Tài liệu Báo cáo khoa học: Fermentative lifestyle in yeasts belonging to the Saccharomyces complex ppt

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:
(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
NRRL-Y-12661 (CBS 4311), S. spencerorum NRRL-Y-17920
(CBS 3019), S. transvaalensis NRRL-Y-17245 (2186), S. uni-
sporus NRRL-Y-1556 (CBS 398), T. iriomotensis NRRL-Y-
27309 (IFO 10929), T. phaffii NRRL-Y-8282 (CBS 4417),
T. delbrueckii NRRL-Y-866 (CBS 1146), T. franciscae
NRRL-Y-17532 (CBS 2926), T. globosa NRRL-Y-12650
(CBS 764), T. pretoriensis NRRL-Y-17251 (CBS 2187),
Z. bisporus NRRL-12626 (CBS 702), Z. florentinus NRRL-
Y-1560 (CBS 746), Z. microellipsoides NRRL-Y-1549
(CBS 427), and Z. rouxii NRRL-Y-229 (CBS 732).
Other species are: a nonfilamentous isolate of E. gossypii,
Y999 (J. Piskur Collection, Lund, Sweden), K. lactis
CBS 2359, S. cerevisiae CEN.PK 113-7D, Z. bailii
ATCC 36947, Z. fermentati CBS 4506, and Z. mrakii
CBS 4219.
Cultivation on agar plates
Frozen cultures were stored at ) 80 °C in 15% glycerol.
Cells were precultured on YPD medium (peptone, 2% w ⁄ v;
yeast extract, 1% w ⁄ v; glucose, 2% w ⁄ v) to the exponential
growth phase, washed, and suspended in distilled water at a
concentration of 10
5
cellsÆmL

)1
. Five-microliter amounts of
this suspension (about 500 cells) were spotted onto different
plates and incubated at 30 °C for a minimum of 7 days to
a maximum of 14 days.
Aerobic culture plates included the following media:
YPD, YPD supplemented with antimycin A (5 lm), SD
medium (yeast nitrogen base, 6.7 gÆL
)1
; glucose, 2% w ⁄ v),
and SD medium supplemented with antimycin A at various
concentrations (0.5, 1, 2, 4, 5, 10, 15, 20 and 25 lm). In this
case, uracil (50 mgÆL
)1
) was also added, because in some
yeasts, de novo pyrimidine biosynthesis is coupled to the
respiratory chain [8]. When specified in the text, lysine and
glutamic acid (both at a concentration of 120 mgÆ L
)1
)or
acetoin (0.6% w ⁄ v) were also added.
Anaerobic culture plates included YPD and SD medium,
both supplemented with antimycin A (5 lm), ergosterol
(10 mgÆL
)1
), Tween-80 (420 mgÆL
)1
) and, in the case of SD,
also uracil (50 mgÆL
)1

). When specified in the text, lysine
and glutamic acid (both at a concentration of 120 mgÆL
)1
)
or acetoin (0.6% w ⁄ v) were added as well.
The anaerobic milieu was obtained through the Anaero-
cult A system and controlled through the Anaerotest strips
(cat. no. 1138290001 and 115112, respectively; Merck, VWR
International, Milano, Italy). Anaerocult A is a sachet con-
taining components that chemically bind oxygen quickly and
completely in the presence of water, thus creating an oxygen-
free milieu and a CO
2
-rich atmosphere. Inoculated plates are
placed in a sealed jar together with the moist Anaerocult A
sachet and a test strip that indicates the anaerobic atmo-
sphere by a color change from blue to white within 4 h.
A. telluris, E. gossypii, K. africanus and S. transvaalensis
showed impaired growth on SD medium. Only for these
strains was the SD medium enriched with peptone at a final
concentration of 0.05% w ⁄ v. All yeast species were tested at
least twice for their growth under all conditions.
Batch cultivations
Aerobic batch cultivations were performed in a Biostat-Q-
system (B-Braun; Sartorius BBI Systems Inc., Bethlehem,
PA) with a working volume of 0.8 L. An air flow of
1LÆmin
)1
and a stirrer speed from 800 to 1400 r.p.m. main-
tained 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 pre-
cultured on the defined synthetic minimal medium des-
cribed in Verduyn et al. [32]. The final concentrations of
the components of the medium were as follows: 20.0 gÆL
)1
of glucose, 5.0 gÆL
)1
of ammonium sulfate, 3.0 gÆL
)1
of
potassium dihydrogen phosphate, 0.5 gÆL
)1
of magnesium
sulfate heptahydrate, 22.5 mgÆL
)1
of EDTA, 6.75 mgÆL
)1
of
zinc sulfate heptahydrate, 1.5 mgÆL
)1
of manganese chloride
tetrahydrate, 0.45 mgÆL
)1
of cobalt(II) chloride hexahy-
drate, 0.45 mgÆL
)1
of copper(II) sulfate pentahydrate,
0.60 mgÆL
)1

of disodium molybdate dihydrate, 6.75 mgÆL
)1
of calcium chloride dihydrate, 4.5 mgÆL
)1
of iron sulfate
heptahydrate, 1.5 mgÆL
)1
of boric acid, 0.15 mgÆL
)1
of
potassium iodide, 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 hydro-
chloride, 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 per-
formed in duplicate.
Anaerobic batch cultivations were performed in the same
synthetic minimal medium as described above, but supple-
mented with ergosterol (10 mgÆL
)1
), Tween-80 (420 mgÆL
)1
)
and uracil (50 mgÆL
)1
). The bioreactor was continuously
flushed with N
2
(containing less than 3 p.p.m. O
2
) 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 diffu-
sion of oxygen into the bioreactor, Norprene tubing
(Cole-Palmer, General Control, Milan, Italy) was used
throughout the setup.
Extracellular metabolites and dry weight
Samples were quickly withdrawn from batch cultures dur-
ing exponential growth at appropriate intervals. The con-
centrations of glucose and ethanol in supernatants were
determined with enzymatic kits (cat. no. 10716251 and
10176290, respectively; Roche, R-Biopharm Italia, Milan,

Italy). All samples were analyzed in triplicate, and the
standard deviation varied between 1% and 2%. For dry
weight determinations, washed culture samples were filtered
on a 0.45 lm glass microfiber GF ⁄ A filter (Whatman,
Fermentative lifestyle in yeasts A. Merico et al.
986 FEBS Journal 274 (2007) 976–989 ª 2007 The Authors Journal compilation ª 2007 FEBS
Biomap, Milano, Italy) and dried for 24 h at 80 °C. Parallel
samples varied by about 3–5%.
Yields of biomass and ethanol relative to consumed
glucose (fructose) were calculated during the glucose
consumption phase of growth as the highest production lev-
els (grams) of biomass and ethanol, respectively, divided by
the corresponding amount of consumed glucose (grams).
Specific consumption rates of glucose (fructose) [q
Glu (Frt)
]
and specific production rates of ethanol (q
EtOH
) were calcu-
lated during the 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 corres-
ponding specific rates of growth (increase of biomass per
hour).
Respiratory-deficient strains
Yeast strains were grown in liquid YPD medium until late
stationary phase, and 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 fermenta-
ble carbon source, whereby colony size strongly depends on
respiratory competence. To look for induced petites, we dilu-
ted the culture 100 times with fresh YPD, and EtBr was
added to a final concentration of 0.01 mgÆmL
)1
. In some
cases, lower concentrations of EtBr were employed. The cul-
tures were incubated for 2–10 days, washed and diluted, and
spread on the petite screening plates. The plates were incuba-
ted 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 plates
and, if viable, replica plated onto glycerol medium (GlyYP)
[31]. Growth on this medium requires respiration. The respir-
atory 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 method and analyzed by restriction
enzyme digestion as previously described [21].
Acknowledgements
We thank M. Kielland-Brandt for critical reading of
the manuscript and K. Wolfe for his useful comments
on our work.
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