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A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

 

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The passage describes the development of a toolset for genetic manipulation of the yeast species Hanseniaspora uvarum, which is commonly found in the early stages of wine fermentations. The genome of the diploid type strain DSM 2768 has been largely elucidated, but until recently, transformation by electroporation was not possible. The toolset includes the isolation of a chromosomal replication origin used for the construction of episomal, self-replicating cloning vectors, as well as the creation of homozygous auxotrophic deletion markers in the diploid genome. These markers, including Huura3, Huhis3, Huleu2, and Huade2, can be used as recipients for gene deletion strategies. A hygromycin resistance cassette and a kanamycin resistance gene were established as dominant markers for selection on G418. Recyclable deletion cassettes with loxP-sites and corresponding Cre-recombinase expression vectors were also developed. Finally, a chemical transformation procedure was established using freeze-competent cells. These techniques and constructs provide a means for efficient and targeted manipulations of H. uvarum.

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