DOI: 10.1002/cbic.200700202 Identification of a Hybrid PKS/NRPS Required for Pseurotin A Biosynthesis in the Human Pathogen Aspergillus fumigatus Shubha Maiya, [a] Alexander Grundmann, [b] Xiang Li, [c] Shu-Ming Li, [b] and Geoffrey Turner* [a] Introduction Filamentous fungi produce a wide range of bioactive secon- dary metabolites, some of which have been exploited commer- cially. [1] They include peptides and polyketides that result from the activity of complex, multidomain enzymes, non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). Re- cently, genome-sequencing projects for Aspergillus fumigatus and other filamentous fungi have revealed the presence of ap- proximately 30 to 50 gene clusters per species, which include genes that encode NRPS and PKS [2] (fungal genome resource list at http://www.fgsc.net). The products of some of these pre- viously unknown clusters have been subsequently determined by gene deletion or overexpression. [3,4,5,6] Many of the filamen- tous fungi sequenced to date also contain at least one gene that encodes a “hybrid” enzyme possessing domains typical of both PKS and NRPS. While such hybrid genes were described some time ago for bacteria, [7] only recently have the products of a few of the predicted fungal hybrid PKS/NRPS genes been determined. These include fusA, which is required for synthesis of a precursor of fusarin C in Fusarium moniliforme and Fusari- um venenatum, [8] a gene required for equisetin biosynthesis in Fusarium heterosporum [9] and a gene that encodes tenellin syn- thetase from Beauveria bassiana. [10] The products of the synthe- tases described to date are 2-pyrrolidinones (tetramic acids). In most cases, fungal genes encoding PKS/NRPS hybrids appear to be paralogues rather than orthologues, and are therefore likely to be making different products, despite similarities in domain structure. Aspergillus fumigatus Af293, the genome reference strain, has a single gene of this type, Afu8g00540, and here we pres- ent evidence that it is involved in the biosynthesis of pseuro- ACHTUNGTRENNUNGtin A. Results Bioinformatics Pseurotin A (1) was originally isolated from Pseudoeurotium ovalis. [11] Together with 8-O-demethylpseurotin A (2) and syner- azol (3), it was also isolated from A. fumigatus. [12,13,14] Azaspir- ene (4), with the same core structure, was obtained from ACHTUNGTRENNUNGNeosartorya sp. [15] Pseurotin A is derived from l-phenylalanine, propionate, malonate and l-methionine, as proven by feeding experiments with 13 C-labelled precursors. [16] The structure and biosynthetic origin of pseurotin A sug- gests the involvement of a hybrid PKS/NRPS. Only one PKS/ NRPS (Afu8g00540) is present in A. fumigatus genome within a cluster of five genes suggestive of secondary metabolism (Figure 1). The putative product of Afu8g00540 is a multido- main protein with b-ketoacylsynthase, acyl transferase, short- chain dehydrogenase, condensation, adenylation, thiolation and dehydrogenase domains. An orthologous gene cluster is also found in A. clavatus but not in the closer relative A. fischeri (W. Nierman, personal communication). Afu8g00540 could be involved in the biosynthesis of pseurotin A. However, an addi- tional gene, Afu8g00620, is located in the genome of A. fumi- [a] S. Maiya, Prof. G. Turner University of Sheffield, Department of Molecular Biology and Biotechnology S10 2TN, Sheffield (UK) Fax:(+ 44)114-222-2800 E-mail:g.turner@sheffield.ac.uk [b] A. Grundmann, Prof. Dr. S.-M. Li Heinrich-Heine-Universität Düsseldorf Institut für Pharmazeutische Biologie und Biotechnologie Universitätsstrasse 1, 40225 Düsseldorf (Germany) [c] Dr. X. Li Agriculture and Agri-Food Canada, Saskatoon Research Centre 107 Science Cresc., Saskatoon, Saskatchewan (Canada) The genome sequence of Aspergillus fumigatus revealed the presence of a single hybrid polyketide synthase–non-ribosomal peptide synthetase (PKS/NRPS) gene that is present within a clus- ter of five genes suggestive of its involvement in secondary me- tabolism. Here, we present evidence that it is required for the bio- synthesis of pseurotin A, a compound with an unusual heterospir- ocyclic g-lactam structure. We have confirmed that the genome reference strain A. fumigatus Af293 produces pseurotin A, a com- pound previously reported to be a competitive inhibitor of chitin synthase and an inducer of nerve-cell proliferation. Deletion or overexpression of the PKS/NRPS gene psoA in A. fumigatus leads to the absence or accumulation of pseurotin A, respectively; this indicates that this gene is essential for the biosynthesis of pseuro- tin A. It is likely that the first product of psoA is converted to pseurotin A by the products of other genes in this cluster. 1736 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2007,8,1736–1743