What can genomics tell us about secondary metabolism in Aspergillus?

Geoffrey Turner University of Sheffield

SO

NH2

COOH

SO

H2N

SH

SO

H2N

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

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

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

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

How many secondary metabolic pathwaysare there in a single species?

What are they producing?

Do these closely related specieshave orthologous pathway genes?

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

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

SO

NH2

COOH

SO

H2N

SH

SO

H2N

A trimodular NRPS: ACVS Synthetase

Amino acidsattached to enzymevia pantotheine arm

α-aminoadipate cysteine valine

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

Fungal PKS architecture (Type I iterative)

KS AT ACP (CYC) or(TE)

(DH) (MT)(ER)(KR)

-Ketoacyl SynthaseAcyltransferaseDehydrataseMethyltransferase-Keto ReductaseEnoyl ReductaseCyclaseThioreductase

Domains:

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

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

A. fumigatus produces many toxic secondary metabolites including peptide derivatives and indole alkaloids

Gliotoxin

Tryprostatins

Fumitremorgens

Fumigaclavines

Can we identify the gene clusters responsible?

TryptophanProlineNRPS?DMAT synthetase

TryptophanDMAT synthetase (X2)

SerinePhenylalanine2 module NRPS?

TerpeneSesquiterpene cyclase

Gliotoxin

Fumagillin

Fumigaclavines

Fumitremorgens

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

TryptophanProlineNRPS?DMAT synthetase

TryptophanDMAT synthetase (X2)

SerinePhenylalanineNRPS?

TerpeneSesquiterpene cyclase

Gliotoxin

Fumagillin

Fumigaclavines

Fumitremorgens

FUMIGACLAVINE BIOSYNTHESIS

Dimethylallyl-tryptophan

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

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

Deletion of DMATSII (= dmaW)leads to loss of fumigaclavine C

Coyle & Panaccione 2005

TryptophanProline2 module NRPS?DMAT synthetase

TryptophanDMAT synthetase (X2)

SerinePhenylalanine2 module NRPS?

TerpeneSesquiterpene cyclase

Gliotoxin

Fumagillin

Fumigaclavines

Fumitremorgens

Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071

prolinetryptophan

Aspergillus fumigatusprenylated alkaloids;fumitremorginstryprostatins

NRPS?

DMATS?

oxidoreductase ankyrin dmats

P450 hydroxylase

dmats

O-methyltransferase

hydroxylase

peptide synthetase

fum9 (hydroxylation)

2 module NRPSDMATSDMATS

oxidoreductase ankyrin dmats

P450 hydroxylase

dmats

O-methyltransferase

hydroxylase

peptide synthetase

fum9 (hydroxylation)

2 module NRPSDMATSDMATS

DMATS expressed in E. coliEnzyme can prenylate cyclo-Trp-ProGrundmann & Li 2005

Von Nussbaum (2003) Angew. Chem. Int. Ed. 2003, 42, 3068 – 3071

prolinetryptophan

Aspergillus fumigatusprenylated alkaloids;fumitremorgenstryprostatins

NRPS?

DMATS?

Dimodular NRPS of A. fumigatus

Deletion

Insertion of additional copies

Introduce strong promoter (PalcA)

Express in naïve host – A. nidulans

Pathway/Species A. oryzae A. fumigatus A. nidulans

Penicillin + - +

Siderophore Sid2 + + +

Siderophore SidC + + +

Conidial pigment WA

+ + +

Aflatoxin/sterigmatocystin

+ - +

Carotenoid + - -

Orthologous secondary metabolic genes

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

Any other orthologues amonst the PKS genes?

Phylogeny done by Resham Kulkani at TIGRUsing KS domain of PKS/Fatty Acid Synthase

wA/alb1 orthologues

PKS for aflatoxin in 2 species only

Possible orthologues amongst unknown PKS?

Hybrid PKS/NRPSKS-AT-CON-AMP-PP

In all 3 species, but not really orthologues?

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

Secondary metabolism – University of Sheffield

Hala MohamedShubha MaiyaJackie PriceDavid Keszenman-Pereyra

Bioinformatics

TIGRBroad InstituteAIST-CBRCUniversity of Tokyo

Chemistry –University of Tubingen

Alexander GrundmannShuming Li