Heterologous expression system in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for fungal biosynthetic gene clusters of secondary metabolites

Fungal secondary metabolites have been considered promising resources in the search for novel bioactive compounds. Given the high potential of fungi as genetic resources, it is essential to find an efficient way to link biosynthetic genes to the product in a heterologous system, because many genes for the secondary metabolite in the original strain are silent under standard laboratory conditions. In a previous study, we constructed a heterologous expression system for a biosynthetic gene cluster using Aspergillus oryzae as the host. To make the host more versatile for the expression of secondary metabolism genes, the expression levels of a global regulator, laeA, were increased by placing the A. oryzae laeA gene under the control of the constitutive active pgk promoter. In the A. oryzae overexpressing laeA, two clusters of heterologous biosynthetic genes [the monacolin K (MK) gene cluster from Monascus pilosus and the terrequinone A (TQ) gene cluster from Aspergillus nidulans] were successfully overexpressed, resulting in the production of the corresponding metabolite, MK or TQ. The successful production of secondary metabolites belonging to different structural groups, namely MK as a polyketide and TQ as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene cluster.     

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.     

Chromatin‐level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum

Genes required for fungal secondary metabolite production are usually clustered, co‐regulated and expressed in stationary growth phase. Chromatin modification has an important role in co‐regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP‐qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin‐level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.     

VeA and MvlA repression of the cryptic orsellinic acid gene cluster in Aspergillus nidulans involves histone 3 acetylation

A perplexing aspect of fungal secondary metabolite gene clusters is that most clusters remain ‘silent’ under common laboratory growth conditions where activation is obtained through gene manipulation or encounters with environmental signals. Few proteins have been found involved in repression of silent clusters. Through multicopy suppressor mutagenesis, we have identified a novel cluster suppressor in Aspergillus nidulans, MvlA (m odulator of v eA l oss). Genetic assessment of MvlA mutants revealed the role of both itself and VeA (but not the VeA partner LaeA) in the suppression of the cryptic ors gene cluster producing orsellinic acid and its F9775 derivatives. Loss of veA upregulates F9775A and F9775B production and this increase is reduced 4–5‐fold when an overexpression mvlA (OE:mvlA) allele is introduced into the ΔveA background. Previous studies have implicated a positive role for GcnE (H3K9 acetyltransferase of the SAGA/ADA complex) in ors cluster expression and here we find expression of gcnE is upregulated in ΔveA and suppressed by OE:mvlA in the ΔveA background. H3K9 acetylation levels of ors cluster genes correlated with gcnE expression and F9775 production in ΔveA and OE:mvlAΔveA strains. Finally, deletion of gcnE in the ΔveA background abolishes ors cluster activation and F9775 production. Together, this work supports a role for VeA and MvlA in modifying SAGA/ADA complex activity.     

Carbendazim-resistance associated β2-tubulin substitutions increase deoxynivalenol biosynthesis by reducing the interaction between β2-tubulin and IDH3 in Fusarium graminearum

Microtubule is a well-known structural protein participating in cell division, motility and vesicle traffic. In this study, we found that β₂-tubulin, one of the microtubule components, plays an important role in regulating secondary metabolite deoxynivalenol (DON) biosynthesis in Fusarium graminearum by interacting with isocitrate dehydrogenase subunit 3 (IDH3). We found IDH3 negatively regulate DON biosynthesis by reducing acetyl-CoA accumulation in F. graminearum and DON biosynthesis was stimulated by exogenous acetyl-CoA. In addition, the expression of IDH3 significantly decreased in the carbendazim-resistant mutant nt167 (Fgβ₂^F167Y). Furthermore, we found that carbendazim-resistance associated β₂-tubulin substitutions reducing the interaction intensity between β₂-tubulin and IDH3. Interestingly, we demonstrated that β₂-tubulin inhibitor carbendazim can disrupt the interaction between β₂-tubulin and IDH3. The decreased interaction intensity between β₂-tubulin and IDH3 resulted in the decreased expression of IDH3, which can cause the accumulation of acetyl-CoA, precursor of DON biosynthesis in F. graminearum. Thus, we revealed that carbendazim-resistance associated β₂-tubulin substitutions or carbendazim treatment increases DON biosynthesis by reducing the interaction between β₂-tubulin and IDH3 in F. graminearum. Taken together, the novel findings give the new perspectives of β₂-tubulin in regulating secondary metabolism in phytopathogenic fungi.     

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM‐gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de‐repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein‐1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM‐specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.     

Activation and identification of five clusters for secondary metabolites in Streptomyces albus J1074

Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S. albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribosomal peptides (NRP) (indigoidine) and glycosylated compounds (paulomycins). By insertion of a strong and constitutive promoter in front of selected genes of two clusters, production of the blue pigment indigoidine and of two novel members of the polycyclic tetramate macrolactam family (6-epi-alteramides A and B) was activated. Overexpression of positive regulatory genes from the same organism also activated the biosynthesis of 6-epi-alteramides and heterologous expression of the regulatory gene pimM of the pimaricin cluster activated the simultaneous production of candicidins and antimycins, suggesting some kind of cross-regulation between both clusters. A cluster for glycosylated compounds (paulomycins) was also identified by comparison of the high-performance liquid chromatography profiles of the wild-type strain with that of a mutant in which two key enzymes of the cluster were simultaneously deleted.