http://repositorium.sdum.uminho.pt/bitstream/1822/7896/1/Paterson_Cordyceps%5b1%5d.pdf

Cordyceps – A traditional Chinese medicine and another fungal therapeutic biofactory?

The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi.

Dereplication, the rapid identification of known compounds present in a mixture, is crucial to the fast discovery of novel natural products. Determining the elemental composition of compounds in mixtures and tentatively identifying natural products using MS/MS and UV/vis spectra is becoming easier with advances in analytical equipment and better compound databases. Here we demonstrate the use of LC-UV/vis-MS-based dereplication using data from UV/vis diode array detection and ESI+/ESI- time-of-flight MS for assignment of 719 microbial natural product and mycotoxin reference standards. ESI+ was the most versatile ionization method, detecting 93% of the compounds, although with 12% ionizing poorly. Using ESI+ alone, 56.1% of the compounds could be unambiguously assigned based on characteristic patterns of multiple adduct ions. Using ESI-, 36.4% of the compounds could have their molecular mass assigned unambiguously using multiple adduct ions, while a further 41% of the compounds were detected only as [M - H]-. The most reliable interpretations of conflicting ESI+ and ESI- data on a chromatographic peak were from the ionization polarity with the most intense ionization. Poor ionization was most common with small molecules (<200 Da). In ESI-, these were often polar and basic, while in ESI+ they were small aromatic acids or anthraquinones. No single ion-source settings could be applied over a m/z 60-2000 range. However, continuous switching among three settings (e.g., for 0.5 s each) during the chromatographic run allowed MS of both small labile molecules and large peptides, and pseudo MS/MS data on labile molecules since the settings for large molecules often induce fragmentation into small molecules.

Dereplication of microbial natural products by LC-DAD-TOFMS.

The genus Metarhizium historically refers to green-spored asexual insect pathogenic fungi. Through culturing and molecular methods, Metarhizium has been linked to Metacordyceps sexual states. Histo...

Clarification of generic and species boundaries for Metarhizium and related fungi through multigene phylogenetics

Gene clusters for biosynthesis of the fungal polyketides hypothemycin and radicicol from Hypomyces subiculosus and Pochonia chlamydosporia, respectively, were sequenced. Both clusters encode a reducing polyketide synthase (PKS) and a nonreducing PKS like those in the zearalenone cluster of Gibberella zeae, plus enzymes with putative post-PKS functions. Introduction of an O-methyltransferase (OMT) knockout construct into H. subiculosus resulted in a strain with increased production of 4-O-desmethylhypothemycin, but because transformation of H. subiculosus was very difficult, we opted to characterize hypothemycin biosynthesis using heterologous gene expression. In vitro, the OMT could methylate various substrates lacking a 4-O-methyl group, and the flavin-dependent monooxygenase (FMO) could epoxidate substrates with a 1',2' double bond. The glutathione S-transferase catalyzed cis-trans isomerization of the 7',8' double bond of hypothemycin. Expression of both hypothemycin PKS genes (but neither gene alone) in yeast resulted in production of trans-7',8'-dehydrozearalenol (DHZ). Adding expression of OMT, expression of FMO, and expression of cytochrome P450 to the strain resulted in methylation, 1',2'-epoxidation, and hydroxylation of DHZ, respectively. The radicicol gene cluster encodes halogenase and cytochrome P450 homologues that are presumed to catalyze chlorination and epoxidation, respectively. Schemes for biosynthesis of hypothemycin and radicicol are proposed. The PKSs encoded by the two clusters described above and those encoded by the zearalenone cluster all synthesize different products, yet they have significant sequence identity. These PKSs may provide a useful system for probing the mechanisms of fungal PKS programming.

Genes for the Biosynthesis of the Fungal Polyketides Hypothemycin from Hypomyces subiculosus and Radicicol from Pochonia chlamydosporia

Pochonins are antiviral and antiparasitic resorcylic acid lactones (RAL) structurally related to monorden. They were found in the invertebrate-associated fungus Pochonia chlamydosporia. Their production and distribution was studied by means of High Performance Liquid Chromatography with UV-visual and mass spectrometric detection (HPLC-UV/Vis and HPLCMS) in cultures of Pochonia species and further conidial fungi with Verticillium-like anamorphs that had until recently been included in Verticillium sect. Prostrata. The results support the recent generic segregation by Gams, Zare and co-workers because pochonins were found to occur exclusively in species of the genus Pochonia. With few exceptions, the production of RAL appeared to be a rather constant feature in cultures of P. chlamydosporia from around the world. According to preliminary results, secondary metabolite profiles in strains of allied genera such as Lecanicillium, Haptocillium and Rotiferophthora are different from those encountered in Pochonia. The alkaloid pseurotin A was found as main metabolite in several of the P. chlamydosporia isolates examined. As inferred from HPLC profiling data, strains of P. suchlasporia clustered into at least three chemotypes. The ex-type strain of P. suchlasporia var. catenata produced monorden, while several other strains produced metabolites whose HPLC-UV and HPLC-MS characteristics were similar to the mycotoxins, aurovertin B and citreoviridin A. Yet different metabolites were detected in a third chemotype of P. suchlasporia. Differences in secondary metabolite profiles were also found in two strains of P. bulbillosa. While the ex-type strain was found devoid of all aforementioned compounds, CBS 247.68 contained the aurovertin-related metabolites detected in part of the P. suchlasporia isolates. The sequence of the ITS nrDNA of CBS 247.68 was different from that of the type strain but identical to the sequences of P. suchlasporia var. catenata. Several strains of the latter variety showed identical sequences, despite considerable variations in their HPLC metabolite profiles. Minisatellite PCR fingerprinting was found useful to segregate Pochonia at species and strain level, pointing toward the existence of further, cryptic species. The possible chemotaxonomical importance and ecological functions of secondary metabolites in these fungi is discussed.

Eleni Katsiou

Papers

Chemotaxonomy of Pochonia and other conidial fungi with Verticillium -like anamorphs