Improved gene-targeting efficiency upon starvation in Saccharomycopsis

Area of Science:

• Mycology
• Molecular Biology
• Yeast Genetics

Background:

• Fungal transformation is crucial for gene-function analyses, particularly in yeasts like Saccharomycopsis schoenii.
• Established methods like lithium acetate/polyethylene glycol/heat shock yield high transformation efficiency but suffer from frequent ectopic integration.
• Identifying virulence pathways in S. schoenii, a mycoparasitic yeast, requires efficient gene targeting.

Purpose of the Study:

• To optimize DNA-mediated gene targeting in Saccharomycopsis schoenii.
• To overcome the challenge of high ectopic integration frequency in fungal transformation protocols.
• To facilitate gene-function analyses for understanding mycoparasitism in S. schoenii.

Main Methods:

• Utilized the lithium acetate/single strand carrier DNA/Polyethylene glycol/heat shock transformation method.
• Implemented overnight cell starvation prior to transformation to enhance homologous recombination.
• Performed gene deletions (CHS1, HIS3, LEU2) and complementation studies in S. schoenii and S. fermentans.

Main Results:

• Overnight starvation significantly increased gene-targeting via homologous recombination in S. schoenii, despite reducing overall transformant numbers.
• Successfully deleted target genes and determined optimal homology region sizes for gene disruption.
• Demonstrated successful complementation of a leu2 mutant using a heterologous gene and generated a new S. fermentans leu2 strain.

Conclusions:

• Overnight starvation is an effective strategy to enhance homologous recombination and gene targeting efficiency in Saccharomycopsis species.
• This optimized approach facilitates molecular studies of virulence and mycoparasitism in CTG-clade yeasts.
• The developed method shows potential for broader application in other yeast species, including Saccharomycopsis fermentans.