Activation of a silent fungal polyketide biosynthesis pathway through regulatory cross talk with a cryptic nonribosomal peptide synthetase gene cluster.

Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion of the asperfuranone activator gene afoA, indicating that ScpR binds to the afoA promoter. To the best of our knowledge, this is the first report of regulatory cross talk between two biosynthesis gene clusters located on different chromosomes.

Activation of fungal silent gene clusters: a new avenue to drug discovery.

The ongoing exponential growth of DNA sequence data will lead to the discovery of many natural-product biosynthesis pathways by genome mining for which no actual product has been characterised. In many cases, these clusters remain silent under laboratory conditions. New technologies based on genetic engineering are available to induce silent genes. Heterologous expression of a silent gene cluster under the control of defined promoters can be applied. Alternatively, promoters of biosynthesis genes within the genome can be exchanged by defined promoters. Most promising, however, is the activation of pathway-specific regulatory genes, which was recently demonstrated. Such regulatory genes are present in many secondary metabolite gene clusters. This approach is rendered feasible by the fact that all of the genes encoding the large number of enzymes required for the synthesis of a typical secondary metabolite are clustered and that in some cases, a single regulator controls the expression of all members of a gene cluster to a certain extent. The advantage of this technique is that only a small gene needs to be handled, and that an ectopic integration is sufficient, bypassing all limitations of homologous recombination. Most conveniently, this strategy can trigger the concerted expression of all pathway genes. The vast amount of DNA sequences in the public database represents only the beginning of this new genomics era. The activation of these gene clusters by genetic engineering will lead to the discovery of many so far unknown products and therefore represents a novel avenue to drug discovery.

Interaction of HapX with the CCAAT-binding complex--a novel mechanism of gene regulation by iron.

Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron-depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.

Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans.

In the postgenomic era it has become increasingly apparent that the vast number of predicted biosynthesis genes of microorganisms is not reflected by the metabolic profile observed under standard fermentation conditions. In the absence of a particular (in most cases unknown) trigger these gene loci remain silent. Because these cryptic gene clusters may code for the biosynthesis of important virulence factors, toxins, or even drug candidates, new strategies for their activation are urgently needed to make use of this largely untapped reservoir of potentially bioactive compounds. The discovery of new microbial metabolites through genome mining has proven to be a very promising approach. Even so, the investigation of silent gene clusters is still a substantial challenge, particularly in fungi. Here we report a new strategy for the successful induction of a silent metabolic pathway in the important model organism Aspergillus nidulans, which led to the discovery of novel PKS-NRPS hybrid metabolites.

PAR-type thrombin receptors in renal carcinoma cells: PAR1-mediated EGFR activation promotes cell migration.

Cross-talk between G-protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signaling systems is established in a wide variety of normal and neoplastic cell types. Here, we show that proteinase-activated receptor 1 (PAR1) mediates the tyrosine phosphorylation of EGFR in human renal carcinoma cells expressing PAR1 and PAR3 endogeneously. This GPCR-EGFR signal transduction pathway cross-talk requires matrix metalloproteinase activity and is involved in the regulation of renal carcinoma cell migration across a collagen barrier as shown using a Boyden chamber type assay. Our data therefore document a regulatory role of PAR1-mediated EGFR transactivation in cancer cell chemotactic migration. Further, our results underline the importance of PAR1-mediated pathways in kidney cancer cells and suggest that the thrombin/PAR1 system mediating EGFR transactivation may play a role in the progression of this tumor entity.