fungi.” Adv. Biochem. Eng. Biotechnol. 27: pp 1–32.
Kurtzman, C. P. (1994). “Molecular Taxonomy of the Yeasts.” Yeast. 10: pp 1727– 1740.
Jin, Y. S., Lee, T. H., Choi, Y. D., Ryu, Y. W., and Seo, J. H. (2000). “Conversion of Xylose to Ethanol by Recombinant Saccharomyces cerevisiae Containing Genes for Xylose Reductase and Xylitol Dehydrogenase from Pichia stipitis.” J. Microbiol. Biotechnol. 10: pp 564–567.
Walfridsson, M., Anderlund, M., Bao, X., and Hahn-Hägerdal, B. (1997). “Expression of Different Levels of Enzymes from the Pichia stipitis XYL1 and XYL2 Genes in Saccharomyces cerevisiae and its Effects on Product Formation during Xylose Utilization.” Appl Microbiol Biotechnol. 48: pp 218–224.
Verduyn, C., Van Kleef, R., Frank, J., Schreuder, H., Van Dijken, J. P., and Scheffers, W. A. (1985). “Properties of the NAD(P)H-Dependent Xylose Reductase from the Xylose-Fermenting Yeast Pichia stipitis.” Biochem. J. 226: pp 669–677.
Rizzi, M., Harwart, K., Erlemann, P., Buithanh, N. A., and Dellweg, H. (1989). “Purification and Properties of the NAD + -Xylitol-Dehydrogenase from the Yeast Pichia stipitis.” J. Ferment. Bioeng. 67: pp 20–24.
Chiang, L.-C., Gong, C.-S., Chem, L.-F., and Tsao, G. T. (1981). “D-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae.” Appl. Environ. Microbiol. 42: pp 284–289.
Tantirungkij, M., Izuishi, T., Seki, T., and Yoshida, T. (1994). “Fed-Batch Fermentation of Xylose by a Fast-Growing Mutant of Xylose-Assimilating Recombinant Saccharomyces cerevisiae.” Appl. Microbiol. Biotechnol. 41: pp 8–12.
Walfridsson, M., Bao, X., Anderlund, M., Lilius, G., Bulow, L., and Hahn-Hägerdal, B. (1996). “Ethanolic Fermentation of Xylose with Saccharomyces cerevisiae Harboring the Thermus thermophilus xylA Gene, which Expresses an Active Xylose (glucose) Isomerase”. Appl. Envir. Microbiol. 62: pp 4648-4651.
Kuyper, M., Harhangi, H.R., Stave, A.K., Winkler, A.A., Jetten, M.S., de Laat, WT., den Ridder, J.J., Op den Camp, H.J., van Dijken, J.P., and Pronk, J.T. (2003). “HighLevel Functional Expression of a Fungal Xylose Isomerase: The Key to Efficient Ethanolic Fermentation of Xylose by Saccharomyces cerevisiae?’’ FEMS Yeast Res. 4(1): pp 69–78.
Ho, N.W.Y., Chen Z., and Brainard A.P. (1998). “Genetically Engineered Saccharomyces Yeast Capable of Effective Cofermentation of Glucose and Xylose”. App Environ Microbiol. 64: pp1852-1859.
Eliasson, A., Christensson, C., Wahlbom, F.C., and Hahn-hägendal, B. (2000). “Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisia Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures.” App Environ Microbiol. 66(8): pp 3381-3386.
Jeppsson, M., Bengtsson, O., Franke, Katja., Lee, H., Hahn-Hägerdal, B., and GorwaGrauslund, M.F. (2006). “The Expression of a Pichia stipitis Xylose Reductase Mutant with Higher K M for NADPH Increases Ethanol Production from Xylose Recombinant Saccharomyces cerevisiae.” Biotechnol Bioeng. 93: pp 665-673.
Metzger, M.H., and Hollenberg, C.P. (1995). “Amino Acid Substitutions in the Yeast Pichia stipitis Xylitol Dehydrogenase Coenzyme-Binding Domain Affect the Coenzyme Specificity.” Eur J Biochem. 228(1): pp 50–54.
Watanabe, S., Kodaki, T., and Makino, K. (2005). “Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc.” J. Biol. Chem. 280: pp 10340-10349.
Kurztman, C.P., (1994). “Molecular Taxonomy of the Yeasts. Yeast”. 10: pp17271740.
Gietz, D., Jean, A.S., Woods, R.A., and Schiestl, R.H. (1992). “Improved Method for High Efficiency Transformation of Intact Yeast Cells.". Nucleic Acids Research. 20(6): pp 1425.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951). “Protein Measurement with the Folin Phenol Reagent.” J. Biol. Chem. 193: pp 265 – 275.
Kang, Y. S., Kane, J., Kurjan, K., Stadel, J. M., and Tipper, D. J. (1990). Effects of Expression of Mammalian Ga and Hybrid Mammalian-Yeast Ga Proteins on the Yeast Pheromone Response Signal Transduction Pathway.” Mol. Cell. Biol. 10: pp 25822590.
Amore, R., Kötter, P., Kuster, C., Ciriacy, M., and Hollenberg, C. P. (1991). “Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent Xylose Reductase-Encoding Gene xyl1 from the Xylose-Assimilating Yeast Pichia stipitis. Gene.” 109: pp 89-97.
Nikawa, J., sass, P., and Wigler, M. (1987). “Cloning and Characterization of the Low-Affinity Cyclic AMP Phosphodiesterase Gene of Saccharomyces cerevisiae.” Mol. Cell. Biol. 7: pp 3629-3636.
Karhumaa, K., Sanchez, R. G., Hahn-Hägerdal, B., and Gorwa-Grauslund, M. F. (2007). “Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae”. Microb Cell Fact”, 6: pp 5.