Terrein is a fungal metabolite with ecological, antimicrobial, antiproliferative, and antioxidative activities. Although it is produced by Aspergillus terreus as one of its major secondary metabolites, not much is known about its biosynthetic pathway. Here, we describe an unexpected discovery of the terrein biosynthesis gene locus made while we were looking for a PKS gene involved in production of conidia coloration pigments common for Aspergilli. The gene, ATEG_00145, here named terA, is essential for terrein biosynthesis and heterologous production of TerA in Aspergillus niger revealed an unusual plasticity in the products formed, yielding a mixture of 4-hydroxy-6-methylpyranone, orsellinic acid, and 6,7-dihydroxymellein. Biochemical and molecular genetic analyses indicate a low extension cycle specificity of TerA. Furthermore, 6-hydroxymellein was identified as a key intermediate in terrein biosynthesis. We find that terrein production is highly induced on plant-derived media, that terrein has phytotoxic activity on plant growth, and induces lesions on fruit surfaces.

Mining the genome of the pathogenic fungus Aspergillus terreus revealed the presence of an orphan polyketide-nonribosomal-peptide synthetase (PKS-NRPS) gene cluster. Induced expression of the transcriptional activator gene adjacent to the PKS-NRPS gene was not sufficient for the activation of the silent pathway. Monitoring gene expression, metabolic profiling, and using a lacZ reporter strain allowed for the systematic investigation of physiological conditions that eventually led to the discovery of isoflavipucine and dihydroisoflavipucine. Phytotoxin formation is only activated in the presence of certain amino acids, stimulated at alkaline pH, but strictly repressed in the presence of glucose. Global carbon catabolite repression by CreA cannot be abolished by positive-acting factors such as PacC and overrides the pathway activator. Gene inactivation and stable isotope labeling experiments unveiled the molecular basis for flavipucine/fruit rot toxin biosynthesis.

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.

Melanins are ubiquitous pigments found in all kingdoms of life. Most organisms use them for protection from environmental stress, although some fungi employ melanins as virulence determinants. The human pathogenic fungus Aspergillus fumigatus and related Ascomycetes produce dihydroxynaphthalene- (DHN) melanin in their spores, the conidia, and use it to inhibit phagolysosome acidification. However, biosynthetic origin of melanin in a related fungus, Aspergillus terreus, has remained a mystery because A. terreus lacks genes for synthesis of DHN-melanin. Here we identify genes coding for an unusual NRPS-like enzyme (MelA) and a tyrosinase (TyrP) that A. terreus expressed under conidiation conditions. We demonstrate that MelA produces aspulvinone E, which is activated for polymerization by TyrP. Functional studies reveal that this new pigment, Asp-melanin, confers resistance against UV light and hampers phagocytosis by soil amoeba. Unexpectedly, Asp-melanin does not inhibit acidification of phagolysosomes, thus likely contributing specifically to survival of A. terreus conidia in acidic environments.

Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts.