Molecular Tools and Protocols for Engineering the Acid-Tolerant Yeast Zygosaccharomyces bailii as a Potential Cell Factory

  • Paola BranduardiEmail author
  • Laura Dato
  • Danilo Porro
Part of the Methods in Molecular Biology book series (MIMB, volume 1152)


Microorganisms offer a tremendous potential as cell factories, and they are indeed used by humans for centuries for biotransformations. Among them, yeasts combine the advantage of unicellular state with a eukaryotic organization, and, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycetales budding yeast, is widely known for its peculiar tolerance to various stresses, among which are organic acids. Despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here, we describe in detail protocols for transformation, for target gene disruption or gene integration, and for designing episomal expression plasmids helpful for developing and further studying the yeast Z. bailii.

Key words

Zygosaccharomyces bailii Yeast transformation Targeted gene deletion Plasmids Promoters 



These works were partially supported by FAR (Fondo di Ateneo per la Ricerca) of the University of Milano-Bicocca to PB and DP.


  1. 1.
    Erickson B, Nelson WP (2012) Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol J 7:176–185CrossRefGoogle Scholar
  2. 2.
    Otero JM, Nielsen J (2010) Industrial systems biology. Biotechnol Bioeng 105:439–460CrossRefGoogle Scholar
  3. 3.
    Porro D, Gasser B, Fossati T, Maurer M, Branduardi P, Sauer M, Mattanovich D (2011) Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems? Appl Microbiol Biotechnol 89:939–948CrossRefGoogle Scholar
  4. 4.
    Sauer M, Porro D, Mattanovich D, Branduardi P (2010) 16 years research on lactic acid production with yeast – ready for the market? Biotechnol Genet Eng Rev 27:229–256CrossRefGoogle Scholar
  5. 5.
    Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D (2012) Recombinant protein production in yeasts. Methods Mol Biol 824:329–358CrossRefGoogle Scholar
  6. 6.
    Johnson EA (2013) Biotechnology of non-Saccharomyces yeasts-the ascomycetes. Appl Microbiol Biotechnol 97(2):503–517Google Scholar
  7. 7.
    Thomas DS, Davenport RR (1985) Zygosaccharomyces bailii – a profile of characteristics and spoilage activities. Food Microbiol 2:157–169CrossRefGoogle Scholar
  8. 8.
    Cole MB, Keenan MHJ (1987) Effects of weak acids and external ph on the intracellular pH of Zygosaccharomyces bailii, and its implications in weak-acid resistance. Yeast 3:23–32CrossRefGoogle Scholar
  9. 9.
    Makdesi AK, Beuchat LR (1996) Evaluation of media for enumerating heat-stressed, benzoate-resistant Zygosaccharomyces bailii. Int J Food Microbiol 33:169–181CrossRefGoogle Scholar
  10. 10.
    Sousa MJ, Miranda L, CorteReal M, Leao C (1996) Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments. Appl Environ Microbiol 62:3152–3157Google Scholar
  11. 11.
    Sousa MJ, Rodrigues F, Corte-Real M, Leao C (1998) Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii. Microbiology 144:665–670CrossRefGoogle Scholar
  12. 12.
    Dang TD, De Maeseneire SL, Zhang BY, De Vos WH, Rajkovic A, Vermeulen A, Van Impe JF, Devlieghere F (2012) Monitoring the intracellular pH of Zygosaccharomyces bailii by green fluorescent protein. Int J Food Microbiol 156:290–295CrossRefGoogle Scholar
  13. 13.
    Guerreiro JF, Mira NP, Sá-Correia I (2012) Adaptive response to acetic acid in the highly resistant yeast species Zygosaccharomyces bailii revealed by quantitative proteomics. Proteomics 12:2303–2318CrossRefGoogle Scholar
  14. 14.
    Mira NP, Teixeira MC, Sá-Correia I (2010) Adaptive response and tolerance to weak acids in Saccharomyces cerevisiae: a genome-wide view. OMICS 14:525–540CrossRefGoogle Scholar
  15. 15.
    Liu ZL (2011) Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates. Appl Microbiol Biotechnol 90:809–825CrossRefGoogle Scholar
  16. 16.
    Sauer M, Porro D, Mattanovich D, Branduardi P (2008) Microbial production of organic acids: expanding the markets. Trends Biotechnol 26:100–108CrossRefGoogle Scholar
  17. 17.
    Branduardi P, Valli M, Brambilla L, Sauer M, Alberghina L, Porro D (2004) The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications. FEMS Yeast Res 4:493–504CrossRefGoogle Scholar
  18. 18.
    Branduardi P, Fossati T, Sauer M, Pagani R, Mattanovich D, Porro D (2007) Biosynthesis of vitamin C by yeast leads to increased stress resistance. PLoS One 2:e1092CrossRefGoogle Scholar
  19. 19.
    Vigentini I, Brambilla L, Branduardi P, Merico A, Porro D, Compagno C (2005) Heterologous protein production in Zygosaccharomyces bailii: physiological effects and fermentative strategies. FEMS Yeast Res 5:647–652CrossRefGoogle Scholar
  20. 20.
    Camattari A, Bianchi MM, Branduardi P, Porro D, Brambilla L (2007) Induction by hypoxia of heterologous-protein production with the KlPDC1 promoter in yeasts. Appl Environ Microbiol 73:922–929CrossRefGoogle Scholar
  21. 21.
    Passolunghi S, Riboldi L, Dato L, Porro D, Branduardi P (2010) Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype. Microb Cell Fact 9:7CrossRefGoogle Scholar
  22. 22.
    Dato L, Branduardi P, Passolunghi S, Cattaneo D, Riboldi L, Frascotti G, Valli M, Porro D (2010) Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant. FEMS Yeast Res 10:894–908CrossRefGoogle Scholar
  23. 23.
    Sauer M, Branduardi P, Valli M, Porro D (2004) Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii. Appl Environ Microbiol 70:6086–6091CrossRefGoogle Scholar
  24. 24.
    Mann C, Davis RW (1986) Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol Cell Biol 6:241–245Google Scholar
  25. 25.
    Araki H, Oshima Y (1989) An autonomously replicating sequence of pSRI plasmid is effective in two yeast species, Zygosaccharomyces rouxii and Saccharomyces cerevisiae. J Mol Biol 207:757–769CrossRefGoogle Scholar
  26. 26.
    Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179–188CrossRefGoogle Scholar
  27. 27.
    Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassette for gene disruption in Saccharomyces cerevisiae. Yeast 15:13Google Scholar
  28. 28.
    Krügel H, Fiedler G, Smith C, Baumberg S (1993) Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei. Gene 127:127–131CrossRefGoogle Scholar
  29. 29.
    Kikuchi Y (1983) Yeast plasmid requires a cis-acting locus and two plasmid proteins for its stable maintenance. Cell 34:7CrossRefGoogle Scholar
  30. 30.
    Toh-e A, Araki H, Utatsu I, Oshima Y (1984) Plasmids resembling 2-micrometers DNA in the osmotolerant yeasts Saccharomyces bailii and Saccharomyces bisporus. J Gen Microbiol 130:8Google Scholar
  31. 31.
    Utatsu I, Sakamoto S, Imura T, Tohe A (1987) Yeast plasmids resembling 2-μm DNA – regional similarities and diversities at the molecular level. J Bacteriol 169:5537–5545Google Scholar
  32. 32.
    Merico A, Rodrigues F, Côrte-Real M, Porro D, Ranzi B, Compagno C (2001) Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii. Yeast 18:775–780CrossRefGoogle Scholar
  33. 33.
    Gonzalez C, Perdomo G, Tejera P, Brito N, Siverio JM (1999) One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha. Yeast 15:1323–1329CrossRefGoogle Scholar
  34. 34.
    Kelly R, Miller SM, Kurtz MB, Kirsch DR (1987) Directed mutagenesis in Candida albicans – one-step gene disruption to isolate ura3 mutants. Mol Cell Biol 7:199–207Google Scholar
  35. 35.
    Guangtao Z, Hartl L, Schuster A, Polak S, Schmoll M, Wang T, Seidl V, Seiboth B (2009) Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol 139:146–151Google Scholar
  36. 36.
    Mollapour M, Piper PW (2001) Targeted gene deletion in Zygosaccharomyces bailii. Yeast 18:173–186CrossRefGoogle Scholar
  37. 37.
    Rodrigues F, Ludovico P, Sousa MJ, Steensma HY, Corte-Real M, Leao C (2003) The spoilage yeast Zygosaccharomyces bailii forms mitotic spores: a screening method for haploidization. Appl Environ Microbiol 69:649–653CrossRefGoogle Scholar
  38. 38.
    Dato L, Sauer M, Passolunghi S, Porro D, Branduardi P (2008) Investigating the multibudded and binucleate phenotype of the yeast Zygosaccharomyces bailii growing on minimal medium. FEMS Yeast Res 8:906–915CrossRefGoogle Scholar
  39. 39.
    Rodrigues F, Zeeman AM, Alves C, Sousa MJ, Steensma HY, Corte-Real M, Leao C (2001) Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2). FEMS Yeast Res 1:67–71Google Scholar
  40. 40.
    Wach A, Brachat A, Pöhlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808CrossRefGoogle Scholar
  41. 41.
    Popolo L, Vai M (1999) The Gas1 glycoprotein, a putative wall polymer cross-linker. Biochim Biophys Acta 1426:385–400CrossRefGoogle Scholar
  42. 42.
    Goldstein A, McCusker J (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541–1553CrossRefGoogle Scholar
  43. 43.
    Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking 6-base pair restriction sites. Gene 74:527–534Google Scholar
  44. 44.
    Ogawa Y, Tatsumi H, Murakami S, Ishida Y, Murakami K, Masaki A, Kawabe H, Arimura H, Nakano E, Motai H (1990) Secretion of Aspergillus oryzae alkaline protease in an osmophilic yeast, Zygosaccharomyces rouxii. Agric Biol Chem 54:2521–2529CrossRefGoogle Scholar
  45. 45.
    Branduardi P (2002) Molecular cloning and sequence analysis of the Zygosaccharomyces bailii HIS3 gene encoding the imidazole glycerol phosphate dehydratase. Yeast 19:1165–1170CrossRefGoogle Scholar
  46. 46.
    Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272CrossRefGoogle Scholar
  47. 47.
    Venturini M, Morrione A, Pisarra P, Martegani E, Vanoni M (1997) In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins. Mol Microbiol 23:997–1007CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  1. 1.Department of Biotechnology and BiosciencesUniversity of Milano-BicoccaMilanItaly

Personalised recommendations