The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.

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Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus. However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA, a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA-encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways.Different classes of ergot alkaloids are produced by members of two distinct fungal lineages. Clavicipitaceous species, which include Claviceps spp. and Neotyphodium spp., are in the order Hypocreales and typically synthesize lysergic acid derivatives (13,16,18). These alkaloids have a double bond in the last assembled of four rings (D ring) of the tetracyclic ergoline ring structure. Ergot alkaloids are also produced by the distantly related opportunistic human pathogen Aspergillus fumigatus, a member of the order Eurotiales (8,14,16,18). Ergot alkaloids of A. fumigatus are of the clavine class and differ from the more complex profile of Claviceps purpurea and Neotyphodium spp. One important distinction between the ergot alkaloids produced by these different fungi is the saturation of the fourth ring of the ergoline structure in A. fumigatus (Fig. 1).Several genes involved in the ergot alkaloid pathways of A. fumigatus and clavicipitaceous fungi are found clustered together in the genome of each species (3,4,6,18,23). These distantly related fungi are hypothesized to share several early pathway steps, after which the pathways diverge to yield distinct sets of ergot alkaloids (3, 13, 16). The gene dmaW, which encodes dimethylallyltryptophan (DMAT) synthase, catalyzes the...

Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms could have potentially flourished in these settings. If so, biomolecules found in microorganisms adapted to high salinity and basic pH environments on Earth may be reliable biomarkers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman (RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore carotenoid molecules in the cell membrane with respect to the various protein and lipid cellular components. RR spectra acquired from all halophilic archaea investigated contained major features at approximately 1000, 1152, and 1505 cm ؊1 . The bands at 1505 cm ؊1 and 1152 cm ؊1 are due to in-phase C¨C ( 1 ) and C-C stretching ( 2 ) vibrations of the polyene chain in carotenoids. Additionally, in-plane rocking modes of CH 3 groups attached to the polyene chain coupled with C-C bonds occur in the 1000 cm ؊1 region. We also investigated the RR spectral differences between bacterioruberin and bacteriorhodopsin as another potential biomarker for hypersaline environments. By comparison, the RR spectrum acquired from bacteriorhodopsin is much more complex and contains modes that can be divided into four groups: the C¨C stretches (1600-1500 cm ؊1 ), the CCH in-plane rocks (1400-1250 cm ؊1 ), the C-C stretches (1250-1100 cm ؊1 ), and the hydrogen out-of-plane wags (1000-700 cm ؊1 ). RR spectroscopy was shown to be a useful tool for the analysis and remote in situ detection of carotenoids from halophilic archaea without the need for large sample sizes and complicated extractions, which are required by analytical techniques such as high performance liquid chromatography and mass spectrometry.

The ergot alkaloids are a diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities. The biosynthetic intermediate chanoclavine-I aldehyde 1 represents a branch point in ergot biosynthesis. Ergot alkaloids festuclavine 2 and agroclavine 3 derive from alternate enzymatic pathways originating from the common biosynthetic precursor chanoclavine-I aldehyde 1. Here we show that while the Old Yellow Enzyme homolog EasA from the ergot biosynthetic gene cluster of Aspergillus fumigatus acts on chanoclavine-I aldehyde 1 to yield festuclavine 2, EasA from Neotyphodium lolii, in contrast, produces agroclavine 3. Mutational analysis suggests a mechanistic rationale for the switch in activity that controls this critical branch point of ergot alkaloid biosynthesis.The ergot alkaloids are a diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities.1 The biosynthetic intermediate chanoclavine-I aldehyde 1 can be enzymatically converted into festuclavine 2, which is further derivatized to form alkaloids such as the fumigaclavines in Aspergillus fumigatus (Scheme 1).1 Alternatively, in certain fungal species such as Claviceps purpurea and Neotyphodium lolii, chanoclavine-I aldehyde 1 is converted to agroclavine 3, which goes on to form lysergic acid-derived ergot alkaloids such as ergopeptines.1 Festuclavine 2 and agroclavine 3 differ only by the degree of unsaturation in the D ring (Scheme 1). Although chanoclavine-I aldehyde 1 had been proposed as an ergot alkaloid biosynthetic intermediate many years ago,2 the enzyme-catalyzed mechanism of D ring formation has remained elusive. Recently, we and others have reported that a homolog of Old Yellow Enzyme from A. fumigatus, EasA_Af, reduces the alkene of the chanoclavine-I aldehyde α, β unsaturated carbonyl moiety (C8-C9, Scheme 1A).3 This reduction facilitates an intramolecular reaction between the aldehyde and the amine moieties to allow formation of the D ring of festuclavine 2 (Scheme 1A). Yet it is still unclear how chanoclavine-I aldehyde 1 is converted into agroclavine 3, which retains the C8-C9 double bond of the starting precursor, but with this alkene in the opposite geometrical configuration. Formation of agroclavine 3 from Dan.Panaccione@mail.wvu.edu, soc@mit.edu. Supporting Information Available: Experimental procedures, sequence alignments and additional experimental data. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript chanoclavine-I aldehyde 1 was investigated previously by Floss and colleagues in whole-cell precursor feeding studies.2 , 4 Results of these studies clearly demonstrated precursorproduct relationships but also raised unanswered questions about the enzymes and mechanisms involved. One particularly puzzling step implicated a cis-trans isomerization involving intermolecular transfer of a hydrogen between successive substrate molecu...