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The conspicuous bright golden to orange-reddish coloration of species of the basidiomycete genus Laetiporus is a hallmark feature of their fruiting bodies, known among mushroom hunters as the “chicken of the woods”. This report describes the identification of an eight-domain mono-modular highly reducing polyketide synthase as sole enzyme necessary for laetiporic acid biosynthesis. Heterologous pathway reconstitution in both Aspergillus nidulans and Aspergillus niger verified that LpaA functions as a multi-chain length polyene synthase, which produces a cocktail of laetiporic acids with a methyl-branched C26–C32 main chain. Laetiporic acids show a marked antifungal activity on Aspergillus protoplasts. Given the multiple products of a single biosynthesis enzyme, our work underscores the diversity-oriented character of basidiomycete natural product biosynthesis.

The oleaginous fungus Mortierella alpina is a safe source of polyunsaturated fatty acids (PUFA) in industrial food and feed production. Besides PUFA production, pharmaceutically relevant surface-active and antimicrobial oligopeptides were isolated from this basal fungus. Both production of fatty acids and oligopeptides rely on the biosynthesis and high turnover of branched-chain-amino acids (BCAA), especially l-leucine. However, the regulation of BCAA biosynthesis in basal fungi is largely unknown. Here, we report on the regulation of the leucine, isoleucine, and valine metabolism in M. alpina. In contrast to higher fungi, the biosynthetic genes for BCAA are hardly transcriptionally regulated, as shown by qRT-PCR analysis, which suggests a constant production of BCAAs. However, the enzymes of the leucine metabolism are tightly metabolically regulated. Three enzymes of the leucine metabolism were heterologously produced in Escherichia coli, one of which is inhibited by allosteric feedback loops: The key regulator is the α-isopropylmalate synthase LeuA1, which is strongly disabled by l-leucine, α-ketoisocaproate, and propionyl-CoA, the precursor of the odd-chain fatty acid catabolism. Its gene is not related to homologs from higher fungi, but it has been inherited from a phototrophic ancestor by horizontal gene transfer.

Secondary metabolites have a great potential as pharmaceuticals, but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer. Here, signals, mediators, and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition. Terrein causes fruit surface lesions and inhibits plant seed germination. Additionally, terrein is moderately antifungal and reduces ferric iron, thereby supporting growth of A. terreus under iron starvation. In accordance, the lack of nitrogen or iron or elevated methionine levels induced terrein production and was dependent on either the nitrogen response regulators AreA and AtfA or the iron response regulator HapX. Independent signal transduction allows complex sensing of the environment and, combined with its broad spectrum of biological activities, terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition. Organisms produce a wide variety of small molecules called metabolites through the break down of food and other chemical reactions. Some of these molecules—known as primary metabolites—are required for growth, reproduction and other vital processes. Other molecules called secondary metabolites are not strictly required by the organism, but generally have other roles that may improve the individual’s ability to survive and reproduce. Fungi and other microbes produce a large variety of secondary metabolites, many of which are used as medicines to treat diseases in humans and other animals. For example, a molecule called lovastatin—which is produced by a fungus known as Aspergillus terreus—can reduce a human patient's risk of heart disease. However, it is not known what role many secondary metabolites play in the microbe that produced them. A. terreus lives in the soil, but it can also infect plants and animals. In addition to lovastatin, it also makes another secondary metabolite called terrein. A recent study identified the genes responsible for making terrein, and discovered that this molecule is harmful to plant cells and may help the fungus to colonize and thrive in the area immediately around plant roots, which is known as the rhizosphere. Here, Gressler et al. studied how terrein may help the fungus to cope with competitors in this environment. The experiments show that terrein increases the availability of iron and inhibits the growth of competing microbes. A shortage of iron or nitrogen-containing nutrients can stimulate the fungus to produce terrein, and elevated levels of a molecule called methionine have the same effect. These conditions are commonly found in the rhizosphere and further experiments identified several proteins in the fungus that are required for sensing them. Gressler et al.'s findings suggest that terrein helps to ensure that the fungus has sufficient nitrogen and iron to thrive in the rhizosphere. Also, this study confirms that the production of secondary metabolites in microbes can happen in response to elaborate cues from the environment, which may explain why only a limited number of secondary metabolites are produced by microbes when they are grown in the laboratory. Future studies will analyze other ways to activate the production of secondary metabolites outside of the microbe's normal environment, which may lead to the discovery of new important drugs.

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.

Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree ( Hevea brasiliensis ) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus , a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales , an understudied group of basidiomycetes. Our analysis further identified pathogen’s genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus . The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.

Background Non-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary diversification into eleven clades further underscores the relevance of these multi-domain enzymes. Following current knowledge, NR-PKSs initiate polyketide assembly by an N-terminal starter unit:acyl transferase (SAT) domain that catalyzes the transfer of an acetyl starter from the acetyl-CoA thioester onto the acyl carrier protein (ACP). Results A comprehensive phylogenetic analysis of NR-PKSs established a twelfth clade from which three representatives, enzymes CrPKS1-3 of the webcap mushroom Cortinarius rufoolivaceus , were biochemically characterized. These basidiomycete synthases lack a SAT domain yet are fully functional hepta- and octaketide synthases in vivo. Three members of the other clade of basidiomycete NR-PKSs (clade VIII) were produced as SAT-domainless versions and analyzed in vivo and in vitro. They retained full activity, thus corroborating the notion that the SAT domain is dispensable for many basidiomycete NR-PKSs. For comparison, the ascomycete octaketide synthase atrochrysone carboxylic acid synthase (ACAS) was produced as a SAT-domainless enzyme as well, but turned out completely inactive. However, a literature survey revealed that some NR-PKSs of ascomycetes carry mutations within the catalytic motif of the SAT domain. In these cases, the role of the domain and the origin of the formal acetate unit remains open. Conclusions The role of SAT domains differs between asco- and basidiomycete NR-PKSs. For the latter, it is not part of the minimal set of NR-PKS domains and not required for function. This knowledge may help engineer compact NR-PKSs for more resource-efficient routes. From the genomic standpoint, seemingly incomplete or corrupted genes encoding SAT-domainless NR-PKSs should not automatically be dismissed as non-functional pseudogenes, but considered during genome analysis to decipher the potential arsenal of natural products of a given fungus.

Background Secondary metabolites (SMs) from mushroom-forming fungi ( Basidiomycota ) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts. Results An expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆ pyrG , resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆ akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus . We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina . Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF. Conclusions The system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi.

Background Fungi are prolific producers of bioactive small molecules of pharmaceutical or agricultural interest. The secondary metabolism of higher fungi (Dikarya) has been well-investigated which led to > 39,000 described compounds. However, natural product researchers scarcely drew attention to early-diverging fungi (Mucoro- and Zoopagomycota) as they are considered to rarely produce secondary metabolites. Indeed, only 15 compounds have as yet been isolated from the entire phylum of the Zoopagomycota. Results Here, we showcase eight species of the order Kickxellales (phylum Zoopagomycota) as potent producers of the indole-3-acetic acid (IAA)-derived compounds lindolins A and B. The compounds are produced both under laboratory conditions and in the natural soil habitat suggesting a specialized ecological function. Indeed, lindolin A is a selective agent against plant-pathogenic oomycetes such as Phytophthora sp. Lindolin biosynthesis was reconstituted in vitro and relies on the activity of two enzymes of dissimilar evolutionary origin: Whilst the IAA–CoA ligase LinA has evolved from fungal 4-coumaryl-CoA synthetases, the subsequently acting IAA-CoA:anthranilate N -indole-3-acetyltransferase LinB is a unique enzyme across all kingdoms of life. Conclusions This is the first report on bioactive secondary metabolites in the subphylum Kickxellomycotina and the first evidence for a non-clustered, two-step biosynthetic route of secondary metabolites in early-diverging fungi. Thus, the generally accepted “gene cluster hypothesis” for natural products needs to be reconsidered for early diverging fungi.

Background The terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin. Results The activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin. Conclusions Our results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.

Galactosaminogalactan (GAG) is an insoluble aminosugar polymer produced by and has anti-inflammatory properties. Here, the minimum glycosidic sequences required for the induction of IL-1Ra by peripheral blood mononuclear cells (PBMCs) was investigated. Using chemical degradation of native GAG to isolate soluble oligomers, we have found that the de- -acetylation of galactosamine residues and the size of oligomer are critical for the immune response. A minimal oligomer size of 20 galactosamine residues is required for the anti-inflammatory response but the presence of galactose residues is not necessary. In a Dextran sulfate induced colitis mouse model, a fraction of de- -acetylated oligomers of 13 < dp < 20 rescue inflammatory damage like the native GAG polymer in an IL-1Ra dependent pathway. Our results demonstrate the therapeutic suitability of water-soluble GAG oligosaccharides in IL-1 mediated hyper-inflammatory diseases and suggest that α-1,4-galactosamine oligomers chemically synthesized could represent new anti-inflammatory glycodrugs.