Carbohydrate and energy-yielding metabolism in non-conventional yeasts1

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Carbohydrate and energy-yielding metabolism in non-conventional yeasts1 Carmen-Lisset Flores 2 , Cristina Rodr|¨guez, Thomas Petit 3 , Carlos Gancedo * Instituto de Investigaciones Biome¨dicas Alberto Sols C.S.I.C.-UAM, Unidad de Bioqu|¨mica y Gene¨tica 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. ß 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

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uptake of sugars . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Common hexoses . . . . . . . . . . . . . . . . . . . . . 2.2. Other sugars . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Disaccharides . . . . . . . . . . . . . . . . . . . . . . . . The common glycolytic trunk . . . . . . . . . . . . . . . 3.1. From intracellular sugar to trioses phosphate 3.2. From trioses phosphate to pyruvate . . . . . . . Metabolic alternatives of intracellular pyruvate . . 4.1. Pyruvate dehydrogenase . . . . . . . . . . . . . . . . 4.2. Pyruvate decarboxylase . . . . . . . . . . . . . . . . . 4.3. The pyruvate bypass . . . . . . . . . . . . . . . . . . . The TCA cycle . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Anaplerotic reactions . . . . . . . . . . . . . . . . . . Oxidation of NADH and energy generation . . . . . The pentose phosphate pathway . . . . . . . . . . . . . 7.1. The genes of xylose metabolism . . . . . . . . . .

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* Corresponding author. Fax: +34 (91) 585 4587; E-mail: [email protected] 1 2 3

This article is dedicated to the memory of Niko van Uden, an enthusiastic yeast researcher and a cultivated person. On leave of absence from Departamento de Bioqu|¨mica, Facultad de Biolog|¨a, Universidad de La Habana, Cuba. Present address: DSM Bakery Ingredient Division, 26000 MA Delft, The Netherlands.

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Ethanol metabolism . . . . . . . . . Glycerol metabolism . . . . . . . . Gluconeogenesis . . . . . . . . . . . Catabolite repression . . . . . . . . Plasma membrane H‡ -ATPase . Concluding remarks . . . . . . . .

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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 di¡erent 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 scienti¢c 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 di¡erent species may also be interesting for a variety of reasons ranging from their use in speci¢c 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 researchers in the last years on di¡erent grounds: Kluyveromyces 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 capacity, 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-

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phosphate or fructose 6-phosphate to pyruvate through the common glycolytic trunk (Fig. 1). The metabolic destiny of pyruvate is then di¡erent 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 e¡ect) [4,5] and therefore, even in the presence of air, S. cerevisiae ferments sugar in batch cultures. Some other yeasts, socalled `Crabtree positive', also behave in this way. However, in most cases in aerobic conditions, pyruvate is oxidized to CO2 through the tricarboxylic acid (TCA) cycle. Glycolysis and the TCA cycle are therefore central pathways of metabolism. They perform a dual role in organisms : 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. cerevisiae. 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 di¡erent aspects of carbohydrate and energy metabolism in S. cerevisiae are available to which the reader may usefully 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 di¡erent 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 reactions 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 implicate 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 di¡erent from glycolysis have been used: 6PG, 6-phosphogluconate; Rul5P, ribulose 5-phosphate; R5P, ribose 5-phosphate ; Xul5P, xylulose 5-phosphate; S7P, sedoheptulose 7-phosphate; E4P, erythrose 4-phosphate; G3P, glycerol 3-phosphate; Pi , `inorganic' phosphate ; OAA, oxaloacetate; Q, ubiquinone ; bc1, cytochromes ; Cyt ox, cytochrome oxidase.

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cycle or morphogenesis may consider it as quite conventional. Some background information and a treatment of several aspects of cultivation, and molecular biology techniques 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 speci¢c transporters, sometimes called permeases. In S. cerevisiae, a family of glucose transporters with Km ranging from 1 to 100 mM, has been characterized. The transcription 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 monosaccharides, glucose, fructose or mannose in S. cerevisiae is a facilitated di¡usion process ; however, the situation may be di¡erent in other yeasts. In K. lactis glucose transport appears to proceed by facilitated di¡usion and two genes encoding transporters with di¡erent a¤nities for glucose have been identi¢ed in this yeast: HGT1 that encodes a high a¤nity glucose transporter, Km 1 mM, [15] and RAG1 that encodes a transporter with low a¤nity for glucose, Km 20^50 mM [16]. Overexpression of HGT1 could not restore the wildtype 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 di¡erent sensitivity to the respiratory 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 Rag3 . Wild-type Rag‡ strains possess RAG1 and HTG1, while Rag3 strains have lost RAG1 [17]. RAG1 seems to have arisen by a recombination event between two genes, KHT1 and KHT2, that encode low a¤nity glucose transporters and are found in tandem in some strains. The sequence of Rag1 is identical to that of Kht1 except for an arginine and a phenylalanine, the last two C-terminal amino acids, which are exchanged for the last three amino acids of Kht2 (alanine^methionine^leucine) [18].

Expression of HGT1 does not vary with culture conditions [15] while transcription of RAG1 that is undetectable during growth in glycerol increases during growth in glucose [17]. It is not known which signal(s) trigger the induction of RAG1 expression [19], but an extended glycolysis does not seem to be required since a rag2 mutant, defective in phosphoglucose isomerase did not show decreased levels of RAG1 mRNA [20,21]. Several genes a¡ect 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 2.0kb 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, suggesting that RAG4 is epistatic over RAG5 for the regulation of the transcription of HGT1 [15]. The RAG8 gene, an apparently essential gene that shows a high degree of identity with the two casein kinases I of S. cerevisiae [23], also abolished expression of RAG1 [19], but did not a¡ect that of HGT1 [15]. These results show that di¡erent controls distinctly a¡ect the transcription of both genes. RAG1 expression was una¡ected in a hgt1 null mutant and vice versa. A double mutant rag1 hgt1 showed a greatly reduced 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 a¤nity 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 levels 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 di¡usion operates when the cells are grown at higher glucose concentrations [25]. Weierstall et al. [27] isolated three genes encoding glucose transporters from the xylose fermenting yeast Pichia stipitis with Km values around 1 mM. Expression of one of these genes, SUT1, was induced by glucose while the expression of the others, SUT2 and SUT3, was constitutive. A transport system for glucose with two components with Km 3 and 10 mM respectively, was found in Y. lipolytica. These components were present with the same activity independently of the glucose concentration in the medium [28]. In Schizosaccharomyces pombe Ho«fer and Nassar [29] identi¢ed hexose transport as a H‡ -symport ; since then

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a family of hexose transporters has been identi¢ed with six components called Ght1 to Ght6 [30]. Ght5 is the most abundant of these transporters. Ght1, Ght2 and Ght5 exhibited higher activities with glucose than with fructose as substrate [30]. Mehta et al. [31] isolated two S. pombe mutants de¢cient in glucose transport; one of them has, apparently, an altered a¤nity 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) a¡ected 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 transport [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 shortened 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 implicates 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 invertase repressed by glucose. In K. lactis, the gene KlINV1 encoding invertase has been isolated. It presents high amino acid homology with the invertase from S. cerevisiae and with the inulinase ^ a fructofuranosidase that hydrolyzes 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]

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whose expression is induced by lactose or galactose [32]. Once transported, lactose is hydrolyzed by a L-galactosidase, 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 mannose are phosphorylated by hexokinases; a glucokinase can phosphorylate glucose and mannose and a galactokinase phosphorylates galactose. The enzymatic equipment for hexose phosphorylation varies among di¡erent yeasts, although no physiological explanation for the di¡erences has been found. While S. cerevisiae possesses two hexokinases and a glucokinase [44], K. lactis has only one hexokinase encoded by the RAG5 gene. rag5 mutants are not only unable to grow in glucose or fructose, but also unexpectedly, in lactose [22]. While the transcription rates of the genes HXK1, HXK2 and GLK1 that encode the hexokinases and the glucokinase of S. cerevisiae are controlled by glucose [45], the levels of RAG5 mRNA did not vary signi¢cantly between cultures in glucose or in glycerol [22]. In Candida tropicalis, two hexokinases and a glucokinase were found in chromatographic studies. The levels of the hexokinases were higher when the yeast was cultured 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 signi¢cantly a¡ect growth in glucose, it has been suggested that the major glucose phosphorylating capacity in vivo is due to glucokinase [47]. Two hexokinases are found in S. pombe, a major one, hexokinase 2, with a Km for glucose around 0.1 mM, the usual range for hexokinases, and a minor one, hexokinase 1, that has a low a¤nity for glucose (Km 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 a¤nity 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 compound that plays an important role in the regulation of glycolysis in S. cerevisiae [51]. The hexokinase of Y. lipo-

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lytica shows the strongest inhibition by trehalose-6-P yet found (Ki 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 di¡erent 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 phosphoglucose 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 Goffrini et al. [20] reported that the rag2 mutant did not produce ethanol during growth in glucose, Gonza¨lez-Siso et al. [54] detected a signi¢cant 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-P2 . The activity of the enzyme is a¡ected by a large variety of e¡ectors and has been the paradigm for `multimodulated enzymes' [55]. Phosphofructokinase 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 composed of two di¡erent 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 phosphofructokinase 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 phosphofructokinase still grow in glucose if respiration is functional, indicating again that the activity of the pentose phosphate 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. Simultaneous 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 contrast with that of S. cerevisiae, but was activated by fructose-2,6-P2 and AMP and inhibited by ATP. Both compounds were equally e¡ective in releasing the ATP inhibition, a di¡erence with S. cerevisiae where fructose2,6-P2 is the most e¡ective one [59]. Lorberg et al. [63] cloned two genes, CaPFK1 and CaPFK2, from the pathogenic yeast C. albicans. They encode proteins of 109- and 104-kDa, respectively, either of which can complement the pfk1 pfk2 phenotype of S. cerevisiae mutants. An interesting feature in the structure of the C. albicans phosphofructokinases is the existence of Nterminal tails of about 180 amino acids that have no counterpart in other phosphofructokinases. The functional implications 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 simultaneously expressed, the hetero-oligomers formed when PFK genes from C. albicans and S. cerevisiae were expressed, 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 e¡ected by fructose-1,6-bisphosphate aldolase that cleaves fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The fructose-1,6bisphosphate 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 puri¢ed; it has a molecular weight of about 70 kDa and contains a Zn2‡ ion as the enzyme from S. cerevisiae [67]. 3.2. From trioses phosphate to pyruvate Triose phosphate isomerase interconverts dihydroxyacetone 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

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[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 lacking 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 behaviour similar to that shown by the rag2 and pfk1 pfk2 mutants (see above). In the step catalyzed by glyceraldehyde-3-phosphate dehydrogenase, 1,3-bisphosphoglycerate is formed from glyceraldehyde 3-phosphate. This is an important step in glycolysis, 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 di¡erent 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, encoding 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 di¡erent carbon sources and apparently encodes a protein that is localized in the cell wall. Transcription of GAP2 is increased in glucose, while transcription of GAP3 was not detected in the conditions used [72]. In C. albicans, a glyceraldehyde-3-phosphate dehydrogenase, 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 glyceraldehyde-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 production of proteins in this yeast as an alternative to the commonly used AOX1 promoter [75]. In the reaction catalyzed by 3-phosphoglycerate kinase the energy-rich acylphosphate from 1,3-bisphosphoglycerate 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

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[78]. In contrast with C. maltosa, the gene encoding 3phosphoglycerate kinase in C. albicans is similarly expressed 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 proline 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-terminus that in S. cerevisiae has been implicated in catalytic activity [81], and also a stretch of 24 aa that in S. cerevisiae appears to be involved in interactions between subunits. Another loop also involved in these types of interactions is absent in the S. pombe protein [82]. Enolase catalyzes the dehydration of 2-phosphoglycerate 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 constitutive, that of ENO2 is stimulated about 20-fold by glucose, suggesting a di¡erential participation of the isoenzymes 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 enolase gene loci that could be distinguished by the localization of particular restriction sites in their 3'£anking regions, 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 di¡erences 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 speci¢c di¡erences between ethanol and pyruvate, although this seems unlikely. In S. pombe a gene, eno1‡ , which could encode an enolase has been identi¢ed ; the corresponding 1.4-kb transcript is more abundant during growth in glucose than during growth in gluconeogenic substrates [87]. Pyruvate kinase catalyzes the second ATP forming reaction in the glycolytic pathway. In S. cerevisiae, two genes exist that encode two di¡erent 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 compound. 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

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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 puri¢cation of the enzyme that behaves as a dimer [89]. The organization 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-bisphosphate 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-1,6bisphosphate 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 produced in glycolysis are either its oxidation to CO2 or its transformation to ethanol. In most yeasts under aerobic conditions, oxidation will be predominant, while transformation to ethanol takes place in anaerobic conditions or at high glucose concentrations even in aerobic conditions in those yeasts that present a Crabtree e¡ect (for a review see [94]). 4.1. Pyruvate dehydrogenase Oxidation of pyruvate to CO2 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 di¡erent components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyl transferase (E2) and dihydrolipoamide 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 localization of the pyruvate dehydrogenase complex implies that cytosolic pyruvate must be transported into the mitochondrion. 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 identi¢ed. In K. lactis, disruption of the gene KlPDA1, that encodes the E1K subunit of the complex, led to a decrease of the speci¢c growth rate in glucose in minimal medium, while growth in rich medium or in ethanol was not affected. Also ethanol was produced in aerobic batch cultures 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 decarboxylase [103]. The E1L subunit of the pyruvate dehydrogenase complex has been cloned from S. pombe, but no

studies on the e¡ects of its disruption have been reported [104]; however, the same authors found that mutants from the ¢ssion yeast with reduced pyruvate dehydrogenase activity were auxotrophic for arginine or glutamine [105]. In P. stipitis, the activity of pyruvate dehydrogenase is constitutive [106]. 4.2. Pyruvate decarboxylase In S. cerevisiae, several genes encoding pyruvate decarboxylase have been found although PDC1 normally accounts 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. cerevisiae lacking pyruvate decarboxylase, K. lactis rag6 mutants 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 mutant (see Section 4.3) and highlights once more the di¡erent 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 decarboxylase 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 ine¡ective [109]. In P. stipitis two genes encoding pyruvate decarboxylase have been found, but detailed studies on their physiological roles are not available. The PsPDC1 gene has an 81bp 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 signi¢cance of this by-pass is shown by the absence of growth of S. cerevisiae pdc mutants 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

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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 concentrations 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 citrate. Three genes encoding functional citrate synthases have been identi¢ed 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 polypeptide has been identi¢ed. 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 containing 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 dependent on the function of the gene YFH1 that encodes frataxin, 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 2oxoglutarate (K-ketoglutarate). In S. cerevisiae an NAD‡ -dependent isocitrate dehydrogenase, whose two di¡erent subunits are encoded by the genes IDH1 and IDH2 [121,122], participates in the TCA cycle [123]. Three di¡erently compartmentalized NADP‡ -dependent isocitrate 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 lacking an NAD‡ -dependent enzyme are glutamate auxotrophs [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

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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 carboxylase 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 glucose when ammonium is the nitrogen source but can grow when glutamate or aspartate is added [129]. The situation is di¡erent from that of S. cerevisiae where glutamate cannot substitute aspartate for growth of a pyruvate carboxylase 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 isocitrate 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 puri¢ed 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 (Pichia) polymorpha has been cloned [140]. The enzyme from C. tropicalis, a homotetrameric protein of molecular mass 250 kDa, was localized in the matrix of the peroxisomes [138], but the protein from H. polymorpha does not have a conventional peroxisomal targeting 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

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two di¡erent 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 cytosol or in the mitochondria. During fermentation, NADH produced in glycolysis is reoxidized to NAD‡ by the formation of ethanol; NADH arising in reactions di¡erent from glycolysis is regenerated mainly by the production of glycerol. During aerobic growth, `external' 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 oxidoreductase [146]. NADH generated in reactions occurring in the mitochondria is regenerated by a NADH dehydrogenase located in the inner mitochondrial membrane facing 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 adequate redox balance in the cytosol [148]. Reoxidation of NADH by the respiratory chain is the main source of energy for cells with a respiratory metabolism. This reoxidation is coupled to the production of ATP in the process known as oxidative phosphorylation. Although we will not discuss in detail oxidative phosphorylation, 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 speci¢c ADP/ATP translocators. In S. cerevisiae, three genes encoding mitochondrial ADP/ATP translocators have been identi¢ed : 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 completely clear; curiously, it has been implicated in the vacuolar 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 ra¤nose [158] and null anc13 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 cytochrome pathway. This pathway is speci¢cally 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 ubiquinone 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 mitochondrial protein of 36 kDa [165] whose synthesis is induced by antimycin A and cyanide or by sulfur-containing 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 logarithmic to stationary growth phase in glucose cultures [170]. In P. stipitis, the alternative pathway seems important 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 a¡ect growth yield; however, both parameters were severely a¡ected during growth in glycerol. The e¡ect was due to competition for ubiquinone between the plant oxidase and the normal cytochrome pathway [173]. The physiological role of the alternative respiratory pathway in yeasts is still not clearly understood; it could act as an energy over£ow 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 metabolic route implicated in the production of NADPH for biosynthetic reactions and of ribose 5-phosphate for synthesis of nucleic acid and nucleotide cofactors; it also provides erythrose 4-phosphate for the synthesis of aromatic amino acids. The ¢rst reactions of the pathway are two

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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 in£uences 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 oxidation of glucose-6-phosphate to 6-phospho-N-gluconolactone. Glucose-6-phosphate dehydrogenase has been puri¢ed from S. pombe [176] and has Km values of 0.66 mM for glucose-6-P and 44 WM for NADP‡ . The enzyme from C. utilis has been puri¢ed [177], it has a molecular weight of 11 or 22 kDa depending on pH and the presence of Mg2‡ . Another group puri¢ed the enzyme from a strain of Pichia jadinii (synonym of C. utilis) and found its N-terminal 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 signi¢cance 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 methionine biosynthesis that require NADPH. It could be interesting to test the phenotype of similar mutants in NCY to ascertain the existence of alternative, e¡ective pathways of NADPH provision. The hydrolysis of 6-phosphate-N-gluconolactone to 6phospho-gluconate occurs spontaneously at neutral pH, but at a very slow rate. A lactonase that accelerated this rate was partially puri¢ed 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 bacterium Zymomonas mobilis [181] and from the fungus Fusarium oxysporum [182]. In those yeasts able to use gluconate, 6-phospho-gluconate may be also formed by the action of 6-phospho-gluconate kinase, an enzyme that has been kinetically characterized in S. pombe [183]. 6-Phospho-gluconate dehydrogenase catalyzes the oxidative decarboxylation of 6-phospho-gluconate to ribulose 5-phosphate. The enzyme has been puri¢ed 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 a¤nity for its substrates (Km 55 WM for 6-P-

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gluconate and 20 WM for NADP‡ ) [185]. In S. cerevisiae, mutants lacking this activity are unable to grow in glucose, but elimination of the glucose-6-phosphate dehydrogenase activity allowed growth in this sugar [186]. The authors concluded that the accumulated 6-phospho-gluconate 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 puri¢ed from C. utilis by Domagk and Doering [187] and by Horitsu et al. [188]. The data about molecular mass and subunit composition di¡er between these authors. Ribulose-5P epimerase catalyzes the epimerization of ribulose 5phosphate 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 carbon) moiety is transferred from a ketose donor to an aldose acceptor. In both reactions, the donor is xylulose 5phosphate, but the acceptor is in one case ribose 5-phosphate and in other erythrose 4-phosphate. Two genes encoding transketolases have been isolated from S. cerevisiae, TKL1 and TKL2. Transketolase has been puri¢ed from C. utilis and shown to have high a¤nity both for xylulose5-P (Km 80 WM) and ribose-5-P (Km 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. cerevisiae [191] (see below). It should be mentioned that in methylotrophic yeasts, a transketolase reaction catalyzed by a di¡erent enzyme (dihydroxyacetone synthase) is responsible for the formation of dihydroxyacetone and glyceraldehyde-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. cerevisiae which functions in the pentose phosphate pathway [192]. Transaldolase catalyzes the reversible formation of glyceraldehyde-3-P and sedoheptulose-7-P from erythrose-4-P and fructose-6-P. Mutants from K. lactis lacking this enzyme, encoded by the gene KlTAL1, have been instrumental to highlight an important di¡erence 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 exclusively 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).

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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 monophosphate pathway [48,183]. However, the ¢nding that mutants lacking hexokinase activity do not grow in glucose is not easily accommodated with this proposed function [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. However, the yeast with the best fermentation capacity, S. cerevisiae 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 implies 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 postulated 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 di¡erent proteins with distinct coenzyme speci¢city [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 puri¢ed [198,199] and the XYL1 gene from Candida guilliermondi 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 e¤cient 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 anaerobiosis, 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. Central to the production or the utilization of ethanol are alcohol dehydrogenases, enzymes that catalyze the reversible 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 reaction catalyzed under physiological conditions. However, in many cases, the precise physiological role of some alcohol dehydrogenases is far from being clearly established. In S. cerevisiae, four genes encode isoenzymes of alcohol dehydrogenases. ADH1 encodes the isoenzyme prominent during glucose fermentation and is induced by glucose [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 de¢cient in alcohol dehydrogenase 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 other 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 alcohol 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 identi¢ed, encoded by genes called PsADH1 and PsADH2 [212,213]; unfortunately 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 anaerobic growth and disruption of the gene brings about an increase of PsADH2 expression on non-fermentable carbon sources [212]. In Y. lipolytica an NAD‡ -dependent alcohol dehydrogenase which acts better on long chain alcohols has been identi¢ed. Three other NADP‡ -dependent dehydrogenases

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were also detected [214]. These authors made the interesting 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 proposed a logical nomenclature and tried to clarify the situation. 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 proteins 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 Mg2‡ . Ald4 and Ald6 seem important for growth in ethanol [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 aldehyde dehydrogenases from NCY is available. The transformation of acetate into acetyl CoA is catalyzed 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 ethanol[217], while disruption of the other, ACS2, caused inability to grow in glucose [113,218]. The subcellular localization 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]. Acetyl 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 dehydrogenase. However, production of glycerol under anaerobic conditions is a secondary, but important, way to regenerate NAD‡ (see Section 6). In S. cerevisiae, mutants lacking alcohol dehydrogenase, still produce ethanol, but increase their glycerol production during glucose fermentation [221]. The situation is di¡erent in K. lactis, where a mutant lacking alcohol dehydrogenase activity did not accumulate much glycerol [222]. Glycerol production

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also plays another important role, namely it allows adaptation of the yeast to situations of osmotic stress. Glycerol formation requires the sequential action of a NADH glycerophosphate dehydrogenase and a glycerophosphatase. In S. pombe ^ as in S. cerevisiae [223] ^ two genes have been identi¢ed 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. cerevisiae, two genes, GPP1 and GPP2, that encode D,L-glycerol3-P phosphatases with relatively low a¤nities, Km about 3^4 mM, for their substrates, have been cloned. Transcription 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 di¡erent pathways of glycerol catabolism have been identi¢ed 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 ubiquinone oxidoreductase [227,228]. In S. pombe dehydrogenation of glycerol by an NAD‡ -dependent glycerol dehydrogenase 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 hyperosmotic, the activity of the channel is reduced and glycerol remains predominantly in the cell [233]. Uptake of glycerol may be by passive di¡usion, 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 di¡erent non-carbohydrate carbon sources. In order to synthesize the sugar phosphates necessary for the synthesis of several cell components, 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 physiological conditions; however, two reactions cannot be reversed due to the unfavorable concentrations of substrates found in the cell. These are the reactions catalyzed by phosphofructokinase and pyruvate kinase. Therefore, to synthesize fructose 6-phosphate and phospho-enol-pyruvate during gluconeogenesis, speci¢c `gluconeogenic' enzymes catalyze reactions that are di¡erent from a simple

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reversal of those catalyzed by phosphofructokinase or pyruvate 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 corresponding 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 puri¢ed 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 differences 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 phosphoenol-pyruvate is phosphoenolpyruvate carboxykinase that synthesizes it from oxaloacetate in an ATP-dependent reaction. In this respect, the yeast enzymes identi¢ed so far, di¡er 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 di¡erence with the localization in some mammalian species. No data exist about its localization in NCY. Transcription 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 Section 5.1. An enzyme that may be also considered as gluconeogenic, is the malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate using NAD(P)‡ . This enzyme is important during growth in malate and has been identi¢ed 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 conditions [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 gluconeogenic substrates, for example organic acids, needs the operation of speci¢c transporters to bring these compounds 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 enzymes 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 repression, some important ¢ndings will be considered. A great amount of work on the mechanism of catabolite repression has been done in S. cerevisiae and a plethora of genes implicated in it has been identi¢ed (for a review see [251]). Much less work exists on catabolite repression in NCY. In di¡erent yeasts, glucose produce an increase in the intracellular concentration of cAMP [143], but in S. cerevisiae cAMP only a¡ects 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]. Interestingly, the cAMP signalling pathway does not seem important for catabolite repression of the inv1‡ gene encoding invertase [38]. Hexokinase II is implicated in S. cerevisiae in catabolite repression of several genes, among then SUC2 that encodes invertase [253]. Mutations in RAG5, the K. lactis gene encoding hexokinase, did not a¡ect repression of the gene encoding invertase [22], but decreased transcription of RAG1 which encodes the low a¤nity glucose transporter [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 glucose 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 nonfermentable carbon sources [251]. Mutants unable to grow in galactose, melibiose, maltose, ra¤nose, 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 glucose repressible enzymes. Identi¢cation of the FOG1 and FOG2 genes revealed that they were related by their mechanism of action. FOG2 is homologous to S. cerevisiae

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SNF1 and can complement the corresponding snf1 mutant. FOG1 encodes a protein with sequence similarity to Gal83, a protein implicated in S. cerevisiae, in the interaction of Snf1 and the regulatory protein Snf4 [251]. However, 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 mutants are unable to grow in a series of substrates di¡erent from glucose [255]. This could be explained by the fact that in S. cerevisiae the Gal83 functions can also be performed 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. cerevisiae is MIG1 which encodes a C2H2 zinc ¢nger repressor [259]. Mig1 exerts its repressive e¡ect by recruiting to the transcription start in the DNA a complex in which the protein Tup1 is the actual repressor [260]. The Mig1 homolog 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 e¡ects of CaMIG1 on repression of CaMAL2 that encodes an Kglucosidase 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 disruption gives rise to a ¢lamentous morphology in all growth conditions tested [263]. 12. Plasma membrane H +-ATPase Although not directly related to the energetic metabolism, we think it is necessary to mention plasma membrane H‡ -ATPase in this review. The activity of this proton pump is critical for di¡erent processes, among them transport 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 di¡erent 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

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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 increased 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 spm13 pzh13 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 membrane 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 Nterminal domain. A change from G to A at position 699 resulted in the substitution of a highly conserved methionine by isoleucine and yielded a protein with a low capacity 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. albicans 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 available on carbon and energy-yielding metabolism in NCY, there are abundant gaps of knowledge in this area. It is clear that di¡erent yeasts species have been selected through evolution to occupy di¡erent ecological niches. Therefore, it is not surprising to ¢nd among NCY a diversity 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 `Eenheid en verscheidenheid in de stofwisseling der microben' [272] are nicely illustrated by these organisms : a similar basic design for the metabolic pathways with di¡erences in sugar transport systems, equipment of sugar phosphorylating enzymes, intracellular localization of certain proteins, or in the regulation of the pathways. The di¡erences between NCY and the classical `yeast' S. cerevisiae do not allow an immediate translation of the available knowledge from this organism to NCY. Certainly, the wealth of information available from S. cerevisiae 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 genome 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-

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terest from NCY, thus opening the doors to the performance 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 di¡erences 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 speci¢c 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 di¡erent organelles ^ mitochondria, peroxisomes, nucleus ^ and the cytosol. Characterization of transport systems implicated in the tra¤c of a series of molecules is of crucial importance to understand the metabolic implications of this tra¤c. Some NCY in which particular organelles are more prominent than in S. cerevisiae will be the objects of choice for such studies. Another area of interest in some NCY yeasts is the relationship between energy metabolism and di¡erentiation processes. In fact this topic is being pursued vigorously at this moment, particularly in those cases where the di¡erentiation process is related with pathogenicity. Readers, particularly non-specialists, will bene¢t from a uniform nomenclature of genes among di¡erent NCY. Genes encoding proteins with the same function are often called di¡erently by scientists working with di¡erent yeast species; hexose transporters are HXT in S. cerevisiae, but ght‡ or std‡ in S. pombe, hexokinases are HXK in S. cerevisiae, but RAG in K. lactis; ACS is acetyl CoA synthetase in S. cerevisiae, but ICL2 in Y. lipolytica ; trehalose 6P synthase is TPS1 in most yeasts, but GGS1 in K. lactis; FOG1 and FOG2 in K. lactis are GAL83 and SNF1, respectively, 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 combined 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, Germany), 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, comments, 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 Project 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, Universidad de La Habana, Cuba, for a leave of absence. C.R. bene¢tted 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).

References [1] Gobetti, I., Corsetti, A., Rossi, J., La Rosa, F. and De Vincenzi, S. (1994) Identi¢cation and clustering of lactic acid bacteria and yeasts from wheat sourdoughs of central Italy. Ital. J. Food. Sci. 6, 85^94. [2] Almeida, M.J. and Pais, C.J. (1996) Characterization of the yeast population from traditional corn and rye bread doughs. Lett. Appl. Microbiol. 23, 154^158. [3] Kurtzman, C.P. and Fell, J.W. (1998) The Yeasts. A Taxonomic Study, Elsevier Science, Amsterdam. [4] De Deken, R.H. (1966) The Crabtree e¡ect: a regulatory system in yeast. J. Gen. Microbiol. 44, 149^156. [5] Fiechter, A. (1981) Regulation of glucose metabolism in growing yeast cells. Adv. Microb. Physiol. 22, 123^183. [6] Sols, A., Gancedo, C. and De la Fuente, G. (1971) Energy-yielding metabolism in yeasts. In: The Yeasts (Rose, A.H. and Harrison, J.S., Eds.), Vol. 2, pp. 271^308. Academic Press, London. [7] Fraenkel, D.G. (1982) Carbohydrate metabolism. In: The Molecular Biology of the Yeast Saccharomyces (Strathern, J.N., Jones, E.W. and Broach, J.R., Eds.), Vol. 1, pp. 1^37. Cold Spring Harbor Laboratory, Cold Spring Harbor. [8] Gancedo, C. and Serrano, R. (1989) Energy yielding metabolism. In: The Yeasts (Rose, A.H. and Harrison, J.S., Eds.), 2nd edn., Vol. 3, pp. 205^260. Academic Press, London. [9] Zimmermann, F.K. and Entian, K.D. (1997) Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology, and Applications, Technomic, Lancaster. [10] Wolf, K. (1996) Nonconventional Yeasts in Biotechnology. A Handbook, Springer, Berlin. [11] Barnett, J.A. (1997) Introduction : a historical survey of the study of yeasts. In: Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology, and Applications (Zimmermann, F.K. and Entian, K.D. Eds.), pp. 1^34. Technomic, Lancaster. [12] Sols, A. (1961) Carbohydrate metabolism. Annu. Rev. Biochem. 30, 213^238. [13] Boles, E. and Hollenberg, C.P. (1997) The molecular genetics of hexose transport in yeasts. FEMS Microbiol. Rev. 21, 85^111. ë zcan, S. and Johnston, M. (1999) Function and regulation of yeast [14] O hexose transporters. Microbiol. Mol. Biol. Rev. 63, 554^569. [15] Billard, P., Menart, S., Blaisonneau, J., Bolotin-Fukuhara, M., Fukuhara, H. and Wesolowski-Louvel, M. (1996) Glucose uptake in Kluyveromyces lactis : role of the HGT1 gene in glucose transport. J. Bacteriol. 178, 5860^5866. [16] Go¡rini, P., Wesolowski-Louvel, M., Ferrero, I. and Fukuhara, H. (1990) RAG1 gene of the yeast Kluyveromyces lactis codes for a sugar transporter. Nucleic Acids Res. 18, 5294.

FEMSRE 689 29-8-00

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529 [17] Wesolowski-Louvel, M., Go¡rini, P., Ferrero, I. and Fukuhara, H. (1992) Glucose transport in the yeast Kluyveromyces lactis. I. Properties of an inducible low-a¤nity glucose transporter gene. Mol. Gen. Genet. 233, 89^96. [18] Weirich, J., Go¡rini, P., Kuger, P., Ferrero, I. and Breunig, K. (1997) In£uence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis. Eur. J. Biochem. 249, 248^257. [19] Chen, X.J., Wesolowski-Louvel, M. and Fukuhara, H. (1992) Glucose transport in the yeast Kluyveromyces lactis. II. Transcriptional regulation of the glucose transporter gene RAG1. Mol. Gen. Genet. 233, 97^105. [20] Go¡rini, P., Wesolowski-Louvel, M. and Ferrero, I. (1991) A phosphoglucose isomerase gene is involved in the Rag phenotype of the yeast Kluyveromyces lactis. Mol. Gen. Genet. 228, 401^409. [21] Wesolowski-Louvel, M., Go¡rini, P. and Ferrero, I. (1988) The RAG2 gene of the yeast Kluyveromyces lactis codes for a putative phosphoglucose isomerase. Nucleic Acids Res. 16, 8714. [22] Prior, C., Mamessier, P., Fukuhara, H., Chen, X.J. and We¨solowskiLouvel, M. (1993) The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis. Mol. Cell. Biol. 13, 3882^3889. [23] Blaisonneau, J., Fukuhara, H. and Wesolowski-Louvel, M. (1997) The Kluyveromyces lactis equivalent of casein kinase I is required for the transcription of the gene encoding the low-a¤nity glucose permease. Mol. Gen. Genet. 253, 469^477. [24] Varma, A., Singh, B.B., Karnani, N., Lichtenberg-Frate, H., Ho«fer, M., Magee, B.B. and Prasad, R. (2000) Molecular cloning and functional characterisation of a glucose transporter, CaHGT1, of Candida albicans. FEMS Microbiol. Lett. 182, 15^21. [25] Peinado, J.M., Cameira-dos-Santos, P.J. and Loureiro-Dias, M.C. (1989) Regulation of glucose transport in Candida utilis. J. Gen. Microbiol. 135, 195^201. [26] van den Broek, P.J.A., van Gompel, A.E., Luttik, M.A.H., Pronk, J.T. and van Leeuwen, C.M. (1997) Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis. Biochem. J. 321, 487^495. [27] Weierstall, T., Hollenberg, C.P. and Boles, E. (1999) Cloning and characterization of three genes (SUT1^3) encoding glucose transporters of the yeast Pichia stipitis. Mol. Microbiol. 31, 871^883. [28] Does, A.L. and Bisson, L.F. (1989) Comparison of glucose uptake kinetics in di¡erent yeasts. J. Bacteriol. 171, 1303^1308. [29] Ho«fer, M. and Nassar, F.R. (1987) Aerobic and anaerobic uptake of sugars in Schizosaccharomyces pombe. J. Gen. Microbiol. 133, 2163^ 2172. [30] Heiland, S., Radovanovic, N., Ho«fer, M., Winderickx, J. and Lichtenberg, H. (2000) Multiple hexose transporters of Schizosaccharomyces pombe. J. Bacteriol. 182, 2153^2162. [31] Mehta, S.V., Patil, V.B., Velmurugan, S., Lobo, Z. and Maitra, P.K. (1998) std1, a gene involved in glucose transport in Schizosaccharomyces pombe. J. Bacteriol. 180, 674^679. [32] Riley, M.I., Srekrishna, K., Bhairi, S. and Dickson, R.C. (1987) Isolation and characterization of mutants of Kluyveromyces lactis defective in lactose transport. Mol. Gen. Genet. 208, 145^151. [33] Hoever, M., Milbradt, B. and Ho«fer, M. (1992) D-Gluconate is an alternative growth substrate for cultivation of Schizosaccharomyces pombe mutants. Arch. Microbiol. 157, 191^193. [34] Caspari, T. (1997) Onset of gluconate-H+ symport in Schizosaccharomyces pombe is regulated by the kinases Wis and Pka1, and requires the gti‡ gene product. J. Cell. Sci. 110, 2599^2608. [35] Caspari, T. and Urlinger, S. (1996) The activity of the gluconate-H‡ symporter of Schizosaccharomyces pombe cells is down-regulated by D-glucose and exogenous cAMP. FEBS Lett. 395, 272^276. [36] Georis, I., Cassart, J.P., Breunig, K.D. and Vandenhaute, J. (1999) Glucose repression of the Kluyveromyces lactis invertase gene KlINV1 does not require Mig1p. Mol. Gen. Genet. 261, 862^870. [37] Cha¨vez, F.P., Pons, T., Delgado, J.M. and Rodr|¨guez, L. (1998)

[38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56] [57]

523

Cloning and sequence analysis of the gene encoding invertase (INV1) from the yeast Candida utilis. Yeast 14, 1223^1232. Tanaka, N., Ohuchi, N., Mukai, Y., Osaka, Y., Ohtani, Y., Tabuchi, M., Bhuiyau, M.S., Fukui, H., Harashima, S. and Takegawa, K. (1998) Isolation and characterization of an invertase and its repressor genes from Schizosaccharomyces pombe. Biochem. Biophys. Res. Commun. 245, 246^253. Williamson, P.R., Huber, M.A. and Bennett, J.E. (1993) Role of maltase in the utilization of sucrose by Candida albicans. Biochem. J. 291, 765^771. Geber, A., Williamson, P.R., Rex, J.H., Sweemey, E.C. and Bennett, J.E. (1992) Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization. J. Bacteriol. 174, 6992^ 6996. Dickson, R.C. and Barr, K. (1983) Characterization of lactose transport in Kluyveromyces lactis. J. Bacteriol. 154, 1245^1251. Sreekrishna, K. and Dickson, R.C. (1985) Construction of strains of Saccharomyces cerevisiae that grow on lactose. Proc. Natl. Acad. Sci. USA 82, 7909^7913. Das, S., Breunig, K.D. and Hollenberg, C.P. (1985) A positive regulatory element is involved in the induction of the beta-galactosidase gene from Kluyveromyces lactis. EMBO J. 4, 793^798. Entian, K.D. (1997) Sugar phosphorylation in yeast. In: Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology, and Applications. (Zimmermann, F.K. and Entian, K.D., Eds.), pp. 67^80. Technomic, Lancaster. Herrero, P., Galindez, J., Ruiz, N., Martinez-Campa, C. and Moreno, F. (1995) Transcriptional regulation of the Saccharomyces cerevisiae HXK1, HXK2 and GLK1 genes. Yeast 11, 137^144. Hirai, M., Ohtani, E., Tanaka, A. and Fukui, S. (1977) Glucosephosphorylating enzymes of Candida yeasts and their regulation in vivo. Biochim. Biophys. Acta 480, 357^366. Petit, T. and Gancedo, C. (1999) Molecular cloning and characterization of the gene HXK1 encoding the hexokinase from Yarrowia lipolytica. Yeast 15, 1573^1584. Petit, T., Bla¨zquez, M.A. and Gancedo, C. (1996) Schizosaccharomyces pombe possesses an unusual and a conventional hexokinase: biochemical and molecular characterization of both hexokinases. FEBS Lett. 378, 185^189. Ca¨rdenas, M.L., Cornish-Bowden, A. and Ureta, T. (1998) Evolution and regulatory role of the hexokinases. Biochim. Biophys. Acta 1401, 242^264. Petit, T., Herrero, P. and Gancedo, C. (1998) A mutation Ser213 /Asn in the hexokinase 1 gene from Schizosaccharomyces pombe increases its a¤nity for glucose. Biochem. Biophys. Res. Commun. 251, 714^ 719. Bla¨zquez, M.A., Lagunas, R., Gancedo, C. and Gancedo, J.M. (1993) Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases. FEBS Lett. 329, 51^54. Bla¨zquez, M.A., Gancedo, J.M. and Gancedo, C. (1994) Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose-6-phosphate. FEMS Microbiol. Lett. 121, 223^228. Maitra, P.K. (1971) Glucose and fructose metabolism in a phosphoglucoseisomerase less mutant of Saccharomyces cerevisiae. J. Bacteriol. 107, 759^769. Gonza¨lez Siso, M.I., Freire Picos, M.A. and Cerda¨n, M.E. (1996) Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants. FEBS Lett. 387, 7^10. Sols, A. (1981) Multimodulation of enzyme activity. Curr. Top. Cell. Regul. 19, 77^101. Schaa¡, I., Heinischm, J., Zimmermann, F.K. (1989) Overproduction of glycolytic enzymes in yeast. Yeast 5, 285-290. Heinisch, J. (1986) Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast. Mol. Gen. Genet. 202, 75^82.

FEMSRE 689 29-8-00

524

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529

[58] Kacser, H. and Burns, J.A. (1979) Molecular democracy: who shares the controls ? Biochem. Soc. Trans. 7, 1149^1160. [59] Bar, J., Schellenberger, W. and Kopperschla«ger, G. (1997) Puri¢cation and characterization of phosphofructokinase from the yeast Kluyveromyces lactis. Yeast 13, 1309^1317. [60] Heinisch, J., Kirchrath, L., Liesen, T., Vogelsang, K. and Hollenberg, C.P. (1993) Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis. Mol. Microbiol. 8, 559^570. [61] Clifton, D. and Fraenkel, D.G. (1982) Mutant studies of yeast phosphofructokinase. Biochemistry 21, 1935^1942. [62] Jacoby, J., Hollenberg, P. and Heinisch, J.J. (1993) Transaldolase mutants in the yeast Kluyveromyces lactis provide evidence that glucose can be metabolized through the pentose phosphate pathway. Mol. Microbiol. 10, 867^876. [63] Lorberg, A., Kirchrath, L., Ernst, J.F. and Heinisch, J.J. (1999) Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans. Eur. J. Biochem. 260, 217^226. [64] Yuan, W., Tuttle, D.L., Shi, Y.J., Ralph, G.S. and Dunn, W.A. (1997) Glucose-induced microautophagy in Pichia pastoris requires the alpha-subunit of phosphofructokinase. J. Cell. Sci. 110, 1935^ 1945. [65] Alvarez, V. and Jime¨nez, J. (1998) Un papel regulador de la fosfofructokinasa 2(PFK2) en el ciclo celular de la levadura S. pombe, XXI Congreso de la Sociedad Espan¬ola de Bioqu|¨mica y Biolog|¨a Molecular, P152. University, Sevilla, Spain. [66] Mutoh, N. and Hayashi, Y. (1994) Molecular cloning and nucleotide sequencing of Schizosaccharomyces pombe homologue of the class II fructose-1, 6-bisphosphate aldolase gene. Biochim. Biophys. Acta 1183, 550^552. [67] Kowal, J., Cremona, T. and Horecker, B.L. (1966) Fructose 1, 6diphosphate aldolase of Candida utilis: puri¢cation and properties. Arch. Biochem. Biophys. 114, 13^23. [68] Russell, P.R. (1985) Transcription of the triose-phosphate-isomerase gene of Schizosaccharomyces pombe initiates from a start point di¡erent from that in Saccharomyces cerevisiae. Gene 40, 125^130. [69] Compagno, C., Boschi, F., Dale¡e, A., Porro, D. and Ranzi, B.M. (1999) Isolation, nucleotide sequence, and physiological relevance of the gene encoding triose phosphate isomerase from Kluyveromyces lactis. Appl. Environ. Microbiol. 65, 4216^4219. [70] McAlister, L. and Holland, M.J. (1985) Di¡erential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenases genes. J. Biol. Chem. 260, 15019^15027. [71] Shuster, J.R. (1990) Kluyveromyces lactis glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase-1 genes are linked and divergently transcribed. Nucleic Acids Res. 18, 4271. [72] Fernandes, P.A., Sena-Esteves, M. and Moradas-Ferreira, P. (1995) Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene family from Kluyveromyces marxianus. Polymerase chain reaction-single-strand conformation polymorphism as a tool for the study of multigenic families. Yeast 11, 725^733. [73] Villamon, E., Gozalbo, D., Mart|¨nez, J.P. and Gil, M.L. (1999) Puri¢cation of a biologically active recombinant glyceraldehyde-3-phosphate dehydrogenase from Candida albicans. FEMS Microbiol. Lett. 179, 61^65. [74] Gil, M.L., Villamon, E., Monteagudo, C., Gozalbo, D. and Mart|¨nez, J.P. (1999) Clinical strains of Candida albicans express the surface antigen glyceraldehyde-3-phosphate dehydrogenase in vitro and in infected tissues. FEMS Immunol. Med. Microbiol. 23, 229^234. [75] Waterham, H.R., Digan, M.E., Koutz, P.J., Lair, S.V. and Cregg, J.M. (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186, 37^44. [76] Chambers, A. (1997) Phosphoglycerate kinase In: Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology, and Applications (Zimmermann, F.K. and Entian, K.D., Eds.), pp. 141^156. Technomic, Lancaster.

[77] Fournier, A., Fleer, R., Yeh, P. and Mayaux, J.F. (1990) The primary structure of the 3-phosphoglycerate kinase (PGK) gene from Kluyveromyces lactis. Nucleic Acids Res. 18, 365. [78] Masuda, Y., Park, S.M., Ohkuma, M., Ohta, A. and Takagi, M. (1994) Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly isolated phosphoglycerate kinase (PGK) gene. Curr. Genet. 25, 412^417. [79] Alloussh, H.M., Lopez-Ribot, J.L., Masten, B.J. and Cha¤n, W.L. (1997) 3-Phosphoglycerate kinase : a glycolytic enzyme protein present in the cell wall of Candida albicans. Microbiology 143, 321^ 330. [80] Le Dall, M., Nicaud, J., Treton, B.Y. and Gaillardin, C.M. (1996) The 3-phosphoglycerate kinase gene of the yeast Yarrowia lipolytica de-represses on gluconeogenic substrates. Curr. Genet. 29, 446^ 456. [81] Walter, R.A., Nairn, J., Duncan, D., Price, N.C., Kelly, S.M., Rigden, D.J. and Fothergill-More, L.A. (1999) The role of the C-terminal region in phosphoglycerate mutase. Biochem. J. 337, 89^95. [82] Nairn, J., Price, N.J., Fothergill-Gilmore, L.A., Walker, G.A., Fothergill, J.E. and Dunbar, B. (1994) The amino acid sequence of the small monomeric phosphoglycerate mutase from the ¢ssion yeast Schizosaccharomyces pombe. Biochem. J. 297, 603^608. [83] Holland, M.J. and Holland, J.P. (1978) Isolation and identi¢cation of yeast messenger nucleic acids coding for enolase, glyceraldehyde-3phosphate dehydrogenase and phosphoglycerate kinase. Biochemistry 17, 4900^4907. [84] McAlister, L. and Holland, M.J. (1982) The targeted deletion of a yeast enolase structural gene. J. Biol. Chem. 257, 7181^7188. [85] Mason, A.B., Buckley, H.R. and Gorman, J.A. (1993) Molecular cloning and characterization of the Candida albicans enolase gene. J. Bacteriol. 175, 2632^2639. [86] Postlethwait, P. and Sundstrom, P. (1995) Genetic organization and mRNA expression of enolase genes of Candida albicans. J. Bacteriol. 177, 1772^1779. [87] Jackson, J.C. and Lopes, J.M. (1995) A cDNA from Schizosaccharomyces pombe encoding a putative enolase. Gene 154, 109^113. [88] Boles, E., Schulte, F., Miosga, T., Freidel, K., Schluter, E., Zimmermann, F.K., Hollenberg, C.P. and Heinisch, J.J. (1997) Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6bisphosphate. J. Bacteriol. 179, 2987^2993. [89] Nairn, J., Smith, S., Allison, P.J., Rigden, D., Fothergill-More, L.A. and Price, N.C. (1995) Cloning and sequencing of a gene encoding pyruvate kinase from Schizosaccharomyces pombe: implications for quaternary structure and regulation of the enzyme. FEMS Microbiol. Lett. 134, 221^226. [90] Strick, C.A., James, L.C., O'Donnell, M.M., Gollaher, M.G. and Franke, A.E. (1992) The isolation and characterization of the pyruvate kinase encoding gene from the yeast Yarrowia lipolytica. Gene 118, 65^72. [91] Strick, C.A., James, L.C., O'Donnell, M.M., Gollaher, M.G. and Franke, A.E. (1994) The isolation and characterization of the pyruvate kinase-encoding gene from the yeast Yarrowia lipolytica. Gene 140, 141^143. [92] Gancedo, J.M., Gancedo, C. and Sols, A. (1967) Regulation of the concentration or activity of pyruvate kinase in yeasts and its relationship to gluconeogenesis. Biochem. J. 102, 23C^25C. [93] Hirai, M., Tanaka, A. and Fukui, S. (1975) Di¡erence in pyruvate kinase regulation among three groups of yeasts. Biochim. Biophys. Acta 391, 282^291. [94] Pronk, J.T., Steensma, H.Y. and van Dijken, J.P. (1996) Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12, 1607^1633. [95] Steensma, H.Y., Holterman, L., Dekker, I., van Sluis, C.A. and Wenzel, T.J. (1990) Molecular cloning of the gene for the E1K subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae. Eur. J. Biochem. 191, 769^774. [96] Behal, R.H., Browning, K.S. and Reed, L.J. (1989) Nucleotide and

FEMSRE 689 29-8-00

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113]

deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase. Biochem. Biophys. Res. Commun. 164, 941^946. Miran, S.G., Lawson, J.E. and Reed, L.J. (1993) Characterization of the PDHL1, the structural gene for the pyruvate dehydrogenase L subunit from Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 90, 1252^1256. Niu, X.-D., Browning, K.S., Behal, R.H. and Reed, L.J. (1988) Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 85, 7546^7550. Ross, J., Reid, G.A. and Dawes, I.A. (1988) The nucleotide sequence of the LDP1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae : comparison between eukaryotic and prokaryotic sequences for related enzymes and identi¢cation of potential upstream control sites. J. Gen. Microbiol. 134, 1131^ 1139. Nalecz, M.J., Nalecz, K.A. and Azzi, A. (1991) Puri¢cation and functional characterization of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae). Biochim. Biophys. Acta 1079, 87^95. Zeeman, A.M., Luttik, M.A., Pronk, J.T., van Dijken, J.P. and Steensma, H.Y. (1999) Impaired growth on glucose of a pyruvate dehydrogenase-negative mutant of Kluyveromyces lactis is due to a limitation in mitochondrial acetyl-coenzyme A uptake. FEMS Microbiol. Lett. 177, 23^28. Tizzani, L., Meacock, P., Frontali, L. and Wesolowski-Louvel, M. (1998) The RAG3 gene of Kluyveromyces lactis is involved in the transcriptional regulation of genes coding for enzymes implicated in pyruvate utilization and genes of the biosynthesis of thiamine pyrophosphate. FEMS Microbiol. Lett. 168, 25^30. Hohmann, S. (1993) Characterisation of PDC2, a gene necessary for high level expression of pyruvate decarboxylase structural genes in Saccharomyces cerevisiae. Mol. Gen. Genet. 241, 657^666. Cavan, G. and MacDonald, D. (1995) Cloning and sequence of a gene encoding the pyruvate dehydrogenase E1 beta subunit of Schizosacchromyces pombe. Gene 152, 117^120. Cavan, G. and MacDonald, D. (1994) Mutations which reduce levels of pyruvate dehydrogenase in Schizosaccharomyces pombe cause a requirement for arginine or glutamine. FEMS Microbiol. Lett. 124, 361^365. Passoth, V., Zimmermann, M. and Klinner, U. (1996) Peculiarities of the regulation of fermentation and respiration in the Crabtreenegative, xylose fermenting yeast Pichia stipitis. Appl. Biochem. Biotechnol. 57^58, 201^212. Hohmann, S. (1997) Pyruvate decarboxylases In: Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology, and Applications (Zimmermann, F.K. and Entian, K.D., Eds.), pp. 187^212. Technomic, Lancaster. Bianchi, M.M., Tizzani, L., Destruelle, M., Frontali, L. and Wesolowski-Louvel, M. (1996) The `petite negative' yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity. Mol. Microbiol. 19, 27^36. Prior, C., Tizzani, L., Fukuhara, H. and Wesolowski-Louvel, M. (1996) RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis. Mol. Microbiol. 20, 765^772. Kaiser, B., Munder, T., Saluz, H.P., Kunkel, W. and Eck, R. (1999) Identi¢cation of a gene encoding the pyruvate decarboxylase gene regulator CaPdc2p from Candida albicans. Yeast 15, 585^591. Lu, P., Davis, B.P. and Je¡ries, T.W. (1998) Cloning and characterization of two pyruvate decarboxylase genes from Pichia stipitis CBS 6054. Appl. Environ. Microbiol. 64, 94^97. Flikweert, M.T., van der Zanden, K., Janssen, W.M.P.T.M., Steensma, H.Y., van Dijken, J.P. and Pronk, J.T. (1996) Pyruvate decarboxylase an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12, 247^257. van den Berg, M.A. and Steensma, H.Y. (1995) ACS2, a Saccharo-

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

525

myces cerevisiae gene encoding acetyl-coenzyme A synthetase, essential for growth on glucose. Eur. J. Biochem. 231, 704^713. Meaden, P.G., Dickinson, F.M., Mifsud, A., Tessier, W., Westwater, J., Bussey, H. and Midgley, M. (1997) The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic, Mg(2+)-activated acetaldehyde dehydrogenase. Yeast 13, 1319^1327. de Jong-Gubbels, P. (1998) Metabolic Fluxes at the Interface of Glycolysis and TCA Cycle in Saccharomyces cerevisiae, Ph.D. Thesis. University Delft, The Netherlands. Velot, C., Lebreton, S., Morgunov, I., Usher, K.C. and Srere, P.A. (1999) Metabolic defects of mislocalized mitochondrial and peroxisomal citrate synthases in the yeast Saccharomyces cerevisiae. Biochemistry 38, 16195^16204. Jia, Y.K., Becam, A.M. and Herbert, C.J. (1997) The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase. Mol. Microbiol. 24, 53^59. Ueda, M., Sanuki, S., Kawachi, H., Shimizu, K., Atomi, H. and Tanaka, A. (1997) Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis : cloning and expression in Saccharomyces cerevisiae. Arch. Microbiol. 168, 8^15. Ganglo¡, S.P., Marguet, D. and Lauquin, G.J. (1990) Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate. Mol. Cell. Biol. 10, 3551^3561. Foury, F. (1999) Low iron concentration and aconitase de¢ciency in a yeast frataxin homologue de¢cient strain. FEBS Lett. 456, 281^ 284. Cupp, J.R. and McAlister-Henn, L. (1992) Cloning and characterization of the gene encoding the IDH1 subunit of NAD(‡ )-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae. J. Biol. Chem. 267, 16417^16423. Cupp, J.R. and McAlister-Henn, L. (1991) NAD(‡ )-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae. J. Biol. Chem. 266, 22199^22205. Zhao, W.N. and McAlister-Henn, L. (1996) Expression and gene disruption analysis of the isocitrate dehydrogenase family in yeast. Biochemistry 35, 7873^7878. Minard, K.I., Jennings, G.T., Loftus, T.M., Xuan, D. and McAlister-Henn, L. (1998) Sources of NADPH and expression of mammalian NADP‡ -speci¢c isocitrate dehydrogenases in Saccharomyces cerevisiae. J. Biol. Chem. 273, 31486^31493. Barel, I. and MacDonald, D.W. (1993) Enzyme defects in glutamate-requiring stains of Schizosaccharomyces pombe. FEMS Microbiol. Lett. 113, 267^272. Stucka, R., Dequin, S., Salmon, J.M. and Gancedo, C. (1991) DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae : analysis of pyruvate carboxylase-de¢cient strains. Mol. Gen. Genet. 229, 307^315. Walker, M.E., Val, D.L., Rohde, M., Devenish, R.J. and Wallace, J.C. (1991) Yeast pyruvate carboxylase: identi¢cation of two genes encoding isoenzymes. Biochem. Biophys. Res. Commun. 176, 1210^ 1217. van Urk, H., Schippe, D., Breedveld, G.J., Mak, P.R., Sche¡ers, W.A. and van Dijken, J.P. (1989) Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim. Biophys. Acta 992, 78^86. Mene¨ndez, J., Delgado, J. and Gancedo, C. (1998) Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identi¢cation of a suppressor of the pyc phenotype. Yeast 14, 647^654. van Dijk, R., van der Heide, M., van der Klei, I.J., Faber, K.N. and Veenhuis, M. (1998) Principles of £avoprotein assembly/activation in the yeast Hansenula polymorpha, EC Framework IV Symposium, Yeast as a Cell Factory, P131. University, Vlaardingen, The Netherlands.

FEMSRE 689 29-8-00

526

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529

[131] Ferna¨ndez, E., Moreno, F. and Rodicio, R. (1992) The ICL1 gene from Saccharomyces cerevisiae. Eur. J. Biochem. 204, 983^990. [132] Duntze, W., Neumann, D., Gancedo, J.M., Atzpodien, W. and Holzer, H. (1968) Studies on the regulation and localization of the glyoxylate cycle enzymes in S. cerevisiae. Eur. J. Biochem. 10, 83^89. [133] McCammon, M.T., Veenhuis, M., Trapp, S.B. and Goodman, J.M. (1990) Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae. J. Bacteriol. 172, 5816^ 5827. [134] Atomi, H., Ueda, M., Hikida, M., Hishida, T., Teranishi, Y. and Tanaka, A. (1990) Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization. J. Biochem. 107, 262^266. [135] Barth, G. and Scheuber, T. (1993) Cloning of the isocitrate lyase gene (ICL1) fromYarrowia lipolytica and characterization of the deduced protein. Mol. Gen. Genet. 241, 422^430. [136] Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M. and Tanaka, A. (1995) A novel promoter, derived from the isocitrate lyase gene of Candida tropicalis, inducible with acetate in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 43, 489^492. [137] Matsuoka, M., Ueda, M. and Aiba, S. (1980) Role and control of isocitrate lyase in Candida lipolytica. J. Bacteriol. 144, 692^697. [138] Okada, H., Ueda, M. and Tanaka, A. (1986) Puri¢cation of peroxisomal malate synthase from alkane-grown Candida tropicalis and some properties of the puri¢ed enzyme. Arch. Microbiol. 144, 137^141. [139] Hikida, M., Atomi, H., Fukuda, Y., Aoki, A., Hishida, T., Teranishi, Y., Ueda, M. and Tanaka, A. (1991) Presence of two transcribed malate synthase genes in an n-alkane-utilizing yeast, Candida tropicalis. J. Biochem. 110, 909^914. [140] Bruinenberg, P.G., Blaauw, M., Kazemier, B. and Ab, G. (1990) Cloning and sequencing of the malate synthase gene from Hansenula polymorpha. Yeast 6, 245^254. [141] Hartig, A., Simon, M.M., Schuster, T., Daugherty, J.R., Yoo, H.S. and Cooper, T.G. (1992) Di¡erentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae. Nucleic Acids Res. 20, 5677^5686. [142] Flury, U., Heer, B. and Fiechter, A. (1974) Regulatory and physicochemical properties of two isoenzymes of malate dehydrogenase from Schizosaccharomyces pombe. Biochim. Biophys. Acta 341, 465^ 483. [143] Eraso, P. and Gancedo, J.M. (1984) Catabolite repression in yeasts is not associated with low levels of cAMP. Eur. J. Biochem. 141, 195^198. [144] Small, W.C. and McAlister-Henn, H. (1998) Identi¢cation of a cytosolically directed NADH dehydrogenase in mitochondria of Saccharomyces cerevisiae. J. Bacteriol. 180, 4051^4055. [145] Luttik, M.A.H., Overkamp, K.M., Ko«tter, P., de Vries, S., van Dijken, J.P. and Pronk, J.T. (1998) The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH. J. Biol. Chem. 273, 24259^24534. [146] Kerschner, S.J., Okun, J.G. and Brandt, U. (1999) A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica. J. Cell Sci. 112, 2347^2354. [147] Marres, C.A.M., de Vries, S. and Grivell, L.A. (1991) Isolation and inactivation of the nuclear gene encoding the rotenone-insensitive internal NADH-ubiquinone oxidoreductase of mitochondira from Saccharomyces cerevisiae. Eur. J. Biochem. 195, 857^862. [148] Larsson, C., Pahlman, I.L., Ansell, R., Rigoulet, M., Adler, L. and Gustafsson, L. (1998) The importance of the glycerol-3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae. Yeast 14, 347^358. [149] de Vries, S. and Marres, C.A. (1987) The mitochondrial respiratory chain of yeast. Structure and biosynthesis and the role in cellular metabolism. Biochim. Biophys. Acta 895, 205^239. [150] Buschges, R., Bahrenberg, G., Zimmermann, M. and Wolf, K.

[151]

[152]

[153] [154]

[155]

[156]

[157]

[158]

[159]

[160]

[161]

[162] [163]

[164]

[165]

[166]

[167]

[168]

[169]

[170]

(1994) NADH:ubiquinone oxidoreductase in obligate aerobic yeasts. Yeast 10, 475^479. Adrian, G.S., McCammon, M.T., Montgomery, D.L. and Douglas, M.G. (1986) Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane. Mol. Cell. Biol. 6, 626^634. Lawson, J.E. and Douglas, M.G. (1988) Separate genes encode functionally equivalent carrier proteins in Saccharomyces cerevisiae. J. Biol. Chem. 263, 14812^14818. Kolarov, J., Kolarova, N. and Nelson, N. (1990) A third ADP/ATP translocator gene in yeast. J. Biol. Chem. 265, 12711^12716. Drgon, T., Sabova, L., Gavurnikova, G. and Kolarov, J. (1992) Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic properties but their deletion produces di¡erent phenotypes. FEBS Lett. 304, 277^280. Viola, A.M., Galeotti, C.L., Go¡rini, P., Ficarelli, A. and Ferrero, I. (1995) A Kluyveromyces lactis gene homologue to AAC2 complements the Saccharomyces cerevisiae op1 mutation. Curr. Genet. 27, 229^233. Nebohacova, M., Mentel, M., Nosek, J. and Kolarov, J. (1999) Isolation and expression of the gene encoding mitochondrial ADP/ ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis. Yeast 15, 1237^1242. Couzin, N., Trezeguet, V., Le Saux, A. and Lauquin, G.J. (1996) Cloning of the gene encoding the mitochondrial adenine nucleotide carrier of Schizosaccharomyces pombe by functional complementation in Saccharomyces cerevisiae. Gene 171, 113^117. Viola, A.M., Lodi, T. and Ferrero, I. (1999) A Klaac null mutant of Kluyveromyces lactis is complemented by a single copy of the Saccharomyces cerevisiae AAC1 gene. Curr. Genet. 36, 29^36. Trezeguet, V., Zeman, I., David, C., Lauquin, G.J. and Kolarov, J. (1999) Expression of the ADP/ATP carrier encoding genes in aerobic yeasts; phenotype of an ADP/ATP carrier deletion mutant of Schizosaccharomyces pombe. Biochim. Biophys. Acta 1410, 229^236. Visser, W., van der Baan, A.A., Batenburg-van der Vegte, W., Sche¡ers, W.A., Kramer, R. and van Dijken, J.P. (1994) Involvement of mitochondria in the assimilatory metabolism of anaerobic Saccharomyces cerevisiae cultures. Microbiology 140, 3039^3046. Schonbaum, G.R., Bonner, W.D., Storey, B.T. and Bahr, J.T. (1971) Speci¢c inhibition of the cyanide-insensitive respiratory pathway in plant mitochondria by hydroxamic acids. Plant. Physiol. 47, 124^128. Henry, M.F. and Nyns, E.J. (1975) Cyanide insensitive respiration. An alternative mitochondrial pathway. Sub-Cell. Biochem. 4, 1^65. Vanlerberghe, G.C. and McIntosh, L. (1997) Alternative oxidase: from gene to function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 703^734. Heritage, J., Tribe, M.A. and Whittaker, P.A. (1981) The respiration pathways of wild-type and petite mutants of Kluyveromyces lactis. Microbios 30, 37^45. Minagawa, N., Sakajo, S., Komiyama, T. and Yoshimoto, A. (1990) A 36 kDa mitochondrial protein is responsible for cyanide resistant respiration in Hansenula anomala. FEBS Lett. 264, 149^152. Minagawa, N., Sakajo, S. and Yoshimoto, A. (1991) Sulfur compounds induce alternative oxidation in Hansenula anomala. Agric. Biol. Chem. 61, 1573^1574. Sakajo, S., Minagawa, N., Komiyama, T. and Yoshimoto, A. (1991) Molecular cloning of cDNA from antimycinA-inducible mRNA and its role in cyanide-resistant respiration in Hansenula anomala. Biochim. Biophys. Acta 1090, 102^108. Huh, W.K. and Kang, S.O. (1999) Molecular cloning and functional expression of alternative oxidase in Candida albicans. J. Bacteriol. 181, 4098^4102. Henry, M.F., Hamaide-Deplus, M.C. and Henry, E.J. (1974) Cyanide insensitive respiration of Candida lipolytica. Antonie van Leeuwenhoek 40, 79^91. Medentsev, A.G. and Akimenko, V.K. (1999) Development and

FEMSRE 689 29-8-00

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529

[171]

[172]

[173]

[174]

[175]

[176]

[177] [178]

[179]

[180] [181]

[182]

[183]

[184]

[185] [186] [187] [188]

[189]

[190]

[191]

activation of cyanide-resistant respiration in the yeast Yarrowia lipolytica. Biochemistry (Moscow) 64, 945^951. Jeppsson, H., Alexander, N.J. and Hahn-Hagerdal, B.C. (1995) Existence of cyanide insensitive respiration in the yeast Pichia stipitis and its possible in£uence on product formation during xylose utilization. Appl. Env. Microb. 61, 2596^2600. Albury, M.S., Dudley, P., Watts, F.Z. and Moore, A.L. (1996) Targeting the plant alternative oxidase protein to Schizosaccharomyces pombe mitochondria confers cyanide-insensitive respiration. J. Biol. Chem. 271, 17062^17066. A¡ourtit, C., Albury, M.S., Krab, K. and Moore, A.L. (1999) Functional expression of the plant alternative oxidase a¡ects growth of the yeast Schizosaccharomyces pombe. J. Biol. Chem. 274, 6212^ 6218. Gancedo, J.M. and Lagunas, R. (1973) Contribution of the pentose phosphate pathway to glucose metabolism in Saccharomyces cerevisiae: a critical analysis on the use of labelled glucose. Plant. Sci. Lett. 1, 193^200. Bruinenberg, P.M., van Dijken, J.P. and Sche¡ers, W.A. (1983) An enzymic analysis of NADPH production and consumption in Candida utilis. J. Gen. Microbiol. 129, 965^971. Tsai, C.S. and Chen, Q. (1998) Puri¢cation and kinetic characterizaton of hexokinase and glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe. Biochem. Cell. Biol. 76, 107^113. Domagk, G.F. and Chilla, R. (1975) Glucose-6-phosphate dehydrogenase from Candida utilis. Methods Enzymol. 41, 205^208. Je¡ery, J., Persson, B., Wood, I., Bergman, T., Je¡ery, R. and Jornvall, H. (1993) Glucose-6-phosphate dehydrogenase. Structure^function relationships and the Pichia jadinii enzyme structure. Eur. J. Biochem. 212, 41^49. Thomas, D., Cherest, H. and Surdin-Kerjan, Y. (1991) Identi¢cation of the structural gene for glucose-6-phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur. EMBO J. 10, 547^553. Brodie, A.F. and Lipmann, F. (1955) Identi¢cation of a gluconolactonase. J. Biol. Chem. 212, 677^685. Kanagasundaram, V. and Scopes, R. (1992) Isolation and characterization of the gene encoding gluconolactonase from Zymomonas mobilis. Biochim. Biophys. Acta 1171, 198^200. Kobayashi, M., Shinohara, M., Sakoh, C., Kataoka, M. and Shimizu, S. (1998) Lactone-ring-cleaving enzyme : genetic analysis, novel RNA editing, and evolutionary implications. Proc. Natl. Acad. Sci. USA 95, 12787^12792. Tsai, C.S., Shi, J.L. and Ye, H.G. (1995) Kinetic studies of gluconate pathway enzymes from Schizosaccharomyces pombe. Arch. Biochem. Biophys. 316, 163^168. Tsai, C.S. and Chen, Q. (1998) Puri¢cation and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe. Biochem. Cell. Biol. 76, 637^644. Rippa, M. and Signorini, M. (1975) 6-Phosphogluconate dehydrogenase from Candida utilis. Methods Enzymol. 41, 237^240. Lobo, Z. and Maitra, P.K. (1982) Pentose phosphate pathway mutants of yeast. Mol. Gen. Genet. 185, 367^368. Domagk, G.F. and Doering, K.M. (1975) D-Ribose-5-phosphate isomerase from Candida utilis. Methods Enzymol. 41, 427^429. Horitsu, H., Sasaki, I., Kikuchi, T., Suzuki, H., Sumida, M. and Tomoyeda, M. (1976) Puri¢cation, properties and structure of ribose-5-phosphate ketol isomerase from Candida utilis. Agric. Biol. Chem. 40, 257^264. Wood, T. (1981) The preparation of transketolase free from D-ribulose-5-phosphate-3-epimerase. Biochim. Biophys. Acta 659, 233^ 243. Jakoby, J.J. and Heinisch, J.J. (1997) Analysis of a transketolase gene from Kluyveromyces lactis reveals that the yeast enzymes are more related to transketolases of prokaryotic origins than to those of higher eukaryotes. Curr. Genet. 31, 15^21. Metzger, M.H. and Hollenberg, C.P. (1994) Isolation and character-

[192]

[193]

[194]

[195]

[196] [197]

[198]

[199]

[200]

[201]

[202]

[203]

[204]

[205]

[206]

[207]

[208]

[209]

[210]

527

ization of the Pichia stipitis transketolase gene and expression in a xylose-utilising Saccharomyces cerevisiae transformant. Appl. Microbiol. Biotechnol. 42, 319^325. Fletcher, T.S., Kwee, I.L., Nakada, T., Largamn, C. and Martin, B.M. (1992) DNA sequence of the yeast transketolase gene. Biochemistry 31, 1892^1896. Bruinenberg, P.M., de Bot, P.H.M., van Dijken, J.P. and Sche¡ers, W.A. (1984) NADH-linked aldose reductase: the key to anaerobic alcoholic fermentation of xylose by yeast. Appl. Microbiol. Biotechnol. 19, 256^260. Mayr, P., Bruggler, K., Kulbe, K.D. and Nidetzky, B. (2000) DXylose metabolism by Candida intermedia: isolation and characterisation of two forms of aldose reductase with di¡erent coenzyme speci¢cities. J. Chromatogr. B Biomed. Sci. Appl. 14, 195^202. Billard, P., Menart, S., Fleer, R. and Bolotin-Fukuhara, M. (1995) Isolation and characterization of the gene encoding xylose reductase from Kluyveromyces lactis. Gene 162, 93^97. Ko«tter, P. and Ciriacy, M. (1993) Xylose fermentation by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 38, 776^783. Kim, Y.S., Kim, S.Y., Kim, J.H. and Kim, S.C. (1999) Xylitol production using recombinant Saccharomyces cerevisiae containing multiple xylose reductase genes at chromosomal delta-sequences. J. Biotechnol. 67, 159^171. Ho, N.W., Lin, F.P., Huang, S., Andrews, P.C. and Tsao, G.T. (1990) Puri¢cation, characterization, and amino terminal sequence of xylose reductase from Candida shehatae. Enzyme Microb. Technol. 12, 33^39. Neuhauser, W., Haltrich, D., Kulbe, K.D. and Nidetzky, B. (1997) NAD(P)H-dependent aldolase reductase from the xylose assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme. Biochem. J. 326, 683^692. Handumrongkul, C., Ma, D.P. and Silva, J.L. (1998) Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris. Appl. Microbiol. Biotechnol. 49, 399^404. Zaragoza, O., Rodr|¨guez, C. and Gancedo, C. (2000) Isolation and characterization of the MIG1 gene from Candida albicans and e¡ect of its disruption on catabolite repression. J. Bacteriol. 182, 320^326. Richard, P., Toivari, M.H. and Penttila, M. (1999) Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase. FEBS Lett. 457, 135^138. Waldfrisson, M., Anderlund, M., Bao, X. and Hahn-Ha«gerdal, B. (1997) Expression of di¡erent levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its e¡ects on product formation during xylose utilisation. Appl. Microbiol. Technol. 48, 218^224. Denis, C.L., Ferguson, J. and Young, E.T. (1983) mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. J. Biol. Chem. 258, 1165^1171. Ciriacy, M. (1997) Alcohol dehydrogenases In: Yeast Sugar Metabolism. Biochemistry, Genetics, Biotechnology, and Applications. (Zimmermann, F.K. and Entian, K.D., Eds.), pp. 213^224. Technomic, Lancaster. Megnet, R. (1967) Mutants partially de¢cient in alcohol dehydrogenase in Schizosaccharomyces pombe. Arch. Biochem. Biophys. 121, 194^201. Russell, P. and Hall, B.D. (1983) The primary structure of the alcohol dehydrogenase gene from the ¢ssion yeast Schizosaccharomyces pombe. J. Biol. Chem. 258, 143^149. Bozzi, A., Saliola, M., Falcone, C., Bossa, F. and Martini, F. (1997) Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis. Biochim. Biophys. Acta 1339, 133^142. Mazzoni, C., Saliola, M. and Falcone, C. (1992) Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis. Mol. Microbiol. 6, 2279^2286. Saliola, M. and Falcone, C. (1995) Two mitochondrial alcohol de-

FEMSRE 689 29-8-00

528

[211]

[212]

[213]

[214]

[215]

[216]

[217]

[218]

[219]

[220]

[221]

[222]

[223]

[224]

[225]

[226]

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529 hydrogenase activities of Kluyveromyces lactis are di¡erently expressed during respiration and fermentation. Mol. Gen. Genet. 249, 665^672. Bertram, G., Swoboda, R.K., Gooday, G.W., Gow, N.A. and Brown, A.J. (1996) Structure and regulation of the Candida albicans ADH1 gene encoding an immunogenic alcohol dehydrogenase. Yeast 12, 115^127. Cho, J.Y. and Je¡ries, T.W. (1998) Pichia stipitis genes for alcohol dehydrogenase with fermentative and respiratory functions. Appl. Environ. Microbiol. 64, 1350^1358. Passoth, V., Schafer, B., Liebel, B., Weierstall, T. and Klinner, U. (1998) Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identi¢cation of the fermentative ADH. Yeast 14, 1311^1325. Barth, G. and Kunkel, W. (1979) Alcohol dehydrogenase (ADH) in yeasts. II. NAD‡ and NADP‡ -dependent alcohol dehydrogenases in Saccharomycopsis lipolytica. Z. Allg. Mikrobiol. 19, 381^390. Navarro-Avin¬o, J.P., Prasad, R., Miralles, V.J., Benito, R.M. and Serrano, R. (1999) A proposal for nomenclature of aldehyde deydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes. Yeast 15, 829^842. Tessier, W.D., Meaden, P.G., Dickinson, F.M. and Midgley, M. (1998) Identi¢cation and disruption of the gene encoding the K‡ activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 164, 29^34. De Virgilio, C., Bu«rckert, N., Barth, G., Neuhaus, J.M., Boller, T. and Wiemken, A. (1992) Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae. Yeast 8, 1043^1051. van den Berg, M.A., de Jong-Gubbels, P., Kortland, C.J., van Dijken, J.P., Pronk, J.T. and Steensma, H.Y. (1996) The two acetylcoenzyme A synthetases of Saccharomyces cerevisiae di¡er with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271, 28953^28959. Kujau, M., Weber, H. and Barth, G. (1992) Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl-coenzyme A synthetase. Yeast 8, 193^203. Tsai, C.S., Mitton, K.P. and Johnson, B.F. (1989) Acetate assimilation by the ¢ssion yeast Schizosaccharomyces pombe. Biochem. Cell. Biol. 67, 464^467. Drewke, C., Thielen, J. and Ciriacy, M. (1990) Ethanol formation in adh0 mutants reveals the existence of a novel acetaldehyde-reducing activity in Saccharomyces cerevisiae. J. Bacteriol. 172, 3909^ 3917. Saliola, M., Bellardi, S., Marta, I. and Falcone, C. (1994) Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis. Yeast 10, 1133^1140. Ansell, R., Granath, K., Hohmann, S., Thevelein, J.M. and Adler, L. (1997) The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J. 16, 2179^2187. Ohmiya, R., Yamada, H., Nakashima, K., Aiba, H. and Mizuno, T. (1995) Osmoregulation of ¢ssion yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth. Mol. Microbiol. 18, 963^ 973. Yamada, H., Ohmiya, R., Aiba, H. and Mizuno, T. (1996) Construction and characterization of a deletion mutant of gpd2 that encodes an isozyme of NADH-dependent glycerol-3-phosphate dehydrogenase in ¢ssion yeast. Biosci. Biotechnol. Biochem. 60, 918^ 920. Norbeck, J., Pahlman, A.K., Akhtar, N., Blomberg, A. and Adler, L. (1996) Puri¢cation and characterization of two isoenzymes of DLglycerol-3-phosphatase from Saccharomyces cerevisiae. Identi¢cation of the corresponding GPP1 and GPP2 genes and evidence for osmotic regulation of Gpp2p expression by the osmosensing mito-

[227]

[228]

[229]

[230]

[231]

[232]

[233]

[234]

[235]

[236]

[237]

[238]

[239]

[240]

[241]

[242]

[243]

[244]

[245]

gen-activated protein kinase signal transduction pathway. J. Biol. Chem. 271, 13875^13881. Gancedo, C., Gancedo, J.M. and Sols, A. (1968) Glycerol metabolism in yeasts. Pathways of utilization and production. Eur. J. Biochem. 5, 165^172. Sprague, G.F. and Cronan, J.E. (1977) Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism. J. Bacteriol. 129, 1335^1342. May, J.W., Marshall, J.H. and Sloan, J. (1982) Glycerol utilization by Schizosaccharomyces pombe: phosphorylation of dihidroxyacetone by a speci¢c kinase as the second step. J. Gen. Microbiol. 128, 1763^1766. Gancedo, C., Llobell, A., Ribas, J.C. and Luchi, F. (1986) Isolation and characterization of mutants from Schizosaccharomyces pombe defective in glycerol catabolism. Eur. J. Biochem. 159, 171^174. Norbeck, J. and Blomberg, A. (1997) Metabolic and regulatory changes associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl. Evidence for osmotic induction of glycerol dissimilation via the dihydroxyacetone pathway. J. Biol. Chem. 272, 5544^5554. Luyten, K., Albertyn, J., Skibbe, W.F., Prior, B.A., Ramos, J., Thevelein, J.M. and Hohmann, S. (1995) Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and e¥ux and is inactive under osmotic stress. EMBO J. 14, 1360^1371. Tamas, M.J., Luyten, K., Sutherland, F.C., Hernandez, A., Albertyn, J., Valadi, H., Li, H., Prior, B.A., Kilian, S.G., Ramos, J., Gustafsson, L., Thevelein, J.M. and Hohmann, S. (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol. 31, 1087^1104. Lages, F. and Lucas, C. (1995) Characterization of a glycerol/H‡ symport in the halotolerant yeast Pichia sorbitophila. Yeast 11, 111^ 119. Lages, F., Silva-Grac°a, M. and Lucas, C. (1999) Active glycerol uptake is a mechanism underlying halotolerance in yeasts: a study of 42 species. Microbiology 145, 2577^2585. Vassarotti, A. and Friessen, J.D. (1985) Isolation of the fructose-1,6bisphosphatase gene of the yeast Schizosaccharomyces pombe. Evidence for transcriptional regulation. J. Biol. Chem. 260, 6348^6353. Zaror, I., Marcus, F., Moyer, D.L., Tung, J. and Shuster, J.R. (1993) Fructose-1, 6-bisphosphatase of the yeast Kluyveromyces lactis. Eur. J. Biochem. 212, 193^199. Traniello, S., Calcagno, M. and Pontremoli, S. (1971) Fructose 1,6diphosphatase and sedoheptulose 1,7-diphosphatase from Candida utilis: puri¢cation and properties. Arch. Biochem. Biophys. 146, 603^610. Ho¡man, C.S. and Winston, F. (1991) Glucose repression of transcription of theSchizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev. 5, 561^571. Stucka, R., Valde¨s-Hevia, M.D., Schwarzlose, C., Feldmann, H. and Gancedo, C. (1988) Nucleotide sequence of the phosphoenolpyruvate carboxykinase gene from Saccharomyces cerevisiae. Nucleic Acids Res. 16, 10926. Valde¨s-Hevia, M.D., de la Guerra, R. and Gancedo, C. (1989) Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae. FEBS Lett. 258, 313^316. Kitamoto, H.K., Ohmono, S. and Imura, Y. (1998) Isolation and nucleotide sequence of the gene encoding phosphoenolpyruvate carboxykinase from Kluyveromyces lactis. Yeast 14, 963^967. Leuker, C.E., Sonneborn, A., Delbruck, S. and Ernst, J.F. (1997) Sequence and promoter regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans. Gene 192, 235^240. Haarasilta, S. and Taskinen, L. (1977) Location of three key enzymes of gluconeogenesis in baker's yeast. Arch. Microbiol. 113, 159^161. Gancedo, C. and Schwerzmann, K. (1976) Inactivation by glucose

FEMSRE 689 29-8-00

C.-L. Flores et al. / FEMS Microbiology Reviews 24 (2000) 507^529

[246] [247]

[248]

[249]

[250]

[251] [252]

[253]

[254]

[255]

[256]

[257]

[258]

[259]

of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae. Arch. Microbiol. 109, 221^225. Osothsilp, C. and Subden, R.E. (1986) Malate transport in Schizosaccharomyces pombe. J. Bacteriol. 168, 1439^1443. Kuczynski, J.T. and Radler, F. (1982) The anaerobic metabolism of malate of Saccharomyces bailii and the partial puri¢cation and characterization of malic enzyme. Arch. Microbiol. 131, 266^270. Baranowski, K. and Radler, F. (1984) The glucose-dependent transport of L-malate in Zygosaccharomyces bailii. Antonie Van Leeuwenhoek 50, 329^340. Viljoen, M., Volschenk, H., Young, R.A. and van Vuuren, H.J. (1999) Transcriptional regulation of the Schizosaccharomyces pombe malic enzyme gene, mae2. J. Biol. Chem. 274, 9969^9975. Boles, E., de Jong-Gubbels, P. and Pronk, J.T. (1998) Identi¢cation and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme. J. Bacteriol. 180, 2875^2882. Gancedo, J.M. (1998) Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62, 334^361. Zaragoza, O., Lindley, C. and Gancedo, J.M. (1999) Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae. J. Bacteriol. 181, 2640^2642. Entian, K.D. (1980) Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast. Mol. Gen. Genet. 178, 633^637. Espinel, A.E., Gomez-Toribio, V. and Peinado, J.M. (1996) The inactivation of hexokinase activity does not prevent glucose repression in Candida utilis. FEMS Microbiol. Lett. 135, 327^332. Go¡rini, P., Ficarelli, A., Donini, C., Lodi, T., Puglisi, P.P. and Ferrero, I. (1996) FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis are homologous to GAL83 and SNF1 of Saccharomyces cerevisiae. Curr. Genet. 29, 316^326. Petter, R., Chang, Y.C. and Kwon-Chung, K.J. (1997) A gene homologous to Saccharomyces cerevisiae SNF1 appears to be essential for the viability of Candida albicans. Infect. Immun. 65, 4909^4917. Kanai, T., Ogawa, K., Ueda, M. and Tanaka, A. (1999) Expression of the SNF1 gene from Candida tropicalis is required for growth on various carbon sources, including glucose. Arch. Microbiol. 172, 256^263. Petter, R. and Kwon-Chung, K.J. (1996) Disruption of the SNF1 gene abolishes trehalose utilization in the pathogenic yeast Candida glabrata. Infect. Immun. 64, 5269^5273. Lundin, M., Nehlin, J.O. and Ronne, H. (1994) Importance of a £anking AT-rich region in target site recognition by the GC box containing zinc ¢nger protein Mig1. Mol. Cell. Biol. 14, 1979^1985.

529

[260] Tzamarias, D. and Struhl, K. (1994) Functional dissection of the yeast Cyc8-Tup1 transcriptional co-repressor complex. Nature 369, 758^760. [261] Dong, J. and Dickson, R.C. (1997) Glucose represses the lactosegalactose regulon in Kluyveromyces lactis through a SNF1 and MIG1 dependent pathway that modulates galactokinase (GAL1) expression. Nucleic Acids Res. 25, 3657^3664. [262] Mukai, Y., Matsuo, E., Roth, S.Y. and Harashima, S. (1999) Conservation of histone binding and transcriptional repressor functions in a Schizosaccharomyces pombe Tup1p homolog. Mol. Cell. Biol. 19, 8461^8468. [263] Braun, B.R. and Johnson, A.D. (1997) Control of ¢lament formation in Candida albicans by the transcriptional repressor TUP1. Science 277, 105^108. [264] Portillo, F. (2000) Regulation of plasma membrane H‡ -ATPase in fungi and plants. Biochim. Biophys. Acta 1469, 31^42. [265] Dufour, J.P. and Go¡eau, A. (1978) Solubilization by lysolecithin and puri¢cation of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe. J. Biol. Chem. 253, 7026^7032. [266] Ghislain, M., Schlesser, A. and Go¡eau, A. (1987) Mutation of a conserved glycine residue modi¢es the vanadate sensitivity of the plasma membrane. H+-ATPase from Schizosaccharomyces pombe. J. Biol. Chem. 262, 17549^17555. [267] Ghislain, M., De Sadeleer, M. and Go¡eau, A. (1992) Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe. Eur. J. Biochem. 209, 275^279. [268] Balcells, L., Martin, R., Ruiz, M.C., Go¨mez, N., Ramos, J. and Arin¬o, J. (1998) The Pzh1 protein phosphatase and the Spm1 protein kinase are involved in the regulation of the plasma membrane H‡ -ATPase in ¢ssion yeast. FEBS Lett. 435, 241^244. [269] Miranda, M., Ramirez, J., Pen¬a, A. and Coria, R. (1995) Molecular cloning of the plasma membrane (H‡ )-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and de¢ciency in K‡ uptake. J. Bacteriol. 177, 2360^2367. [270] Gupta, P., Mahanty, S.K., Ansari, S. and Prasad, R. (1991) Isolation, puri¢cation and kinetic characterization of plasma membrane H(‡ )-ATPase of Candida albicans. Biochem. Int. 24, 907^915. [271] Monk, B.C., Niimi, M. and Sheperd, M.G. (1993) The Candida albicans plasma membrane and H(‡ )-ATPase during yeast growth and germ tube formation. J. Bacteriol. 175, 5566^5574. [272] Kluyver, A.J. (1924) Eenheid en verscheidenheid in de stofwisseling der microben. Chem. Weekblad 21, 266^277. [273] Ho, N.W.Y., Chen, Z. and Brainard, A.P. (1998) Genetically engineered Saccharomyces yeast capable of e¡ective cofermentation of glucose and xylose. Appl. Environm. Microbiol. 64, 1852^1859.

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