*3.2. Sugar Transport*

The transport of sugars into the cytosol of cells is a key step of the glycolytic pathway. *S. cerevisiae* is able to detect extracellular nutrients and make metabolic adjustments that rapidly facilitate the use of extracellular compounds [39].

Of the multiple sensors described in *S. cerevisiae*, *H. vineae* possessed the following genes Hv*SNF3*, Hv*GPA2*, Hv*GPR1,* and Hv*ASC1*, which were determined to be associated with the hexose sensing capacity of both T02/19AF and T02/05AF strains (Table 2). Sc*SNF3* encodes a low glucose sensor present in the plasma membrane that is involved in the regulation of glucose transport and also has the capacity to sense fructose and mannose in *S. cerevisiae*. Expression of the gene in *H. vineae* increases throughout fermentation (Figure 1). Sc*GPA2*, Sc*GPR1,* and Sc*ASC1* are hexose sensors that have been reported to be necessary for fermentation and are part of the "fermentome" in *S. cerevisiae*. Deletion of the genes was previously reported to induce protracted fermentation [28]. Hv*GPA2* and Hv*GPR1* have similar expression patterns throughout the fermentation process. The genes are maximally expressed on day 4 and their expression levels decrease at day 10. On the other hand, Hv*ASC1* is most highly expressed on the first day of fermentation and levels were drastically reduced both on day 4 and 10 relative to day 1 (Figure 2).

*S. cerevisiae* possesses 20 sequences putatively associated with the hexose transport [40]. *H. guilliermondii* UTAD222 possess 22 sugar transporters, and based on their DNA sequences, ten were predicted to be associated with the hexose transport, all of them were most similar to *HXT2* [27]. A comparison of sugar transporters of both sequenced strains of *H. vineae* with *S. cerevisiae* revealed that T02/05AF had two copies of the Hv*HXT6* gene and one copy of Hv*HXT1*. Sequences homologous to Sc*HXT2* were not found in the species. However, strain T02/19AF was determined to have a single copy of Hv*HXT6*. Expression levels of the gene increased after day 10 of fermentation. No sequences homologous to *HXT1* were identified. Sc*HXT1* is a low affinity hexose and pentose transmembrane transporter and is paralogous to Sc*HXT6* [41]. The Sc*HXT6* gene encodes a high affinity hexose transmembrane transporter that transports glucose, fructose, and mannose [42]. Tondoni et al. [43] revealed that in *S. cerevisiae* and *Torulaspora delbrueckii*, *HXT6* is most highly expressed throughout late stages of fermentation (Figure 2). In addition, both T02/05AF and T02/19AF strains have one sequence that is homologous to the *S. cerevisiae* Sc*HXT7* gene. Sc*HXT7* is a high-affinity glucose transporter that is very similar to Sc*HXT6* [42,44]. This gene is maximally expressed at day 4 at the end of the exponential phase of fermentation in *H. vineae*. Both *H. vineae* strains sequenced lacked polyol transporters (such as Sc*HXT13*, Sc*HXT17,* or Sc*HXT16* of *S. cerevisiae*) needed for the uptake of sorbitol and mannitol.

**Figure 2.** Heatmap depicting the expression levels of genes putatively involved in sugar detection and transport in *H. vineae* after 1, 4, and 10 d of fermentation. The green colour indicates elevated expression levels and the red colour indicates reduced expression levels. Data are shown in triplicate.

Other homologs of sugar transporters have been found in *H. vineae*. Three tandem copies of Hv*STL1* were identified in both T02/19AF and T02/05AF strains that shared homology with a glycerol proton symporter of the plasma membrane, which has been shown to be inactivated in response to glucose in *S. cerevisiae* [45]. Other *Hanseniaspora* species sequenced, such as *H. osmophila*, *H. opuntiae*, *H. guilliermondii*, *H. uvarum,* and *H. valbyensis* also possessed between two and four copies of the gene. According to the transcriptomic analyses, just one of the copies identified was differentially expressed throughout fermentation in *H. vineae* (Figure 2).

Hv*FPS1*, a putative plasma membrane channel involved in glycerol and xylitol movement, is present in the genome of both T02/19AF and T02/05AF. Expression of the gene is elevated near the beginning of fermentation reactions (days 1 and 4) and decreased at day 10 (Figure 1). One copy of Hv*GUP1* was present in each strain analyzed as well as Hv*JEN1*. Moreover, it was suggested that Sc*GUP1* participates in glycerol transport and Sc*JEN1* mediates the high-affinity uptake of lactate, pyruvate, and acetate so that they can be used as carbon sources in *S. cerevisiae* [46,47].

#### *3.3. Glycolytic Pathway in H. vineae Strains*

The first enzyme of the glycolysis pathway is a hexokinase (Figure 3). Sc*HXK2* phosphorylates glucose in the cytosol. In *S. cerevisiae,* this isoform is principally responsible for glucose activation, which is needed to initiate glycolysis when glucose is provided as a carbon source and inhibits Sc*HXK1* [41]. However, in *H. vineae,* Hv*HXK2* was the only enzyme identified with putative hexokinase activity, amino acid homology was higher compared to other species of this genus.

**Figure 3.** Glycolysis and alcoholic fermentation pathways in yeast. Genes putatively predicted to be involved in the catabolic pathway based on sequence data from genomic analyses of *Hanseniaspora vineae* strains are presented.

Phosphofructokinase activity was determined to be the second-most important glycolytic enzyme. The enzyme determines fermentation capacity and is indispensable for anaerobic growth. In *S. cerevisiae*, the enzyme is composed of two alpha and beta subunits that are encoded by Sc*PFK1* and Sc*PFK2*, respectively. *Hanseniaspora* strains possess sequences homologous to both Sc*PFK1* and Sc*PFK2* subunits and similar to *S. cerevisiae*, the subunits form a hetero-octameric complex [29]. Protein

sequences of both Hv*PFK1* and Hv*PFK2* were most similar to *S. cerevisiae* (76.78% and 79.24%) and *H. osmophila* (76.46% and 77.50%) relative to the other *Hanseniaspora* species assessed (Figure 4A). Phosphofructokinase only works in the forward direction and is not involved in gluconeogenesis. In fact, three activities are required for gluconeogenesis: Pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase. No genes encoding the key gluconeogenic enzymes have been identified in *H. vineae*, *H. guilliermondii*, *H. uvarum*, *H. osmophila,* or *H. valbyensis* [27]. This explains why *Hanseniaspora* species are not able to grow when non-carbohydrate precursors such as pyruvate, amino acids, or glycerol are provided as energy sources. This is different than *S. cerevisiae*, which is able to grow on a variety of carbon sources including ethanol and lactate [48].

**Figure 4.** Genes involved in glycolysis. (**A**) Dendrograms showing the genetic distances between predicted amino acid sequences of enzymes involved in glycolysis from seven *Hanseniaspora* species and the *Saccharomyces cerevisiae* S288c strain. Amino acid homology was calculated for each *Hanseniaspora* strain against *S. cerevisiae*. (**B**) Amino acid sequences that correspond to the binding domains of fructose

1,6-bisphosphate inducer of pyruvate kinase in *S. cerevisiae.* Sc*CDC19* and Sc*PYK2* genes were compared with predicted sequences of *CDC19* from *H. uvarum* and *H. vineae*. Amino acids corresponding to the region that differ from *CDC19* and *PYK2* are highlighted in green and the position of residues are marked with an asterisk (\*). ( **C**) A heatmap describing the expression levels of genes putatively determined to be involved in glycolytic pathways of *H. vineae* 1, 4, and 10 days after the initiation of fermentation. Green and red colours indicate high and low levels of expression, respectively. Data are shown in triplicate.

Predicted amino acid sequences of phosphoglucose isomerase (*PGI1*) from *H. vineae* and *H. osmophila* were 86% similar to that of *S. cerevisiae*. Predicted *PGI1* amino acid sequences from *H. uvarum*, *H. valbyensis*, *H. guilliermondii,* and *H. opuntiae* were approximately 71% similar to *S. cerevisiae*. This tetrameric enzyme is involved in the interconversion of glucose-6-phosphate and fructose-6-phosphate. Phosphoglucose isomerase activity has also been associated with the regulation of the cell cycle and gluconeogenic events of sporulation in *S. cerevisiae* [49,50].

Two copies of the *S. cerevisiae* Sc*ENO1* gene that encodes an enolase were identified in *H. osmophila*, while only one copy was identified in other sequenced *Hanseniaspora* species. This enzyme catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a tetramer that catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate. Three unlinked genes, Sc*TDH1*, Sc*TDH2*, and Sc*TDH3*, encode related, but not identical, polypeptides that form catalytically active homotetramers with di fferent specific glyceraldehyde 3-phosphate dehydrogenase activities in *S. cerevisiae* [51,52]. In *H. vineae*, only *TDH2* and *TDH3* homologues have been identified, and both were di fferentially expressed throughout fermentation (Figure 4C).

*H. vineae* strains T02/19AF and T02/05AF possess Hv*GUT1* and Hv*GUT2* genes. Both Sc*GUT1* and Sc*GUT2* are associated with glycerol kinase activities in the cytoplasm and mitochondria, respectively. Glycerol degradation is a two-step process that is mediated by *GUT1* and/or *GUT2*. Under aerobic conditions, *S. cerevisiae* is able to utilize glycerol as a sole carbon and energy source [53]. Both of the enzymes have homologs that have been identified in *H. vineae* and *H. osmophila*, other *Hanseniaspora* species such as *H. uvarum*, *H. guilliermondii,* and *H. opuntiae* lack homologous of these genes [27].

Several specific activities associated with glycolytic enzymes of *S. cerevisiae* and *H. uvarum* have high degrees of similarity, which highlights the general conservation of glycolytic pathways and the downstream reactions involved in ethanol production [29]. Pyruvate kinase is a key enzyme that catalyses an irreversible step of the glycolytic pathway. The position of the enzyme at the branchpoint between fermentation and respiration makes it a key determinant energy metabolism [54]. Recent work revealed that the pyruvate kinase activity enhanced the capacity of *S. cerevisiae* to ferment sugars versus *H. uvarum* [29]. The predicted proteins, Cdc19p, of *H. vineae* and *H. osmophila* are more homolog to the corresponding Cdc19p of *S. cerevisiae* than those of *H. uvarum* and other *Hanseniaspora* species (Figure 4A). When residues of the catalytic domain of ScCdc19p [55] are compared with those of *H. uvarum* and *H. vineae,* only one amino acid di fference was identified; Asp<sup>265</sup> was substituted with Gly<sup>269</sup> in *H. uvarum* and *H. vineae* (Figure 4B). However, in the binding site of the allosteric activator, fructose 1,6-bisphosphate, two amino acid di fferences between *H. uvarum* and *S. cerevisiae* and one between *H. vineae* and *S. cerevisiae* were identified. The two di fferences identified between *H. uvarum* and *S. cerevisiae* are at the same positions (Figure 4B) as those identified in the *PYK*2 gene of *S. cerevisiae,* a paralog of *CDC*19 that is characterized by its low pyruvate kinase activity compared with the pyruvate kinase protein encoded by *CDC19* (formerly *PYK1*) [54].

Expression levels of 13 *H. vineae* genes involved in the glycolytic pathway mainly decreased from day 1 to day 4 of fermentation and were maintained throughout the stationary phase (Figure 4C). This finding is in agreemen<sup>t</sup> to previous observations in *S. cerevisiae* [43]. However, levels of Hv*TDH2* expression remained high at both days 1 and 4 and decreased expression levels were observed at day 10. Additionally, expression of Hv*GUT2* peaked at days 4 and 10, and increased expression levels of the gene were not detected at day 1. Finally, Hv*GPM2* was not expressed under the conditions assessed.

#### *3.4. Alcoholic Fermentation in H. vineae Strains*

The pyruvate decarboxylase activity plays a key role in the alcoholic fermentation pathway. Three di fferent pyruvate decarboxylase isozymes have been identified in the genome of *S. cerevisiae*: Sc*PDC1*, Sc*PDC5,* and Sc*PDC6*. The function of pyruvate decarboxylase is the degradation of pyruvate into acetaldehyde and carbon dioxide. The enzyme is responsible for transferring the final product of glycolysis (pyruvate) to ethanol production [56]. In *H. vineae*, no sequences homologous to Sc*PDC5* and Sc*PDC6* were found and Hv*PDC1* was the only pyruvate decarboxylase isozyme identified in the species. In *S. cerevisiae*, Sc*PDC1* was strongly expressed in fermenting cells. The enzyme is conserved among yeast, bacteria, and plants. It is regulated by glucose and ethanol concentrations and also by itself [57]. The active enzyme has a homotetrameric structure and the enzyme has two known cofactors: Thiamin diphosphate (ThDP) and Mg<sup>2</sup>+ [58–60]. In *H. vineae*, genes involved in thiamine biosynthesis have not been identified and a similar finding was also reported in *H. guilliermondii* [27] and most other *Hanseniaspora* species [36]. It has been suggested that this may contribute to the low alcoholic fermentative capacity of *Hanseniaspora* species, the phenotype has been shown to be related to the weak pyruvate kinase activity of *H. uvarum* [29]. *S. cerevisiae* genes associated with thiamine production are upregulated in the stationary phase of growth. Oenological strains with improved expression levels of the genes have corresponding elevated rates of fermentation [61]. This phenomenon may result from vitamin depletion that occurs after the exponential phase.

Alcohol dehydrogenases, which catalyse the conversion of acetaldehyde to ethanol are key fermentative enzymes. Many alcohol dehydrogenases have been identified in *S. cerevisiae* including Sc*ADH1*, Sc*ADH2*, Sc*ADH3*, Sc*ADH4*, Sc*ADH6,* and Sc*ADH7*. Many homologues of *S. cerevisiae* alcohol dehydrogenases have been found in the *H. vineae* genome. *H. vineae* has the same number of copies of the genes as *S. cerevisiae*. Eight alcohol dehydrogenase genes are present in *H. vineae* species, compared to six in *H. osmophila,* and four in other sequenced species of *Hanseniaspora* such us *H. uvarum*, *H. guilliermondii*, *H. valbyensis,* and *H. opunt*iae. This may explain the improved adaptation of *H. vineae* to alcohol fermentation relative to other *Hanseniaspora*. It is noteworthy that of the eight Hv*ADH* sequences found in the genome of *H. vineae*, at least three Hv*ADH6* genes are encoded in tandem. Increased copies of the gene may be associated with increased fermentation capacity, indicating that the alcohol dehydrogenase activity might be a key feature of alcoholic fermentation adaptations [62]. *H. vineae* has an enhanced tolerance to ethanol (Figure 5B) versus *H. uvarum* and *H. osmophila*, which are unable to grow in media containing 10% ethanol.

*H. vineae* and *H. osmophila* genes encoding putative alcohol dehydrogenases were grouped in two main clusters that contained either *ADH1*, *ADH2* and *ADH3* or *ADH6* and *ADH7* (Figure 5A), this is in agreemen<sup>t</sup> with the two multigenic families reported by Giorello et al. [22]. The clusters were formed according to the clustal alignment of predicted protein sequences, however, regarding adscription by a single homology with *S. cerevisiae ADH*s in the databases [22] produced some discrepancies. Therefore, Hv*ADH6* homologs from *H. vineae* and *H. osmophila* were removed from the Hv*ADH6* and Hv*ADH7* cluster. Moreover, the Hv*ADH1* homologous sequence of *H. vineae* is grouped in the cluster of Sc*ADH6* and Sc*ADH7*.

Hv*ADH* genes display di fferent expression patterns (Figure 5C). Two of four paralogous copies of Hv*ADH6* were not di fferentially expressed at the time points analysed. Expression of one copy of *ADH6* significantly declined between days 1 and 4 of fermentation. In addition, the expression of one copy of *ADH3* was elevated on day 4 relative to day 1 (Figure 5C). These behaviours are similar to those of aryl alcohol dehydrogenases that facilitate the production of increased levels of alcohol by *S. cerevisiae* [63]. Therefore, Hv*ADH*s may be important for reducing levels of fusel aldehydes by producing increased levels of alcohol in *H. vineae* [22].

**Figure 5.** Characteristics that facilitate fermentation. (**A**) Dendrogram depicting relationships between the predicted amino acid sequences of several putative *ADH* genes. *Hanseniaspora vineae* sequences are indicated in red. (**B**) Growth of *Hanseniaspora* species and *Saccharomyces cerevisiae* in synthetic wine containing 5% of ethanol (solid line) and 10% ethanol (dotted line) for 48 h. Error bars are not shown to enhance clarity. SD < 0.05 for all samples. (**C**) A heatmap depicting expression levels of genes putatively involved in glycolytic pathways in *H. vineae* after 1, 4, and 10 days of fermentation. Green and red colours indicate high and low levels of expression, respectively. Data are shown in triplicate.

#### *3.5. Hanseniaspora Genus as an Evolution Model for Alcoholic Fermentation Adaptations*

The glycolytic potential of two strains of *H. vineae* were analysed using genetic, transcriptomic, and phenotypic data. Results explained the good performance of the species with respect to fermenting wine [7,21]. Findings also showed that the *H. vineae* behaviour was similar to traditional *S. cerevisiae* strains used in winemaking. Due to the outstanding capacity of *H. vineae* to produce aromatic metabolites, it was necessary to compare the capacities of the *H. vineae* strains to produce ethanol with *S. cerevisiae*. The high degree of similarity between glycolytic and alcoholic fermentation enzymes of *H. vineae* and *H. osmophila* with *S. cerevisiae* showed that the two species should be classified as fermenters, while the remaining *Hanseniaspora* species assessed were adapted to the fruit niche and were correspondingly included in the fruit group. In our experience, *H. vineae* strains cannot be isolated from the fresh grape fruits [19]. A dendrogram of concatenated DNA sequences from seven glycolytic and fermentation genes (Figure 6) indicated the presence of two clades of *Hanseniaspora* species, similar to findings of Steenwyk et al. [36] determined using genes from the DNA repair processes present within the genus. Interestingly, the fruit and fermentation clades shown in Figure 6 were correlated with the slow and fast evolution lineages defined by these authors. Branches were in agreemen<sup>t</sup> with phylogenetic classifications that were based on ribosomal genes [19]. It might be interesting to use the group as an evolution model to determine the mechanism by which the fermentation group diverged separately from the fruit group [36], giving less species diversity probably due to slow evolution mechanisms. Further work will be needed to understand whether the process might be an example of domestication, as has been proposed for *S. cerevisiae* wine and beer strains [64].

Previous studies have compared the fermentation capacity of two species belonging to the fruit group: *H. guillermondi* and *H.uvarum* [27,29], and the work presented here is the first assessment of a member of the fermentation group of *Hanseniaspora*.

**Figure 6.** Dendrogram of seven concatenated DNA sequences from *Hanseniaspora* species constructed using the neighbour-joining method. The robustness of branching is indicated by bootstrap values (%) calculated for 1000 subsets. The entries in brackets correspond to NCBI BioSample identifiers.
