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Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2 a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The human fungal pathogen Aspergillus fumigatus causes life-threatening invasive aspergillosis in immunocompromised individuals. Adaptation to the host environment is integral to survival of A. fumigatus and requires the coordination of short- and long-distance vesicular transport to move essential components throughout the fungus. We previously reported the importance of MyoE, the only class V myosin, for hyphal growth and virulence of A. fumigatus. Class V myosins are actin-based, cargo-carrying motor proteins that contain unique binding sites for specific cargo. Specific cargo carried by myosin V has not been identified in any fungus, and previous studies have only identified single components that interact with class V myosins. Here we utilized a mass spectrometry-based whole proteomic approach to identify MyoE interacting proteins in A. fumigatus for the first time. Several proteins previously shown to interact with myosin V through physical and genetic approaches were confirmed, validating our proteomic analysis. Importantly, we identified novel MyoE-interacting proteins, including members of the cytoskeleton network, cell wall synthesis, calcium signaling and a group of coat protein complex II (COPII) proteins involved in the endoplasmic reticulum (ER) to Golgi transport. Furthermore, we analyzed the localization patterns of the COPII proteins, UsoA (Uso1), SrgE (Sec31), and SrgF (Sec23), which suggested a potential role for MyoE in ER to Golgi trafficking., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tuning the electrocatalytic properties of high surface area porous metallic frameworks like Nanoporous Gold (NPG) by tailoring the structure is a convenient strategy to design electrochemical sensors. Accordingly, an NPG-based sensitive, selective and robust electroanalytical platform was designed for the detection of ascorbic acid (AA) in acidic extracts of Aspergillus fumigatus fungus and Arabidopsis thaliana leaves. NPG films were electrodeposited on a gold microelectrode by potentiostatic electrodeposition and characterized by electron microscopy techniques, which confirmed the morphology and highly porous structure resembling nanowires-type pure gold fractals. The electrodeposition parameters, particularly deposition potential and time, were optimized to achieve large and selective amperometric detection of AA on the NPG modified electrodes. Faster electron transfer kinetics was manifested on the 0.3 V shift in overpotential and remarkable enhancement of the oxidation peak current as compared with bare gold electrode. Amperometric measurements were performed at 0.3 V vs. Ag/AgCl (sat. KCl) in the highly acidic electrolyte solution employed to extract ascorbate from biological samples and minimize its autoxidation. The sensitivity of conventional Au-microelectrodes was increased about one thousand-fold upon modification with NPG film, reaching 2 nA μmol -1  L -1 . The detection limit for AA based on a linear current-concentration calibration plot was found to be 2 μmol L -1 . The NPG-based microsensor was demonstrated to be selective, reproducible and stable, and was employed for determinations of AA concentration in highly acidic biological extracts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aspergillus fumigatus is a major pathogen of invasive pulmonary aspergillosis with high mortality rate. The nucleoside diphosphate kinase of A. fumigatus, AfNDK (also called SwoH) is essential for its viability, however, its structural characteristic was unknown. In this study, we solved the crystal structure of AfNDK and found that it exists predominantly in form of tetramer in solution. Oligomeric form rather than dimeric form was essential for its kinase activity. The Arg30 and the C terminal amino acids were crucial for dimer-dimer interaction and the viability of A. fumigatus. Mutation V83F might make the secondary structure α5 helix protrude outward so that the whole protein structure became unstable at higher temperature, which might subsequently result to the inviability of A. fumigatus under 44 °C. In conclusion, the crystal structure of AfNDK was for the first time analyzed and the stability of the tetrameric form with dimer-dimer interaction were crucial for its function in A. fumigatus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Demethoxyfumitremorgin C, a secondary metabolite of the marine fungus, Aspergillus fumigatus, had been reported to demonstrate cytotoxic effect on mouse tsFT210 cells. However, no information is available regarding its functional mechanism and the chemo-sensitization effects on different kinds of human cancer cells. We found that treatment of demethoxyfumitremorgin C inhibited the cell viability of PC3 human advanced prostate cancer cells, induced apoptosis as determined by Annexin V/propidium iodide double staining, and decreased mitochondrial membrane potential. Demethoxyfumitremorgin C induced apoptosis was associated with downregulation of anti-apoptotic proteins: Ras, PI3K, Akt, Bcl-xL, and Bcl-2, and upregulation of pro-apoptotic Bax. Demethoxyfumitremorgin C activated caspase-3, -8, and -9, leading to PARP cleavage. Additionally, caspase inhibitors blocked demethoxyfumitremorgin C-induced apoptosis of PC3 cells. These results suggest that demethoxyfumitremorgin C from Aspergillus fumigatus inhibits the proliferation of PC3 human prostate cancer cells via the intrinsic (mitochondrial) and extrinsic pathway, followed by downstream events leading to apoptotic cell death. Demethoxyfumitremorgin C could therefore, serve as a useful agent to treat human advanced prostate cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection., Significance: Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chaetominine (CHA) is a quinazolinone alkaloid with strong anti-cancer activity produced by Aspergillus fumigatus CY018. For recovering CHA from A. fumigates efficiently, adsorption and desorption capacities of eight macroporous resins were tested in this work. Based on batch experiments, XAD-16 resin was revealed the best adsorption and desorption performance among all the tested resins. Then, adsorption kinetics and adsorption isotherms were constructed on XAD-16 resin, and the experimental data were fitted well to the pseudo first-order kinetics and Freundlick isotherm model. In the dynamic adsorption and desorption, the purity of CHA increased from 0.0314% (w/w) in the crude extract to 57.86% in the final product with recovery yield of 70.56% by a one-step treatment. Moreover, the experiments were also performed in a lab scale-up scale, in which the purity and recovery of CHA were 56.12% (w/w) and 68.02%, respectively. In addition, XAD-16 resin could be recycled 3 times for CHA separation after regeneration without adverse effects on adsorption/desorption performance. These results suggested that XAD-16 resin adsorption could act as a useful and economic method for recovering CHA from A. fumigatus., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this work, the separation and purification of fumigaclavine C (FC), an ergot alkaloid with strong anti-inflammatory activity from fermented mycelia of Aspergillus fumigatus was systematically evaluated. Among the eight tested resins, the non-polar resin D101 displayed the best adsorption and desorption based on of static adsorption and desorption tests. Adsorption isotherms were constructed on D101 resin and fitted well to the Freundlich model. Dynamic adsorption and desorption tests on a column packed with D101 resin have been investigated for optimization of chromatographic parameters. Under optimized conditions, the contents of FC increased from 7.32% (w/w) in the crude extract to 67.54% in the final product with a recovery yield of 90.35% (w/w) via one run. Furthermore, a lab scale-up separation was carried out, in which the FC content and recovery yield were 65.83% and 90.13%, respectively. These results demonstrated that this adsorption-desorption strategy by using D101 resin was simple and efficient, thus showing potential for large scale purification and preparation of FC in the future., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Indole prenyltransferases AnaPT, CdpC3PT, and CdpNPT are known to catalyze the formation of prenylated pyrroloindoline diketopiperazines from tryptophan-containing cyclic dipeptides in one-step reactions. In this study, we investigated the different stereoselectivities of these enzymes toward all the stereoisomers of cyclo-Trp-Ala and cyclo-Trp-Pro. The stereoselectivities of AnaPT and CdpC3PT mainly depend on the configuration of the tryptophanyl moiety in the substrates, and they usually introduce the prenyl moiety from the opposite sides. CdpNPT showed lower stereoselectivity, and the structure of the second amino acid moiety in the substrates is important for the stereospecificity in its enzyme catalysis. Moreover, we determined the crystal structure of AnaPT in complex with thiolodiphosphate and compared it with the known structures of CdpNPT. Our results clearly revealed the presence of an indole binding mode that has so far not been characterized., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aspergillus fumigatus causes life-threatening infections in immunocompromised patients. We have found that extracts of mycelial mats of A. fumigatus contain a potent hemagglutinin. To clarify the characteristics of this factor, the hemagglutinin was purified from late-stage cultures and characterized at the molecular level. The hemagglutinin is a 32-kilodalton protein that shows activity as an L-fucose lectin. The gene encoding this protein, AfufleA, was identified from a genomic DNA library utilizing consensus primers designed for amino acid sequences obtained from peptides following limited trypsin proteolysis. An open reading frame was found that consists of 942 nucleotides encoding 314 amino acids with a deduced molecular mass of 34,498 and contains all seven of trypsin-digested peptide sequences; four short introns, 49-63 bp, were also identified. AfufleA shares homology with a fucose-specific lectin produced by the orange peel mushroom, Aleuria aurantia. The role of AfufleA fucose-specific lectin is not clear, but this lectin may enhance attachment of fungal spores to mammalian cell membranes and contribute to the pathogenicity of A. fumigatus.

The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus.

Macrophage differentiation and polarization occur in vivo under the influence of the localized cytokine milieu. In vitro studies frequently rely on cellular differentiation in culture; hence, unrecognized variables could have a large influence on the observed cellular phenotype. We measured macrophage in vitro responses to fungal ligands (Aspergillus germ tubes and zymosan), focusing on the degree to which culture conditions impact stimulatory responses through the C-type lectin receptor, dectin-1, which is involved in both MyD88-dependent and MyD88-independent signaling in response to fungal beta1,3 glucan. Results show that macrophages harvested from different murine anatomic sites exhibit varying degrees of MyD88-dependence, with bone marrow-derived macrophages (BMDM) cultured in L929 conditioned medium (L929 CM) exhibiting the largest degree of MyD88-independence. After differentiation in recombinant MCSF (rMCSF), MyD88(-/-) macrophages have decreased surface expression of dectin-1 compared to wild type macrophages; however, culture in L929CM results in higher, and equivalent expression of dectin-1 on both MyD88(-/-) and wild type BMDM. In addition to MCSF, L929CM contains high amounts of VEGF, MCP-1, KC, and MIG, and low amounts of FGF-beta, Eotaxin, IL-10, IL-9, and IL-12. Thus, methods of in vitro maturation dictate variable inflammatory responses by MyD88(-/-) macrophages in association with altered expression of cell surface dectin-1. L929 conditioned medium is a suboptimal alternative to rMCSF for in vitro studies. As MyD88(-/-) BMDM exhibit low surface expression of dectin-1 after in vitro culture in rMCSF, differences in dectin-1 dependent, MyD88-independent signaling may account for some of the phenotypes currently ascribed to MyD88-deficiency alone.

Gliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10min with 30-100ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin-induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia.

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.

Aspergillus fumigatus (AF) is a ubiquitous mold and is the most common cause of invasive aspergillosis, an important source of morbidity and mortality in immunocompromised hosts. Using cytokine flow cytometry, we assessed the magnitude of functional CD4+ and CD8+ T-cell responses following stimulation with Aspergillus antigens. Relative to those seen with cytomegalovirus (CMV) or superantigen stimulation, responses to Aspergillus antigens were near background levels. Subsequently, we confirmed that gliotoxin, the most abundant mycotoxin produced by AF, was able to suppress functional T-cell responses following CMV or staphylococcal enterotoxin B (SEB) stimulation. Additional studies demonstrated that crude AF filtrates and purified gliotoxin inhibited antigen-presenting cell function and induced the preferential death of monocytes, leading to a marked decrease in the monocyte-lymphocyte ratio. Analysis of caspase-3 activation confirmed that gliotoxin preferentially induced apoptosis of monocytes; similar effects were observed in CD83+ monocyte-derived dendritic cells. Importantly, the physiologic effects of gliotoxin in vitro were observed below concentrations recently observed in the serum of patients with invasive aspergillosis. These studies suggest that the production of gliotoxin by AF may constitute an important immunoevasive mechanism that is mediated by direct effects on antigen-presenting cells and both direct and indirect effects on T cells.

beta-Galf-(1-->5)-beta-Galf-(1-->6)-alpha-Manp-(1-->6)-alpha-Manp, the immunodominant epitope in the cell-wall galactomannan of Aspergillus fumigatus, was synthesized for the first time as its allyl glycoside. The key disaccharide glycosyl donor, 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranosyl-(1-->5)-2-O-acetyl-3,6-di-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (10), was constructed by 5-O-glycosylation of 1,2-O-isopropylidene-3,6-di-O-benzoyl-alpha-D-galactofuranose (4) with 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (5), followed by 1,2-O-deacetonation, acetylation, selective 1-O-deacetylation, and trichloroacetimidation. The target tetrasaccharide 16 was obtained by the condensation of allyl 2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside (14) as glycosyl acceptor with the disaccharide glycosyl donor 10, followed by deprotection.

Two oligosaccharides, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp-(1-->4)-alpha-D-GlcpNAc (I) and alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp-(1-->4)-alpha-D-GlcpNAc (II), the glycosylphosphatidylinositol (GPI) anchor glycans from S. cerevesiae and A. fumigatus were synthesized as their methyl glycosides in a regio- and stereoselective manner. The pentasaccharide I was obtained from 6-O-selective glycosylation of methyl 2,3-di-O-benzoyl-alpha-D-mannopyranosyl-(1-->4)-2-acetamido-3,6-di-O-benzoyl-2-deoxy-alpha-D-glucopyranoside (8) with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9), followed by benzoylation, deacetylation, and mannosylation, and then by deprotection. The hexasaccharide (II) was obtained via condensation of allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (17) with 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (16), followed by deallylation, trichloroacetimidation, and coupling with acceptor (8), and finally by deprotection.

A new chroman derivative, named ruakuric acid, was isolated from a strain of Aspergillus fumigatus growing in conjunction with a coral lichen. The structure was determined as 6-acetyl-5-hydroxy-4-methoxy-chroman-2-carboxylic acid (mixture of 2,4-cis,trans isomers).

The use of supercritical fluids for the extraction of biologically active compounds from the biomass of microbial fermentations has been compared with extraction using the organic solvents methanol and dichloromethane. Compounds representing a range of structural types were selected for investigation. All the extracts obtained were examined using reversed-phase high-performance liquid chromatography. The extractability of metabolites using unmodified and methanol-modified supercritical-fluid carbon dioxide was examined in particular detail for six microbial metabolites: chaetoglobosin A, mycolutein, luteoreticulin, 7,8-dihydro-7,8-epoxy-1-hydroxy-3-hydroxymethyl-xanthone-8-carboxyl ic acid methyl ester, sydowinin B and elaiophylin. The extraction strength of supercritical-fluid carbon dioxide alone appeared to be lower than that of dichloromethane. All the components of interest that were extractable with dichloromethane and methanol were also extractable with methanol-modified carbon dioxide.