Decoding Secondary Metabolism in Aspergillus: A Genomics Perspective

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

2. H Peptides penicillin G gliotoxin cyclosporin Alkaloids A. nidulans ergopeptides fumitremorgin C Terpenes fusarin C lovastatin trichothecene T2 toxin aristolochene gibberellin GA3 aflatoxin B1 WA Polyketides

3. H Peptides penicillin G gliotoxin cyclosporin Alkaloids A. fumigatus ergopeptides fumitremorgin C Terpenes fusarin C lovastatin trichothecene T2 toxin aristolochene gibberellin GA3 aflatoxin B1 WA Polyketides

4. Fungal secondary metabolites Non-ribosomal peptides (NRPS)Terpenes b-lactam antibiotics Trichodiene - toxin Cyclosporin – immunosuppressant Aristolochene - 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 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. L-a-aminoadipic acid L-cysteine L-valine acvA ACV synthetase H SH H CH NH H N 3 2 CH 3 H O COOH NH COOH O H LLD-ACV tripeptide ipnA IPN synthase H H NH H N CH S 2 3 CH 3 H O COOH N O H COOH isopenicillin N penDE phenylacetyl CoA a-aminoadipic acid Acyl transferase CoA H H NH CH S 3 CH O 3 O N O H COOH penicillin V NRPS

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

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

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

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

13. Aspergillus nidulans annotated genome (Broad Institute) PKS

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

15. Gene type A. oryzae A. fumigatus A. nidulans PKS 30 14 27 NRPS 18 14 14 FAS 5 1 6 Sesquiterpene cyclase 1 not detected 1 DMATS 2 7 2 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. Serine Phenylalanine 2 module NRPS? Gliotoxin Terpene Sesquiterpene cyclase Fumagillin Tryptophan DMAT synthetase (X2) Fumigaclavines Tryptophan Proline NRPS? DMAT synthetase 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. Serine Phenylalanine NRPS? Gliotoxin Terpene Sesquiterpene cyclase Fumagillin Tryptophan DMAT synthetase (X2) Fumigaclavines Tryptophan Proline NRPS? DMAT synthetase Fumitremorgens

20. FUMIGACLAVINE BIOSYNTHESIS Dimethylallyl-tryptophan

22. Gene type A. oryzae A. fumigatus A. nidulans PKS 30 14 27 NRPS 18 14 14 FAS 5 1 6 Sesquiterpene cyclase 1 (1) (1) DMATS 2 7 2 “DMATS” hits probably reflect a variety of prenyltransferases - add dimethylallyl side chain to a variety of compounds

23. Conserved hypothetical protein Oxidoreductase “Cp ox2” Conservedd Hypothetical protein Cytochrome P450, monooxygenase Acetyl transferase Oxidoreductase “Cp ox1” Hypothetical protein Pisatin demethylase, putative Putative dimethyl- allyl-tryptophan- synthetase Dimethylallyl trryptophan synthetase monooxygenase- related protein NADP- dehydrogenase Catalase DMATII DMATIII 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

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

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

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

27. DMATS DMATS 2 module NRPS fum9 (hydroxylation) P450 hydroxylase hydroxylase dmats peptide synthetase O-methyltransferase oxidoreductase ankyrin dmats

28. DMATS DMATS 2 module NRPS fum9 (hydroxylation) P450 hydroxylase hydroxylase dmats peptide synthetase O-methyltransferase oxidoreductase ankyrin dmats

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

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

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

32. Pathway/Species A. oryzae A. fumigatus A. nidulans Penicillin + - + Siderophore Sid2 + + + Siderophore SidC + + + Conidial pigment WA + + + Aflatoxin /sterigmatocystin + - + Carotenoid + - - Orthologous secondary metabolic genes

33. Melanin cluster in A. fumigatus alb-1 (=wA) • 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 Chemistry – University of Tubingen Alexander Grundmann Shuming Li Bioinformatics TIGR Broad Institute AIST-CBRC University of Tokyo