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Yeasts belonging to the genus of Hanseniaspora are widespread in nature and are generally part of the microbiome of ripe fruits, such as apples, pears and grapes. Hanseniaspora spp. form 50-75% of the microorganisms found at the onset of grape must fermentations, H. uvarum often being the most abundant. H. uvarum is also a common isolate from other fruits, such as oranges, pineapples, mangoes, apples and pears. Since H. uvarum, known for producing high amounts of acetic acid and ethyl acetate, dominates the onset of spontaneous grape must fermentations, it greatly influences the aroma profile of wines. Besides its important role in fermentations, more biotechnological research has been reported in recent years, highlighting the universal value of H. uvarum beyond the beverage industry.
In this thesis, I characterized the aroma-production profile of seven different Hanseniaspora spp. in simultaneous co-fermentations with Saccharomyces cerevisiae. Significant differences could be detected between fermentations using Hanseniaspora spp. belonging to the fast-evolving lineage (FEL) and the slow-evolving lineage (SEL). Fermentations with yeasts of FEL showed increased amounts of ethyl acetate, whereas yeasts belonging to SEL revealed no meaningful difference compared to the pure S. cerevisiae fermentation. Furthermore, co-fermentations resulted in lactic acid depletion (H. osmophila), extremely high isoamyl acetate levels (H. guilliermondii), reduction in glycerol (H. occidentalis), increased terpenols (H. opuntiae) as well as esterified terpenes (H. guilliermondii and H. uvarum). This is the first study to investigate the use of H. nectarophila and H. meyerii in relation to wine, of which fermentations using H. nectarophila revealed high levels of acetate esters.
Nitrogen sources in form of amino acids have a major impact on the resulting aroma-production profile of yeasts as they serve as backbones for several volatile organic compounds. I explored amino acid uptake preferences of Hanseniaspora spp. of both lineages (H. uvarum = FEL; H. osmophila = SEL), S. cerevisiae (all glucophilic), as well as the fructophilic yeasts Starmerella bacillaris and Zygosaccharomyces rouxii in synthetic must, spiked with single amino acids. Among yeasts investigated, H. uvarum showed the most rapid amino acid uptake. Additional research on yeast fermentations with synthetic must supplemented with an amino acid mix resulted in amino acid-dependent synthesis of specific aroma compounds by the respective yeast. This study reveals similarities in both preferences in amino acid uptake and nitrogen source depending aroma compound synthesis of H. osmophila and S. cerevisiae.
I investigated the transcriptomic profile of H. uvarum and S. cerevisiae to gain insight into the different regulation mechanisms involved in the synthesis of aroma compounds. Furthermore, I explored the impact of different levels of nitrogen (nitrogen deficiency/ diammonium hydrogen phosphate (DAHP) supplemented must) within the grape must on the gene expression profile and therefore on the resulting aroma profile. Even though the different nitrogen regimes did not significantly affect transcriptional levels, a major impact on both fermentation kinetics and the aroma profile could be monitored. H. uvarum unravelled possessing additional genes including alcohol acetyltransferase activity leading to a rapid esterification of higher alcohols to acetate esters. In addition, the lack of genes involved in the synthesis of higher alcohols and ethyl esters, as well as an up-regulation of genes responsible for the synthesis of acetyl-coA and acetate esters favour the high acetate ester production.
In this study, I developed the first genetic modification tool for H. uvarum by constructing a synthetic marker gene encoding the hygromycin marker under the control of a H. uvarum promoter, flanked by ~1 000 bp homology region to the target locus, enabling the knock-out of one copy of the alcohol acetyltransferase 1 (ATF1) gene. By increasing the concentration of the antibiotic during selection, I could identify a knock-out of both ATF1 alleles in the diploid yeast, generating a homozygotic atf1 mutant. Fermentations resulted in a significant reduction of the acetate ester synthesis, proving the function of ATF1 as an important gene involved in the ester synthesis in H. uvarum.
Genetic modification of H. uvarum was optimized by establishing a rapid PCR-based gene targeting approach for one-step gene replacement. Generation of two novel synthetic antibiotic resistance genes (pFA-hygXL and pFA-clnXL) provided resistance against hygromycin and nourseothricin, respectively. Short flanking-homology regions of 56-80 bp added to these selection markers via PCR enabled gene targeting and the deletion of both the H. uvarum LEU2 and LYS2 genes via two rounds of consecutive transformations, generating a leucine auxotrophic leu2/leu2 mutant and a lysine auxotrophic lys2/lys2 mutant, respectively. The genetic modification approach of H. uvarum using PCR-based gene targeting was further validated by targeted complementation of the auxotrophic leu2/leu2 strain.