Exploring future applications of the apiculate yeast Hanseniaspora

Abstract

As a metaphor, lemons get a bad rap; however the proverb ‘if life gives you lemons, make lemonade’ is often used in a motivational context. The same could be said of Hanseniaspora in winemaking. Despite its predominance in vineyards and grape must, this lemon-shaped yeast is underappreciated in terms of its contribution to the overall sensory profile of fine wine. Species belonging to this apiculate yeast are known for being common isolates not just on grape berries, but on many other fruits. They play a critical role in the early stages of a fermentation and can influence the quality of the final product. Their deliberate addition within mixed-culture fermentations shows promise in adding to the complexity of a wine and thus provide sensorial benefits. Hanseniaspora species are also key participants in the fermentations of a variety of other foodstuffs ranging from chocolate to apple cider. Outside of their role in fermentation, Hanseniaspora species have attractive biotechnological possibilities as revealed through studies on biocontrol potential, use as a whole-cell biocatalyst and important interactions with Drosophila flies. The growing amount of ‘omics data on Hanseniaspora is revealing interesting features of the genus that sets it apart from the other Ascomycetes. This review collates the fields of research conducted on this apiculate yeast genus.

Keywords:

• Hanseniaspora
• fermentation
• wine
• biocontrol
• whole-cell biocatalyst
• aroma
• Drosophila

Introduction

In Pasteur’s sketches and notes on the early stages of a grape must fermentation he observed small, lemon-shaped yeast cells that he named Saccharomyces apiculatus, which disappeared as fermentation progressed and were replaced with bigger, round-shaped yeast cells [1]. The term ‘lemon-shaped’ is not often used to describe ‘wine yeast’ (i.e., members of the Saccharomyces genus) and it is evident that Pasteur observed what we now call Hanseniaspora (Figure 1). The lemon-shape denotes the bipolar budding that is a distinctive feature of the apiculate yeast which, besides Hanseniaspora, also include the related genera of Nadsonia, Saccharomycodes and Wickerhamia. Hanseniaspora yeasts are widely known for their prevalence on ripe fruits, following a comprehensive survey cataloguing yeast isolation campaigns from several continents and viniviticultural settings and spanning decades [2]. Although the majority of isolation studies were conducted in wine milieu, Hanseniaspora spp. are also commonly isolated from apple and pear cider [3–6], as well as from a variety of other fruits [7–10]. Not just their prevalence, but their general abundance is quite noteworthy, as at least 50-75% of the general yeast population at the onset of a grape must fermentation can be comprised of members of the Hanseniaspora genus [11,12]. One species in particular is that of H. uvarum, which is generally considered to be the most commonly-isolated yeast from grapes [2]. In general, Hanseniaspora spp. are not good fermenters and are often dominated by fermenting yeast belonging to the Saccharomyces genus. Assessing the influence that Hanseniaspora can have on a fermentation is complex and dependent on many variables.

Figure 1. Species belonging to the genus Hanseniaspora. These lemon-shaped yeasts are prevalent on ripe fruits and have been isolated from a diverse range of environments. Some of these apiculate species are associated with grapes and wine; grains and beer; agave plants and tequila; and cacao and coffee beans; as well as apple and pear cider.

There is a stark contrast in the trajectories of scientific interest in Hanseniaspora and Saccharomyces, the most common and the most important yeast genera found in must, respectively. Saccharomyces is by far the best-studied yeast. Not only because it is the main player in beer, wine and bread production, along with myriad other biotechnological applications, but these studies laid the groundwork for our fundamental understanding of how a eukaryotic cell operates [13]. With Hanseniaspora, research has been focused on its role in fermentation beverages and as a possible copartner with Saccharomyces cerevisiae [14], along with possible application in biocontrol trials as well as other biotechnological ventures. Despite a blooming interest in applications for Hanseniaspora there is still a big gap in understanding its biology. Recent genomic analyses have unveiled interesting features of this genus that will provide insights into its lifestyle, which we will discuss.

Members of the genus

The name Hanseniaspora was first coined in 1912 to describe the apiculate yeasts commonly found in fermenting fruit juices [15]. Cells tend not to be uniform but often display a distinct lemon-shape morphology in routine culturing media. Cells can also be ovoid, long-ovoid or elongate and sometimes form pseudohyphae. The use of standard molecular techniques for the classification of yeast species resolved the issue of its inconsistent ability to form ascospores and allowed for the merging with the anamorphic genus Kloeckera. At the time of writing (2022), 22 species have been recognized including four new species described during the past four years [16–19]. Phylogenetic analyses further divide the genus into two major clades: the ‘fermentation clade’ and the ‘fruit clade’ (Figure 2). The ‘fermentation clade’, which has fewer members, includes H. vineae and H. osmophila for their stronger fermentative capabilities, whereas the ‘fruit clade’ contains members mostly adapted to fruit niches [20].

Figure 2. A phylogenetic tree of members of the genus Hanseniaspora. Using maximum-likelihood analysis of the internal transcribed sequences of one representative of each Hanseniaspora species divides the genus into two large clades, a fast-evolving fruit clade and a slow-evolving fermentation clade. MEGA 11 software was used to draw the tree [218].

Role in fermentations

Impact on wine

By far the most studied aspect of Hanseniaspora is its role within a grape must fermentation as discussed in previous reviews [14,21,22]. Due to their sheer numbers at the onset of a fermentation, Hanseniaspora could have an impactful role in the early development of a wine. It should be noted that although trends exist regarding the general role of Hanseniaspora within fermentation settings, their contribution is shrouded with contradictions. Hanseniaspora has been labeled a ‘spoilage yeast’ as it can produce compounds like acetic acid, acetaldehyde and ethyl acetate in excess amounts that could cause wine faults [23,24]. Hanseniaspora species are known to consume sufficient amounts of thiamin or other nutrients early in a fermentation leading to a stuck or sluggish fermentation [25,26]. Conversely, different Hanseniaspora species have been successfully implemented as a copartner with a Saccharomyces starter culture in a mixed-culture fermentation, with many reports showing a positive impact on the final wine product. Figure 3 shows some of the beneficial attributes of using Hanseniaspora spp. as a starter culture for winemaking.

Figure 3. Effect of Hanseniaspora-initiated cultures in mixed-culture wine fermentations. Increases and decreases in metabolite concentrations are shown relative to the levels of those metabolites produced in fermentations conducted with a pure culture of Saccharomyces cerevisiae. References for Hanseniaspora uvarum [67,76,85,104,219–224], Hanseniaspora guilliermondii [31,220,221,225], Hanseniaspora vineae [68,69,75,79,85,105,226] and other Hanseniaspora species [75,223,227–229].

When inoculated alone, Hanseniaspora spp. tend to display a glucophilic nature and do not completely consume the sugars in grape must [27]. There are, however, members of the ‘fermentation clade’, particularly H. osmophila, which is able to ‘finish’ fermentation, and can produce a significant amount of ethanol of over 11% [28]. Hanseniaspora’s production of the compounds acetic acid, acetoin, acetaldehyde, ethyl acetate and glycerol is often significantly higher than the amounts produced by any wine strain of S. cerevisiae, yet the interspecies and inter-strain variation is striking [29–36].

An important consideration is their interaction with Saccharomyces spp. within a fermentation. In spontaneous fermentations, i.e., where no starter culture is added, a dramatic shift in the populations of Hanseniaspora (as well as other non-Saccharomyces yeast or NSY) and Saccharomyces is often observed. As Hanseniaspora cell numbers decline rapidly in the first couple of days of fermentation, those of Saccharomyces increase [37–41]. This is, of course, exacerbated when a Saccharomyces starter culture is added [42,43]. Most wine strains of Saccharomyces produce a wide array of so-called ‘killer factors’ that limit the viability of other members of the wine microbiota [44]. These factors include antimicrobial peptides encoded on RNA virus-like particles [45–47]. Killer activity requires cell-to-cell contact as a membrane separation between Saccharomyces and non-Saccharomyces strains within a fermentation vessel curbs cell death of the non-Saccharomyces yeast cells [48–51]. Although ethanol production by Saccharomyces can also be considered as a ‘zymostatic agent’, Hanseniaspora strains can often withstand ethanol levels of ∼10%, and it is thus unclear to what extent ethanol affects Hanseniaspora viability [52–56]. Standard SO₂ additions at the onset of a fermentation have a negative impact on the viability of most non-Saccharomyces yeasts, yet studies on how it specifically affects Hanseniaspora spp. have shown contradictory results [28,57–62]. Killer ability, particularly RNA viruses, have also been identified in H. uvarum strains. These killer H. uvarum cultures inhibit a number of yeasts in vitro, including S. cerevisiae, yet its role in a fermentation setting remains unexplored [63–66].

Strains from three Hanseniaspora species have been extensively explored in a mixed-culture fermentation namely H. guilliermondii, H. uvarum and H. vineae (Figure 3). Although many trials have been conducted in which the performance of these Hanseniaspora species have been studied and tested on a pilot-scale [67–71], at the time of writing, no Hanseniaspora strain has been commercially sold as a starter culture [72,73]. There are many beneficial aspects attributed to the addition of Hanseniaspora starter cultures, ranging from lowering the ethanol levels [74–76], modulating the acid composition [77] to an increase in anthocyanin content [78,79], but arguably their biggest contribution is toward the aroma profile. Although every aroma component has been modulated by the addition of Hanseniaspora starter cultures, the majority of mixed-culture fermentations highlight a meaningful increase in the acetate ester content (Figure 4). Acetate ester synthesis is largely catalyzed by alcohol acetyltransferases (AAFs or ATFs), which condenses primary alcohols with the carboxylic group from acetyl-coenzyme A. Three of the five main acetate esters found in wine: 2-methyl butyl acetate, isoamyl acetate (both imparting a fruity, banana-like aroma) and 2-phenethyl acetate (honey, flower-like aroma) obtain their alcohol part from the breakdown of the corresponding amino acids via the Ehrlich pathway [80]. Hexyl acetate (apple-like aroma) obtains its alcohol from hexanol, which is found in grape must and the most common acetate ester in wine, ethyl acetate, is largely synthesized via the condensation of ethanol and acetyl-coenzyme A. This ester lends pleasant fruity aromas at concentrations below 50 mg/L, but above this concentration can mask other aromas and is considered a sensory defect at concentrations above 150 mg/L, imparting a solvent-like, nail polish aroma [81]. These levels are often exceeded with Hanseniaspora additions. Alcohol acetyltransferases can also catalyze the esterification of other wine alcohols, most notably varietal thiols like 3‑sulfanylhexan-1-ol or terpene alcohols like geraniol, leading to their corresponding acetate esters [82,83].

Figure 4. Biosynthesis pathways of fermentation-derived aroma compounds produced by grape-related Hanseniaspora species during winemaking [14,80].

All Hanseniaspora species are capable of growing in media containing cellobiose as the sole carbohydrate source, implying that they possess β-glucosidase activity [15,84]. β-Glucosidases are useful enzymes in winemaking as they can release certain aroma compounds from bound sugar moieties like terpenes [85–88]. β-Glucosidases from some Hanseniaspora release important, but non-aromatic, compounds from glycosylated precursors like the polyphenol resveratrol [89,90] and anthocyanins [78]. Strong β-xylosidase, polygalacturonase and protease activities have been identified in Hanseniaspora strains which could also enhance the flavor profile of wines [90–93]. In addition, the protease activity could be useful in the reduction of haze formation [94].

The deliberate use of non-Saccharomyces yeast strains as starter cultures have enjoyed a large appeal in winemaking and are a key force in the current elaboration of wine profiles [95–97]. The Hanseniaspora species with arguably the most potential in winemaking is that of H. vineae, as has been discussed in several reviews [98–100]. Even though the general acetate ester production of H. vineae strains are still pronounced, high levels of desirable benzenoids, including 2-phenyl ethanol and 2-phenethyl acetate, have often been stated [69,101,102].

The mode of inoculation, i.e., whether the Hanseniaspora starter culture is added at the same time as Saccharomyces or prior (normally three days before Saccharomyces addition), has a dramatic influence on the final wine composition [102–105]. The addition of oxygen also plays an important role, particularly due to most Hanseniaspora spp. showing limited fermentative capabilities [106]. Thus, fine-tuning the culturing conditions, along with strain and/or species selection, are key determinants in order to reliably display beneficial wine attributes in mixed-culture fermentations.

Impact on other fermentations

Like with grape berries, members of the Hanseniaspora genus are also frequently isolated on a wide variety of ripe fruit and would be present at the onset of the respective fruit wine fermentation. These include the fruit or resulting juices from agave [107], apple [10,108–111], mango [112], orange [7,113,114], pineapple [113,115,116] and plum [10,117] – by no means an exhaustive list. Their role in these different fruit juice matrices are not as extensively studied as in wine, but, in general, their populations decline as the fermentation progresses, as seen in grape must [5,118].

Researchers have also explored the effect of the deliberate addition of Hanseniaspora spp. to the start of a fermentation in order to augment the aromatic content of the resulting fruit wine. Figure 5 gives examples of outcomes of different Hanseniaspora starter cultures when added to a given non-grape starting material. As observed in wine, every aroma compound can be affected when compared to the pure inoculation of the fermenting Saccharomyces strain, but what stands out is the consistent increase in acetate ester content [4,108,119].

Figure 5. Effect of the deliberate addition of Hanseniaspora starter cultures on a diverse set of foodstuffs. References for beer [4,128,129,230–232], cocoa [233–235], coffee [236,237], citrus [238–240], apple [241–245], tequila [118,246,247], other [248–252].

Populations of Hanseniaspora spp. (particularly H. guilliermondii, H. opuntiae and H. uvarum) are also frequently isolated from beans of coffee and cocoa, as has been discussed in previous reviews [120–125]. Yeasts are critical for the successful fermentation of these beans, as suppressing their growth via the addition of an antizymotic agent led to a much lower quality bean prior to roasting [126,127]. The complex role of yeasts within bean fermentation includes ethanol production; acid modulation; and the production of volatile components, proteases and pectinases. Although both bean fermentations are still largely driven by actions of the natural microbiota, starter cultures have been added to accelerate the fermentation or to direct the flavor profile into a certain direction, including Hanseniaspora cultures which are also mentioned in Figure 5.

Hanseniaspora spp. lack the ability to utilize maltose and lactose and only a few members (belonging to the ‘fermentation clade’) can utilize sucrose [15]. Thus, they do not normally form part of the natural yeast microbiota of fermentations that are grain- or dairy-based, or fermentations where sucrose or molasses are added. Recently though, Hanseniaspora spp. have been added to beer fermentations owing to its ester-producing abilities, yet they were unable to utilize the maltose within the fermentation (Figure 5). This forms part of the growing trend in beer brewing where low or non-alcohol beers are preferred among consumers [128,129].

Hanseniaspora as a biocontrol agent

Fruits and vegetables farmed on a large-scale and in monoculture are particularly susceptible to the colonization by undesired microbiota or phytopathogens of fungal origin, which negatively impact the quality and yield of the harvest and their subsequent storage [130]. Current practices to mitigate the growth of these microbiota consist mainly of chemical interventions or fungicides. Despite success in controlling disease, their negative long-term impact on the environment [131], resistance being developed by fungi [132], as well as growing consumer pressure to reduce their usage [133], prompted investigations into alternative solutions [134]. Yeast associated with fruit and that show antagonistic action against the growth of these fungal phytopathogens have long been considered as potential biocontrol agents [135]. As common isolates from these niches, Hanseniaspora strains have been extensively employed in biocontrol trials to prevent fungal disease on a variety of fruit. Table 1 lists examples where Hanseniaspora yeast cultures were successfully implemented in a range of biocontrol experiments to prevent fungal disease, especially caused by Penicillium spp., Aspergillus spp. and Botrytis spp.

Table 1. Summary of biocontrol trials where Hanseniaspora strains were employed.

Species	Effect	Reference
H. clermontiae	Strong protease activity along with inhibitory action against B. cinerea and P. expansum growth on lab media	[135]
H. guilliermondii	Inhibits mycelial growth of Aspergillus spp.-infected grapes	[136]
H. guilliermondii	Inhibits the growth of the pineapple pathogen F. guttiforme	[253]
H. guilliermondii	Reduces incidence of disease caused by P. digitatum on various citrus fruits	[254]
H. guilliermondii	Displays in vitro antifungal activity against F. graminearum	[255]
H. opuntiae	Releases compounds inhibiting B. cinerea and C. cassiicola growth on Arabidopsis thaliana and Glycine max	[143]
H. opuntiae	Inhibits growth and mycotoxin production of A. flavus attributed to its volatile organic compounds	[151]
H. opuntiae	Inhibits the growth of A. flavus which is enhanced when co-cultured with lactic acid bacteria	[256]
H. opuntiae	Produces volatiles inhibiting mycelial growth of P. italicum and B. cinerea	[257]
H. opuntiae	Reduces radial growth of fig pathogens P. expansum, C. cladosporoides, B. cinerea, and M. laxa on lab media	[258]
H. opuntiae	Displays in vitro antifungal activity against B. cinerea	[259]
H. osmophila	Produces an array of diffusible compounds capable of inhibiting the growth of several fungal pathogens of table grapes	[260]
H. osmophila	Together with G. cerinus inhibits in vitro mycelial growth of Botrytis, Aspergillus, Penicillium, and Rhizopus	[261]
H. uvarum	Exhibits strong inhibitory action against A. carbonarius, B. cinerea, and P. expansum growth on lab media	[135]
H. uvarum	Inhibits growth of P. digitatum and P. italicum attributed to its phenyl ethanol production promoting adhesion	[137]
H. uvarum	Inhibits P. digitatum and P. italicum growth attributed to its 2-phenylethanol production	[138]
H. uvarum	Produces trans-cinnamaldehyde which were shown to inhibit B. cinerea-infection in cherry tomatoes	[139]
H. uvarum	Supplemented with trehalose induces defence-related enzyme activities in table grapes combatting A. tubingensis	[140]
H. uvarum	Produces volatile organics and induce defence‐related enzyme production in strawberries	[141]
H. uvarum	With β-aminobutyric acid shows inhibition against the growth of B. cinerea and A. alternata in kiwifruit	[144]
H. uvarum	Together with phosphatidylcholine inhibits spore germination and mycelial growth of P. digitatum in oranges	[145]
H. uvarum	With the addition of ammonium molybdenum reduces grey mold caused by B. cinerea in grapes	[146]
H. uvarum	With the addition of trehalose reduces grape rot caused by A. tubingensis and P. commune	[147]
H. uvarum	Along with salicylic acid or sodium bicarbonate shows decreased disease incidence of grape berries infected with B. cinerea	[148]
H. uvarum	Certain strains reduce ochratoxin levels within a grape must fermentation	[150]
H. uvarum	Inhibits growth and mycotoxin production of A. flavus attributed to its volatile organic compounds	[151]
H. uvarum	Inhibits the growth of mycotoxigenic fungi and detoxify the major mycotoxins aflatoxin B1 and ochratoxin in grape berries	[152]
H. uvarum	Reduces incidence of disease caused by P. digitatum on various citrus fruits	[254]
H. uvarum	Displays in vitro antifungal activity against B. cinerea	[259]
H. uvarum	Reduces postharvest mold decay in strawberries	[262]
H. uvarum	Reduces natural decay development caused by B. cinerea on grape berries	[263]
H. uvarum	Limits blue mold decay caused by P. italicum on navel orange fruit	[264]
H. uvarum	Produces volatile compounds capable of inhibiting growth of A. ochraceaus in coffee beans	[265]
H. uvarum	Provides effective control against rot caused by Rhizopus of postharvest stone fruits	[266]
H. uvarum	Reduces decay caused by the green and blue molds caused by P. digitatum and P. italicum on a commercial scale	[267]
H. uvarum	Reduces postharvest decay of grapes and peach caused by R. stolonifera and grey mold by B. cinerea	[268]
H. uvarum	Controls brown rot in postharvest plum caused by M. fructicola attributing to chitinase activity and volatile organic compounds	[269]
H. uvarum	Reduces disease caused by P. expansum, M. fructicola and B. cinerea in peaches and nectarines	[270]
H. uvarum	Reduces the incidence of decay, caused by B. cinerea in strawberries	[271]
H. uvarum	Shows inhibition of B. cinerea-infections on grape, apples, and peaches	[272]
H. uvarum	Shows reduction in chili fruit rot caused by C. capsici	[273]
Full species names of fungal pathogens Aspergillus  A. alternata  A. carbonarius  A. flavus  A. ochraceaus  A. tubingensis Botrytis  B. cinerea Cladosporium  C. cladosporoides	Colletotrichum  C. capsici Corynespora  C. cassiicola Fusarium  F. graminearum  F. guttiforme Monilinia  M. fructicola  M. laxa	Penicillium  P. commune  P. digitatum  P. expansum  P. italicum Rhizopus  R. stolonifera

Although the majority of these reports was primarily concerned in the outcome, i.e., whether a meaningful reduction in phytopathology was observed, some reports have attempted to explain the mode of inhibition caused by Hanseniaspora. The killer activity identified in Hanseniaspora has been attributed to its biocontrol ability [136]. Interestingly, multiple reports have attributed the biocontrol to the production of volatiles, particularly phenylethanol [137,138]. Another volatile, trans-cinnamaldehyde, produced by a H. uvarum strain, was shown to be the main volatile responsible for inhibiting fungal growth [139]. Moreover, the inoculation of Hanseniaspora induced the expression of defence-related genes in table grapes, strawberries, oranges and Arabidopsis thaliana [140–143]. The inhibition effect was enhanced with the addition of other compounds like β-aminobutyric acid [144], phosphatidylcholine [145], ammonium molybdate [146], trehalose [140,147], salicylic acid and sodium bicarbonate [148]. Interestingly, with the combination of an acetic acid bacteria, Gluconobacter cerinus, H. osmophila showed an increased biocontrol effect leading to a bioproduct patent [149]. Hanseniaspora cultures also have been shown to degrade mycotoxins, which are important carcinogenic compounds produced by infections caused by strains belonging to Aspergillus [150–153].

Despite the promise of using Hanseniaspora or any other yeast antagonist in the biocontrol of fungal growth, it should be noted that strain-specific variability do exist, because Hanseniaspora cultures can also display no meaningful fungal inhibition [154,155]. Most of the reports, especially on post-harvest applications on fruit, appear to show a clear path forward in this research field, yet it would be interesting to see how effective and realistic it would be for large-scale implementations of spraying Hanseniaspora yeast cultures in a field.

Associations with Drosophila

Members of the fruit-fly genus Drosophila co-inhabit the same ephemeral fruit niches as yeast [156]. Moreover, yeast cells are an important food source for both larva and adult drosophila and greatly influence its development time, body weight, egg-laying behavior (or oviposition) and survival [157–159]. Two noteworthy members of this genus include the vinegar fly (D. melanogaster)—a well-studied commensal fruit fly that swarms vineyards and orchards as fruit ripens and lays their eggs in rotten fruit—and the spotted wing fruit-fly (D. suzukii). The latter is a major insect pest capable of ovipositing in healthy ripe fruit. This causes major damage to the harvest of a wide range of fruit crops, including grapes, where it contributes to sour rot disease [160–162].

As one of the more common yeasts found on fruit, it is not a surprise that members of the Hanseniaspora genus are also often isolated from Drosophila fruit-flies with H. uvarum reported as being the most common isolate [156,163–166]. Although not conclusively proven, it is conceivable that Drosophila fruit-flies are an important vector for the dispersal of the non-motile Hanseniaspora cells within their environment, which could explain their relative abundance on ripe fruit. Some members, such as H. occidentalis, survive the alimentary tract of Drosophila adults [167]. Several studies have looked at the effect of single-yeast diets on life traits of Drosophila fruit-flies including that of H. uvarum. A H. uvarum-rich diet shortened the ‘egg-to-pupa’ period [168], increased larva survival [159], and promoted fecundity [169], yet has also shown poor larval development despite H. uvarum being the most attractive [170]. These results underscore the pivotal role this yeast has on the life quality of Drosophila.

It is well-established that Drosophila fruit-flies are significantly more attracted to partially-fermented juices than sterile or unfermented juices [158,171–173] suggestive of the important role that volatile organic compounds, produced by yeast, can have on the olfactory attraction of Drosophila. As a pure culture, H. uvarum-initiated fermentations significantly attract Drosophila fruit-flies [169,171,174,175].

The infestation of Drosophila peaks at fruit maturity, thus just prior to harvest, making chemical management undesirable, and leading to many investigations to develop microbial-based traps or lures [176–178]. Exploiting the attraction that Drosophila fruit-flies have for Hanseniaspora-inoculated substrates, various studies have shown the successful implementation of attract-and-kill strategies using Hanseniaspora-cultures as ‘bait’ in both laboratory and field experiments [172,179–183]. These findings show formulations using Hanseniaspora cultures as a promising and cost-effective ‘attracticide’ and can dramatically lower the use of insecticides to control D. suzukii-infestations [184]. Research still needs to be done to improve selectivity as current formulations attract all species of Drosophila.

Whole-cell biotransformation of chemical compounds

Using intact cells to act as so-called whole-cell biocatalysts has been useful in providing alternatives for the chemical synthesis or conversion of many compounds [185]. This method provides many benefits, as it can be highly selective, catalytically efficient and conducted in milder conditions. In addition, whole-cell catalysts can conduct multi-step reactions within ‘one pot’ and is also preferred for its environmentally-friendly approaches. Hanseniaspora cells (particularly H. guilliermondii) are often candidates to be screened for the bioconversion of many different chemicals [186]. H. guilliermondii has been used for the reduction of the flavor molecules (4 R)-(−)-carvone and (1 R)-(−)-myrtenal, presumably by the catalytic action of ene-reductases and/or carbonyl reductases [187–189]. H. guilliermondii cells reduce the C=C bonds of some chalcones [190]. In one study, a H. guilliermondii strain was shown to possess high levels of phytase activity and was able to effectively break down phytate in a cereal-based gruel [191]. In another study, it was reported that a yeast consortium consisting of, amongst others, a H. valbyensis and/or a H. opuntiae strain degraded the 5- and 6-ring structures of perylene and benzo[ghi]perylene often found at oil spills [192,193]. Interestingly, a H. uvarum strain was found to possess exceptional denitrification capabilities and could be used in the removal of nitrogen from wastewater systems [194].

‘Omics-based research

As with all biological research, large-scale genomic sequencing of Hanseniaspora strains, coupled with transcriptomic, proteomic, and biochemical data, have greatly contributed to our understanding of this genus and made studies on this yeast far more intriguing due to some surprising discoveries.

The mitochondrial genome sequence of a H. uvarum strain was first published in 2005 [195] and showed a strikingly compact linear molecule, which is the smallest among sequenced Ascomycetes with only 5.1% of non-coding regions [196]. In 2014, the first whole-genome sequence of a H. vineae strain was published [197] followed by many other well-known Hanseniaspora members in subsequent years: H. guilliermondii, H. opuntiae, H. osmophila, H. uvarum and H. valbyensis [198–201]. Most of the accepted species within Hanseniaspora have at least one represented genome sequenced.

Perhaps the most instructive genomic work on Hanseniaspora was done by Steenwyk and colleagues [202]. Their phylogenomic analyses divided Hanseniaspora into two main lineages: the faster-evolving lineage (FEL), which encompasses the ‘fruit clade’, and a slower-evolving lineage (SEL), which corresponds to the ‘fermentation clade’. Closer inspection on the genomic content of Hanseniaspora indicated that many genes associated with DNA repair pathways and maintenance, i.e., genes involved in cell cycle-checkpoints, were absent from the genomes – especially from members belonging to the FEL. It was already shown with comparative genome-scale analyses of members within the subphylum Saccharomycotina that species within Hanseniaspora are characterized by very long branches [203,204]. This feature resembles the long branches of fungal hypermutator strains belonging to cryptococci [205,206]. Hanseniaspora spp. also possess a low guanine-cytosine content, small genome sizes as well as lower gene numbers compared to other members of Saccharomycotina [203,204]. These features are quite remarkable and could explain the lifestyle of these yeasts, as they are abundant on ripe fruits or flowers where simple sugars are available as a food source for one period in a year [16]. A loss of cell-cycle checkpoint genes has also been suggested to give Hanseniaspora spp. a rapid growth advantage over competitors. There could also be a link to the lack of sporulation that has been reported for H. uvarum strains, yet this remains unestablished [207]. Interestingly, H. vineae strains have been shown to undergo arrest at G2 cell cycle during the stationary phase, an extremely uncommon phenomenon among eukaryotes in general [208].

In one study it was shown that multigene comparisons of different strains belonging to the same species displayed a sizable level of intraspecific nucleotide diversity [209]. Flow cytometry analysis has also shown a fairly dynamic ploidy architecture within the genus with allodiploids, allotriploids and allotetraploids being identified. Even interspecies hybridization between H. opuntiae and H. pseudoguilliermondii has been reported. Upon comparison among eight genomes of H. uvarum strains, genes involved in flocculation as well as oligopeptide transport were found to be missing in some strains, which could predict their fermentation performance, but reaffirmed the remarkable genomic variation found within one species [210].

In another study, it was demonstrated that in H. uvarum, the enzymatic function of a key enzyme within glycolysis—pyruvate kinase—is ∼10-fold lower than its counterpart in S. cerevisiae, which could explain its low fermentative capacity [198]. This was further substantiated with a study that showed that differences in pyruvate kinases could be the reason for H. vineae’s limited fermentative capability [20]. Yet, interestingly, members of the SEL have enzymes involved in the glycolytic pathway that are more closely related to S. cerevisiae than members belonging to the FEL. Transcriptome analysis on H. vineae also showed a marked upregulation of the aroma-related genes [211]. This, combined with genome data, which showed duplications of the ARO8 and ARO9 genes, which encodes amino acid aminotransferases, and the ARO10 gene phenylpyruvate decarboxylase, along with multiple ATF-like genes, could explain H. vineae’s capability of producing meaningful amounts of phenyl ethanol and phenethyl acetate. Within the genome of H. guilliermondii, four putative ATF-like genes have been identified which could explain the high acetate ester content often associated with H. guilliermondii-initiated cultures [200]. Owing to its strong ester-forming capabilities, the ethyl acetate transferase gene of H. uvarum was overexpressed in S. cerevisiae in order to improve its ethyl acetate production capability [212].

Transcriptomic data of a H. uvarum strain used in biocontrol studies have identified genes involved in ABC transport and ribosome biosynthesis to be upregulated when cultivated at a low temperature. The data also identified genes involved in the synthesis of the amino acids arginine, phenylalanine and lysine to be upregulated when co-incubated with P. digitatum [213]. Transcriptomic data of H. vineae strains revealed that the genes upregulated upon amino acid and ammonium consumption are similar to what is known from S. cerevisiae under similar conditions, suggesting a shared nitrogen catabolism mechanism [214].

Using H. vineae as model organism, an alternative pathway for the synthesis of benzenoid-derived compounds was discovered via the mandelate pathway [215]. This could account for the lack of phenylalanine ammonia lyase and tyrosine ammonia lyase pathways in ascomycetes yet they are capable of synthesizing benzenoids.

Attempts have been made to genetically modify H. uvarum by constructing plasmids containing autonomous replicating sequences from the species [216]. Recently, the first tools to knock out the genes of H. uvarum have been developed [217]. The two copies of the ATF-encoding gene (HuATF) were genetically removed, which led to a strain with much lower acetate ester production. This paves the way for further gene function studies in the Hanseniaspora genus.

Conclusions

A surge in applied research exploiting the interesting qualities of Hanseniaspora has occurred over the past decade – not just in fermentation settings, but also in the biocontrol of fungal and insect phytopathogens and as a whole-cell biocatalyst. This underlines the tremendous biotechnological potential of this genus that is still poorly understood regarding its ecological function, interstrain and intraspecies variation. The emergence of ‘omics-based data has already shown fascinating aspects of this genus and will continue to help elucidate this apiculate yeast.

Acknowledgements

The authors thank Geisenheim University and Macquarie University for co-funding of the research fellowship of N.V.W. I.S.P. is a team member of the Macquarie-led national Centre of Excellence in Synthetic Biology funded by the Australian Government thorough its agency, the Australian Research Council. The authors thank the Hessen State Ministry of Higher Education, Research and the Arts for the financial support within the Hessen initiative for scientific and economic excellence (LOEWE) in the framework of AROMAplus (https://www.hs-geisenheim.de/aromaplus/).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was jointly funded by Geisenheim University and Macquarie University.