Scholarly article on topic ' The wine and beer yeast Dekkera bruxellensis '

The wine and beer yeast Dekkera bruxellensis Academic research paper on "Industrial Biotechnology"

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Academic research paper on topic " The wine and beer yeast Dekkera bruxellensis "


Mini-review: Yeast-Primer April 2014

The wine and beer yeast Dekkera bruxellensis

Schifferdecker1, Sofia Dashko2, Olena P. Ishchuk1# and Jure Piskur

Department of Biology, Lund University, Solvegatan 35, Lund SE-223 62, Sweden

ïne Research Centre, University of Nova Gorica, Vipava, Slovenia

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/yea.3023

#Corresponding author: Mailing address: Department of Biology, Lund University, Sölvegatan 35, Lund SE-22362, Sweden. Phone: +46462221445. E-mail:


Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae, and especially known for two important characteristics: on one hand it is considered to be one of the main spoilage organisms in the wine and bioethanol industry, on the other hand it is "indispensable" as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model to study yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas which are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history.

Yeast, Food Sciences, Wine, Beer, Bioethanol, Genomics

Introduction to basic characteristics

Dekkera bruxellensis is considered to be a major cause of wine spoilage world wide (Boulton et al., 1996; Fugelsang, 1996; Delfini and Formica, 2001; Loureiro and Malfeito-Ferreira, 2003). Infected wines develop dinstinctive, unpleasant aromas due to volatile phenols produced by this species (Woolfit et al., 2007), also called "Brett" taints (Chatonnet et al., 1995), and normally associated with aromas of barnyard, burnt plastic, wet animal and horse-sweat (Licker et al., 1998). However, this species is also known for its positive contribution of acetic acid flavour to Belgian lambic beers (Dequin et al., 2003; Dufour et al.,

2003) and to the fermented and sweetened tea Kombucha (Mayser et al., 1995; Teoh et al.,

2004). Occurence of the species in feta cheese (Fadda et al., 2001) and sourdough (Meroth et al., 2003) has also been reported. This yeast also provides the characteristic aroma profile in some wines, such as the French Château de Beaucastel wines. D. bruxellensis is also often associated with high-ethanol biotechnological habitats (de Souza Liberal et al., 2007; Passoth et al, 2007). It has previously been isolated from Belgian stout, lambic beer, grape must as well as sparkling wine, sherry and porter. Its presence on the surface of grape berries has been shown by Renouf and Lonvaud-Funel (2007). This yeast is now also becoming a model to deduce yeast evolution processes (Rozpedowska et al., 2011).

It is s till common in the current literature to use both, Dekkera and Brettanomyces, as the genus name. The anamorphs Brettanomyces/Dekkera anomala, B./ D. bruxellensis, B. custersianus, B. naardenensis and B. nanus build this genus. Teleomorphs have been reported for two out of these five species: D. anomala and D. bruxellensis. The first reference to the genus Brettanomyces dates back to 1904, when Hjelte Claussen first isolated the yeast from British beers (Claussen, 1904). The flavours produced by this yeast became characteristic of certain British beers of that time and the name "Brettanomyces" was derived from "British

brewing fungus" (greek, "Brettano" means British brewer, "Myces" means fungus). First in 1940 the genus Brettanomyces was proven to occur in wine (Custers, 1940). The yeast

940 th


Mycotorulum intermedia, which was isolated from French wine by Krumholz and Tauschanoff in 1933, was named as a Brettanomyces species and later renamed as Brettanomyces intermedius (van der Walt and van Kerken, 1959). In 1960, Johannis van der Walt and Amelia van Kerken reported ascospore formation in B. bruxellensis, which led to reclassification of the genus Brettanomyces (van der Walt and van Kerken, 1960). The new genus Dekkera was proposed to accomodate the ascosporogenous forms - the name was chosen in honour of Nellie Margaretha Stelling-Dekker for her contribution to the taxonomy of the ascosporogenous yeasts (van der Walt, 1964). After the first description of spore formation (van der Walt and van Kerken, 1960), spores have, to our knowledge, never been reported again.

D. br uxellensis has adapted to harsh and limiting environmental conditions, like very high ethanol concentrations, low pH values (Fugelsang, 1996; Rozpedowska et al., 2011) and "poor" nitrogen sources. For example, D. bruxellensis preferentially uses amonium ions but can also use nitrate (de Barros et al., 2011; Galafassi et al., 2013). Renouf et al. (2006) have shown a higher adaption rate of D. bruxellensis, in comparison to other wild yeasts, to survive in must and during alcoholic fermentation. D. bruxellensis yeast grows between 19°C and 35°C, shows variable growth between 37°C and 42°C and cannot grow at 45°C (, May 2013 and Fig. 1). Van der Walt (1964) has also characterized the colony colours ranging from cream to light brown, usually shiny and smooth.

The first attempt to determine the D. bruxellensis genome sequence was in 2007 (Woolfit et al., 2007) and provided almost half of the open reading frames. In 2012, the whole genome of two different Dekkera bruxellensis strains were determined and are now publically available ( by Piskur et al.,

2012, and GenBank: AHIQ01000137.1 by Curtin et al., 2012). Piskur et al. (2012) have determined the whole genome sequence of the strain Y879 (CBS2499) and used it to deduce several "food relevant" properties of this yeast. Up ot date there are 5.636 predicted genes based on the sequenced strain CBS2499 (Y879) (DOE Joint Genome Institute), and so far the UniProt database comprises of 4.929 protein sequences. Phylogenetic analyses, based on 3930 individual gene trees in the context of 21 closely related fungal species, placed D. bruxellensis as a sister-group to Pichia (Komagataella) pastoris. The Komatagaella genus and its closest relatives are known as aerobic poor ethanol producer yeasts (de Schutter et al., 2009), which is just opposite to D. bruxellensis and S. cerevisiae. Analyses of growth parameters and carbon metabolism of several D. bruxellensis isolates have demonstrated that this yeast produces ethanol under aerobiosis, and has the ability to grow without oxygen, similar to S. cerevisiae (Nissen et al., 2000) and its closest relatives. D. bruxellensis can thus be described as a Crabtree-positive and facultative anaerobic yeast (Rozpedowska et al., 2011), which can particularly dominate in harsh environments.

Genetic and molecular tools

Although spores have been observed previously (van der Walt and van Kerken, 1960), neither mating types or mating events nor crosses of two haploid strains have been observed so far. Thus, it could be that this yeast is asexual. There is just little known about variation within the whole Dekkera genus at genomic level but preliminary investigations suggest that differences may be large and the D. bruxellensis clade could consist of several sister species (Hellborg and Piskur, 2009; Galafassi et al., 2011).

Multiple D. bruxellensis strains have been screened for auxotrophic mutants after mutagenesis by UV or ethane methyl sulfonate (EMS) treatment. The frequency of auxotrophs was lower than 0.1% (Siurkus, 2004). A ura3 deficient mutant has been isolated,

which promotes the use of the URA3 gene as a selection marker (Siurkus, 2004). For subsequent transformation experiments the URA3 gene has been sub-cloned from the genomic DNA (originating from the strain CBS2499) into the pUC57 vector, resulting in plasmid P892. This plasmid has been used to develop the first transformation protocol. A lithium acetate electrotransformation procedure (based on Becker and Guarente, 1991; Boretsky et al., 2007) has been modified to obtain transformants and the plasmids integrated at random sites into the genome (Hagstrom, 2008; Ishchuk et al., in preparation). Genomic libraries from two different strains (Y879 and Y881) have been constructed to find putative autonomous replication elements. Three types of replicating loci have been deduced, CIGO 1, d have been tused for the construction of autonomously replicating plasmids al., in preparation). A reporter system based on the K. lactis LAC4 gene under the control of the D. bruxellensis promoter from YPR100W (MRLP51) and the sub-cloned us (Fig. 2), have been used to determine the plasmid copy number, which is approximately 10 to 15. This reporter system can also be used for screening of different promoter elements. Apart from the URA3 gene, also the Sh ble gene can be used as a dominant selection marker (Fig. 3). This marker and URA3 have also been used for targeted deletions. The length of the homologous end sequences, which promoted homologous recombination and integration, varied from 350 to 600 bp (unpublished data).

D. bruxellensis shows much greater diversity among strains in chromosome number and ploidy than does S. cerevisiae. The proposed genome size ranges from under 20 to over 30 Mb (Hellborg and Piskur, 2009). Analysis of thirty D. bruxellensis isolates showed a range in chromosome sizes from less than 1 Mb to over 6 Mb, the different strains contained between 4 and 9 chromosomes, suggesting that the genome has been rearranged very fast upon the separation of single lineages. Almost all strains had polymorphic sites at the analysed loci, suggesting a higher than one ploidy status (Hellborg and Piskur, 2009). Curtin

et al. (2012) have suggested that the sequenced AWRI 1499 strain has a triploid genome, and the strain speciation has occurred through inter-specific hybridisation. Native D. bruxellensis

ie strain sp xains inde

strains indeed exhibit a large diversity of genotypes and phenotypes. Fugelsang and

Zoecklein (2003) have shown that not all D. bruxellensis wine isolates are able to grow in 0 \

Pinot Noir wine. Analyses of 244 wine isolates obtained from wineries located in 31 winemaking regions of Australia by using the multilocus AFLP fingerprinting method revealed large diversity and the presence of three major genotypes in Australian wine. Differences in the 26S rRNA gene sequence imply that some differences between the groups might be highly conserved, showing the existence of isolate-specific genotypes (Curtin et al., 2007).

Although the whole genomes of two D. bruxellensis strains have been deduced in 2012, commercial microarrays are still not available. Tiukova et al. (2013) have introduced an RNAseq approach and detected the expression of 3715 out of 4861 annotated genes in the D. bruxellensis CBS11270 strain. They have analyzed the transcriptome by using the AB SOLiD sequencing technique in conditions of sugar limitation and low oxygen concentrations, which are similar to those in industrial fermentations, where D. bruxellensis is able to outcompete S. cerevisiae. Several genes associated with sugar import as well as glycolysis were highly expressed. Their results indicate a high frequency of transcription events also outside the reading frames (Tiukova et al., 2013).

Methods of detection

For the wine industry it is important to have reliable methods to detect the spoilage yeast D. bruxellensis. Some of these methods are time consuming (microbiological), some are less (molecular detection). By using differencial media with ethanol as carbon source, bromcresol green and phenolic precursors, it is possible to distinguish Dekkera genus after a

relatively long period of cultivation (Rodrigues et al., 2001). Much faster but also more costly is a method based on fluorescent in situ hybridization using peptide nucleic acid probes that has been introduced by Stender et al. (2001). A PCR method for the species based identification of Dekkera, based on polymorphisms on the ITS regions, has been published by Egli and Henick-Kling (2001). Phister and Mills (2003) have developed a quantitative realtime PCR that targets only species within the Dekkera/Brettanomyces genus, while yeast and bacteria common to winery environment are not targeted. Cocolin et al. (2004) developed a PCR-restriction enzyme analysis protocol to detect and identify D. bruxellensis and D. anomala directly in wine samples. This technique allows much faster identification of these species isolated from wine. Molecular detections include a nested PCR method (Ibeas et al., 1996) and amplification of 26S rDNA region with further resolving of the PCR product by denaturing gradient gel electrophoresis (Manzano et al., 2004). The spoilage activity is strain dependent, thus methods for detection on strain level are of great oenological importance. Vigentini et al. (2012) has developed tools to assess the genetic intraspecific variation through the use of introns as molecular targets and designed specific primers annealing to introns 5'-splice site sequence (ISS), where they found a conserved pattern. For the interstrain discrimitation, restriction enzyme analysis and pulse field electrophoresis have been introduced by Miot-Sertier and Lonvaud-Funel (2007).

Carbon metabolism and the ability of anaerobic growth

Dekkera/Brettanomyces yeast can utilize several different sugars (Galafassi et al., 2011). Sugars, like glucose, are broken down into smaller molecules to become a source of energy and building blocks for the synthesis of other molecules. Glycolysis, the major process for sugar degradation, breaks down a glucose molecule into two molecules of pyruvate. Yeasts, depending on conditions, can use pyruvate by fermentation and/ or by

respiration. Since respiration of sugars is energetically more favorable than fermentation, most organisms use fermentation only when respiration is impaired, for example when oxygen availability decreases. However, in several yeast species, like S. cerevisiae and D. bruxellensis, the metabolic destiny of pyruvate formed at a high rate is largely switched from respiration to fermentation even when oxygen is abundant (for review see Pronk et al. 1996; Rozpedowska et al. 2011). In other words, these two yeasts may ferment sugars also under aerobic conditions, showing the so called "Crabtree-positive" phenotype (Fig. 4). In contrast, "Crabtree-negative" yeasts, like Kluyveromyces lactis and B. naardenensis, lack fermentative products and, under aerobic conditions, biomass and carbon dioxide are the sole products (Fig. 5).

The availability of oxygen varies among different niches. One of the main problems organisms face under anaerobic conditions is the lack of the final electron acceptor in the respiratory chain. The ability of yeasts to grow under oxygen-limited conditions seems to be strictly dependent on the ability to perform alcoholic fermentation. According to the dependence on oxygen during the life cycle, yeasts are classified as: (i) obligate aerobes displaying exclusively respiratory metabolism, (ii) facultative fermentatives (or facultative anaerobes), displaying both respiratory and fermentative metabolism; and (iii) obligate fermentatives (or obligate anaerobes) (Merico et al., 2007). S. cerevisiae and D. bruxellensis are both facultative anaerobes (Rozpedowska et al., 2011), while K. lactis and B. naardenensis are obligate aerobes (Fig. 5).

D. bruxellensis can spontanously generate mitochondrial petite mutants (McArthur and Clark-Walter, 1983). Thus, just like in S. cerevisiae, the active respiratory chain is not necessary for survival. However, what is different between these two yeasts is that the mitochondrial genome of D. bruxellensis encodes the NADH dehydrogenase (respiratory complex I) (Prochazka et al., 2010). The Dekkera/ Brettanomyces mDNAs exhibit a large

size polymorphism (Hoeben and Clark-Walter, 1986, and Prochazka et al., 2010), and mitochondrial loci represent an efficient tool to "easily" identify different species of this complex (Hoeben et al., 1993).

Wine and beer aroma associated aspects £ ^Dekkera yeasts are often causing wine spoilage. Odors of wine contaminated with Dekkera could be described as "pharmaceutical", "smoky", "wet horse" and are mainly caused by two groups of chemical compounds. Mousiness can be the consequence of carbonyl compounds. Among these nitrogenous compounds are 2-acetyl-3,4,5,6-tetrahydropyridine, 2-acetyl-1,2,5,6-tetrahydropyridine and 2-ethyl-3,4,5,6-tetrahydropyridine. The second group is presented by volatile phenols such as 4-vinylphenol, 4-vinylguiacol, 4-ethylphenol, 4-ethylguiacol (Pretorius, 2000). Dekkera is almost unique among other yeast because of its ability to convert hydroxycinnamic acids - antimicrobial

nonvolatile compounds present in grape must - into ethyl derivatives. Also the wine yeast

Meyerozyma guilliermondii can perform a similar metabolic activity (Barata et al., 2006).

Many other microorganisms with hydroxycinnamate activity would only form vinyl derivatives with no further conversion to ethyl derivatives (Chatonnet et al., 1992).

The origin of phenolic metabolites and enzymes involved in this hydroxycinnamic acid conversion pathway has been described only recently. Godoy et al. (2008) have purified the corresponding p-coumarate decarboxylase (CD) and vinylphenol reductase (VR). It has also been shown that the PAD gene is present in the Dekkera and Saccharomyces genus and it encodes the CD activity. However, the sequence of PAD in Dekkera is much more similar to bacterial phenolic acid decarboxylase than to the S. cerevisiae PAD1 gene. Only M. guilliermondii has the D. bruxellensis PAD homolog among all budding yeast described (Curtin et al., 2012). On the other hand, VR activity has not been found in Saccharomyces.

Harris et al. (2009) have published the sequence of the D. anomala gene with putative decarboxylase activity, and de Souza Liberal et al. (2012) have described the existence of two genes in D. bruxellensis, which might be paralogues of phenyl pyruvate decarboxylase (Db ARO10).

Dekkera species are also important in sourdough and beer industry. When present at high levels food spoilage occurs. However, low amounts of Dekkera contribute metabolites desirable in bread, lambic beer, ale and kombucha tea. In beer, concentration of 4-ethylphenol (medicinal aroma) is lower than 4-ethylguiacol (clove, spicy smell). In case of the wine situation, it is opposite. Thus, relative concentrations determine different effects in different food products (Curtin et al., 2012). In contrast to S. cerevisiae, some Dekkera isolates possess P-glucosidase activity, which catalyzes release of desirable bounded phenolic compounds from hops in beer (Daenen et al., 2008). The kinetic properties of a-glucosidases, and their role during beer fermentation, have also been described (Shantha Kumara et al.,

1993). 1993).

Fermentation characteristics of Dekkera

During fermentation process, yeasts need to adapt to high osmotic pressure and high sugar concentrations, and partially anaerobic conditions, deficiency of nitrogen and ethanol presence. It has been noted before, that the Dekkera genus shows the Custer effect during fermentation (van Dijken and Scheffers, 1986; Vigentini et al., 2008). When growing partially anaerobically, fermentation is partially inhibited because of the acetic acid production and redox imbalance (Vigentini et al., 2008). Blomqvist et al. (2010) described fermentation properties of D. bruxellensis using full factorial design. It has been noted that growth rate and ethanol yield on maltose are lower than that on glucose as a carbon source. When comparing D. bruxellensis with industrial S. cerevisiae strains, the latter grew five

times faster but with lower ethanol yields. S. cerevisiae was also the "winner" in glycerol amounts produced (six fold higher). After a while however S. cerevisiae biomass levels reached 72 - 84% of D. bruxellensis biomass (Blomqvist et al., 2010). Nardi et al. (2010) have demonstrated that Dekkera consume sugar and grow much slower than S. cerevisiae. The roles are changed at the end of fermentation, when sugar is depleted and only low amounts of nitrogen are available. The expression of MSN4 (transcription factor involved in activation of heat shock, osmotic stress and high ethanol stress) in Dekkera has been activated at higher ethanol concentrations than in S. cerevisiae. When comparing response on sugar, situation has been opposite. Late activation of MSN4 might mean a different ability of Dekkera to survive on alternative carbon source under glucose starvation conditions. For VPS34, ERG6 and ATP1, essential in the presence of ethanol, expression patterns in S. cerevisiae and Dekkera have been different. In Dekkera, ERG6 has not been repressed in

■fNl p

stationary phase and ATP1 expression level at the start of fermentation has been extremely high. Nardi et al. (2010) have explained this by not well-established anaerobiosis. In general, some stress response genes in D. bruxellensis have been expressed at the beginning of fermentation but some of the genes have been expressed much later than the ones in S. cerevisiae. It seemed that just like the conventional yeast, Dekkera is well adapted to fermentation conditions through changes in expression of its genome or some morpho-physiological features. For example, it has been demonstrated that D. bruxellensis is able to form biofilms which improves the attachment on the barrel surface (Joseph et al., 2007).

Rozpedowska et al. (2011) have studied several isolates of D. bruxellensis under controlled batch cultivation for their growth parameters and carbon metabolism. Under aerobic conditions D. bruxellensis produced substantial amounts of ethanol and, as Aguillar Uscanaga et al. (2003) have described before, good aeration stimulated acetate production. After glucose depletion both ethanol and acetate were completely consumed. This yeast could

also grow under anaerobiosis if the minimal medium was supplemented with Tween 80, ergosterol and amino acids (see also Blomquist et al., 2012). In contrast, in the presence of oxygen B. naardenensis exhibited a completely respiratory metabolism and could not grow under anaerobiosis. The D. bruxellensis ethanol yield in aerobiosis and the ability to grow without oxygen are very similar to those reported for S. cerevisiae (Nissen et al., 2000) and its sister species. During anaerobic growth glucose is converted into biomass at lower yields than in aerobiosis due to a higher ethanol production.

Biofuel production

Dekkera yeasts are often present during the biofuel production process. While Basilio et al. (2008) have considered presence of these yeasts as spoilers of the production processes, other authors refer to Dekkera genus as alternative yeast to S. cerevisiae for ethanol production (Passoth et al., 2007). There are a few important features that make Dekkera suitable for bioethanol production. D. bruxellensis is a Crabtree-positive yeast, so it is possible to obtain ethanol from yeast culture when high sugar concentrations are available even under aerobiosis. It has also been revealed that the Custer effect takes place under increasingly anaerobic conditions. In this case alcoholic fermentation is inhibited due to a redox imbalance. The other special feature is high acetic acid production during aerobic alcoholic fermentation (Leite et al., 2013).

When scaling up, the aeration in bioreactor is becoming a problematic and expensive procedure. Under these conditions S. cerevisiae is the most preferable organism. However, S. cerevisiae is able to use only ammonium ions as a source of the nitrogen. In this case, nitrate assimilation by D. bruxellensis is a superior advantage in lignocelluslose media, which are rich in nitrate. Recently, a strict correlation between acetic acid production and nitrate utilization has been reported (Galafassi et al., 2013). Glucose consumption on the media with

nitrate has also been improved. It seems that NADPH and NADH, which are available in the cell under anaerobic conditions, could neutralize the redox imbalance. Both cofactors play a role of electron donors for nitrate reductase, which is active under anaerobic conditions.

Evolution aspects

A majority of ascomycotic fungi under aerobic conditions convert sugar-based substrates into CO2. However, at least three groups, including budding and fission yeasts, have apparently independently evolved the metabolic ability to produce ethanol in the presence of oxygen and excess of glucose (reviewed in Rozpedowska et al., 2011; Rhind et al., 2011). This metabolic "invention" represents in nature a possible tool to poison/ outcompete other microbes. Crabtree-positive budding yeasts S. cerevisiae and D. bruxellensis can efficiently catabolize ethanol and therefore their corresponding life style has been named as "make-accumulate-consume (ethanol)" strategy (Thomson et al., 2005; Piskur et al., 2006; Rozpedowska et al., 2011). On the other hand, the third Crabtree-positive group, including the fission yeast Schizosaccharomycespombe, only poorly metabolizes ethanol.

The phylogenetic analysis of Ascomycetes suggests that the Saccharomyces/ Kluyveromyces and Dekkera/ Brettanomyces lineages separated at least 200 mya (Rozpedowska et al., 2011). In other words, the divergence took place long before the Whole Genome Duplication (WGD), promoter rewiring, URA1 horizontal transfer and ADH duplication events that occurred in the S. cerevisiae lineage and are thought to be involved in the evolution of the "make-accumulate-consume" strategy (reviewed in Piskur et al., 2006). The S. cerevisiae and D. bruxellensis lineages, but not the K. lactis and B. naardenensis lineages, have apparently independently acquired the ability to accumulate ethanol in the presence of oxygen and resistance to high ethanol concentration, which is a crucial trait to accompany efficient ethanol production and accumulation (Rozpedowska et al., 2011) (Fig.

origin of "make-accumulate-consume" strategy coinsides with origin of modern

plants with fruits, which more than 125 mya brought to microbial communities a new larger

and increasingly abundant source of food based on simple sugars (reviewed in Piskur et al., 0 \

2006). Ancient yeasts could hardly produce the same amount of new biomass as bacteria during the same time interval, and could therefore be out-competed. One can speculate that slower growth rate could in principle be counter-acted by production of compounds like ethanol and acetate that could inhibit the growth rate of competitors. Therefore, it is not surprising that several similar "winning" traits, like the ability to grow without oxygen and the Crabtree effect (or "make-accumulate-consume" strategy), can be found among not so closely related modern yeasts, like the Saccharomyces and Dekkera clades. Surprisingly, both lineages used the same tool, global promoter rewiring, to change the regulation pattern of respiration-associated genes resulting in ethanol accumulation and consequently in the development of the "make-accumulate-consume" strategy (Rozpedowska et al., 2011). An interesting aspect is also that both yeasts have independently duplicated their alcohol dehydrogenase encoding ADH genes (Piskur et al., 2012).

In conclusion, D. bruxellensis will in the following years represent one of the central model organisms to understand evolution of the yeasts alcoholic fermentations and will also be in focus from the wine and beer makers perspective.


We would like to thank the Marie Curie Initial Training Network Cornucopia (FP 7), the Fysiografen and Lindstrom Foundations and the Slovenian Research Agency (ARRS) for their interest in this project and their financial support.


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Figure 1 Dekkera bruxellensis Y879 (CBS 2499) in the minimum medium (the figure is a courtesy of Concetta Compagno, Milan, Italy).

Figure 2 A linear presentation of the plasmid P1017. The D. bruxellensis URA3 gene is shown as a grey box, K. lactis LAC4 reporter gene as red box, D. bruxellensis promoter YPR100W (MRLP51, mitochondrial ribosomal protein large subunit) as yellow box, the CIGO1 motif (for autonomous replication) is shown as a black box, and pUC57 part as a thin line (Ishchuk et al., in preparation).

Figure 3 A scheme of the development of molecular and genetic tools for D. bruxellensis, from auxotrophic mutants to targeted deletions, based on Siurkus (2004), Hagstrom (2008), and Ishchuk et al. (in preparation).

Figure 4 Batch culture of the Crabtree-positive yeast D. bruxellensis Y879 (CBS 2499) under

aerobic conditions in the defined minimal medium. Optical density, OD6oonm (orange line),

glucose (black) and ethanol (violet) concentrations are shown (right-side scale, presented as

g/l), adopted from Rozpedowska et al. (2011).

Figure 5 A simplified phylogenetic relationship among the Saccharomyces/ Kluyveromyces and Dekkera/ Brettanomyces clades, which separated more than 200 mya. The Saccharomyces and Dekkera lineages have independently evolved (red arrows) the abvility to produce ethanol in the presence of oxygen (Crabtree effect) and the ability to propagate under anaerobic conditions.


Dekkera bruxellensis is known as both a spoilage yeast and a contributor to flavour and aroma in some beers and wines. Current research focusses on these processes, carbon metabolism and yeast evolution.