About different mechanisms of the biochemical adaptation of mycelial fungi to temperature stress: changes in cytosol carbohydrate composition

The effect of temperature stress on the cytosol carbohydrate composition of fungi belonging to various systematic groups was investigated. In Mucorales representatives (subkingdom Eomycota, phylum Archemycota, class Zygomycetes), adaptation to hypo- and hyperthermia occurs via the regulation of trehalose synthesis, although inositol is also involved in these processes in Blakeslea trispora. Basidiomycota (subkingdom Neomycota) use two pathways of biochemical adaptation, depending on the cytosol carbohydrate composition. In the absence of sucrose, glycerol and arabitol are involved in the adaptation to hyperthermia; trehalose accumulates under hypothermic conditions (type I of regulation). Type II regulation (revealed in Pleurotus ostreatus) involves sucrose rather than glycerol or arabitol. The data obtained are discussed in terms of fungal systematics and phylogeny.     

Alterations in the carbohydrate composition of the cytosol of fungal spores caused by temperature variations and the storage process

Differences in the carbohydrate composition were revealed among spores of fungi belonging toZygomycetes, Ascomycota, Basidiomycota, andOomycota, part of the novel kingdomChromista. It was shown for the first time thatPhytophthora infestons contains arabitol in addition to glucose and trehalose. Sucrose was detected inPleurotus ostreatus basidiospores. It was established thatBlakeslea trispora stylospores contain inositol. The dependence of the spore carbohydrate composition on the temperature of the habitat of the corresponding species is discussed. It was shown that the cytosol of the conidia is dominated by trehalose and inositol under hypothermic conditions and by mannitol and glucose under hyperthermic conditions.Neomycota andEomycota were shown to differ in their responses to stress (starvation), which correlated with the differences in the carbohydrate composition of the spore cytosols. Assuming that cytosol carbohydrates perform a protective function, we explain the higher viability of conidia compared to stylo- and sporangiospores.     

Changes in the carbohydrate composition of cytosol from fungal spores in connection with environmental temperature and during storage

Differences in the carbohydrate composition were revealed among spores of fungi belonging to Zygomycetes, Ascomycota, Basidiomycota, and Oomycota, part of the novel kingdom Chromista. It was shown for the first time that Phytophthora infestans contains arabitol in addition to glucose and trehalose. Sucrose was detected in Pleurotus ostreatus basidiospores. It was established that Blakeslea trispora stylospores contain inositol. The dependence of the spore carbohydrate composition on the temperature of the habitat of the corresponding species is discussed. It was shown that the cytosol of the conidia is dominated by trehalose and inositol under hypothermic conditions and by mannitol and glucose under hyperthermic conditions. Neomycota and Eomycota were shown to differ in their responses to stress (starvation), which correlated with the differences in the carbohydrate composition of the spore cytosols. Assuming that cytosol carbohydrates perform a protective function, we explain the higher viability of conidia compared to stylo- and sporangiospores.     

Dynamics of the cytosol soluble carbohydrates and membrane lipids in response to ambient pH in alkaliphilic and alkalitolerant fungi

Comparative composition of lipids and cytosol soluble carbohydrates at different ambient pH values was studied for two obligately alkaliphilic fungi (Sodiomyces magadii and S. alkalinus) and for two alkalitolerant ones (Acrostalagmus luteoalbus and Chordomyces antarcticus). The differences and common patterns were revealed in responses to pH stress for the fungi with different types of adaptation to ambient pH. While trehalose was one of the major cytosol carbohydrates in alkaliphilic fungi under optimal growth conditions (pH 10.2), pH decrease to 7.0 resulted in doubling its content. In alkalitolerant fungi trehalose was a minor component and its level did not change significantly at different pH. In alkalitolerant fungi, arabitol and mannitol were the major carbohydrate components, with their highest ratio observed under alkaline conditions and the lowest one, under neutral and acidic conditions. In alkaliphiles, significant levels of arabitol were revealed only under alkaline conditions, which indicated importance of trehalose and arabitol for alkaliphily. Decreased pH resulted in the doubling of the proportion of phosphatidic acids among the membrane lipids, which was accompanied by a decrease in the fractions of phosphatidylcholines and sterols. Alkalitolerant fungi also exhibited a decrease in sterol level at decreased pH, but against the background of increased proportion of one of phospholipids. Decreased unsaturation degree in the fatty acids of the major phospholipids was a common response to decreased ambient pH.     

Carbohydrate metabolism of the sugarcane smut fungusUstilago scitaminea

Ustilago scitaminea synthesizes specific carbohydrates, mainly erythritol and mannitol, plus smaller quantities of arabitol, inositol, and trehalose. Intensive secretion of erythritol is obtained when the fungus is grown in liquid culture. Glucose, fructose, or sucrose can be used interchangeably as carbon sources by the fungus, sucrose being quickly inverted.     

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose), and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures — highly homogeneous with regard to the respective cell form produced — contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5–2% to the biomass of M. rouxii and 1.5–6% to that of C. albicans; the values for trehalose ranged from 0.2–11% in the former and 1–3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions — regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.     

Adaptation of Flammulina velutipes to hypothermia in natural environment: the role of lipids and carbohydrates

The role of lipids and carbohydrates in the adaptation of Flammulina velutipes to hyperthermia (5 to -5 degrees C) in natural environments has been studied for the first time. The main changes are were found to occur in membrane lipids: the levels of sterols and glycolipids decreased, and the proportion of phospholipids with a high degree of nonsaturation (2.2) increased, which was due to predominance of two fatty acids, linoleic (35% of the total) and linolenic (50%). Phosphatidylcholine became the major phospholipid. Under hypothermic conditions, glycerol, known to have antifreeze properties, accumulated in the cell cytosol along with arabitol and trehalose. The significance of this biochemical strategy in the resistance of the fungus to temperatures below zero (i.e., to cryobiotic conditions) is discussed.     

Carbohydrates in the hypothallus and areolae of the crustose lichen <Emphasis Type="Italic">Rhizocarpon geographicum</Emphasis> (L.) DC.

Carbohydrate concentrations in the marginal hypothallus and areolae of the crustose lichen Rhizocarpon geographicum (L.) DC. were measured in north Wales, U.K. using gas chromatography. Ribitol, arabitol, and mannitol were the most abundant carbohydrates while α- glucose β-glucose, fructose, sucrose, and trehalose were present in smaller amounts. The concentrations of arabitol, ribitol, mannitol, fructose, and α-glucose were greater in the areolae while the concentration of trehalose was greater in the hypothallus. Concentrations of carbohydrates varied little between sample days. Concentrations of polyols in the hypothallus were not correlated with those in the areolae. These results suggest: 1) the hypothallus has a lower demand for carbohydrates than the areolae or there is limited transport from areolae to hypothallus, 2) increased trehalose in the non-lichenised hypothallus may be an adaptation to withstand stress and desiccation, and 3) polyols are partitioned differently in the hypothallus and areolae.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Carbohydrate Composition and Acid Invertase Activity in Rice Leaves Infected with Pyricularia oryzae

Ethanol-soluble carbohydrates in healthy and blast-infected leaves and also cultures of Pyricularia oryzae were analyzed using gas chromatography. Arabitol, mannitol, and trehalose occurred in infected leaf tissue, but not in healthy controls. Cultures of P. oryzae contained mannitol and trehalose, but not arabitol and sucrose. The presence of arabitol only in blast-infected rice leaves suggests that arabitol synthesis may be induced in infected leaf tissue as a result of the rice-blast fungus interaction, but may not be within blast fungus itself. The amounts of glucose, fructose, and sucrose in infected leaves were slightly increased until 5 days, and greatly enhanced at 7 days after inoculation, There were no differences in amounts of these sugars between the cultivars Nakdong and Dobong. At 7 days after inoculation, increases in amounts of arabitol, mannitol, and trehalose were pronounced in the susceptible cultivar Nakdong than in the moderately susceptible cultivar Dobong. The increased amounts of glucose and fructose in infected plants of the two cultivars were closely correlated with the presence of a very active invertase.     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

产肌醇毕赤酵母细胞工厂的优化

【背景】肌醇是一种B族维生素,广泛应用于食品、医药、饲料等领域。微生物发酵法是最具前景的肌醇生产方法,但使用大肠杆菌生产的肌醇在食品及医药领域中的使用受到限制。毕赤酵母作为生物安全菌株是工业上生产异源蛋白的良好宿主,其本身含有天然的肌醇合成途径,具有被改造成为高效生产肌醇细胞工厂的潜力。【目的】通过代谢工程改造毕赤酵母工程菌株,降低副产物的生成并提高肌醇的产量。【方法】以实验室前期构建的产肌醇毕赤酵母工程菌株为出发菌株,确定副产物阿拉伯糖醇、核糖醇和甘露糖合成相关基因。通过关键基因敲除、发酵液中葡萄糖浓度控制降低副产物的产量。通过过表达甘油转运蛋白、甘油激酶和甘油-3-磷酸脱氢酶基因实现产肌醇毕赤酵母对甘油和葡萄糖的共利用,得到重组菌Z10。经过发酵条件优化,进一步提高Z10的肌醇产量。【结果】在最优条件下,重组菌Z10的肌醇产量达到36.7 g/L,是目前酵母类细胞工厂生产肌醇的最高值,副产物总产量与出发菌株相比降低了63.1%。【结论】在毕赤酵母中建立了降低阿拉伯糖醇、核糖醇和甘露糖合成的有效策略,并通过甘油、葡萄糖共利用及相对应的发酵条件优化提高了肌醇产量,为肌醇及其他高价值生物...     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Polyol concentrations in Aspergillus repens grown under salt stress

Na⁺, K⁺ and the ratio of Na⁺/K⁺ were higher in cells of the halotolerant Aspergillus repens grown with 2 M NaCl than without NaCl. The osmolytes, proline, glycerol, betaine and glutamate, did not affect the Na⁺/K⁺ ratio, nor the polyol content of cells under any conditions. The concentrations of polyols, consisting of glycerol, arabitol, erythritol and mannitol, changed markedly during growth, indicating that they have a crucial role in osmotic adaptation.     

Adaptation of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> to Hypothermia in Natural Environments: The Role of Lipids and Carbohydrates

The role of lipids and carbohydrates in the adaptation of Flammulina velutipes to hypothermia (5 to −5°C) in natural environments has been studied for the first time. The main changes are were found to occur in membrane lipids: the levels of sterols and glycolipids decreased, and the proportion of phospholipids with a high degree of nonsaturation (2.2) increased, which was due to predominance of two fatty acids, linoleic (35% of the total) and linolenic (50%). Phosphatidylcholine became the major phospholipid. Under hypothermic conditions, glycerol, known to have antifreeze properties, accumulated in the cell cytosol along with arabitol and trehalose. The significance of this biochemical strategy in the resistance of the fungus to temperatures below zero (i.e., to cryobiotic conditions) is discussed.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 329–334.Original Russian Text Copyright © 2005 by Tereshina, Memorskaya.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Carbohydrate Storage in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana was grown in 1% (wt/vol) gelatin-liquid media singly supplemented with a monosaccharide (glucose or fructose), a disaccharide (maltose or trehalose), a polyol (glycerol, mannitol, or sorbitol), or the amino sugar N-acetyl-d-glucosamine. The relative contributions of the carbohydrate, protein, and water contents in the fungal biomass were determined. Carbohydrates composed 18 to 42% of the mycelial dry weight, and this value was lowest in unsupplemented medium and highest in medium supplemented with glucose, glycerol, or trehalose. Biomass production was highest in liquid cultures supplemented with trehalose. When liquid cultures were grown in medium supplemented with 0 to 1% (wt/vol) glucose, trehalose, or N-acetyl-d-glucosamine, there was an increase in the biomass production and the contribution of carbohydrate to mycelial dry weight. Regardless of the glucose concentration in the culture, water content of the mycelia remained about 77.5% (wt/wt). Mycelial storage carbohydrates were determined by capillary gas chromatography. In gelatin-liquid medium supplemented with 1% (wt/vol) glucose, B. bassiana stored glycogen (12.0%, wt/dry wt) and the polyols mannitol (2.2%), erythritol (1.6%), glycerol (0.4%), and arabitol (0.1%). Without glucose, B. bassiana stored glycogen (5.4%), mannitol (0.8%), glycerol (0.6%), and erythritol (0.6%) but not arabitol. To our knowledge, this is the first report of carbohydrate storage in an entomopathogenic fungus, and the results are discussed in relation to other fungi and the potential implications to commercial formulation and insect-fungus interactions.     

Carbohydrate Spectrum of Extremophilic Yeasts <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under pH Stress

Alterations in the concentrations of cell cytosol carbohydrates of polyextremophilic yeasts Yarrowia lipolytica under stresses of diverse nature were observed. Under pH stress, mannitol was the main storage carbohydrate (up to 89% of the total cytosol carbohydrates), while arabitol, glucose, and inositol were present in insignificant amounts (3 to 6%). Experiments with inhibition of de novo mannitol synthesis by bis(p-nitrophenyl) disulfide revealed that the cytoprotective effect of mannitol was most noticeable in the cells grown under acidic conditions (pH 4.0), while the role of catalase and superoxide dismutase, the enzymes of the first line of antioxidant protection, increased under alkaline conditions (pH 9.0). The constitutively high mannitol content in Y. lipolytica cells was hypothesized to be a part of the core mechanism of stress resistance in this yeast species.     

Membrane lipids and soluble sugars dynamics of the alkaliphilic fungus Sodiomyces tronii in response to ambient pH

Alkaliphily, the ability of an organism to thrive optimally at high ambient pH, has been well-documented in several lineages: archaea, bacteria and fungi. The molecular mechanics of such adaptation has been extensively addressed in alkaliphilic bacteria and alkalitolerant fungi. In this study, we consider an additional property that may have enabled fungi to prosper at alkaline pH: altered contents of membrane lipids and cytoprotectant molecules. In the alkaliphilic Sodiomyces tronii, we showed that at its optimal growth pH 9.2, the fungus accumulates abundant cytosolic trehalose (4–10% dry weight) and phosphatidic acids in the membrane lipids, properties not normally observed in neutrophilic species. At a very high pH 10.2, the major carbohydrate, glucose, was rapidly substituted by mannitol and arabitol. Conversely, lowering the pH to 5.4–7.0 had major implications both on the content of carbohydrates and membrane lipids. It was shown that trehalose dominated at pH 5.4. Fractions of sphingolipids and sterols of plasma membranes rapidly elevated possibly indicating the formation of membrane structures called rafts. Overall, our results reveals complex dynamics of the contents of membrane lipids and cytoplasmic sugars in alkaliphilic S. tronii, suggesting their adaptive functionality against pH stress.

     

Acquired thermotolerance,membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.