Polysaccharides of the fungus Cunninghamella japonica mycelium

Preliminary data on the polysaccharide composition of mycelium of the submerged grown fungus Cunninghamella japonica (synonymous with C. echinulata) were obtained. Mild acid hydrolysis of the mycelium led to formation of glucose, mannose and galactose, whereas acid treatment under drastic conditions afforded glucosamine as the hydrolysis product of chitin and chitosan, the summary content of both glucosaminoglycans being estimated as about 35%. Sequential treatment of the mycelium with hot water, 2% aqueous NaOH and 10% AcOH gave rise to several polysaccharide fractions, which were characterized by their monosaccharide composition. The yield ofchitosan extracted by AcOH was negligible. Additional purification of the fraction obtained by the action of alkali afforded a polysaccharide preparation, which was shown to be a linear (1-->3)-alpha-D-glucopyranan according to the data of chemical methods of structural analysis and NMR spectroscopy. It was concluded that Cunninghamella japonica differs from several other known representatives of Mucorales by the presence of this alpha-D-glucan, as well as by low content of chitosan and polyuronides.     

Effect of cycloheximide and actinomycin D on the phospholipid and neutral lipid content of Cunninghamella japonica

Changes in the composition of neutral lipids and phospholipids were studied during the growth of Cunninghamella japonica under the action of cycloheximide and actinomycin D. The data were used to discuss how the synthesis of enzymes catalysing the formation of individual lipid classes was regulated and whether it would be possible to control the composition of phospholipids and neutral lipids using inhibitors of RNA and protein synthesis. The results are also indicative of a certain correlation between growth phases of the fungus and changes in certain characteristics of membrane lipids.     

Effect of nitrogen limitation and sporulation on sterol and lipid formation inSaccharomyces cerevisiae

The content of sterols and lipids was compared in the cells ofSaccharomyces cerevisiae cultivated in sporulation and the sterol-induction nitrogen-limited media. After 24 h the measured values in the two cultivations did not significantly differ. However, after subsequent 24 h, further formation of lipid globules and a corresponding increase of lipid and sterol content was detected only in the sterol-induction medium. To demonstrate the similarity of physiological state during the first day of the two cultivations, the combined cultivations were performed. Maximum sporulation, suggesting maximum similarity, of the two processes was achieved when the cells were grown in the sterol-induction medium for 15 h and then transferred to a sporulation medium.     

Effect of culture conditions on lipid and gamma-linolenic acid production by mucoraceous fungi

The effect of various culture conditions on growth, lipid production and fatty acid composition in Mucor rouxii and Mucor sp.1b were studied. Total lipid production was higher in media containing potassium nitrate for both the cultures (30%) and cultures grown on plant seed oil produced more than 44% lipid. Among the carbon sources tested, γ-linolenic acid (GLA) production was maximal in cultures grown on glucose. The major fatty acids produced by these two cultures were palmitic, stearic and oleic acids. Levels of GLA in M. rouxii and M. sp.1b was in the range of 3–17% under different culture conditions. Lactose was a poor promoter for biomass and lipid production in both cultures. No GLA was found in fungal cultures grown on sesame oil. The optimal conditions for the production of GLA was standardised in these cultures.     

Effect of nitrogen sources on the activities of lipogenic enzymes in oleaginous fungus Cunninghamella echinulata

Various inorganic and organic nitrogen sources were used to compare their effects on the lipogenesis and the activities of lipogenic enzymes (providing acetyl-CoA and donating NADPH) in gamma-linolenic acid-producing fungus Cunninghamella echinulata. Lipid accumulation was enhanced by organic nitrogen, among them the presence of corn-steep led to almost 40% oil in the biomass. While organic nitrogen increased activities of acetyl-CoA carboxylase (ACC) and malic enzyme (ME), ATP:citrate lyase (ACL) was rapidly enhanced by ammonium ion. The use of NaNO(3) resulted in high activities of glucose 6-phosphate dehydrogenase (GPD) and 6-phosphogluconate dehydrogenase (PGD). NADP-isocitrate dehydrogenase (NADP-ICD) was more active when the fungus utilized all inorganic N-compounds. The rise of nitrogen concentration in medium was accompanied with reduced lipid accumulation and a fall of ACL, ACC, and ME. In contrast, N-sufficient conditions favored biomass growth and elevated activities of GPD and PGD. Kinetic experiments also suggest that a significant portion of the required acetyl-CoA was being provided via ACL and ACC, and ME (probably coupled with GPD) channeled the NADPH into the fatty acid biosynthesis. The contribution of the lipogenic enzymes to metabolic pathways other than lipogenesis is also discussed.     

Effect of light intensity on sporulation of Botryosphaeria ribis

Pycnidia were produced by six of seven isolates ofB. ribis at one or more intensity levels of continuous illumination at 21 °C. Under conditions of alternating light (12 h–27 °C) and darkness (12 h–21 °C) pycnidia formed in cultures of six isolates at three or more intensity levels, while one isolate failed to form pycnidia at any intensity level. Pycnidia did not develop when cultures were incubated in complete darkness. Exposure periods as brief as 2 days under continuous illumination at 21 °C induced pycnidial formation. In alternating light (12 h–27 °C) and darkness (12 h–21 °C), the shortest period of exposure which induced pycnidial formation was 4 days. Continuous illumination at 21 °C favored development of uniloculate pycnidia, while alternating light (12 h–27 °C) and darkness (12 h–21 °C) favored formation of multiloculate pycnidia.Contribution No. 22 from The Botany Section of The Department of Biology, The Pennsylvania State University.     

The role of exogenous lipids in lycopene synthesis in the mucoraceous fungus Blakeslea trispora

The addition of plant oils to the growth medium stimulated growth and lipid synthesis in the fungus Blakeslea trispora. However, only oils with high content of linoleic and especially linolenic acid enhanced lycopene formation. The increase in lycopene formation was accompanied by accumulation in the neutral lipid fraction of the fatty acids prevailing in plant oils. In contrast, the influence of exogenous lipids on the fatty acid composition of bulk fungal phospholipids was insignificant. Nonetheless, a marked increase in the content of membrane lipids and of their phosphatidylethanolamine content was revealed. Presumably, the main mechanism of stimulation of lycopene formation by the oils involves an increase in the solubility of lycopene in the triacylglycerols of the lipid bodies, which is due to an increase in the desaturation degree of their fatty acids. The predominance of linoleic and especially of linolenic fatty acid in plant oils is regarded as a criterion for selecting the oil species for the purpose of intensifying lycopene synthesis.     

Importance of lipidic acyl chains in the process of biochemical thermal adaptation of <Emphasis Type="Italic">Cunninghamella japonica</Emphasis> mucoraceous fungus

The temperature of C. japonica cultivation influences the lipid content and composition of acyl chains, especially the content of such polyunsaturated acids as linoleic and linolenic. Thermal adaptation is accompanied by the modulation of fatty acid isomeric composition and acyl chain length and, at low temperatures, promotes the appearance of fatty acids uncommon to the fungus, in particular, arachidonic acid. The changes occur on a background of significant alterations in the fungus metabolism (in glucose uptake, ATP content, economic coefficient value, etc.). In experiments on the inhibition of translation with cycloheximide, abrupt temperature change (supraoptimal to cold) did not lead to desaturase de novo synthesis, but rather stimulated the activity of the named enzymes, except for palmitoleoyl-CoA desaturase. In the process of temperature adaptation, polar lipid microviscosity modulating compounds influenced fatty acid acyl chain composition. Microviscosity differences between polar and neutral lipids and correlation to the degree of fatty acid unsaturation during temperature fluctuation were established.     

Biotransformation of fluorene by the fungus Cunninghamella elegans.

The metabolism of fluorene, a tricyclic aromatic hydrocarbon, by Cunninghamella elegans ATCC 36112 was investigated. Approximately 69% of the [9-14C]fluorene added to cultures was metabolized within 120 h. The major ethyl acetate-soluble metabolites were 9-fluorenone (62%), 9-fluorenol, and 2-hydroxy-9-fluorenone (together, 7.0%). Similarly to bacteria, C. elegans oxidized fluorene at the C-9 position of the five-member ring to form an alcohol and the corresponding ketone. In addition, C. elegans produced the novel metabolite 2-hydroxy-9-fluorenone.     

Biotransformation of malachite green by the fungus Cunninghamella elegans

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.     

Effect of light and phosphate on conidiation in the fungus Verticillium agaricinum

Verticillium agaricinum (Link) Corda, grown in a yeast extract-sucrose medium, conidiated abundantly in darkness after irradiation with near ultraviolet (290–400 nm) for 15 min or blue light (400–550 nm) for 60 min. Few conidia were formed in total darkness. Exposure to 30 min of near ultraviolet light suppressed conidiation. Conidiation was also suppressed by phosphate in excess of 10⁻⁴ M irrespective of light condition. After irradiation with near ultraviolet light for more than 30 min, there was a cessation of growth and a change in colony color from yellow to reddish. The color does not appear to be due to a carotenoid because the colonies turned from red to yellow when covered with acid. At pH lower than 6.0 the pigment has an absorption maximum around 390 nm, whereas at higher pH it is around 540 nm. Thus, it appears that irradiation of V. agaricinum with near ultraviolet may cause an increase in pH, which in turn produces the change of colony color from yellow to reddish.     

Inducing sporulation in Alternaria solani II. Effect of light

Among the three different light sources viz. incandescent electric light, infra-red light and sunlight, only sunlight was effective in inducing sporulation in petri dish cultures ofA. solani. The intensity of sporulation depended upon the age and growth stage of the cultures, duration and number of exposures to light and the presence or absence of a sporulating zone in the culture. Maximum sporulation was obtained in the case of 6 days old partially grown cultures by inducing the formation of sporulating zone which appeared in 24 hours after every exposure of 60 minutes to sunlight.     

Growth and sporulation of the fungus Claviceps purpurea in batch fermentation

Summary The sporulation of the fungus Claviceps purpurea is connected with the change in its respiration rate which is effected by deficiency of dissolved oxygen tension. It stops the vegetative growth of the fungus and induces the formation of conidiophores with conidia production until glucose is exhausted. With exhaustion of glucose the conidiophores continue to produce conidia by transforming vegetative cell material into conidia. Therefore final conidial concentration in batch fermentation depends on these two processes which can be regulated by oxygen input.     

Influence of lignin and oxygen on the growth and the lipid formation of the fungus Lentinus tigrinus

During cultivation of the filamentous fungus Lentinus tigrinus on a medium containing lignin, a high oxygen content stimulated the growth of the fungus and contributed to the yield of lipids. A high content of phosphatidic acid and a reduction in the level of phosphatidylethanolamine and phosphatidylserine were first detected in the composition of phospholipids. Changes in the composition of neutral lipids, such as variation in the ratio of esterified and free sterols, have occurred; thus, the amount of sterol esters reduced simultaneously with a decrease in the content of free fatty acids. Based on the obtained results, the possible role of phosphatidic acid as a second messenger in the process of the consumption of lignin by the fungus Lentinus tigrinus is discussed.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.