Hanseniaspora uvarum (anamorph Kloeckera apiculata) is a predominant yeast on wine grapes and other fruits and has a strong influence on wine quality, even when Saccharomyces cerevisiae starter cultures are employed. In this work, we sequenced and annotated approximately 93% of the H. uvarum genome. Southern and synteny analyses were employed to construct a map of the seven chromosomes present in a type strain. Comparative determinations of specific enzyme activities within the fermentative pathway in H. uvarum and S. cerevisiae indicated that the reduced capacity of the former yeast for ethanol production is caused primarily by an ~10-fold-lower activity of the key glycolytic enzyme pyruvate kinase. The heterologous expression of the encoding gene, H. uvarumPYK1 (HuPYK1), and two genes encoding the phosphofructokinase subunits, HuPFK1 and HuPFK2, in the respective deletion mutants of S. cerevisiae confirmed their functional homology.IMPORTANCEHanseniaspora uvarum is a predominant yeast species on grapes and other fruits. It contributes significantly to the production of desired as well as unfavorable aroma compounds and thus determines the quality of the final product, especially wine. Despite this obvious importance, knowledge on its genetics is scarce. As a basis for targeted metabolic modifications, here we provide the results of a genomic sequencing approach, including the annotation of 3,010 protein-encoding genes, e.g., those encoding the entire sugar fermentation pathway, key components of stress response signaling pathways, and enzymes catalyzing the production of aroma compounds. Comparative analyses suggest that the low fermentative capacity of H. uvarum compared to that of Saccharomyces cerevisiae can be attributed to low pyruvate kinase activity. The data reported here are expected to aid in establishing H. uvarum as a non-Saccharomyces yeast in starter cultures for wine and cider fermentations. Copyright © 2017 American Society for Microbiology.