Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

Regulation and Role of Fungal Secondary Metabolites.

Fungi have the capability to produce a tremendous number of so-called secondary metabolites, which possess a multitude of functions, e.g., communication signals during coexistence with other microorganisms, virulence factors during pathogenic interactions with plants and animals, and in medical applications. Therefore, research on this topic has intensified significantly during the past 10 years and thus knowledge of regulatory mechanisms and the understanding of the role of secondary metabolites have drastically increased. This review aims to depict the complexity of all the regulatory elements involved in controlling the expression of secondary metabolite gene clusters, ranging from epigenetic control and signal transduction pathways to global and specific transcriptional regulators. Furthermore, we give a short overview on the role of secondary metabolites, focusing on the interaction with other microorganisms in the environment as well as on pathogenic relationships.

Advances in Synthetic Biology of Fungi and Contributions to the Discovery of New Molecules.

The sequencing of fungal genomes is becoming increasingly accessible, with a wealth of data already available. In parallel, the prediction of the putative biosynthetic pathways responsible for the synthesis of potential new natural products is also increasing. The difficulty of translating computational analyses into available compounds is becoming evident, slowing down a process that was thought to be faster with the advent of the genomic era. Advances in gene techniques made it possible to genetically modify a wider range of organisms, including fungi typically considered recalcitrant to DNA manipulation. However, the possibility of screening many gene cluster products for new activities in a high-throughput manner remains unfeasible. Nonetheless, some updates on the synthetic biology of fungi could provide interesting insights that could help to achieve this goal in the future.

Chemical Diversity and Biosynthesis of Drimane-Type Sesquiterpenes in the Fungal Kingdom.

Drimane-type sesquiterpenes are a class of compounds produced by a wide range of organisms, initially isolated and characterized in plants. Meanwhile, in the past 20-30 years, a large number of novel structures from many divergent fungi have been elucidated. Recently, the biosynthesis of drimane-type sesquiterpenes and their esters has been explained in two filamentous fungi, namely Aspergillus oryzae and Aspergillus calidoustus, disclosing the basic biosynthetic principles needed to identify similar pathways in the fungal kingdom.

Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid.

BACKGROUND: The availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoeba Dictyostelium discoideum represents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineered D. discoideum strain. RESULTS: In this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (εmax) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerable εmax of 3.9 W/kg for D. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 µg/L, a productivity of 0.04 µg/L/h and a yield of 0.56 µg/g glucose. CONCLUSION: We developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level for D. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass.

The fungal CCAAT-binding complex and HapX display highly variable but evolutionary conserved synergetic promoter-specific DNA recognition.

To sustain iron homeostasis, microorganisms have evolved fine-tuned mechanisms for uptake, storage and detoxification of the essential metal iron. In the human pathogen Aspergillus fumigatus, the fungal-specific bZIP-type transcription factor HapX coordinates adaption to both iron starvation and iron excess and is thereby crucial for virulence. Previous studies indicated that a HapX homodimer interacts with the CCAAT-binding complex (CBC) to cooperatively bind bipartite DNA motifs; however, the mode of HapX-DNA recognition had not been resolved. Here, combination of in vivo (genetics and ChIP-seq), in vitro (surface plasmon resonance) and phylogenetic analyses identified an astonishing plasticity of CBC:HapX:DNA interaction. DNA motifs recognized by the CBC:HapX protein complex comprise a bipartite DNA binding site 5'-CSAATN12RWT-3' and an additional 5'-TKAN-3' motif positioned 11-23 bp downstream of the CCAAT motif, i.e. occasionally overlapping the 3'-end of the bipartite binding site. Phylogenetic comparison taking advantage of 20 resolved Aspergillus species genomes revealed that DNA recognition by the CBC:HapX complex shows promoter-specific cross-species conservation rather than regulon-specific conservation. Moreover, we show that CBC:HapX interaction is absolutely required for all known functions of HapX. The plasticity of the CBC:HapX:DNA interaction permits fine tuning of CBC:HapX binding specificities that could support adaptation of pathogens to their host niches.

Intrinsic Ability of the ß-Oxidation Pathway To Produce Bioactive Styrylpyrones.

Naturally occurring α-pyrones with biological activities are mostly synthesised by polyketide synthases (PKSs) via iterative decarboxylative Claisen condensation steps. Remarkably, we found that some enzymes related to the fatty acid ß-oxidation pathway in Escherichia coli, namely the CoA ligase FadD and the thiolases FadA and FadI, can synthesise styrylpyrones with phenylpropionic acids in vivo. The two thiolases directly utilise acetyl-CoA as an extender unit for carbon-chain elongation through a non-decarboxylative Claisen condensation, thus making the overall reaction more efficient in terms of carbon and energy consumption. Moreover, using a cell-free approach, different styrylpyrones were synthesised in vitro. Finally, targeted feeding experiments led to the detection of styrylpyrones in other species, demonstrating that the intrinsic ability of the ß-oxidation pathway allows for the synthesis of such molecules in bacteria, revealing an important biological feature hitherto neglected.

Biosynthesis of Fungal Drimane-Type Sesquiterpene Esters.

Drimane-type sesquiterpenes exhibit various biological activities and are widely present in eukaryotes. Here, we completely elucidated the biosynthetic pathway of the drimane-type sesquiterpene esters isolated from Aspergillus calidoustus and we discovered that it involves a drimenol cyclase having the same catalytic function previously only reported in plants. Moreover, since many fungal drimenol derivatives possess a γ-butyrolactone ring, we clarified the functions of the cluster-associated cytochrome P450 and FAD-binding oxidoreductase discovering that these two enzymes are solely responsible for the formation of those structures. Furthermore, swapping of the enoyl reductase domain in the identified polyketide synthase led to the production of metabolites containing various polyketide chains with different levels of saturation. These findings have deepened our understanding of how fungi synthesize drimane-type sesquiterpenes and the corresponding esters.

Correction: Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina).

[This corrects the article DOI: 10.1371/journal.pgen.1004496.].

Facile assembly and fluorescence-based screening method for heterologous expression of biosynthetic pathways in fungi.

Heterologous expression of multi-gene biosynthetic pathways in eukaryotic hosts is limited by highly regulated individual monocistrons. Dissimilar to prokaryotes, each eukaryotic gene is strictly controlled by its own regulatory elements, such as promoter and terminator. Consequently, parallel transcription can occur only when a group of genes is synchronously activated. A strategy to circumvent this limitation is the concerted expression of multiple genes as a polycistron. By exploiting the "stop-carry on" mechanism of picornaviruses, we have designed a sophisticated, yet easy-to-assemble vector system to heterologously express multiple genes under the control of a single promoter. For facile selection of correctly transformed colonies by basic fluorescence microscopy, our vector includes a split gene for a fluorescent reporter protein. This method was successfully applied to produce the psychotropic mushroom alkaloid psilocybin in high yields by heterologous expression of the entire biosynthetic gene cluster in the mould Aspergillus nidulans.

Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

The Aspergillus fumigatus conidial melanin production is regulated by the bifunctional bHLH DevR and MADS-box RlmA transcription factors.

Melanins play a crucial role in defending organisms against external stressors. In several pathogenic fungi, including the human pathogen Aspergillus fumigatus, melanin production was shown to contribute to virulence. A. fumigatus produces two different types of melanins, i.e., pyomelanin and dihydroxynaphthalene (DHN)-melanin. DHN-melanin forms the gray-green pigment characteristic for conidia, playing an important role in immune evasion of conidia and thus for fungal virulence. The DHN-melanin biosynthesis pathway is encoded by six genes organized in a cluster with the polyketide synthase gene pksP as a core element. Here, cross-species promoter analysis identified specific DNA binding sites in the DHN-melanin biosynthesis genes pksP-arp1 intergenic region that can be recognized by bHLH and MADS-box transcriptional regulators. Independent deletion of two genes coding for the transcription factors DevR (bHLH) and RlmA (MADS-box) interfered with sporulation and reduced the expression of the DHN-melanin gene cluster. In vitro and in vivo experiments proved that these transcription factors cooperatively regulate pksP expression acting both as repressors and activators in a mutually exclusive manner. The dual role executed by each regulator depends on specific DNA motifs recognized in the pksP promoter region.

Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.

Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial mucorales (Mucoromycotina).

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1-4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparison to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae.

Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation.

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores.

Hitting the caspofungin salvage pathway of human-pathogenic fungi with the novel lasso peptide humidimycin (MDN-0010).

Fungal infections have increased dramatically in the last 2 decades, and fighting infectious diseases requires innovative approaches such as the combination of two drugs acting on different targets or even targeting a salvage pathway of one of the drugs. The fungal cell wall biosynthesis is inhibited by the clinically used antifungal drug caspofungin. This antifungal activity has been found to be potentiated by humidimycin, a new natural product identified from the screening of a collection of 20,000 microbial extracts, which has no major effect when used alone. An analysis of transcriptomes and selected Aspergillus fumigatus mutants indicated that humidimycin affects the high osmolarity glycerol response pathway. By combining humidimycin and caspofungin, a strong increase in caspofungin efficacy was achieved, demonstrating that targeting different signaling pathways provides an excellent basis to develop novel anti-infective strategies.

Identification of the antiphagocytic trypacidin gene cluster in the human-pathogenic fungus Aspergillus fumigatus.

The opportunistic human pathogen Aspergillus fumigatus produces numerous different natural products. The genetic basis for the biosynthesis of a number of known metabolites has remained unknown. The gene cluster encoding for the biosynthesis of the conidia-bound metabolite trypacidin is of particular interest because of its antiprotozoal activity and possible role in the infection process. Here, we show that the genes encoding the biosynthesis enzymes of trypacidin reside within an orphan gene cluster in A. fumigatus. Genome mining identified tynC as an uncharacterized polyketide synthase with high similarity to known enzymes, whose products are structurally related to trypacidin including endocrocin and fumicycline. Gene deletion of tynC resulted in the complete absence of trypacidin production, which was fully restored when the mutant strain was complemented with the wild-type gene. When confronted with macrophages, the tynC deletion mutant conidia were more frequently phagocytosed than those of the parental wild-type strain. This was also found for phagocytic amoebae of the species Dictyostelium discoideum, which showed increased phagocytosis of ΔtynC conidia. Both macrophages and amoebae were also sensitive to trypacidin. Therefore, our results suggest that the conidium-bound trypacidin could have a protective function against phagocytes both in the environment and during the infection process.

The isoprenyl chain length of coenzyme Q mediates the nutritional resistance of fungi to amoeba predation.

Amoebae are environmental predators feeding on bacteria, fungi, and other eukaryotic microbes. Predatory interactions alter microbial communities and impose selective pressure toward phagocytic resistance or escape which may, in turn, foster virulence attributes. The ubiquitous fungivorous amoeba Protostelium aurantium has a wide prey spectrum in the fungal kingdom but discriminates against members of the Saccharomyces clade, such as Saccharomyces cerevisiae and Candida glabrata. Here, we show that this prey discrimination among fungi is solely based on the presence of ubiquinone as an essential cofactor for the predator. While the amoeba readily fed on fungi with CoQ presenting longer isoprenyl side chain variants CoQ8-10, such as those from the Candida clade, it failed to proliferate on those with shorter CoQ variants, specifically from the Saccharomyces clade (CoQ6). Supplementing non-edible yeast with CoQ9 or CoQ10 rescued the growth of P. aurantium, highlighting the importance of a long isoprenyl side chain. Heterologous biosynthesis of CoQ9 in S. cerevisiae by introducing genes responsible for CoQ9 production from the evolutionary more basic Yarrowia lipolytica complemented the function of the native CoQ6. The results suggest that the use of CoQ6 among members of the Saccharomyces clade might have originated as a predatory escape strategy in fungal lineages and could be retained in organisms that were able to thrive by fermentation. IMPORTANCE: Ubiquinones (CoQ) are universal electron carriers in the respiratory chain of all aerobic bacteria and eukaryotes. Usually 8-10 isoprenyl units ensure their localization within the lipid bilayer. Members of the Saccharomyces clade among fungi are unique in using only 6. The reason for this is unclear. Here we provide evidence that the use of CoQ6 efficiently protects these fungi from predation by the ubiquitous fungivorous amoeba Protostelium aurantium which lacks its own biosynthetic pathway for this vitamin. The amoebae were starving on a diet of CoQ6 yeasts which could be complemented by either the addition of longer CoQs or the genetic engineering of a CoQ9 biosynthetic pathway.

The spatial organization of sphingofungin biosynthesis in Aspergillus fumigatus and its cross-interaction with sphingolipid metabolism.

Sphingofungins are sphinganine analog mycotoxins acting as inhibitors of serine palmitoyl transferases, enzymes responsible for the first step in the sphingolipid biosynthesis. Eukaryotic cells are highly organized with various structures and organelles to facilitate cellular processes and chemical reactions, including the ones occurring as part of the secondary metabolism. We studied how sphingofungin biosynthesis is compartmentalized in the human-pathogenic fungus Aspergillus fumigatus, and we observed that it takes place in the endoplasmic reticulum (ER), ER-derived vesicles, and the cytosol. This implies that sphingofungin and sphingolipid biosynthesis colocalize to some extent. Automated analysis of confocal microscopy images confirmed the colocalization of the fluorescent proteins. Moreover, we demonstrated that the cluster-associated aminotransferase (SphA) and 3-ketoreductase (SphF) play a bifunctional role, supporting sphingolipid biosynthesis, and thereby antagonizing the toxic effects caused by sphingofungin production.IMPORTANCEA balanced sphingolipid homeostasis is critical for the proper functioning of eukaryotic cells. To this end, sphingolipid inhibitors have therapeutic potential against diseases related to the deregulation of sphingolipid balance. In addition, some of them have significant antifungal activity, suggesting that sphingolipid inhibitors-producing fungi have evolved mechanisms to escape self-poisoning. Here, we propose a novel self-defense mechanism, with cluster-associated genes coding for enzymes that play a dual role, being involved in both sphingofungin and sphingolipid production.

Optimized psilocybin production in tryptophan catabolism-repressed fungi.

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an in vivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.