accessing the diversity of pks genes in fungi isolates of thailand … · 2011-06-02 · accessing...

Accessing the diversity of PKS genes in fungi isolates of Thailand:

potential use in production of novel natural compounds

Supapon Cheevadhanarak

Combinatorial Biosynthesis Lab, BIOTEC

KMUTTKMUTT

Why we need new bioactive compound ?Why we need new bioactive compound ?

Drug resistance: can emerge and disseminate rapidlyIn France, penicillin-resistant isolates of Streptococcus pneumoniae

has increased from 5% to 25% within five years.

Emerging diseases Severe acute respiratory syndrome (SARS) is a respiratory illness that has recently been reported in Asia, North America, and Europe.

Bird flu (Avian influenza viruses)

(Drlica, 2001)

To overcome drug resistance

Develop new drug: is one approach- natural screening- artificial synthesizing

Polyketides (compound of choice)

Filamentous fungi (organism of choice)

Novel uNovel unnaturalnnatural naturalnaturalproductsproducts

‘‘Combinatorial BiosynthesisCombinatorial Biosynthesis’’by gene technologyby gene technology

Drug leads Chemical structure ~ 98%

already known

Natural product Natural product from microbefrom microbe

((mostmost; ; soil microbesoil microbe))

Screening Screening ProgrammesProgrammes

Strategy for novel compound discoveryStrategy for novel compound discoveryCompound ScreeningCompound Screening

((compound appcompound app))Molecular ScreeningMolecular Screening

((genetic resource appgenetic resource app))

Genetic resourceGenetic resourcefrom naturefrom nature

Gibberella genomeContains 35 PKS genes

Genetic screeningGenome searchMetagenomics

Why polyketides ? Why polyketides ?

• Diverse structure and function.

• Synthesized by a wide range of organisms, including bacteria, fungi, marine organisms (mollusks, sponges) and plants.

• Known to possess a wealth of pharmacologically important activities.

• Great commercial interest for drug discovery and account for medicinal sales exceeding $20 billion per year.

(Du and Ben shen, 2000 )

http://linux1.nii.res.in/~pksdb/polyketide.html

Applications of polyketidesApplications of polyketides

(Staunton and Weissman, 2001)

Spinosad

insecticide

Type I PKSs

Iterative type I

Type II PKSs

Actinorhodin synthase (act PKS)

Modular type I

Deoxyerythronolide B synthase (DEBS)

Type III PKSs

Chalcone/Stilbene synthases (CHS/STS)

Three Types of Polyketide SynthasesThree Types of Polyketide Synthases

KS AT KR ACP

Where to find new polyketides?

PlantMollusk

FungiBacteria

Shen, 2003, Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms

Protist

High diversity of molecular structure

Diverse range of metabolites, potent biological activitiesAntibiotics, toxins, dyes, anticancer drugs, immunosuppressants

Many of filamentous fungi can produce polyketides such asAspergillus sp.Penicillium sp.Cochliobolus heterostrophusXylariaceae

The polyketides are the large group of fungal metabolites.

Why polyketides in filamentous fungi ?Why polyketides in filamentous fungi ?

19,20-epoxycytochalasin Q

(Whalley and Edwardsb, 1999)

Potentials of fungi in polyketide synthesisPotentials of fungi in polyketide synthesis

Genome search Genome search

Genetic screeningGenetic screening

Aspergillus nidulans : 15-20 putative PKS

A. fumigatus : 14 putative PKS

A. terreus : 7 putative PKS

Cochliobolus sp. : 40 putative PKS

Fusarium sp. : 42 putative PKS

Neurospora crassa : 7 putative PKS

Aspergillus sp.more than 20 different polyketide synthase

(Vargar et al,2003)

by bioinformatics methodby bioinformatics method

by PCRby PCR

Partial reduced PKSsPartial reduced PKSs

MSAMSA

Non reduced PKSsNon reduced PKSs

PigmentsPigments

aflatoxinaflatoxin

Highly Highly reduced reduced PKSsPKSs

LovastatinLovastatin

TT--toxintoxin

FumonisinsFumonisins

KS Ketoacyl synthase ACP Acyl carrier protein AT Acyl transferase KR Keto reductaseMT Methyltransferase ER Enoyl reducthaseDH Dehydratase TE ThioesteraseCyc Cyclase

Fungal polyketides synthase geneFungal polyketides synthase gene

Polyketide biosynthesisPolyketide biosynthesis

11

22

ObjectivesObjectives- Identify and clone novel polyketide synthase genes

from filamentous fungi- Express the cloned gene in a well-defiend heterologous host

e.g., Pichia pastoris

AimAim- Increase accessibility to novel polyketide structure in nature and

by combinatorial biosynthesis using gene technology- Produce valuable polyketide compounds with enhanced biological

activities

Thailand populated with undiscovered fungi.

Tanticharoen and Hywel, 2003, Bioresources initiatives: Thailand, Asia Pacific natural products conference

Biodiversity in Thailand

9000 species of birds in the world with 918 species known from Thailand

ca. 250,000 higher plants in the World with 20-30,000 species recorded for

Thailandca. 4500 mammals in the World with ca.

470 species known from Thailand

10%National parks in Thailand

David Hawksworth estimates1,500,000 fungi for the World

Thailand could therefore have about 150,000 species of

fungi

(10%)

Hawksworth, D. L., 2001

Fungi: sources of polyketides

Not all of fungi can produce diverse polyketides

Recent study by genome search and PCR of each group of fungi shows that

“Subphylum Pezizomycotina” (Phylum Acomycota)=

a diverse group of fungi, has most potential to produce wide array of polyketides

Gibberella zeae Aspergillus terreus Hirsutella saussurei Pertusaria albescensKroken et al., 2003, Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes

BIOTEC Culture Collection - 2004

Phylogenetic and BIOTEC culture collection laboratory, 2005

Culture in collection of 9809 fungus

2322

1450

1102973

759

710

693

482

391320 307 142 109 49

Insect pathogenic fungi

Endophytic fungi

Wood decay fungi

Soil fungi

Marine fungi

Basidiomycetes

Aquatic fungi

Alkaline tolerant fungi

Seed fungi

Palm fungi

Lichenised fungi

Sand fungi

Dung fungi

Bamboo fungi

StrategyStrategyXylaria sp. BCC1067

(model)polyketide producer potential

Functional analysis

& compound structure identification

1. Group with interesting metabolites

2. New groups (unknown)

Phylogenetic analysis

Interesting genes

PKS gene screening(PCR tech)

Fungal selection

30 species

Phylogenetic analysis

PKS gene screening(PCR tech)

1. Group with interesting metabolites

2. New groups (unknown)

Interesting genes

Characterization and cloning of amplified PKS genes

Classify amplified PKS using

phylogenetic analysis

Select the interested genes

(up to hypothesis and rational

from phylogeny)

Cloning for full length PKS genes from genomic

library using PCR fragments as probe Couch et al., 2004, Rational elimination of Aspergillus terreus sulochrin production

Xylariceae model: Xylaria sp. BCC1067

OH

OHO O

1O

OOH OH

O

2

O

OOH OH

3

NH

O

O

OH

O

O OAc

4

OCO 2Me

OMe 5

Depudecin

Phaseolinone

Phomenone

19,20-epoxycytochalasin Q

(E)-methyl 3-(4-methoxyphenoxy)propenoate

5 metabolites was observed using chemical screening

method

2 of them are polyketides and posses drug activity

Isaka et al, 2000, Antiplasmodial compounds from the wood-decayed fungus Xylaria sp. BCC1067

NR PKS screening primers

LC series primer

Bingle et al., 1999

KS series primer

Nicholson et al., 2001Bingle et al., 1999, Nicholson et al., 2001

65.4224CCAYTGIGCICCYTGICCTGTRAAKAR28375

59.3820GARKSICAYGCIACITCIACKAF28374

65.4224CCAYTGIGCICCRTGICCIGARAAKAR18373

61.4220GARKSICAYGGIACIGGIACKAF18372

TmLenSeq 5’ to 3’NameSeq#

Design from multiple alignment of highly and partially reduced PKS at conserved sites in KS and AT designed by Amnuaykanjanasin et. al., 2005

KS forward primer

AT reverse primer

KS AT

KA series primers: for HR PKS

Amnuaykanjanasin et al, 2005, Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC1067

Screening for polyketide synthases“Diversity of highly reducing PKS genes in fungi isolates in Thailand”

Phylogenetic analysisDNA sequencing

KS forward primerAT reverse primer

PKSs genesKS AT

Cloned into vector

Combinatorial biosynthesis laboratory, 2005

Diversity of Xylaria sp. BCC1067 PKS

PKSKA1PKSMTPKSX1PKS3PKS12PKS35

XyX12A11XyF1R1A3XyF1R2A3XyF1R2A5XyF1R2A10

11 PKS genes were screened using primer pairs and classified

by phylogenetic analysisAmnuaykanjanasin et al, 2005, Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC1067

Characterization of Xylaria sp. BCC1067 full length PKS

Results confirm hypothesis of phylogenetic analysis about genes architecture and some function

Amnuaykanjanasin et al, 2005, Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC1067

Fungal genomes exhibit high numbers of HR PKS

30 fungal strains selected(Pezizomycotina)

Insect pathogenic fungi (15)1. Akanthosmyces novoguineensis

2. Akanthosmyces pistillariformis

3. Aschersonia tubulata

4. Beauveria bassina

5. Cordyceps nipponica

6. Cordyceps pseudomillitaris

7. Cordyceps unilateralis

8. Gibbellula sp.

9. Hymenostilbe sp.

10. Hypocrella discoidea

11. Hypocrella raciborskii

12. Metarhizium anisopliae

13. Metarhizium flavoviride

14. Paecillomyces tenuipes

Wood decay fungi (2015. Phomopsis sp.

16. Xylaria sp.

Marine endophyte (9)17. Acremonium sp.

18. Aigialus parvus

19. Corollospora besarispora

20. Corollospora maritima

21. Epicoccum sp.

22. Halorosellinia oceanica

23. Halosarphiea cf. kandeliae

24. Kallichroma glabrum

25. Massarina sp.

Lichenized fungi (5)26. Coniothyrium sp.

27. Graphis afzelii

28. Microsphaeropsis sp.

29. Phaeographina sp.

30. Trypethelium sp.

Possible use of polyketide from selected group of fungi

Marine endophyteFrom BCC, many of them are already analyzed for some activity and polyketide compounds, this is an

interesting point to access to all producing potential for more natural secret from them

Acremonium sp.

Kallichroma glabrum

Corollospora maritima

Halosarpheia cf. kandeliae


Lichenized fungiExtreme condition induce them to produce

polyketide for many purpose

1. Irradiation screening

2. Bitter tasting compounds against herbivore grazing

3. Antifungal and antibacterial agent against

competitive organisms

4. Improvement of gas exchange

5. Maintenance of symbiotic equilibrium

6. Weathering of rock for better attachment to

substratesSchmitt, 2005

Lichenized fungi

Graphis afzelii

Trypethelium sp.

Phaeographina sp.

Colorful group of fungi which has high number of non reduced

polyketide


Insect pathogenic fungiThey are interesting by their bioactivity and

relationship to insect host.Their produced polyketide may be used as

drugs or biological control agents as pesticidesWe are interested in strains cover 6 host-fungi specific

association in pezizomycotina


Diversity of highly reduce PKS genes in fungi isolates in Thailand: Phylogenetic analysis

GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C

BebF1R2B2CopF1R2DHydF1R2A2

HaoF1R2BHaoF1R1BXylF1R2A3

BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2

Atat1NBChPK18NR2BfPK12NR2

Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

The tree was generated using various methods

such as NJ, MP, and ML. And select the best method which can be supported by

bootstrap value

Using KS-AT interdomain region, completely classifyPKS with their architecture

Combinatorial biosynthesis laboratory, 2005,

Clade 1


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

With MT

Non MT

Product

“Diketide clade”lovastatin, compactin,

T-toxin

Clade 2


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

Product

“Nonaketide

and Hybrid PKS”lovastatin, compactin(cholesterol-lowering agent)

Clade 3


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

Product

“Nonaketide”T-toxin

(phytoxin: maize)

Clade 4


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

With MT

No MT

Product“fumonisins”(carcinogen)

NR Clade


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

Product

“Toxin and

pigments”

PR Clade


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

Product

“MSAS”

Interesting point from marine endophyte PKS phylogeny

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

C

GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

There are 3 genes from marine endophyte that locate in novel clade.

These genes are organized with non-reduced PKS

while they were amplified by reducing primers.

They might lose some domains during evolution

and interesting metabolites might be produced Combinatorial biosynthesis laboratory, 2005

Interesting point of insect pathogenic fungi PKS was the group in clade 3 PKS.

These genes have high identity together.

Hypothesized that these genes may produce the

same metabolites which are important among insect

pathogenic fungi

Interesting point from insect pathogenic PKS phylogeny

M e fF 1 R 2 BP a tF 1 R 2 C

B e b F 1 R 2 B 2C o p F 1 R 2 D

H yd F 1 R 2 A 2


GmPKS5C1

HaoF1R1AChPKS10C1

PhoF1R2EXylPKSMT

AcrF1R2A16

ChPKS9C1AipF1R2A2

ChPKS8C1KagF1R1D

BfPKS1C1GmPKS2C1

XylF1R2A10

ChPKS4C1GmPKS7C1

BfPKS11C4CopF1R1XA

ChPKS7C1DmPKS1C1

ChPKS1C1ChPKS2C3

MefF1R2BPatF1R2C



BfPKS8C3

GmPKS13C4ChPKS3C1

NcPKS2C3KagF1R1A

AcrF1R2A5PcmlcBC1

AtlovFC1

GmPKS15C4KagF1R2A

AipF1R2A5GmPKS14C4

NcPKS3C1

KagF1R1EKagF1R2E

ChPKS15C4

GmPKS12C4

ChPKS11C4GmFUM1C4

ChPKS12C4

ChPKS14C4EpiF1R2A2

HaoF1R2DAcrF1R2B1

XylPKSKA1MicF1R

EpiF1R2B1BfPKS10C4

BebF1R1A2

PhoF1R2BXylF1R1A3

BebF1R2B3

AipF1R1A5EpiF1R1A1

GmPKS8C2HydF1R1A2

AstF1R1A1XylPKS3

AstF1R1B2CopF1R1B1

BfPKS4C2BfPKS3C2

HaoF1R2CChPKS16C2

PatF1R1BConF1R2B3

CobF1R2A4AstF1R2B2

CobF1R2A2AcrF1R1B5

BfPKS5C2CopF1R2A1

ConF1R1B3NcPKS1C3

AtlovBC2PcmlcAC2

GmPKS9C2GmPKS1C2

BfPKS6C2GmPKS10C2

ChPKS17C2

AcrF1R1B7AcrF1R1A12

KagF1R2YBfPKS2C1

BfPK17NR3ChPK21NR3

BfPK20NR3BfPK18NR3

ChPK22NR3BfPK16NR3

ChPK23NR3

BfPKS15NBAtat5NR2

ChPKS19NBChPKS20NB

BfPKS14NBGmPKS3NR1

AppkL1NR1EnpkSTNR1

AnwANR1Afalb1NR1

GmPKS4NR1Atat4NR1

GzPK12NR1BfPK13NR2


Glpks1NR2ClPKS1NR2

XylPK12NR2Nspks1NR2

Mppks1C2

MtmasBacMtppsCBac

MlPKSEBacMtppsDBac

AtpksMPRAppksL2PR

Pg6MSASPRPp6MSASPR

Bn6MSASPRXylPKS35PR

ChPKS25PRMamcyEBac

'PUFA-PKS'ChPKS24Hb

AkpF1R1A8

GmPKS6C1XylPKSX1

ChPKS5C1

XylF1R2A5

0.5

““Diversity of highly reducing PKS genes in Diversity of highly reducing PKS genes in fungi isolates in Thailandfungi isolates in Thailand””

Total species: 16 speciesTotal amplified genes: 57 genes (Average = 3.56 genes/species)New clade: 3 clades (Non MT in clade 1, novel HR in clade 4, and unknown clade)Unknown: 3 genes (which cannot be grouped to any clade)

Total species: 16 species

Total amplified genes: 57 genes (Average = 3.56 genes/species)

Unknown: 3 genes

New clade: 3 clades (Non MT clade 1, novel HR clade 4, and unknown clade)

““Diversity of highly reducing PKS genes in Diversity of highly reducing PKS genes in fungi isolates in Thailandfungi isolates in Thailand”” (cont’d)

Characterization and cloning of amplified PKS genes

Classify amplified PKS using

phylogenetic analysis

Select the interested genes

(up to hypothesis and rational

from phylogeny)

Cloning for full length PKS genes from genomic

library using PCR fragments as probe Couch et al., 2004, Rational elimination of Aspergillus terreus sulochrin production

Functional analysis of cloned PKS (future work)

Cloned PKS

Gene disruption in wild type organisms Heterologous expression in new hostCouch et al., 2004, Pfeifer, 2001

Pichia pastoris

Combinatorial biosynthesis (future work)

Domain addition

Domain shuffling

Domain deletionGenerate genes library which increase possibility of novel drug discovery

Leadlay, 1997, Combinatorial approaches to polyketide biosynthesis, and our lab’s picture

• Further increase the diversity of polyketides(via combinatorial biosynthesis using gene technology)

• Create a large pool of polyketides• Generate more natural drugs with enhanced pharmacological properties

Future prospectsFuture prospects

Applications:•Antibacterial, antifungal•Anticancer drugs•Insecticides•Cosmetic use, food coloring•Engineer plants for self production of fungicides and pesticides

PK Company list PK Company list

Mixis, Paris, FR

LibraGen, Toulouse, FR

Kosan, California, USA

Biotica, little Chesterford, UK

AcknowledgementsAcknowledgementsPhylogenetic Lab, BIOTEC

Mycology Lab, BIOTEC

BIOTEC Culture Collection, BIOTEC

Bioresouces Research Unit, BIOTEC

Bioinformatics Lab, BIOTEC

Researchers in Combinatiorial Biosynthesis

Graduate Students from King Mongkut’s Uni Tech Thonburi

Research Grant:

BIOTEC, KMUTT

accessing the diversity of pks genes in fungi isolates of thailand … · 2011-06-02 · accessing...

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