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Different aldolase activities were demonstrated in cell-free extracts of Aspergillus ustus grown on d-glucose or d-gluconate as the only source of carbon. Pyruvic, oxalacetic, or glycolic acids were used as substrates for enzymatic condensation with different aldehydes. 2-Keto-3-deoxy arabonic acid (KDA) or 2-keto-3-deoxy gluconic acid (KDG) was the product of such condensation. No activity could be detected when glycolic acid, formaldehyde, or acetaldehyde was used as substrate. Maximum pH and temperature were found to be 7.5 and 50°C for KDA aldolase, and 7.0 and 50°C for KDG adolase. Decreased activity was obtained when d-glucose replaced d-gluconate in the fermentation medium. Thermal stability behavior of KDG aldolase indicates that temperature has a stimulating effect on enzyme activity at 50°C. HgCl2, para-mercurychlorobenzoate, ZnSO4, and CuSO4 were potent inhibitors of this enzyme. The Km values were determined for pyruvate and glyceraldehyde and were found to be 4.1 × 10−3 and 7.1 × 10−3m, respectively.

The structures of austalides A–E, five new mycotoxins isolated from cultures of Aspergillus ustus, are based on the X-ray crystallographic study of austalide A, chemical derivatization, and correlation of their 13C and 1H n.m.r spectral data.

Nine genera of fungi were isolated from more than 200 insects representing eight species of corn pests. They are Beauveria, Metarrhizium, Fusarium, Aspergillus, Penicillium, Paecilomyces, Rhizopus, Scopulariopsis , and Mycoderma . The European corn borer, Ostrinia nubilalis (Hubner), was used as a test animal for pathogenicity tests with the fungus organisms. Beauveria bassiana and Metarrhizium anisopliae were the most pathogenic strains recovered. Aspergillus ustus, Fusarium neoceras, paecilomyces varioti, Penicillium decumbens, P. puberulum , and Scopulariopsis brevicaulis had not previously been reported from insects.

Aspergillus ustus is one of the most prevalent fungi in the soil. There are now two reports of the occurrence of toxin-producing strains of this fungus on stored foodstuffs. In addition, strains of A. ustus have been isolated along with Penicillium species from samples of South African cheeses. All A. ustus isolates tested were judged to be highly toxic to ducklings when grown on maize meal, however, the toxins involved were not isolated. Austin is the trivial name of one of the toxins made by the fungus found on stored food. Preliminary work to studying the biosynthesis of this compound using /sup 13/C-labeled sodium acetate is reported here. The feasibility of the biosynthetic study was determined by feeding (1-/sup 14/C)-sodium acetate to A. ustus cultures. The assignments made in the /sup 13/C-nmr spectrum of Austin are shown. The lowest dilution factor obtained in (1-/sup 14/C)-sodium acetate feeding experiments was 14. This dilution factor is sufficiently low to allow a successful feeding of (1,2-/sup 13/C/sub 2/)-sodium acetate. A new metabolite of A. ustus, deacetylaustin, was isolated and identified. An alkaloid of unknown structure was also isolated from the fungus.

A MOULD, isolated by Kurung1 in 1944 and considered to be a strain of Aspergillus ustus (Bain.) Thom and Church, was shown to produce a metabolic liquor which possessed a marked antibiotic activity towards Mycobacterium tuberculosis and M. ranae. Afterwards, Hogeboom and Craig2, by application of a counter-current technique, were able to isolate, from an ethereal extract of this mould and its substrate, two crystalline products, called compound I and compound II, which showed a very high activity against M. ranae. Doering, Dubos, Noyce and Dreyfus3, using the same strain of mould, isolated a number of chlorine-containing metabolic products, one of which was proved to be identical with Hogeboom and Craig's compound I. Doering et al. proposed the name ‘ustin’ for this compound, established its empirical formula (C19H15O5Cl3) and published a superficial account of some of its chemical properties.

Aspergillus fumigatus was isolated with high frequency from samples of ornithogenic soil collected from an Adélie penguin rookery at Hallett Station, Antarctica. While A. fumigatus has been well documented as the principal incitant of aspergillosis among penguins in captivity, this represents the first report of its presence in antarctic materials. Aspergillus flavus and Aspergillus ustus were also found in the Hallett samples. In a comparable number of soil and guano samples from isolated Adélie rookeries on Foyn Island and Possession Island, these fungi were not encountered. It is suggested that their presence at Hallett Station may be the result of human contamination. A brief consideration of epidemiological implications is given.

Twenty four fungal species were screened for their ability to produce alkaline L-methioninase on methionine-glucose liquid medium. Aspergillus ustus AUMC 10151 displayed the highest yield of enzyme (10.8 U/mg protein), followed by A. ochraceus and Fusarium proliferatum. Upon optimization of the submerged fermentation (SmF)conditions, the maximum enzyme yield (18.23 U/mg protein) was obtained on a medium containing L-methionine (0.5%), sucrose (0.95%), KH2PO4 (0.1%) and 175 rpm. Seven agro-industrial by-products were screened as substrates for L-methioninase production under solid-state fermentation (SSF). Wheat bran resulted 38.1 U/mg protein, followed by rice bran (27.6 U/mg protein) and soya bean meal (26.6 U/mg protein). Maximum alkaline L-methioninase (99.56U/mg protein) was achieved at initial moisture content of 71.5%, inoculum size of 2.0 mL of spore suspension, initial pH 8.5, incubation period eight days at 30°C and supplementation of the salt basal medium with pyridoxine(100 μg/mL) and beet molasses (20% v/v). The productivity of L-methioninase by A. ustus under SSF was higher than that of SmF about 5.45 fold under optimum conditions.

Incorporation studies on austdiol (1), a metabolite from Aspergillus ustus were carried out with [1,2-13C2]-acetate, [Me-13C]-, and [Me-2H3]-methionine. Specific incorporation of 4,6-dihydroxy-3,5-dimethyl-2-(2-oxopropyl)benzaldehyde (2) into austdiol (1) was demonstrated using 2H n.m.r. spectroscopy. The total synthesis of the ketoaldehyde (2), allowing the specific insertion of a label, is described.

Secondary metabolites of 22 fungal strains (genus Aspergillus, section Usti) isolated at diverse geographic regions, including the Arctic permafrost deposits, were studied. The studied strains were found to synthesize a variety of biologically active compounds, structurally identified as drimane sesqueterpenoids, isoquinoline alkaloids (TMC-120 A−C, derivative 1), meroterpenoids (austalides О and J), and anthraquinone pigments (averufin, versicolorin C). Desferritriacetylfusigen production by A. calidoustus isolates is reported for the first time. The individual spectra of secondary metabolites were used for reidentification of 17 strains, of which 15 were identified as A. calidoustus and two, as A. pseudodeflectus.

Incorporations of [1-13C]-, [2-13C]-, [1,2-13C2]- acetates and [Me-13C] methionine into austin, a metabolite of Aspergillus ustus, indicate its formation by a mixed polyketide-terpenoid biosynthetic pathway.

Incorporations of 13C-labelled acetates and methionine into the mycotoxin austin in cultures of Aspergillus ustus give a labelling pattern consistent with a mixed polyketide-terpenoid pathway. Incorporations of 14C and 2H labelled 3,5-dimethylorsellinate confirm the intermediary of a preformed tetraketide-derived phenolic precursor. Further information on the mechanisms involved in the modifications of both the farnesyl- and tetraketide-derived portions of the molecule are provided by incorporation studies with [1-13C,18O2]acetate, [methyl-13C,2H3] methionine, 13C,18O-labelled dimethylorsellinate,18O2 gas and [6-13C,6-2H3]mevalonic acid lactone.

Ethyl 3,5-dimethylorsellinate (3) doubly labelled with 13C and 18O in the carbonyl of the carboxy group and at the C-6 position has been synthesised using a four-step procedure from sodium [1-13C, 18O2]acetate, and incorporated into austin (1) by cultures of Aspergillus ustus.

Mass spectral and 13C n.m.r. analyses of austin (1) produced by fermentation of Aspergillus ustus in the presence of 18O2 and [Me-13C,2H3]methionine showed that all labelled methionine-derived hydrogens were retained, determined that five of nine oxygens were introduced by aerobic oxidation, and elucidated the general mechanism of late biosynthetic stages.

The fungal metabolite dihydrodeoxy-8-epi-austdiol, elaborated by Aspergillus ustus, has been identified as (7R,8R)-7,8-dihydro-7,8-dihydroxy-3,5,7-trimethyl-2-benzopyran-6-one. The absolute configuration of the azaphilones has been deduced.

The assignment of the natural-abundance 13C n.m.r. spectrum of austocystin D, a mycotoxin produced by Aspergillus ustus MRC 1163, allowed a study of its biosynthetic origin. The complete 13C–13C coupling pattern in austocystin D enriched from [1,2-13C2]acetate has been determined and confirms that no randomisation of labelling in ring A occurs during the anthraquinone → xanthone conversion in the biosynthetic pathway.

The fungal metabolite austdiol, elaborated by Aspergillus ustus, has been identified as (7R,8S)-7,8-dihydro-7,8-dihydroxy-3,7-dimethyl-6-oxo-6H-2-benzopyran-5-carbaldehyde. The absolute configuration was deduced by the ‘partial resolution’ method of Horeau.

Interpretation of the incorporation data of 18O2 into austdiol in Aspergillus ustus suggests a mono-oxygenase mechanism.

Incorporation of [1-13C]- and [1,2-13C2]-acetate, and (3RS)-[2-13C]mevalonate into austalide D, a metabolite of Aspergillus ustus, and the isolation of the cometabolites austalide J, K, and L, indicate its formation via a mixed polyketide–terpenoid biosynthetic pathway.

14 C and 2H Labelling experiments together with 2H n.m.r. spectroscopy show that 3,5-dimethylorsellinic acid is a specific precursor of austin and terretonin in Aspergillus ustus and Aspergillus terreus respectively and so substantiate the mixed polyketide–terpenoid origin proposed for these metabolites from the incorporation of 13C-labelled simple precursors.

Incorporationof [1-13C]-, [1,2-13C2]-and [1-13C,2,2,2-2H3]-acetate, and (3RS)-[2-13C]mevalonolactone into austalide D(2), a metabolite of Aspergillus ustus, shows that the austalides are derived from 6[(2E,6E)farnesyl]-5,7-dihydroxy-4-methylphthalide. The proposed mechanism for the subsequent cyclisation and oxidative modifications of the farnesyl moiety, consistent with the relative stereochemistry of the austalides and the structures of the cometabolites, austalides J (4), K (5), and L (6), is supported by the incorporation of austalide K into austalide D. The addition of ethanol to growing cultures of A. ustus severely inhibits normal metabolite production and induces the formation of a new metabolite, ethyl 6-ethyl-2,4-dihydroxy-3-methylbenzoate (11). The biosynthetic origin of this metabolite was studied using [1-13C]- and [1-13C,2,2,2-2H3]acetate and (2S)-[methyl-13C]methionine as precursors.

The 13C n.m.r, spectrum of austin has been fully assigned by inter alia analysis of long range 1H–13C couplings in the fully 1H-coupled 13C n.m.r. spectrum. Further related metabolites, dehydroaustin, austinol, and isoaustin, have been isolated from Aspergillus ustus, Aspergillus variecolor, and Penicillium diversum. Their structures have been assigned by detailed analysis of their 1H and 13C n.m.r. spectra.

A study of the fate of the hydrogen atoms in the biosynthesis of austalide D using both (13C,2H)- and 2H-labelled mevalonolactones established, in conjunction with absolute configuration as determined by an X-ray crystallographic study, the stereochemical course of cyclisation of the farnesyl-derived moiety.

The almost uniformly fatal outcome ofAspergillusendocarditis, most of which has occurred on prosthetic cardiac valves, has been well documented.1,2The difficulty in the diagnosis of this type of infection, the problems of therapy of prosthetic valve endocarditis, and the relative ineffectiveness of antifungal chemotherapeutic agents account for the high mortality. We describe a patient withAspergillusendocarditis on a prosthetic valve who was cured with a combination of surgery, amphotericin B therapy, and long-term therapy with flucytosine. (This patient was included in a previous report of two patients withAspergillusendocarditis,3but the details and results of therapy were not included in that report.) A review of the literature onAspergillusendocarditis is also included. Patient Summary Patient 1. —A 50-year-old black man was first admitted to the Philadelphia Veterans Administration Hospital in February 1963 because of increasing symptoms of heart failure. He had had rheumatic