Chemotypic and genotypic diversity in the ergot alkaloid pathway of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogen that synthesizes a group of mycotoxins via a branch of the ergot alkaloid pathway. This fungus is globally distributed, and genetic data indicate that isolates recombine freely over that range; however, previous work on ergot alkaloids has focused on a limited number of isolates. We hypothesized that A. fumigatus harbors variation in the chemotype of ergot alkaloids and genotype of the ergot alkaloid gene cluster. Analysis of 13 isolates by high performance liquid chromatography revealed four distinct ergot alkaloid profiles or chemotypes. Five isolates completed the A. fumigatus branch of the ergot alkaloid pathway to fumigaclavine C. Six independent isolates accumulated fumigaclavine A, the pathway intermediate immediately before fumigaclavine C. One isolate accumulated only the early pathway intermediates chanoclavine-i and chanocla-vine-i aldehyde, and one isolate lacked ergot alkaloids altogether. A genetic basis for each of the observed chemotypes was obtained either by PCR analysis of the ergot alkaloid gene cluster or through sequencing of easL, the gene encoding the prenyl transferase that reverse prenylates fumigaclavine A to fumigaclavine C. Isolates also exhibited differences in pigmentation and sporulation. The ergot alkaloid chemotypes were widely distributed geographically and among substrate of origin.     

Effect of phosphate on ergot alkaloid synthesis inAspergillus fumigatus

High concentration of inorganic phosphate in the culture medium ofAspergillus fumigatus inhibited ergot alkaloid synthesis. Addition ofl-tryptophan but not mevalonate or 5-methyltryptophan to the above culture restored the alkaloid synthesis to the level found in normal cultures. The decrease in alkaloid synthesis in the fungus accompanies an increase in cell mass, cellular protein and sterol content. Aspartate aminotransferase and alanine aminotransferase activities were significantly increased in the high-phosphate culture. Part of the work was presented at the seminar on “Enzymatic Methods in Mycology” organised by the Czechoslovak Microbiological Society in Brno, Czechoslovakia, in June 1975.     

A model study of ergot alkaloid biosynthesis

A hypothesis of the mechanistic mode of prenylation of tryptophan, an early phase of ergot alkaloid biosynthesis, occurring by way of 3-(α,α-dimethylally)indolenine and its ring-tautomer indoline intermediates and a Cope rearrangement of the latter has been tested in a model study. 3-(α,α-Dimethylallyl)indolines derived from tetrahydrocarbazole and its cyclopentano equivalent have been synthesized by a five-step procedure. The compounds are stable up to ca. 200°C and liberate no benz-prenylated products even on pyrolysis beyond this temperature, thus diminishing the viability of the above biosynthetic hypothesis. The chemistry of the thermolyses is described.     

Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.     

Association of ergot alkaloids with conidiation in Aspergillus fumigatus

Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festucla-vine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (brlA), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation.     

Ergosterol biosynthesis pathway in Aspergillus 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.     

Production of ergot alkaloids by Aspergillus fumigatus (Fresenius)

Summary Nutritional requirements for the production of ergot alkaloids were studied with Aspergillus fumigatus under submerged conditions of fermentation, in a chemically defined medium. Glucose in combination with mannitol and triammonium citrate were found to be the best sources of carbon and nitrogen for the production of alkaloids. Carbon to nitrogen ratio of 4.16 : 1 was found optimum. Phosphate at elevated concentration inhibited alkaloid production.     

Bidirectional gene transfer between Aspergillus fumigatus and Aspergillus nidulans

Abstract The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes β-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased β-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli .     

In vitro biosynthesis of glycosylphosphatidylinositol in Aspergillus fumigatus

Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells. GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells. Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall. An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism. In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI. Using GDP-Man, two types of mannosylation are observed. First, one or two mannose residues are added to GlcN-PI. This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor. Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor. The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed. This latter series of GPI intermediates identified in the A. fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems. Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A. fumigatus.     

Correlation between growth and ergot alkaloid biosynthesis in Claviceps purpurea batch fermentation

Summary Following the growth kinetics of a C. purpurea ergotoxine-producing strain it was observed that alkaloid synthesis and alkaloid distribution depend on fungal growth velocity during idiophase. Formation of extracellular alkaloids is favoured by higher fungal growth velocity, while intracellular alkaloids begin to accumulate at a lower rate of growth. Simple lysergic acid derivatives prevali among extracellular alkaloids, whereas ergotoxines predominate among intracellular alkaloids. By varying cultivation conditions, by feeding the culture, or by varying the inoculum size, alkaloid composition can be influenced within the limits of strain capabilities.     

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.     

Factors affecting the fermentation of ergot alkaloids by Aspergillus fumigatus (Fresenius)

Summary Six strains of Aspergillus fumigatus were studied for their alkaloid producing capacity. With one strain (NCIM 902) high yields (162mg/l) of ergot alkaloids were obtained after seven days in a new medium.     

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway. Received: 26 August 1998 / Accepted: 19 October 1998     

The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).     

Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.     

The biosynthesis of 4-hydroxycoumarin and dicoumarol by Aspergillus fumigatus Fresenius

A strain of Aspergillus fumigatus Fresenius, isolated from spoiled hay, converts melilotic acid (o-hydroxyphenylpropionic acid) and o-coumaric acid into 4-hydroxycoumarin and dicoumarol. The sequence is shown to be melilotic acid (I) [Formula: see text] coumaric acid (IV) [Formula: see text] beta-hydroxymelilotic acid (II) [Formula: see text] beta-oxomelilotic acid (III) [Formula: see text] 4-hydroxycoumarin (VI), on the basis of (1) studies on the formation of postulated intermediates, (2) experiments with isotopically labelled materials and (3) sequential enzyme induction. In the presence of semicarbazide, o-coumaraldehyde is formed from o-coumaric acid: there is no evidence, however, that this lies on the normal metabolic pathway.     

Carbon and nitrogen source nutrition of fumagillin biosynthesis by Aspergillus fumigatus

The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L(-1) xylan and 50 g L(-1) mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L(-1) was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings.