Journal of Life Science (생명과학회지)

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

DOI QR Code

Breeding of Ethanol-producing and Ethanol-tolerant Saccharomyces cerevisiae using Genome Shuffling

Genome shuffling을 이용한 에탄올 생산 및 내성 효모 균주의 육종

  • Park, A-Hwang (Department of Biomaterial Control, Dong-Eui University) ;
  • Kim, Yeon-Hee (Department of Biomaterial Control, Dong-Eui University)
  • 박아황 (동의대학교 바이오물질제어공학과) ;
  • 김연희 (동의대학교 바이오물질제어공학과)
  • Received : 2013.07.18
  • Accepted : 2013.10.23
  • Published : 2013.10.30
Download PDF
⟨ Previous Next ⟩

Abstract

To improve yeast strains for bioethanol production, yeasts with ethanol tolerance, thermotolerance, and ${\beta}$-1,3-glucanase activity were bred using yeast genome shuffling. Saccharomyces cerevisiae $BY4742{\Delta}exg1$/pAInu-exgA, which has extracellular ${\beta}$-1,3-glucanase activity, and the Aspergillus oryzae and S. cerevisiae YKY020 strains, which exhibit ethanol tolerance and thermotolerance, were fused by yeast protoplast fusion. Following cell fusion, four candidate cells (No. 3, 9, 11, and 12 strains) showing thermotolerance at $40^{\circ}C$ were selected, and their ethanol tolerance (7% ethanol concentration) and ${\beta}$-1,3-glucanase activity were subsequently analyzed. All the phenotypes of the two parent cells were simultaneously expressed in one (No. 11) of the four candidate cells, and this strain was called BYK-F11. The BYK-F11 fused cell showed enhanced cell growth, ethanol tolerance, ${\beta}$-1,3-glucanase activity, and ethanol productivity compared with the $BY4742{\Delta}exg1$/pAInu-exgA and YKY020 strains. The results prove that a new yeast strain with different characters and the same mating type can be easily bred by protoplast fusion of yeasts.

바이오 에탄올 생산을 위한 최적 효모균주의 개량을 위해 효모 genome shuffling 법을 이용하여 에탄올내성, 내열성 및 ${\beta}$-1,3-glucanase 활성을 가진 효모균주의 육종을 계획하였다. 본 연구에서는 세포 외 ${\beta}$-1,3-glucanase 활성을 가진 Saccharomyces cerevisiae $BY4742{\Delta}exg1$/pAInu-exgA 균주와 에탄올내성 및 내열성을 가진 S. cerevisiae YKY020 균주를 효모 protoplast fusion을 통하여 융합시켰다. 세포융합에 의해 $40^{\circ}C$에서 내열성을 보이는 네 개의 후보 균주(No. 3, 9, 11, 12)를 선별한 다음, 7% 에탄올 농도에서의 에탄올내성 및 ${\beta}$-1,3-glucanase 활성을 조사하였다. 두 모균주의 모든 표현형을 보이는 하나의 균주(No. 11)가 선별되었고, 이 균주를 BYK-F11이라고 명명하였다. BYK-F11 융합균주는 $BY4742{\Delta}exg1$/pAInu-exgA와 YKY020균주에 비해서 증가된 세포성장속도, 에탄올 내성, ${\beta}$-1,3-glucanase 활성 및 에탄올 생산성을 보임을 알 수 있었다. 따라서 본 연구에서는 다양한 특성을 가지지만 같은 접합형을 가진 효모균주들을 protoplast fusion법을 사용하여 손쉽게 새로운 산업용 효모균주로 육종시킬 수 있다는 것을 증명하였다.

Keywords

References

  1. Attfield, P. V. 1997. Stress tolerance: the key to effective strains of industrial bakers yeast. Nat Biotechnol 15, 1351-1357. https://doi.org/10.1038/nbt1297-1351
  2. Bajwa, P. K., Pinel, D., Martin, V. J., Trevors, J. T. and Lee, H. 2010. Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling. J Microbiol Methods 81, 179-186. https://doi.org/10.1016/j.mimet.2010.03.009
  3. Bara, M. T. F., Lima, A. L. and Ulhoa, C. J. 2003. Purification and characterization of an exo-${\beta}$-1,3-glucanase produced by Trichoderma asperellum. FEMS Microbiol Lett 219, 81-85. https://doi.org/10.1016/S0378-1097(02)01191-6
  4. Dai, M. H. and Copley, S. D. 2004. Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Appl Environ Microbiol 70, 2391-2397. https://doi.org/10.1128/AEM.70.4.2391-2397.2004
  5. Guegen, Y., Chemardin, P., Janbon, G., Arnaud, A. and Galzy, P. 1996. A very efficient ${\beta}$-glucosidase catalyst for the hydrolysis of flavor precursors of wines and fruit juices. J Agric Food Chem 44, 2336-2340. https://doi.org/10.1021/jf950360j
  6. Harashima, S., Takagi, A. and Oshima, Y. 1984. Transformation of protoplasted yeast cells is directly associated with cell fusion. Mol Cell Biol 4, 771-778. https://doi.org/10.1128/MCB.4.4.771
  7. Hida, H., Yamada, T. and Yamada, Y. 2007. Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Appl Microbio Biotech 73, 1387-1393. https://doi.org/10.1007/s00253-006-0613-1
  8. Hou, L. 2010. Improved production of ethanol by novel genome shuffling in Saccharomyces cerevisiae. Appl Biochem Biotechnol 160, 1084-1093. https://doi.org/10.1007/s12010-009-8552-9
  9. Jeon, H. T., Park, U. M. and Kim, K. 2011. The use of aureobasidin A resistant gene as the dominant selectable marker for the selection of industrial yeast hybrid. Korean J Microbiol Biotechnol 39, 111-118.
  10. Jijakli, M. H. and Lepoivre, P. 1998. Characterization of an exo-${\beta}$-1,3-glucanase produced by Pichia anomala strain K, antagonist of Botrytiscinerea on apples. Phytopathology 88, 335-343. https://doi.org/10.1094/PHYTO.1998.88.4.335
  11. Kim, M. J., Nam, S. W., Tamano, K., Machida, M., Kim, S. K. and Kim, Y. H. 2011. Optimazation for production of exo-${\beta}$-1,3-glucanase (laminarase) from Aspergillus oryzae in Saccharomyces cerevisiae. Korean Soc Biotech Bioeng 26, 427-432.
  12. Lee, S. M., Kim, J. H., Cho, H. Y., Joo, H. and Lee, J. H. 2009. Production of bio-ethanol from brown algae by physicochemical hydrolysis. J Korean Ind Eng Chem 20, 517-521.
  13. Lin, Y., Zhang, W., Li, C., Sakakibara, K., Tanaka, S. and Kong, H. 2012. Factors affecting ethanol fermentation using Saccharomyces cerevisiae BY4742. Biomass Bioenergy 47, 395- https://doi.org/10.1016/j.biombioe.2012.09.019
  14. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal Chem 31, 426-428. https://doi.org/10.1021/ac60147a030
  15. Patnaik, R., Louie, S., Gavrilovic, V., Stemmer, W. P. C., Ryan, C. M. and Cardayre, S. 2002. Genome shuffling of lactobacillus for improved acid tolerance. Nature Biotech 20, 707-712. https://doi.org/10.1038/nbt0702-707
  16. Pitson, S. M., Seviour, R. J. and McDougall, B. M. 1993. Noncellulolytic fungal beta-glucanases: their physiology and regulation. Enzyme Microb Technol 15, 178-192. https://doi.org/10.1016/0141-0229(93)90136-P
  17. Sheehan, C. and Weiss, A. S. 1990. Yeast artificial chromosome: rapid extraction for high resolution analysis. Necleic Acids Res 18, 2193. https://doi.org/10.1093/nar/18.8.2193
  18. Shi, D. J., Wang, C. L. and Wang, K. M. 2009. Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae. J Mircobiol Biotechnol 36, 139-147.
  19. Shoseyov, O., Bravdo, A. B., Ikan, R. and Chet, I. 1990. Immobilized endo-${\beta}$-glucosidase enriches flavor of wine and passion fruit juice. J Agric Food Chem 27, 1973-1976.
  20. Spencer, J. F. T. and Spencer, D. M. 1983. Genetic improvement of industrial yeast. Ann Rev Microbiol 37, 121-142. https://doi.org/10.1146/annurev.mi.37.100183.001005
  21. van Rensburg, P., van Zyl, W. H. and Pretorius, I. S. 1997. Over-expression of the Saccharomyces cerevisiae exo-${\beta}$-1,3-glucanase gene together with the Bacillus subtilis endo-${\beta}$- 1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-${\beta}$-1,4-glucanase gene in yeast. J Biotechnol 55, 43-53. https://doi.org/10.1016/S0168-1656(97)00059-X
  22. Wei, P., Li, Z., He, P., Lin, Y. and Jiang, N. 2008. Genome shuffling of ethanologenic yeast Candida krusei for improved acetic acid tolerance. Biotech Appl Biochem 49, 113-128. https://doi.org/10.1042/BA20070072
  23. Zhang, Y. X., Perry, K., Vinci, V. A., Powell, K., Stemmer, W. P. and del Cardayre, S. B. 2002. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 644-646. https://doi.org/10.1038/415644a
  24. Zheng, D. Q., Wu, X. C., Tao, X. L., Wang, P. M., Li, P., Chi, X. Q., Li, Y. D., Yan, Q. F. and Zhao, Y. H. 2011. Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance. Bioresour Technol 102, 3020-3027. https://doi.org/10.1016/j.biortech.2010.09.122

Cited by

  1. Investigation into the Ethanol Tolerance Mechanism by Regulation of Gene Expression vol.26, pp.1, 2016, https://doi.org/10.5352/JLS.2016.26.1.17
  2. Influence of Chromosome Number on Cell Growth and Cell Aging in Yeast vol.26, pp.6, 2016, https://doi.org/10.5352/JLS.2016.26.6.646