Basidiomycete non-reducing polyketide synthases function independently of SAT domains.

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.

Bifurcate evolution of quinone synthetases in basidiomycetes.

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.

Blue Light-Dependent Pre-mRNA Splicing Controls Pigment Biosynthesis in the Mushroom Terana caerulea.

Light induces the production of ink-blue pentacyclic natural products, the corticin pigments, in the cobalt crust mushroom Terana caerulea. Here, we describe the genetic locus for corticin biosynthesis and provide evidence for a light-dependent dual transcriptional/cotranscriptional regulatory mechanism. Light selectively induces the expression of the corA gene encoding the gateway enzyme, the first described mushroom polyporic acid synthetase CorA, while other biosynthetic genes for modifying enzymes necessary to complete corticin assembly are induced only at lower levels. The strongest corA induction was observed following exposure to blue and UV light. A second layer of regulation is provided by the light-dependent splicing of the three introns in the pre-mRNA of corA. Our results provide insight into the fundamental organization of how mushrooms regulate natural product biosynthesis. IMPORTANCE The regulation of natural product biosyntheses in mushrooms in response to environmental cues is poorly understood. We addressed this knowledge gap and chose the cobalt crust mushroom Terana caerulea as our model. Our work discovered a dual-level regulatory mechanism that connects light as an abiotic stimulus with a physiological response, i.e., the production of dark-blue pigments. Exposure to blue light elicits strongly increased transcription of the gene encoding the gateway enzyme, the polyporic acid synthetase CorA, that catalyzes the formation of the pigment core structure. Additionally, light is a prerequisite for the full splicing of corA pre-mRNA and, thus, its proper maturation. Dual transcriptional/cotranscriptional light-dependent control of fungal natural product biosynthesis has previously been unknown. As it allows the tight control of a key metabolic step, it may be a much more prevalent mechanism among these organisms.

Secretion of Iron(III)-Reducing Metabolites during Protein Acquisition by the Ectomycorrhizal Fungus Paxillus involutus.

The ectomycorrhizal fungus Paxillus involutus decomposes proteins using a two-step mechanism, including oxidation and proteolysis. Oxidation involves the action of extracellular hydroxyl radicals (•OH) generated by the Fenton reaction. This reaction requires the presence of iron(II). Here, we monitored the speciation of extracellular iron and the secretion of iron(III)-reducing metabolites during the decomposition of proteins by P. involutus. X-ray absorption spectroscopy showed that extracellular iron was mainly present as solid iron(III) phosphates and oxides. Within 1 to 2 days, these compounds were reductively dissolved, and iron(II) complexes were formed, which remained in the medium throughout the incubation. HPLC and mass spectrometry detected five extracellular iron(III)-reducing metabolites. Four of them were also secreted when the fungus grew on a medium containing ammonium as the sole nitrogen source. NMR identified the unique iron(III)-reductant as the diarylcyclopentenone involutin. Involutin was produced from day 2, just before the elevated •OH production, preceding the oxidation of BSA. The other, not yet fully characterized iron(III)-reductants likely participate in the rapid reduction and dissolution of solid iron(III) complexes observed on day one. The production of these metabolites is induced by other environmental cues than for involutin, suggesting that they play a role beyond the Fenton chemistry associated with protein oxidation.

The Laetiporus polyketide synthase LpaA produces a series of antifungal polyenes.

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.

S-Adenosyl-l-Methionine Salvage Impacts Psilocybin Formation in "Magic" Mushrooms.

Psychotropic Psilocybe mushrooms biosynthesize their principal natural product psilocybin in five steps, among them a phosphotransfer and two methyltransfer reactions, which consume one equivalent of 5'-adenosine triphosphate (ATP) and two equivalents of S-adenosyl-l-methionine (SAM). This short but co-substrate-intensive pathway requires nucleoside cofactor salvage to maintain high psilocybin production rates. We characterized the adenosine kinase (AdoK) and S-adenosyl-l-homocysteine (SAH) hydrolase (SahH) of Psilocybe cubensis. Both enzymes are directly or indirectly involved in regenerating SAM. qRT-PCR expression analysis revealed an induced expression of the genes in the fungal primordia and carpophores. A one-pot in vitro reaction with the N-methyltransferase PsiM of the psilocybin pathway demonstrates a concerted action with SahH to facilitate biosynthesis by removal of accumulating SAH.

Enzymatic Route toward 6-Methylated Baeocystin and Psilocybin.

Psilocybin and its direct precursor baeocystin are indole alkaloids of psychotropic Psilocybe mushrooms. The pharmaceutical interest in psilocybin as a treatment option against depression and anxiety is currently being investigated in advanced clinical trials. Here, we report a biocatalytic route to synthesize 6-methylated psilocybin and baeocystin from 4-hydroxy-6-methyl-l-tryptophan, which was decarboxylated and phosphorylated by the Psilocybe cubensis biosynthesis enzymes PsiD and PsiK. N-Methylation was catalyzed by PsiM. We further present an in silico structural model of PsiM that revealed a well-conserved SAM-binding core along with peripheral nonconserved elements that likely govern substrate preferences.

Melleolides from Honey Mushroom Inhibit 5-Lipoxygenase via Cys159.

5-Lipoxygenase (5-LO) initiates the biosynthesis of pro-inflammatory leukotrienes from arachidonic acid, which requires the nuclear membrane-bound 5-LO-activating protein (FLAP) for substrate transfer. Here, we identified human 5-LO as a molecular target of melleolides from honey mushroom (Armillaria mellea). Melleolides inhibit 5-LO via an α,ß-unsaturated aldehyde serving as Michael acceptor for surface cysteines at the substrate entrance that are revealed as molecular determinants for 5-LO activity. Experiments with 5-LO mutants, where select cysteines had been replaced by serine, indicated that the investigated melleolides suppress 5-LO product formation via two distinct modes of action: (1) by direct interference with 5-LO activity involving two or more of the cysteines 159, 300, 416, and 418, and (2) by preventing 5-LO/FLAP assemblies involving selectively Cys159 in 5-LO. Interestingly, replacement of Cys159 by serine prevented 5-LO/FLAP assemblies as well, implying Cys159 as determinant for 5-LO/FLAP complex formation at the nuclear membrane required for leukotriene biosynthesis.

Enzymatic Synthesis of Psilocybin.

Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production.

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.

Injury-induced biosynthesis of methyl-branched polyene pigments in a white-rotting basidiomycete.

A stereaceous basidiomycete was investigated with regard to its capacity to produce yellow pigments after physical injury of the mycelium. Two pigments were isolated from mycelial extracts, and their structures were elucidated by ESIMS and one- and two-dimensional NMR methods. The structures were identified as the previously undescribed polyenes (3Z,5E,7E,9E,11E,13Z,15E,17E)-18-methyl-19-oxoicosa-3,5,7,9,11,13,15,17-octaenoic acid (1) and (3E,5Z,7E,9E,11E,13E,15Z,17E,19E)-20-methyl-21-oxodocosa-3,5,7,9,11,13,15,17,19-nonaenoic acid (2). Stable-isotope feeding with [1-(13)C]acetate and l-[methyl-(13)C]methionine demonstrated a polyketide backbone and that the introduction of the sole methyl branch is most likely S-adenosyl-l-methionine-dependent. Dose-dependent inhibition of Drosophila melanogaster larval development was observed with both polyenes in concentrations between 12.5 and 100 µM. GI50 values for 1 and 2 against HUVEC (K-562 cells) were 71.6 and 17.4 µM (15.4 and 1.1 µM), respectively, whereas CC50 values for HeLa cells were virtually identical (44.1 and 45.1 µM).

Melleolides induce rapid cell death in human primary monocytes and cancer cells.

The melleolides are structurally unique and bioactive natural products of the basidiomycete genus Armillaria. Here, we report on cytotoxic effects of melleolides from Armillaria mellea towards non-transformed human primary monocytes and human cancer cell lines, respectively. In contrast to staurosporine or pretubulysin that are less cytotoxic for monocytes, the cytotoxic potency of the active melleolides in primary monocytes is comparable to that in cancer cells. The onset of the cytotoxic effects of melleolides was rapid (within <1 h), as compared to the apoptosis inducer staurosporine, the protein biosynthesis inhibitor cycloheximide, and the DNA transcription inhibitor actinomycin D (>5 h, each). Side-by-side comparison with the detergent triton X-100 and staurosporine in microscopic and flow cytometric analysis studies as well as analysis of the viability of mitochondria exclude cell lysis and apoptosis as relevant or primary mechanisms. Our results rather point to necrotic features of cell death mediated by an as yet elusive but rapid mechanism.

In vitro cytotoxicity of melleolide antibiotics: structural and mechanistic aspects.

Melleolide sesquiterpene aryl esters are secondary products of the mushroom genus Armillaria. We compared the cytotoxicity of eleven melleolides--five thereof are new natural products--against four human cancer cell lines. Armillaridin, 4-O-methylarmillaridin, and dehydroarmillylorsellinate were most active, at IC(50) = 3.0, 4.1 and 5.0 µM, respectively, against Jurkat T cells for the former two compounds, and K-562 cells for the latter. Dehydroarmillylorsellinate did not inhibit respiration and RNA-synthesis of K-562 cells at 5 µM. However, replication of DNA dropped to 35% after 120 min at this concentration, and translational activity also decreased.

Structure and cytotoxicity of arnamial and related fungal sesquiterpene aryl esters.

We report on the structure elucidation of arnamial, a new Delta(2,4)-protoilludane everninate ester from the fungus Armillaria mellea, and on the apoptotic activity of arnamial as well as the cytotoxic activity of structurally related compounds on selected human cancer cells. Arnamial showed cytotoxicity against Jurkat T cells, MCF-7 breast adenocarcinoma, CCRF-CEM lymphoblastic leukemia, and HCT-116 colorectal carcinoma cells at IC50 = 3.9, 15.4, 8.9, and 10.7 microM, respectively, and the related aryl ester melledonal C showed cytotoxic activity against CCRF-CEM cells (IC50 = 14.75 microM). [1,2-13C2]Acetate feeding supports a polyketide origin of the orsellinic acid moiety of arnamial.

The Aspergillus nidulans enzyme TdiB catalyzes prenyltransfer to the precursor of bioactive asterriquinones.

The asterriquinones represent a class of ascomycete metabolic products whose significance stems from remarkable and useful pharmacological activities, among those antiretroviral (e.g., against the HI-virus), antitumor, and antidiabetes properties. Recently, the first genetic locus for an asterriquinone, the clustered terrequinone genes tdiA-E, was identified during a genome-wide screen in Aspergillus nidulans for "orphan" natural product biosynthesis loci. Here, we describe overexpression and characterization of TdiB, which catalyzes the reverse prenylation event during terrequinone A biosynthesis, which is the transfer of dimethylallyl diphosphate to carbon atom 2' of the intermediate didemethylasterriquinone D, to yield asterriquinone C-1. TdiB does not depend on the presence of divalent metal cations for catalysis and lacks the canonical prenyl diphosphate binding motif (D/N)DXXD.

Accurate prediction of the Aspergillus nidulans terrequinone gene cluster boundaries using the transcriptional regulator LaeA.

LaeA is a regulatory protein of the genus Aspergillus which controls global expression of secondary metabolism gene clusters. During a previous genome-wide transcriptional profiling screen, numerous novel gene clusters were found to be regulated by LaeA including a cluster required for the production of the antitumor agent terrequinone A. Beginnings and ends of secondary metabolite gene clusters are difficult to predict by gene sequence alone, but sharp demarcation of transcriptional control by LaeA suggested this protein might present a tool to identify cluster ends. To address this possibility in a first test case, we created null mutants of three genes, one regulated and two not regulated by LaeA, lying at the first, sixth and seventh position of a contiguous seven gene region where the third gene in this continuum was already shown to be required for terrequinone A production. Only the gene regulated by LaeA, tdiE, sixth in the series, was required for terrequinone A. In this pilot experiment, these findings experimentally validate LaeA-based predictions of the terrequinone biosynthetic locus boundaries. Further, this study identifies TdiE as a protein of unknown yet essential function for terrequinone A biosynthesis.

Fungal genetics, genomics, and secondary metabolites in pharmaceutical sciences.

Filamentous fungi produce a plethora of bioactive natural products. These metabolites display a broad range of useful activities for pharmaceutical purposes, exemplified best by the antibiotic penicillin. Yet, many more have been isolated, characterised, and tested, and some have made their way in clinical trials and into pharmaceutical practice. Through genomics, we become increasingly aware that the biosynthetic abilities for natural products are much richer than expected. The first part of our review highlights selected metabolites that filamentous fungi offer to pharmacists for drug development. This is followed by a summary on the potential of fungal genetics and genomics for pharmaceutical sciences and natural product research.

Genomic mining for Aspergillus natural products.

The genus Aspergillus is renowned for its ability to produce a myriad of bioactive secondary metabolites. Although the propensity of biosynthetic genes to form contiguous clusters greatly facilitates assignment of putative secondary metabolite genes in the completed Aspergillus genomes, such analysis cannot predict gene expression and, ultimately, product formation. To circumvent this deficiency, we have examined Aspergillus nidulans microarrays for expressed secondary metabolite gene clusters by using the transcriptional regulator LaeA. Deletion or overexpression of laeA clearly identified numerous secondary metabolite clusters. A gene deletion in one of the clusters eliminated the production of the antitumor compound terrequinone A, a metabolite not described, from A. nidulans. In this paper, we highlight that LaeA-based genome mining helps decipher the secondary metabolome of Aspergilli and provides an unparalleled view to assess secondary metabolism gene regulation.

Generation of novel landomycins M and O through targeted gene disruption.

Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.

The C-Glycosyltransferase UrdGT2 is unselective toward d- and l-configured nucleotide-bound rhodinoses.

UrdGT2 is a d-olivosyltransferase from the metabolic pathway of urdamycin A, an angucycline antitumor and antimicrobial drug. The remarkable feature of this biocatalyst is its ability to set up C-glycosidic bonds. Using an in vivo system suitable to deliver the trideoxysugar rhodinose in both d- and l- configuration we could verify that both have been accepted as substrates and attached to the urdamycin polyketide backbone via a C-glycosidic bond. Regardless of the stereochemistry, these C-glycosides served as acceptor for a subsequent glycosylation step to yield the novel urdamycins R and S with di-rhodinosyl side chains at C-9 of the polyketide moiety.