3 Yeast Yeast Metabol Metabol ism Metabolism refers to the biochemical assimilation (in anabolic pathways) and dissimilation (in catabolic pathways) of nutrients by a cell. Like in other organisms, in yeast these processes are mediated by enzymic reactions, and regulation of the underlying pathways have been studied in great detail in yeast. An abol ic pat hw ays ays include reductive processes leading to the production of new cellular material, while catabolic pathways are pathways are oxidative processes which remove electrons from substrates or intermediates that are used to generate energy. Preferably, these processes use NADP or NAD, respectively, as co-factors. Although all yeasts are ar e microorganisms that derive their chemical energy, in the from of ATP, from the breakdown of organic compounds, there is metabolic diversity in how these organisms generate and consume energy from these substrates. Knowledge of the underlying regulatory mechanisms is not only valuable in the understanding of general principles of regulation but also of great importance in biotechnology, if new metabolic capabilities of particular yeasts have to be exploited. It is now well established that most yeasts employ sugars as their main carbon and hence energy source, but there are particular yeasts which can utilize non-conventional carbon sources. With regard to nitrogen metabolism, most yeasts are capable of assimilating simple nitrogenous sources to biosynthesize amino acids and proteins (Table 3-1). Aspects of phosphorus and sulphur metabolism as well as aspects of metabolism of other inorganic compounds have been studied in some detail, predominantly in the yeast, Saccharomyces Saccharomyces cerevisiae. cerevisiae. Table 3-1: Nutrients for growth of yeast (S. cerevisiae) cells. Substrate Saccharose Maltose Melibiose Glucose Ethanol
Lactate Glycerol
Intermediates
Acetaldehayde > Acetyl-CoA> Oxaloacetate> Pyruvate> Glycerol-3phosphate> Dihydroxyacetonphosphate
Enzymes Invertase Maltase Melibiase Alcohol-Dehydrogenase
Lactate-Dehydrogenase
Product s Glucose + Fructose Glucose Glucose + Galactose Products of Glycolysis Glucose by gluconeogenesis
Glucose by gluconeogenesis Glucose by gluconeogenesis
Amino acids Glutamate Ammonium
3.1 3. 1 Sugar Cataboli Cataboli sm in Ye Yeast ast 3.1. 3. 1.1 1 Princ ipal Pa Pathways thways The major source for energy production in the yeast, Saccharomyces cerevisiae, cerevisiae, is glucose and glycolysis is the general pathway for conversion of glucose to pyruvate, whereby production of energy in form of ATP is coupled to the generation of intermediates and reducing power in form of NADH for biosynthetic pathways. Two principal modes of the use of pyruvate pyruvate in further energy production can be distinguished: respiration and fermentation (Figure 3-1). In the presence of oxygen and absence of repression,
pyruvate enters the mitochondrial matrix where it is oxidatively decarboxylated to acetyl CoA by the pyruvate dehydrogenase multi enzyme complex. This reaction links glycolysis to the citric acid cycle, in which the acetyl CoA is completely oxidized to give two molecules of CO2 and reductive equivalents in form of NADH and FADH2. However, the citric acid cycle is an amphibolic pathway, since it combines both catabolic and anabolic functions. The latter results, for example, from the production of intermediates for the synthesis of amino acids and nucleotides. Replenishment of compounds necessary to drive the citric acid cycle, such as oxaloacetate and α-ketoglutarate, are (i) the fixation of CO2 to pyruvate by the actions of the enzymes pyruvate carboxylase (ATP-dependent) and phosphoenolpyruvate carboxykinase and (ii) the glyoxalate cycle (a shortcut across the citric acid cycle), which is important when yeasts are grown on two-carbon sources, such as acetate or ethanol.
Figure 3-1: Metabolism in yeast under aerobic and anaerobic conditions. During alcoholic fermentation of sugars, yeasts re-oxidize NADH to NAD in a two-step reaction from pyruvate, which is first decarboxylated by pyruvate decarboxylase followed by the reduction of acetaldehyde, catalyzed by alcohol dehydrogenase (ADH). Concomitantly, glycerol is generated from dihydroyacetone phosphate to ensure production of this important compound. An alternative mode of glucose oxidation is the hexose phosphate pathway also known as the pentose phosphate cycle, which provides the cell with pentose sugars and cytosolic NADPH, necessary for biosynthetic reactions, such as the production of fatty acids, amino acids and sugar alcohols. The first step in this pathway is the dehydrogenation of glucose-6-phosphate to 6phosphogluconolactone dehydrogenase).
and
generation
Subsequently,
of
one
mole
6-phosphogluconate
of is
NADPH
(by
decarboxlated
glucose-6-phosphate by
the
action
of
phosphogluconate dehydrogenase to give ribulose-5-phosphate and a second mole of NADPH. Thus, besides generating NADPH, the other major function of this pathway is the production of ribose sugars which serve in the biosynthesis of nucleic acid precursors and nucleotide coenzymes. The redox carriers, NAD and FAD, which become reduced during the breakdown of sugars to NADH and FADH2, respectively, are reoxidized in the respiratory (electron transport) chain located in the inner mitochondrial membrane. The energy released during the transfer of electrons is coupled to the
process of oxidative phosphorylation, which is effected by ATP synthase, an enzyme complex which is also located in the inner mitochondrial membrane and designed to synthesize ATP from ADP and inorganic phosphate. These pathways will be considered separately in chapter 9: Transport.
3.1.2 Regulation of B ioch emical Pathw ays Biochemical pathways in yeasts are regulated at various levels: (i) Enzyme synthesis - induction, repression and derepression of gene expression; (ii) Enzyme activity - allosteric activation, inhibition, or interconversion of isoenzymes; (iii) Cellular compartmentalization - localization of particular pathways to the cytosol, mitochondria, peroxisomes, or the vacuole; (iv) Transport mechanisms - internalization, secretion, trafficking of compounds between the various cellular compartments. Like in the studies of many biochemical aspects, yeast as a versatile system has contributed significantly to decipher a number of important regulatory circuits, which in many instances have been conserved among all eukaryotes investigated thus far. Examples will be presented in chapters ‘Transport’ and ‘Regulation’.
3.1.3 Respir ation versus Fermentati on Yeasts can be catagorized in several groups according to their modes of energy production, utilizing respiration or fermentation (Table 3-2). It is important to note that these processes are mainly regulated by environmental factors, the best documented being the availability of glucose and oxygen. Thus yeasts can adapt to varying growth environments, and even within a single species, the prevailing pathways will depend on the actual growth conditions. For example, glucose can be utilized in several different ways by S. cerevisiae, depending on the presence of oxygen and other carbon sources. Table 3-2: Principal modes of respiration in yeasts. Types
Examples
Respiration
Fermentation
Anaerobi c growth
Obligate respirers
Rhodotorula spp. Cryptococcus spp.
YES
NO
NO
Anaerobic respirers
Candida spp. Kluyveromyces spp. Pichia spp. S. pombe
YES
Anaerobic in pregrown cells
NO
Limited
Aerobic and anaerobic
NO
Facultative aerobic fermenters
S. cerevisiae
Limited
Aerobic and anaerobic
Facultative
Obligate fermenters
Torulopsis
NO
Anaerobic
YES
Aerobic fermenters
Figure 3-2: Sugar metabolism in different yeasts. Catabolite repression occurs when glucose or an initial product of glucose metabolism represses the synthesis of various respiratory and gluconeogenic enzymes. Catabolite inactivation results in the rapid disappearance of such enzymes on addition of glucose. In catabolite repression, enzyme activity is lost by dilution with cell growth. Although enzymes are still present, they are no longer synthesized
due to gene repression by signals derived from glucose or other sugars. However, the nature of the signal(s) is not clear at present. Glucose repression in yeast describes a long-term regulatory adaptation to degrade glucose exclusively to ethanol and CO2. Therefore, when S. cerevisiae is grown aerobically on high concentrations of glucose, fermentation will account for the bulk of glucose consumption. In batch culutures, when the levels of glucose decline, cells become gradually derepressed, resulting in the induction of respiratory enzyme synthesis. This in turn results in oxidative consumption of ethanol, when cells enter a second phase of growth known as the diauxic shift. Catabolite inactivation is more rapid than repression and is thought to be due to deactivation by glucose of a limited number of key enzymes, such as fructose 1,6-bisphosphatase. Inacivation occurs primarily by enzyme phosphorylation, followed by slower vacular degradation of the enzyme. It has been established that cAMP as a second messenger plays a central role in regulating catabolite repression and inactivation in S. cerevisiae.
3.1.4 Other Sugars - Galact ose Galactose is a 'non-conventional' nutrient for yeast, which however can be used as a sole carbon source when glucose is absent from the medium. In yeast cells supplied with glucose, the GAL genes are repressed. They are activated a thousand fold in cells that are starved for glucose, and this one of the few pathways in yeast which is regulated in a nearly 'all-or-nothing' mode. The three enzymes involved are depicted in Figure 3-2.
Figure 3-2: Metabolism of galactose.
3.1.5 Metaboli sm of Non-Hexose Carbon Sources In addition to hexose sugars, yeasts can utilize a number of 'non-conventional' carbon sources , such as biopolymers, pentoses, alcohols, polyols, hydrocarbons, fatty acids and organic acids. This is of particular interest for biotechnological processes, the most prominent being the use ofS. cerevisiae in fermentation. One should also remember that free glucose is scarce in natural environments or in natural products used to feed yeast cells. For example, disaccharides, such as maltose, sucrose, melibiose, lactose or cellobiose can easily be accepted as nutrients by the action of corresponding hydrolases which break these disaccharides down into their constituent monosaccharides (Table 3-3). Notably, hydrolysis is coupled to transport of either the disaccharide or the resulting monosaccharides. Table 3-3: Disaccharides as substrates in yeasts. Disaccharide
Extracellular hydrolysis
Maltose
Intracellular hydrolysis
Products
Organism
Maltase
2 Glucose
S. cerevisiae
Sucrose
Invertase
Glucose + Fructose
S. cerevisiae
Melibiose
α−galactopyranosidase
Glucose + Galactose
S. carlsbergensis
Glucose + Galactose
Kluyveromyces
2 Glucose
Brettanomyces
Lactose Cellobiose
ß-galactosidase ß-glucosidase
Other saccharide biopolymers, like starch, inulin, cellulose, hemicellulose, or pectin, can be metabolized by some specialized yeasts directly, while for the use of carbon sources to other species they have to be hydrolyzed by non-yeast enzymes before utilization. Pentose sugars can be fermented to ethanol by only very few yeast species, although many yeasts can grow aerobically on pentoses. The inability of S. cerevisiae to ferment xylose (e.g. derived from hemicellulose) could be circumvented by introducing genes for xylose reductase and xylitol dehydrogenase from xylose-fermenting species (Pichia) by recombinant DNA technology. However, the efficiency of xylose fermentation remains low. Many yeasts have the capability of metabolizing ethanol (Table 3-4) or methanol , an approach used in biomass production of yeasts of biotechnological interest. Methanol-utilizing (methylotropic) yeasts are found, for example, in Hansenula polymorpha, Pichia pastoris, several Candida species, and Torulopsis sonorensis. In these organisms, methanol is first metabolized by an O2-dependent oxidase to formaldehyde, which is then converted into dihydroxy acetone (DAH) by a DAH synthase. DHA and GAP can be utilized to synthesize fructose-6-phosphate. Glycerol functions as a compatible solute in osmoregulation in osmotolerant yeasts that are capable of growing in high sugar or salt environments. Many yeasts can grow on glycerol as a sole carbon source under aerobic conditions, but glycerol is a non-fermentable carbon source for many yeast species, including S. cerevisiae. To serve as a carbon source, glycerol after internalization has to converted by glycerol kinase to glycerol-3-phosphate, which is then transformed into DAH phosphate by glycerol-3-phosphate dehdrogenase that is a substrate in gluconeogenesis.
Table 3-4: Use of unusual nutrients in yeasts. Carbon sour ce
Metabol ites
Examples
Starch
Glucose
Candida spp.; Pichia spp.
Cellulose
Glucose
Hemicellulose
Glucose, Xylose
Candida spp. ; Pichia spp.
Pectin
Galacturonic acid
Candida spp. ; Kluyveromyces
Inulin
Fructose
Candida spp. ; Kluyveromyces
Xylose
Pyruvate > Ethanol
Candida; Pichia; Kluyveromyces
Organic acids
Acetyl-CoA
Many yeasts
Protein
Amino acids
Candida spp. ; Kluyveromyces spp.; S. cerevisiae
Lipids
Fatty acids + glycerol
Candida spp.; Pichia spp.; Yarrowia lipolytica
Alkanes
Fatty acids
Candida; Pichia; Yarrowia lipolytica
Methanol
GAP + DAP
Hansenula; Pichia pastoris; Candida
Nitrog en sour ce
Metabol ites
Examples
Urea
Ammonium (urea ammonium
Many yeasts
hydrolase) Nitrate
> Nitrite > Ammonium
Candida spp.; Hansenula spp.
3.2 Gluconeogenesis and Carbohydrate Biosynthesis The growth of yeast on non-carbohydrate substrates as sole carbon sources necessitates the synthesis of sugars required for macromolecular biosynthesis, especially that of complex polysaccharides. Like in other organisms, gluconeogenesis, the conversion of pyruvate to glucose is dependent on ATP as an energy donor and NADH as a reducing power. Structural polysaccharide synthesis in yeast is associated with the cell and the spore wall and include mannans, glucans and chitin. Like in other organisms, all sugar polymerization reactions employ sugar nucleotides as substrates, which are formed via activation by UTP or GTP, depending on the substrate. A major activity in yeast is the synthesis of storage carbohydrates: glycogen and trehalose. Like in other organisms, glycogen is formed by sequential addition of glucose units from UDP-glucose, employing glycogen synthase for the linear enzyme in the formation of
α-1,6
α-1,4-linkage
of the backbone chain, and branching
branches. Degradation of glycogen to glucoese-1-phosphate is
effected by glycogen phosphorylase. cAMP is known to be involved in the regulation of glycogen metabolism. An unconventional storage disaccharide found in yeast is trehalose (α,α-1,1-diglucose), present in particularly high concentrations in resting and in stressed cells. Trehalsoe-phosphate is synthesized in yeast from glucose-6-phosphate and UDP-glucose by trehalose-6-phosphate synthase and converted
to terhalose by a phosphatase. The breakdown of trehalose to glucose is mediated by trehalase. Both synthesis and degradation are regulated via cAMP.
3.3 Fatty Acid and Li pid Metabolism Fatty acids available to yeasts for catabolism include those derived from microsomal alkane oxidation or extracellular lipolysis of fats or those exogenously supplied in the growth medium. The fatty acids are catabolized by ß-oxidation in peroxisomes, which differs from the system in the mitochondria in the involvement of catalase in re-oxidizing FADH2 and in the mechanism of re-oxidizing NADH (Figure 33).
Figure 3-3: Fatty acid utilization. The series of reactions leading to the synthesis of long-chain fatty acids, starting from acetyl-CoA is achieved by a multi-enzyme complex, the fatty acid synthase. The subsequent formation of unsaturated fatty acids, which are needed for membrane integrity, involves an oxidative desaturation by a fatty acid desaturase. Synthesis of lipids is similar to the reactions known in other organisms, starting from glycerolphosphate and fatty acids. Breakdown of lipids effected by a yeast lipase that generates long-chain fatty acids and glycerol, which latter is catabolized in the glycolytic pathway. The metabolic pathways of glycerophospholipids in yeast are depicted in Figure 3-4.
Figure 3-4: Metabolic pathways of glycerophospholipids in yeast [Kohlwein et al., 1996]. Pink box: synthesis and activation of fatty acids; blue box: de nova pathway of phospholipid synthesis, synthesis of bulk membrane lipids; yellow box: phospholipid degradation and recycling of amino-alcohol head groups (salvage pathway); green box: phospholipid remodelling, deacylation and reacylation of phospholipids ( fatty acid specificity in s n-1 and sn-2 positions); red box: phosphatidylinositol (Ptdlns) phosphorylation, signalling and membrane vesicle fusion. Precursors and lipids: CDP-DAC, cytidine diphosphate-diacylglycerol; Cho, choline; Cho-CDP, cytidine diphosphate-choline; Cho-P, choline phosphate; CL, cardiolipin; DAG, diacylglycerol; DAC-PP, diacylglycerol pyrophosphate; Etn, ethanolamine; EtnCDP, cytidine diphosphate-ethanolamine; Etn-P, ethanolamine phosphate; FFA, free fatty acid; Clc-6-P, glucose 6-phosphate; 00-3-P, glycerol 3-phosphate; Ins, inositol; Ins-l-P, inositol 1 -phosphate; PtdCho, phosphatidylcholine; PtdDMEtn, phosphatidyldimethylethanolamine; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdGro-P, phosphatidylglycerol-phosphate; Ptdlns, phosphatidylinositol; PtdMMEtn, phosphatidylmonomethylethanolamine; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; TAG, triacylglycerol. Enzymes and genes (italic): ACCI, acetyl-CoA carboxylase; CCTI, CWcholinephosphate cytidylyltransferase; CDSI, CDPdiacylglycerol synthase; CHO7, phosphatidylserine synthase; Cff02, phosphatidylethanolamine N-methyltransferase; CKII, choline kinase; CPTl, cholinephosphotransferase; CTR7, choline transporter; ECT?, ClFethanolaminephosphate cytidylyltransferase; EPT7, ethanolaminephosphotransferase; FAA7-4, acyl-CoA synthetases l-4; FAS1,2, fatty acid synthase subunits p and cl; CAT, glycerol-3-phosphate acyltransferase; IN07, inositol-1 -phosphate synthase; NMJ?, myristoyl-CoA protein N-myristoyltransferase; O/I?, acyl-CoA desaturase; OP13, phospholipid-N-methyltransferase; PAP, phosphatidate phosphatase; /X7, phosphatidylinositol synthase; PLB7, phospholipase B; PLC7, Ptdlns-specific phospholipase C; PSD7,2, phosphatidylserine decarboxylase; PLD7/SPO74, phospholipase D.
3.4 Nitr ogen Metaboli sm 3.4.1 Catabolic Pathways Yeasts are capable of utilizing a range of different inorganic and organic sources of nitrogen for incorporation into the structural and functional nitrogenous components of the cell, such as amino acids (and consequently peptides and proteins), polyamines, nucleic acids and vitamins. Growth media are often supplemented with complex mixtures of amino acids. However, yeasts can also live on ammonium ions as a sole nitrogen source, because they posses a whole repertoire of genes encoding enzymes to the biosynthesis of all amino acids.
Ammonium ions , either supplied as nutrient or derived from the catabolism of other nitrogenous compounds, can be directly assimilated into a couple of amino acids, notably glutamate and glutamine, which can then serve as donors of the amino group in other amino acids. The major route for assimilation of ammonium is the reaction of the NADPH-dependent glutamate dehydrogenase (GDH) which forms glutamate from
α-ketoglutarate
and ammonium. Whenever ammonium ion
concentration is low, but also as a prerequisite for the synthesis of many nitrogenous compounds glutamine synthase is activated, which forms glutamine from
α−ketoglutarate
and ammonium in an
ATP-dependent reaction. Glutamine is absolutely required as a prominent precursor in several important pathways, such as the synthesis of asparagine, tryptophane, histidine, arginine, carbamoyl phosphate, CTP, AMP, GMP, glucosamine, and NAD. Whereas S. cerevisiae is incapable of utilizing nitrate as a nitrogen source, there are a couple of other yeast species that have this capability. Nitrate assimilation occurs by the action of NADPH-dependent nitrate reductase, forming nitrite. Subsequently, nitrite is reduced to ammonium by NADPH-dependent nitrite reductase. Urea is widely used by yeasts as a nitrogen source. In urease-negative S. cerevisiae, urea aminohydrolase (ATP-dependent urea carboxylase plus allophanate hydrolase), hydrolyses urea to ammonium and carbonate.
Figure 3-5: Scheme of amino acid biosynthesis in yeast.
3.4.2 Amino Acid Biosynthesis Pathways All of these pathways and their regulation in yeast have been studied in great detail. For example, the metabolism of methionine and S-adenosyl methionine is mediated by nearly 20 different enzymes. Because of their complexity, we will not summarize the pathways in this brief overview. Figure 3-5 just summarizes the major reactions.
3.4.3 Prot ein Bios ynth esis Protein biosynthesis has been studied in yeast as one of the first eukaryotic model organisms. Many basal findings on the structure and function of tRNAs, tRNA synthetases, 80S ribosomes, and the initiation, elongation and termination factors mediating translation have been identified in yeast and studied in great detail (see chapter ‘Expression’).
3.5 Phosphate Metabolism
Figure 3-6: Phosphate acquisition and storage system in yeast.
Phosphorus requirements of yeast cells are met by the uptake of inorganic phosphate from the nutrient media (Figure 3-6). The phosphate taken up can be utilized for incorporation of major cell constituents, such as phospholipids, nucleic acids and proteins, and is needed for the many transphosphorylation reactions in intermediary metabolism. The intracellular concentration of free phosphate is generally maintained at very low levels. Only when yeast cells switch from respiratory to fermentative metabolism following a glucose pulse, dynamic fluctuations in cellular phosphate have been observed. The bulk of phosphate in yeast is in organic linkage and in the form of polyphosphates. These latter are linear polymeres of orthophosphate in anhydrous linkage. As high concentrations of polyphosphates are accumulated and their hydrolysis yields the same amount of free energy as the hydrolysis of ATP to ADP and Pi, they are important for both phosphorus and energy supply in the cell. In addition to membrane-associated ATPases, yeast cells contain many important enzymes involved in phosphorylation and dephosphorylation: kinases and phosphatases are crucial in governing a multitude of cellular processes, like in other eukaryotes. A peculiarity of yeast is the presence of alkaline (PHO 8) and acid phosphatases (PHO3, PHO5, PHO10, PHO11) in the periplasm which act non-specifically on several phosphate esters of sugars, alcohols and nucleosides, to supplement phosphorus supply. One of the acidic phosphatases genes, PHO3, is constituvely expressed, while the PHO5 gene is highly regulated and turned on at low phosphate concentrations (see chapter ‘Regulation’).
3.6 Sulph ur Metabolism The sulphur requirement of yeast can be met by the uptake of sulphates, which can be assimilated through reduction into sulphur amino acids.
3.7 Transitio n Metals All eukaryotes and most prokaryotes require transition metals, such asiron, copper, zinc, and manganese. These metals have to be acquired by cells via specific transport systems that mediate uptake across the plasma membrane. Much of this understanding has resulted from genetic and biochemical studies in yeast, and the regulation has been defined at both the transcriptional and posttranscriptional level. These aspects will be dealt with in chapters 8 (Transport) and 13 (Signalling and Regulation).
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