Dimorphic Fungus Mucor Rouxii Research Articles

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Sustainable and Effective Chitosan Production by Dimorphic Fungus Mucor rouxii via Replacing Yeast Extract with Fungal Extract

The effects of switching morphology and replacing supplementary nutrients with fungal extract (5 and 10g/L) on the production of major metabolites and chitosan by Mucor rouxii were investigated. This approach was supposed to promote sustainability of the fermentation process and improve its economic feasibility. Different fungal morphologies, i.e., purely filamentous (PF), purely yeast-like (PY), mostly filamentous (MF), and mostly yeast-like (MY), were evaluated. The highest ethanol yields were obtained from the media supplemented with 10g/L fungal extract for all morphologies, while adding nutrient salts did not make any improvements in these yields, except a slight decrease in the fermentation time. Except for PF morphology, the replacement of yeast extract favored the biomass production yields. Moreover, the alkali insoluble material (AIM) yields were higher as a result of the replacement for most cases. Furthermore, the replacement resulted in increased glucosamine and decreased N-acetyl-glucosamine content of AIM for almost all the morphologies. AIM yields of at least 0.25g/g-glucose and maximum chitin/chitosan yield of 0.78g/g-AIM were obtained from the solids remaining after autolysis process, which were higher than that obtained from the raw biomass. The maximum yield of 0.135g/g-AIM purified chitosan with intact molecular weight was obtained from the biomass with PF morphology supplemented with 10g/L fungal extract plus nutrients.

Up-regulated expression of desaturase genes of Mucor rouxii in response to low temperature associates with pre-existing cellular fatty acid constituents

Transcriptional response of desaturase genes to low temperature was investigated in the dimorphic fungus Mucor rouxii. The two morphological forms of M. rouxii, yeast-like and mycelial cells containing different fatty acid profiles were shifted from 30 to 10°C. Both cultures exhibited significantly altered fatty acid composition, whose content in polyunsaturated fatty acids increased as consequence of the temperature shift and was accompanied by a reduction of C18:1Δ(9) about 2 h after the temperature shift. These changes were particularly significant in phosphatidylcholine and phosphatidylethanolamine fractions. Moreover, the fatty acid profiles of monoacylglycerol and diacylglycerol were also modulated in response to the lower temperature of incubation. The changes of membrane lipids of M. rouxii were due to the cold-induced expression of Δ(9)-, Δ(12)- and Δ(6)-desaturase genes. Although the mRNA levels of the three desaturases were transiently induced by lowering the temperature, the pre-existing composition of fatty acid profiles of mycelial and yeast-like forms of M. rouxii may have lead to different expression profiles of desaturase genes that modified their membrane physical state under cold shock. While expression of Δ(12)-desaturase gene contributed mainly to cold acclimation of mycelia, Δ(9)-desaturase expression was the main transcript identified in the yeast-like culture after temperature shift.

Mucor rouxii ultrastructure: cyclic AMP and actin cytoskeleton

A comparative analysis of the effect of two compounds, dibutyryl-cyclic-AMP (dbcAMP) and latrunculin B, on the morphology and ultrastructure of the dimorphic fungus Mucor rouxii under aerobic growth conditions is presented. dbcAMP acts through the sustained activation of protein kinase A, and latrunculin B through the disruption of the actin cytoskeleton. Upon addition of these compounds to the growth medium at any stage of the germination process, cells lost polarised growth and switched to isodiametric growth. The effect was reversible. The morphologies, visualised by light microscopy or scanning electron microscopy (SEM), were alike. A switch from a rough to a smooth surface was observed by SEM when cells were repolarised by removal of the added compound. Ultrastructural changes under both conditions, as observed by transmission electron microscopy, were similar, the main feature being the enlargement of the cell wall, with irregular depositions, and detachment from the cell membrane. dbcAMP-treated cells showed a decrease in the number of glycogen granules compared with control and latrunculin B-treated cells. F-actin staining with fluorescein isothiocyanate-phalloidin showed that both dbcAMP- and latrunculin B-treated cells displayed a much lower fluorescence than control cells, with only a few pale plaques. The results suggest that the sustained activation of protein kinase A, which impairs polarised growth, might exert its effect through a modification of actin cytoskeleton organisation, very probably also involving an integrinlike pathway, as judged by the cell wall detachment and loss of cell adhesiveness of the dbcAMP-treated isodiametric cells.

Chemo- and stereoselective reduction of β-keto esters by spores and various morphological forms of Mucor rouxii

We report the efficient enantioselective reduction of a number of β-ketoesters by mycelial and yeast-like forms of the dimorphic fungus Mucor rouxii in a whole-cell process. Mycelial cells, grown in aerobiosis, were efficient in water, whereas the yeast-like cells, grown in anaerobic medium, were both efficient in water and in organic solvents. Almost 100% of conversion with 97% of enantiomeric excess of the ( S) forms of the reduced β-ketoesters was obtained at 15 g biomass (wet weight)/mmol substrate in two hours. The fungal spores, which are the physiologically resistant form of the fungus, also catalyzed the reductive process efficiently and stereoselectively. The freeze-dried as well as the warm-air dried yeast-like cells, rehydrated in a small volume of water, maintained the same efficiency and selectivity of the reaction in organic solvents as the fresh biomass up to at least 4 months.

Protein Kinase A Activity in Permeabilized Cells as an Approximation to in Vivo Activity

Permeabilized germlings from the dimorphic fungus Mucor rouxii were used for in situ measurement of protein kinase A (PKA) activation, to compare the results with those obtained in vitro at low or high (nonlinear) enzyme concentrations. The apparent total activity per cell when measured in situ is 5- to 10-fold lower than the in vitro measured activity in crude extracts from those cells. Polyamines and NaCl stimulate the activity in situ. The apparent relative specific activity of the in situ measured PKA toward four peptide substrates is similar to the results obtained in vitro at high holoenzyme concentration and not to those obtained with the free catalytic subunit. Saturation in the activation of PKA by cAMP in situ is attained at low concentrations (2 to 10 μM), while in vitro, at high holoenzyme concentration, no saturation was attained up to 1 mM cAMP (V. Zaremberg et al. Arch. Biochem. Biophys. 381, 74–82, 2000). Activation of PKA by site-selective cAMP analogs is assayed in situ and in vitro at two enzyme concentrations. Site B–selective cAMP analogs are good activators of PKA at low enzyme concentration in vitro but poor activators either at high enzyme concentration in vitro or in permeabilized cells. A physiological correlation with the behavior of site-selective analogs in situ is demonstrated in vivo when assaying the effect of increasing concentrations of site-selective cAMP analogs on the impairment of polarized growth of M. rouxii spores.

Mechanism of Activation of cAMP-Dependent Protein Kinase: In Mucor rouxii the Apparent Specific Activity of the cAMP-Activated Holoenzyme Is Different than That of Its Free Catalytic Subunit

Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater Km, the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: substrate participation in the dissociation process and/or holoenzyme activation without dissociation.

Stimulation of hyphal growth in anaerobic cultures of Mucor rouxii by extracellular trehalose. Relevance of cell wall-bound activity of acid trehalase for trehalose utilization.

In fungi, the hydrolysis of extracellular trehalose is carried out by acid trehalases. These secretory glycoproteins may be more abundant either in the vacuolar compartment, like in yeast, or at the cell surface, such as in many filamentous fungi. The relative efficiency of these two compartments for the utilization of extracellular trehalose was investigated using as a model the dimorphic fungus Mucor rouxii, which produces yeast-like cells under a CO(2) atmosphere, or hyphae in the presence of air. Under CO(2), cultures supplemented with glucose produced yeast-like cells devoid of acid trehalase activity. On the other hand, trehalose-supplemented cultures developed hyphae exhibiting cell wall-bound and intracellular acid trehalase activity. Glucose-grown yeast-like cells supplemented with trehalose after glucose exhaustion, induced intracellular activity of acid trehalase, but no activity was detected at the cell surface. Even endowed of significant intracellular activity of acid trehalase, these cells did not grow further. When exposed to air these yeast-like produced germ tubes exhibiting cell wall-bound acid trehalase activity. These results suggest that the utilization of extracellular trehalose as a source of carbon for growth requires the localization of acid trehalase activity at the cell surface. Our results also show that extracellular trehalose elicits a morphogenetic phenomenon, inducing the formation of hyphae which are the physiological support for acid trehalase activity.

Stimulation of hyphal growth in anaerobic cultures of Mucor rouxii by extracellular trehalose. Relevance of cell wall-bound activity of acid trehalase for trehalose utilization

In fungi, the hydrolysis of extracellular trehalose is carried out by acid trehalases. These secretory glycoproteins may be more abundant either in the vacuolar compartment, like in yeast, or at the cell surface, such as in many filamentous fungi. The relative efficiency of these two compartments for the utilization of extracellular trehalose was investigated using as a model the dimorphic fungus Mucor rouxii, which produces yeast-like cells under a CO 2 atmosphere, or hyphae in the presence of air. Under CO 2, cultures supplemented with glucose produced yeast-like cells devoid of acid trehalase activity. On the other hand, trehalose-supplemented cultures developed hyphae exhibiting cell wall-bound and intracellular acid trehalase activity. Glucose-grown yeast-like cells supplemented with trehalose after glucose exhaustion, induced intracellular activity of acid trehalase, but no activity was detected at the cell surface. Even endowed of significant intracellular activity of acid trehalase, these cells did not grow further. When exposed to air these yeast-like produced germ tubes exhibiting cell wall-bound acid trehalase activity. These results suggest that the utilization of extracellular trehalose as a source of carbon for growth requires the localization of acid trehalase activity at the cell surface. Our results also show that extracellular trehalose elicits a morphogenetic phenomenon, inducing the formation of hyphae which are the physiological support for acid trehalase activity.

Copper Resistance and Accumulation in the Zygomycete Mucor rouxii

Two copper-resistant (Copr) mutants, strains P1 and P3, were obtained from the dimorphic fungus Mucor rouxii. They were characterized as to their ability to take up copper in a growth medium supplemented with this metal ion. Detection of copper by linear sweep striping voltammetry in cell walls and in the cell wall-free fraction of disrupted cells revealed a higher content of the metal in both mutant fractions, as compared with those of the copper-sensitive (Cops) parental strain. Copper binding by M. rouxii growing cells was also studied through the use of a cytochemical method based on the compounds neocuproine (NCP) and sodium diethyldithiocarbamate (DTC). This method indicated that the P1 Copr strain accumulated more metal than the parental Cops strain, both on the cellular surface and in the intracellular milieu.

Environmental and developmental regulation of carotenogenesis in the dimorphic fungus Mucor rouxii

Aerobic mycelium of wild-type Mucor rouxii accumulated about ten times higher amounts of the carotenoid pigment β-carotene when grown continuously in the presence of light than the corresponding cultures grown in the dark. Carotenoid accumulation was dependent on light intensity, with the threshold located at about 10-2 W.m-2. Photocarotenogenesis in complex medium was more efficient with glucose as a carbon source. Carotenoid synthesis by M. rouxii mycelium was unaffected by both retinol acetate and retinal, which are stimulators of carotenogenesis in other zygomycetes. Carotenogenesis was significant in aerobic mycelium but was almost undetectable in anaerobic mycelium as well as in aerobic or anaerobic yeast cells. This suggested an involvement of oxygen in carotenoid synthesis by M. rouxii and the existence of developmental regulation of the expression or operation of the pathway.

Genetic evidence for independence between fermentative metabolism (ethanol accumulation) and yeast-cell development in the dimorphic fungus Mucor rouxii

Three allyl-alcohol-resistant mutants were isolated in the dimorphic fungus Mucor rouxii and characterized with regard to their alcohol dehydrogenase (ADH) activity in vitro and in vivo as well as their ability to execute the morphological alternatives of dimorphism under different environmental stimuli, either in the absence or in the presence of oxygen. These studies indicated that fermentation and yeast-cell development are independent events and that ADH activity is essential for growth of the fungus in the absence of oxygen. Heterokaryon construction and analysis indicated that in the three mutant strains the corresponding genetic alterations are recessive nuclear mutations which behave as allelic in complementation tests.

Regulation of protein kinase A subunits during germination of Mucor rouxii sporangiospores.

Levels of protein kinase A (PKA) subunits and of cAMP have been measured during aerobic germination of the sporangiospores of the dimorphic fungus Mucor rouxii; further, the holoenzyme and its catalytic (C) and regulatory (R) subunits have been visualized through sucrose gradient centrifugation. Sporangiospores contain around 0.06 microM of a dimeric holoenzyme species of 5.5 S and a sixfold excess of a free R subunit of 2.7 S. Both these species are proposed to be derived by proteolysis from the native forms. Enzymic activity at this stage is highly inhibited, as demonstrated with permeabilized cells. Immediately upon germination, and after a transient increase in cAMP concentration from 10 microM to 90 microM, C-subunit levels fall to 30%. After the onset of germination, the specific activity and concentration of both the 5.5 S holoenzyme species and the 2.7 S species of free R subunit decrease in parallel to the increase in total protein and volume. Net synthesis of C and R subunits to form a native holoenzyme species of 8.8 S is apparent 4 h onwards after germination. A significant increase in cellular concentration is observed at 6 h. At 7 h of growth, when germ-tube emission is complete, the holoenzyme concentration is around 0.23 microM; there is almost no free R subunit and the intracellular concentration of cAMP is around 3 microM. A role for PKA during germination and morphogenesis is discussed.

Two different intrachain cAMP sites in the cAMP‐dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.

Regulatory role of δ-aminolevulic acid dehydratase in the dimorphic fungus Mucor rouxii

1. 1. With the aim of finding a possible relationship between the known dimorphism phenomenon existing in the fungus Mucor rouxii and the biosynthesis of respiratory pigments, the activity of aminolevulic acid synthetase (ALA-S) and ALA-dehydratase (ALA-D) was studied in crude extracts and in 15,000 g supernatants of both mycelium and yeast-like cells. 2. 2. The activity of ALA-S was unusually high (3 nmol ALA/hr/mg protein) compared with that reported for other tissues and did not vary with the fungus morphology. 3. 3. Instead, ALA-D specific activity was found to be 16.5 nmol PBG/hr/mg protein in mycelium extracts, that is 7-fold greater than that measured in the yeast-like morphology (2.6 nmol PBG/hr/mg protein). 4. 4. It was of importance to determine the activity levels of ALA-D along with the morphogenic transition from yeast to mycelium. It was observed that the greatest change and enhancing of specific activity occurred 2 hr before the emergence of the germ tubes and was held constant up to the complete development of mycelium. 5. 5. Both hyphae formation and enhancement of ALA-D activity were diminished when cAMP was added to the culture shifted from the anaerobic atmosphere to air. 6. 6. These findings and preliminary studies on the characterization of M. rouxii ALA-D indicate that this enzyme plays a regulatory role in porphyrin biosynthesis in this fungus as well as a key function in the characteristic morphogenic transition.

Environmental Control of the Respiratory System in the Dimorphic Fungus Mucor rouxii

Spores of Mucor rouxii had low levels of respiratory activity partially sensitive to cyanide and salicylhydroxamic acid (SHAM). They contained a full set of cytochromes but in low concentrations, especially cytochromes aa 3. Aerobic growth induced a rapid increase in respiratory activity resistant to SHAM but fully sensitive to cyanide, and in the levels of cytochromes, mainly cytochromes aa 3. These processes were subject to catabolite repression. Incubation under anaerobic conditions, independent of cell morphology, favoured the persistence of SHAM-sensitive respiration, and the maintenance of low levels of b and c cytochromes. No cytochromes aa 3 were formed under these conditions. CO-difference spectra suggested the presence of cytochrome o in higher levels in aerobic than in anaerobic cells.

Processing of asparagine-linked saccharides in Mucor rouxii

Asparagine-linked Glc 1Man 9GlcNAc 2, Glc 1Man 8GlcNAc 2 and Glc 1Man 7GlcNAc 2 were detected in mycelial-form cells of the dimorphic fungus Mucor rouxii inculbated with [U- 14C]glucose for 3 min. The oligosaccharides were absent from glycoproteins isolated from cells chased for 15 min with the unlabed monosaccharide. This was due to deglucosylation of the oligosaccharides and not to further addition of mannose residues to them. The half-lives of the glucosylated compounds were much shorter, therefore, in M. rouxii than in other eucaryotic cells. Further processing of N-linked saccharides led to the synthesis of mannan-like glycoproteins, some of whch contained methyl groups in position 3 or the mannose residues. Methylation occurred only at the non-reducing ends and prevented further elongation of the branches.

Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit.

The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R2C2 of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behavior towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.

3-O-methylation of mannose residues. A novel reaction in the processing of N-linked oligosaccharides occurring in Mucor rouxii.

Yeast- and mycelial-form cells of the dimorphic fungus Mucor rouxii incubated with [U-14C]glucose were found to synthesize Man-P-dolichol, Glc-P-dolichol, and Glc3Man9GlcNAc2-P-P-dolichol. The structure of the oligosaccharide moiety of the latter was similar to that of the same compound isolated from other eucaryotic cells. Oligosaccharides that migrated on paper chromatography as Man6-30GlcNAc standards were obtained upon treatment of delipidated proteins with a protease and endo-beta-N-acetylglucosaminidase H. The oligosaccharides that migrated apparently as single substances on paper chromatography could be separated into three different populations by paper electrophoresis in sodium borate buffer. The fastest migrating substances contained only mannose and N-acetylglucosamine residues, whereas the other two contained, in addition, different proportions of 3-O-methylmannose units. The oligosaccharides with the highest content of 3-O-methylmannose residues appeared to be completely resistant to alpha-mannosidase degradation; they were, however, cleaved by endo-beta-N-acetylglucosaminidase H. Mycelial cells synthesized a much higher proportion of 3-O-methylmannose-containing oligosaccharides than yeast cells. Cells incubated with [methyl-14C]methionine were found to label only the N-linked oligosaccharides containing 3-O-methylmannose residues. It is concluded that transfer of Glc3Man9GlcNAc2 to protein is followed by excision of glucose and probably one or two mannose residues, followed by further mannosylation and in some cases also methylation of oligosaccharides. This represents a novel reaction in the processing of N-linked oligosaccharides.

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I ( M r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K 0.5 for MnCl 2 of 2 m m. Enzyme II ( M r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn 2+. This enzyme was strongly inhibited by 50 m m NaF or 1 m m ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn 2+, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn 2+. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.