Fungi produce numerous low molecular weight molecules endowed with a multitude of biological activities. However, mining the fullgenome sequences of fungi indicates that their potential to produce secondary metabolites is greatly underestimated. Because most of the biosynthesis gene clusters are silent under laboratory conditions, one of the major challenges is to understand the physiological conditions under which these genes are activated. Thus, we cocultivated the important model fungus Aspergillus nidulans with a collection of 58 soil-dwelling actinomycetes. By microarray analyses of both Aspergillus secondary metabolism and full-genome arrays and Northern blot and quantitative RT-PCR analyses, we demonstrate at the molecular level that a distinct fungal-bacterial interaction leads to the specific activation of fungal secondary metabolism genes. Most surprisingly, dialysis experiments and electron microscopy indicated that an intimate physical interaction of the bacterial and fungal mycelia is required to elicit the specific response. Gene knockout experiments provided evidence that one induced gene cluster codes for the long-sought after polyketide synthase (PKS) required for the biosynthesis of the archetypal polyketide orsellinic acid, the typical lichen metabolite lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B. A phylogenetic analysis demonstrates that orthologs of this PKS are widespread in nature in all major fungal groups, including mycobionts of lichens. These results provide evidence of specific interaction among microorganisms belonging to different domains and support the hypothesis that not only diffusible signals but intimate physical interactions contribute to the communication among microorganisms and induction of otherwise silent biosynthesis genes.genome mining ͉ lecanoric acid ͉ orsellinic acid ͉ Streptomyces

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Filamentous fungi produce a multitude of bioactive natural products, which cover a broad range of useful pharmaceutical activities. Many of these compounds were discovered by traditional natural product screening approaches and have found various applications in modern medicine. 1,2 Through recent whole-genome sequencing projects, however, we become increasingly aware that the biosynthetic potential of microorganisms is much higher than expected. 3 In many cases, the number of putative biosynthetic genes of fungi and bacteria is not reflected by the metabolic profile observed under laboratory culture conditions. Several gene loci encoding diverse metabolic pathways seem to lack expression in the absence of particular physical or chemical stimuli. 4 Mining the genome of Aspergillus nidulans for putative biosynthesis gene clusters revealed biosynthetic abilities for the production of up to 28 polyketides and 24 nonribosomally synthesized peptides. 5,6 However, this abundance of gene clusters clearly outnumbers the known secondary metabolites of this fungus. To gain access to this untapped reservoir of potentially bioactive natural products, various strategies to induce the expression of silent genes have been developed. 4,7 Important recent examples involve the ectopic expression of a regulatory gene, 8 the induction of a transcription activator by promotor exchange, 9 as well as modulation of the epigenetic regulation of biosynthetic genes at the chromatin level. [10][11][12][13] Furthermore, it was shown that the intimate contact between A. nidulans and a soil-dwelling actinomycete may lead to the specific induction of a cryptic polyketide gene cluster. 14 These strategies resulted in the discovery of a set of novel secondary metabolites, which could not be located by classical screening methods. Another option often preceding genomic approaches is the systematic investigation of the microbial secondary metabolome under various growth conditions. Since the early days of fermentations, it is known that the choice of the cultivation parameters is critical to the number and type of secondary metabolites produced by microorganisms. 4 Thus, the formation of cryptic natural products can up to a certain extent be triggered by a systematic variation of standard fermentation parameters to increase the number of secondary compounds produced in the bacterial or fungal culture. 15,16 On the basis of the assumption that changing environmental conditions can shift the metabolic profile of an organism, we systematically varied the cultivation parameters to reinvestigate the metabolome of A. nidulans. In this study, we report the discovery of two new isoindole alkaloids, aspernidine A (1) and B (2), as an addition to the metabolic data of this important model fungus (Figure 1a). An A. nidulans extract library was prepared by adjusting 45 different culture conditions (variation of culture media, cultivation period, temperature and oxygen supply) and the metabolic profiles were screened by HPLC-DAD-MS. Investigation of the cultur...

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