PCR-based gene targeting in Hanseniaspora uvarum.

Area of Science:

• Microbiology
• Molecular Biology
• Yeast Genetics

Background:

• Hanseniaspora uvarum is a prevalent yeast in winemaking environments.
• Limited tools for gene-function analysis hinder the study of H. uvarum biology.
• Understanding H. uvarum is crucial for wine fermentation and spoilage processes.

Purpose of the Study:

• To develop and validate a rapid PCR-based gene targeting system for H. uvarum.
• To create tools for one-step gene replacement in this diploid yeast.
• To facilitate genetic manipulation for studying H. uvarum gene functions.

Main Methods:

• Generation and validation of two synthetic antibiotic resistance genes (pFA-hygXL, pFA-clnXL) for hygromycin and nourseothricin resistance.
• Utilizing short flanking-homology regions (56-80 bp) with selection markers for PCR-based gene targeting.
• Performing sequential transformations to delete LEU2 and LYS2 genes, creating auxotrophic strains (leu2/leu2, lys2/lys2).
• Complementation of the leu2/leu2 auxotrophic strain to validate the gene targeting approach.

Main Results:

• Successful development of PCR-based gene targeting in H. uvarum.
• Creation of two novel selection markers (pFA-hygXL, pFA-clnXL) for H. uvarum.
• Generation of auxotrophic H. uvarum strains (leu2/leu2 and lys2/lys2) through targeted gene deletion.
• Demonstration of successful targeted complementation of the leu2/leu2 strain.
• Gene targeting efficiency in H. uvarum was found to be lower compared to Saccharomyces cerevisiae.

Conclusions:

• A rapid PCR-based gene targeting method has been established for Hanseniaspora uvarum.
• The developed synthetic marker genes provide essential tools for directed gene manipulation.
• This approach significantly advances the study of gene function in H. uvarum, despite lower efficiency than in S. cerevisiae.