Abstract
Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. Further, its association with traditional cheese and meat products imparting special flavors to these products project this yeast with enormous biotechnological potential in the agrofood sector. However, lack of an efficient transformation system in D. hansenii still direct the complementation based assay in S. cerevisiae mutants for functional analysis of D. hansenii genes. Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain, DBH9 (generated by UV induced random mutagenesis), and the DhHIS4 gene as the selectable marker (Minhas et al., 2009). Moreover, the same method has also been employed for gene disruption in D. hansenii by homologous recombination.
Keywords: Debaryomyces hansenii, Transformation, Gene disruption, Halotolerant yeast, Electroporation, Auxotroph
Background
Debaryomyces hansenii CBS767 is one of the most osmotolerant and halotolerant yeasts and can tolerate salinity levels up to 4 M NaCl, whereas growth of Saccharomyces cerevisiae is inhibited when salinity reaches above 1.7 M NaCl. Debaryomyces hansenii is frequently associated with traditional cheese and meat products imparting special flavors to these products. Therefore, this yeast is not only an excellent model to study salt tolerance mechanisms in eukaryotic cells but it also has enormous biotechnological potential in the agrofood sector. However, lack of efficient transformation system and gene disruption tools required for gene manipulation in D. hansenii is a big lacuna behind the molecular analysis of its halotolerant feature. Ricaurte and Govind (1999) developed an ura3 mutant based transformation system for D. hansenii; however this system exhibited low-efficiency. Despite the development of transformation system for Candida famata (Voronovsky et al., 2002), an anamorph of D. hansenii, functional studies of the genes from D. hansenii are still based on complementation of S. cerevisiae mutants. Our transformation system is based on a histidine auxotrophic strain, DBH9 and the DhHIS4 gene as the selectable marker. The same method has also been demonstrated for gene disruption in D. hansenii by homologous recombination (Minhas et al., 2009).
Materials and Reagents
Equipment
Software
Procedure
Notes:
Data analysis
For details, refers to Minhas et al. (2009).
Recipes
Acknowledgments
The research was supported by Council of Scientific and Industrial Research, New Delhi, India. Special gratitude to Dr. Alok Mondal for guiding the original work and writing the published manuscript. We would like to acknowledge our original publication “Minhas, A., Biswas, D. and Mondal, A. K. (2009). Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii. FEMS Yeast Res 9 (1): 95-102.
Competing interests
Authors have no competing interest.
References
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