Survey
* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project
Download Document
Document related concepts
no text concepts found
Transcript
What can genomics tell us about secondary metabolism in Aspergillus? Geoffrey Turner University of Sheffield O S O S H2N H2N NH2 COOH O SH S Peptides H penicillin G Alkaloids gliotoxin cyclosporin A. nidulans ergopeptides fumitremorgin C Terpenes fusarin C lovastatin trichothecene T2 toxin aristolochene gibberellin GA3 aflatoxin B1 WA Polyketides Peptides H penicillin G Alkaloids gliotoxin cyclosporin A. fumigatus ergopeptides fumitremorgin C Terpenes fusarin C lovastatin trichothecene T2 toxin aristolochene gibberellin GA3 aflatoxin B1 WA Polyketides Fungal secondary metabolites Non-ribosomal peptides (NRPS) Terpenes b-lactam antibiotics Cyclosporin – immunosuppressant Echinocandin – antifungal drug Trichodiene - toxin Aristolochene - toxin Polyketides (PKS) Lovastatin – cholesterol lowering agent Aflatoxin – carcinogen Indole alkaloids Ergotamine – migraine treatment – control of post partum bleeding 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 cinereus Rhizopus oryzae TIGR/Sanger Centre Aspergillus fumigatus – completed 2004 Japanese consortium Aspergillus oryzae – completed 2004 How many secondary metabolic pathways are there in a single species? What are they producing? Do these closely related species have orthologous pathway genes? 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 L-a-aminoadipic acid L-cysteine L-valine acvA ACV synthetase H NH H2N H COOH O SH H CH 3 CH 3 COOH NH O H LLD-ACV tripeptide NRPS ipnA IPN synthase NH H2 N H COOH H H CH 3 CH 3 S O N O H COOH isopenicillin N penDE phenylacetyl CoA a-aminoadipic acid CoA Acyl transferase NH O O O H H CH 3 CH 3 S N H penicillin V COOH A trimodular NRPS: ACVS Synthetase O S O S H2N NH2 O S H2N SH COOH α-aminoadipate cysteine valine Amino acids attached to enzyme via pantotheine arm 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 Fungal PKS architecture (Type I iterative) KS Domains: AT (DH) (MT)(ER)(KR) ACP b-Ketoacyl Synthase Acyltransferase Dehydratase Methyltransferase b-Keto Reductase Enoyl Reductase Cyclase Thioreductase (CYC) or (TE) Aspergillus nidulans annotated genome (Broad Institute) 5 module NRPS PKS Aspergillus nidulans annotated genome (Broad Institute) PKS Aspergillus nidulans annotated genome (Broad Institute) Hybrid PKS-NRPS Summary of secondary metabolic gene classes in Aspergillus 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 A. fumigatus produces many toxic secondary metabolites including peptide derivatives and indole alkaloids Gliotoxin Tryprostatins Fumitremorgens Fumigaclavines Can we identify the gene clusters responsible? Serine Phenylalanine 2 module NRPS? Gliotoxin Terpene Sesquiterpene cyclase Fumagillin Tryptophan DMAT synthetase (X2) Fumigaclavines Tryptophan Proline NRPS? DMAT synthetase Fumitremorgens 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 Serine Phenylalanine NRPS? Gliotoxin Terpene Sesquiterpene cyclase Fumagillin Tryptophan DMAT synthetase (X2) Fumigaclavines Tryptophan Proline NRPS? DMAT synthetase Fumitremorgens FUMIGACLAVINE BIOSYNTHESIS CO 2H NH 2 CO 2H CO 2H OPP DMAT synthetase SAM N-methylation NH 2 N H L-tryptophan NH Me N H N H oxidations + decarboxylative cyclisation HO HO 2C Dimethylallyl-tryptophan N N H oxidations peptide synthetase H H N H N O H N R2 OH O R 1 O O N H lysergic acid oxidation + reductive cyclisation agroclavine C16H18N2 Exact Mass: 238.15 oxidation H reduction H N H N H N HN Me H H N O H N R2 OH O R 1 O O N H lysergic acid agroclavine C16H18N2 Exact Mass: 238.15 N oxidation H reduction N N ergot peptides H R1-Pro-R2 eg ergocryptine Val-Pro-Leu ergotamine Ala-Pro-Phe H HO N H H H oxidation N H fumigaclavine B C16H20N2O Exact Mass: 256.16 N H festuclavine C16H20N2 Exact Mass: 240.16 Mol. Wt.: 240.34 acetyl transferase O N O H O N H O H OPP DMAT synthetase H N H fumigaclavine C C23H30N2O2 Exact Mass: 366.23 N H fumigaclavine A C18H22N2O2 Exact Mass: 298.17 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 Hypothetical protein monooxygenaserelated protein Conserved hypothetical protein NADPdehydrogenase Oxidoreductase “Cp ox2” Conservedd Oxidoreductase Hypothetical protein “Cp ox1” Cytochrome P450, Acetyl monooxygenase transferase Pisatin Putative dimethyldemethylase, allyl-tryptophanputative synthetase Dimethylallyl trryptophan Catalase synthetase DMATIII 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 Deletion of DMATSII (= dmaW) leads to loss of fumigaclavine C Coyle & Panaccione 2005 Serine Phenylalanine 2 module NRPS? Gliotoxin Terpene Sesquiterpene cyclase Fumagillin Tryptophan DMAT synthetase (X2) Fumigaclavines Tryptophan Proline 2 module NRPS? DMAT synthetase Fumitremorgens NRPS? proline tryptophan DMATS? Aspergillus fumigatus prenylated alkaloids; fumitremorgins tryprostatins Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071 DMATS DMATS 2 module NRPS fum9 (hydroxylation) P450 hydroxylase hydroxylase dmats oxidoreductase ankyrin dmats O-methyltransferase peptide synthetase DMATS DMATS 2 module NRPS fum9 (hydroxylation) P450 hydroxylase hydroxylase dmats oxidoreductase ankyrin dmats O-methyltransferase peptide synthetase DMATS expressed in E. coli Enzyme can prenylate cyclo-Trp-Pro Grundmann & Li 2005 NRPS? proline tryptophan DMATS? Aspergillus fumigatus prenylated alkaloids; fumitremorgens tryprostatins Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071 Dimodular NRPS of A. fumigatus Deletion Insertion of additional copies Introduce strong promoter (PalcA) Express in naïve host – A. nidulans Orthologous secondary metabolic genes Pathway/Species A. oryzae A. fumigatus A. nidulans Penicillin + - + Siderophore Sid2 + + + Siderophore SidC + + + Conidial pigment WA Aflatoxin /sterigmatocystin + + + + - + Carotenoid + - - Melanin cluster in A. fumigatus 72.t00906 72.t00907 72.t00908 72.t00909 72.t00910 72.t00911 72.t00912 72.t00913 72.t00914 72.t00907 72.m19833 72.m19834 72.m19835 72.m19836 72.m19837 72.m19838 72.m19839 72.m19840 72.t00908 brown 1 brown 1 yellowish-green 1 1,3,6,8-tetrahydroxynaphthalene reductase hypothetical protein Scytalone dehydratase hypothetical protein polyketide synthetase 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 Any other orthologues amonst the PKS genes? Phylogeny done by Resham Kulkani at TIGR Using KS domain of PKS/Fatty Acid Synthase wA/alb1 orthologues PKS for aflatoxin in 2 species only Possible orthologues amongst unknown PKS? Hybrid PKS/NRPS KS-AT-CON-AMP-PP In all 3 species, but not really orthologues? 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 Secondary metabolism – University of Sheffield Chemistry – University of Tubingen Hala Mohamed Shubha Maiya Jackie Price David Keszenman-Pereyra Alexander Grundmann Shuming Li Bioinformatics TIGR Broad Institute AIST-CBRC University of Tokyo
Related documents