Docstoc

What can genomics tell us about secondary metabolism - The

Document Sample
What can genomics tell us about secondary metabolism - The Powered By Docstoc
					What can genomics tell us about secondary metabolism
                   in Aspergillus?
                         Geoffrey Turner
                      University of Sheffield




                  O    S      O    S     O    S


                             H2N        H2N

                             NH2   SH

                           COOH
   Peptides                                     H




                                penicillin G                                   gliotoxin


                                                      cyclosporin
                               Alkaloids

                                                      A. nidulans
                ergopeptides
                                    fumitremorgin C



                                    Terpenes               fusarin C              lovastatin
       trichothecene T2 toxin




aristolochene          gibberellin GA3                 aflatoxin B1                 WA

                                                                       Polyketides
   Peptides                                     H




                                penicillin G                                   gliotoxin


                                                      cyclosporin
                               Alkaloids


                                                                    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                       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
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
ACV synthetase                                                           acvA
                                          H   SH       H
           HN
           2                  NH                                  CH 3
                                                                  CH 3
           H COOH         O
                                              NH                  COOH
                                  O                    H


                LLD-ACV tripeptide                                                   NRPS
                                                                         ipnA
IPN synthase
                                          H    H
            H2 N                  NH                   S          CH 3
             H COOH           O                                   CH 3
                                               N
                                      O
                                                   H       COOH

                      isopenicillin N
                                                                         penDE
phenylacetyl CoA
Acyl transferase                                            a-aminoadipic acid
                                          H    H
                                                                  CoA
                                  NH                   S          CH 3
                      O       O                                   CH 3
                                               N
                                      O
                                                   H       COOH


                      penicillin V
     A trimodular NRPS: ACVS Synthetase



 O     S            O    S     O    S


                   H2N        H2N
                         SH
             NH2

           COOH
                                        Amino acids
α-aminoadipate cysteine valine          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         AT    (DH) (MT)(ER)(KR) ACP        (CYC)
                                                or
Domains:    b-Ketoacyl Synthase                (TE)
            Acyltransferase
            Dehydratase
            Methyltransferase
            b-Keto Reductase
            Enoyl Reductase
            Cyclase
            Thioreductase
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   1                not detected    1
cyclase

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
                                             OPP                         CO 2H                                        CO 2H
                    NH 2     DMAT                                                    SAM
                             synthetase                                  NH 2        N-methylation                     NH
           N                                                                                                           Me
           H                                                     N                                               N
                                                                 H                                               H
    L-tryptophan
                                                                                                             oxidations +
                                                                                                             decarboxylative
                                                                                                             cyclisation

                         HO 2C                                                                          HO
                        Dimethylallyl-tryptophan                                                                     HN Me
                                     N                                       N
                                         H                                                oxidation +                   H
                                                                                 H        reductive
                                                   oxidations                                                H
            peptide                                                  H                    cyclisation
            synthetase
                                         N                                                                             N
                                         H                                       N                                     H
                                                                                 H
    N           OH          lysergic acid
                                                                   agroclavine
        H        O R
O                   1 O                                             C16H18N2
            N                                                   Exact Mass: 238.15
                    N
    R2              H
                O            N
                                 H                                                             oxidation
                                                                 reduction
                                                                      H
    N           OH        lysergic acid
                                                           agroclavine
        H        O R
O                   1 O                                     C16H18N2
            N                                           Exact Mass: 238.15
                    N
    R2              H
                O          N
                               H                                                 oxidation
                                                         reduction


ergot peptides              N                                                                   N
R1-Pro-R2                   H                           N                        HO                 H
eg ergocryptine Val-Pro-Leu                                 H                         H
ergotamine Ala-Pro-Phe                                               oxidation
                                                    H

                                                                                           N
                                                         N                                 H
                                                         H                         fumigaclavine B
                                              festuclavine                           C16H20N2O
                                               C16H20N2
                                           Exact Mass: 240.16                     Exact Mass: 256.16
                                            Mol. Wt.: 240.34
                                                                                          acetyl
                                                                                          transferase


                                                                                  O
                                                                                                 N
                                   O                                                  O              H
                                            N                                             H
                                   O            H                         OPP
                                       H                        DMAT
                                                                synthetase                  N
                                                                                            H
                                                N                                  fumigaclavine A
                                                H                                    C18H22N2O2
                                fumigaclavine C                                   Exact Mass: 298.17
                                  C23H30N2O2
                               Exact Mass: 366.23
Gene type       A. oryzae         A. fumigatus     A. nidulans

PKS             30                14               27

NRPS            18                14               14

FAS             5                 1                6

Sesquiterpene   1                 (1)              (1)
cyclase

DMATS           2                 7                2


“DMATS” hits probably reflect a variety of prenyltransferases
- add dimethylallyl side chain to a variety of compounds
                                           Conserved                                                                                                   Conservedd
                                          hypothetical                      Oxidoreductase   Cytochrome P450, Acetyl                    Oxidoreductase Hypothetical
       Hypothetical protein                                                 “Cp ox2”
                                            protein                                          monooxygenase    transferase               “Cp ox1”         protein

                                                       Pisatin  Putative dimethyl-                                                   Dimethylallyl
monooxygenase-                   NADP-              demethylase, allyl-tryptophan-                                                   trryptophan
                                                      putative      synthetase                                              Catalase synthetase
related protein               dehydrogenase

                                                                  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


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

                                    P450                   hydroxylase              hydroxylase

                                                                     dmats


                                                                             O-methyltransferase    peptide synthetase
oxidoreductase   ankyrin    dmats
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.t00907     72.m19833        brown 1
72.t00908     72.m19834        brown 1
72.t00909     72.m19835        yellowish-green 1
72.t00910     72.m19836        1,3,6,8-tetrahydroxynaphthalene reductase
72.t00911     72.m19837        hypothetical protein
72.t00912     72.m19838        Scytalone dehydratase
72.t00913     72.m19839        hypothetical protein
72.t00914     72.m19840        polyketide synthetase

     • This cluster is not conserved in Ana/Aoa        alb-1 (=wA)
     • 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 –    Chemistry –
University of Sheffield   University of Tubingen

Hala Mohamed              Alexander Grundmann
Shubha Maiya              Shuming Li
Jackie Price
David Keszenman-Pereyra



Bioinformatics

TIGR
Broad Institute
AIST-CBRC
University of Tokyo

				
DOCUMENT INFO
Shared By:
Categories:
Stats:
views:40
posted:4/13/2011
language:English
pages:40