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

• Published in: International journal of molecular sciences Vol. 24; no. 3; p. 1859
• Main Authors: Heinisch, Jürgen J, Murra, Andrea, Jürgens, Kai, Schmitz, Hans-Peter
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 17-01-2023; MDPI
• Subjects: Antibiotics; Aroma compounds; auxotrophic markers; Cassettes; Cloning; Cloning vectors; Cre/loxP system; Diploids; dominant selection markers; E coli; Electroporation; Enzymes; Expression vectors; Fruits; Gene deletion; Genes; Genetic engineering; Genetic transformation; Genomes; Hanseniaspora - genetics; Hanseniaspora uvarum; Hygromycin; Kanamycin; Kinases; Metabolism; Nucleotide sequence; Plasmids; Polymerase Chain Reaction; Replication origins; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; transformation; Wine; wine yeast; Yeast; wine yeast; dominant selection markers; Cre/loxP system; auxotrophic markers; transformation
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24031859

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 (
,
,
,
) 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
-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
.