PCR-based gene targeting in Hanseniaspora uvarum

This article describes a PCR-based gene targeting approach with two marker genes as tools for directed gene manipulations in the apiculate yeast Hanseniaspora uvarum. Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

Automated summary

This article presents a PCR-based gene targeting approach using two marker genes for directed gene manipulations in Hanseniaspora uvarum. The lack of gene-function analysis tools limits the study of this yeast, which is abundant in grapes and must. The authors developed synthetic antibiotic resistance genes as markers and successfully achieved gene replacement in H. uvarum. Although the efficiency was lower compared to Saccharomyces cerevisiae, this approach provides important tools for directed gene manipulations in H. uvarum.