Targeted gene deletion in Zygosaccharomyces bailii

Yeasts of the genus Zygosaccharomyces are notable agents of large-scale foo d spoilage. Despite the economic importance of these organisms, little is k nown about the stress adaptations whereby they adapt to many of the more se vere conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservativ es and ethanol, can be isolated by complementation of the corresponding mut ant strains of Saccharomyces cerevisiae. it was also discovered that the ac quisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was su fficient for the former yeast to acquire the ability to degrade two major f ood preservatives, benzoic acid and sorbic acid. Using DNA cassettes contai ning dominant selectable markers and methods originally developed for perfo rming gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. b ailii genome were sequentially deleted. The resulting Zbyme2/ Zbyme2 homozy gous deletant strain had lost any ability to utilize benzoate as sole carbo n source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono- oxygenase function, is essential for Z. bailii to degrade food preservative s. This ability to catabolize weak acid preservatives is a significant fact or contributing to the preservative resistance of Z, bailii under aerobic c onditions, This study is the first to demonstrate that it is possible to de lete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of f urther mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Acc ession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170). Copyright (C) 2000 John Wiley & Sons, Ltd.