Genome-Based Deletion Analysis Reveals the Prenyl Xanthone Biosynthesis Pathway in Aspergillus nidulans

Abstract

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.

Accurate prediction of secondary metabolite gene clusters in filamentous fungi

“…If a given detected metabolite is not the end product of the biosynthetic pathway, gene deletions will only identify a part of an SM cluster as being relevant for that metabolite, thus missing genes. An example of this is seen in the emodin/monodictyphenone cluster (PKS AN0150), where a subset of the genes is only required for some of the metabolites, resulting in a two-step elucidation of the gene cluster (7,8). The CS method correctly calls the full cluster.…”

“…The production of austinol and derived compounds (the meroterpenoid pathway) has been shown to be dependent on two separate clusters (11,30), and the biosynthesis of prenyl xanthones is dependent on three separate clusters (8). We were interested in seeing whether this is a general phenomenon and whether such cross-chromosomal "superclusters" could be detected using our expression data.…”

“…This effort has been especially noticeable in the model fungus Aspergillus nidulans, which is presently the fungal species with the largest number (n = 25) of characterized SM synthases/synthetases, due to a massive effort by several groups . In recent studies, this fungus has also been shown to have cross-chemistry between gene clusters on separate chromosomes (8,30). Although these reactions are highly interesting for combinatorial chemistry, the identification of gene clusters involved in cross-chemistry is cumbersome because it involves combinatorial deletion of SM synthetic genes, thus greatly increasing the potential number of candidates.…”

“…In agreement with this scenario, it is not uncommon that newly detected compounds are linked to known PKS pathways. For example, shamixanthones and arugusins were recently discovered to be products derived from the monodictyphenone cluster (8,11), and this cluster has been redefined several times (9,10). For the remaining case, the apdG gene of the aspyridone cluster (20), misprediction of the cluster members is due to a complete divergence between the transcription profiles of apdG and the remainder of the gene cluster.…”

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization

“…A large gene cluster is also involved in the production of monodictyphenone in A. nidulans, which consists of the core PKS gene mdpG and at least seven tailoring genes (7,8). In the latter case, it has been shown that these genes also belong to a three-loci supercluster that produces prenyl xanthones (10,12). Together, these examples illustrate how structurally related but functionally distinct compounds can result from the diversification of tailoring genes within a conserved biosynthetic gene cluster.…”