Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

Incorporation of the histone variant H2A.Z counteracts gene silencing mediated by H3K27 trimethylation in Fusarium fujikuroi.

BACKGROUND: Fusarium fujikuroi is a pathogen of rice causing diverse disease symptoms such as 'bakanae' or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin. RESULTS: Through the combination of diverse '-omics' methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism. CONCLUSION: In this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms.

H3K27me3 is vital for fungal development and secondary metabolite gene silencing, and substitutes for the loss of H3K9me3 in the plant pathogen Fusarium proliferatum.

Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer involved in secondary metabolite (SM) gene silencing and crucial for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in Δfpkmt6, indicating crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In Δfpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that may explain the subtle phenotype of the Δfpkmt1 strain which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain and loss of H3K27me3 results in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in Δfpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production is largely restricted to F. fujikuroi, possibly outlining the distinct lifestyles of these notorious plant pathogens. We show that H3K27me3 is involved in GA gene silencing in F. proliferatum and at least one additional FFSC member, and thus, may serve as a regulatory layer for gene silencing under non-favoring conditions.

Filling out the gaps - identification of fugralins as products of the PKS2 cluster in Fusarium graminearum.

As one of the grain crop pathogenic fungi with the greatest impacts on agricultural economical as well as human health, an elaborate understanding of the life cycle and subsequent metabolome of Fusarium graminearum is of great interest. Throughout the lifetime of the fungus, it is known to produce a wide array of secondary metabolites, including polyketides. One of the F. graminearum polyketides which has remained a mystery until now has been elucidated in this work. Previously, it was suggested that the biosynthetic product of the PKS2 gene cluster was involved in active mycelial growth, the exact mechanism, however, remained unclear. In our work, disruption and overexpression of the PKS2 gene in F. graminearum enabled structural elucidation of a linear and a cyclic tetraketide with a double methyl group, named fugralin A and B, respectively. Further functional characterization showed that the compounds are not produced during infection, and that deletion and overexpression did not affect pathogenicity or visual growth. The compounds were shown to be volatile, which could point to possible functions that can be investigated further in future studies.

Genome analysis of Cephalotrichum gorgonifer and identification of the biosynthetic pathway for rasfonin, an inhibitor of KRAS dependent cancer.

BACKGROUND: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells. RESULTS: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is indeed responsible for rasfonin biosynthesis. CONCLUSIONS: This study identified a biosynthetic gene cluster containing a key-gene responsible for rasfonin production. Additionally, molecular tools were established for the non-model fungus Cephalotrichum gorgonifer which allows strain engineering and heterologous expression of the BGC for high rasfonin producing strains and the biosynthesis of rasfonin derivates for diverse applications.

Biosynthesis of the Isocoumarin Derivatives Fusamarins is Mediated by the PKS8 Gene Cluster in Fusarium.

Fusarium mangiferae causes the mango malformation disease (MMD) on young mango trees and seedlings resulting in economically significant crop losses. In addition, F. mangiferae produces a vast array of secondary metabolites (SMs), including mycotoxins that may contaminate the harvest. Their production is tightly regulated at the transcriptional level. Here, we show that lack of the H3 K9-specific histone methyltransferase, FmKmt1, influences the expression of the F. mangiferae polyketide synthase (PKS) 8 (FmPKS8), a so far cryptic PKS. By a combination of reverse genetics, untargeted metabolomics, bioinformatics and chemical analyses including structural elucidation, we determined the FmPKS8 biosynthetic gene cluster (BGC) and linked its activity to the production of fusamarins (FMN), which can be structurally classified as dihydroisocoumarins. Functional characterization of the four FMN cluster genes shed light on the biosynthetic pathway. Cytotoxicity assays revealed moderate toxicities with IC50 values between 1 and 50 µM depending on the compound.

How to Completely Squeeze a Fungus-Advanced Genome Mining Tools for Novel Bioactive Substances.

Fungal species have the capability of producing an overwhelming diversity of bioactive substances that can have beneficial but also detrimental effects on human health. These so-called secondary metabolites naturally serve as antimicrobial "weapon systems", signaling molecules or developmental effectors for fungi and hence are produced only under very specific environmental conditions or stages in their life cycle. However, as these complex conditions are difficult or even impossible to mimic in laboratory settings, only a small fraction of the true chemical diversity of fungi is known so far. This also implies that a large space for potentially new pharmaceuticals remains unexplored. We here present an overview on current developments in advanced methods that can be used to explore this chemical space. We focus on genetic and genomic methods, how to detect genes that harbor the blueprints for the production of these compounds (i.e., biosynthetic gene clusters, BGCs), and ways to activate these silent chromosomal regions. We provide an in-depth view of the chromatin-level regulation of BGCs and of the potential to use the CRISPR/Cas technology as an activation tool.

Ten decadal advances in fungal biology leading towards human well-being.

Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.

Dissimilarity of Airway and Lung Tissue Microbiota in Smokers undergoing Surgery for Lung Cancer.

Human airways are continuously colonized by microaspiration of microbiota. Less is known about the presence, origin and composition of microbiota in the lung parenchyma. In a study of 13 patients undergoing surgery for peripheral lung cancer microbiota composition was comparatively evaluated in upper airway, lower airway and lung tissue samples using 16S rDNA analysis. Bacterial density decreased stepwise from upper to lower airways and tissue. On a taxonomic level upper and lower airway microbiota were similar whereas lung tissue showed marked dissimilarities compared to lower airways that may reflect different environmental conditions shaping local microbiota and host immunity.