avena strigosa from metabolic gene clusters to genomics 2.4/anne osbourn.pdf · avena strigosa...

Avena strigosa – from metabolic gene clusters to genomics

Anne [email protected]

CH2OH

Rha-Gal-Glc-O

O O

O

OH

OHC

CO.O-Fuc-O

Xyl-GlcA-O

Gal

OH

O O-Ara

O OH O

Rha-Xyl-Api

Chromosaponin I

QS21

CH2OH

Rha-Gal-Glc-O

O O

O

OH

OHC

CO.O-Fuc-O

Xyl-GlcA-O

Gal

OH

O O-Ara

O OH O

Rha-Xyl-Api

Chromosaponin I

QS21

CO2H

H

HO

H

HO

R

H

O

NC

O

CO2HH

HO

H

HO

H

O-Glc

Glc-OHO

H

CO2H

H

CO2H

GlcA-GlcA-O

O

CH2OH

CHO

Glc-Ara-O

O

O

O HNMe

OH

CH2OH

Rha-Gal-GlcA-O

OH

CO.O-Glc-Rha-Ara

Xyl-Fuc-GlcNAc-O

OH

O

O

OHO

Xyl O

O

OHOH

Glc

Oleanolic acid Beta-Amyrin R = CO2H, CO2Me, CO.NHMe, CNSynthetic oleananes

Betulinic acid Ginsenoside Rg1Glycyrrhizin

Avenacin A-1 Soyasaponin I

Avicin D

Glc

CO2H

H

HO

H

HO

R

H

O

NC

O

CO2HH

HO

H

HO

H

O-Glc

Glc-OHO

H

CO2H

H

CO2H

GlcA-GlcA-O

O

CH2OH

CHO

Glc-Ara-O

O

O

O HNMe

OH

CH2OH

Rha-Gal-GlcA-O

OH

CO.O-Glc-Rha-Ara

Xyl-Fuc-GlcNAc-O

OH

O

O

OHO

Xyl O

O

OHOH

Glc




Avicin D

Glc

CO2H

H

HO

H

HO

R

H

O

NC

O

CO2HH

HO

H

HO

H

O-Glc

Glc-OHO

H

CO2H

H

CO2H

GlcA-GlcA-O

O

CH2OH

CHO

Glc-Ara-O

O

O

O HNMe

OH

CH2OH

Rha-Gal-GlcA-O

OH

CO.O-Glc-Rha-Ara

Xyl-Fuc-GlcNAc-O

OH

O

O

OHO

Xyl O

O

OHOH

Glc




Avicin D

Glc

CO2H

H

HO

H

HO

R

H

O

NC

O

CO2HH

HO

H

HO

H

O-Glc

Glc-OHO

H

CO2H

H

CO2H

GlcA-GlcA-O

O

CH2OH

CHO

Glc-Ara-O

O

O

O HNMe

OH

CH2OH

Rha-Gal-GlcA-O

OH

CO.O-Glc-Rha-Ara

Xyl-Fuc-GlcNAc-O

OH

O

O

OHO

Xyl O

O

OHOH

Glc




Avicin D

Glc

Metabolic diversity – a snapshot

CO2H

H

HO

H

HO

R

H

O

NC

O

CO2HH

HO

H

HO

H

O-Glc

Glc-OHO

H

CO2H

H

CO2H

GlcA-GlcA-O

O

CH2OH

CHO

Glc-Ara-O

O

O

O HNMe

OH

CH2OH

Rha-Gal-GlcA-O

OH

CO.O-Glc-Rha-Ara

Xyl-Fuc-GlcNAc-O

OH

O

O

OHO

Xyl O

O

OHOH

Glc




Avicin D

Glc

SweetenerBitterness/antifeedant

Antifungal

Adjuvant

Anti-cancer

Plant growth stimulant

“Adaptogen”

Avenacins – Antimicrobial defence compounds produced by oat (Avena spp.)

O

O

OOH

O

OH

CHO

NMe

Avenacin A-1

-D-glu (1 2)

-D-glu (1 4)

-L-ara (1 )3

O

3

H

-Amyrin

Avenacin A-1

Papadopoulou et al. (1999) PNAS 96:12923

The genes for avenacin synthesis are clustered

~3.5 cM:Sad3

Sad1 Sad2Sad7Sad9 Sad10 Sad6Sad8

400 kb

Roots Shoots Leaves Flowers

Sad2

Sad1

Sad7

Sad10

Sad9

GAPDH

Sad2

Sad1

Sad7

Sad9

OSC CYP51

Qi et al (2004) PNAS 101:8233; Qi et al (2006) PNAS 103:18848; Mylona et al (2008) Plant Cell 20:201; Mugford et al (2009)

Plant Cell 21:2473; Wegel et al (2009) Plant Cell 21:3926; Qin et al (2010) Phytochemistry 71:1245; Owatworakit et al

(2012) JBC, 288:3696; Mugford et al Plant Cell (2013) 25:1078; Geisler et al. (2013) PNAS 110:E3360-67

2,3 Oxidosqualene

Shikimate pathway

Sad1(AsβAS1)

Sad2(AsCYP51H10)

Sad7

(AsSCPL1)

Sad9(AsMT1)

Sad10(AsUGT74H5)

Avenacin A-1

NMA-Glc

NMA-Glc

NMA

Anth

β-Amryin

? ?

Cytosol

Vacuole

Plastid

ER

ER derivedvesicles

MVA pathway

Des-acyl avenacin A

Des-acylavenacin A

OH

NH

OH

O

Glc

NH

O

OCH3

Glc

NH

O

OCH3

OHGlc

Glc

CHO

OH

O

O

Ara

NH

O

OCH3

Glc

Glc

CHO

OH

OH

OH

O

O

Ara

Glc

Glc

CHO

OH

OH

OH

O

O

Ara

NH

HO

OCH3

Owatworakit et al (2012)

JBC, 288:3696; Mugford et

al Plant Cell (2013) 25:1078

Metabolic engineering for disease resistance

Take-all disease of wheat

-L-Ara--D-Glc(1-4)

-D-Glc(1-2)

HO

O

HO HO

CO2H

O

O

HO

HOCH2OH

OH

O

CHOO

O NHMe

OH

OH

O

2, 3-oxidosqualene

squalene

squaleneepoxidase

cycloartenol

plant steroids

-amyrin -amyrin lupeol

oxidation

glycosylation

oxidation

glycosylation

avenacin A1

eclabatin etc

lupane betulinicacid etc

soyasapogenol B

cycloartenol synthaseoxidosqualene

cyclases

over 100different scafoldsdiscovered so far

multiplehydroxylation

patterns

greatest diversityintroduced by

functionalisation ofhydroxyl groups

mevalonate farnesyl pyrophosphate

acylation

-D-GlcA

-D-GlcA(1-2)

30

21

16

1213

3

23 24

H

H21

30

16

13

12

24H

H21

30

16

13

12

24

-L-Ara--D-Glc(1-4)

-D-Glc(1-2)

HO

O

HO HO

CO2H

O

O

HO

HOCH2OH

OH

O

CHOO

O NHMe

OH

OH

O

2, 3-oxidosqualene

squalene

squaleneepoxidase

cycloartenol

plant steroids


oxidation

glycosylation

oxidation

glycosylation

avenacin A1

eclabatin etc


soyasapogenol B


cyclases



patterns




acylation

-D-GlcA

-D-GlcA(1-2)

30

21

16

1213

3

23 24

-L-Ara--D-Glc(1-4)

-D-Glc(1-2)

HO

O

HO HO

CO2H

O

O

HO

HOCH2OH

OH

O

CHOO

O NHMe

OH

OH

O

2, 3-oxidosqualene

squalene

squaleneepoxidase

cycloartenol

plant steroids


oxidation

glycosylation

oxidation

glycosylation

avenacin A1

eclabatin etc


soyasapogenol B


cyclases



patterns




acylation

-D-GlcA

-D-GlcA(1-2)

30

21

16

1213

3

23 24

A) Scaffolds (OSCs):

B) Scaffold oxygenation:(CYP450s)

AsCYPA

AsCYPB

GmCYP93E1

GgCYP72A154

AsbAS1 LjAMY2

21

30

16

13

12

24

BvC10,C12

GmUGT73P2

GmUGT91H4

AsGTs

Glycosylation

(GTs)

Acylation

AsMT1

AsGT2

AsAT1

C) Further modification:

-L-Ara--D-Glc(1-4)

-D-Glc(1-2)

HO

O

HO HO

CO2H

O

O

HO

HOCH2OH

OH

O

CHOO

O NHMe

OH

OH

O

2, 3-oxidosqualene

squalene

squaleneepoxidase

cycloartenol

plant steroids


oxidation

glycosylation

oxidation

glycosylation

avenacin A1

eclabatin etc


soyasapogenol B


cyclases



patterns




acylation

-D-GlcA

-D-GlcA(1-2)

30

21

16

1213

3

23 24

Arabidopsis RiceOats Arabidopsis RiceOats Arabidopsis RiceOats Arabidopsis RiceOats

Oat Arabidopsis Rice

Medicago truncatula

The oat promoters are active in other species

Kemen et al. (2014) PNAS 111:8679

AB 36.12AB 36.11AB 36.7

pEW286 (GG CONSTRUCT -pSad1-Gus with 2 introns)

The oat promoters are active in other species

Aymeric Leveau, Gemma Farres, with National

Institute of Agricultural Botany, Cambridge

The HyperTrans system – Rapid high level

transient expression of protein in plants

Sainsbury F and Lomonossoff G. (2008) Plant Phys. 148:1212

Stigmasterol

Stigmasterol

Beta-Amyrin

Stigmasterol

Control

Sad1

Sad1 +

Sad2

Control

Sad1

Sad1 +

Sad2

New product

OH

O

OH

12,13-epoxy-

16-hydroxy-

β-amyrin

1213

16

OH

O

OH

12,13-epoxy-

16-hydroxy-

β-amyrin

1213

16

Production of a functionalised triterpene

Geisler et al. (2013) PNAS 110:E3360-67

OSC

1GT1 MT6 AT5KANRLB

R

B

Level 2 Golden Gate binary vector

Standardized assembly of gene parts from a library, using Golden Gate technology

O

OH

CH3 CH3

CH3 CH3

CH3 CH3

CH3

CH3

O

OHOOH

OH

OH

O

OHOOH

OH

OH

O

CH3

OH

CH3CH3

CH3

CH3

O

ONH

CH3

CH3

O

OH

O

O

OH

Scaffolds

Scaffold oxygenation

Further modification

Precursor

Glycosylation (GTs)

Acylation (MTs, GTs, ATs )

Pathway steps

CYP1 CYP4 GT3

Promoters

CDS

Terminators

GT

1

GT

2

GT

3

GT

4

GT

5

GT

6

GT

7

OSCs

CYPs

GTs

MTs

ATsAT

1

AT

2

AT

3

AT

4

AT

5

AT

6

AT

7

P1 P2 P3 P4 P5 P6 P7

T1 T2 T3 T4 T5 T6 T7

CYP1

MT1 MT2 MT3 MT4 MT5 MT6

OSC1 OSC2 OSC3 OSC4 OSC5 OSC6 OSC7

MT7

CYP2 CYP3 CYP4 CYP5 CYP6 CYP7CYP1

OSCs

CYPs

MTs

ATs

GTsOH

CH3

OH

CH3CH3

CH3

CH3

OH

CH3

O

OH

O

Golden Gate: Weber et al. (2011) PLoS ONE 6(2): e16765. doi:10.1371/journal.pone.0016765; Thomas Leveau unpub.

TS

CYP450

CYP450

MT

GTs

AT Accumulation of acylated terpene in the vacuole

+

+

+

+

+

Functional assembly of a terpene pathway- Proof of concept

The thalianol pathway – The first example of an

“operon-like” gene cluster from Arabidopsis

Field & Osbourn (2008) Science 320 534-547

Thalianol

Thalian-diol

Desaturatedthalian-diol

TS

2,3-Oxidosqualene

CYP708A2

CYP705A5

O

H

OH

OH

H

OH

OH

H

OH

TS KO TS KO

CYP708A2 KO CYP708A2 KO


wild type wild type

thalia

nol

thalia

n-d

iol

desat-

thalia

n-d

iol

SIC 229 amu SIC 227 amu

Thalianol

Thalian-diol

Desaturatedthalian-diol

TS

2,3-Oxidosqualene

CYP708A2

CYP705A5

O

H

OH

OH

H

OH

OH

H

OH

OO

H

OH

H

OH

OH

H

OH

OH

H

OH

H

OH

OH

H

OH

OH

H

OH

H

OH

TS KO TS KO



wild type wild type

thalia

nol

thalia

n-d

iol

desat-

thalia

n-d

iol


TS KO TS KO



wild type wild type

thalia

nol

thalia

n-d

iol

desat-

thalia

n-d

iol

TS KO TS KO



wild type wild type

thalia

nol

thalia

n-d

iol

desat-

thalia

n-d

iol


Examples of gene

clusters for specialised

metabolic pathways in

a range of plants

Nutzmann & Osbourn (2014) Curr Opin.

Biotechnol. 26:91

Arabidopsis thaliana

Thalianol

Marneral

Lotus japonicusLotaustralin Linamarin

Zea mays

Solanum lycopersicum

Sorghum bicolor

Papaver somniferum

DIMBOA-Glc

α-Tomatine

Noscapine

Dhurrin

Oryza sativaMomilactone A Phytocassanes A-E

Avena strigosa

Avenacin A-1

A

F

G

E

C

H

B

D

Nutzmann & Osbourn, Curr Opin. Biotechnol. 2014

Arabidopsis thalianaBrassica rapaCucumis sativusGlycine maxLotus japonicusMimulus guttatusMedicago truncatulaPopulus trichcarpaRicinis communisSolanum lycopersiconSolanum tuberosumVitis viniferaBrachypodiumdistachyonmOryza sativaSorghum bicolorSetaria indicaZea mays

Mining the terpenome

Boutanaev et al., (2015) PNAS

Functional analysis of candidate pathways

Boutanaev et al., (2015) PNAS

Avenacin cluster (A. strigosa)

Clustering may allow a ‘belts and braces’ approach to pathway regulation

Thalianol cluster (A. thaliana)

Chromatin modifications at plant metabolic gene clusters

Nutzmann and Osbourn (2015); Yu, Nutzmann et al., submitted.

Genome mining for new pathways and

chemistries

Nutzmann & Osbourn, Curr Opin. Biotechnol. 2014

Avena strigosa (S75) – A diploid model

Genome Size ~3.92 Gb (1C=4.00)

Diploid: AA

2n=2x=14

Chromosome number : 7

Photosynthetic pathway: C3

Bin Han

Qiang Zhao

Ying Lu

Yan Li

Hengyun Lu

Xuehui Huang

Qi Feng

Danling Fan

Wenjun Li

Qilin Tian

Aymeric Leveau

Thomas Louveau

Daisy Orme

Preparation of genomic DNA and samples for RNA-seq,

454 resources, BAC libraries, genome mining, functional

analysis, metabolic biology

Anne Osbourn

Project leader (JIC)

Bin Han

Genome assembly and annotation

Annotation and comparative genomics

Annotation

Genome assembly

Material and Genome sequencing

Genome and transcriptome sequencing

Genome sequencing

Transcriptome sequencing

Director & project leader

Annotation

Genome sequencing ofAvena strigosa S75April 2013-

mate pair(3k, 8k, 20k)

S75 raw reads

Contigs

Scaffolds

Super scaffolds

Fosmid ends(40k)

raw reads

Fosmid

Super scaffolds

scaffolds

Assembly strategy Bin Han

v6 – 90% coverage

RNAseq

Head emergenceSeedling and root Root tips

Boot stage

a

a

a

b

b

a Identical mutations (G1912 → A)b Identical mutations (G7249 → A)

Mutant Mutation

event

Predicted

amino acid

change

Predicted

Mutation type

A1 G1912A Tyr165

STOP

Premature

termination of

translation

B1 G1912A Tyr165

STOP

109 G3417A Tyr380

STOP

610 G1912A Tyr165

STOP

1146 G4169A Tyr471

STOP

1293 G39A Tyr13

STOP

110 G6689A -

Splicing error225 G3302

A -

589 G3914A -

1001 G4365A -

297 G3939A Glu419

Lys

Amino acid

substitution

358 G5234A Cys563

Tyr

384 C7249T Ser728

Phe

532 G549A Gly121

Glu

599 G2809A Gly277

Glu

1023 C7249T Ser728

Phe

1217 G2025A Gly203

Glu

a

a

a

b

b

a Identical mutations (G1912 → A)b Identical mutations (G7249 → A)

Mutant Mutation

event

Predicted

amino acid

change

Predicted

Mutation type

A1 G1912A Tyr165

STOP

Premature

termination of

translation

B1 G1912A Tyr165

STOP

109 G3417A Tyr380

STOP

610 G1912A Tyr165

STOP

1146 G4169A Tyr471

STOP

1293 G39A Tyr13

STOP

110 G6689A -

Splicing error225 G3302

A -

589 G3914A -

1001 G4365A -

297 G3939A Glu419

Lys

Amino acid

substitution

358 G5234A Cys563

Tyr

384 C7249T Ser728

Phe

532 G549A Gly121

Glu

599 G2809A Gly277

Glu

1023 C7249T Ser728

Phe

1217 G2025A Gly203

Glu

Characterization of sad1 mutants

S75 29

735

838

453

259

910

23

1217

S75 11

022

558

910

01A B C

GAPDH

Sad1

S75 A1 B1 10

961

011

46

1293

S75 29

735

838

453

259

910

23

1217

S75 29

735

838

453

259

910

23

1217

S75 11

022

558

910

01

S75 11

022

558

910

01A B C

GAPDH

Sad1

S75 A1 B1 10

961

011

46

1293

S75 A1 B1 10

961

011

46

1293

Stop codons Splicing errors Amino acid substitutions

Transcript

188

98

62

49

38

28

17

14

kDa

Protein

D

F259

F725

S728

I554

T260Y264

D

F259

F725

S728

I554

T260Y264

F259

F725

S728

I554

T260Y264D484

C563

C485

C

D484

C563

C485

D484

C563

C485

C

Characterization of sad1 mutants

Areas for future work

• Avena strigosa genome annotation• Comparative genomics with other species

– Why are oats different?

• Analysis of important metabolic pathways – Agriculture, health, industrial biotechnology

• Establish a reverse genetics platform for diploid oat- Exome capture (validate with allelic series of characterised mutants; then test with 50 uncharacterized avenacin-deficient mutants)

• Longer term - Resequencing of other Avena accessions, GWAS analysis…

Osbourn Group:Ramesh Thimmappa Hans Neutzmann Aymeric LeveauGemma FarresTessa MosesThomas Louveau James Reed Rachel Melton Daisy OrmeZheyong XueDiego Batista

OpenPlant Project Manager:Colette Matthewman

In collaboration with:

Lionel Hill, Paul Brett, Rob Field, Martin Rejzek, George Lomonossoff, Paul O’Maille, Martin Trick, Vinod Kumar (JIC)Andy Greenland, Emma Wallingford (NIAB) Bin Han and team (SIPPE)Tim Langdon (IBERS)The Syntegron team: Susan Rosser (Univ. Glasgow), Jay Keasling (UC Berkeley), Paul Freemont (Imperial), Declan Bates (Univ. Exeter), Josh Leonard (Northwestern) and co-workersUnilever, Croda, AlgenuityChristina Smolke, Beth Sattely, Sue Rhee

Jenni Rant (The SAW Trust)

NIH

avena strigosa from metabolic gene clusters to genomics 2.4/anne osbourn.pdf · avena strigosa...

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