Generation of stable homozygous transformants of diploid yeasts such as Xanthophyllomyces dendrorhous

The nonconventional yeast Xanthophyllomyces dendrorhous is an established platform for genetic pathway modification. A genetic tool box is available and can be used extensively to select from for different engineering strategies. Due to the diploid nature of X. dendrorhous, genetic transformation typically results in heterozygous lines. They are genetically unstable and lose their phenotypes caused by mitotic recombination. In addition, targeted integration for inactivation of genes of the carotenoid pathway resulted in an intermediary phenotype of incomplete pathway disruption. This issue is the main scope of this review. It is illustrated by using genetic modification of the carotenoid pathway of X. dendrorhous as a model system with a focus on the demonstration of how to solve these problems by generation of homozygous lines. They can be selected from heterozygous transformants after spontaneous mitotic recombination and selection or after induced meiotic recombination. Corresponding methods of how to proceed including the initiation of a sexual cycle are described. The selected segregated lines are stable in fermenter cultures without the need of selection pressure. This is an essential requirement for any industrial application. KEY POINTS: • Genetic interventions of diploid yeasts result in heterozygous transformants that are unstable without selection pressure. • This is due to mitotic recombination leading to the elimination of inserted DNA. • Stable homozygous lines can be obtained and selected after either meiotic or mitotic recombination.