Philipp Wiemann

FfVel1 and FfLae1, components of a velvet‐like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence

Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigments bikaverin and neurosporaxanthin and the mycotoxins fumonisins and fusarin C. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here, we report on the characterization of two components of the F. fujikuroi velvet‐like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA orthologue outside the class of Eurotiomycetidae is upregulated in ΔFfvel1 microarray‐studies and FfLae1 interacts with FfVel1 in the nucleus. Deletion of Ffvel1 and Fflae1 revealed for the first time that velvet can simultaneously act as positive (GAs, fumonisins and fusarin C) and negative (bikaverin) regulator of secondary metabolism, and that both components affect conidiation and virulence of F. fujikuroi. Furthermore, the velvet‐like protein FfVel2 revealed similar functions regarding conidiation, secondary metabolism and virulence as FfVel1. Cross‐genus complementation studies of velvet complex component mutants between Fusarium, Aspergillus and Penicillium support an ancient origin for this complex, which has undergone a divergence in specific functions mediating development and secondary metabolism.

Biosynthesis of the red pigment bikaverin in Fusarium fujikuroi: genes, their function and regulation

Fusarium secondary metabolites are structurally diverse, have a variety of activities and are generally poorly understood biosynthetically. The F. fujikuroi polyketide synthase gene bik1 was previously shown to be responsible for formation of the mycelial pigment bikaverin. Here we present the characterization of five genes adjacent to bik1 as encoding a putative FAD‐dependent monooxygenase (bik2), an O‐methyltransferase (bik3), an NmrA‐like protein (bik4), a Zn(II)2Cys6 transcription factor (bik5) and an MFS transporter (bik6). Deletion of each gene resulted in total loss or significant reduction of bikaverin synthesis. Expression studies revealed that all bik genes are repressed by high amounts of nitrogen in an AreA‐independent manner and are subject to a time‐ and pH‐dependent regulation. Deletion of the pH regulatory gene pacC resulted in partial derepression while complementation with a dominant active allele resulted in repression of bik genes at acidic ambient pH. Transcription of all bik genes in strains lacking bik1, bik2 or bik3 was essentially eliminated, while transcription of some bik genes was detected in strains lacking bik4, bik5 or bik6. Thus, bikaverin synthesis is regulated by a complex regulatory network. Understanding how different factors influence the synthesis of this model secondary metabolite will aid understanding secondary metabolism in general.

Strategies for mining fungal natural products

Fungi are well known for their ability to produce a multitude of natural products. On the one hand their potential to provide beneficial antibiotics and immunosuppressants has been maximized by the pharmaceutical industry to service the market with cost-efficient drugs. On the other hand identification of trace amounts of known mycotoxins in food and feed samples is of major importance to ensure consumer health and safety. Although several fungal natural products, their biosynthesis and regulation are known today, recent genome sequences of hundreds of fungal species illustrate that the secondary metabolite potential of fungi has been substantially underestimated. Since expression of genes and subsequent production of the encoded metabolites are frequently cryptic or silent under standard laboratory conditions, strategies for activating these hidden new compounds are essential. This review will cover the latest advances in fungal genome mining undertaken to unlock novel products.

Biosynthesis of Fusarubins Accounts for Pigmentation of Fusarium fujikuroi Perithecia

ABSTRACT Fusarium fujikuroi produces a variety of secondary metabolites, of which polyketides form the most diverse group. Among these are the highly pigmented naphthoquinones, which have been shown to possess different functional properties for the fungus. A group of naphthoquinones, polyketides related to fusarubin, were identified in Fusarium spp. more than 60 years ago, but neither the genes responsible for their formation nor their biological function has been discovered to date. In addition, although it is known that the sexual fruiting bodies in which the progeny of the fungus develops are darkly colored by a polyketide synthase (PKS)-derived pigment, the structure of this pigment has never been elucidated. Here we present data that link the fusarubin-type polyketides to a defined gene cluster, which we designate fsr, and demonstrate that the fusarubins are the pigments responsible for the coloration of the perithecia. We studied their regulation and the function of the single genes within the cluster by a combination of gene replacements and overexpression of the PKS-encoding gene, and we present a model for the biosynthetic pathway of the fusarubins based on these data.

Genetic evidence for natural product‐mediated plant–plant allelopathy in rice (Oryza sativa)

• There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). • However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. • The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). • Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.

Genetic Manipulation of the Fusarium fujikuroi Fusarin Gene Cluster Yields Insight into the Complex Regulation and Fusarin Biosynthetic Pathway

In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively.

Lae1 regulates expression of multiple secondary metabolite gene clusters in Fusarium verticillioides

The filamentous fungus Fusarium verticillioides can cause disease of maize and is capable of producing fumonisins, a family of toxic secondary metabolites linked to esophageal cancer and neural tube defects in humans and lung edema in swine and leukoencephalomalacia in equines. The expression of fumonisin biosynthetic genes is influenced by broad-domain transcription factors (global regulators) and Fum21, a pathway-specific transcription factor. LaeA is a global regulator that in Aspergillus nidulans, affects the expression of multiple secondary metabolite gene clusters by modifying heterochromatin structure. Here, we employed gene deletion analysis to assess the effect of loss of a F. verticillioides laeA orthologue, LAE1, on genome-wide gene expression and secondary metabolite production. Loss of Lae1 resulted in reduced expression of gene clusters responsible for synthesis of the secondary metabolites bikaverin, fumonisins, fusaric acid and fusarins as well as two clusters for which the corresponding secondary metabolite is unknown. Analysis of secondary metabolites revealed that, in contrast to a previously described Fusarium fujikuroi lae1 mutant, bikaverin production is reduced. Fumonisin production is unchanged in the F. verticillioides lae1 mutant. Complementation of the F. verticillioides mutant resulted in increased fumonisin production.

An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium

Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

The Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1 of Fusarium fujikuroi Controls Development, Secondary Metabolism and Pathogenicity

The heterothallic ascomycete Fusarium fujikuroi is a notorious rice pathogen causing super-elongation of plants due to the production of terpene-derived gibberellic acids (GAs) that function as natural plant hormones. Additionally, F. fujikuroi is able to produce a variety of polyketide- and non-ribosomal peptide-derived metabolites such as bikaverins, fusarubins and fusarins as well as metabolites from yet unidentified biosynthetic pathways, e.g. moniliformin. The key enzymes needed for their production belong to the family of polyketide synthases (PKSs) and non-ribosomal peptide synthases (NRPSs) that are generally known to be post-translationally modified by a Sfp-type 4′phosphopantetheinyl transferase (PPTase). In this study we provide evidence that the F. fujikuroi Sfp-type PPTase FfPpt1 is essentially involved in lysine biosynthesis and production of bikaverins, fusarubins and fusarins, but not moniliformin as shown by analytical methods. Concomitantly, targeted Ffppt1 deletion mutants reveal an enhancement of terpene-derived metabolites like GAs and volatile substances such as α-acorenol. Pathogenicity assays on rice roots using fluorescent labeled wild-type and Ffppt1 mutant strains indicate that lysine biosynthesis and iron acquisition but not PKS and NRPS metabolism is essential for establishment of primary infections of F. fujikuroi. Additionally, FfPpt1 is involved in conidiation and sexual mating recognition possibly by activating PKS- and/or NRPS-derived metabolites that could act as diffusible signals. Furthermore, the effect on iron acquisition of Ffppt1 mutants led us to identify a previously uncharacterized putative third reductive iron uptake system (FfFtr3/FfFet3) that is closely related to the FtrA/FetC system of A. fumigatus. Functional characterization provides evidence that both proteins are involved in iron acquisition and are liable to transcriptional repression of the homolog of the Aspergillus GATA-type transcription factor SreA under iron-replete conditions. Targeted deletion of the first Fusarium homolog of this GATA-type transcription factor-encoding gene, Ffsre1, strongly indicates its involvement in regulation of iron homeostasis and oxidative stress resistance.

Perturbations in small molecule synthesis uncovers an iron-responsive secondary metabolite network in Aspergillus fumigatus.

Iron plays a critical role in survival and virulence of the opportunistic pathogen Aspergillus fumigatus. Two transcription factors, the GATA-factor SreA and the bZip-factor HapX oppositely monitor iron homeostasis with HapX activating iron acquisition pathways (e.g., siderophores) and shutting down iron consumptive pathways (and SreA) during iron starvation conditions whereas SreA negatively regulates HapX and corresponding pathways during iron sufficiency. Recently the non-ribosomal peptide, hexadehydroastechrome (HAS; a tryptophan-derived iron (III)-complex), has been found important in A. fumigatus virulence. We found that HAS overproduction caused an iron starvation phenotype, from alteration of siderophore pools to regulation of iron homeostasis gene expression including sreA. Moreover, we uncovered an iron dependent secondary metabolism network where both SreA and HapX oppositely regulate multiple other secondary metabolites including HAS. This circuitry links iron-acquisition and consumption pathways with secondary metabolism—thus placing HAS as part of a metabolic feedback circuitry designed to balance iron pools in the fungus and presenting iron availability as one environmental trigger of secondary metabolism.