Abstract
Fermentation properties under the control of multiple genes of industrial Saccharomyces cerevisiae strain are difficult to alter with traditional methods. Here, we describe efficient and reliable genome shuffling to increase ethanol production through the rapid improvement of stress resistance. The strategy is carried out using yeast sexual and asexual reproduction by itself instead of polyethylene glycol-mediated protoplast fusion. After three rounds of genome shuffling, the best performing strain S3-10 was obtained on the special plate containing a high ethanol concentration. It exhibits substantial improvement in multiple stress tolerance to ethanol, glucose, and heat. The cycle of fermentation of S3-10 was not only shortened, but also, ethanol yield was increased by up to 10.96% compared with the control in very-high-gravity (VHG) fermentations. In total, S3-10 possesses optimized fermentation characteristics, which will be propitious to the development of bioethanol fermentation industry.
Similar content being viewed by others
Abbreviations
- PEG:
-
polyethylene glycol
- EMS:
-
ethyl methane sulfonate
- CFU:
-
colony-forming units
- HPLC:
-
high-performance liquid chromatography
- FCAS:
-
flow-cytometry analysis
- VHG:
-
very high gravity
References
Steinmeta, L. M., Sinha, H., Richaeds, D. R., Spiegelman, J. I., Oefner, P. J., McCusker, J. H., et al. (2002). Dissecting the architecture of a quantitative trait locus in yeast. Nature, 416, 326–330. doi:10.1038/416326a.
Boulton, B., Singleton, V. L., Bisson, L. F., & Kunkee, R. E. (1996). Yeast and biochemistry of ethanol fermentation. In B. Boulton, V. L. Singleton, L. F. Bisson, & R. E. Kunkee (Eds.), Principles and practices of winemaking (pp. 139–172). New York: Chapman and Hall.
Stephanopoulos, G. (2002). Metabolic engineering by genome shuffling. Nature Biotechnology, 20, 666–668. doi:10.1038/nbt0702-666.
Alper, H., Moxley, J., Nevoigt, E., Fink, G. R., & Stephanopoulos, G. (2006). Engineering yeast transcription machinery for improved ethanol tolerance and production. Science, 314, 1565–1568. doi:10.1126/science.1131969.
Zhang, Y. X., Perry, K., Vinci, V. A., Powell, K., Stemmer, W. P. C., & Cardayre, S. B. (2002). Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature, 415, 644–646. doi:10.1038/415644a.
Patnaik, R., Louie, S., Gavrilovic, V., Stemmer, W. P. C., Ryan, C. M., & Cardayre, S. (2002). Genome shuffling of lactobacillus for improved acid tolerance. Nature Biotechnology, 20, 707–712. doi:10.1038/nbt0702-707.
Dai, M. H., & Copley, S. D. (2004). Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Applied and Environmental Microbiology, 70, 2391–2397. doi:10.1128/AEM.70.4.2391-2397.2004.
Hida, H., Yamada, T., & Yamada, Y. (2007). Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Applied Microbiology and Biotechnology, 73, 1387–1393. doi:10.1007/s00253-006-0613-1.
Wei, P., Li, Z., He, P., Lin, Y., & Jiang, N. (2008). Genome shuffling of ethanologenic yeast Candida krusei for improved acetic acid tolerance. Biotechnology and Applied Biochemistry, 49, 113–128. doi:10.1042/BA20070072.
Giudici, P., Solieri, L., Pulvirenti, A. M., & Cassanelli, S. (2005). Strategies and perspectives for genetic improvement of wine yeasts. Applied Microbiology and Biotechnology, 66, 622–628. doi:10.1007/s00253-004-1784-2.
Yu, L., Pei, X., Lei, T., Wang, Y., & Feng, Y. (2008). Genome shuffling enhanced l-lactic acid production by improving glucose tolerance of lactobacillus rhamnosus. Journal of Biotechnology, 134, 154–159. doi:10.1016/j.jbiotec.2008.01.008.
Herman, P. K., & Rine, J. (1997). Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle. The EMBO Journal, 16, 6171–6181. doi:10.1093/emboj/16.20.6171.
Kong, Q. X., Cao, L. M., Zhang, A. L., & Chen, X. (2007). Overexpressing GlT1 in gpd1Δ mutant to improve the production of ethanol of Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 73, 1382–1386. doi:10.1007/s00253-006-0610-4.
Lawrence, C. W. (2004). Guide to yeast genetics and molecular and cell biology. Methods in enzymology Part A. Elesevier academic press NK, 194.
Houston, P., Simon, P. J., & Broach, J. R. (2004). The saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination. Genetics, 166, 1187–1197. doi:10.1534/genetics.166.3.1187.
Spencer, J. F. T., & Spencer, D. M. (1996). Yeast Protocols: Methods in Cell and Molecular Biology, Mutagenesis in yeast. 17–18.
Carlson, C. R., Grallert, B., Bernander, R., Stokke, T., & Boye, E. (1997). Measurement of nuclear DNA content in fission yeast by flow cytometry. Yeast (Chichester, England), 13, 1329–1335. doi:10.1002/(SICI)1097-0061(199711)13:14<1329::AID-YEA185>3.0.CO;2-M.
Ormerod, M. G., & Kubbies, M. (1992). Cell cycle analysis of asynchronous cells by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain. Cytometry, 13, 678–685. doi:10.1002/cyto.990130703.
Rautio, J. J., Huuskonen, A., Vuokko, H., Vidgren, V., & Londesborough, J. (2007). Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression. Yeast (Chichester, England), 24, 741–760. doi:10.1002/yea.1510.
Cardona, F., Carrasco, P., Perez-Ortin, J. E., Olmo, M., & Aranda, A. (2007). A novel approach for the improvement of stress resistance in wine yeasts. International Journal of Food Microbiology, 28, 83–91. doi:10.1016/j.ijfoodmicro.2006.10.043.
Jones, R. P. (1989). Biological principles for the effects of ethanol. Enzyme and Microbial Technology, 11, 130–153. doi:10.1016/0141-0229(89)90073-2.
Zhao, K., Ping, W., Zhang, L., Liu, J., Lin, Y., Jin, T., et al. (2008). Screening and breeding of high taxol producing fungi by genome shuffling. Science in China Series C-Life Sciences, 51, 222–231. doi:10.1007/s11427-008-0037-5.
Mayer, W. V., & Aguilera, A. (1990). High levels of chromosome instability in polyploids of Saccharomyces cerevisiae. Mutation Research, 231, 177–186. doi:10.1016/0027-5107(90)90024-X.
Devantier, R., Pedersen, S., & Olsson, L. (2005). Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain. Applied Microbiology and Biotechnology, 68, 622–629. doi:10.1007/s00253-005-1902-9.
Acknowledgments
The author particularly thanks Prof. P. Ma for constructive advice on this work. The research was supported by the National Natural Science Foundation of China (no. 30470849).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hou, L. Improved Production of Ethanol by Novel Genome Shuffling in Saccharomyces cerevisiae . Appl Biochem Biotechnol 160, 1084–1093 (2010). https://doi.org/10.1007/s12010-009-8552-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-009-8552-9