Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Genetic Transformation of the Yeast Dekkera/Brettanomyces bruxellensis with Non-Homologous DNA

  • Miklenic, Marina (Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb) ;
  • Stafa, Anamarija (Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb) ;
  • Bajic, Ana (Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb) ;
  • Zunar, Bojan (Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb) ;
  • Lisnic, Berislav (Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb) ;
  • Svetec, Ivan-Kresimir (Laboratory for Biology and Microbial Genetics, Faculty of Food Technology and Biotechnology, University of Zagreb)
  • Received : 2012.11.19
  • Accepted : 2012.12.31
  • Published : 2013.05.28
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Abstract

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/${\mu}g$ DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

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References

  1. Abdel-Banat, B. M., S. Nonklang, H. Hoshida, and R. Akada. 2010. Random and targeted gene integrations through the control of non-homologous end joining in the yeast Kluyveromyces marxianus. Yeast 27: 29-39.
  2. Blomqvist, J., E. South, I. Tiukova, M. H. Momeni, H. Hansson, J. Stahlberg, et al. 2011. Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis. Lett. Appl. Microbiol. 53: 73-78. https://doi.org/10.1111/j.1472-765X.2011.03067.x
  3. Chatonnet, P., D. Dubourdieu, J. N. Boidron, and M. Pons. 1992. The origin of ethylphenols in wines. J. Sci. Food Agric. 60: 165-178. https://doi.org/10.1002/jsfa.2740600205
  4. Chatonnet, P., D. Dubourdieu, and J. N. Boidron. 1995. The influence of Brettanomyces/Dekkera spp. yeasts and lactic acid bacteria on the ethylphenol content of red wines. Am. J. Enol. Vitic. 46: 463-468.
  5. Chatonnet, P., C. Viala, and D. Dubourdieu. 1997. Influence of polyphenolic components of red wines on the microbial synthesis of volatile phenols. Am. J. Enol. Vitic. 48: 443-448.
  6. Chua, G., L. Taricani, W. Stangle, and P. G. Young. 2000. Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe. Nucleic Acids Res. 28: E53. https://doi.org/10.1093/nar/28.11.e53
  7. Curtin, C. D., A. R. Borneman, P. J. Chambers, and I. S. Pretorius. 2012. De-novo assembly and analysis of the heterozygous triploid genome of the wine spoilage yeast Dekkera bruxellensis AWRI1499. PLoS One 7: e33840. https://doi.org/10.1371/journal.pone.0033840
  8. de Barros Pita, W., F. C. Leite, A. T. de Souza Liberal, D. A. Simoes, and M. A. de Morais Jr. 2011. The ability to use nitrate confers advantage to Dekkera bruxellensis over S. cerevisiae and can explain its adaptation to industrial fermentation processes. Antonie Van Leeuwenhoek. 100: 99-107. https://doi.org/10.1007/s10482-011-9568-z
  9. de Souza Liberal, A. T., A. C. Basilio, A. do Monte Resende, B. T. Brasileiro, E. A. da Silva-Filho, J. O. de Morais, et al. 2007. Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation. J. Appl. Microbiol. 102: 538-547.
  10. Farah, J. A., E. Hartsuiker, K. Mizuno, K. Ohta, and G. R. Smith. 2002. A 160-bp palindrome is Rad50.Rad32-dependent mitotic recombination hotspot in Schizosaccharomyces pombe. Genetics 161: 461-468.
  11. Freer, S. N., B. Dien, and S. Matsuda. 2003. Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH. World J. Microbiol. Biotechnol. 19: 101-105. https://doi.org/10.1023/A:1022592810405
  12. Fugelsang, K. C. 1997. Wine Microbiology. Chapman and Hall New York, NY.
  13. Galafassi, S., A. Merico, F. Pizza, L. Hellborg, F. Molinari, J. Piskur, and C. Compagno. 2011. Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygenlimited and low-pH conditions. J. Ind. Microbiol. Biotechnol. 38: 1079-1088. https://doi.org/10.1007/s10295-010-0885-4
  14. Gietz, R. D. and R. A. Woods. 2001. Genetic transformation of yeast. Biotechniques 30: 816-820, 822-826, 28 passim.
  15. Gietz, R. D., R. H. Schiestl, A. R. Willems, and R. A. Woods. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11: 355-360. https://doi.org/10.1002/yea.320110408
  16. Gietz, R. D. and R. H. Schiestl. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2: 31-34. https://doi.org/10.1038/nprot.2007.13
  17. Gjura i , K. and Z. Zgaga. 1996. Illegitimate integration of single stranded DNA in Saccharomyces cerevisiae. Mol. Gen. Genet. 253: 173-181. https://doi.org/10.1007/s004380050310
  18. Gray, M. and S. M. Honigberg. 2001. Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces. Nucleic Acids Res. 29: 5156-5162. https://doi.org/10.1093/nar/29.24.5156
  19. Grigoriev, I. V., H. Nordberg, I. Shabalov, A. Aerts, M. Cantor, D. Goodstein, et al. 2012. The genome portal of the Department of Energy Joint Genome Institute. Nucleic Acids Res. 40(Database issue): 26-32.
  20. Hastings, P. J., C. McGill, B. Shafer, and J. N. Strathern. 1993. Ends-in vs. ends-out recombination in yeast. Genetics 135: 973-980.
  21. Hinnen, A., J. B. Hicks, and G. R. Fink. 1978. Transformation of yeast. Proc. Natl. Acad. Sci. USA 75: 1929-1933. https://doi.org/10.1073/pnas.75.4.1929
  22. Kawai, S., T. A. Pham, H. T. Nguyen, H. Nankai, T. Utsumi, Y. Fukuda, and K. Murata. 2004. Molecular insights on DNA delivery into Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 317: 100-107. https://doi.org/10.1016/j.bbrc.2004.03.011
  23. Kawai, S., W. Hashimoto, and K. Murata. 2010. Transformation of Saccharomyces cerevisiae and other fungi: Methods and possible underlying mechanism. Bioeng. Bugs 1: 395-403. https://doi.org/10.4161/bbug.1.6.13257
  24. Kegel, A., P. Martinez, S. D. Carter, and S. U. Astrom. 2006. Genome wide distribution of illegitimate recombination events in Kluyveromyces lactis. Nucleic Acids Res. 34: 1633-1645. https://doi.org/10.1093/nar/gkl064
  25. Klinner, U. and B. Schafer. 2004. Genetic aspects of targeted insertion mutagenesis in yeasts. FEMS Microbiol. Rev. 28: 201-223. https://doi.org/10.1016/j.femsre.2003.10.002
  26. Licker, J. L., T. E. Acree, and T. Henick-Kling. 1999. What is "Brett" (Brettanomyces) flavour? A preliminary investigation, pp. 96-115. In A. L. Waterhouse and S. E. Ebeler (eds.). Chemistry of Wine Flavour. American Chemical Society, Washington, DC.
  27. Lisni , B., I. K. Svetec, A. Stafa, and Z. Zgaga. 2009. Sizedependant palindrome-induced intrachromosomal recombination in yeast. DNA Repair 8: 383-389. https://doi.org/10.1016/j.dnarep.2008.11.017
  28. Mezard, C. and A. Nicolas. 1994. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae. Mol. Cell. Biol. 14: 1278-1292.
  29. Piskur, J., Z. Ling, M. Marcet-Houben, O. P. Ishchuk, A. Aerts, K. LaButti, et al. 2012. The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties. Int. J. Food Microbiol. 157: 202-209. https://doi.org/10.1016/j.ijfoodmicro.2012.05.008
  30. Rose, M. D. 1987. Isolation of genes by complementation in yeast. Methods Enzymol. 152: 481-504. https://doi.org/10.1016/0076-6879(87)52056-0
  31. Rozp dowska, E., L. Hellborg, O. P. Ishchuk, F. Orhan, S. Galafassi, A. Merico, et al. 2011. Parallel evolution of the makeaccumulate- consume strategy in Saccharomyces and Dekkera yeasts. Nat. Commun. 2: 302. https://doi.org/10.1038/ncomms1305
  32. Sambrook, J. and D. W. Russel. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, 3th Ed, Cold Spring Harbour, NY.
  33. Schiestl, R. H. and R. D. Gietz. 1989. High efficency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16: 339-346. https://doi.org/10.1007/BF00340712
  34. Schiestl, R. H. and T. D. Petes. 1991. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88: 7585-7589. https://doi.org/10.1073/pnas.88.17.7585
  35. Serpaggia, V., F. Remize, G. Recorbet, E. Gaudot-Dumas, A. Sequeira-Le Grand, and H. Alexandre. 2012. Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiol. 30: 438-447. https://doi.org/10.1016/j.fm.2011.12.020
  36. Svetec, I. K., A. Stafa, and Z. Zgaga. 2007. Genetic side effects accompanying gene targeting in yeast: The influence of short heterologous termini. Yeast 24: 637-652. https://doi.org/10.1002/yea.1497
  37. Svetec, I. K., B. Lisni , and Z. Zgaga. 2002. A 110 bp palindrome stimulates plasmid integration in yeast. Period. Biol. 104: 421-424.
  38. Stafa, A., I. K. Svetec, and Z. Zgaga. 2005. Inactivation of the SGS1 and EXO1 genes synergistically stimulates plasmid integration in yeast. Food Technol. Biotechnol. 43: 103-108.
  39. Tatebayashi, K., J. Kato, and H. Ikeda. 1994. Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe. Mol. Gen. Genet. 244: 111-119.
  40. Wang, T. T., Y. L. Choi, and B. H. Lee. 2001. Transformation systems of non-Saccharomyces yeasts. Crit. Rev. Biotechnol. 21: 177-218. https://doi.org/10.1080/20013891081719

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