Fermentative lifestyle in yeasts belonging to the Saccharomyces complex Annamaria Merico 1 , Pavol Sulo 2 , Jure Pis ˇ kur 3 and Concetta Compagno 1 1 Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita ` degli Studi di Milano, Milan, Italy 2 Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia 3 Department of Cell and Organism Biology, Lund University, Sweden The concentration of oxygen in the environment is one of the most important factors that regulate energy conversion in living cells. Organisms have developed multiple processes to optimize the utiliza- tion of oxygen when its availability is reduced. Accord- ing to the role of oxygen in their metabolism, yeasts can be classified as: (a) obligate aerobes, displaying an exclusively respiratory metabolism; (b) facultative fermentatives, disp laying both respiratory and fermenta- tive metabolism; and (c) obligate fermentatives. The ability of yeasts to grow in very oxygen-limited condi- tions is strictly dependent on the ability to perform alcoholic fermentation, allowing reoxidation of NADH generated during glycolysis. In Saccharomyces cerevisiae, fermentation predominates over respiration when glucose concentrations are high, even under aerobic conditions. Depending on this characteristic, yeasts are classified as Crabtree-positive or Crabtree- negative. Thus, in Crabtree-positive yeasts, such as S. cerevisiae, NADH is mainly oxidized in glucose- Keywords evolution; fermentation; petite mutants; redox metabolism; respiration Correspondence C. Compagno, Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita ` degli Studi di Milano, via Celoria, 26 20133 Milan, Italy Fax: +39 02503 14912 E-mail: concetta.compagno@unimi.it (Received 9 October 2006, revised 24 November 2006, accepted 11 December 2006) doi:10.1111/j.1742-4658.2007.05645.x The yeast Saccharomyces cerevisiae is characterized by its ability to: (a) degrade glucose or fructose to ethanol, even in the presence of oxygen (Crabtree effect); (b) grow in the absence of oxygen; and (c) generate respir- atory-deficient mitochondrial mutants, so-called petites. How unique are these properties among yeasts in the Saccharomyces clade, and what is their origin? Recent progress in genome sequencing has elucidated the phylo- genetic relationships among yeasts in the Saccharomyces complex, providing a framework for the understanding of the evolutionary history of several modern traits. In this study, we analyzed over 40 yeasts that reflect over 150 million years of evolutionary history for their ability to ferment, grow in the absence of oxygen, and generate petites. A great majority of isolates exhibited good fermentation ability, suggesting that this trait could already be an intrinsic property of the progenitor yeast. We found that lineages that underwent the whole-genome duplication, in general, exhibit a fermentative lifestyle, the Crabtree effect, and the ability to grow without oxygen, and can generate stable petite mutants. Some of the pre-genome duplication lin- eages also exhibit some of these traits, but a majority of the tested species are petite-negative, and show a reduced Crabtree effect and a reduced abil- ity to grow in the absence of oxygen. It could be that the ability to accumu- late ethanol in the presence of oxygen, a gradual independence from oxygen and ⁄ or the ability to generate petites were developed later in several line- ages. However, these traits have been combined and developed to perfection only in the lineage that underwent the whole-genome duplication and led to the modern Saccharomyces cerevisiae yeast. Abbreviation EtBr, ethidium bromide. 976 FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS rich media by fermentation rather than by respiration, even in the presence of oxygen. This has been attrib- uted to a limited capacity and ⁄ or saturation of the respiratory route of pyruvate dissimilation [1,2]. Glu- cose metabolism and oxygen can also be related by the Pasteur effect, which has been defined as the inhi- bition of fermentative metabolism by oxygen, but in S. cerevisiae this phenomenon is only observable at low glycolytic fluxes [3]. In the Kluyver effect, the absence of oxygen impairs the utilization of particular disaccharides, although one or both of the monosac- charide components can be used anaerobically in fer- mentation [4]. This characteristic seems to be determined mainly by the activity of sugar carriers [5]. The inhibition of fermentation of glucose as well as other sugars in the absence of oxygen has been described as the Custer effect, found in Brettanomyces intermedius and in Candida utilis [6], and has been proposed to be due to a redox imbalance. The regula- tory mechanisms behind these phenomena appear to influence energy metabolism in different ways among different yeast species. Apart from alcoholic fermentation, the ability to grow under anaerobic conditions also determined by other factors. Some metabolic pathways require the presence of molecular oxygen. This is true to various extents for the biosynthesis of sterols and fatty acids, heme ⁄ hemoproteins, NAD, and uracil [7,8]. The abil- ity to translocate ATP produced in the cytoplasm into mitochondria, and the ability to adjust the redox bal- ance, play a very important role in independence from oxygen [9–14]. In S. cerevisiae, three genes encode for ATP transporters, AAC1, AAC2 and AAC3. Deletion of AAC2 and AAC3 is anaerobically lethal [9–11]. Under anaerobic conditions, yeast cells can achieve redox balance by production of glycerol [12–14]. This means that the nutritional conditions also have a strong influence on the ability to grow anaerobically. Comparison of species belonging to several yeast genera for their ability to grow anaerobi- cally in complex and synthetic minimal media revealed a superiority of S. cerevisiae for growth under restrictive conditions in terms of strict anaero- biosis and minimal presence of organic nutrients [15]. The use of cDNA arrays recently provided new insights into gene networks and pointed out the essen- tial role of the regulation of gene expression underly- ing the physiologic response of S. cerevisiae to oxygen deprivation [16,17]. Saccharomyces cerevisiae constantly produces mutants that are stable during vegetative reproduction and are characterized by a reduced colony size on solid media in which a fermentable carbon source is the limiting factor [18]. These mutants are called ‘petites’, and are a special class of respiratory-deficient mutants characterized by large deletions in their mtDNA or a complete lack of the mitochondrial gen- ome [19,20]. Several Saccharomyces yeasts readily give rise to petites [21], but a majority of other yeasts fail to yield stable petite mutants, and are therefore called ‘petite-negative’ yeasts [22]. So far, the origin of and the biochemical and physiologic requirements for the occurrence of petites in yeast have been unclear. It has previously been suggested that the petite-positive character might coincide with the ability to grow in the absence of oxygen [22–24]. However, Saccharo- myces kluyveri is an example of a yeast that can grow anaerobically, but cannot generate true petite mutants [25]. The origin of different responses to the pres- ence ⁄ absence of oxygen has so far been poorly under- stood [26]. Among the reasons are that few yeasts have been studied, and that the phylogenetic relation- ships among these yeasts were unclear at the time. Recently, phylogenetic relationships among yeasts have been determined from a multigene sequence ana- lysis, which placed 75 species of the Saccharomyces complex into 14 well-supported clades [27]. In many cases, these clades do not correspond to the circum- scribed genera: species of Kluyveromyces as well as of Zygosaccharomyces are found in different clades, indi- cating the polyphyly of these genera as presently defined. According to this analysis, it was proposed to reassign the species into five new genera [28]. The S. cerevisiae lineage underwent a whole-genome dupli- cation about 100 million years ago [29–31], and the Saccharomyces clade can therefore be subdivided into pre- and post-genome duplication lineages. Appar- ently, the duplication took place after the separation of Saccharomyces, Kazachstania, Naumovia, Nakesimia and Tetrapisispora from the rest of Saccharomyces complex genera (Fig. 1). Another problem for comparative studies on the regulation of energy metabolism in aerobic and anaer- obic growth is caused by differences among the experi- mental conditions used, such as composition of media and adequate control of anaerobic conditions. The purpose of the present work was to study the fermen- tative capacity, the ability to grow in anaerobic condi- tions and the occurrence of the petite phenotype in a large set of strains belonging to the ‘Saccharomyces complex’. Our study includes more than 40 strains, and provides a basis for speculation on how these metabolic traits evolved within the Saccharomyces clade, which originated approximately 150 million years ago. A. Merico et al. Fermentative lifestyle in yeasts FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS 977 Results Glucose metabolism and ethanol production in aerobiosis (Crabtree effect) In order to look for the presence of the Crabtree effect in species belonging to the Saccharomyces complex, we performed batch cultivations in a fermenter under well- controlled aerobic conditions. As a consequence of respirofermentative glucose metabolism (Crabtree effect), leading to the production of ethanol and other byproducts (pyruvate, acetate, succinate, and glycerol), S. cerevisiae growing in batch on glucose under aerobic conditions gave a low biomass yield (Table 1). Species belonging to the genera Naumovia (Saccharomyces castellii) and Nakaseomyces (Candida glabrata) showed (Table 1) high specific ethanol production rates (20.5 mmolÆg )1 Æh )1 and 16.8 mmolÆg )1 Æh, respectively) as well as a low biomass yield (0.08 gÆg )1 and 0.11 gÆg )1 ) during the exponential phase of growth, with values very similar to those reported for S. cerevisiae [35]. These data indicate that these species behave as typical Crab- tree-positive yeasts. In the Torulaspora genus, we found one species, T. globosa, that showed a typical Crabtree effect, with a high specific ethanol production rate (18.6 mmolÆg )1 Æh )1 ) and a low biomass yield (0.08 gÆg )1 ). On the other hand, T. delbrueckii showed a less pronounced Crabtree effect, with a lower specific ethanol production rate (6.13 molÆg )1 Æh )1 ) and a higher biomass yield (0.27 gÆg )1 ), as previously observed in S. kluyveri (Table 1) [36]. A similar situation was detec- ted in species belonging to the Hanseniaspora genus. Hanseniaspora vinae and Hanseniaspora occidentalis did in fact exhibit the ability to produce ethanol under aero- bic conditions, but to a lower extent than S. cerevisiae (Table 1). In the Zygosaccharomyces genus, Z. bailii has been reported to show a reduced Crabtree effect [37]. In our experiments, Z. rouxii showed the lowest ethanol production rate (1.51 mmolÆg )1 Æh )1 ) of all tested species. Species belonging to the Kluyveromyces genus, such as K. wickerhamii, behaved like the Crabtree-negative yeast K. lactis, being quite unable to produce ethanol under aerobic conditions (Table 1), in spite of high glucose consumption rates. As a consequence of a purely respiratory metabolism, the two Kluyveromyces species showed the highest biomass yields (0.45 gÆg )1 and 0.4 gÆg )1 , respectively). In conclusion, even though a limited number of species was tested, our data indi- cate that the Crabtree effect is present in several spe- cies of the Saccharomyces complex, but is expressed at significantly different levels. Growth in aerobic conditions in the presence of antimycin A To further assess fermentative capacity, we tested for growth when respiration becomes gradually more impaired, by increasing the concentration of anti mycin A. This drug is a well-known inhibitor of elec- tron transfer from quinone to cytocrome b [38]. Yeast strains were cultivated in aerobic conditions on plates containing rich or synthetic minimal medium (Table 2). All but two of the species analyzed grew on rich medium plus antimycin A, indicating that they are able to grow through fermentative metabo- lism, and most likely produce ethanol. Most of the species, 29 out of 49, were able to grow on synthetic minimal medium at the highest antimycin A concen- Table 1. Occurrence of respirofermentative metabolism in aerobic batch cultures: specific rates of growth (l ),specific consumption rates of glucose (fructose) [q Glu (Frt) ], specific production rates of ethanol (q EtOH ) and the yields of biomass and ethanol relative to consumed glucose (fructose) for several yeasts of the Saccharomyces complex. The data for S. cerevisiae, Z. bailii and S. kluyveri are from the literature [35–37]. Strain Carbon source l (h )1 ) q Glu (Frt) (mmolÆg )1 Æh )1 ) q EtOH (mmolÆg )1 Æh )1 ) Biomass yield (gÆg )1 ) Ethanol yield (gÆg )1 ) S. cerevisiae [35] Glc 0.7% 0.37 14.80 22.00 0.13 0.40 S. castellii Glc 0.7% 0.22 16.93 20.54 0.08 0.30 C. glabrata Glc 2% 0.28 13.94 16.80 0.11 0.31 Z. rouxii Glc 2% 0.10 2.34 1.51 0.28 0.16 Z. bailii [37] Fru 0.7% 0.30 7.82 6.00 0.29 0.22 T. globosa Glc 2% 0.23 15.03 18.60 0.08 0.32 T. delbrueckii Glc 2% 0.38 4.31 6.13 0.27 0.26 S. kluyveri [36] Glc 2% 0.47 8.7 3.4 0.29 0.08 K. wickerhamii Glc 2% 0.43 9.89 0 0.45 0 K. lactis Glc 2% 0.50 11.95 0 0.40 0 H. vineae Glc 2% 0.41 13.05 7.11 0.16 0.16 H. occidentalis Glc 2% 0.33 6.23 4.86 0.23 0.18 Fermentative lifestyle in yeasts A. Merico et al. 978 FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS Table 2. Analysis of growth under aerobic conditions in the presence of increasing concentrations of antimycin A. The analysis refers to the Saccharomyces complex: the species are listed according to their phylogenetic relationship with S. cerevisiae (the lowest species in the col- umn is the least related) as reported by Kurtzman & Robnett [27]. –, no growth; +, growth within 7 days; NT, not tested. Numbers indicate the maximal tolerated dose of antimycin A. Clade Strain Antimycin A concentration (l M) Rich medium: 5 Synthetic minimal medium: 0.5–25 Synthetic minimal medium plus lysine and glutamic acid: 0.5–25 Synthetic minimal medium plus acetoin: 0.5–25 Saccharomyces S. cerevisiae +25NT NT S. paradoxus +25NT NT S. pastorianus +2025 20 S. bayanus + 4 25 5 Kazachstania S. servazii + 5 25 5 S. unisporus +25NT NT A. telluris +25NT NT S. transvaalensis +25NT NT K. africanus +25NT NT S. spencerorum +25NT NT K. lodderae +25NT NT K. piceae + 5 NT NT S. exiguus +2025 25 S. barnettii +4– – C. humilis +25NT NT Naumovia S. castellii +25NT NT S. dairensis + 5 NT NT Nakaseomyces C. glabrata +25NT NT K. delphensis +25NT NT K. bacillisporus +25NT NT Tetrapisispora K. blattae +25NT NT Te. phaffii +25NT NT Te. iriomotensis +25NT NT Zygosaccharomyces Z. rouxii +– – – Z. bailii + 5 25 25 Z. bisporus – –NT NT Zygotorulaspora Z. florentinus +25NT NT Z. mrakii +45 – Torulaspora Tor. globosa + 5 25 25 Tor. franciacae +25NT NT Tor. pretoriensis +25NT NT Tor. delbrueckii +25NT NT Z. microellipsoides + 2 25 25 Lachancea Z. fermentati +25NT NT K. thermotolerans +25NT NT K. waltii +25NT NT S. kluyveri +25NT NT Kluyveromyces K. aestuarii +25NT NT K. nonfermentans – –NT NT K. wickerhamii +1– – K. lactis + 5 25 25 K. marxianus +25NT NT Eremothecium E. gossypii +– – – Hanseniaspora H. valbyensis + 2 25 – A. Merico et al. Fermentative lifestyle in yeasts FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS 979 tration tested, whereas the rest could grow at lower levels of the drug. For some of these species, such as Z. bailii, T. globosa, Zygosaccharomyces microellipso- ides, K. lactis, and H. occidentalis, the addition of acetoin, as well as the addition of lysine plus glutam- ate, restored growth in the presence of high concen- trations of antimycin A. This suggests that for these species the inability to grow in the presence of anti- mycin A is mainly due to an impaired redox balance. This balance is substantially affected on synthetic minimal medium because of the high level of NADH generation, due to amino acid biosynthesis. Much of the generation of NADH during amino acid biosyn- thesis takes place in the mitochondria. Because of the block in the respiratory chain caused by the addition of antimycin A, NADH should then be reoxidized through shuttle mechanisms with the cytoplasm [14]. Acetoin acts as a redox sink at the cytoplasmic level, being reduced to 2,3-butanediol by the cytosolic NAD + -linked 2,3-butanediol dehydrogenase [39]. In some species (Saccharomyces bayanus, Saccharomyces servazii, Hanseniaspora valbyensis), we observed that the inability to grow in the presence of high concen- trations of antimycin A was actually due to an impairment in the reoxidation of NADH at the mit- ochondrial level, because in this case the addition of acetoin did not help to restore the redox balance (Table 2). This could indicate that, in these yeasts, the mechanisms for shuttling NADH reducing equiva- lents from mitochondria to cytosol are inefficient. For other species, such as S. barnettii, Z. rouxii, K. wick- erhamii, Eremothecium gossypii, and Kloeckera lindner- i, the inability to grow on synthetic minimal medium when respiration is at least partially impaired was not alleviated by the addition of acetoin or of amino acids. In this case, the very low fermentative capacity does not provide sufficient energy for growth when respiration is limited. These data seem to indicate that most of the species belonging to the Saccharomyces complex possess a good fermentative capacity, being able to generate suf- ficient energy to grow when respiration is impaired. Nevertheless, we observed that redox problems can, in some cases, limit the ability of the yeast to grow when the respiration chain is blocked. Growth under strict anerobic conditions All strains were cultivated on plates containing rich or synthetic minimal medium, and incubated under strict anerobic conditions. Under these conditions, most of the species were able to grow after 7 days on both complex and synthetic minimal media (Fig. 1). All an- alyzed species belonging to the Saccharomyces, Kaz- achstania, Naumovia, Nakaseomyces and Tetrapisispora genera were able to grow under the most stringent conditions, i.e. on synthetic minimal medium under strict anaerobiosis (Fig. 1, species in red). Z. microellipsoides (Torulaspora genus) and S. kluyveri (Lachancea genus) were able to grow after 7 days only on rich medium. However, the addition of acetoin ⁄ amino acids restored growth on synthetic minimal medium after 14 days of incubation (Fig. 1, in blue). This suggests that the growth problems of these strains on synthetic minimal medium are mainly caused by inefficient homeostasis of the redox cofactors under these conditions. Species belonging to the genera Zygosaccharomyces (Z. bailii), Torulaspora (T. globosa), Kluyveromyces (K. lactis, K. marxianus) and Hanseniaspora (H. guiller- mondii and H. occidentalis) showed growth on rich medium only after 14 days of incubation, but failed to grow on synthetic minimal medium, even in the pres- ence of acetoin (Fig. 1, in green). This may reflect a strong redox problem that can completely impair growth in anaerobic conditions on synthetic minimal Table 2. (Continued). Clade Strain Antimycin A concentration (l M) Rich medium: 5 Synthetic minimal medium: 0.5–25 Synthetic minimal medium plus lysine and glutamic acid: 0.5–25 Synthetic minimal medium plus acetoin: 0.5–25 Klo. lindneri +– – – H. guilliermondii +25NT NT H. vineae +25NT NT H. osmophila +25NT NT H. occidentalis + 5 25 25 Fermentative lifestyle in yeasts A. Merico et al. 980 FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS medium, where NADH production is high. Z. bailii is known to produce more ethanol on fructose than on glucose [37], and fructose is taken up by facilitated transport [40]. We then tested whether the presence of fructose (instead of glucose) as carbon source could allow for growth in anaerobic conditions. However, this was not the case. Other species belonging to the genera Zygosaccha- romyces (Z. rouxii, Z. bisporus), Zygotorulaspora (Z. mrakii), Kluyveromyces (K. aestuarii, K. nonfermen- tans, K. wickerhamii), Eremothecium (E. gossypii) and Hanseniaspora (K. lindneri) (Fig. 1, in black) were unable to grow on both rich and synthetic minimal media in anaerobic conditions, even after addition of acetoin. The ability of some species to grow under anaerobic conditions on synthetic minimal medium was also tes- ted in batch cultures. K. lactis was used as a negative control, because it was previously found to be unable to grow under these conditions [13]. The species ana- lyzed, S. castellii and C. glabrata, showed the same behavior as observed in plate experiments, and were able to grow at high specific growth rates: 00.18 h )1 and 0.2 h )1 , respectively (Fig. 2). In short, the upper five genera on the phylogenetic tree (post-genome duplication genera) showed a clear Fig. 1. Growth under strict anaerobic condi- tions: yeast species in red grow both on rich and on synthetic minimal medium within 7 days; species in blue grow on rich med- ium within 7 days and on synthetic minimal medium enriched with lysine and glutamic acid or acetoin within 14 days; species in green grow on rich medium within 14 days, but fail to grow on the synthetic minimal medium; species in black do not grow on either rich or on synthetic minimal medium. The phylogenetic tree is adapted from Kurtzman & Robnett [27]. The timing, approximately 100 million years ago, of the whole-genome duplication [29] is indicated by an arrow. A. Merico et al. Fermentative lifestyle in yeasts FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS 981 potential to grow under strictly anaerobic conditions. On the other hand, the lower genera (pre-genome duplication species) represent a mosaic of phenotypes; some species being able and others being unable to grow in the absence of oxygen. Petite generation The ability to generate respiratory-deficient mutants with grossly rearranged mtDNA molecules, sometimes referred to as ‘the petite phenotype’, has often been associated with the ability to grow anaerobically [25]. The following species, belonging to the Saccharomyces clade, have previously been studied in detail for petite generation ability and mtDNA structure: several Sac- charomyces spp. sensu stricto, Kazachstania genus (S. servazzii, S. unisporus, S. transvaalensis, S. exiguus), Naumovia genus (S. castellii and S. dairenensis), and Nakeseomyces genus (C. glabrata). They were found to be petite-positive [21,41]. On the other hand, S. kluyveri (belonging to the Lachancea genus) and K. lactis (belonging to the Kluyveromyces genus) do not easily produce viable and stable petite clones [25]. Over 30 species ⁄ strains, mainly belonging to the groups that have so far not been tested for petite generation, were analyzed in at least two independent experiments (Fig. 3). The aim of this experiment was to determine whether a certain strain ⁄ species can exist as a petite mutant (which represents a special physio- logic state) and not to study the mechanisms behind the generation of petite mutants. Kazachstania species (Arxiozyma telluris, S. transvaalensis, K. africanus, S. spencerorum, K. lodderae, K. piceae, S. barnettii and C. humilis) could all generate spontaneous respiratory- deficient colonies, and also generated petites at a high frequency when exposed to ethidium bromide (EtBr). In the Nakeseomyces genus, C. glabrata and K. bacilli- sporus generated spontaneous petites and EtBr- induced petites, but petites could not be detected in K. delphensis. In the Tetrapisispora genus, two species, T. phaffii and T. iriomotensis, were sensitive to EtBr and could therefore not be tested for petite induction, but K. blattae easily generated petites upon exposure to EtBr. T. phaffii and T. iriomotensis did not generate spontaneous petites, or induced petites at lower EtBr concentrations. The tested members of the genera Zygosaccharomyces (Z. bisporus, Z. rouxii), Zygotoru- laspora (Z. mrakii), Torulaspora (T. delbrueckii, T. glo- bosa), Lachancea (Z. fermentati, K. thermotolerans and S. kluyveri) and Kluyveromyces (K. aestuarii, K. nonfer- mentans and K. lactis) did not generate any sponta- neous or induced petites under the employed conditions, and are therefore considered to be petite- negative. However, two species, Z. florentinus and K. wickerhamii, generated petites upon prolonged exposure (10 days) to EtBr. A few K. wickerhamii petites were analyzed, and were found to contain grossly rearranged mtDNA with an elevated A + T content (data not shown). E. gossypii was very sensi- tive to EtBr, and its ability to produce petites could therefore not be tested, but spontaneous petites could not be detected. In the Hanseniospora genus, H. occi- dentalis and H. vinae did not generate petites spontane- ously or upon induction with EtBr, but H. osmophila generated petites upon prolonged exposure to EtBr. Again, post-genome duplication species, except for the Tetrapisispora group, showed an almost uniform phe- notype with regard to the ability to generate petite mutants. On the other hand, a majority of the pre-gen- ome duplication species could not generate viable petites, except for three species belonging to three dif- ferent genera (Fig. 3). Discussion The fundamental physiologic characteristics of the yeast S. cerevisiae can be summarized as the ability to: (a) degrade glucose or fructose to ethanol, even in the presence of oxygen (Crabtree effect); (b) grow in the A B Fig. 2. Anaerobic batch cultures on glucose synthetic minimal med- ium of (A) S. castellii and (B) C. glabrata : r, biomass measured as D 600 ⁄ mL; j, glucose; m, ethanol; h, glycerol. Both species show behavior similar to that of S. cerevisiae [25]. Fermentative lifestyle in yeasts A. Merico et al. 982 FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS absence of oxygen; and (c) generate respiratory- deficient mitochondrial mutants, so-called petites [42]. However, how unique are these properties among clo- sely related yeasts, and what is their origin? Recent progress in genome sequencing has elucidated phylo- genetic relationships among yeasts belonging to the Saccharomyces clade, and thereby provides a frame- work for an understanding of the evolutionary history of several modern traits. For example, the whole-gen- ome duplication took place approximately 100 million years ago in the S. cerevisiae lineage [29–31], and we can therefore talk about pre- and post-whole-genome duplication yeasts within the Saccharomyces clade. In this study, we analyzed over 40 yeasts for their ability to ferment, grow in the absence of oxygen, and gener- ate stable petites, and we attempted to determine whether these traits were expressed in the progenitor yeasts, and whether they are related to the whole- genome duplication. A good fermentative capacity is the condi- tio sine qua non for the development of the ability to grow in strictly anaerobic conditions. Under anaerobic conditions, the respiration-based biochemical pathways are shut down, and substrate-level phosphorylation is the only way for the cell to produce energy. However, homeostasis of the redox cofactors is also important for continuation of metabolic activities. Under anaer- obic conditions, yeast cells achieve such a redox bal- ance through the production of glycerol, mainly through the action of glycerol 3-phosphate dehydrogen- ase (Gpd2) [12], and through the production of succi- nate, by fumarate reductase [43]. Under these conditions, the mitochondria do not play a role in energy metabolism, but they are still essential for some Fig. 3. Distribution of petite-positive and petite-negative species in a phylogenetic tree of the Saccharomyces complex, adap- ted from Kurtzman & Robnett [27]. The examined species are indicated by different colors: red, petite-positive species; green, petite-negative species. The timing, approxi- mately 100 million years ago, of the whole- genome duplication [29] is indicated by an arrow. A. Merico et al. Fermentative lifestyle in yeasts FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS 983 assimilatory reactions, as in amino acid biosynthesis, and the generation of NADH [44]. The ability to grow in anaerobic conditions is therefore also strictly dependent on the nutritional conditions. In our experiments, all but two of the analyzed spe- cies belonging to the Saccharomyces complex could grow on rich media when mitochondrial respiration was partially impaired with antimycin A (Table 2). Thus, the progenitor of the Saccharomyces complex yeast probably had a well-developed fermentative meta- bolism, which was sufficient to support growth in the absence of oxygen. When we made the conditions more stringent, by increasing the concentration of antimy- cin A and testing on the synthetic minimal medium (Table 2), different yeast groups showed different growth properties. A high fermentative activity is, in fact, essential in this case to cope with this situation. If the fermentative activity is too low, energy problems can arise. ATP is consumed by glucose uptake in the case of yeasts having H + -symport mechanisms for glu- cose transport, and in all cases ATP is used for phos- phorylation of the hexose before ATP can be produced in later metabolism. Moreover, glycerol production leads to reduced ATP production. In these cases, the presence of alternative respiration mechanisms, such as cyanide-resistant salicyl hydroxamate-sensitive respir- ation associated with the presence of complex I, can operate and provide additional ATP when respiration is blocked by antimycin A [45]. Nevertheless, in some cases we observed that the main problem for growth, when respiration is impaired, seems to be insufficient homeostasis of redox cofactors. In these cases, the addition of a redox sink, at the cytosolic as well as at the mitochondrial level, efficiently promoted growth. This means that, in addition to high-level fermentative metabolism, efficient mechanisms to maintain redox balance are important for the ability to grow at low levels of oxygen. Among the analyzed species belonging to the genera Saccharomyces, Kazachstania, Naumovia, Nakaseomy- ces and Tetrapisispora, those that showed high resist- ance to antimycin A were also able to grow under the most stringent conditions, i.e. on the synthetic minimal medium and under strict anaerobic conditions (Table 2 and Fig. 1). Interestingly, some species, such as S. bay- anus, S. servazii, and S. barnettii, which showed severely impaired growth in aerobic conditions in the presence of antimycin A, were perfectly able to grow under strict anaerobic conditions. This seems to reflect an inhibitory effect exerted by oxygen on fermentative activity, the so-called Pasteur effect [3]. Such an inhibi- tory effect of oxygen could be a more recently acquired trait, originating independently in several yeast lineages. In contrast, whereas the upper four post-genome duplication genera generated respiratory- deficient petite mutants, Tetrapisispora exhibited a transition petite phenotype. This group deserves more study to determine the details of respiratory, fermenta- tive and mtDNA metabolism. In the other yeast groups (pre-genome duplication genera), the situation is more heterogeneous. Among the analyzed species belonging to the genera Zygotoru- laspora, Torulaspora, Lachancea and Hanseniaspora, some of those that, in aerobic conditions, showed good resistance to antimycin A were able to grow under strict anaerobic conditions, like the above-mentioned genera (Table 2 and Fig. 1). Some species belonging to the Zygosaccharomyces, Torulaspora, Kluyveromyces and Hanseniaspora groups were able to grow in anaer- obic conditions, but only on rich media, where the presence of amino acids can remedy the redox imbal- ance problems, and at low growth rates (detection requiring 14 days). Other species belonging to the gen- era Zygosaccharomyces, Zygotorulaspora, Kluyveromy- ces, Eremothecium and Hanseniaspora showed a much reduced level of resistance to antimycin A, and were quite unable to grow in anaerobic conditions, both on rich and on synthetic minimal media. In these cases, the main growth problem appeared to be lack of energy, because an insufficient amount of ATP could be generated by fermentation. This interpretation is supported by the fact that S. cerevisiae mutants in which glycolytic enzyme levels are low, such as gcr1 or gcr2, or in which hexose transport is inefficient, are sensitive to low concentrations of antimycin A and are unable to grow in anaerobic conditions [46,47]. The ability to grow in anaerobic conditions is a result of fine-tuning of several metabolic pathways. This trait is not only dependent on the presence of genes encoding specific enzyme activities; these must also be a part of a well-regulated network. The phylo- genetic tree (Fig. 1) suggests that lineages that under- went whole-genome duplication exhibit a fermentative lifestyle, the presence of the Crabtree effect, the ability to grow without oxygen, and the ability to generate petites (Table 1, Figs 1 and 3). Whereas a majority of pre-genome duplication species showed a reduced Crabtree effect, could not generate viable petite mutants, and needed some oxygen for their growth, some lineages exhibited similar traits as the post-gen- ome duplication lineage (Fig. 4). However, it should be noted that none of the pre-genome duplication spe- cies had all these traits expressed to the same quantita- tive level as the post-genome duplication species. The presence of these traits in at least one species in each genus suggests that the Saccharomyces complex Fermentative lifestyle in yeasts A. Merico et al. 984 FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS progenitor had the basic capacity to ferment, and this was probably an adaptation to an environment with a low oxygen concentration. The mosaic distribution of the studied phenotypes in the phylogenetic tree may, then, reflect independent adaptations to changes in environmental conditions that occurred many millions of years ago. The end of the Cretaceous period provi- ded an excess of fruits, and thereby increased amounts of sugars. Different lineages of yeast, able to ferment, entered into a fierce competition for these sugars with different bacteria. The independence from oxygen and the ability to generate spontaneous petites, which can only ferment and therefore produce ethanol, were likely to strengthen the competitive advantages of yeast. Horizontal transfer of bacterial genes could also have contributed to the increase in level of oxygen independence [48]. The ability to accumulate ethanol in the presence of oxygen was exploited by several yeasts as an additional weapon to inhibit the growth of other microbes. The appearance of an elevated fre- quency of spontaneous petites helped to increase the production of ethanol. However, other evolutionary strategies could also have contributed to the evolution of these traits in yeasts [49,50]. Alternatively, it could be that the progenitor was already Crabtree-positive, petite-positive and able to grow without oxygen, but these properties were later independently lost in several pre-genome duplication lineages. However, it is difficult to find a rationale for this and imagine environmental conditions that would promote this evolutionary scenario. Experimental procedures Yeast strains The yeast species analyzed in this study belong to the Saccharomyces complex described by Kurtzman & Robnett [27]. Most of these strains were kindly provided by C. Kurtzman (Microbial Genomics and Bioprocessing Research Unit, US Department of Agriculture, Peoria, IL, USA). A majority of the studied species are represented by their type strains: A. telluris NRRL-YB-4302 (CBS 2685), C. glabrata NRRL-Y-65 (CBS 138), C. humilis NRRL- Y-17074 (CBS 5658), H. guillermondii NRRL-Y-1625 (CBS 465), H. occidentalis NRRL-Y-7946 (CBS 2592), H. osmophila NRRL-Y-1613 (CBS 313), H. valbyensis NRRL-Y-1626 (CBS 479), H. vineae NRRL-Y-17529 (CBS 2171), Klo. lindneri NRRL-Y-17531 (CBS 285), K. aestuarii NRRL-YB-4510 (CBS 4438), K. africanus NRRL-Y-8276 (CBS 2517), K. bacillisporus NRRL-Y-17846 (CBS 7720), K. blattae NRRL-Y-10934 (CBS 6284), K. del- phensis NRRL-Y-2379 (CBS 2170), K. lodderae NRRL-Y- 8280 (CBS 2757), K. marxianus NRRL-Y-8281 (CBS 712), K. nonfermentans NRRL-Y-27343 (JCM 10232), K. piceae NRRL-Y-17977 (CBS 7738), K. thermotolerans NRRL-Y- 8284 (CBS 6340), K. waltii NRRL-Y-8285 (CBS 6430), K. wickerhamii NRRL-Y-8286 (CBS 2745), S. barnettii NRRL-Y-27223 (CBS 6946), S. bayanus NRRL-Y-12624 (CBS 380), S. castellii NRRL-Y-12630 (CBS 4309), S. dairensis NRRL-Y-12639 (CBS 421), S. exiguus NRRL-Y-12640 (CBS 379), S. kluyveri NRRL-Y-12651 (CBS 3082), S. paradoxus NRRL-Y-17217 (CBS 432), S. pastorianus NRRL-Y-27171 (CBS 1538), S. servazii Fig. 4. A simple phylogenetic relationship between the yeasts analyzed in aerobic batch cultures is shown, and the size of their Crabtree effect is quantified as the yields of biomass in relation to consumed glucose (in brackets, gÆg )1 ). The S. cerevisiae and K. lactis lineages separated more than 100 million years ago; the S. cerevisiae and S. pombe lineages separated more than 200 million years ago. The timing, approxi- mately 100 million years ago, of the whole- genome duplication [29] is indicated by an arrow. A. Merico et al. Fermentative lifestyle in yeasts FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS 985 [...]... performed in the same synthetic minimal medium as described above, but supplemented with ergosterol (10 mgÆL)1), Tween-80 (420 mgÆL)1) and uracil (50 mgÆL)1) The bioreactor was continuously flushed with N2 (containing less than 3 p.p.m O2) at a flow rate of 0.1 LÆmin)1 per liter of medium, and a stir rate of 500 r.p.m was maintained In order to minimize the diffusion of oxygen into the bioreactor, Norprene... replica plated onto glycerol medium (GlyYP) [31] Growth on this medium requires respiration The respiratory potential of the putative mutants was tested on the YPD plates using the tetrazolium method [34] The color of tetrazolium in the medium ⁄ colony changes from white to red if an active respiratory chain is present in the colony In some cases, mtDNA was isolated from petite strains using a CsCl centrifugation-based... BM & Compagno C (2003) Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii¢ FEMS Yeast Res 4, 277–283 38 Kaniuga Z, Bryla J & Slater EC (1969) Inhibitors around the antimycin-sensitive site in the respiratory chain In Inhibitors ) Tools in Cell Research (Bucher Th & Sies H, eds), pp 282–300 Springer, Berlin 39 Gonzalez E, Fernandez MR, Larroy C, Sola L, Pericas MA, Pares X & Biosca... exponential phase of growth as the amount (millimoles) of glucose consumed, and ethanol produced, divided by the corresponding amount of the produced biomass (grams of dry weight) and multiplied by the corresponding specific rates of growth (increase of biomass per hour) Fermentative lifestyle in yeasts 2 3 4 5 6 7 Respiratory-deficient strains Yeast strains were grown in liquid YPD medium until late... added to a final concentration of 0.01 mgÆmL)1 In some cases, lower concentrations of EtBr were employed The cultures were incubated for 2–10 days, washed and diluted, and spread on the petite screening plates The plates were incubated for 1–2 weeks, and examined for the presence of small colonies, representing putative respiratory-deficient mutants The obtained small colonies were transferred to YPD... 239– 245 46 Sasaki H & Uemura H (2005) In uence of low glycolytic activities in gcr1 and gcr2 mutants on the expression of other metabolic pathway genes in Saccharomyces cerevisiae Yeast 22, 111–127 Fermentative lifestyle in yeasts 47 Otterstedt K, Larsson C, Bill RM, Stahlberg A, Boles E, Hohmann S & Gustafsson L (2004) Switching the mode of metabolism in the yeast Saccharomyces cerevisiae EMBO Reports... seeded onto the petite screening plates, containing GGlyYP (peptone, 1% w ⁄ v; yeast extract, 0.1% w ⁄ v; glycerol, 2% w ⁄ v; glucose, 0.1% w ⁄ v) [33], to determine the appearance and frequency of spontaneous petites GGlyYP plates contain only a limited amount of a fermentable carbon source, whereby colony size strongly depends on respiratory competence To look for induced petites, we diluted the culture... were performed in a Biostat-Qsystem (B-Braun; Sartorius BBI Systems Inc., Bethlehem, PA) with a working volume of 0.8 L An air flow of 1 LÆmin)1 and a stirrer speed from 800 to 1400 r.p.m maintained a dissolved oxygen concentration above 30% of air saturation The temperature was kept at 30 °C and the pH at 5.0 by automatic addition of 2 m KOH Cells were precultured on the defined synthetic minimal medium... 0.08 mgÆL)1 of d-(–)-biotin, 1.5 mgÆL)1 of calcium d-(+)-panthotenate, 1.5 mgÆL)1 of nicotinic acid, 37.5 mgÆL)1 of myoinositol, 1.5 mgÆL)1 of thiamine hydrochloride, 1.5 mgÆL)1 of pyridoxol hydrochloride, and 0.3 mgÆL)1 of p-aminobenzoic acid The cell biomass was washed and used to inoculate batch cultures onto the same synthetic minimal medium Batch experiments were performed in duplicate Anaerobic... Genomic analysis of anaerobically induced genes in Saccharomyces cerevisiae: functionsl roles of Rox1 and other factors in mediating the anoxic response J Bacteriol 184, 250–265 18 Ephrussi B, Hottinguer H & Chimenes AM (1949) Action de l’acriflavine sur les levures I La mutation ‘petite colonie’ Ann Inst Pasteur 76, 351–357 19 Pisˇ kur J (1994) Inheritance of the yeast mitochondrial genome Plasmid 31, . due to amino acid biosynthesis. Much of the generation of NADH during amino acid biosyn- thesis takes place in the mitochondria. Because of the block in the. limited. These data seem to indicate that most of the species belonging to the Saccharomyces complex possess a good fermentative capacity, being able to generate