Carbohydrate and Energy-Yielding Metabolism in Non-Conventional Yeasts

Carbohydrate and energy-yielding metabolism in non-conventional
yeasts
1
Carmen-Lisset Flores
2
, Cristina Rodr| guez, Thomas Petit
3
, Carlos Gancedo *
Instituto de Investigaciones Biomedicas Alberto Sols C.S.I.C.-UAM, Unidad de Bioqu| mica y Genetica de Levaduras, 28029 Madrid, Spain
Received 3 April 2000; received in revised form 13 June 2000; accepted 14 June 2000
Abstract
Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of
sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the
pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most
non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review
provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts:
glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and,
briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and
Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many
components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be
important to generate genetically engineered yeast strains for certain industrial uses. 2000 Federation of European Microbiological
Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: Non-conventional yeast; Carbohydrate metabolism; Respiration; Tricarboxylic acid cycle; Catabolite repression
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
2. Uptake of sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
2.1. Common hexoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
2.2. Other sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
2.3. Disaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
3. The common glycolytic trunk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
3.1. From intracellular sugar to trioses phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
3.2. From trioses phosphate to pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
4. Metabolic alternatives of intracellular pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
4.1. Pyruvate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
4.2. Pyruvate decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
4.3. The pyruvate bypass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
5. The TCA cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
5.1. Anaplerotic reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
6. Oxidation of NADH and energy generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
7. The pentose phosphate pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
7.1. The genes of xylose metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
0168-6445 / 00 / $20.00 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 0 0 ) 0 0 0 3 7 - 1
* Corresponding author. Fax: +34 (91) 585 4587; E-mail : cgancedo@iib.uam.es
1
This article is dedicated to the memory of Niko van Uden, an enthusiastic yeast researcher and a cultivated person.
2
On leave of absence from Departamento de Bioqu| mica, Facultad de Biolog| a, Universidad de La Habana, Cuba.
3
Present address: DSM Bakery Ingredient Division, 26000 MA Delft, The Netherlands.
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FEMS Microbiology Reviews 24 (2000) 507^529
www.fems-microbiology.org
8. Ethanol metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
9. Glycerol metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
10. Gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
11. Catabolite repression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
12. Plasma membrane H
-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
13. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
1. Introduction
Yeasts are microorganisms for which we have some of
the oldest bibliographic references. Already in Exodus 12
and 13 there are mentions of leaven, likely a mixture of
dierent yeast species and bacteria similar to that found
nowadays in those rare places where bread is still baked by
truly artisanal means [1,2]. For historical reasons, one
yeast species, Saccharomyces cerevisiae, has dominated
the scientic stage and has become a synonym for yeast.
However, S. cerevisiae is but one among the almost 800
yeast species described in a recent taxonomy treatise [3]. It
is logical to think that many of these dierent species may
also be interesting for a variety of reasons ranging from
their use in specic technological applications to the threat
of the infections caused by some of them. It is perhaps
time for zymologists to dedicate more attention to those
non-conventional yeasts (NCY) or non-Saccharomyces
yeasts. In fact, some species have already attracted re-
searchers in the last years on dierent grounds: Kluyver-
omyces lactis as a possible utilizer of the residual whey in
dairy industries, some methylotrophic yeasts for their e-
ciency as hosts for the production of heterologous proteins
or for their special qualities for the study of peroxisome
biogenesis, Yarrowia lipolytica for its ability to grow on
particular substrates and its high protein excretion ca-
pacity, not to mention Candida albicans or Criptococcus
neoformans due to their relevance in some health issues.
In spite of the importance of this group of yeasts, basic
knowledge about enzymes of fundamental pathways, their
regulation and the genes encoding them is relatively
scarce.
Sugars are utilized by all known yeasts. The common
theme in their metabolism is the conversion of glucose 6-
phosphate or fructose 6-phosphate to pyruvate through
the common glycolytic trunk (Fig. 1). The metabolic des-
tiny of pyruvate is then dierent depending on the yeast
species and the cultivation conditions. In S. cerevisiae (the
conventional yeast) glucose and related sugars cause a
strong impairment in respiratory capacity (Crabtree eect)
[4,5] and therefore, even in the presence of air, S. cerevisiae
ferments sugar in batch cultures. Some other yeasts, so-
called `Crabtree positive', also behave in this way. How-
ever, in most cases in aerobic conditions, pyruvate is oxi-
dized to CO
2
through the tricarboxylic acid (TCA) cycle.
Glycolysis and the TCA cycle are therefore central path-
ways of metabolism. They perform a dual role in organ-
isms: to produce energy and reducing equivalents in the
form of ATP, NADH or NADPH and to provide building
blocks to synthesize other biomolecules.
The purpose of this review is to survey the existing
knowledge on these pathways and related ones in some
NCY and contrast it with what is established in S. cere-
visiae. It will become apparent that, in a great number of
cases, the absence of information is the dominant note and
we hope that this awareness will provide an incitation to
ll in the corresponding gaps. Detailed reviews concerning
dierent aspects of carbohydrate and energy metabolism
in S. cerevisiae are available to which the reader may use-
fully refer [6^9].
We have restricted this review to a number of species of
the genera Kluyveromyces, Pichia, Candida, Yarrowia and
Schizosaccharomyces, although occasional references to
other genera will be made. This choice was dictated by
the already well-established importance of species of these
genera in some area of research or technology. To place
Schizosaccharomyces among the non-conventional yeasts
may be a matter of opinion; researchers working on cell
C
Fig. 1. Scheme of dierent pathways implicated in carbon and energy metabolism in yeasts. The gure represents an hypothetical yeast cell with features
from S. cerevisiae and other yeasts. The thick line with black dots represents the common glycolytic trunk and the TCA cycle. Dashed lines denote re-
actions that are used during growth in non-fermentable carbon sources. Lines with `dots and dashes' indicate that, depending of the yeast genera, some
of the disaccharides represented may be either hydrolyzed outside the cell or transported as such into it and hydrolyzed in the cytoplasm. Circles in
membranes indicate transport systems or proteins located in such structures. Lines going through the mithochondrial membrane do not necessarily im-
plicate that the corresponding metabolite freely crosses it. The malic enzyme is not represented. No organelles besides the mitochondrion are drawn.
The following non-standard abbreviations in pathways dierent from glycolysis have been used: 6PG, 6-phosphogluconate; Rul5P, ribulose 5-phos-
phate; R5P, ribose 5-phosphate; Xul5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; G3P, glycerol 3-phos-
phate; P
i
, ìnorganic' phosphate; OAA, oxaloacetate; Q, ubiquinone; bc1, cytochromes; Cyt ox, cytochrome oxidase.
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C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529 508
FEMSRE 689 29-8-00
cycle or morphogenesis may consider it as quite conven-
tional. Some background information and a treatment of
several aspects of cultivation, and molecular biology tech-
niques pertaining to certain NCY may be found in [10].
2. Uptake of sugars
``Don't talk to me of permeability, this is the last resort of
the biochemist who cannot nd any better explanation'' M.
Stephenson (quoted in [11])
``The realization that the membranes are not sievelike
bags but barriers to a large extent impermeable to glucose,
makes it obvious that some sort of mechanism is likely to
intervene to enable an adequate supply of glucose to reach
the intracellular glycolytic machinery'' A. Sols [12]
2.1. Common hexoses
Cell membranes are not freely permeable for a variety of
solutes, sugars among them. Therefore, transport is the
rst step in carbohydrate metabolism, except in those
cases in which a di- or tri-saccharide is hydrolyzed outside
the cell. Transport across the membrane is carried out by
specic transporters, sometimes called permeases. In S.
cerevisiae, a family of glucose transporters with K
m
rang-
ing from 1 to 100 mM, has been characterized. The tran-
scription of the genes encoding these transporters is nely
regulated by the glucose concentration of the medium (for
reviews see [13,14]. Transport of the common monosac-
charides, glucose, fructose or mannose in S. cerevisiae is a
facilitated diusion process; however, the situation may be
dierent in other yeasts.
In K. lactis glucose transport appears to proceed by
facilitated diusion and two genes encoding transporters
with dierent anities for glucose have been identied in
this yeast: HGT1 that encodes a high anity glucose
transporter, K
m
1 mM, [15] and RAG1 that encodes a
transporter with low anity for glucose, K
m
20^50 mM
[16]. Overexpression of HGT1 could not restore the wild-
type phenotype to a rag1 mutant indicating that HGT1
and RAG1 have distinct functions in glucose transport
[15]. Wild-type strains of K. lactis are divided into two
groups with regard to their dierent sensitivity to the res-
piratory inhibitor antimicyn A: those that grow in glucose
in the presence of the inhibitor are called Rag
(resistance
to antimycin on glucose) and those that do not, are termed
Rag
3
. Wild-type Rag
strains possess RAG1 and HTG1,

while Rag
3
strains have lost RAG1 [17]. RAG1 seems to
have arisen by a recombination event between two genes,
KHT1 and KHT2, that encode low anity glucose trans-
porters and are found in tandem in some strains. The se-
quence of Rag1 is identical to that of Kht1 except for an
arginine and a phenylalanine, the last two C-terminal ami-
no acids, which are exchanged for the last three amino
acids of Kht2 (alanine^methionine^leucine) [18].
Expression of HGT1 does not vary with culture condi-
tions [15] while transcription of RAG1 that is undetectable
during growth in glycerol increases during growth in glu-
cose [17]. It is not known which signal(s) trigger the in-
duction of RAG1 expression [19], but an extended glycol-
ysis does not seem to be required since a rag2 mutant,
defective in phosphoglucose isomerase did not show de-
creased levels of RAG1 mRNA [20,21]. Several genes aect
the expression of RAG1 and HGT1. Mutations in RAG5
encoding the unique hexokinase of K. lactis [22] abolish
expression of RAG1 [19] and decrease the level of the 2.0-
kb mRNA species of HGT1 [15]. Mutations in RAG4, a
gene of unknown function, eliminated transcription of
RAG1 [19] but increased the 2.0-kb mRNA species of
HGT1 [15]. A rag4 rag5 double mutant showed a level
of HGT1 transcript similar to that of the wild-type, sug-
gesting that RAG4 is epistatic over RAG5 for the regula-
tion of the transcription of HGT1 [15]. The RAG8 gene, an
apparently essential gene that shows a high degree of iden-
tity with the two casein kinases I of S. cerevisiae [23], also
abolished expression of RAG1 [19], but did not aect that
of HGT1 [15]. These results show that dierent controls
distinctly aect the transcription of both genes. RAG1 ex-
pression was unaected in a hgt1 null mutant and vice
versa. A double mutant rag1 hgt1 showed a greatly re-
duced level of glucose uptake, but still grew in glucose,
although it stopped growth earlier than the wild-type
[15]. It seems, therefore, that some alternative system
with low glucose anity is able to transport this sugar
in the double mutant.
A gene from C. albicans that restores growth in glucose
of a S. cerevisiae strain deleted in the seven physiologically
relevant glucose transporters has been isolated and named
CaHGT1. The gene encodes a protein of 545 amino acids
with 12 transmembrane domains and a 51% identity with
the K. lactis glucose transporter, Hgt1. The transcript lev-
els of CaHGT1 were enhanced in response to a variety of
drugs suggesting an implication of the encoded protein in
drug resistance [24].
In Candida utilis, the popular `fodder yeast', glucose
appears to be transported by a proton symport when the
organism is grown at low glucose concentration [25,26],
but a facilitated diusion operates when the cells are
grown at higher glucose concentrations [25].
Weierstall et al. [27] isolated three genes encoding glu-
cose transporters from the xylose fermenting yeast Pichia
stipitis with K
m
values around 1 mM. Expression of one of
these genes, SUT1, was induced by glucose while the ex-
pression of the others, SUT2 and SUT3, was constitutive.
A transport system for glucose with two components
with K
m
3 and 10 mM respectively, was found in Y. lipo-
lytica. These components were present with the same ac-
tivity independently of the glucose concentration in the
medium [28].
In Schizosaccharomyces pombe Ho fer and Nassar [29]
identied hexose transport as a H
-symport ; since then

FEMSRE 689 29-8-00
a family of hexose transporters has been identied with six
components called Ght1 to Ght6 [30]. Ght5 is the most
abundant of these transporters. Ght1, Ght2 and Ght5 ex-
hibited higher activities with glucose than with fructose as
substrate [30]. Mehta et al. [31] isolated two S. pombe
mutants decient in glucose transport; one of them has,
apparently, an altered anity for glucose and both present
defects in the regulation of the expression of the enzymes
of the pentose phosphate pathway. No information is
available on the gene(s) aected in these mutants.
2.2. Other sugars
Galactose seems to be transported in K. lactis by the
same protein that transports lactose, since mutations in
the LAC12 gene encoding the lactose transporter abolish
growth both in lactose and galactose [32].
S. pombe is able to grow on gluconate and -much better
^ in N-gluconolactone. Gluconate is taken up by a proton
motive-force driven transport [33]. The transporter protein
Ght3 (see above) is the one implicated in gluconate trans-
port [30]. Transport of gluconate is repressed by glucose
and its derepression seems dependent on the activity of a
gene named gti1
[34]. Overexpression of this gene short-

ened the time of derepression and increased the transport
activity with respect to the wild-type. Glucose inactivates
rapidly and reversibly the transport in a process that im-
plicates cAMP [35].
2.3. Disaccharides
Metabolism of disaccharides is preceded by a hydrolysis
to their monomers. Depending on the yeasts species, and
the nature of the sugar, this hydrolysis may occur outside
the cell membrane, in the periplasmic space, or inside the
cell after the transport of the disaccharide.
Sucrose is hydrolyzed in most cases by an external in-
vertase repressed by glucose. In K. lactis, the gene KlINV1
encoding invertase has been isolated. It presents high ami-
no acid homology with the invertase from S. cerevisiae
and with the inulinase ^ a fructofuranosidase that hydro-
lyzes sucrose ^ from Kluyveromyces marxianus [36]. Genes
encoding periplasmic invertases have been isolated from
C. utilis, [37] and S. pombe [38].
In C. albicans, sucrose is transported into the cell by a
sucrose inducible H
-symport [39] and hydrolyzed by an

intracellular K-glucosidase, also able to hydrolyze maltose
[39]. This enzyme is encoded by the CaMAL2 gene [39]
whose expression is induced by sucrose and maltose and
repressed by glucose [40]. While no maltose transport has
yet been described in C. albicans, maltose is transported in
C. utilis by a proton symport as in S. cerevisiae [26]. In
contrast, in S. pombe, maltose is hydrolyzed by a external
maltase; details on this enzyme are not available [31].
Lactose uptake in K. lactis occurs by an inducible active
transport system [41], encoded by the LAC12 gene [42]
whose expression is induced by lactose or galactose [32].
Once transported, lactose is hydrolyzed by a L-galactosi-
dase, also induced by lactose, encoded by the LAC4 gene
[43].
3. The common glycolytic trunk
3.1. From intracellular sugar to trioses phosphate
The intracellular hexoses enter the glycolytic pathway
after a phosphorylation step. Glucose, fructose and man-
nose are phosphorylated by hexokinases; a glucokinase
can phosphorylate glucose and mannose and a galactoki-
nase phosphorylates galactose. The enzymatic equipment
for hexose phosphorylation varies among dierent yeasts,
although no physiological explanation for the dierences
has been found. While S. cerevisiae possesses two hexoki-
nases and a glucokinase [44], K. lactis has only one hexo-
kinase encoded by the RAG5 gene. rag5 mutants are not
only unable to grow in glucose or fructose, but also un-
expectedly, in lactose [22]. While the transcription rates of
the genes HXK1, HXK2 and GLK1 that encode the hex-
okinases and the glucokinase of S. cerevisiae are con-
trolled by glucose [45], the levels of RAG5 mRNA did
not vary signicantly between cultures in glucose or in
glycerol [22].
In Candida tropicalis, two hexokinases and a glucoki-
nase were found in chromatographic studies. The levels
of the hexokinases were higher when the yeast was cul-
tured in glucose or fructose than when grown in alkanes
[46]. In Y. lipolytica a hexokinase and a glucokinase were
detected [46,47]. The gene encoding the hexokinase has an
intron of 436 bp located 39 bp after the adenine of the rst
ATG [47]. Taking into account the kinetic characteristics
of both enzymes and the fact that disruption of the
YlHXK1 gene does not signicantly aect growth in glu-
cose, it has been suggested that the major glucose phos-
phorylating capacity in vivo is due to glucokinase [47].
Two hexokinases are found in S. pombe, a major one,
hexokinase 2, with a K
m
for glucose around 0.1 mM, the
usual range for hexokinases, and a minor one, hexokinase
1, that has a low anity for glucose (K
m
9 mM) [48].
Hexokinases and glucokinase show, in general, extensive
homology in sequence [49] ; however, hexokinase 1 from S.
pombe is peculiar in having a serine in the glucose binding
site at a position where an asparagine is usually present.
The low anity for glucose of this enzyme is due, in part,
to this substitution [50]. The physiological role of this
minor hexokinase is not clear.
In all cases studied, the genes encoding hexokinases
from NCY were able to complement the hxk2 mutation
from S. cerevisiae [22,47,48].
Most hexokinases are inhibited by trehalose-6-P, a com-
pound that plays an important role in the regulation of
glycolysis in S. cerevisiae [51]. The hexokinase of Y. lipo-
FEMSRE 689 29-8-00
lytica shows the strongest inhibition by trehalose-6-P yet
found (K
i
3.6 mM) and has been used in a method for the
quantitative determination of this compound [52]. The
hexokinases from S. pombe are however, peculiar with
regard to the inhibition by trehalose-6-P: hexokinase 2 is
not inhibited, and hexokinase 1 is only poorly inhibited
[48]. Apparently the regulation of glycolysis in this yeast
has dierent characteristics from that of S. cerevisiae.
While it is known that hexokinase 2 from S. cerevisiae
plays a role in the catabolite repression of certain genes,
not much information on the participation of hexokinases
on catabolite repression in NCY is available (see Section
11).
Phosphoglucose isomerase converts glucose-6-P into
fructose-6-P. In K. lactis the gene RAG2 encodes phospho-
glucose isomerase. A K. lactis rag2 mutant is able to grow
in glucose, but does not produce ethanol and this growth
is dependent upon an active respiration [20]. This behavior
contrasts with the lack of growth in glucose found in the
equivalent pgi mutant in S. cerevisiae [53] and indicates
that in K. lactis, the capacity of the pentose phosphate
pathway is enough for glucose utilization. Although Gof-
frini et al. [20] reported that the rag2 mutant did not
produce ethanol during growth in glucose, Gonzalez-Siso
et al. [54] detected a signicant ethanol production and
postulated that the dependence on respiratory activity
for growth in glucose of the rag2 mutant is related to
the need of function of the external mitochondrial
NAD(P)H dehydrogenase for reoxidation of the NADPH
produced in the hexosephosphate pathway.
Phosphofructokinase catalyzes the phosphorylation of
fructose-6-P to fructose-1,6-P
2
. The activity of the enzyme
is aected by a large variety of eectors and has been the
paradigm for `multimodulated enzymes' [55]. Phosphofruc-
tokinase received much attention in S. cerevisiae because it
was thought to be the `bottleneck' of glycolysis. This view
has been weakened by the nding that overexpression of
the genes encoding phosphofructokinase did not increase
fermentation in S. cerevisiae [56,57] and by the realization
that the control of a pathway may be rarely ascribed to a
single step [58]. In all yeast species in which the genetics of
phosphofructokinase have been studied, two genes have
been found. In K. lactis the enzyme is an octamer com-
posed of two dierent types of subunits of molecular
masses 119 and 102 kDa [59] encoded by the genes
KlPFK1 and KlPFK2 [60]. Mutants lacking KlPFK1 or
KlPFK2 grew in glucose, although no phosphofructoki-
nase activity was detected in vitro, a behavior similar to
that observed in S. cerevisiae [61]. However, in contrast
with the behavior of pfk1 pfk2 mutants in S. cerevisiae,
Klpfk1 pfk2 mutants that lack both subunits of phospho-
fructokinase still grow in glucose if respiration is function-
al, indicating again that the activity of the pentose phos-
phate is enough for glucose utilization (see behavior of the
rag2 mutant). This view is strengthened by the fact that a
disruption of the gene KlTAL1, that encodes a transaldo-
lase, abolishes this growth [62] (see also Section 7). Either
KlPFK1 or KlPFK2 can restore the ability to grow in
glucose to pfk1 pfk2 mutants of S. cerevisiae. Simultane-
ous expression of one of the PFK genes from K. lactis and
one from S. cerevisiae did not produce an enzyme with
activity in an in vitro assay [60]. The K. lactis enzyme
did not exhibit cooperativity towards fructose-6-P, in con-
trast with that of S. cerevisiae, but was activated by fruc-
tose-2,6-P
2
and AMP and inhibited by ATP. Both com-
pounds were equally eective in releasing the ATP
inhibition, a dierence with S. cerevisiae where fructose-
2,6-P
2
is the most eective one [59].
Lorberg et al. [63] cloned two genes, CaPFK1 and
CaPFK2, from the pathogenic yeast C. albicans. They en-
code proteins of 109- and 104-kDa, respectively, either of
which can complement the pfk1 pfk2 phenotype of S. ce-
revisiae mutants. An interesting feature in the structure of
the C. albicans phosphofructokinases is the existence of N-
terminal tails of about 180 amino acids that have no coun-
terpart in other phosphofructokinases. The functional im-
plications of these extensions are unknown. In contrast
with the absence of enzymatic activity observed when
PFK genes from K. lactis and of S. cerevisiae were simul-
taneously expressed, the hetero-oligomers formed when
PFK genes from C. albicans and S. cerevisiae were ex-
pressed, produced an enzyme with detectable activity in
the in vitro assay [63].
In a search for mutants of Pichia pastoris that did not
degrade peroxisomes in certain conditions, a mutant
named gsa1 was isolated. The corresponding gene turned
out to be the K subunit of phosphofructokinase; how it
participates in the autophagic process is unknown [64].
The P. pastoris phosphofructokinase is kinetically similar
to classical phosphofructokinases [64].
In S. pombe, overexpression of a cDNA encoding a
truncated phosphofructokinase inhibited the transition
G2/M and produced an atypical septation in those cells.
The results suggested a link between metabolism and cell
cycle [65].
Transformation of a six carbon molecule into trioses is
eected by fructose-1,6-bisphosphate aldolase that cleaves
fructose 1,6-bisphosphate into dihydroxyacetone phos-
phate and glyceraldehyde 3-phosphate. The fructose-1,6-
bisphosphate aldolase encoding gene from S. pombe has
been cloned and encodes a polypeptide of 358 amino acids
which is 63% homologous with the S. cerevisiae protein
[66]. The enzyme from C. utilis has been puried; it has a
molecular weight of about 70 kDa and contains a Zn
2
ion as the enzyme from S. cerevisiae [67].
3.2. From trioses phosphate to pyruvate
Triose phosphate isomerase interconverts dihydroxyace-
tone phosphate and glyceraldehyde 3-phosphate. The gene
encoding this enzyme in S. pombe has been cloned and
presents a high sequence homology to that of S. cerevisiae
FEMSRE 689 29-8-00
[68]. The corresponding gene from K. lactis has been
cloned and disrupted; in contrast with the behavior of a
tpi mutant of S. cerevisiae, the mutant from K. lactis lack-
ing the gene encoding triose phosphate isomerase is able to
grow in glucose [69]. The growth is not possible in the
presence of the respiratory inhibitor antimycin A, a behav-
iour similar to that shown by the rag2 and pfk1 pfk2
mutants (see above).
In the step catalyzed by glyceraldehyde-3-phosphate de-
hydrogenase, 1,3-bisphosphoglycerate is formed from glyc-
eraldehyde 3-phosphate. This is an important step in gly-
colysis, since the oxidation of the aldehyde generates an
energy-rich acyl phosphate bond. The NADH generated in
the reaction has to be reoxidized for glycolysis to proceed
(see Section 6). In S. cerevisiae, three genes named TDH1,
TDH2 and TDH3 encode three dierent isoenzymes, with
TDH3 accounting for 50^60% of the enzyme found in the
cell [70]. Mutants with only TDH1 are not viable and
those lacking either TDH2 or TDH3 grow more slowly
in glucose than the wild-type [70]. A gene, KlGAP1, encod-
ing glyceraldehyde-3-P dehydrogenase has been cloned
from K. lactis. This gene and the one encoding alcohol
dehydrogenase 1 are transcribed in opposite directions
and separated by only 1.2 kb, suggesting that they share
common elements in the promoter [71]. In contrast to the
single gene reported in K. lactis, a family of three genes
encoding glyceraldehyde-3-phosphate dehydrogenases is
found in K. marxianus [72]. KmGAP1 is transcribed at
similar rates in dierent carbon sources and apparently
encodes a protein that is localized in the cell wall. Tran-
scription of GAP2 is increased in glucose, while transcrip-
tion of GAP3 was not detected in the conditions used [72].
In C. albicans, a glyceraldehyde-3-phosphate dehydro-
genase, encoded by the gene TDH1 [73], also appears in
the cell wall and is able to bind to laminin and bronectin
[74].
In P. pastoris the GAP gene, that encodes a glyceralde-
hyde-3-phosphate dehydrogenase with a molecular mass
of 35.4 kDa, is constitutively expressed. The promoter of
this gene has been used to direct the heterologous produc-
tion of proteins in this yeast as an alternative to the com-
monly used AOX1 promoter [75].
In the reaction catalyzed by 3-phosphoglycerate kinase
the energy-rich acylphosphate from 1,3-bisphosphoglycer-
ate is used to produce ATP. In S. cerevisiae, the PGK1
gene that encodes phosphoglycerate kinase, is one of the
most highly expressed and as such it has been widely
studied both to understand why this is so and to use its
promoter to direct expression of foreign proteins [76].
Genes encoding 3-phosphoglycerate kinase have been
cloned from several NCY. In K. lactis the corresponding
gene encodes a protein of 416 aa with strong codon bias
[77]. In Candida maltosa, a gene encoding a protein of
similar size has been cloned. Its expression was higher in
glucose than in alkanes or oleic acid and its promoter has
been used to direct expression of heterologous proteins
[78]. In contrast with C. maltosa, the gene encoding 3-
phosphoglycerate kinase in C. albicans is similarly ex-
pressed under various growth conditions. A certain
amount of the protein has been found attached to the
cell wall [79].
In Y. lipolytica, expression of the PGK1 gene was higher
on gluconeogenic substrates than on glycolytic ones [80].
Curiously, strains with a disrupted PGK1 gene were pro-
line auxotrophs when grown in glucose. No immediate
explanation exist for this phenotype that has not been
observed, to our knowledge, in other yeasts.
Phosphoglycerate mutase catalyzes the interconversion
between 3- and 2-phosphoglycerate. In contrast with the
protein of S. cerevisiae which is a tetrameric enzyme, the
phosphoglycerate mutase of S. pombe appears to be
monomeric; it lacks an amino acid segment at the C-ter-
minus that in S. cerevisiae has been implicated in catalytic
activity [81], and also a stretch of 24 aa that in S. cerevi-
siae appears to be involved in interactions between sub-
units. Another loop also involved in these types of inter-
actions is absent in the S. pombe protein [82].
Enolase catalyzes the dehydration of 2-phosphoglycer-
ate to phospho-enol-pyruvate. Two genes, ENO1 and
ENO2, encode the subunits of this homodimeric enzyme
in S. cerevisiae [83]. While transcription of ENO1 is con-
stitutive, that of ENO2 is stimulated about 20-fold by
glucose, suggesting a dierential participation of the iso-
enzymes in glycolysis or in gluconeogenesis [84]. Although
it was reported that C. albicans contained only one gene
(ENO1) encoding enolase, a protein of 440 amino acids
[85], Postlethwait and Sundstrom [86] later found two eno-
lase gene loci that could be distinguished by the localiza-
tion of particular restriction sites in their 3'anking re-
gions, while the promoter and coding region were
identical[86]. There have been also discrepancies regarding
expression of the enolase encoding gene(s) in C. albicans.
The corresponding mRNA level was reported to be 6 and
13 times higher in glucose than in ethanol [85] ; however,
other authors did not nd dierences between glucose and
pyruvate grown cells [86]. This discrepancy could be due
either to a lack of normalization to a reference RNA in
the rst case, or to specic dierences between ethanol and
pyruvate, although this seems unlikely.
In S. pombe a gene, eno1
, which could encode an eno-

lase has been identied; the corresponding 1.4-kb tran-
script is more abundant during growth in glucose than
during growth in gluconeogenic substrates [87].
Pyruvate kinase catalyzes the second ATP forming re-
action in the glycolytic pathway. In S. cerevisiae, two
genes exist that encode two dierent proteins. One of
these, encoded by the PYK1 gene, is strongly activated
by fructose-1,6-bisphosphate, while the other one encoded
by the PYK2 (YOR347c), does not respond to this com-
pound. This last enzyme is repressed by glucose and in
certain conditions may allow function of glycolysis at a
reduced rate [88]. There are no reports of the existence
FEMSRE 689 29-8-00
of more than one pyruvate kinase encoding gene in NCY.
Only the genes encoding pyruvate kinases from S. pombe
and Y. lipolytica have been cloned [89,90]. Overexpression
of the pyk1
gene from S. pombe has allowed purication

of the enzyme that behaves as a dimer [89]. The organiza-
tion of the YlPYK1 gene from Y. lipolytica is curious as it
presents two introns separated by a short stretch of 45 bp.
[90,91]
In general, pyruvate kinase from diverse origins presents
a sigmoidal kinetics with respect to PEP; fructose-1,6-bis-
phosphate activates the enzyme and converts its kinetic to
an hyperbolic one. In contrast, the enzyme from C. utilis
[92] and Y. lipolytica [93] are not activated by fructose-1,6-
bisphosphate and in this last yeast the kinetics towards
PEP is hyperbolic [93].
4. Metabolic alternatives of intracellular pyruvate
The two quantitative major fates of the pyruvate pro-
duced in glycolysis are either its oxidation to CO
2
or its
transformation to ethanol. In most yeasts under aerobic
conditions, oxidation will be predominant, while transfor-
mation to ethanol takes place in anaerobic conditions or
at high glucose concentrations even in aerobic conditions
in those yeasts that present a Crabtree eect (for a review
see [94]).
4.1. Pyruvate dehydrogenase
Oxidation of pyruvate to CO
2
occurs via the TCA cycle.
To enter the TCA cycle, pyruvate undergoes an oxidative
decarboxylation catalyzed by pyruvate dehydrogenase, a
mitochondrial multienzyme complex that is formed by
three dierent components: pyruvate dehydrogenase
(E1), dihydrolipoamide acetyl transferase (E2) and dihy-
drolipoamide dehydrogenase (E3). The E1 element, in
turn, consists of two subunits E1K and E1L. The respective
genes in S. cerevisiae are named PDA1 [95] or PDHK1
[96], PDHL1 [97], LAT1 [98] and LPD1 [99]. The local-
ization of the pyruvate dehydrogenase complex implies
that cytosolic pyruvate must be transported into the mi-
tochondrion. A mitochondrial pyruvate carrier was puri-
ed from S. cerevisiae by Nalecz et al. [100], but up to now
the gene encoding it has not been identied.
In K. lactis, disruption of the gene KlPDA1, that en-
codes the E1K subunit of the complex, led to a decrease
of the specic growth rate in glucose in minimal medium,
while growth in rich medium or in ethanol was not af-
fected. Also ethanol was produced in aerobic batch cul-
tures of the mutant [101]. Expression of KlPDA1 was
partly repressed during growth in glucose by the product
of the Rag3 protein [102] a homologue of the PDC2 gene
of S. cerevisiae that controls transcription of pyruvate de-
carboxylase [103]. The E1L subunit of the pyruvate dehy-
drogenase complex has been cloned from S. pombe, but no
studies on the eects of its disruption have been reported
[104] ; however, the same authors found that mutants from
the ssion yeast with reduced pyruvate dehydrogenase ac-
tivity were auxotrophic for arginine or glutamine [105]. In
P. stipitis, the activity of pyruvate dehydrogenase is con-
stitutive [106].
4.2. Pyruvate decarboxylase
In S. cerevisiae, several genes encoding pyruvate decar-
boxylase have been found although PDC1 normally ac-
counts for about 80% of the enzymatic activity. Enzyme
activity increases in glucose due to enhanced transcription
of the PDC1 and PDC5 genes [107]. The product of the
PDC2 gene is involved in the control of expression of the
structural PDC genes [107]. In contrast, in K. lactis only
the gene RAG6 appears to encode pyruvate decarboxylase
[108]. Contrary to what happens in mutants from S. cere-
visiae lacking pyruvate decarboxylase, K. lactis rag6 mu-
tants grew in glucose at the same rate as the wild-type.
This fact raises the question of the origin of the acetyl
CoA needed for biosynthesis in the cytoplasm in this mu-
tant (see Section 4.3) and highlights once more the dier-
ent lifestyles of these two yeasts. Transcription of RAG6 is
stimulated by glucose and autoregulated. The product of
the gene RAG3 is necessary for glucose induction [109].
Mutations in RAG1, RAG5 or RAG2 reduce the induction
by glucose of RAG6 [108].
In C. albicans, the gene encoding pyruvate decarboxy-
lase has not been yet cloned, but a homologue of ScPDC2
has been found [110]. CaPDC2 complemented the defect
of S. cerevisiae pdc2 mutants [110] in contrast with what
happened with RAG3 that was ineective [109].
In P. stipitis two genes encoding pyruvate decarboxylase
have been found, but detailed studies on their physiolog-
ical roles are not available. The PsPDC1 gene has an 81-
bp stretch that is absent from other known PDC genes
[111].
4.3. The pyruvate bypass
Acetyl CoA can also be formed in the cytosol through
the so-called pyruvate-bypass, that involves the synthesis
of acetyl CoA through the concerted action of pyruvate
decarboxylase, acetaldehyde dehydrogenase and acetyl
CoA synthetase. These reactions and transport of the so
formed acetyl CoA to the mitochondria could in principle
`by-pass' the action of pyruvate dehydrogenase ([94], see
Fig. 1). The physiological signicance of this by-pass is
shown by the absence of growth of S. cerevisiae pdc mu-
tants in mineral medium with glucose unless ethanol or
acetate is added [112] and by the observation that acs1
acs2 mutants devoid of acetyl CoA synthetase activity
do not grow in glucose [113].
The enzymes participating in the pyruvate bypass are
localized in the cytosol in S. cerevisiae [114,115] but no
FEMSRE 689 29-8-00
information about their localization in other yeasts is
available.
The fact that in K. lactis, disruption of the gene
KlPDA1, causes production of ethanol in aerobic batch
cultures indicates that either the pyruvate bypass does
not produce enough acetyl CoA in those conditions or
that the mitochondria has a limited capacity to import
acetyl CoA from the cytosol. Since addition of low con-
centrations of L-carnitine restored the aerobic growth in
glucose the second possibility appears the most likely
[101].
5. The TCA cycle
Acetyl CoA generated either by pyruvate dehydrogenase
or by the pyruvate bypass, is the link between glycolysis
and the TCA cycle. Acetyl CoA reacts with oxaloacetate
in a reaction catalyzed by citrate synthase to produce cit-
rate. Three genes encoding functional citrate synthases
have been identied in S. cerevisiae: CIT1 and CIT3
that encode mitochondrial enzymes and CIT2 that encodes
the isoenzyme involved in the glyoxylate cycle [116,117]. In
C. tropicalis a gene, CtCIT, encoding a 45-kDa polypep-
tide has been identied. This gene has an intron of 79 bp
in the region encoding the N-terminal part of the protein;
this intron can be correctly spliced in S. cerevisiae [118].
The next step of the pathway is the conversion of citrate
to isocitrate catalyzed by aconitase, a 4Fe^4S cluster con-
taining protein, that has not been much studied. In S.
cerevisiae, the gene ACO1 encoding it, is synergistically
repressed by glucose and glutamate and its disruption
led to glutamate auxotrophy [119]. Its activity is depen-
dent on the function of the gene YFH1 that encodes fra-
taxin, a protein that seems implicated in the regulation of
mitochondrial iron transport and that is implicated in the
human degenerative disease Friedreich ataxia [120].
Isocitrate dehydrogenase transforms isocitrate into 2-
oxoglutarate (K-ketoglutarate). In S. cerevisiae an
NAD
-dependent isocitrate dehydrogenase, whose two

dierent subunits are encoded by the genes IDH1 and
IDH2 [121,122], participates in the TCA cycle [123]. Three
dierently compartmentalized NADP
-dependent isoci-
trate dehydrogenases, encoded by the genes IPD1, IPD2
and IPD3 exist. At least some of them are important in
the production of NADPH [124]. S. pombe mutants lack-
ing an NAD
-dependent enzyme are glutamate auxo-

trophs [125].
Apparently no other studies on genes encoding enzymes
implicated in the TCA cycle have been done in NCY. This
lack of information contrasts with the importance of this
pathway in the aerobic metabolism of these yeasts.
5.1. Anaplerotic reactions
Intermediates from the TCA cycle are removed during
growth for biosynthetic purposes; therefore, reactions that
replenish the cycle are needed to keep it running. In yeasts
growing in a minimal medium with a sugar as carbon
source and NH
4
as nitrogen source, pyruvate carboxylase
is the main anaplerotic reaction (see Fig. 1). Two genes
PYC1 and PYC2 encode isoenzymes of pyruvate carbox-
ylase in S. cerevisiae [126,127], these proteins are located
in the cytosol as in C. utilis, in contrast with the situation
in other fungi and in mammals [128]. One unique gene
encoding pyruvate carboxylase has been isolated from P.
pastoris. Mutants lacking this enzyme do not grow in glu-
cose when ammonium is the nitrogen source but can grow
when glutamate or aspartate is added [129]. The situation
is dierent from that of S. cerevisiae where glutamate can-
not substitute aspartate for growth of a pyruvate carbox-
ylase mutant, likely due to the repression exerted by sugars
on glutamate dehydrogenase (C. Gancedo, unpublished
results). An interesting observation is that of van Dijk et
al. [130] who found that pyruvate carboxylase appears
implicated in the assembly of alcohol oxidase in Hansenula
polymorpha. Up to now, no indications are available about
the possible mechanism of participation of this protein in
the process.
Another important anaplerotic device is the glyoxylate
cycle required for growth in minimal medium in carbon
sources of less than three carbon atoms, such as ethanol or
acetate. Two characteristic enzymes of the cycle are isoci-
trate lyase and malate synthase. Isocitrate lyase catalyzes
the cleavage of isocitrate to succinate and glyoxylate and
malate synthase catalyzes the condensation of glyoxylate
with a molecule of acetyl CoA.
The gene ICL1 encodes isocitrate lyase in S. cerevisiae
[131], where the protein is not located in the mitochondria
[132,133]. The isocitrate lyase from C. tropicalis and that
of Y. lipolytica have putative peroxisomal targeting signals
[134,135]. The gene CtICL1, encoding isocitrate lyase from
C. tropicalis has been expressed to relatively high levels
from its own promoter in S. cerevisiae growing in acetate,
glycerol, lactate, ethanol or oleate [136]. Mutants of Y.
lipolytica lacking isocitrate lyase, are unable to grow on
acetate, ethanol or fatty acids, but utilize succinate or
glycerol [137] ; curiously growth is also impaired in glucose
[135].
Malate synthase was puried from C. tropicalis [138].
Two identical genomic regions ^ except for one amino
acid replacement in the encoded protein ^ were found,
although only one RNA species was detected [139]. A
gene, MAS1, encoding the enzyme from Hansenula (Pi-
chia) polymorpha has been cloned [140].
The enzyme from C. tropicalis, a homotetrameric pro-
tein of molecular mass 250 kDa, was localized in the ma-
trix of the peroxisomes [138], but the protein from H.
polymorpha does not have a conventional peroxisomal tar-
geting sequence [140]. There is no report of the existence
of more than one malate synthase in NCY as it happens in
S. cerevisiae, where two genes MLS1 and DAL7 encode
FEMSRE 689 29-8-00
two dierent enzymes, one involved in carbon and the
other in nitrogen metabolism [141].
S. pombe lacks the enzymes of the glyoxylate cycle and
this could explain the lack of growth in ethanol of this
yeast [142,143].
6. Oxidation of NADH and energy generation
Catabolic reactions generate NADH either in the cyto-
sol or in the mitochondria. During fermentation, NADH
produced in glycolysis is reoxidized to NAD
by the for-
mation of ethanol ; NADH arising in reactions dierent
from glycolysis is regenerated mainly by the production of
glycerol. During aerobic growth, èxternal' mitochondrial
NADH dehydrogenases participate in the reoxidation of
cytosolic NADH. In S. cerevisiae they are encoded by two
genes called either NDH1 and NDH2 [144] or NDE1 and
NDE2 [145]. The existence of this external dehydrogenases
seems consistent with the apparent absence of a malate
aspartate shuttle in this yeast [145]. In Y. lipolytica, a
gene named YlNDH2, encodes an external NADH oxido-
reductase [146]. NADH generated in reactions occurring
in the mitochondria is regenerated by a NADH dehydro-
genase located in the inner mitochondrial membrane fac-
ing the matrix space. The protein is encoded by the gene
NDI1 in S. cerevisiae [147]. During growth on reduced
substrates like ethanol, the L-glycerol-3-phosphate shuttle
(see Fig. 1) appears to be important to maintain an ad-
equate redox balance in the cytosol [148].
Reoxidation of NADH by the respiratory chain is the
main source of energy for cells with a respiratory metab-
olism. This reoxidation is coupled to the production of
ATP in the process known as oxidative phosphorylation.
Although we will not discuss in detail oxidative phosphor-
ylation, it should be mentioned that while in S. cerevisiae
phosphorylation at site I of the electron transfer chain is
absent [149], it appears to be operative in other yeasts
[146,150].
The ATP generated in the mitochondrion is delivered to
the cytosol via specic ADP/ATP translocators. In S. ce-
revisiae, three genes encoding mitochondrial ADP/ATP
translocators have been identied: AAC1 [151], AAC2
[152] and AAC3 [153]. The major protein involved in
translocation appears to be Aac2, but Aac3 is necessary
for anaerobic growth [153]. The role of Aac1 is not com-
pletely clear; curiously, it has been implicated in the vac-
uolar accumulation of the red pigment formed by ade2
mutants [154]. Gene homologues to those encoding ATP
translocators in S. cerevisiae have been cloned from K.
lactis [155], Candida parasilopsis [156] and S. pombe. In
this last yeast, the gene has been termed anc1
[157]. There
is only one gene encoding an ATP/ADP translocator in K.
lactis and S. pombe. A null mutation of AAC2 in K. lactis
made the yeast unable to grow on glycerol, galactose,
maltose and ranose [158] and null anc1
3
mutants of S.
pombe were unable to grow under oxygen limitation [159].
In some conditions, e.g. during anaerobic growth, ATP
should be translocated into the mitochondria. Due to
the expression pattern of the ATP/ADP translocators in
S. cerevisiae it has been hypothesized that AAC3 is the one
that carries out this function [160].
It ought to be mentioned here, that a number of yeasts
possess a respiration pathway alternative to the cyto-
chrome pathway. This pathway is specically inhibited
by hydroxamic acids, but is not inhibited by cyanide
[161,162]. Cyanide insensitive respiration is mediated by
an alternative oxidase that accept electrons from the ubi-
quinone pool and transfer them to oxygen that is reduced
to water. However, this electron ow is not accompanied
by synthesis of ATP [163].
The existence of the alternative respiration pathways in
K. lactis was demonstrated by Heritage et al. [164]. In the
yeast Hansenula anomala, the alternative oxidase is a mi-
tochondrial protein of 36 kDa [165] whose synthesis is
induced by antimycin A and cyanide or by sulfur-contain-
ing amino acids; a cDNA encoding it has been isolated
[166,167]. The gene AOX1 encodes the alternative oxidase
in C. albicans, the putative protein has about 44 kDa
[168]. It is a non-essential gene and it is functional in S.
cerevisiae, a genus that lacks the alternative respiratory
pathway. Y. lipolytica possesses the pathway [169] whose
activity appears to increase during the transition from log-
arithmic to stationary growth phase in glucose cultures
[170]. In P. stipitis, the alternative pathway seems impor-
tant during oxygen-limited xylose fermentations [171].
Since the alternative pathway is absent in S. pombe this
yeast has been used to express the alternative oxidase from
the plant Sauromatum guttatum under the control of the
thiamine-repressible nmt1 promoter [172]. Expression of
the plant oxidase decreased slightly the growth rate of
the yeast in glucose, but did not aect growth yield; how-
ever, both parameters were severely aected during growth
in glycerol. The eect was due to competition for ubiqui-
none between the plant oxidase and the normal cyto-
chrome pathway [173].
The physiological role of the alternative respiratory
pathway in yeasts is still not clearly understood; it could
act as an energy overow device or be related to particular
stress situations. The alternative respiration pathway and
its underlying molecular basis and regulation is a topic
that deserves further consideration.
7. The pentose phosphate pathway
The pentose phosphate pathway is an important meta-
bolic route implicated in the production of NADPH for
biosynthetic reactions and of ribose 5-phosphate for syn-
thesis of nucleic acid and nucleotide cofactors; it also pro-
vides erythrose 4-phosphate for the synthesis of aromatic
amino acids. The rst reactions of the pathway are two
FEMSRE 689 29-8-00
oxidative reactions that are physiologically irreversible,
while the other ones are non-oxidative and reversible.
The partition of hexose metabolism between the glycolytic
and the pentose phosphate pathway occurs at the level of
glucose 6-phosphate (see Fig. 1). It has been observed that
the nitrogen source of the medium inuences the amount
of sugar directed to the pentose phosphate pathway. In S.
cerevisiae, growth in a medium supplemented with amino
acids decreased the ux through the pentose phosphate
pathway [174]. In those yeasts that use nitrate as nitrogen
source, an increase of the carbon ux through the pentose
phosphate pathway shall be expected due to the increased
NADPH requirement caused by the operation of nitrate
and nitrite reductase; indeed this has been found in C.
utilis [175].
Glucose-6-phosphate dehydrogenase directs glucose into
the pentose phosphate pathway by catalyzing the oxida-
tion of glucose-6-phosphate to 6-phospho-N-gluconolac-
tone. Glucose-6-phosphate dehydrogenase has been puri-
ed from S. pombe [176] and has K
m
values of 0.66 mM
for glucose-6-P and 44 WM for NADP
. The enzyme from

C. utilis has been puried [177], it has a molecular weight
of 11 or 22 kDa depending on pH and the presence of
Mg
2
. Another group puried the enzyme from a strain of
Pichia jadinii (synonym of C. utilis) and found its N-ter-
minal amino acid acetylated and a 15 amino acid stretch
rich in Ser and Thr (411^425 in a protein of 495 residues)
that is absent in other related glucose-6-P dehydrogenases
[178]. The signicance of this stretch, if any, is unknown.
Mutants in the gene ZWF1 from S. cerevisiae that lack
glucose-6-phosphate dehydrogenase activity, turned out to
require an organic sulfur source [179]. This requirement
may be explained by the existence of two steps in methio-
nine biosynthesis that require NADPH. It could be inter-
esting to test the phenotype of similar mutants in NCY to
ascertain the existence of alternative, eective pathways of
NADPH provision.
The hydrolysis of 6-phosphate-N-gluconolactone to 6-
phospho-gluconate occurs spontaneously at neutral pH,
but at a very slow rate. A lactonase that accelerated this
rate was partially puried from baker's yeast by Brodie
and Lipmann [180] ; however, no further studies on this
enzyme have been performed. Up to now, genes encoding
gluconolactonases has been isolated only from the bacte-
rium Zymomonas mobilis [181] and from the fungus Fusa-
rium oxysporum [182].
In those yeasts able to use gluconate, 6-phospho-gluco-
nate may be also formed by the action of 6-phospho-gluc-
onate kinase, an enzyme that has been kinetically charac-
terized in S. pombe [183].
6-Phospho-gluconate dehydrogenase catalyzes the oxi-
dative decarboxylation of 6-phospho-gluconate to ribulose
5-phosphate. The enzyme has been puried from S. pombe
as a tetramer and its kinetic characteristics studied [184].
In C. utilis the enzyme appears to be a dimer of 100 kDa
with high anity for its substrates (K
m
55 WM for 6-P-
gluconate and 20 WM for NADP
) [185]. In S. cerevisiae,
mutants lacking this activity are unable to grow in glu-
cose, but elimination of the glucose-6-phosphate dehydro-
genase activity allowed growth in this sugar [186]. The
authors concluded that the accumulated 6-phospho-gluco-
nate was toxic for the cell.
Ribulose-5-P can be either isomerized to ribose-5-P or
epimerized to xylulose-5-P. The rst reaction is catalyzed
by ribose-5-P isomerase. This enzyme has been puried
from C. utilis by Domagk and Doering [187] and by Ho-
ritsu et al. [188]. The data about molecular mass and sub-
unit composition dier between these authors. Ribulose-5-
P epimerase catalyzes the epimerization of ribulose 5-
phosphate to xylulose 5-phosphate. No data for this step
in NCY are available at this time.
Transketolase catalyzes two reactions in the pentose
phosphate pathway in which a glycolaldehyde (two car-
bon) moiety is transferred from a ketose donor to an al-
dose acceptor. In both reactions, the donor is xylulose 5-
phosphate, but the acceptor is in one case ribose 5-phos-
phate and in other erythrose 4-phosphate. Two genes en-
coding transketolases have been isolated from S. cerevisi-
ae, TKL1 and TKL2. Transketolase has been puried from
C. utilis and shown to have high anity both for xylulose-
5-P (K
m
80 WM) and ribose-5-P (K
m
430 WM) [189]. A
gene, KlTKL1, encoding transketolase has been isolated
from K. lactis. It complements an S. cerevisiae tkl1 tkl2
mutant for its requirement of aromatic amino acids. The
amino acid sequence of KlTKL1 appeared to be more
related to the corresponding prokaryotic transketolases
than to those of eukaryotes [190]. The corresponding
gene, TKT1, from P. stipitis has also been cloned and
used to enhance xylose utilization by strains of S. cerevi-
siae [191] (see below). It should be mentioned that in
methylotrophic yeasts, a transketolase reaction catalyzed
by a dierent enzyme (dihydroxyacetone synthase) is re-
sponsible for the formation of dihydroxyacetone and glyc-
eraldehyde-3-P from xylulose-5-P and formaldehyde. The
gene encoding this enzyme from Hansenula anomala has a
high degree of homology with the transketolase of S. ce-
revisiae which functions in the pentose phosphate pathway
[192].
Transaldolase catalyzes the reversible formation of glyc-
eraldehyde-3-P and sedoheptulose-7-P from erythrose-4-P
and fructose-6-P. Mutants from K. lactis lacking this en-
zyme, encoded by the gene KlTAL1, have been instrumen-
tal to highlight an important dierence between this yeast
and S. cerevisiae concerning the capacity of the pentose
phosphate pathway for glucose utilization. It was shown
that K. lactis double mutants lacking phosphofructokinase
and transaldolase activities could not grow in glucose,
while those lacking only phosphofructokinase could [62].
This result indicates that K. lactis can utilize glucose ex-
clusively via the pentose phosphate pathway, in contrast
to the situation in S. cerevisiae, and is consistent with the
behavior of rag2 mutants (see Section 3).
FEMSRE 689 29-8-00
An enzyme that could be related with the pentose
phosphate pathway, but that, up to now, lacks a clear
physiological function, is the glucose dehydrogenase
from S. pombe. This enzyme produces gluconate in a
reaction dependent of NADP
and could, in principle,

allow metabolism of glucose through the hexose mono-
phosphate pathway [48,183]. However, the nding that
mutants lacking hexokinase activity do not grow in glu-
cose is not easily accommodated with this proposed func-
tion [48].
7.1. The genes of xylose metabolism
Xylose is a pentose that is a quantitatively important
constituent of hemicellulose and as such is a sugar with
high biotechnological potential. It can be used for the
production of xylitol, a non-cariogenic sweetener with
low caloric value or for the production of ethanol. How-
ever, the yeast with the best fermentation capacity, S. ce-
revisiae does not metabolize xylose, although it grows on
xylulose. Since some NCY grow in xylose, attempts have
been made to express in S. cerevisiae the genes required to
transform xylose into xylulose. Utilization of xylose im-
plies a reduction to xylitol by an NAD(P)H dependent
xylose reductase followed by an oxidation to xylulose by
an NAD
-dependent xylitol dehydrogenase. Xylulose is

then phosphorylated by xylulose kinase and enters the
pentose phosphate pathway (Fig. 1). It has been postu-
lated that fermentation of xylose to ethanol requires the
action of an NADH xylose reductase to avoid a serious
redox imbalance in the yeast [193].
Xylose reductases utilize NADPH or NADH; it is not
completely clear if some yeasts produce dierent proteins
with distinct coenzyme specicity [194]. The gene XYL1
encoding xylose reductase has been cloned from several
yeasts. A single gene exists in K. lactis that is expressed
constitutively; its disruption leads to inability to grow
in xylose [195]. The gene has also been cloned from
P. stipitis [196]. Expression of this gene integrated at about
40 copies in the genome of S. cerevisiae, resulted in e-
cient production of xylitol [197]. The xylose reductase
from Candida shehatae and Candida tenuis, have been pu-
ried [198,199] and the XYL1 gene from Candida guillier-
mondi has been cloned [200]. A genomic region with high
sequence similarity with the C-terminal region of genes
encoding xylose reductases has been found in C. albicans
[201].
Xylitol dehydrogenase is encoded in P. stipitis by the
gene XYL2 [196]. Although in S. cerevisiae, ORF
YLR070c has the characteristics of an NADH-dependent
xylitol dehydrogenase, this activity is only measurable
when the gene is overexpressed [202]. Expression of the
P. stipitis XYL1 and XYL2 genes allows S. cerevisiae to
utilize xylose; however, an ecient fermentation with high
production of ethanol appears to require overexpression of
xylulokinase [196,203,273].
8. Ethanol metabolism
Production of ethanol during growth in sugars by NCY
is not as important as it is in S. cerevisiae due to their
predominantly aerobic metabolism. Nevertheless, in anae-
robiosis, ethanol is produced by those NCY able to thrive
in these conditions. On the other hand, ethanol may be
used as a carbon and energy source by most yeasts. Cen-
tral to the production or the utilization of ethanol are
alcohol dehydrogenases, enzymes that catalyze the revers-
ible reduction of acetaldehyde to ethanol. The kinetic and
regulatory characteristics of these enzymes, the regulation
of the transcription of the genes that encode them, or their
subcellular localization determine the direction of the re-
action catalyzed under physiological conditions. However,
in many cases, the precise physiological role of some al-
cohol dehydrogenases is far from being clearly established.
In S. cerevisiae, four genes encode isoenzymes of alco-
hol dehydrogenases. ADH1 encodes the isoenzyme prom-
inent during glucose fermentation and is induced by glu-
cose [204], while ADH2 is repressed by glucose and is
implicated in the utilization of ethanol. The physiological
role of the other isoenzymes is not completely clear [205].
In S. pombe, mutants partially decient in alcohol de-
hydrogenase were isolated by Megnet [206] by selection of
strains able to grow in glucose in the presence of 1 mM
allyl alcohol. This work seems to be the rst one that tried
to isolate mutants in a step of the glycolytic chain. A gene
encoding the S. pombe alcohol dehydrogenase was isolated
16 years later [207].
Four genes encoding alcohol dehydrogenases have been
characterized in K. lactis and the proteins encoded studied.
KlAdhI and II are NAD
-dependent, cytosolic enzymes.

KlAdhIII and IV are mitochondrial and may use NAD
or NADP
. These enzymes also showed activity with oth-

er aliphatic alcohols [208]. The genes KlADH1 and
KlADH2 are preferentially expressed in glucose grown
cells [209]. On the contrary, the KlADH3 gene is expressed
at low glucose concentration and strongly repressed by
ethanol, while KlADH4 is induced by it [210].
In C. albicans, a gene, CaADH1, that encodes an alco-
hol dehydrogenase has been isolated. The corresponding
mRNA was less abundant during growth in glucose or
ethanol than during growth in other carbon sources [211].
In the xylose-utilizing yeast P. stipitis, two cytoplasmic
alcohol dehydrogenases have been identied, encoded by
genes called PsADH1 and PsADH2 [212,213] ; unfortu-
nately the numbers assigned to the genes by these two
groups are not the same. PsADH1 expression (gene
name from [212]) increases about 10 times during anaero-
bic growth and disruption of the gene brings about an
increase of PsADH2 expression on non-fermentable car-
bon sources [212].
In Y. lipolytica an NAD
-dependent alcohol dehydro-

genase which acts better on long chain alcohols has been
identied. Three other NADP
-dependent dehydrogenases
FEMSRE 689 29-8-00
were also detected [214]. These authors made the interest-
ing observation that Y. lipolytica is extremely tolerant, up
to 1 M, to allyl alcohol. This result contrasts with what
has been found in other genera like S. pombe [206].
Aldehyde dehydrogenases are important enzymes not
only for the metabolism of acetaldehyde produced from
ethanol, but also for that of toxic aldehydes produced in
some stress situations. In S. cerevisiae the information
about the number of these enzymes and the corresponding
genes was confused. Navarro-Avin o et al. [215] have pro-
posed a logical nomenclature and tried to clarify the sit-
uation. According to these authors, ve related ORFs exist
in the sequenced S. cerevisiae genome: YMR170c,
YMR169c, YOR374w, YER073w and YPL061w. They
proposed to name the corresponding genes encoding
them ALD2, ALD3, ALD4, ALD5 and ALD6. The pro-
teins Ald2, Ald3 and Ald6, are cytoplasmic while Ald4
and Ald5 are mitochondrial. Ald2 and Ald3 use NAD
as cofactor, are stress-induced and repressed by glucose;

Ald4 uses NAD
or NADP
and is also repressed by

glucose, while Ald5 uses NADP
and is constitutive.
Ald6 uses NADP
, is constitutive and activated by

Mg
2
. Ald4 and Ald6 seem important for growth in etha-
nol [216] and other data point to an important role of
some of the aldehyde dehydrogenases in the synthesis of
cytoplasmic acetyl CoA [215]. No information on alde-
hyde dehydrogenases from NCY is available.
The transformation of acetate into acetyl CoA is cata-
lyzed by acetyl CoA synthetase (see Section 4.3). In S.
cerevisiae, two genes encoding acetyl CoA synthetases
have been isolated. Disruption of one of them, ACS1,
caused inability to grow in acetate, but not in etha-
nol[217], while disruption of the other, ACS2, caused in-
ability to grow in glucose [113,218]. The subcellular local-
ization of the enzyme(s) is not known. In Y. lipolytica,
mutants lacking acetyl CoA synthetase, encoded by the
gene ICL2, did not grow in acetate. Interestingly, mutants
in the gene encoding ICL1 that encodes isocitrate lyase,
increased the amount of acetyl CoA synthetase [219]. Ace-
tyl CoA synthetase has also been detected in S. pombe;
although this yeast does not grow in acetate, the levels of
the enzyme increased in its presence [220].
9. Glycerol metabolism
As already stated, during anaerobiosis, reoxidation of
most NADH is carried out in yeasts by alcohol dehydro-
genase. However, production of glycerol under anaerobic
conditions is a secondary, but important, way to regener-
ate NAD
(see Section 6). In S. cerevisiae, mutants lack-

ing alcohol dehydrogenase, still produce ethanol, but in-
crease their glycerol production during glucose
fermentation [221]. The situation is dierent in K. lactis,
where a mutant lacking alcohol dehydrogenase activity did
not accumulate much glycerol [222]. Glycerol production
also plays another important role, namely it allows adap-
tation of the yeast to situations of osmotic stress.
Glycerol formation requires the sequential action of a
NADH glycerophosphate dehydrogenase and a glycero-
phosphatase. In S. pombe ^ as in S. cerevisiae [223] ^
two genes have been identied that encode two NADH
glycerophosphate dehydrogenases. One of them, gpd1
,
is transcribed in response to osmotic upshift, while the
other one, gpd2
, is constitutively transcribed although

to a low level [224]. Curiously, deletion of gpd2
caused
a requirement for histidine and lysine [225]. In S. cerevisi-
ae, two genes, GPP1 and GPP2, that encode D,L-glycerol-
3-P phosphatases with relatively low anities, K
m
about
3^4 mM, for their substrates, have been cloned. Transcrip-
tion of GPP2 is under the control of the HOG pathway
and is induced by high osmolarity [226]. No information
on these enzymes is available from NCY.
Glycerol may be also utilized as a carbon source under
aerobic conditions by many yeasts. Two dierent path-
ways of glycerol catabolism have been identied in yeasts.
In S. cerevisiae or C. utilis glycerol is phosphorylated by a
glycerol kinase and the L-glycerol-3-phosphate formed is
oxidized by a mitochondrial glycerol phosphate ubiqui-
none oxidoreductase [227,228]. In S. pombe dehydrogen-
ation of glycerol by an NAD
-dependent glycerol dehy-

drogenase forms dihydroxyacetone which is then
phosphorylated by a dihydroxyacetone kinase [229,230].
Genes encoding these enzymes have also been found in
S. cerevisiae; they are expressed in response to high salt
concentration in the medium [231].
One interesting point related to glycerol metabolism is
that of its transport. In S. cerevisiae, glycerol produced
intracellularly leaves the cell through a channel protein
encoded by the gene FPS1 [232]. If the medium is hyper-
osmotic, the activity of the channel is reduced and glycerol
remains predominantly in the cell [233]. Uptake of glycerol
may be by passive diusion, although active transport
systems have been described in some yeasts acting, mainly,
in conditions of osmotic stress [234,235].
10. Gluconeogenesis
NCY are able to grow in dierent non-carbohydrate
carbon sources. In order to synthesize the sugar phos-
phates necessary for the synthesis of several cell compo-
nents, many glycolytic steps, or all of them, need to be
reversed depending on the non-sugar carbon source used.
Most of the glycolytic reactions are reversible under phys-
iological conditions; however, two reactions cannot be
reversed due to the unfavorable concentrations of sub-
strates found in the cell. These are the reactions catalyzed
by phosphofructokinase and pyruvate kinase. Therefore,
to synthesize fructose 6-phosphate and phospho-enol-py-
ruvate during gluconeogenesis, specic `gluconeogenic' en-
zymes catalyze reactions that are dierent from a simple
FEMSRE 689 29-8-00
reversal of those catalyzed by phosphofructokinase or py-
ruvate kinase.
Fructose-1,6-bisphosphatase catalyzes the hydrolysis of
fructose-1-6-bisphosphate to fructose-6-P. This reaction is
the terminal step of gluconeogenesis and therefore the cor-
responding enzyme is required for metabolism of every
non-sugar carbon source. The gene encoding this enzyme
has been isolated from S. pombe [236] and from K. lactis
[237] and the enzyme has also been puried from C. utilis
[238]. The protein from K. lactis contains a short amino
acid insert sequence near the binding site of the allosteric
inhibitor AMP absent in the S. cerevisiae protein. This
amino acid stretch seems to be responsible for the lower
sensitivity to AMP inhibition of this enzyme with respect
to that of S. cerevisiae [237]. The expression of the gene is
usually repressed in the presence of glucose, although dif-
ferences in the degree of repression are observed among
species. In the case of S. pombe, the pathway of repression
implicates a cAMP signalling pathway [239] (see Section
11).
The enzyme responsible for the synthesis of phospho-
enol-pyruvate is phosphoenolpyruvate carboxykinase that
synthesizes it from oxaloacetate in an ATP-dependent re-
action. In this respect, the yeast enzymes identied so far,
dier from those of other organisms that require GTP. In
addition to the gene PCK1 isolated from S. cerevisiae
[240,241] genes encoding this enzyme have been isolated
from K. lactis [242] and C. albicans [243] and present a
high sequence homology with the gene from S. cerevisiae.
In this yeast, the enzyme is localized in the cytosol [244] a
dierence with the localization in some mammalian spe-
cies. No data exist about its localization in NCY. Tran-
scription of the C. albicans gene is repressed by glucose
[243], a situation similar to that found in S. cerevisiae
[241,245].
Isocitrate lyase and malate synthase that may be also
necessary during gluconeogenesis when the organisms
grow on 2C or 1C carbon sources are considered in Sec-
tion 5.1.
An enzyme that may be also considered as gluconeogen-
ic, is the malic enzyme that catalyzes the oxidative decar-
boxylation of malate to pyruvate using NAD(P)
. This
enzyme is important during growth in malate and has
been identied in S. pombe [246] and Zygosaccharomyces
bailii [247,248]. In Z. bailii the enzyme seems constitutive
[248], while in S. pombe, transcription of the gene mae2
,
encoding malic enzyme is induced when cells are grown in
high concentrations of glucose or under anaerobic condi-
tions [249]. It has been suggested that this enzyme may
play a role in the provision of pyruvate or in maintaining
the redox potential under certain conditions [249,250].
It should be remembered that growth on certain gluco-
neogenic substrates, for example organic acids, needs the
operation of specic transporters to bring these com-
pounds into the cell. Also, a series of transporters that
interconnect metabolically the mitochondria and the cyto-
sol need to be operative during gluconeogenesis. We have
not dealt with those proteins in this review, but they are
important components for the operation of the respective
pathways.
11. Catabolite repression
In S. cerevisiae metabolism of easily used sugars causes
repression of the transcription of genes that encode en-
zymes necessary for the use of alternative carbon sources;
this process is known as catabolite repression. Although
we will not go into a deep treatment of catabolite repres-
sion, some important ndings will be considered. A great
amount of work on the mechanism of catabolite repres-
sion has been done in S. cerevisiae and a plethora of genes
implicated in it has been identied (for a review see [251]).
Much less work exists on catabolite repression in NCY.
In dierent yeasts, glucose produce an increase in the
intracellular concentration of cAMP [143], but in S. cere-
visiae cAMP only aects the repression of some genes and
even for those, redundant regulatory mechanisms exist
[252]. In contrast, in S. pombe, catabolite repression of
the fbp1
gene, encoding fructose-1,6-bisphosphatase, is

dependent on a cAMP signalling pathway. Several genes
named git (glucose insensitive transcription) participate in
the repression process. One of them, git2
/cyr1
, encodes
an adenylate cyclase. Loss of function of git2
, causes
constitutive fbp1
transcription, and addition of cAMP

to the growth medium restores the repression [239]. Inter-
estingly, the cAMP signalling pathway does not seem im-
portant for catabolite repression of the inv1
gene encod-
ing invertase [38].
Hexokinase II is implicated in S. cerevisiae in catabolite
repression of several genes, among then SUC2 that en-
codes invertase [253]. Mutations in RAG5, the K. lactis
gene encoding hexokinase, did not aect repression of
the gene encoding invertase [22], but decreased transcrip-
tion of RAG1 which encodes the low anity glucose trans-
porter [19]. In C. utilis, high hexokinase activity is not
related to glucose repression; in fact, hexokinase activity
seemed inversely proportional to glucose repression, since
cells growing in a chemostat at low concentration of glu-
cose had a higher level of hexokinase than in repressed
cells [254].
A gene that plays a central role in catabolite repression
in S. cerevisiae is SNF1 that encodes a protein kinase that
participates in a complex necessary for growth in non-
fermentable carbon sources [251]. Mutants unable to
grow in galactose, melibiose, maltose, ranose, glycerol,
ethanol or lactate have been isolated in K. lactis and
named fog (fermentative and oxidative growth negative)
[255]. These mutants were unable to derepress several glu-
cose repressible enzymes. Identication of the FOG1 and
FOG2 genes revealed that they were related by their mech-
anism of action. FOG2 is homologous to S. cerevisiae
FEMSRE 689 29-8-00
SNF1 and can complement the corresponding snf1 mu-
tant. FOG1 encodes a protein with sequence similarity to
Gal83, a protein implicated in S. cerevisiae, in the inter-
action of Snf1 and the regulatory protein Snf4 [251]. How-
ever, no evidence for the existence of an equivalent of Snf4
in K. lactis is available. While S. cerevisiae gal83 mutants
did not show a noticeable phenotype, K. lactis fog1 mu-
tants are unable to grow in a series of substrates dierent
from glucose [255]. This could be explained by the fact
that in S. cerevisiae the Gal83 functions can also be per-
formed by the proteins Sip1 or Sip2 that are absent in K.
lactis [251].
In contrast with the situation in other yeasts, SNF1 is
an essential gene in C. albicans or C. tropicalis, but not in
Candida glabrata [256^258].
Another central gene in catabolite repression in S. cere-
visiae is MIG1 which encodes a C2H2 zinc nger repressor
[259]. Mig1 exerts its repressive eect by recruiting to the
transcription start in the DNA a complex in which the
protein Tup1 is the actual repressor [260]. The Mig1 ho-
molog from K. lactis plays a role in the repression of
lactose metabolism [261], but does not participate in the
repression of invertase [36]. In C. albicans, no eects of
CaMIG1 on repression of CaMAL2 that encodes an K-
glucosidase or CaGAL1 that encodes galactokinase, have
been found [201]. On the contrary, in S. pombe, an intact
src1+ gene, a homolog of MIG1, is required for repression
of inv1
[38]. Genes encoding proteins similar to Tup1

have been cloned in K. lactis and in S. pombe [262]. The
gene found in K. lactis complemented the tup1 phenotype
in S. cerevisiae [262]. Two genes were found in S. pombe,
tup11
and tup12
; disruption of both caused a defect in

glucose repression of fbp1
[262]. In C. albicans, TUP1 has

not been related with catabolite repression, but its disrup-
tion gives rise to a lamentous morphology in all growth
conditions tested [263].
12. Plasma membrane H
+
-ATPase
Although not directly related to the energetic metabo-
lism, we think it is necessary to mention plasma membrane
H
-ATPase in this review. The activity of this proton

pump is critical for dierent processes, among them trans-
port of several nutrients and maintenance of intracellular
pH. The enzyme from S. cerevisiae, encoded by the PMA1
gene, is an essential protein and has been studied in great
detail as it is subject to several regulatory mechanisms at
dierent levels [264].
In S. pombe two genes, pma1
and pma2
, that encode
plasma membrane H
-ATPases have been isolated. The

most abundant of these proteins with a molecular mass
of 105 kDa is that encoded by pma1
[265,266]. The
pma2
gene is weakly expressed and is not essential for

growth; it encodes a protein about the same size as Pma1
(molecular mass 119 216 Da). When the coding region of
pma2
is expressed from a strong promoter, it is able to

complement the lethal phenotype of an S. pombe pma1
mutant [266]. In S. pombe, as it happens in S. cerevisiae,

the activity of the plasma membrane H
-ATPase is in-
creased by glucose [267]. This activation is likely due to
a phosphorylation and could implicate the MAP kinase
encoded by the spm1
/pmk1
and the protein phosphatase

encoded by the pzh1
gene. This is suggested by the fact

that an spm1
3
pzh1
3
mutant exhibits a lower level of H
-
ATPase activity that cannot be activated by glucose [268].
In K. lactis a gene (KlPMA1) encoding a plasma mem-
brane H
-ATPase has been isolated [269]. The protein is

899 amino acids long and is structurally related to other
H
-ATPases except for the presence of a non-homolog N-

terminal domain. A change from G to A at position 699
resulted in the substitution of a highly conserved methio-
nine by isoleucine and yielded a protein with a low ca-
pacity to pump protons [269].
The plasma membrane H
-ATPase has been also puri-

ed from C. albicans [270]. In contrast with what happens
in S. cerevisiae or in S. pombe, the enzyme from C. albi-
cans responds weakly to glucose and its activity appears to
change mainly during morphogenetic changes [271].
13. Concluding remarks
This review was written as an attempt to systematize the
existing knowledge on some central metabolic pathways in
NCY. Although a certain amount of information is avail-
able on carbon and energy-yielding metabolism in NCY,
there are abundant gaps of knowledge in this area. It is
clear that dierent yeasts species have been selected
through evolution to occupy dierent ecological niches.
Therefore, it is not surprising to nd among NCY a di-
versity in the nature of the components or the regulation
of the pathways examined. The pioneering ideas of Albert
Kluyver about unity and diversity in the metabolism of
microorganisms presented in his classical lecture Èenheid
en verscheidenheid in de stofwisseling der microben' [272]
are nicely illustrated by these organisms: a similar basic
design for the metabolic pathways with dierences in sug-
ar transport systems, equipment of sugar phosphorylating
enzymes, intracellular localization of certain proteins, or in
the regulation of the pathways.
The dierences between NCY and the classical `yeast' S.
cerevisiae do not allow an immediate translation of the
available knowledge from this organism to NCY. Cer-
tainly, the wealth of information available from S. cerevi-
siae will be a good starting point when considering NCY;
however, it shall be kept in mind that these organisms may
present surprising and interesting variations.
The completion of the sequence of the S. cerevisiae ge-
nome and the availability of disruptions, or easy methods
to perform them, in practically every gene has opened
enormous possibilities to identify and isolate genes of in-
FEMSRE 689 29-8-00
terest from NCY, thus opening the doors to the perform-
ance of reverse genetics in these organisms. Such a study
of the components of central pathways in NCY will be
fruitful and rewarding both from a pure and an applied
point of view. Regulatory problems in carbon and energy
metabolism have not been much studied in NCY,
although data in the literature indicate important dier-
ences in many cases, both among them and with respect to
S. cerevisiae. Precise knowledge of regulatory mechanisms
is of extreme interest when trying to modify organisms for
specic uses, or when using some genes encoding regulated
proteins to be expressed in other yeasts. This knowledge is
also important when trying to model the behavior of NCY
systems for industrial settings.
A topic that needs deeper study is that of the relations
between the dierent organelles ^ mitochondria, peroxi-
somes, nucleus ^ and the cytosol. Characterization of
transport systems implicated in the trac of a series of
molecules is of crucial importance to understand the met-
abolic implications of this trac. Some NCY in which
particular organelles are more prominent than in S. cere-
visiae will be the objects of choice for such studies.
Another area of interest in some NCY yeasts is the
relationship between energy metabolism and dierentia-
tion processes. In fact this topic is being pursued vigo-
rously at this moment, particularly in those cases where
the dierentiation process is related with pathogenicity.
Readers, particularly non-specialists, will benet from a
uniform nomenclature of genes among dierent NCY.
Genes encoding proteins with the same function are often
called dierently by scientists working with dierent yeast
species; hexose transporters are HXT in S. cerevisiae, but
ght
or std
in S. pombe, hexokinases are HXK in S. ce-

revisiae, but RAG in K. lactis; ACS is acetyl CoA synthe-
tase in S. cerevisiae, but ICL2 in Y. lipolytica; trehalose 6-
P synthase is TPS1 in most yeasts, but GGS1 in K. lactis;
FOG1 and FOG2 in K. lactis are GAL83 and SNF1, re-
spectively, in S. cerevisiae, to name but a few examples.
Perhaps it is time to reach a consensus on how to avoid
this chaos.
It should be kept in mind, that some problems presented
by NCY will not be solved without a detailed, general
appreciation of the organism. This will require the com-
bined knowledge of modern molecular biology tools and
that of the more classical physiology.
Acknowledgements
We thank Juana M. Gancedo and Oscar Zaragoza for
critical reading of the manuscript and useful comments.
Thanks are also due to J.T. Pronk (Delft, Netherlands),
E. Boles (Du sseldorf, Germany), K. Breunig (Halle, Ger-
many), M. Ho fer (Bonn, Germany), D. Porro (Milan,
Italy), H.Y. Steensma (Leiden, Netherlands), H. Fukuhara
(Orsay, France), T. Caspari (Brighton, UK), C. Gaillardin
(Grignon, France), G. Barth (Dresden, Germany) and J.
D| az Brito (La Habana) who provided information, com-
ments, or both during the writing of the review. Work in
the laboratory of the authors was supported by Grant
PB97-1213-CO2-01 from the Spanish CICYT and by Proj-
ect BIO4-CT95-0132 `From gene to products in yeasts: a
quantitative approach' from the European Union (DGXII
Framework IV, Program of Cell Factories). C.L.F. thanks
the academic authorities of the Facultad de Biolog| a, Uni-
versidad de La Habana, Cuba, for a leave of absence.
C.R. benetted from a Fellowship from the Fundacio n
Ramo n Areces (Madrid, Spain). The stay of T.P. in Delft
is supported by the Marie Curie Biotechnology Program
from the European Union (Grant ERB-4001GT980575/
Project BIO4-CT98-5096).
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Carbohydrate and Energy-Yielding Metabolism in Non-Conventional Yeasts

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Carbohydrate and Energy-Yielding Metabolism in Non-Conventional Yeasts

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Carbohydrate and energy-yielding metabolism in non-conventional

strains possess RAG1 and HTG1,

-symport ; since then

[34]. Overexpression of this gene short-

-symport [39] and hydrolyzed by an

, which could encode an eno-

gene from S. pombe has allowed purication

-dependent isocitrate dehydrogenase, whose two

-dependent enzyme are glutamate auxo-

. The enzyme from

and could, in principle,

-dependent xylitol dehydrogenase. Xylulose is

-dependent, cytosolic enzymes.

. These enzymes also showed activity with oth-

-dependent alcohol dehydro-

as cofactor, are stress-induced and repressed by glucose;

and is also repressed by

, is constitutive and activated by

(see Section 6). In S. cerevisiae, mutants lack-

, is constitutively transcribed although

-dependent glycerol dehy-

gene, encoding fructose-1,6-bisphosphatase, is

transcription, and addition of cAMP

[38]. Genes encoding proteins similar to Tup1

; disruption of both caused a defect in

[262]. In C. albicans, TUP1 has

-ATPase in this review. The activity of this proton

-ATPases have been isolated. The

gene is weakly expressed and is not essential for

is expressed from a strong promoter, it is able to

mutant [266]. In S. pombe, as it happens in S. cerevisiae,

and the protein phosphatase

gene. This is suggested by the fact

-ATPase has been isolated [269]. The protein is

-ATPases except for the presence of a non-homolog N-

-ATPase has been also puri-

in S. pombe, hexokinases are HXK in S. ce-

zcan, S. and Johnston, M. (1999) Function and regulation of yeast

gene product. J. Cell. Sci. 110, 2599^2608.

symporter of Schizosaccharomyces pombe cells is down-regulated by

-specic isocitrate dehydrogenases in Saccharomyces

-dependent alcohol dehydrogenases

symport in the halotolerant yeast Pichia sorbitophila. Yeast 11, 111^

-ATPase in ssion yeast. FEBS Lett. 435, 241^244.

)-ATPase from Kluyveromyces

uptake. J. Bacteriol. 177,

)-ATPase of Candida albicans. Biochem. Int. 24, 907^915.

)-ATPase during yeast growth

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