1. What can genomics tell us about secondary metabolism in Aspergillus? Geoffrey Turner University of Sheffield

2. gibberellin GA3 penicillin G fusarin C lovastatin aristolochene gliotoxin H fumitremorgin C cyclosporin ergopeptides aflatoxin B1WA trichothecene T2 toxin Peptides Polyketides Terpenes Alkaloids A. nidulans

3. gibberellin GA3 penicillin G fusarin C lovastatin aristolochene gliotoxin H fumitremorgin C cyclosporin ergopeptides aflatoxin B1WA trichothecene T2 toxin Peptides Polyketides Terpenes Alkaloids A. fumigatus

4. Fungal secondary metabolites Non-ribosomal peptides (NRPS)Terpenes -lactam antibioticsTrichodiene - toxin Cyclosporin – immunosuppressantAristolochene - toxin Echinocandin – antifungal drug Polyketides (PKS) Lovastatin – cholesterol lowering agent Aflatoxin – carcinogen Indole alkaloids Ergotamine – migraine treatment – control of post partum bleeding

5. Public genome sequencing projects in filamentous fungi: Broad Institute Neurospora crassa – completed and published 2003 Aspergillus nidulans – completed and annotated 2003 Other genomes 2003-5 Podospora anserina Magnaporthe grisea Fusarium graminearum Cryptococcus neoformans Ustilago maydis Coprinus cinereusPodospora anserinaMagnaporthe griseaFusarium graminearumCryptococcus neoformansUstilago maydisCoprinus cinereus Rhizopus oryzae TIGR/Sanger Centre Aspergillus fumigatus – completed 2004 Japanese consortium Aspergillus oryzae – completed 2004

6. How many secondary metabolic pathways are there in a single species? What are they producing? Do these closely related species have orthologous pathway genes?

7. Automated annotation by sequencing institutes makes gene finding much easier: Multienzyme complexes like NRPS and PKS have recognisable domains Secondary metabolic pathway genes tend to be clustered

8. NRPS

9. A trimodular NRPS: ACVS Synthetase Amino acids attached to enzyme via pantotheine arm α-aminoadipate cysteine valine

10. adenylationN-methylationthioester formation ACV synthetase Cyclosporin synthetase Non-ribosomal peptide synthetase (NRPS): large multifunctional enzymes with a modular structure - One module per amino acid Recent record: Peptaibol synthetase: 18 modules Trichoderma virens (Kenerly 2002)2.3 Mda

11. Fungal PKS architecture (Type I iterative) KSATACP(CYC) or (TE) (DH) (MT)(ER)(KR) -Ketoacyl Synthase Acyltransferase Dehydratase Methyltransferase -Keto Reductase Enoyl Reductase Cyclase Thioreductase Domains:

12. Aspergillus nidulans annotated genome (Broad Institute) 5 module NRPSPKS

13. Aspergillus nidulans annotated genome (Broad Institute) PKS

14. Aspergillus nidulans annotated genome (Broad Institute) Hybrid PKS-NRPS

15. Gene typeA. oryzaeA. fumigatusA. nidulans PKS301427 NRPS1814 FAS516 Sesquiterpene cyclase 1not detected1 DMATS272 Summary of secondary metabolic gene classes in Aspergillus

16. A. fumigatus produces many toxic secondary metabolites including peptide derivatives and indole alkaloids Gliotoxin Tryprostatins Fumitremorgens Fumigaclavines Can we identify the gene clusters responsible?

17. Tryptophan Proline NRPS? DMAT synthetase Tryptophan DMAT synthetase (X2) Serine Phenylalanine 2 module NRPS? Terpene Sesquiterpene cyclase Gliotoxin Fumagillin Fumigaclavines Fumitremorgens

18. Gardiner et al. Mol. Microbiol. (2004) Sirodesmin cluster of Leptophaeria maculans Sirodesmin Gliotoxin Putative A. fumigatus gliotoxin cluster identified in genome by homology: Includes 2 module NRPS

19. Tryptophan Proline NRPS? DMAT synthetase Tryptophan DMAT synthetase (X2) Serine Phenylalanine NRPS? Terpene Sesquiterpene cyclase Gliotoxin Fumagillin Fumigaclavines Fumitremorgens

20. FUMIGACLAVINE BIOSYNTHESIS Dimethylallyl-tryptophan

22. Gene typeA. oryzaeA. fumigatusA. nidulans PKS301427 NRPS1814 FAS516 Sesquiterpene cyclase 1(1) DMATS272 DMATS hits probably reflect a variety of prenyltransferases - add dimethylallyl side chain to a variety of compounds

23. monooxygenase- related protein Hypothetical protein NADP- dehydrogenase Conserved hypothetical protein Pisatin demethylase, putative Putative dimethyl- allyl-tryptophan- synthetase Oxidoreductase Cp ox2 Cytochrome P450, monooxygenase Acetyl transferase Catalase Dimethylallyl trryptophan synthetase Oxidoreductase Cp ox1 Conservedd Hypothetical protein DMATII DMATII 72. M19886 shows 52 % identity with Claviceps purpurea DMAT gene. DMATIII 72. m19881 shows 30 % identity with Claviceps purpurea DMAT gene. Oxidoreductase 72.m19888 has best hit (43%) with C. purpurea ox1 NADPH dehydrogenase 19877 (cpox3?), one of a family, best hit with Claviceps, 57%. Catalase 19885, 72% with Claviceps cluster Short chain dehydrogenase, 19882, 60% with Claviceps cluster DMATIII

24. Deletion of DMATSII (= dmaW) leads to loss of fumigaclavine C Coyle & Panaccione 2005

25. Tryptophan Proline 2 module NRPS? DMAT synthetase Tryptophan DMAT synthetase (X2) Serine Phenylalanine 2 module NRPS? Terpene Sesquiterpene cyclase Gliotoxin Fumagillin Fumigaclavines Fumitremorgens

26. Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071 proline tryptophan Aspergillus fumigatus prenylated alkaloids; fumitremorgins tryprostatins NRPS? DMATS?

27. oxidoreductaseankyrindmats P450hydroxylase dmats O-methyltransferase hydroxylase peptide synthetase fum9 (hydroxylation) 2 module NRPSDMATS

28. oxidoreductaseankyrindmats P450hydroxylase dmats O-methyltransferase hydroxylase peptide synthetase fum9 (hydroxylation) 2 module NRPSDMATS

29. DMATS expressed in E. coli Enzyme can prenylate cyclo-Trp-Pro Grundmann & Li 2005

30. Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071 proline tryptophan Aspergillus fumigatus prenylated alkaloids; fumitremorgens tryprostatins NRPS? DMATS?

31. Dimodular NRPS of A. fumigatus Deletion Insertion of additional copies Introduce strong promoter (P alcA ) Express in naïve host – A. nidulans

32. Pathway/SpeciesA. oryzaeA. fumigatusA. nidulans Penicillin+-+ Siderophore Sid2+++ Siderophore SidC+++ Conidial pigment WA +++ Aflatoxin /sterigmatocystin +-+ Carotenoid+-- Orthologous secondary metabolic genes

33. alb-1 (=wA) Melanin cluster in A. fumigatus This cluster is not conserved in Ana/Aoa More like N. crassa melanin genes wA is in a conserved syntenic region in both species deletions do affect spore pigment colour pigment biosynthesis pathway may be different in Afu1

34. Any other orthologues amonst the PKS genes? Phylogeny done by Resham Kulkani at TIGR Using KS domain of PKS/Fatty Acid Synthase

35. wA/alb1 orthologues

36. PKS for aflatoxin in 2 species only

37. Possible orthologues amongst unknown PKS?

38. Hybrid PKS/NRPS KS-AT-CON-AMP-PP In all 3 species, but not really orthologues?

39. Conclusions Few true orthologues across the genus Aspergillus Each species has its own repertoire Orthologues often found in less closely related species Gene/product relationship requires functional analysis in most cases

40. Secondary metabolism – University of Sheffield Hala Mohamed Shubha Maiya Jackie Price David Keszenman-Pereyra Bioinformatics TIGR Broad Institute AIST-CBRC University of Tokyo Chemistry – University of Tubingen Alexander Grundmann Shuming Li