Fungal Secondary Metabolite Gene Clusters Research Articles

Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.

Microbial communication leading to the activation of silent fungal secondary metabolite gene clusters.

Microorganisms form diverse multispecies communities in various ecosystems. The high abundance of fungal and bacterial species in these consortia results in specific communication between the microorganisms. A key role in this communication is played by secondary metabolites (SMs), which are also called natural products. Recently, it was shown that interspecies “talk” between microorganisms represents a physiological trigger to activate silent gene clusters leading to the formation of novel SMs by the involved species. This review focuses on mixed microbial cultivation, mainly between bacteria and fungi, with a special emphasis on the induced formation of fungal SMs in co-cultures. In addition, the role of chromatin remodeling in the induction is examined, and methodical perspectives for the analysis of natural products are presented. As an example for an intermicrobial interaction elucidated at the molecular level, we discuss the specific interaction between the filamentous fungi Aspergillus nidulans and Aspergillus fumigatus with the soil bacterium Streptomyces rapamycinicus, which provides an excellent model system to enlighten molecular concepts behind regulatory mechanisms and will pave the way to a novel avenue of drug discovery through targeted activation of silent SM gene clusters through co-cultivations of microorganisms.

Prototype of an intertwined secondary-metabolite supercluster

The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.

VeA and MvlA repression of the cryptic orsellinic acid gene cluster in Aspergillus nidulans involves histone 3 acetylation

A perplexing aspect of fungal secondary metabolite gene clusters is that most clusters remain 'silent' under common laboratory growth conditions where activation is obtained through gene manipulation or encounters with environmental signals. Few proteins have been found involved in repression of silent clusters. Through multicopy suppressor mutagenesis, we have identified a novel cluster suppressor in Aspergillus nidulans, MvlA (modulator of veA loss). Genetic assessment of MvlA mutants revealed the role of both itself and VeA (but not the VeA partner LaeA) in the suppression of the cryptic ors gene cluster producing orsellinic acid and its F9775 derivatives. Loss of veA upregulates F9775A and F9775B production and this increase is reduced 4-5-fold when an overexpression mvlA (OE:mvlA) allele is introduced into the ΔveA background. Previous studies have implicated a positive role for GcnE (H3K9 acetyltransferase of the SAGA/ADA complex) in ors cluster expression and here we find expression of gcnE is upregulated in ΔveA and suppressed by OE:mvlA in the ΔveA background. H3K9 acetylation levels of ors cluster genes correlated with gcnE expression and F9775 production in ΔveA and OE:mvlAΔveA strains. Finally, deletion of gcnE in the ΔveA background abolishes ors cluster activation and F9775 production. Together, this work supports a role for VeA and MvlA in modifying SAGA/ADA complex activity.

The chromatin code of fungal secondary metabolite gene clusters.

Secondary metabolite biosynthesis genes in fungi are usually physically linked and organized in large gene clusters. The physical linkage of genes involved in the same biosynthetic pathway minimizes the amount of regulatory steps necessary to regulate the biosynthetic machinery and thereby contributes to physiological economization. Regulation by chromatin accessibility is a proficient molecular mechanism to synchronize transcriptional activity of large genomic regions. Chromatin regulation largely depends on DNA and histone modifications and the histone code hypothesis proposes that a certain combination of modifications, such as acetylation, methylation or phosphorylation, is needed to perform a specific task. A number of reports from several laboratories recently demonstrated that fungal secondary metabolite (SM) biosynthesis clusters are controlled by chromatin-based mechanisms and histone acetyltransferases, deacetylases, methyltransferases, and proteins involved in heterochromatin formation were found to be involved. This led to the proposal that establishment of repressive chromatin domains over fungal SM clusters under primary metabolic conditions is a conserved mechanism that prevents SM production during the active growth phase. Consequently, transcriptional activation of SM clusters requires reprogramming of the chromatin landscape and replacement of repressive histone marks by activating marks. This review summarizes recent advances in our understanding of chromatin-based SM cluster regulation and highlights some of the open questions that remain to be answered before we can draw a more comprehensive picture.

Chromatin-level regulation of biosynthetic gene clusters

Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone H3 lysine 4 methylation, activated the expression of cryptic secondary metabolite clusters in A. nidulans. One new cluster generated monodictyphenone, emodin and emodin derivatives, whereas a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal secondary metabolite clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.

The GC and window-averaged DNA curvature profile of secondary metabolite gene cluster in Aspergillus fumigatus genome

An immense variety of complex secondary metabolites is produced by filamentous fungi including Aspergillus fumigatus, a main inducer of invasive aspergillosis. The identification of fungal secondary metabolite gene cluster is essential for the characterization of fungal secondary metabolism in terms of genetics and biochemistry through recombinant technologies such as gene disruption and cloning. Most of the prediction methods for secondary metabolite gene cluster severely depend on homology searches. However, homology-based approach has intrinsic limitation to unknown or novel gene cluster. We analyzed the GC and window-averaged DNA curvature profile of 26 secondary metabolite gene clusters in the A. fumigatus genome to find out potential conserved features of secondary metabolite gene cluster. Fifteen secondary metabolite gene clusters showed a conserved pattern in window-averaged DNA curvature profile, that is, the DNA regions including secondary metabolic signature genes such as polyketide synthase, nonribosomal peptide synthase, and/or dimethylallyl tryptophan synthase consisted of window-averaged DNA curvature values lower than 0.18 and these DNA regions were at least 20 kb. Forty percent of secondary metabolite gene clusters with this conserved pattern were related to severe regulation by a transcription factor, LaeA. Our result could be used for identification of other fungal secondary metabolite gene clusters, especially for secondary metabolite gene cluster that is severely regulated by LaeA or other proteins with similar function to LaeA.

Characterisation of a novel endophyte NRPS gene and its role in endophyte-grass symbioses

Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism