Abstract
Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.
Highlights
The term apiculate yeast signifies a distinctive lemon-shaped cell structure of yeast species belonging to the genus of Hanseniaspora and closely-related genera, such as Saccharomycodes and Nadsonia [1]
It was initially thought that the gradual increase in ethanol concentration was primarily responsible for the decline in the Hanseniaspora population, but there is growing scientific evidence suggesting that a variety of metabolites secreted by S. cerevisiae significantly impacts the viability of Hanseniaspora within a grape must fermentation [12]
Hanseniaspora populations have been regarded to be spoilage yeasts when a significant proportion of these apiculate yeasts persists within a fermentation, as some strains are capable of producing significant amounts of acetaldehyde, acetic acid, and ethyl acetate
Summary
The term apiculate yeast signifies a distinctive lemon-shaped cell structure of yeast species belonging to the genus of Hanseniaspora and closely-related genera, such as Saccharomycodes and Nadsonia [1]. Hanseniaspora populations have been regarded to be spoilage yeasts when a significant proportion of these apiculate yeasts persists within a fermentation, as some strains are capable of producing significant amounts of acetaldehyde, acetic acid, and ethyl acetate Winemaking practices, such as the addition of SO2 or even the utilisation of yeasts, which produce killer toxins that have antimicrobial activity against apiculate yeasts [15], are used to limit the proliferation of Hanseniaspora yeasts. This hypothesis of Hanseniaspora as purely a spoilage yeast has been challenged and re-evaluated in recent years as wine researchers are reporting many beneficial oenological contributions that Hanseniaspora strains can provide when co-cultured with S. cerevisiae in a mixed-culture type of must fermentation. We targeted the H. uvarum ATF1 homolog encoding an alcohol acetyltransferase, successfully deleted both alleles in this diploid yeast, and compared the fermentation performance and flavour production of the mutant with the wild type
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