Metabolic engineering of tomato aroma.

The sounds of silent metabolism.

Efraim Lewinsohn
Newe Ya'ar Research Center
Agricultural Research Organization
Israel

The Newe Yaar

Research
Center
Chemical, Biological and
Genetic Factors Affecting
Aromas of Fruits, Vegetables,
Israel
Flowers and Spices
Agricultural Research
Organization,
Ministry of Agriculture
State of Israel

Projects

watermelon
tomato
citrus
roses
basil
melon
fennel
koubo
apple
lilly
strawberry
cactus pear
banana
khat
ephedra

?Where have all the flavors gone

 saltiness
acidity
sweetness
bitterness
umami

CHO
O

nootkatone
(grapefruit)

cinnamaldehyde
(cinnamon)

O C CH3
OH

isoamyl acetate
(banana)
CHO benzaldeyde

(almonds)

geraniol
(roses)

enantiomers
O

)+(-Carvone

caraway

)-(-Carvone

spearmint

The aroma of coffee

300 volatile constituents
contributing to the smell of
green coffee
850 volatile constituents
contributing to the flavor of
roasted coffee

Aroma Chemicals
Mixtures of volatile compounds
Dominant and subtle components
Often small chemical differences have big
impact on aroma properties

Can we use genomic approaches to

?study aroma formation in plants

CO2

OH

R
OH
R-Linalool

R = OCH 3
Eugenol

R
OCH3
R=H
R = OCH 3

Estragole
Methyl Eugenol

H2O + minerals

Estimated number of natural products

Biosynthetic pathways
25,000 terpenes

mevalonic acid pathway

deoxyxylulose phosphate pathway

12,000 alkaloids

mostly from amino acids

8,000 phenolics

shikimic acid pathway

malonate-acetate pathway
Croteau et al., 2000

IPP
Menthol
OPP

Pinene
Limonene

Linalool

Geranyl diphosphate
(GPP)

Monoterpenes

IPP
Farnesene
OPP

Farnesyl diphosphate
(FPP)

Sesqui-Bisabolol
and Triterpenes Valencene
Nerolidol

IPP
OPP

Geranylgeranyl diphosphate
(GGPP)

Di- and
Tetraterpenes

Carotenoids
Chlorophyll
)Phytol(
Tocopherols
Gibberellin

S-Adenosyl methionine (SAM)

Furaneol: O-Methyl transferase
O

OH

OCH3

SAM +

+ SAH
O

furaneol

methoxyfuraneol

Alcohol dehydrogenases
OH

Geraniol

(Roses)

Geraniol )alcohol(
dehydrogenase

+
NADPH
NADP

NADP+
NADPH

Geranial

(Citrus)

Clarkia breweri (Onagraceae)

.

OH

OC
H
3
OH

OH

O C

CH3

OC
H

H
OC

OH

OC
H

C OCH
3

H
OC

OCH3

H
OC

OCH3

OC
H
3
OH

O C

O
CH3

OCH3

OH

OH

C OCH
3

OH

OC
H

OC
H

H
OC

OH

O
.

OH

OC
H
3
OH

OH

O C

OCH3
OH

CH3

OCH3
OCH3

C OCH
3

H
OC

OC
H

OC
H

H
OC

OH

OCH3
OH

Monoterpenes

Volatiles emitted by
Clarkia breweri
flowers

Benzenoids

Metabolic engineering
Clarkia breweri
of the terpenoid
S-linaloolpathway
synthasein)LIS(
tomato fruit
OH

O
OH
8-hydroxylinalool

Tomato

NerolOH
OPP Geraniol
OPP
synthase
LIS

Geranyl diphosphate
)GDP(
Geranyl
diphosphate

Geranial
O Glc

OH

S-linalool

OH

Petunia

Neral

OH

Geraniol

linalyl-glucoside

Citronellol

Carnation
OCOCH3
Carotenoids
)Lycopene(

Geranyl
acetate

O
HO

O
OCOCH
3
HO

linalool oxides

Citronellyl
acetate

Lewinsohn et al., 2001

:Conclusions
Ectopic expression of LIS brings about to:
Increases in S-linalool
Formation of 8-hydroxylinalool )probably
due to an endogenous hydroxylase activity(
Yet unknown functionality acting coincidentally
on S-linalool
Relic from the past
)a trait conserved during domestication
and breeding(

 Expression of ONE gene brings about

many expected and unexpected
phenotypical changes.

Silent Metabolism

OH

Tomato

8-hydroxylinalool

Lewinsohn et al., 2001

OH
OH

O Glc

OPP

LIS

Geranyl
diphosphate

Petunia

S-linalool

Silent
metabolism

linalyl-glucoside

Carnation

O
HO

Lcker et al., 2001

O
HO

linalool oxides

Lavy et al., 2002

 The SAME gene, overexpressed in

different plants
)genetic and metabolic backgrounds(
resulted in different metabolic profiles

Silent Metabolism

Geranial:
Lemon scented acyclic monoterpene
aldehyde, an aroma constituent of many
fruits, flowers and herbs.
Antifungal, antibacterial, insect repellant,
insecticidal and anticarcinogenic properties.

O
geranial

 Phenotypic
Phenotypic Diversity
Diversity in
in Basil
Basil (Ocimum
(Ocimum spp.)
spp.)

Major Components of Basil Lines

EMX:
EMX: mainly
mainly estragole
estragole (methylchavicol)
(methylchavicol)
SW:
SW: mainly
mainly eugenol
eugenol and
and RR-linalool
-linalool
Sweet
Sweet Dani:
Dani: citral
citral (geranial
(geranial and
and neral)
neral)
OH
O
O

OCH3
OCH3
estragole

EMX

OH
eugenol

R-linalool

SW

geranial

SD

neral

SEM

Ocimum basilicum (Lamiaceae)

BALUTOT

Werker et al.,
Ann. of Bot. 71,42, 1993

Thymus vulgaris,
before and after gland isolation

Micrographs: Courtesy of J. Gershenzon

Metabolic pathways
in
glandular trichomes

Gang et al.,
Plant Physiol 125: 539, 2001

Relative abundance of ESTs in basil peltate glands

(by putative function)
No Identification Due to
Insignificant Similarity to
Database Sequences
13%

Match to Functionally
Undefined Protein
22%

Phenylpropanoid Pathway
13%
Terpenoid Pathways
1%

Miscellaneous Functions
4%

SAM Biosynthesis and

Regeneration
14%

Oxidation/Stress
Protection
4%

Glycolysis /
Pentosphosphate
Pathways
3%

Structural Proteins
1%
Protein Synthesis and
Turnover
7%
Gene Expression and
Regulation
7%

Lipid Transfer Protein

1%

Small Molecule
Transporters
3%

Other Metabolism
Enzymes
4%
ATP / NAD)P(H Formation
3%

Expression of a putative terpene synthase

in two basil lines

L: Leaf
G: Gland

Iijima et al., 2004

: Functional Expression Approach

Putative
aroma gene

Catalysis

Heterologously
Expressed
Protein

Substrate

Bacterial
Lysate

Product

Geraniol synthase
from lemon basil glandular trichomes

OH

OPP
Geraniol synthase

Geranyldiphosphate

Geraniol
Iijima et al.,
Plant Physiol 134: 370, 2004

isopentenyl
diphosphate )IPP(

dimethylallyl
diphosphate )DMAPP(

OPP

Geraniol Synthase

Geranyldiphosphate )GPP(

OH

Geraniol dehydrogenase

Geraniol

Geranial

Farnesyldiphosphate )FPP(
Geranylgeranyldiphosphate )GGPP(

Carotenoids

pBIN19
pg promoter

OcGES

pg terminator

GES trangenesis resulted in

lower levels of lycopene
T

carotene

C
Transgenic lines

WT

Silent Metabolism
geranial and neral
Geraniol dehydrogenase

citronellol, citronellal
Geraniol reductase

geranyl acetate, citronellyl acetate

Geraniol / citronellol acetyltransferase

All present in tomato fruit cell-free extracts

Silent Metabolism

)Lewinsohn & Gijzen 2009(

Silent Metabolism
Unknown Function:
The silent enzymes have a different function
but happen to act on new substrates
Evolutionary Relics:
In crop domestication, traits that are not
disadvantageous are not always lost

Silent Metabolism
Ecological and Evolutionary Advantages
Evolution favors diversity
Easy to modify an existing biochemical
pathway to produce novel compounds
Easier to restore a lost trait

Taste and smell panelists

Control

GES-transgenic
Transgenic better
Control better
No preference

Not only tomatoes

Carotenoid containing fruits
and vegetables:
melon, carrot, potato, apricots
Flowers: Gerbera, Ornithogalum
Spice plants: basil, lavender
Seed crops: Rice, maize

Potential of Metabolic Engineering to

Improve and Modify Aroma and Taste
of Agricultural Produce

 We have not forgotten the flavor of tomatoes

Special Thanks
Rachel
Rachel Davidovich-Rikanati,
Davidovich-Rikanati, Newe
Newe Yaar
Yaar
Yaron
Yaron Sitrit,
Sitrit, Ben
Ben Gurion
Gurion University
University
Yaakov
Yaakov Tadmor,
Tadmor, Newe
Newe Yaar
Yaar
Einat
Einat Bar,
Bar, Newe
Newe Yaar
Yaar
Eran
Eran Pichersky,
Pichersky, University
University of
of Michigan
Michigan
Natalia
Natalia Dudareva,
Dudareva, Purdue
Purdue University
University

Newe Yaar
Research Center,
Nurit Katzir
Uzi Ravid
Nativ Dudai
Yossi Burger
Ari Schaffer
Eli Putievsky
Olga Larkov
Ayala Meir
Mwafaq Ibdah
Yaniv Azulay
Karin Rand
Ilan Butnik
Bentsi Carmona
Raz Krizevski

ARO

Ack

Jim Simon, Rutgers University

Yoko Iijima, Kazusa DNA Research
Institute, Japan
David Gang, University of Arizona

This Project was funded by

BARD: US-ISRAEL AGRICULTURAL
RESEARCH FUND

!!!Thanks