Isolation and identification of two new compounds from marine-derived fungus Acremonium fusidioides RZ01.

Two new compounds, (22E)-25-carboxy-8ß,14ß-epoxy-4α,5α-dihydroxyergosta-2,22-dien-7-one (1) and fusidione (3), along with two known compounds, 5α,8α-epidioxy ergosta-6,22-diene-3ß-ol (2) and microperfuranone (4), were isolated from the fermentation products of the marine-sourced fungus Acremonium fusidioides RZ01. The structures of compounds 1 and 3 were elucidated by extensive spectroscopic methods, especially 2D NMR, and their absolute configurations were suggested on the basis of the circular dichroism spectral analysis and the NOESY data. Both new compounds showed inhibitory activity against HL-60 cells with IC50 values being16.6 and 44.9 µmol·L-1, respectively.

Bioaugmentation of soil contaminated with high-level crude oil through inoculation with mixed cultures including Acremonium sp.

Heavy contamination of soil with crude oil has caused significant negative environmental impacts and presents substantial hazards to human health. To explore a highly efficient bioaugmentation strategy for these contaminations, experiments were conducted over 180 days in soil heavily contaminated with crude oil (50,000 mg kg(-1)), with four treatments comprised of Bacillus subtilis inoculation with no further inoculation (I), or reinoculation after 100 days with either B. subtilis (II), Acremonium sp.(III), or a mixture of both organisms (IV). The removal values of total petroleum hydrocarbons were 60.1 ± 2.0, 60.05 ± 3.0, 71.3 ± 5.2 and 74.2 ± 2.7 % for treatment (I-IV), respectively. Treatments (III-IV) significantly enhanced the soil bioremediation compared with treatments (I-II) (p < 0.05). Furthermore, significantly (p < 0.05) greater rates of degradation for petroleum hydrocarbon fractions were observed in treatments (III-IV) compared to treatments (I-II), and this was especially the case with the degradative rates for polycyclic aromatic hydrocarbons and crude oil heavy fractions. Dehydrogenase activity in treatment (III-IV) containing Acremonium sp. showed a constant increase until the end of experiments. Therefore reinoculation with pure fungus or fungal-bacterial consortium should be considered as an effective strategy in bioaugmentation for soil heavily contaminated with crude oil.

[Screening of endophytic fungi from Huperzia serrata for acetylcholinesterase inhibitory activity and its taxonomic identification].

OBJECTIVE: To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata. METHOD: Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics. RESULT: Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium. CONCLUSION: Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Production of cephalosporin C using crude glycerol in fed-batch culture of Acremonium chrysogenum M35.

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.

Synthesis of a natural gamma-butyrolactone from nerylacetone by Acremonium roseum and Fusarium oxysporum cultures.

Natural gamma-butyrolactone - (4R, 5R)-5-(4'-methyl-3'pentenyl)-4-hydroxy-5-methyl-dihydrofuran-2-one (2) was isolated as the product of microbial transformation of nerylacetone (1) by fungal strains. This product was obtained as the enantiomer (+) in high yields 24% and 61% with ee=94% and 82% by the biotransformation in the cultures of Acremonium roseum AM336 and Fusarium oxysporum AM13 respectively.

Generation of two new macrolactones through sequential biotransformation of dihydroresorcylide.

Biotransformation is an effective method to generate new derivatives from natural products. Combination of various enzymes or whole-cell biocatalysts creates new opportunities for natural product biosynthesis. Dihydroresorcylide (1) is a phytotoxic macrolactone from Acremonium aeae. It was first chlorinated at C-11 by an engineered Escherichia coli BL21-CodonPlus (DE3)-RIL/pJZ54 strain that overexpresses a fungal flavin-dependent halogenase, and subsequently glycosylated at 12-OH by Beauveria bassiana ATCC 7159, giving rise to a novel derivative, 11-chloro-4'-O-methyl-12-O-beta-D-glucosyl-dihydroresorcylide (3). Although 1 can be converted into a new 4'-O-methyl-glucosylated derivative 4 by B. bassiana, this product cannot be further chlorinated by E. coli BL21-CodonPlus (DE3)-RIL/pJZ54 to afford 3. The sequence of these two biotransformation steps was thus restricted and not interchangeable. This sequential biotransformation approach can be applied to other structurally similar natural products to create novel derivatives.

Quantitative metabolic flux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chrysogenum fed with soybean oil.

A metabolic network was constructed for the Acremonium chrysogenum cultivation fed with soybean oil. Metabolic flux analysis indicated that the shift from exponential growth to rapid cephalosporin C (CPC) formation was accompanied by 1.63- and 5-fold carbon flux enlargement in TCA cycle and glyoxylate by-pass, respectively. The flux via pentose phosphate pathway branch was little affected during the rapid CPC formation period; the contributory explanation was that 35.6% of NADPH was consumed in the dissimilation of fatty acids. Estimation of NADPH, ATP generation, and consumption demonstrated that, with soybean oil as carbon source in rapid CPC formation phase, the NADPH consumed in fatty acid catabolism was fourfold greater than that used in the CPC biosynthesis-relevant part; simultaneously, more than 90% energy spent was not directly related to the CPC formation. Therefore, the improvement of CPC production yield through optimization of the NADPH, ATP generation, and consumption was put forward.

Characterisation of uranium(VI) sorption by two environmental fungal species using gamma spectrometry.

Uranium(VI) sorption by two highly uranium-resistant air-borne fungi in a low-pH medium was measured by means of gamma spectrometry. Growth kinetics and stoichiometry of the two fungal species were also studied. Results show acceptable growth rates in synthetic medium with glucose and ammonia as carbon and nitrogen sources, respectively. Typical oxidative metabolism of carbohydrates was found. In vivo uptake of the radionuclide was negligible, but biosorption dry biomass presented a remarkable performance. The fungal strains showed potential for use in bioremediation.

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs.

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.

Accumulation of copper by Acremonium pinkertoniae, a fungus isolated from industrial wastes.

Acremonium pinkertoniae isolated from zinc wastes was studied to understand the mechanisms that allow living organisms to thrive in polluted environments and the possible role of the fungus in the redistribution and cycling of copper. The fungus was cultured on solid media supplemented with copper sulfate at increasing concentrations. At high doses it was observed that the mycelia acquired a characteristic blue color. This was accompanied by morphological changes and the formation of crystalloid structures within thickened cell walls. The material was further analysed with EDX, X-ray powder diffraction and IR spectroscopy. It was demonstrated that A. pinkertoniae is able to accumulate over 20% dry weight of copper by what probably is a chitin-glucan complex.

A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides.

The maize endophyte Acremonium zeae is antagonistic to kernel rotting and mycotoxin producing fungi Aspergillus flavus and Fusarium verticillioides in cultural tests for antagonism, and interferes with A. flavus infection and aflatoxin contamination of preharvest maize kernels. Chemical studies of an organic extract from maize kernel fermentations of Acremonium zeae (NRRL 13540), which displayed significant antifungal activity against Aspergillus flavus and F. verticillioides, revealed that the metabolites accounting for this activity were two newly reported antibiotics pyrrocidines A and B. Pyrrocidines were detected in fermentation extracts for 12 NRRL cultures of Acremonium zeae isolated from maize kernels harvested in Illinois (4/4 cultures), North Carolina (5/5), Georgia (1/2) and unrecorded locations within the USA (2/2). Pyrrocidine B was detected by LCMSMS in whole symptomatic maize kernels removed at harvest from ears of a commercial hybrid that were wound-inoculated in the milk stage with A. zeae (NRRL 13540) or (NRRL 13541). The pyrrocidines were first reported from the fermentation broth of an unidentified filamentous fungus LL-Cyan426, isolated from a mixed Douglas Fir hardwood forest on Crane Island Preserve, Washington, in 1993. Pyrrocidine A exhibited potent activity against most Gram-positive bacteria, including drug-resistant strains, and was also active against the yeast Candida albicans. In an evaluation of cultural antagonism between 13 isolates of A. zeae in pairings with A. flavus (NRRL 6541) and F. verticillioides (NRRL 25457), A. zeae (NRRL 6415) and (NRRL 34556) produced the strongest reaction, inhibiting both organisms at a distance while continuing to grow through the resulting clear zone at an unchanged rate. Maximum colony diameters for A. zeae (NRRL 6415) and (NRRL 13540), on potato dextrose agar after 14 d, were attained within the range of 25-30 degrees C, with less growth recorded at 15 degrees and 37.5 degrees and no growth at 5 degrees. Potential interactions between A. zeae and other maize endophytes are considered and the significance of these interactions relative to the aflatoxin and fumonisin contamination of preharvest maize is presented. This is the first report of natural products from Acremonium zeae.

[Studies on antibacterial activities of secondary metabolites from fungus Cephalosporium sp. AL031].

The antibacterial activity of fourteen identified compounds from secondary metabolites of fungus Cephalosporium sp. AL031 against eleven gram-positive or gram-negative bacteria in vitro was investigated by agar diffusion assay. The results suggested that two new compounds [S-(+)-5-hydroxymellein, S-(+)-7-hydroxymellein], together with four known compounds [S-(+)-mellein, 2,4-dihydroxy-3,6-dimethylbenzoic acid, 3,6-dihydroxy-2,4-dimethylbenzoic acid and succinic acid] had broad spectrum antibacterial activity. The minimum inhibitory concentrations (MIC) of these six compounds against Shigella sonnei, Mycobacterium tuberculosis and Streptococcus pneumoniae were determined. Their MIC were 0.03125 to approximately 0.5 mg/ml.

Analysis of the relationship between growth, cephalosporin C production, and fragmentation in Acremonium chrysogenum.

Mycelial fragmentation in submerged cultures of the cephalosporin C (CPC) producing fungus Acremonium chrysogenum was characterized by image analysis. In both fed-batch and chemostat cultures, the proportion of mycelial clumps seemed to be the most sensitive morphological indicator of fragmentation. In a fed-batch fermentation culture, this declined from roughly 60% at inoculation to less than 10% after 43 h. Subsequent additions of glucose resulted in a sharp increase back to near the initial value, an increase that reversed itself a few hours after glucose exhaustion. Meanwhile CPC production continued to decline steadily. On the other hand, the addition of soybean oil enhanced CPC production, but had no significant effect on the morphology. Although it may sometimes appear that morphology and productivity are related in batch or fed-batch cultures, this study suggests that this is because both respond simultaneously to more fundamental physiological changes, dependent on the availability of carbon. In circumstances, such as supplementary carbon source addition, the relationship is lost. Chemostat cultures supported this belief, as CPC-production rates were hardly affected by the specific growth rate, but the morphology showed significant differences, i.e., lower dilution rates resulted in a lower proportion of clumps and in smaller clumps.

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum.

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed.

Alkaloids from endophyte-infected Festuca argentina.

Five loline-type alkaloids, the known lolinine, N-formylloline, loline, N-methylloline and the new 5,6-dehydro-N-acetylloline, were characterized from the pooid-grass endophyte-infected Festuca argentina. These compounds are not responsible for the toxicity of the plant, as shown upon i.p. injection into mice. Their probable phytoalexine role and chemotaxonomical significance are discussed.

Recombinant Acremonium chrysogenum strains for the industrial production of cephalosporin.

Conventional strain improvement programs based on random mutagenesis and rational screening have meant valuable results to the antibiotic producing companies. The development of recombinant DNA techniques and their applications to the industrially-used cephalosporin-producing fungus Acremonium chrysogenum has provided a new tool, complementary to classical mutation, promoting the design of alternative biosynthetic pathways making it possible to obtain new antibiotics and to improve cephalosporin production. Yield increases have been achieved by increasing the dosage of the biosynthetic genes cefEF (deacetoxycephalosporin C expandase/hydroxylase) and cefG (deacetylcephalosporin C acetyltransferase) or enhancing the oxygen uptake by expressing a bacterial oxygen-binding heme protein (Vitreoscilla hemoglobin). New biosynthetic capacities such as the production of 7-aminocephalosporanic acid (7-ACA) or penicillin G have been achieved through the expression of the foreign genes dao (D-amino acid oxidase) coupled with cephalosporin acylase or penDE(acyl-CoA:6-APA acyltransferase) respectively. Confined manipulation of the above-mentioned recombinant strains must be performed according to standing rules.

Equine fescue toxicosis: signs and solutions.

Gravid mares grazing endophyte-infested (E+) tall fescue exhibit increased gestation lengths, agalactia, foal and mare mortality, tough and thickened placentas, weak and dysmature foals, increased sweating during warm weather, reduced serum prolactin and progesterone, and increased serum estradiol-17 beta levels. Also, E+ tall fescue hay is less digestible than endophyte-free (E-) hay. Unlike many other species, horses consuming E+ tall fescue do not exhibit increased body temperature. Young horses consuming only E+ pasture do not gain as well as those consuming E- pasture. There is little difference in gain when the pasture is supplemented with enough concentrate to meet NRC requirements for growth. Neither selenium injections nor supplementing with corn at 50% of the NRC requirements for energy reduces the effects of toxic tall fescue on reproduction and lactation in gravid mares. It seems that the alkaloids of E+ tall fescue are serving as D2 dopamine receptor agonists. This activity would explain their prolactin-lowering effect. Domperidone, a dopamine receptor antagonist, is effective in preventing the signs of tall fescue toxicosis in horses without neuroleptic side effects.

Direct determination of the cephalosporin transforming activity of immobilized cells with use of an enzyme thermistor. 1. Verification of the mathematical model.

A simple method for the direct measurement of the catalytic properties of immobilized cells in the flow minicalorimeter, the enzyme thermistor (ET), is presented. A Trigonopsis variabilis strain with cephalosporin C-transforming activity was used as the model system. The yeast cells were immobilized either by crosslinking with a homobifunctional reagent or by entrapment in gels. The actual activity of the immobilized cells used in the ET was estimated by means of a stirred-batch reactor measurement in conjunction with HPLC analysis of substrate and products. Similar results were also obtained using D-amino acid oxidase (EC 1.4.3.3) isolated from T. variabilis cells and immobilized by gel entrapment. This calibration procedure was found to be appropriate for all biocatalyst systems used. The thermometric signal was proportional to the amount of biocatalyst immobilized in the ET minicolumn. It was shown that the rate of reaction catalyzed by T. variabilis entrapped in calcium pectate gel was limited by internal diffusion to an extent depending on the cell concentration in the biocatalyst particle. This approach offers a direct method for studying the kinetic properties of immobilized cells.

Effects of hydrated sodium calcium aluminosilicate on fescue toxicosis and mineral absorption.

The possibility of supplementing livestock diets with an aluminosilicate to protect them from fescue toxicosis was investigated. An in vitro study showed that hydrated sodium calcium aluminosilicate (HSCAS) removed greater than 90% of the ergotamine from aqueous solutions at pH 7.8 or lower, indicating a high affinity of ergotamine for HSCAS in vitro. Rats fed diets containing tall fescue seed infested (E+) with the endophytic fungus Acremonium coenophialum had lower (P less than .05) feed intakes and weight gains than did rats fed diets containing uninfested (E-) tall fescue seed. When feed intake by rats fed the E- seed diet was limited to that of rats fed the E+ seed diet, weight gains did not differ, but testes weights and serum prolactin (PRL) concentrations were lower (P less than .05 and .10, respectively) in rats receiving E+ seed. Supplementing E+ seed diets with HSCAS did not eliminate effects of E+ seed on intake, PRL, or testes weights. Sheep fed E+ tall fescue hay had higher (P less than .05) rectal temperatures than did sheep fed an equal amount of E- tall fescue hay, but OM and N digestion coefficients did not differ between the two hays. Supplementing E+ hay diets with HSCAS did not eliminate the effect of E+ hay on rectal temperatures. Addition of 2% HSCAS to tall fescue hay diets did not affect apparent absorption by sheep of OM, N, Ca, P, Na, K, or Cu, but it reduced (P less than .05) the apparent absorption of Mg, Mn, and Zn.(ABSTRACT TRUNCATED AT 250 WORDS)