Transcriptional, translational and post-translational control
Plasma membrane and cell wall modulation
The yeast Saccharomyces cerevisiae is one of the few yeasts with the capacity to grow rapidly under anaerobic conditions73. This property has made it the most abundantly used yeast in industry. Anaerobic incubation of S. cerevisiae plays a major part in the production of both alcoholic beverages and bread.
Another industrial interest in anaerobic growth arises because of the problems with oxygen gradients encountered in voluminous aerobic fermentations. High cell densities required for the production of heterologous proteins may lead to gradients in the oxygen concentration as a result of imperfect mixing. In general, full levels of oxygenation are almost impossible to maintain in large-scale fermenters. Local and transient hypoxic or anaerobic conditions will trigger transcriptional and metabolic changes in the cell, which could lead to fermentation and thus disturb the production process. Manipulating the activity of a transcription factor that controls key enzymes of specific metabolic pathways could be a solution. This method has been applied for overexpression of Hap4, resulting in partial relief of glucose repression of respiration9. and for disruption of MIG1, alone or in combination with MIG2, which results in the partial alleviation of glucose control of sucrose and galactose metabolism35. Because other mechanisms may also control the intended pathway, the effects are often only partial.
Yet another possible industrial application of anaerobic growth lies in the transfer of this ability to other organisms. For example, the yeast Kluyveromyces lactis can utilize lactose as a sole carbon source. This sugar is the major component of whey, which is a waste product of cheese industry. Conversion of whey to ethanol would greatly reduce the costs and environmental strain of this industry. K. lactis is able to ferment, but can not grow under anaerobic conditions12. Transfer of the genetic information for anaerobic growth from S. cerevisiae could be a solution to this problem.
A similar case can be made for the bioethanol production from lignocellulosic hydrolysates, which mainly contain xylose. In this case, the organism that would be subjected to a transplantation of the ability for anaerobic growth is Pichia stipitis56. Bioethanol is most commonly produced by anaerobic fermentations with S. cerevisiae. Many attempts have been made to increase the overall conversion yield from glucose to ethanol. Recently, Bro et al. (2005) have used a genome-scale metabolic network model in order to find target genes for metabolic engineering13.
Even in humans, genes that are specifically intended for adaptation to conditions of oxygen depletion are present. Expression of the hypoxia-inducible factor-1α (HIF-1α) has been implicated in the induction of metastasis in breast cancer53, 80, whereas the expression of the tumour suppressor p53 and the cytoprotector molecule HO-1 are induced by stress factors, including hypoxia30, 42.
Apart from being fundamentally interesting, insights into the processes that are important for anaerobic growth and in the mechanisms that control them can help to solve problems industry is facing with respect to the anaerobic growth of organisms.
The ability to ferment sugars is a necessity for growth under anaerobic conditions. Although few yeast species are able to grow without oxygen73, most of them are able to ferment69. When a hexose is imported into the cell, it is broken down by glycolysis into two molecules of pyruvate. During glycolysis there is a net production of two molecules of ATP and two molecules of NADH.
Under aerobic conditions NAD+ is regenerated by transfer of the electrons of NADH to the first protein of the respiratory chain. In S. cerevisiae the main entry point of NADH into the respiratory chain is the NADH-Q oxidoreductase Ndi1, which faces the matrix of the mitochondria79. The subsequent process of respiration results in the reduction of molecular oxygen to water and to the generation of a proton gradient along the mitochondrial membrane. This gradient, which is also called the proton-motive force, is then used to drive ATP-synthase, a mitochondrial membrane–enzyme complex44. Also, the pyruvate produced by glycolysis can be further dissimilated to carbon dioxide and water via the pyruvate dehydrogenase complex and the tricarboxylic acid cycle, which results in an additional ATP molecule as well as five redox equivalents. In total, the complete respiratory dissimilation of one molecule of glucose results in 16 ATP molecules71.
In the absence of molecular oxygen, the enzymes pyruvate decarboxylase and alcohol dehydrogenase convert pyruvate into ethanol and carbon dioxide to reoxidize the two molecules of NADH which were produced in glycolysis7. This process is known as alcoholic fermentation. As a consequence, only two ATP molecules are formed from one molecule of glucose.
Oxygen may be a key factor in the regulation of pyruvate decarboxylase activity. High levels of this enzyme are present under aerobic conditions in Crabtree-positive yeasts (see below), such as S. cerevisiae, while in Crabtree-negative yeasts such as Candida utilis and K. lactis the levels increase only under oxygen-limited conditions33, 76. Thus, fermentation would likely be a response to oxygen limitation, which indeed it is in many cases. Interestingly, K. lactis could be turned into a Crabtree-positive yeast by inactivation of the pyruvate dehydrogenase complex81.
When alcoholic fermentation occurs under aerobic conditions, this is called the Crabtree effect23 The long-term Crabtree effect is the occurrence of aerobic fermentation under fully adapted, steady-state conditions at high growth rates, which has been explained in terms of a limited respiratory capacity of the yeast27, 32, and an uncoupling effect of acetate, formed at high growth rates52. The short-term Crabtree effect is the sudden fermentative response under fully aerobic conditions upon addition of excess sugar to yeasts that did not ferment before this addition72. The increased flux of sugar entering the cell results in an increased production of NADH, which cannot be completely oxidized by the respiratory chain. Thus, the production of ethanol by fermentation is needed to remove the excess NADH32, 37. Crabtree-positive yeasts, such as S. cerevisiae and K. lactis, have facilitated-diffusion glucose-transport systems with much higher Km values for glucose than the high-affinity proton-symport mechanisms that are common in Crabtree-negative yeasts70.
A related phenomenon is the Pasteur effect, which is defined as the inhibition of the sugar consumption rate by aerobiosis. Lagunas raises doubts about the common explanation of this phenomenon, that fermentation cannot effectively compete with respiration, in terms of ATP yield, and that this in turn leads to a reduced fermentation rate under aerobic conditions39. In S. cerevisiae the Pasteur effect occurs in aerobic sugar-limited chemostat cultures and in resting-cell suspensions because of low sugar consumption rates74.
Not all yeasts are capable of fermenting all sugars. The Kluyver effect, widespread among yeasts, describes the phenomenon that any given yeast may be able to ferment certain sugars but not others58. There are several factors that are proposed to cause this effect: oxygen requirement for sugar transport, activity of the pyruvate decarboxylase6 and product inhibition76. A Kluyver effect has not been observed for S. cerevisiae.
Even when a particular yeast species is capable of fermenting different sugars, the results of these fermentations may be different. For example, in S. cerevisiae, maltose is co-transported with protons in a one-to-one stoichiometry—proton symport. This import requires the hydrolysis of one molecule ATP per molecule maltose imported. Therefore, anaerobic growth on maltose yields a higher specific ethanol production as compared to the fermentation of glucose75.
Fermentation is a redox-neutral process and any redox equivalents produced in other processes should be reoxidized by the production of glycerol or other highly reduced compounds. The Custers effect occurs in the genera Brettanomyces, Dekkera and Eeniella. These yeasts show an anaerobic inhibition of fermentation of glucose to ethanol and acetate, which is thought to be the result of redox problems55.
Non-respiratory oxygen-utilizing pathways
Molecular oxygen is also required in several biosynthetic pathways, such as those for haem, sterols, unsatured fatty acids, pyrimidines and deoxyribonucleotides3, 19, 45. These reactions have been reviewed recently59 but are briefly summarized here for completeness.
The synthesis of haem is dependent on traces of molecular oxygen and there is no known way to eliminate this requirement. It has been suggested that in anaerobically growing cells, the haem released by degradation of respiratory cytochromes is recycled in the cytoplasm20, 38. The dependency of the biosynthesis of haem on oxygen also implies that production of haemoproteins, most of which are cytochromes, requires oxygen. There may be anaerobic alternatives for these proteins25, 38, 60. However, these proteins still need haem and thus oxygen. If the cells are growing, recycled haem cannot account for it all and cells should have alternative solutions to this problem.
A second pathway that requires oxygen is the biosynthesis of sterols. Under aerobic circumstances sterols are produced in an oxygen-dependent way, through the activities of six Erg enzymes (Figure 1). For the synthesis of one molecule of ergosterol, 12 molecules of molecular oxygen are needed54. Under anaerobic conditions the cells no longer synthesize sterols, but instead import them. This sterol uptake is essential under anaerobic conditions77. Transfer depends on the cellular levels of ergosterol and oleate17, 46. Oleate is added to media for anaerobic growth in the form of Tween 80, and can be used as a source for unsatured fatty acids (UFAs), the production of which is also oxygen-dependent. The transport might be a result of the permeability of the membrane, combined with specific transporters2, 26, 47, 66, 67.
Synthesis of pyrimidines also requires oxygen. The fourth step in the process, the conversion of dihydro-orotate to orotate, is catalysed by dihydro-orotate dehydrogenase (DHDODase), which is a respiratory chain-dependent mitochondrial protein in most yeasts. However, S. cerevisiae, which is able to grow anaerobically, has a cytosolic DHDODase. This enzyme is not dependent on the functionality of the respiratory chain28. Indeed, transfer of the S. cerevisiae DHODase gene (encoded by URA1) into Pichia stipitis transformed this yeast into a facultative anaerobe56.
Biosynthesis of deoxyribonucleotides is catalysed by ribonucleotide reductases (RNRs)37. These enzymes convert the ribonucleotides into their deoxyribonucleotide counterparts. There are three major classes of RNRs. Members of class I are dependent on the presence of oxygen, members of class III function in the absence of oxygen and members of class II can reduce ribonucleotides under both conditions. Until now only class I RNRs have been found in yeast species. However, since the three-dimensional structures of the three classes are quite similar, while the sequence homology is very low, it could be that a class II or III RNR is present in yeasts that are able to grow without oxygen.
Nicotinic acid is required for the synthesis of NAD+ and S. cerevisiae can synthesize it from tryptophan via the kynurenine pathway. The nicotinate moiety can also be recycled and incorporated in NAD+ directly by the activity of nicotinate phosphoribosyl transferase (Npt1). Only the second pathway is oxygen-independent. Since there is no other way to synthesize NAD+, the NPT1 gene is essential under anaerobic conditions49.
Under aerobic conditions, the reoxidation of NADH formed during glycolysis occurs through the respiratory chain, transferring the reducing equivalents to oxygen. This is not possible during anaerobiosis. Several ways to reoxidize NADH are known in S. cerevisiae. Apart from alcoholic fermentation, the genes FRDS and OSM1 encode fumarate reductases, which irreversibly catalyse the reduction of fumarate to succinate, thereby reoxidizing NADH. Other ways to reoxidize excess NADH are through the actions of the Gpd2, which is a glycerol 3-phosphate dehydrogenase, and Adh3, which is a mitochondrial alcohol dehydrogenase4, 5.
Genes essential for anaerobic growth
Despite the large number of S. cerevisiae genes, ca. 500, with significant change transcription levels when aerobic and anaerobic cultures are compared38, 50, 61, 63, only 23 are essential for anaerobic growth (Table 1) and are dispensable under aerobic conditions59. Apart from ARV1, NPT1 and GUP1, the 20 other genes have no obvious function in anaerobic metabolism. Similarly, of the ca. 1300 genes that are essential for aerobic growth, only 33 are not required for anaerobic growth, whereas disruptions of 32 genes leads to retarded anaerobic growth when growth on rich medium, YEPD supplemented with ergosterol and Tween 80, is compared. Apart from genes involved in ergosterol metabolism and some mitochondrion-related genes, there is no clear relationship with aerobic metabolism59. The transcription levels of none of the 23 anaerobic essential genes and the 65 genes that are only essential or required for aerobic growth are significantly affected by the presence or absence of oxygen. It thus appears that there is not a correlation between essential genes and their transcriptional regulation, as has been reported for genes involved in protection against DNA-damaging agents8. When the S. cerevisiae genes that are implicated in anaerobic metabolism, either because of being essential, regulated, or known anaerobic transcription factors, are compared to the K. lactis genome, 20 genes are missing from K. lactis, a species that is not able to grow anaerobically. Seven of these genes are involved in sterol uptake and this suggests that this might be an important factor for the ability to grow under anaerobic conditions59. A similar conclusion was reached when the genomes of hemiascomycetous yeasts were compared10.
Table 1. Genes essential for anaerobic growth and not essential for aerobic growth
The necessity of sterol and unsaturated fatty acids is not as self-evident as it appears, since already in 1971 Bulder16 reported that a strain of Schizosaccharomyces japonicus grew and sporulated under anaerobic conditions without addition of ergosterol and Tween 80 to the medium, as well as aerobically, and that this particular strain was exceptional, as it has a very low sterol content in the absence of oxygen. The anaerobic sporulation is especially remarkable. S. cerevisiae is not able to sporulate in the absence of molecular oxygen, even when Tween 80 and ergosterol are added to the sporulation medium or when the acetate is replaced by low concentrations (0.2%) of glucose, fructose, galactose, maltose or raffinose, sugars that allow sporulation with a frequency of 2–5% under aerobic conditions (H.Y.S., unpublished results).
Transcriptional, translational and post-translational control
The adaptation of yeasts to an anaerobic environment, as compared to conditions in which oxygen is present, takes place at different levels in the cell. First, there is the evolutionary adaptation. Since S. cerevisiae has been used in anaerobic processes for centuries, it has adapted to living without oxygen more than any other known yeast strain. The ability to grow anaerobically is believed to originate from whole-genome duplication around 100 000 000 years ago51, 78. Species such as K. lactis, which diverged from a common ancestor before this event, are not able to grow without oxygen. Today the evolutionary favouring of a predominantly fermentative metabolism of S. cerevisiae in the wild can still be seen in its codon bias, and it is therefore termed a ‘translationally biased’ organism18.
A second level of adaptation is the transcriptional changes of genes that are differentially needed under anaerobic and aerobic conditions. Several factors for transcriptional regulation of anaerobic metabolism have been proposed83. One of those is the upregulation of aerobic genes by a homodimer of Hap1, bound to haem82, and the derepression by Hap1 when haem is absent29. ROX1 is one of the targets of Hap1 and Rox1, together with the Tup1–Ssn6 complex, represses hypoxic genes in the presence of oxygen24. Mot3 is needed for efficient normoxic repression of a subset of anaerobic genes1. In another regulatory system, UPC2 and ECM22 are implicated in a dual role in the induction of anaerobic sterol import21, 57, 62. Upon sterol depletion, Upc2 levels increase, as does the amount of Upc2 bound to promoters. Ecm22, however, shows a decrease in both the total amount of protein in the cell and the fraction bound to promoters22. The induction of UPC2 upon anaerobiosis appears to be the result of haem depletion. Mox1 and Mox2 could interact with Upc2 to fine-tune its regulatory function1. Another factor that has been implicated in the sterol import system, needed under anaerobic conditions, is Sut1. This factor, and perhaps also Sut2, has a regulatory effect on the permeability of the membrane2. The expression of Sut1 increased following a shift to anaerobic conditions.
Other genes have also been implicated in anaerobic regulation, either because of their effect on transcriptional levels or because of their haem dependency, such as ORD141 and HAP2/3/4/583. All of these genes together regulate the expression of aerobically and anaerobically specific genes in a complex way. However, the transcriptional responses to anaerobiosis of many genes are still unexplained, such as the PAU genes, which are genes of unknown function that are strongly and consistently upregulated under anaerobic conditions61. Also, the transcriptional changes of the cell wall proteins Dan1 and Tir1, when aerobic conditions are compared to anaerobic ones, cannot be explained by the alleviation of aerobic repression by Rox1 alone34, 64. It has been shown that for the DAN/TIR genes, activation through Upc2 is necessary. Repression seems to be mediated by Rox1, Mot3, Mox1, Mox2 and the Tup1–Ssn6 complex1. Repression of ANB1 is not completely abolished by deletion of ROX1, suggesting that in this case activation is also needed64. Furthermore, the promoter of the anaerobically upregulated YML083C gene does carry a Rox1 binding site, but deletion of these bases has no effect on transcription levels65.
Finally, in a screen for anaerobic transcription factors, Snf7 was found. The SNF7 gene, originally identified because of its regulating effect on SUC2 expression, is involved in endo- and exocytosis. Snf7 is a component of the ESCRT III complex, which is part of the multivesicular body sorting pathway for recycling or degradation of membrane proteins11. Microarray data of anaerobic chemostat cultures of a snf7 mutant showed that 13.5% of the 155 ‘true’ anaerobic genes, as defined by Tai et al61, changed transcription significantly when compared to the wild-type strain. Half of these encode proteins that are designated as cell wall or plasma membrane proteins, suggesting that Snf7 is involved in the remodelling of the cell envelope which occurs in anaerobic conditions (see below) (Snoek et al., manuscript in preparation).
The third level of regulation is translation of the mRNA into protein. Not all changes observed in behaviour can be attributed to transcriptional control. Although regulation of haemoproteins such as catalases is found at the mRNA level, (post)translational control has to be postulated to explain the fact that double mutants of ole3, which in itself gives a haem-deficient phenotype, together with cgr4 or cas1, contain mature catalase mRNA31.
Iron regulatory proteins (IRPs) modulate mRNA utilization by binding to iron-responsive elements (IREs) in the 5′ or 3′ untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. In yeast, Irp1 can function as an oxygen-modulated posttranscriptional regulator of gene repression. This is the result of the stability of the 4Fe–4S cluster in the protein, which is dependent on phosphorylation. Unphosphorylated clusters decay fast when exposed to oxygen. Only with an intact 4Fe–4S cluster is the protein able to bind IREs14.
Closely related to the translational regulation are the posttranslational mechanisms. This interaction is very clear in the regulation of the ANB1 gene. Transcription of the anaerobic gene ANB1 is regulated by oxygen and haem via Rox1. Anb1 is probably the yeast homologue of the eukaryotic translation initiation factor eIF-4D. Apart from influencing translation initiation, the protein itself undergoes a posttranslational modification of the Lys-50 residue to the amino acid hypusine43. SOD1 is also posttranslationally activated through the functioning of the copper chaperone for SOD1 (CCS) to accommodate a fast response to a sudden elevation of oxygen availability15.
Plasma membrane and cell wall modulation
The plasma membrane forms a relatively impermeable barrier for hydrophilic molecules. It consists of a bilayer of polar lipids and proteins. These proteins are often associated with other proteins in the plasma membrane, with the cytoskeleton or with proteins in the extracellular matrix. They can be either intrinsic, spanning the whole membrane, or extrinsic, embedded in part of the membrane and protruding from one side. Functions of these proteins vary from amino acid and sugar transporters to ATPases, cell wall synthesis, signal transduction, or they can be part of the cytoskeleton. The lipids are disposed asymmetrically across the bilayer and are greatly diverse in size and composition, which is tightly regulated. This suggests that lipids play a role in the activity of the embedded proteins. Some membrane-associated processes, such as amino acid transport and membrane ATPase activity, are affected by a changed lipid composition. The rigidity of the membrane is largely determined by the sterol content. This may affect the lateral movement and activity of membrane proteins. Alternatively, sterols may also create patches into which polypeptides can insert68.
The lipid composition of the membrane under anaerobic conditions is different from that of cells grown under aerobic conditions. Anaerobically, the plasma membrane contains more saturated fatty acids, less total sterol, less ergosterol and less squalene48. These differences can be explained by the inability of the cell to synthesize these compounds without oxygen.
The cell wall is a rigid structure that surrounds the cell and gives it its shape. It protects the cell from the effects of outside conditions, such as heat, cold and osmotic stress. It also works as a selection filter for the entrance of substances into the cell. Most compounds, both organic, such as sugars, and inorganic, such as metal ions, need a facilitator to get across the cell wall and an active transporter in the plasma membrane.
The cell wall is composed of several layers, the first of which contains β 1,3-glucan and chitin. These compounds are responsible for the mechanical strength of the cell wall. The outer layer consists of heavily glycosylated mannoproteins. These make the inner layer less accessible to cell wall-degrading enzymes and are involved in cell–cell recognition events. The porosity of the cell wall is mainly determined by this outer layer, because of the long and highly branched carbohydrate side chains linked to asparagine residues. The inner layer is highly porous and limits only the passage of very large molecules. The way in which the mannoproteins are linked to the inner layer divides them into two groups. GPI-dependent cell wall proteins (GPI-CWPs) are linked indirectly through a β1,6-glucan moiety. Pir proteins (Pir-CWPs) are directly linked to β1,3-glucan. The cell seems to be able to repair cell wall damage, among other ways, through the Slt2 MAP kinase pathway, which is rapidly induced upon stress. Sensing of the damage is probably the result of plasma membrane stretch. The sensors, such as Mid2, are linked to the β1,3-glucan network in a Pir-like fashion. Generally the activation of the Slt2 MAP kinase pathway leads to the activation of several cell wall reinforcing reactions, one of which is the elevation of chitin levels. Another MAP kinase pathway, the Hog1 pathway, is also implicated in cell wall construction, both under stress and in non-stress conditions36.
Upon anaerobiosis there is a general remodelling activity associated with the cell wall and plasma membrane. This remodelling is required, in part, for the efficient import and processing of the supplements needed under these conditions, such as oleate and ergosterol, in order to combat the compromised ability to regulate membrane fluidity38. However, these changes are slow to occur and take several generations for completion40. Generally, transcript levels of CWP1 and CWP2 decrease, while those of the seripauperin family genes, such as DAN, TIR and PAU, increase36. These changes are quite drastic and suggest a complete switch from one set of GPI-CWPs to another. It is not known how this change facilitates the import of supplements or whether perhaps it has some additional functions.
Growth in the absence of molecular oxygen requires adaptation of the cell for at least three reasons: first, energy yield is usually much lower than under aerobic conditions; second, several biosynthetic pathways require molecular oxygen; and third, different molecules have to be transported into and out of the cell (Figure 2). In S. cerevisiae this adaptation appears mainly on the regulatory level, in particular transcription. Only 23 of the genes are specifically essential for anaerobic growth, whereas the transcription levels of ca. 500 genes differ significantly when aerobic and anaerobic cultures are compared. For many of these genes, it is unclear why they are essential or why their expression levels are up- or downregulated under anaerobic conditions. This means that the requirements for life in the absence of molecular oxygen are still largely unknown and that the study of anaerobic organisms, including yeasts, may have some surprises in store.
This work was supported by a grant from the Netherlands Organization for Scientific Research (NWO/ALW, No. 811.35.004). We would also like to thank our colleagues in Delft and Leiden for helpful discussions.