Hans-Wilhelm Nützmann

Intimate bacterial–fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans

Fungi produce numerous low molecular weight molecules endowed with a multitude of biological activities. However, mining the full-genome sequences of fungi indicates that their potential to produce secondary metabolites is greatly underestimated. Because most of the biosynthesis gene clusters are silent under laboratory conditions, one of the major challenges is to understand the physiological conditions under which these genes are activated. Thus, we cocultivated the important model fungus Aspergillus nidulans with a collection of 58 soil-dwelling actinomycetes. By microarray analyses of both Aspergillus secondary metabolism and full-genome arrays and Northern blot and quantitative RT-PCR analyses, we demonstrate at the molecular level that a distinct fungal-bacterial interaction leads to the specific activation of fungal secondary metabolism genes. Most surprisingly, dialysis experiments and electron microscopy indicated that an intimate physical interaction of the bacterial and fungal mycelia is required to elicit the specific response. Gene knockout experiments provided evidence that one induced gene cluster codes for the long-sought after polyketide synthase (PKS) required for the biosynthesis of the archetypal polyketide orsellinic acid, the typical lichen metabolite lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B. A phylogenetic analysis demonstrates that orthologs of this PKS are widespread in nature in all major fungal groups, including mycobionts of lichens. These results provide evidence of specific interaction among microorganisms belonging to different domains and support the hypothesis that not only diffusible signals but intimate physical interactions contribute to the communication among microorganisms and induction of otherwise silent biosynthesis genes.

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

Gene clustering in plant specialized metabolism.

Physically linked clusters of genes that encode the enzymatic information for the synthesis of specialized metabolites are a well-established feature of microbial secondary metabolism. In contrast, the biosynthesis of plant specialized metabolites has until recently been thought to be almost exclusively encoded by genes that are randomly scattered in the genome. However, recent reports highlight the growing number of examples of gene clusters for specialized metabolic pathways in plants. Numerous gene clusters that encode for the biosynthesis of different classes of metabolite have now been discovered in a variety of plant species. Comparison of these characterized clusters now enables us to begin to define their salient features and to exploit plant biosynthetic gene clusters for synthetic biology applications.

Plant metabolic clusters – from genetics to genomics

Contents 771 I. 771 II. 772 III. 780 IV. 781 V. 786 786 References 786 SUMMARY: Plant natural products are of great value for agriculture, medicine and a wide range of other industrial applications. The discovery of new plant natural product pathways is currently being revolutionized by two key developments. First, breakthroughs in sequencing technology and reduced cost of sequencing are accelerating the ability to find enzymes and pathways for the biosynthesis of new natural products by identifying the underlying genes. Second, there are now multiple examples in which the genes encoding certain natural product pathways have been found to be grouped together in biosynthetic gene clusters within plant genomes. These advances are now making it possible to develop strategies for systematically mining multiple plant genomes for the discovery of new enzymes, pathways and chemistries. Increased knowledge of the features of plant metabolic gene clusters - architecture, regulation and assembly - will be instrumental in expediting natural product discovery. This review summarizes progress in this area.

Cytotoxic Pheofungins from an Engineered Fungus Impaired in Posttranslational Protein Modification

What makes a fungus blush? The deletion of a gene that is required for global protein N-acetylation triggers the production of unprecedented metabolites in Aspergillus nidulans. The pronounced red pigmentation of the engineered mutant is caused by pheofungins (benzothiazinone chromophores), the biogenesis of which is strikingly similar to those of pheomelanins found in red bird feathers and hair of Celtic origin.

Regulation of metabolic gene clusters in Arabidopsis thaliana

Recent discoveries have revealed that the genes for the biosynthesis of a variety of plant specialized metabolites are organized in operon-like clusters within plant genomes. Here we identify a regulatory process that is required for normal expression of metabolic gene clusters in Arabidopsis thaliana. Comparative gene expression analysis of a representative clustered gene was performed in a set of chromatin mutant lines. Subsequently, metabolite levels were analysed by GC-MS and the local chromatin structure was investigated by chromatin immunoprecipitation and nucleosome positioning. We show that the transcript levels of genes within two metabolic clusters are coordinately reduced in an arp6 and h2a.z background. We demonstrate that H2A.Z enrichment in the clusters is positively correlated with active cluster expression. We further show that nucleosome stability within the cluster regions is higher in the arp6 background compared with the wild-type. These results implicate ARP6 and H2A.Z in the regulation of metabolic clusters in Arabidopsis thaliana through localized chromatin modifications that enable the coordinate expression of groups of contiguous genes. These findings shed light on the complex process of cluster regulation, an area that could in the future open up new opportunities for the discovery and manipulation of specialized metabolic pathways in plants. See also the Commentary by Fernie and Tohge

Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans

ABSTRACT Chromatin remodelling events play an important role in the secondary metabolism of filamentous fungi. Previously, we showed that a bacterium, Streptomyces rapamycinicus, is able to reprogram the histone-modifying Spt-Ada-Gcn5-acetyltransferase/ADA (SAGA/ADA) complex of the model fungus Aspergillus nidulans. Consequently, the histone H3 amino acids lysine 9 and lysine 14 at distinct secondary metabolism genes were specifically acetylated during the bacterial fungal interaction, which, furthermore, was associated with the activation of the otherwise silent orsellinic acid gene cluster. To investigate the importance of the histone modifications for distinct gene expression profiles in fungal secondary metabolism, we exchanged several amino acids of histone H3 of A. nidulans. These amino acids included lysine residues 9, 14, 18, and 23 as well as serine 10 and threonine 11. Lysine residues were replaced by arginine or glutamine residues, and serine/threonine residues were replaced by alanine. All generated mutant strains were viable, allowing direct analysis of the consequences of missing posttranslational histone modifications. In the mutant strains, major changes in the expression patterns at both the transcriptional and metabolite levels of the penicillin, sterigmatocystin, and orsellinic acid biosynthesis gene clusters were detected. These effects were due mainly to the substitution of the acetylatable lysine 14 of histone H3 and were enhanced in a lysine 14/lysine 9 double mutant of histone H3. Taken together, our findings show a causal linkage between the acetylation of lysine residue 14 of histone H3 and the transcription and product formation of secondary metabolite gene clusters.

Delineation of metabolic gene clusters in plant genomes by chromatin signatures

Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.

Regulatory cross talk and microbial induction of fungal secondary metabolite gene clusters.

Filamentous fungi are well-known producers of a wealth of secondary metabolites with various biological activities. Many of these compounds such as penicillin, cyclosporine, or lovastatin are of great importance for human health. Genome sequences of filamentous fungi revealed that the encoded potential to produce secondary metabolites is much higher than the actual number of compounds produced during cultivation in the laboratory. This finding encouraged research groups to develop new methods to exploit the silent reservoir of secondary metabolites. In this chapter, we present three successful strategies to induce the expression of secondary metabolite gene clusters. They are based on the manipulation of the molecular processes controlling the biosynthesis of secondary metabolites and the simulation of stimulating environmental conditions leading to altered metabolic profiles. The presented methods were successfully applied to identify novel metabolites. They can be also used to significantly increase product yields.

Embracing the next generation of plant scientists

July 2017 saw the second New Phytologist next generation scientists meeting (https://www.newphytologist.org/nextgenevent/2017) take place at the John Innes Conference Centre in the grounds of the Norwich Research Park (UK). Surrounded by the eminent plant scientists of the John Innes Centre, The Sainsbury Laboratory and the Earlham Institute, over 100 early career researchers (ECRs) and established scientists from around the world gathered to present and discuss their work, as well as to forge new links for their future careers (Fig. 1). Fully funded by the New Phytologist Trust (https://www.newphytologist.org/) and Wiley (http://eu.wiley.c om/WileyCDA/), the meeting’s aim was to create a stimulating environment to share and highlight current topics of note across the plant science community, and to support interdisciplinary scientific endeavour. Selected ECRs presented their work in talks, and established academics and past winners of the New Phytologist Tansley medal (https://www.newphytologist.org/ta nsleymedal) were invited to present plenary and keynote lectures. The scientific programme was supplemented with workshops on scientific publishing and ethics. The talks and posters showcased the diversity and breadth of plant sciences. The research presented ranged from adaptation of ectomycorrhizas in southern Patagonia, through immune receptor activation in Arabidopsis thaliana, to the genetics of floral heteromorphy in Primula. Hot topics in plant research, such as climate change, developmental evolution and synthetic biology were covered by different presenters, and from various angles across the scientific programme, as well as by the ECRs themselves. Caroline Dean (John Innes Centre, Norwich, UK) opened the meeting by explaining the intricate epigenetic regulation of flowering in response to prolonged cold during winter. The presentation highlighted how small molecular modifications of a single gene are of crucial importance for plants to balance the timing of flowering initiation (Whittaker & Dean, 2017). Her group’s latest research suggests that plants measure fluctuations in temperature peaks instead of averages in temperatures. These findings may prove important for modelling the outcomes of climate change, as peak temperatures alter in a faster and more extreme manner than averages. Further presentations by ECRs highlighted the roles of light signalling and energy metabolism in the adaptation to elevated temperatures, of potato (poster 7 (P7)) and Arabidopsis thaliana (P83), respectively. The New Phytologist Tansley Medal 2014 winner William Anderegg (University of Utah, USA; Lennon & Dolan, 2015), brought the climate agenda from the gene to the ecosystem level. He discussed the ability to predict the impact of drought stress on vegetation, and how variabilities in such models can affect predictions on carbon absorption and temperature increases (Schwalm et al., 2017). Other aspects of climate change were presented by ECRs, with