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What can genomics tell us about secondary metabolism in Aspergillus?
Geoffrey Turner University of Sheffield
SO
NH2
COOH
SO
H2N
SH
SO
H2N
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gibberellin GA3
penicillin G
fusarin C lovastatin
aristolochene
gliotoxin
H
fumitremorgin C
cyclosporin
ergopeptides
aflatoxin B1 WA
trichothecene T2 toxin
Peptides
Polyketides
Terpenes
Alkaloids
A. nidulans
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gibberellin GA3
penicillin G
fusarin C lovastatin
aristolochene
gliotoxin
H
fumitremorgin C
cyclosporin
ergopeptides
aflatoxin B1 WA
trichothecene T2 toxin
Peptides
Polyketides
Terpenes
Alkaloids
A. fumigatus
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Fungal secondary metabolites
Non-ribosomal peptides (NRPS) Terpenes
-lactam antibiotics Trichodiene - toxinCyclosporin – immunosuppressant Aristolochene - toxinEchinocandin – antifungal drug
Polyketides (PKS)
Lovastatin – cholesterol lowering agentAflatoxin – carcinogen
Indole alkaloids
Ergotamine – migraine treatment – control of post partum bleeding
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Public genome sequencing projects in filamentous fungi:
Broad InstituteNeurospora crassa – completed and published 2003Aspergillus 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 CentreAspergillus fumigatus – completed 2004
Japanese consortiumAspergillus oryzae – completed 2004
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How many secondary metabolic pathwaysare there in a single species?
What are they producing?
Do these closely related specieshave orthologous pathway genes?
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Automated annotation by sequencing institutesmakes gene finding much easier:
Multienzyme complexes like NRPS and PKShave recognisable domains
Secondary metabolic pathway genes tend to be clustered
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L--aminoadipic acid L-cysteine L-valine
LLD-ACV tripeptide
H N2
H COOH
NH
NH
SH H CH3
O
CH3COOH
O
H
H
H N2
H COOH
NH CH3
O
CH3N
S
COOHH
HH
O
isopenicillin N
NH CH3
O
CH3ON
S
COOHH
HH
O
penicillin V
penDE
ipnA
acvA
phenylacetyl CoA
-aminoadipic acidCoA
ACV synthetase
IPN synthase
Acyl transferase
NRPS
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SO
NH2
COOH
SO
H2N
SH
SO
H2N
A trimodular NRPS: ACVS Synthetase
Amino acidsattached to enzymevia pantotheine arm
α-aminoadipate cysteine valine
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adenylation N-methylation thioester 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 modulesTrichoderma virens (Kenerly 2002) 2.3 Mda
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Fungal PKS architecture (Type I iterative)
KS AT ACP (CYC) or(TE)
(DH) (MT)(ER)(KR)
-Ketoacyl SynthaseAcyltransferaseDehydrataseMethyltransferase-Keto ReductaseEnoyl ReductaseCyclaseThioreductase
Domains:
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Aspergillus nidulans annotated genome (Broad Institute)
5 module NRPS PKS
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Aspergillus nidulans annotated genome (Broad Institute)
PKS
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Aspergillus nidulans annotated genome (Broad Institute)
Hybrid PKS-NRPS
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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
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A. fumigatus produces many toxic secondary metabolites including peptide derivatives and indole alkaloids
Gliotoxin
Tryprostatins
Fumitremorgens
Fumigaclavines
Can we identify the gene clusters responsible?
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TryptophanProlineNRPS?DMAT synthetase
TryptophanDMAT synthetase (X2)
SerinePhenylalanine2 module NRPS?
TerpeneSesquiterpene cyclase
Gliotoxin
Fumagillin
Fumigaclavines
Fumitremorgens
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Gardiner et al. Mol. Microbiol. (2004)Sirodesmin cluster of Leptophaeria maculans
Sirodesmin
Gliotoxin
Putative A. fumigatus gliotoxin clusteridentified in genome by homology:Includes 2 module NRPS
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TryptophanProlineNRPS?DMAT synthetase
TryptophanDMAT synthetase (X2)
SerinePhenylalanineNRPS?
TerpeneSesquiterpene cyclase
Gliotoxin
Fumagillin
Fumigaclavines
Fumitremorgens
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FUMIGACLAVINE BIOSYNTHESIS
Dimethylallyl-tryptophan
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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
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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
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
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Deletion of DMATSII (= dmaW)leads to loss of fumigaclavine C
Coyle & Panaccione 2005
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TryptophanProline2 module NRPS?DMAT synthetase
TryptophanDMAT synthetase (X2)
SerinePhenylalanine2 module NRPS?
TerpeneSesquiterpene cyclase
Gliotoxin
Fumagillin
Fumigaclavines
Fumitremorgens
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Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071
prolinetryptophan
Aspergillus fumigatusprenylated alkaloids;fumitremorginstryprostatins
NRPS?
DMATS?
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oxidoreductase ankyrin dmats
P450 hydroxylase
dmats
O-methyltransferase
hydroxylase
peptide synthetase
fum9 (hydroxylation)
2 module NRPSDMATSDMATS
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oxidoreductase ankyrin dmats
P450 hydroxylase
dmats
O-methyltransferase
hydroxylase
peptide synthetase
fum9 (hydroxylation)
2 module NRPSDMATSDMATS
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DMATS expressed in E. coliEnzyme can prenylate cyclo-Trp-ProGrundmann & Li 2005
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Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071
prolinetryptophan
Aspergillus fumigatusprenylated alkaloids;fumitremorgenstryprostatins
NRPS?
DMATS?
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Dimodular NRPS of A. fumigatus
Deletion
Insertion of additional copies
Introduce strong promoter (PalcA)
Express in naïve host – A. nidulans
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Pathway/Species A. oryzae A. fumigatus A. nidulans
Penicillin + - +
Siderophore Sid2 + + +
Siderophore SidC + + +
Conidial pigment WA
+ + +
Aflatoxin/sterigmatocystin
+ - +
Carotenoid + - -
Orthologous secondary metabolic genes
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72.t00906 72.t00907 72.t0090872.t00907 72.m19833 brown 172.t00908 72.m19834 brown 172.t00909 72.m19835 yellowish-green 172.t00910 72.m19836 1,3,6,8-tetrahydroxynaphthalene reductase72.t00911 72.m19837 hypothetical protein72.t00912 72.m19838 Scytalone dehydratase72.t00913 72.m19839 hypothetical protein72.t00914 72.m19840 polyketide synthetase
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
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Any other orthologues amonst the PKS genes?
Phylogeny done by Resham Kulkani at TIGRUsing KS domain of PKS/Fatty Acid Synthase
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wA/alb1 orthologues
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PKS for aflatoxin in 2 species only
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Possible orthologues amongst unknown PKS?
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Hybrid PKS/NRPSKS-AT-CON-AMP-PP
In all 3 species, but not really orthologues?
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Conclusions
Few true orthologues across the genus AspergillusEach species has its own repertoireOrthologues often found in less closely related species
Gene/product relationship requires functional analysisin most cases
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Secondary metabolism – University of Sheffield
Hala MohamedShubha MaiyaJackie PriceDavid Keszenman-Pereyra
Bioinformatics
TIGRBroad InstituteAIST-CBRCUniversity of Tokyo
Chemistry –University of Tubingen
Alexander GrundmannShuming Li