Split-marker recombination for efficient targeted gene deletions in Candida intermedia

Övrigt konferensbidrag, 2018

Candida intermedia is a non-conventional yeast species with a natural ability to produce ethanol from xylose, making it an attractive non-GMO alternative for lignocellulosic biomass conversion in biorefineries and/or gene donor to Saccharomyces cerevisiae to improve its xylose fermentation capacity. We have de novo genome sequenced the C. intermedia strain CBS 141442, previously isolated in our lab, which allows us to study the yeast at a genomic and molecular level. The aim of this project was to develop a molecular toolbox for C. intermedia to enable also targeted genome editing and subsequent mutant phenotyping.

C. intermedia is a haploid yeast belonging to the CTG clade of fungal species, and thus requires drug-resistant markers adapted for the alternative codon usage of these organisms. Transformation of linearized plasmid containing the CaNAT1 marker flanked by the TEF1 promoter and terminator from Ashbya gossypii [1] resulted in hundreds of Nourseothricin-resistant transformants. We then constructed an ADE2-deletion cassette, where the CaNAT1 marker was fused to the upstream and downstream sequences (1000bp) of CiADE2. Transformations resulted in less than 1% of ade2 mutants with the characteristic red pigmentation, which indicates that the non-homologous end joining pathway (NHEJ) is dominant over the homologous recombination (HR) pathway in this yeast. Using the cell cycle inhibitor hydroxyurea to arrest cells in the S-phase has been shown to improve the HR/NHEJ ratio in other yeasts [2], and increased the ADE2 deletion efficiency to 4% in C. intermedia. To further improve the targeted deletion rate, we applied the "split-marker” strategy previously developed for Saccharomyces cerevisiae [3]. Here, the selectable marker gene is truncated in two different fragments, and the gene is not functional until homologous recombination takes place between the two overlapping parts of the fragments. The truncated marker gene fragments were flanked by homologous sequences (1000 bp) upstream and downstream of the target gene using fusion PCR, thereby avoiding a tedious cloning step. This approach increased the targeted gene disruption of ADE2 to 56%. As proof of concept, the method was also used to delete KU70, the xylose reductase gene XYL1_2 as well as a large gene cluster in C. intermedia, with allele-specific HR efficiencies between 87 and 100%. The split-marker approach for targeted gene-disruptions will pave the way for high throughput genetic analysis in C. intermedia as well as in other yeasts where NHEJ is the predominant form of recombination.