A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins.

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases.

Numerous polyketides are known from bacteria, plants, and fungi. However, only a few have been isolated from basidiomycetes. Large scale genome sequencing projects now help anticipate the capacity of basidiomycetes to synthesize polyketides. In this study, we identified and annotated 111 type I and three type III polyketide synthase (PKS) genes from 35 sequenced basidiomycete genomes. Phylogenetic analysis of PKS genes suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged. A comparison of genomic and metabolomic data shows that the number of polyketide genes exceeds the number of known polyketide structures by far. Exploiting these results to design degenerate PCR primers, we amplified and cloned the complete sequence of armB, a PKS gene from the melleolide producer Armillaria mellea. We expect this study will serve as a guide for future genomic mining projects to discover structurally diverse mushroom-derived polyketides.

Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria.

The armA-gene, encoding a tridomain enzyme reminiscent of nonribosomal peptide synthetases, was identified in the genome of the basidiomycete Armillaria mellea. Heterologously expressed enzyme and the ATP-pyrophosphate exchange assay were used for the in vitro biochemical characterisation of the ArmA adenylation domain. l-leucine was the preferred substrate, while l-threonine, l-valine, l-alanine, and l-isoleucine were turned over at lower rates (83 %, 62 %, 56 %, and 44 %, respectively). Other proteinogenic amino acids, 2-oxo acids, and benzoic acid derivatives were not accepted. As the substrate range of ArmA is incompatible with the secondary metabolites known from the genus Armillaria, our results imply greater natural product diversity in this genus. This is the first biochemical characterisation of a basidiomycete amino acid-adenylating domain, and our results may help refine computer algorithms to predict substrate specificities for basidiomycete nonribosomal peptide synthetases whose genes are discovered through genome sequencing.

Structure and cytotoxicity of arnamial and related fungal sesquiterpene aryl esters.

We report on the structure elucidation of arnamial, a new Delta(2,4)-protoilludane everninate ester from the fungus Armillaria mellea, and on the apoptotic activity of arnamial as well as the cytotoxic activity of structurally related compounds on selected human cancer cells. Arnamial showed cytotoxicity against Jurkat T cells, MCF-7 breast adenocarcinoma, CCRF-CEM lymphoblastic leukemia, and HCT-116 colorectal carcinoma cells at IC50 = 3.9, 15.4, 8.9, and 10.7 microM, respectively, and the related aryl ester melledonal C showed cytotoxic activity against CCRF-CEM cells (IC50 = 14.75 microM). [1,2-13C2]Acetate feeding supports a polyketide origin of the orsellinic acid moiety of arnamial.

In vivo and in vitro production options for fungal secondary metabolites.

Microbial natural products, among them a vast diversity of fungal origin, represent a major source for new drug candidates. Focusing on fungal metabolites, our review covers recent advances in the field of biotransformation, heterologous expression, in vivo production approaches, genomics, and the metabolism of unexplored fungal groups as options to generate and identify new compounds or optimize known ones.

Processing sites involved in intron splicing of Armillaria natural product genes.

We analysed the structure of four genes whose transcriptional products are likely to be involved in the small molecule metabolism of the homobasidiomycete Armillaria mellea with the aim of verifying splice sites. To this end we experimentally validated in silico predicted intron/exon junctions for accuracy. Based on 78 verified junctions, a consensus for donor and acceptor sites in Armillaria is presented, along with experimental evidence for non-canonical splice sites, introns with alternative donor or acceptor junctions, and allele-selective splicing. The investigated reading frames show significant homologies to: (1) antibiotic and other small molecule efflux transporter genes; (2) phenoloxidase/laccase genes; (3) genes for dual Cys2His2/Zn(II)2Cys6 transcriptional regulators. For all of these gene categories, this is the first report on examples from the genus Armillaria.

Fungal genetics, genomics, and secondary metabolites in pharmaceutical sciences.

Filamentous fungi produce a plethora of bioactive natural products. These metabolites display a broad range of useful activities for pharmaceutical purposes, exemplified best by the antibiotic penicillin. Yet, many more have been isolated, characterised, and tested, and some have made their way in clinical trials and into pharmaceutical practice. Through genomics, we become increasingly aware that the biosynthetic abilities for natural products are much richer than expected. The first part of our review highlights selected metabolites that filamentous fungi offer to pharmacists for drug development. This is followed by a summary on the potential of fungal genetics and genomics for pharmaceutical sciences and natural product research.