Mollapour, Mehdi; (2000) Molecular genetic analysis of preservative resistance in zygosaccharomyces bailii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The ascomycetous yeast Zygosaccharomyces bailii (Z. bailii) is one of the microorganisms most commonly associated with the spoilage of foods and beverages. This yeast can adapt to become resistant to the highest levels of those weak acid food preservatives (primarily bisulphite, sorbic, benzoic, acetic and propionic acids) allowed in food preservation. Also, under aerobic conditions, it can catabolise some of these preservatives. Z. bailii is also tolerant of in excess of 20% ethanol and a major cause of serious spoilage of bottled wine. This project has been the first to isolate genes of Z. bailii and prove that it is possible to investigate genetically the mechanism of weak acid adaptation in the food spoilage yeast Z. bailii. Z. bailii sequences that complement the ura3 and trp1 mutations of Saccharomyces cerevisiae were initially isolated and sequenced. Also isolated was a Z. bailii gene (ZbYME2) that confers upon S. cerevisie cells the ability to utilise the preservative benzoic acid as sole carbon source. Using DNA cassettes containing dominant selectable markers (KanMX4, hphMX4 and new gene disruption cassette (SFA1MX4)), together with methods that were originally devised for gene deletion in S. cerevisiae, the two copies of ZbYME2 in the genome of Z. bailii strain 1427 were sequentially deleted. Unlike the original wild type isolate, the homozygous Zbyme2/Zbyme2 deletant strain lacks the ability to catabolise benzoate. This is a proof of the principle that it is possible to delete genes in Z. bailii, especially those genes that are suspected to be important for the growth of this yeast in preserved foods and beverages. Physiological analysis of this and other Z. bailii deletant strains should reveal the molecular mechanisms that provide this yeast with an exceptional capacity to adapt to conditions of food preservation. This in turn will allow the rational design of new food preservation strategies. Genes from both S. cerevisiae and Z. bailii that act as the multicopy suppressors of the S. cerevisie weak acid sensitive pdrl2 mutant were also isolated. The isolated S. cerevisie genes (YOR114w, YPL246C) are ORFs of unknown function. The Z. bailii DNA fragments contained a YOR114w homologue and the gene for a small internal fragment of apocytochrome b. The later appears to be a fragment of mitochondrial DNA that has "escaped" to the nucleus. These may represent new genes of weak acid resistance in Z. bailii.

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