Phomenoic acid and phomenolactone, antifungal substances from Phoma lingam (Tode) Desm.: kinetics of their biosynthesis, with an optimization of the isolation procedures.

Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.     

Phomenolactone,an antifungal substance from phoma lingam

The structure of phomenolactone, an antifungal substance isolated from the fungus Phoma lingam was established by reaction of phomenoic acid with dipyrridyl 2,2′-isulphide in the presence of triphenylphosphine (yield 90%). Anhydrophomenolactone is obtained as a secondary product during this synthesis in a yield of 10%.     

Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI

Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces, have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g l(-1)) was obtained by constructing a T. reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium, which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCl precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 15 l of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also, to some extent, to non-specific proteases although its thermostability was excellent.     

Effect of fatty acids on aflatoxin production byAspergillus parasiticus

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.     

Studies on the effects of carbon:nitrogen ratio, inoculum type and yeast extract addition on jasmonic acid production by Botryodiplodia theobromae Pat. strain RC1

Jasmonic acid is a native plant growth regulator produced by algae, microorganisms and higher plants. This regulator is involved in the activation of defence mechanisms against pathogens and wounding in plants. Studies concerning the effects of carbon: nitrogen ratio (C/Nr: 17, 35 and 70), type of inoculum (spores or mycelium) and the yeast extract addition in the media on jasmonic acid production by Botryodiplodia theobromae were evaluated. Jasmonic acid production was stimulated at the carbon: nitrogen ratio of 17. Jasmonic acid productivity was higher in the media inoculated with mycelium and in the media with yeast extract 1.7 and 1.3 times, respectively.     

Enhanced Production of Ganoderic Acids and Cytotoxicity of Ganoderma lucidum Using Solid-Medium Culture

Submerged cultures of Ganoderma lucidum are used to produce fungal mycelium, which is used as a functional food and in the production of various triterpenoids, including ganoderic acids (GAs). Specific culture approaches that produce fungal mycelium with high levels of GAs and good biological activity are critical in the functional food industry. In this study, a solid-medium culture approach to producing mycelium was compared to the submerged culture system. Production of GAs, biomass, intracellular polysaccharides, and cytotoxicity of the cultured mycelium were compared as between solid and submerged culture. Growing G. lucidum strains on solid potato dextrose agar medium increased biomass, the production of ganoderic acid 24 (lanosta-7,9(11), 24-trien-3α-o1-26-oic acid), GAs, and total intracellular polysaccharides as compared to fungi grown in submerged culture. Triterpenoid-enriched methanol extracts of mycelium from solid-medium culture showed higher cytotoxicity than those from submerged culture. The IC(50) values of methanol extracts from solid-medium culture were 11.5, 8.6, and 9.9 times less than submerged culture on human lung cancer cells CH27, melanoma cells M21, and oral cancer cells HSC-3 respectively. The squalene synthase and lanosterol synthase coding genes had higher expression on the culture of solid potato dextrose medium. This is the first report that solid-medium culture is able to increase GA production significantly as compared to submerged culture and, in the process, produces much higher biological activity. This indicates that it may be possible to enhance the production of GAs by implementing mycelium culture on solid medium.     

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi-preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H-, 13C-NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.     

Conditions of cultivation, composition, and biological activity of mycelium of Flammulina velutipes (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karat. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5-10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6-78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages--central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Continuous itaconic acid production by immobilized biocatalysts.

The continuous itaconic acid production from sucrose with Aspergillus terreus TKK 200-5-3 mycelium immobilized on polyurethane foam cubes was optimized in column bioreactors using statistical experimental design and empirical modelling. The highest itaconic acid product concentration calculated on the basis of the obtained model was 15.8 g l-1 in the investigated experimental area, when sucrose concentration was 13.5%, aeration rate 150 ml min-1 and residence time 178 h. From sucrose with immobilized A. terreus TKK 200-5-3 mycelium itaconic acid production was stable for at least 4.5 months in continuous column bioreactors. In comparison, using glucose as substrate and immobilized A. terreus TKK 200-5-1 mycelium as biocatalyst similar stability was obtained with higher product concentration. The omission of copper sulphate from the production medium gave the highest itaconic acid product concentration (26 g l-1) from 9% glucose with 0.25% ammonium nitrate and 0.095% magnesium sulphate.     

The production of ectomycorrhizal mycelium in forests: Relation between forest nutrient status and local mineral sources

Due to acid rain and nitrogen deposition, there is growing concern that other mineral nutrients, primarily potassium and phosphorus, might limit forest production in boreal forests. Ectomycorrhizal (EcM) fungi are important for the acquisition of potassium and phosphorus by trees. In a field investigation, the effects of poor potassium and phosphorus status of forest trees on the production of EcM mycelium were examined. The production of EcM mycelium was estimated in mesh bags containing sand, which were buried in the soil of forests of different potassium and phosphorus status. Mesh bags with 2% biotite or 1% apatite in sand were also buried to estimate the effect of local sources of nutrients on the production of EcM mycelium. No clear relation could be found between the production of EcM mycelium and nutrient status of the trees. Apatite stimulated the mycelial production, while biotite had no significant effect. EcM root production at the mesh bag surfaces was stimulated by apatite amendment in a forest with poor phosphorus status. The contribution of EcM fungi to apatite weathering was estimated by using rare earth elements (REE) as marker elements. The concentration of REE was 10 times higher in EcM roots, which had grown in contact with the outer surface of apatite-amended mesh bags than in EcM roots grown in contact with the biotite amended or sand-filled mesh bags. In a laboratory study, it was confirmed that REE accumulated in the roots with very low amounts <1 translocated to the shoots. The short-term effect of EcM mycelium on the elemental composition of biotite and apatite was investigated and compared with biotite- and apatite-amended mesh bags buried in trenched soil plots, which were free from EcM fungi. The mesh bags subjected to EcM fungi showed no difference in chemical composition after 17 months in the field. This study suggests that trees respond to phosphorus limitation by increased exploitation of phosphorus-containing minerals by ectomycorrhiza. However, the potential to ameliorate potassium limitation in a similar way appears to be low.     

Itaconic acid production by immobilizedAspergillus terreus on sucrose medium

Summary The itaconic acid production by immobilizedAspergillus terreus TTK 200-5-3 mycelium was optimized in shake flask fermentations using statistical experimental design and empirical modelling. The maximum itaconic acid concentration was calculated to be 13.3 g/l in the investigated experimental area when initial sucrose concentration was 10%, ammonium nitrate concentration 0.275% and initial pH 3. The itaconic acid product concentration using immobilized mycelium was about double of that obtained with the free mycelium.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Conditions of cultivation,composition, and biological activity of mycelium of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karst. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5–10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6–78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages—central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Protoplasts fromPodospora anserina: Isolation,purification, and transformation

Protoplasts fromPodospora anserina mycelium were produced using the commercially available enzyme Novozym 234. Different parameters involved in protoplast isolation were analyzed in order to establish optimal conditions, and protoplast production was notably increased. For the purification of protoplasts, several techniques based on both centrifugation and filtration were assayed, with filtration yielding the best results. Regeneration of protoplasts was studied on different media and osmostic stabilizers, and about 80% regeneration was obtained. The good physiological condition of the protoplasts produced with this method is demonstrated by the lack of cell wall and high regeneration rate and transformation frequencies.     

Factors contributing to roquefortine yield variability during cultivation of penicillium roquefortii

Variability in the roquefortine yield was shown to be associated with its consumption by the mycelium during isolation of the end product, which depended on temperature, time of culture liquid storage, and biomass concentration. This was also related to the presence in chloroform of chlorocarbonic acid ethyl ester that reacted with roquefortine.     

Factors Contributing to Roquefortine Yield Variability during Cultivation of Penicillium roquefortii

Variability in the roquefortine yield was shown to be associated with its consumption by the mycelium during isolation of the end product, which depended on temperature, time of culture liquid storage, and biomass concentration. This was also related to the presence in chloroform of chlorocarbonic acid ethyl ester that reacted with roquefortine.     

Production of laccase by membrane-surface liquid culture of Trametes versicolor using a poly(l-lactic acid) membrane

Membrane-surface liquid culture (MSLC) is a promising method for the bioproduction of highly aerobic filamentous fungi [A. Ogawa, A. Yasuhara, T. Tanaka, T. Sakiyama, K. Nakanishi, Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704, J. Ferment. Bioeng. 80 (1995) 35–40]. This paper reports on the production of laccase by Trametes versicolor on a microporous membrane of poly(l-lactic acid) (PLLA), which can be biodegraded via composting after use. The membrane was stable as a support for 24 days at 30 °C. During the first 9 days in MSLC, the fungus produced half as much laccase as it did in liquid-surface culture (LSC); however, the mycelium on the membrane was able to be re-used five times for laccase production. The laccase production was accelerated in the repeated use of the culture while the mycelium in LSC ceased to produce the enzyme. This study shows that compostable PLLA microporous membranes can be used for enzyme production by MSLC of filamentous fungi.     

The rpoZ gene,encoding the RNA polymerase omega subunit,is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis

The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (omega), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete omega of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.