Analysis of Rhizonin Biosynthesis Reveals Origin of Pharmacophoric Furylalanine Moieties in Diverse Cyclopeptides.

Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3-furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non-ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l-DOPA as Fua precursors and provided the first mechanistic insight. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme-dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining.

High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space.

Coenzyme F420 is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F420-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F420 levels are produced in microorganisms, F420 availability is a serious bottleneck for research and application. Recent advances in our understanding of the F420 biosynthesis enabled heterologous overproduction of F420 in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F420 biosynthesis in E. coli. However, it still led to the production of low amounts of F420 and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module ("core") addressed parts of the riboflavin biosynthesis pathway and FO synthase for the conversion of GTP to the stable F420 intermediate FO. The enzymes of the second module ("decoration") were chosen to turn FO into F420. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F420-derived fluorescence. This approach yielded the highest titer of coenzyme F420 produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F420-dependent bioprocesses as well as applications of F420-dependent enzymes in biocatalysis.

Bacteria-induced production of the antibacterial sesquiterpene lagopodin B in Coprinopsis cinerea.

Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase-encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell-free supernatants of axenic cultures of the Gram-positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram-positive bacteria including B. subtilis but not Gram-negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite.

Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes.

Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420 Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future.IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges.

The as-yet uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina" live associated with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum "Tectomicrobia," Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca Entotheonella phylotypes contain numerous additional genes for as-yet unknown metabolites. Here, we provide insights into the biology of these remarkable bacteria using genomic, (meta)proteomic, and chemical methods. The data suggest a metabolic model of Ca Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F420 The latter likely accounts for the strong autofluorescence of Ca Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of organic molecules indicates roles in host-bacterial interaction. In addition, a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chemically most prolific microorganisms known from microbial dark matter.

Injury-Triggered Blueing Reactions of Psilocybe "Magic" Mushrooms.

Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ 13 C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.

Multi-genome analysis identifies functional and phylogenetic diversity of basidiomycete adenylate-forming reductases.

Among the invaluable benefits of basidiomycete genomics is the dramatically enhanced insight into the potential capacity to biosynthesize natural products. This study focuses on adenylate-forming reductases, which is a group of natural product biosynthesis enzymes that resembles non-ribosomal peptide synthetases, yet serves to modify one substrate, rather than to condense two or more building blocks. Phylogenetically, these reductases fall in four classes. The phylogeny of Heterobasidion annosum (Russulales) and Serpula lacrymans (Boletales) adenylate-forming reductases was investigated. We identified a previously unrecognized phylogenetic branch within class III adenylate-forming reductases. Three representatives were heterologously produced and their substrate preferences determined in vitro: NPS9 and NPS11 of S. lacrymans preferred l-threonine and benzoic acid, respectively, while NPS10 of H. annosum accepted phenylpyruvic acid best. We also investigated two class IV adenylate-forming reductases of Coprinopsis cinerea, which each were active with l-alanine, l-valine, and l-serine as substrates. Our results show that adenylate-forming reductases are functionally more diverse than previously recognized. As none of the natural products known from the species investigated in this study includes the identified substrates of their respective reductases, our findings may help further explore the diversity of these basidiomycete secondary metabolomes.

Precursor-Directed Diversification of Cyclic Tetrapeptidic Pseudoxylallemycins.

Cyclic peptides containing non-proteinogenic amino acids often exhibit a broad bioactivity spectrum and many have entered clinical trials with good prospects for drug development. We recently reported the discovery of six cyclic tetrapeptides, pseudoxylallemycins A-F (1-6), from a termite-associated Pseudoxylaria sp. X802. These compounds contain a rare O-homoallenyl-l-tyrosine moiety and show promising antimicrobial activity against the Gram-negative pathogenic bacterium Pseudomonas aeruginosa. To perform more detailed structure-activity studies, we pursued a precursor-directed diversification strategy. Herein, we report the purification, identification, and testing of 21 new pseudoxylallemycin derivatives.

A Highly Conserved Basidiomycete Peptide Synthetase Produces a Trimeric Hydroxamate Siderophore.

The model white-rot basidiomycete, Ceriporiopsis (Gelatoporia) subvermispora B, encodes putative natural product biosynthesis genes. Among them is the gene for the seven-domain nonribosomal peptide synthetase CsNPS2. It is a member of the as-yet-uncharacterized fungal type VI siderophore synthetase family, which is highly conserved and widely distributed among the basidiomycetes. These enzymes include only one adenylation (A) domain, i.e., one complete peptide synthetase module, and two thiolation/condensation (T-C) didomain partial modules which together constitute an AT1C1T2C2T3C3 domain setup. The full-length CsNPS2 enzyme (274.5 kDa) was heterologously produced as a polyhistidine fusion in Aspergillus niger as a soluble and active protein. N 5-acetyl-N 5-hydroxy-l-ornithine (l-AHO) and N 5-cis-anhydromevalonyl-N 5 -hydroxy-l-ornithine (l-AMHO) were accepted as the substrates, based on results of an in vitro substrate-dependent [32P]ATP-pyrophosphate radioisotope exchange assay. Full-length holo-CsNPS2 catalyzed amide bond formation between three l-AHO molecules to release the linear l-AHO trimer, called basidioferrin, as the product in vitro, which was verified by liquid chromatography-high-resolution electrospray ionization-mass spectrometry analysis. Phylogenetic analyses suggested that type VI family siderophore synthetases are widespread in mushrooms and evolved in a common ancestor of basidiomycetes.IMPORTANCE The basidiomycete nonribosomal peptide synthetase CsNPS2 represents a member of a widely distributed but previously uninvestigated class (type VI) of fungal siderophore synthetases. Genes orthologous to CsNPS2 are highly conserved across various phylogenetic clades of the basidiomycetes. Hence, our work serves as a broadly applicable model for siderophore biosynthesis and iron metabolism in higher fungi. Also, our results on the amino acid substrate preference of CsNPS2 support a further understanding of the substrate selectivity of fungal adenylation domains. Methodologically, this report highlights the Aspergillus niger/SM-Xpress-based system as a suitable platform to heterologously express multimodular basidiomycete biosynthesis enzymes in the >250-kDa range in soluble and active form.

Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins.

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors.

Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose-degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition 'toolbox' has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

A Lanthipeptide-like N-Terminal Leader Region Guides Peptide Epimerization by Radical SAM Epimerases: Implications for RiPP Evolution.

Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes.

Activity of α-Aminoadipate Reductase Depends on the N-Terminally Extending Domain.

L-α-Aminoadipic acid reductases catalyze the ATP- and NADPH-dependent reduction of L-α-aminoadipic acid to the corresponding 6-semialdehyde during fungal L-lysine biosynthesis. These reductases resemble peptide synthetases with regard to their multidomain composition but feature a unique domain of elusive function--now referred to as an adenylation activating (ADA) domain--that extends the reductase N-terminally. Truncated enzymes based on NPS3, the L-α-aminoadipic acid reductase of the basidiomycete Ceriporiopsis subvermispora, lacking the ADA domain either partially or entirely were tested for activity in vitro, together with an ADA-adenylation didomain and the ADA domainless adenylation domain. We provide evidence that the ADA domain is required for substrate adenylation: that is, the initial step of the catalytic turnover. Our biochemical data are supported by in silico modeling that identified the ADA domain as a partial peptide synthetase condensation domain.

Structure, genetics and function of an exopolysaccharide produced by a bacterium living within fungal hyphae.

The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.

Genetic engineering activates biosynthesis of aromatic fumaric acid amides in the human pathogen Aspergillus fumigatus.

The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

Functional and phylogenetic divergence of fungal adenylate-forming reductases.

A key step in fungal L-lysine biosynthesis is catalyzed by adenylate-forming L-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized L-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.

Biosynthesis of the halogenated mycotoxin aspirochlorine in koji mold involves a cryptic amino acid conversion.

Aspirochlorine (1) is an epidithiodiketopiperazine (ETP) toxin produced from koji mold (Aspergillus oryzae), which has been used in the oriental cuisine for over two millennia. Considering its potential risk for food safety, we have elucidated the molecular basis of aspirochlorine biosynthesis. By a combination of genetic and chemical analyses we found the acl gene locus and identified the key role of AclH as a chlorinase. Stable isotope labeling, biotransformation, and mutational experiments, analysis of intermediates and an in vitro adenylation domain assay gave totally unexpected insights into the acl pathway: Instead of one Phe and one Gly, two Phe units are assembled by an iterative non-ribosomal peptide synthetase (NRPS, AclP), followed by halogenation and an unprecedented Phe to Gly amino acid conversion. Biological assays showed that both amino acid transformations are required to confer cytotoxicity and antifungal activity to the mycotoxin.

Deazaflavin metabolite produced by endosymbiotic bacteria controls fungal host reproduction.

The endosymbiosis between the pathogenic fungus Rhizopus microsporus and the toxin-producing bacterium Mycetohabitans rhizoxinica represents a unique example of host control by an endosymbiont. Fungal sporulation strictly depends on the presence of endosymbionts as well as bacterially produced secondary metabolites. However, an influence of primary metabolites on host control remained unexplored. Recently, we discovered that M. rhizoxinica produces FO and 3PG-F420, a derivative of the specialized redox cofactor F420. Whether FO/3PG-F420 plays a role in the symbiosis has yet to be investigated. Here, we report that FO, the precursor of 3PG-F420, is essential to the establishment of a stable symbiosis. Bioinformatic analysis revealed that the genetic inventory to produce cofactor 3PG-F420 is conserved in the genomes of eight endofungal Mycetohabitans strains. By developing a CRISPR/Cas-assisted base editing strategy for M. rhizoxinica, we generated mutant strains deficient in 3PG-F420 (M. rhizoxinica ΔcofC) and in both FO and 3PG-F420 (M. rhizoxinica ΔfbiC). Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is maintained upon reinfection with wild-type M. rhizoxinica or M. rhizoxinica ΔcofC. In contrast, R. microsporus is unable to sporulate when co-cultivated with M. rhizoxinica ΔfbiC, even though the fungus was observed by super-resolution fluorescence microscopy to be successfully colonized. Genetic and chemical complementation of the FO deficiency of M. rhizoxinica ΔfbiC led to restoration of fungal sporulation, signifying that FO is indispensable for establishing a functional symbiosis. Even though FO is known for its light-harvesting properties, our data illustrate an important role of FO in inter-kingdom communication.

Bimodular peptide synthetase SidE produces fumarylalanine in the human pathogen Aspergillus fumigatus.

The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[(32)P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus.