Some microbial species, including D. hansenii, produce and accumulate high amounts of lipids. In analogy to oil-bearing plant seeds these microorganisms have been termed oleaginous80. The capacity to accumulate lipids is found in less than 30 of about 600 yeast species155. There is no official definition of oleaginous microorganisms, but operationally microorganisms capable of accumulating more than 20–25% of their biomass as oil are regarded as oleaginous. For yeasts, moulds and eukaryotic algae, but not bacteria, a biochemical definition of oleagenicity based on the presence of the enzyme ATP : citrate lyase (EC 22.214.171.124) has been proposed154, 155. This enzyme generates acetyl-CoA, the key substrate for fatty acid biosynthesis, in the cytoplasm from citrate. Oleaginous microorganisms transport citrate out of the mitochondria into the cytoplasm via a malate–citrate translocase system, and cleave citrate by ATP-citrate lyase to form acetyl-CoA:
Non-oleaginous organisms do not possess the enzyme and rely on less effective means of producing acetyl-CoA in the cytoplasm. The ATP-citrate lyase is undoubtedly crucial for the lipid accumulation but not the only enzyme controlling and directing the metabolism towards storage lipids. Malate dehydrogenase [oxaloacetate-decarboxylating; NADP(+); malic enzyme E.C. 126.96.36.199) seems to control the activity governing the extent of lipids which are accumulated in oleaginous microorganisms155:
The first investigators to analyse the lipids of D. hansenii were Merdinger and Devine120. They showed that neutral lipids and phospholipids comprised 67% and 33% of the organism's total lipids, respectively. The neutral lipids, which are present as microdroplets, were almost entirely composed of triacylglycerols. The fatty acyl groups of both the neutral and phospholipids are usually conventional plant-like entities in the following order of relative abundance: oleic (18 : 1) > palmitic (16 : 0) > linoleic (18 : 2) = stearic acid (18 : 0)156. Some variations occur between various yeast strains. Ergosterol, together with smaller amounts of stigmasterol and another unidentified sterol, were also found in the cells, as were saturated hydrocarbons containing 16–39 carbon atoms, C22 being the most prevalent. The major phospholipid was phosphatidylcholine, followed by phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phophatidylglycerol and cardiolipin. The overall fatty acid composition of the phospholipids was only slightly changed when D. hansenii was grown at high NaCl concentrations. Similarly, the fatty acid profiles of the individual phospholipids were only slightly affected by increases in salinity, the most notable change being an increasing proportion of polyenoic C18 acids in cardiolipin. Increases in salinity caused increases in the proportion of phosphatidylserine, while those of phophatidylglycerol, phosphatidyl-ethanolamine, and phosphatidylinositol decreased. Only minor changes were observed in the relative abundance of phosphatidylcholine and cardiolipin178.
Yeasts do not possess rare fatty acids that could be of commercial interest. Hence, microbial oils will not be able to compete with cheap plant seed oils unless very cheap substrates or waste substrates can be identified. However, yeasts can produce other potentially useful lipids that are usually not found in plants, including glycolipid surfactants, such as a sophortose lipid produced by Candida bombicola, and carotenoids, e.g. astaxanthin from Phaffia rhodozyma156. It is possible that yeast-based biotechnological production of such lipids could be commercially competitive with their chemical production, if the organisms can be induced or manipulated to produce them cost-effectively. Genetic modification may pave the way to such syntheses, as will be illustrated in detail later.
D. hansenii is notably extremophilic, due to its osmo- and xerotolerance as well as its halotolerance (Table 2)9, 40. These features have prompted many investigations on D. hansenii, and its growth dependency on high salt regimes has been repeatedly shown40, 76, 150, 176. These investigations have shown that D. hansenii grows faster in the presence of up to 1 M NaCl or KCl than in lower concentration of salts. Debaryomyces can grow in a wide range of water activities, and natural habitats for that yeast include sea water and habitats such as meat, wine, beer, cheeses, fruits and soil.11 According to a widely accepted definition by Kushner98, one can distinguish slight halophiles (many marine organisms; optimal growth at about 3% w/v NaCl), moderate halophiles (optimal growth at 3–15% w/v salt), extreme halophiles (optimal growth at 25% w/v NaCl; halobacteria and halococci) and borderline extreme halophiles (which require at least 12% w/v salt). According to these definitions D. hansenii can be classified as moderately halophilic, because this species grows optimally at 3–5% w/v salt (unpublished studies in the authors' laboratory).
Table 2. Osmotolerant and osmophilic yeasts111, 177
Since it is one of the most common yeasts in salty environments and can tolerate salinity levels up to 25%, D. hansenii is an important model organism for salt tolerance studies, and its mechanism of osmotolerance has been studied in detail.23, 82, 133
A major component of the mechanisms that allow halophilic organisms to maintain their osmotic balance and to grow in high-salt environments is the production and accumulation of substances named compatible solutes22. In fungi, the alditols glycerol, mannitol, arabinitol and erythritol are the principal organic solutes used to adjust the internal osmotic potential as a prerequisite for survival and growth15. The dominant solute in growing cells of D. hansenii was found to be glycerol, while arabinitol was the main solute found in stationary phase cells in studies by Nobre and Da Costa132 and Larsson and Gustafsson101. These observations are consistent with other investigations showing that the concentration of glycerol declines when cells enter the stationary phase84. Salt stress induces changes in two key parameters related to the yeast's energy metabolism (the ATP pool and the heat production rate) and enhances glycerol production in D. hansenii. This yeast has the ability to regulate its alditol metabolism in response to high salt concentration and thus optimize its growth101. There are also reports indicating that trehalose is used as a compatible solute in Debaryomyces (see below). Unlike in some bacteria, amino acids play no role in the osmoprotection of D. hansenii2. As early as 1985, Adler and co-workers described links between osmoregulation and glycerol metabolism in a glycerol non-utilizing mutant of D. hansenii. Their study indicated that glycerol was synthesized via dihydroxyacetone phosphate, which was reduced to glycerol 3-phosphate, followed by dephosphorylation to glycerol.1 The intracellular glycerol concentration increased with the solute concentration of the growth medium, while accumulation of arabinitol was less pronounced101, 102, 132. At low salinity, arabinitol was the most prominent intracellular solute throughout the growth cycle2, 132. The yeast's ability to synthesize two different solutes under different growth conditions may be important for its practical application. In addition, D. hansenii produces more trehalose than S. cerevisiae76. Gonzalez-Hernandez and co-workers76 found that under saline stress (2.0 and 3.0 M salts) D. hansenii accumulated more glycerol than trehalose, whereas the opposite held true under moderate NaCl stress, leading to the suggestion that trehalose serves as a reserve carbohydrate, as it does in other microorganisms.
Unlike S. cerevisiae, D. hansenii has the capacity to regulate its glycerol metabolism under hyperosmolaric conditions101. It also has superior transport capacities, with twice as many amino acid and carbohydrate transporters and more genes involved in osmo-sensing than Saccharomyces species84.
As a rule, sodium and potassium ions seem to be very important in the mechanisms involved in maintaining osmobalance. The halotolerance mechanisms of D. hansenii include the accumulation of higher concentrations of either K+ or Na+ than in S. cerevisiae, indicating that haloadaptation does not involve the capacity to extrude sodium ions, as assumed by Norkrans and co-workers134, 135, but probably an intrinsic resistance to their toxic effects150. Further evidence supporting the hypothesis that D. hansenii has such intrinsic resistance unknown in other yeasts77, was provided by Gonzalez-Hernandez et al.75.
Norkrans and Kylin found that although Debaryomyces accumulated large concentrations of Na+, it rapidly extruded Na+ in exchange for K+ when incubated in the presence of K+, leading them to suggest the involvement of an Na+/K+ antiporter134, 135. This putative K+/Na+ exchanger does not seem to be the main efflux mechanism, because effluxes of both cations were also observed in the absence of the other ion134, 135, 176. The proposed cation channel was thought to be unspecific, since it was found to promote effluxes of both cations with very similar kinetic parameters75. Gonzalez-Hernandez and co-workers also observed non-competitive inhibition of K+ uptake by Na+ uptake, and vice versa, while other investigators found that Na+ substituted for K+, but only when K+ was scarce150. In addition, Gonzalez-Hernandez et al.75 postulated the existence of a uniporter that is responsible for the transport of either cation to the interior of the cell, with higher affinity for K+ than for Na+, when D. hansenii is incubated with the other cation. Armstrong and Rothstein8 had previously described a similar uniporter in S. cerevisiae.
A mechanism for the transport of Na+ and K+ in D. hansenii has been proposed, in which an AP type H+-ATPase, similar to that encoded by the PMA1 gene of S. cerevisiae, putatively energizes the plasma membrane and generates a transmembrane potential and a pH gradient by massive proton efflux75, 169, 176. The membrane potential was proposed to drive the uptake of Na+ or K+ via one or more uniporters. Na+ might also be slowly expelled in D. hansenii by another P-type ATPase, encoded by ENA1, in a manner that appears to be independent of the membrane potential,5 and an electroneutral exchanger similar to that encoded by NHA1 in S. cerevisiae75, 149. The DhENA1 and DhENA2 genes of D. hansenii are similar to genes encoding Na+-ATPases in S. cerevisiae, and appear to be involved in Na+ extrusion. They also exhibit high homology to the corresponding ENA genes of Schwanniomyces occidentalis and Zygosaccharomyces rouxii84. DhENA1 is expressed in the presence of high Na+ concentrations, while the expression of DhENA2 also requires high pH values according to Almagro et al.5, who suggested that the proteins encoded by the ENA genes in D. hansenii do not cause Na+ extrusion, unlike those in S. cerevisiae, but play an important role in maintaining balanced levels of intracellular cations and ionic homeostasis in the cell.
Maintenance of osmotic balance in D. hansenii has been proposed to involve the activity of several transporters under salt stress conditions (see above). However, views about the transporters involved have repeatedly changed in the past, resulting in some confusion. Therefore, an attempt is made here to briefly describe the key transporters (Figure 2). The first detailed investigation concerning the transporters was presented in 1990114. For D. hansenii growing exponentially on glucose medium containing sodium chloride, it was proposed that the osmotic balance is maintained by coupling sodium and glycerol gradients of opposite signs across the plasma membrane through a sodium–glycerol co-transport mechanism. According to this hypothesis, the transmembrane sodium gradient is the force that drives active glycerol transport, which maintains the endogenous glycerol gradient and thus osmotic balance. The symporter also purportedly accepts potassium ions instead of sodium ions as co-substrates for glycerol transport, while glycerol uptake in the presence of extracellular sodium chloride is accompanied by proton uptake, and the sodium gradient is maintained by sodium-proton antiport activity114.
Figure 2. Model showing important transport pathways for maintaining osmotic balance in D. hansenii. Right box, transporters proposed by Lucas et al.114 Middle box, transporters proposed by Prista et al.150 Left box, transporters proposed by Serrano169, Thome-Ortiz et al.176 and Gonzalez-Hernandez et al.75 1, Nha1 exchanger; 2, AP-type H+-ATPase similar to Pma1 gene; 3, AP-type ATPase similar to Ena1 gene. Lower box, transporters proposed by Norkrans134 and Norkrans and Kylin135
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Further investigations focused on the influence of salt stress on the regulation of enzyme synthesis. It was shown that rapid transcriptional up-regulation of glycerol 3-phosphate dehydrogenase (GDP) occurred in response to salt challenges84, 114, 175. The specific activity of several glycolytic enzymes was inhibited and, in agreement with the GDP stimulation, a general glycolytic flux deviation during salt stress towards glycerol as the major compatible solute occurred126. In order to overcome the negative effects of salt stress, the expression of genes encoding enzymes of the central metabolic pathways can also be modulated to increase transcription and enzyme synthesis. In D. hansenii the activity of NADP-glutamate dehydrogenase (NADP-GDH) was found to be five-fold higher in the presence of 1 M NaCl than in salt-free media4. However, amino acids did not seem to be involved in the process of xero-resistance, because glutamate was not accumulated when D. hansenii was grown in the presence of NaCl. The increased NADP-GDH activity under these conditions suggested that higher enzyme activity was needed to counteract the inactivation resulting from the accumulated ions. The increased enzyme activity in this case could be attributed to amplified enzyme synthesis4. Whether this is only true for this enzyme or reflects a general mechanism involved in osmoadaptation needs further investigation. Guerrero and co-workers77 investigated the hitherto unknown mechanism of the increased enzyme synthesis and demonstrated that the salt-dependent regulation of the studied genes involved mechanisms that are present in D. hansenii but not in S. cerevisiae. They showed that expression of the DhGDH1-encoded NADP+-glutamate dehydrogenase was increased when D. hansenii was grown in the presence of high salt concentrations, while that of DhGLN1 (encoding glutamine synthetase) was reduced.
The osmotolerant yeast D. hansenii could be also very important as a parental strain in protoplast fusion experiments. This technique can be used to obtain genetically modified industrial yeast strains of Saccharomyces cerevisiae with increased tolerance to stress factors such as elevated concentrations of salts113. In future studies it would be interesting to investigate whether the increased tolerance to salts affects or changes the production of secondary metabolites (e.g. esters or higher alcohols) or the ability to metabolize organic acids62.
In addition, there is a growing interest in making plants resistant to salinity in order to increase the area of cultivable land. In regions with extremely arid climates, it has been impossible to cultivate robust and fast growing forage plants to date. The development of halotolerant or halophilic plants should solve this problem. Expressing the genes conferring salt resistance in D. hansenii in plants could be an effective strategy and could make a substantial contribution to reducing hunger in the world168.
We suggest that the ability to withstand high concentrations of salt, and grow at high rates in their presence, makes D. hansenii very valuable for biotechnological applications. D. hansenii can be cultivated without stringent sterility measures (due to the yeast's ability to withstand high salt concentrations in the medium the possibility of a Debaryomyces cultivation contaminated with unwanted organisms is reduced) and can use cheap salt waste products as substrates, e.g. salt-containing glycerine/water mixtures resulting from the trans-esterification of rapeseed oil. In addition it can withstand high educt and product concentrations occurring in the process (i.e. it has high chemostress tolerance) and, thus, can be highly productive (data from the authors' laboratory, not shown). Further potential applications stem from its ability to produce compatible solutes of commercial interest.