Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans

Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of Aspergillus nidulans, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins alternariol (8) and cichorine (19).     

Effect of vitamin E on autolysis and sporulation of Aspergillus nidulans

The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulans FGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly, progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulans FGSC26 and a loss-of-function FlbA autolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative effect on the sporulation of strain FGSC26 and the FadA ^G203R hypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross autolysis should be avoided.     

Proteome-Based Profiling of Hypercellulase-Producing Strains Developed Through Interspecific Protoplast Fusion Between Aspergillus nidulans and Aspergillus tubingensis

Thirty heterokaryons, formed by protoplast fusion of Aspergillus nidulans and Aspergillus tubingensis, were selected on the basis of their ability to grow on 2-deoxyglucose (0.2 %, w/v) and intermediate spore color. These heterokaryons were studied for cellulase production using shake flask and solid substrate cultures at 40 °C. Fusants 51 and 28 exhibited appreciably higher levels of endoglucanase, cellobiohydrolase, β-glucosidase, and FPase activities when compared with parental strains. Employing proteomic-based approaches, the differential expression of proteins in secretome of fusants and parental strains were analyzed using two-dimensional electrophoresis. The expression of some of the proteins in the fusants was found to be up/downregulated. The upregulated proteins in the fusant 51 were identified by liquid chromatography–mass spectroscopy as endoxylanase, endochitinase, β-glucosidase, as well as hypothetical proteins. The cellulases produced by fusants 28 and 51 showed improved saccharification of alkali treated rice straw when compared with the parental strains.     

Immobilization of Aspergillus nidulans SU04 cellulase on modified activated carbon

The present study deals with the immobilization of Aspergillus nidulans SU04 cellulase onto modified activated carbon (MAC). The effect of contact time, cellulase concentration, MAC dosage, and temperature for maximum immobilization percentage and immobilization capacity is investigated. The equilibrium nature of immobilization is described by Langmuir and Freundlich isotherms. The kinetic data were tested using the pseudo first order. The activation energy of immobilization was evaluated to be 11.78?J?mol^?1. Results of the thermodynamic investigation indicate the spontaneity (?G <0), slightly endothermic (?H >0), and irreversible (?S >0) nature of the sorption process. Entropy and enthalpy were found to be 41.32 J?mol^?1?mg^?1 and 10.99?kJ?mol^?1, respectively. The Gibbs free energy was found to be ?22.79?kJ?mol^?1. At 80?rpm, 323?K, 2?h, 5?mg of MAC, immobilization capacity was 4.935?mg cellulase per mg of MAC from an initial cellulase concentration of 16?mg?ml^?1 with retention of 70% of native cellulase activity up to 10 cycles of batch hydrolysis experiments. The diffusion studies that were carried out revealed the reaction rate as ??mol?min^?1. At optimized conditions, immobilized cellulase had a higher Michaelis?CMenten constant, K _m of 1.52?mmol and a lower reaction rate, V _max of 42.2???mol?min^?1, compared with the free cellulase, the K _m and V _max values of which were 0.52?mmol and 18.9???mol?min^?1, respectively, indicating the affinity of cellulase for MAC matrix.     

Reengineering an azaphilone biosynthesis pathway in Aspergillus nidulans to create lipoxygenase inhibitors

Sclerotiorin, an azaphilone polyketide, is a bioactive natural product known to inhibit 15-lipoxygenase and many other biological targets. To readily access sclerotiorin and analogs, we developed a 2-3 step semisynthetic route to produce a variety of azaphilones starting from an advanced, putative azaphilone intermediate (5) overproduced by an engineered strain of Aspergillus nidulans. The inhibitory activities of the semisynthetic azaphilones against 15-lipoxygenase were evaluated with several compounds displaying low micromolar potency.     

Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.     

Evaluation of Strategies to Improve the Production of Alkaline Protease PrtA from Aspergillus nidulans

Aspergillus nidulans produces several proteases. The prtA gene encodes a major protease, and two approaches were explored to achieve the overproduction of this enzyme. Molecular cloning of the mature form of this enzyme in Pichia pastoris resulted in the production of an inactive form. In addition, the presence of this enzyme was toxic for the host and resulted in cell lysis. The modification of the culture medium constituents resulted in a 6.4-fold increase in enzyme production. The main effect was achieved through the use of organic nitrogen sources. Although it was previously shown that the PrtA protease shows promiscuous esterase activity, the production of this enzyme was not induced by lipidic sources.     

Engineering of an "unnatural" natural product by swapping polyketide synthase domains in Aspergillus nidulans

An StcA-AfoE hybrid polyketide synthase (PKS), generated by swapping the AfoE (asperfuranone biosynthesis) SAT domain with the StcA (sterigmatocystin biosynthesis) SAT domian, produced a major new metabolite with the same chain length as the native AfoE product. Structure elucidation allowed us to propose a likely pathway, and feeding studies supported the hypothesis that the chain length of PKS metabolites may be under precise control of KS and PT domains.     

Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans

BACKGROUND: Based on the homology with fatty acid synthases and bacterial polyketide synthases (PKSs), thioesterase domains have been assigned at the C-terminus regions of fungal iterative type I PKSs. We previously overexpressed Aspergillus nidulans wA PKS gene in a heterologous fungal host and identified it to encode a heptaketide naphthopyrone synthase. In addition, expression of C-terminus-modified WA PKS gave heptaketide isocoumarins suggesting that the C-terminus region of WA PKS is involved in the cyclization of the second aromatic ring of naphthopyrone. To unravel the actual function of the C-terminus region, we carried out functional analysis of WA PKS mutants by C-terminus deletion and site-directed mutagenesis. RESULTS: Only the 32 amino acid deletion from the C-terminus of WA PKS caused product change to heptaketide isocoumarins from heptaketide naphthopyrone, YWA1 1, a product of intact WA PKS. Further C-terminus deletion mutant of WA PKS up to Ser(1967), an active site residue of so far called thioesterase, still produced isocoumarins. Site-directed mutagenesis of amino acid residues in this C-terminus region showed that even a single mutation of S1967A or H2129Q caused production of isocoumarin instead of naphthopyrone. Furthermore, the role of tandem acyl carrier proteins (ACPs), a typical feature of fungal aromatic PKSs, was examined by site-directed mutagenesis and the results indicated that both ACPs can function as ACP independently. CONCLUSIONS: Claisen-type cyclization is assumed to be involved in formation of aromatic compounds by some fungal type I PKSs. These PKSs have a quite identical architecture of active site domain organization, beta-ketoacyl synthase, acyltransferase, tandem ACPs and thioesterase (TE) domains. Since the C-terminus region of WA PKS of this type was determined to be involved in Claisen-type cyclization of the second ring of naphthopyrone, we propose that the so far called TE of these PKSs work not just as TE but as Claisen cyclase.     

Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans

Meroterpenoids are a class of fungal natural products that are produced from polyketide and terpenoid precursors. An understanding of meroterpenoid biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has previously been found to produce two meroterpenoids, austinol and dehydroaustinol. Using targeted deletions that we created, we have determined that, surprisingly, two separate gene clusters are required for meroterpenoid biosynthesis. One is a cluster of four genes including a polyketide synthase gene, ausA. The second is a cluster of 10 additional genes including a prenyltransferase gene, ausN, located on a separate chromosome. Chemical analysis of mutant extracts enabled us to isolate 3,5-dimethylorsellinic acid and 10 additional meroterpenoids that are either intermediates or shunt products from the biosynthetic pathway. Six of them were identified as novel meroterpenoids in this study. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans meroterpenoids.     

Regulation of Nonribosomal Peptide Synthesis: bis-Thiomethylation Attenuates Gliotoxin Biosynthesis in Aspergillus fumigatus

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Discovery of aspoquinolones A-D, prenylated quinoline-2-one alkaloids from Aspergillus nidulans, motivated by genome mining

Motivated by the observation that the Aspergillus nidulans genome bears multiple anthranilic acid synthase gene copies, the fungal metabolome was reinvestigated under various fermentation conditions, resulting in the discovery of novel prenylated quinolin-2-one alkaloids, two of which bear unprecedented terpenoid side chains.     

Production and Characterization Te-Peptide by Induced Autolysis of Saccharomyces Cerevisiae

Recently, the interest in mimicking functions of chalcogen-based catalytic antioxidants like selenoenzymes, has been increased. Various attempts had been done with selenium, but very few attempts were carried out with tellurium. Bio-complex formation and characterization of tellurium was not tried earlier by using any organism. The present study was focused on tellurium peptide production, characterization, and bioactivity assessment especially Mimetic to glutathione peroxidase (GPx). The production was achieved by the autolysis of total proteins obtained from Saccharomyces cerevisiae ATCC 7752 grown with inorganic tellurium. The GPx-like activity of the hydrolyzed tellurium peptide was increased when prepared by autolysis, but decreased when prepared by acid hydrolysis. Tellurium peptide produced by autolysis of the yeast cell showed increased GPx-like activity as well as tellurium content. Tellurium peptide showed little toxicity, compared to highly toxic inorganic tellurium. The results showed the potential of tellurium peptide as an antioxidant that can be produced by simple autolysis of yeast cells.     

Metabolites of microorganisms. 143. Echinocandin B, a novel polypeptide-antibiotic from Aspergillus nidulans var. echinulatus: isolation and structural components

From a strain of Aspergillus nidulans var. echinulatus, A 32204, echinocandin, a novel polypeptide antibiotic complex with high and specific anti-yeast activity was isolated. The main component, echinocandin B, was separated by counter-current distribution. A tentative molecular formula C₅₂H₈₁N₇O₁₈ is discussed. The antibiotic is characterized by its IR. and NMR. spectra and by its characteristic hydrolytic degradation products: linoilic acid, L -4-hydroxyproline, L -4-oxoproline, (2S, 3S, 4S)-4-methyl-3-hydroxy-proline, L -threonine (2 mols), and 1-(p-hydroxyphenyl)-3-amino-propan-2-one.     

Antifungal activity of Bignoniaceae plants on Aspergillus carbonarius and Aspergillus niger

Abstract

Twenty four extracts from Bignoniaceae plants of northwest Argentina were tested for antifungal activity against Aspergillus species responsible of the grape black rot. Stems and leaves of Amphilophium cynanchoides, Macfadyena cynanchoides, Tecoma stans and Jacaranda mimosifolia were separately extracted with solvents of increasing polarity to obtain the dichloromethane (fCH₂Cl₂), ethyl acetate (fEtOAc) and methanol extracts (fMeOH). The fCH₂Cl₂ from stem of M. cynanchoides had the lowest IC₅₀ (1.0–1.2?mg/mL) and MID values (0.6–1.2?mg) and the highest ID values (5.0–6.8?mm) on A. niger and A. carbonarius. The main contributors of the antifungal activity of fCH₂Cl₂ were identified as lapachol (MIC?=?0.25–1.00?mg/ml) and 1-hydroxy-4-methylanthraquinone (MIC?=?0.0625–0.125?mg/mL). These compounds synergized the antifungal activity of sodium metabisulfite and showed an additive effect in mixtures with propiconazol. They might be used as additives of commercial antifungals to protect grapes against A. niger and A. carbonarius.     

Comparison of the Effects of Aspergillus niger and Aspergillus ficuum on the Removal of Impurities in Feldspar by Bio-beneficiation

Applied Biochemistry and Biotechnology - Depending on its purity, feldspar has a wide application in industry. Fungi play an important role in the feldspar purification. A bacterial leaching study...     

Butyrolactones from Aspergillus terreus

In the process development of lovastatin using Aspergillus terreus DRCC 152 in solid state fermentation, we have isolated a new butyrolactone-IV (3) along with the previously reported butyrolactone-I (1) and butyrolactone-II (2) produced under submerged conditions. The structure of compound 3 has been characterized as 3-hydroxy-5-[2-(1-hydroxy-1-methylethyl)-2(R)-2,3-dihydro-benzo[b]furan- 5 ylmethyl]-4-(4-hydroxyphenyl)-5-methoxycarbonyl-(5R)-2,5-dihydro-2 -furanone on the basis of spectroscopic studies. The absolute stereochemistry has been determined by single crystal X-ray diffraction studies. The cytotoxic and antibacterial activities of these compounds were determined.