Genomic and Transcriptomic Basis of Hanseniaspora vineae's Impact on Flavor Diversity and Wine Quality

Abstract

Hanseniaspora is the main genus of the apiculate yeast group that represents approximately 70% of the grape-associated microflora. Hanseniaspora vineae is emerging as a promising species for quality wine production compared to other non-Saccharomyces species. Wines produced by H. vineae with Saccharomyces cerevisiae consistently exhibit more intense fruity flavors and complexity than wines produced by S. cerevisiae alone. In this work, genome sequencing, assembling, and phylogenetic analysis of two strains of H. vineae showed that it is a member of the Saccharomyces complex and it diverged before the whole-genome duplication (WGD) event from this clade. Specific flavor gene duplications and absences were identified in the H. vineae genome compared to 14 fully sequenced industrial S. cerevisiae genomes. The increased formation of 2-phenylethyl acetate and phenylpropanoids such as 2-phenylethyl and benzyl alcohols might be explained by gene duplications of H. vineae aromatic amino acid aminotransferases (ARO8 and ARO9) and phenylpyruvate decarboxylases (ARO10). Transcriptome and aroma profiles under fermentation conditions confirmed these genes were highly expressed at the beginning of stationary phase coupled to the production of their related compounds. The extremely high level of acetate esters produced by H. vineae compared to that by S. cerevisiae is consistent with the identification of six novel proteins with alcohol acetyltransferase (AATase) domains. The absence of the branched-chain amino acid transaminases (BAT2) and acyl coenzyme A (acyl-CoA)/ethanol O-acyltransferases (EEB1) genes correlates with H. vineae's reduced production of branched-chain higher alcohols, fatty acids, and ethyl esters, respectively. Our study provides sustenance for understanding and potentially utilizing genes that determine fermentation aromas.IMPORTANCE The huge diversity of non-Saccharomyces yeasts in grapes is dominated by the apiculate genus Hanseniaspora Two native strains of Hanseniaspora vineae applied to winemaking because of their high oenological potential in aroma and fermentation performance were selected to obtain high-quality genomes. Here, we present a phylogenetic analysis and the complete transcriptome and aroma metabolome of H. vineae during three fermentation steps. This species produced significantly richer flavor compound diversity than Saccharomyces, including benzenoids, phenylpropanoids, and acetate-derived compounds. The identification of six proteins, different from S. cerevisiae ATF, with diverse acetyltransferase domains in H. vineae offers a relevant source of native genetic variants for this enzymatic activity. The discovery of benzenoid synthesis capacity in H. vineae provides a new eukaryotic model to dilucidate an alternative pathway to that catalyzed by plants' phenylalanine lyases.

Keywords: Illumina; flavor compounds; genome; metabolome; transcriptome; wine aroma.

FIG 1

Metabolic pathways studied in this work involved in wine aroma formation. Ehrlich pathway for higher alcohol production, acetate ester biosynthesis, and ethyl ester biosynthesis from amino acids (AA) and sugars.

FIG 2

Maximum likelihood phylogeny of Saccharomyces complex species from concatenation of 227 genes. H. vineae is framed in red inside the Saccharomyces complex and outside the whole-genome duplication (WGD) clade. The clade CTG groups yeasts with alternative genetic codes. Numbers close to the node match bootstrap support (BS) for those values above 70 and internode certainty (IC). The scale bar represents units of amino acid substitutions per site. The tree has a midpoint root for easier visualization.

FIG 3

Overview of transcriptomic analysis. (a) Venn diagram showing the differentially expressed genes shared between each fermentation point. (b) Venn diagram showing the genes shared between each fermentation point for the top 100 most highly expressed genes. (c) Main biochemical cascades of the most expressed genes at each sampled day of fermentation. GPI, glycosylphosphatidylinositol.

FIG 4

Higher alcohols and 2-phenylethanol production and putatively related genes. (a) Comparison of total higher alcohols, 2-phenyelthanol, and 2-phenylethyl acetate produced in H. vineae and S. cerevisiae at day 10 of fermentation. (b) Three steps of metabolic pathway of higher alcohols biosynthesis with putative enzymes involved in H. vineae. (c) Production of total higher alcohols and 2-phenylacetate by H. vineae at 1, 4, and 10 days of fermentation. (d) Expression heatmap of genes putatively involved in higher alcohols and 2-phenylethanol production from H. vineae at 1, 4, and 10 days of fermentation. Lighter colors indicate higher expression values, and data are shown for triplicates. Significant changes in expression of each gene are indicated with arrows to the right of the heatmap as analyzed using the package edgeR (FDR < 0.05). 1–4, differential expression between days 1 and 4; 1–10, differential expression between days 1 and 10; 4–10, differential expression between days 4 and 10.

FIG 5

Acetate ester production and putatively related genes. (a) Comparison of total acetate esters and 2-phenylethyl acetate produced in H. vineae and S. cerevisiae at day 10 of fermentation. (b) Metabolic pathway of acetate esters biosynthesis with putative enzymes involved in H. vineae. (c) Production of total acetate esters and 2-phenylethyl acetate by H. vineae at 1, 4, and 10 days of fermentation. (d) Expression heatmap of genes putatively involved in total acetate esters and 2-phenylethyl acetate production from H. vineae at 1, 4, and 10 days of fermentation. Lighter colors indicate higher expression values, and data are shown for triplicates. Significant changes in expression of each gene are indicated with arrows to the right of the heatmap as analyzed using the package edgeR (FDR < 0.05). 1–4, differential expression between days 1 and 4; 1–10, differential expression between days 1 and 10; 4–10, differential expression between days 4 and 10.

FIG 6

Ethyl esters production and putatively related genes. (a) Comparison of ethyl esters produced in H. vineae and S. cerevisiae at day 10 of fermentation. (b) Metabolic pathway of acetate ester biosynthesis with putative enzymes involved in H. vineae. (c) Production of ethyl esters by H. vineae at 1, 4, and 10 days of fermentation. (d) Expression heatmap of genes putatively involved in ethyl ester production from H. vineae at 1, 4, and 10 days of fermentation. Lighter colors indicate higher expression values, and data shown are of triplicates. Significant changes in expression of each gene are indicated with arrows to the right of the heatmap as analyzed using the package edgeR (FDR < 0.05). 1–4, differential expression between days 1 and 4; 1–10, differential expression between days 1 and 10; 4–10, differential expression between days 4 and 10.