Honoo Satake

Metabolic engineering of lignan biosynthesis in Forsythia

OH

OCH3

OH

HO

O

O

H H

OH

OCH3

OCH3

OH

O

OH

CH3

coniferyl alcohol x 2 (+)-pinoresinol

HO

O

H H

OH

OCH3

OCH3

OH

(+)-lariciresinol (-)-matairesinol

HO

OH

H3CO

H

H

OCH3

O

O

Forsythia(+)-pinoresinol glucoside

HO

O

O

H H

O-Glc

OCH3

OCH3

(-)-podophyllotoxin Linum

H

O

O

OCH3

OCH3OCH3

H O

O

O

H

HH

O

O

OCH3

OCH3OCH3

H O

O

O

O

O

HH

(-)-matairesinol

HO

OH

H3CO

H

H

OCH3

O

O

O

O

H H

O

O

O

O

O

O

H H

OH

OCH3

O

O

O

O

H H

O

O

O

O

(+)-piperitol (+)-sesamin

OH

(+)-sesaminol Sesamum

Lignans are species-unique plant secondary compounds

Sesamin: A sesame lignan exerting beneficial actions on humans

•A furofuran lignan• Most abundantly contained in sesame seeds (~0.5% of sesame oil)

O

O

H H

O

O

O

O

(+)-sesamin(+)-sesamin

O

O

H H

OH

OCH3

O

O

(+)-piperitol(+)-piperitol

HO

O

O

H H

OH

OCH3

OCH3

(+)-pinoresinol(+)-pinoresinol

CYP81QCYP81Q CYP81QCYP81Q

•Sesamin is biosynthesized from pinoresinol via the formation of two methylenedioxy bridges by a unique enzyme, CYP81Q.

•Sesamin has been shown to exert a wide variety of biological effects:such as anti-oxidative activity, anti-hypertensive activity, and protection of the liver from alcohol.

•Sesamin has become commercially available as a new health supplement.•The demand for sesamin has been markedly increasing.•Sesamin has become commercially available as a new health supplement.•The demand for sesamin has been markedly increasing.

Metabolic engineering of lignan biosynthesis(=Generation of sesamin-producing transgenic plants)

・ Very small amounts: sesamin comprises at most 0.5% component of sesame oil, which most abundantly contains sesamin.

・ Sesame seeds are cultivated only once every year.

・ Japan imports 99% of its sesame seeds.

Convert “agricultural production” into industrial production using a transgenic plant in a “plant factory”

Low efficiency in acquisition of sesamin via extraction from sesame seeds

Which plant is the best transgenic host for sesamin production?

Forsythia spp. as a transgenic host for sesamin production

・ Perennial woody plants

・ Their leaves and fruits are used as Chinese medicines because they contain various lignans.

・ Sesamin is not produced in Forsythia.

・ They produce large amounts of various lignans, including pinoresinol, a direct precursor of sesamin.

<Our idea>Construction of “sesamin-producing Forsythia”

by metabolic engineering of the lignan biosynthesis pathways

HO

O

O

H H

OH

OCH3

OCH3

(+)-pinoresinol

O

O

H H

O

O

O

O

(+)-sesamin

HO

O

H H

OH

OCH3

OCH3

OH

PLR

(+)-lariciresinol

Strategy for production of sesamin by metabolic engineering of Forsythia spp.

Suppression of PLR by RNAi

Engineering: CYP81Q

Introduction of Sesamum CYP81Q

Examination of sesamin production using Forsythia suspension cell cultures

CPi-Fk WT

PLR

CYP81Q1

nptⅡ

rRNA

#1 #2 #1

Generation of F. koreana transgenic cell, CPi-Fk

Callus formationCallus formation

Suspension cultureSuspension culture

30 days30 days 30 days30 days

selectionselection

CPi-FkCPi-Fk

agrobacterium-based transformationagrobacterium-based transformation

• CYP81Q (and nptII) is expressed in CPi-Fk.• PLR expression is suppressed in CPi-Fk.

• Transgenic Forysthia can produce sesamin.• This is the first report of metabolic engineering of a lignan.

CPi-Fk produces approx. 0.8 mg/g DW of sesamin

0.0

0.2

0.4

0.6

0.8

1.0

WT CPi-Fk

ND

Ses

amin

(

mg

g-1 D

W)

WT CPi-Fk

Production of sesamin by CPi-Fk

MASSMASS

quantificationquantification

Effect of light on sesamin production by CPi-Fk

• CPi-Fk produces 0.8 mg/g DW under dark conditions.

• Several other secondary metabolites have been found to be regulated.

• The effects of light on lignan production have never been reported.

Examination of the effect of LED or fluorescent light on sesamin production by CPi-Fk

• Red light moderately reduces CPi-Fk growth.

• CPi-Fk grows under blue LED or white fluorescent light as well as in the dark.

Effects of light on the growth of CPi-Fk

WT

0

50

100

150

Dark White Blue Red

Gro

wth

rate

(%)

CPi-Fk

0

50

100

150

Dark White Blue Red

Gro

wth

rate

(%)

CPi-FkWildtype

red LED, 450-550 nm, 470 nm-peak; blue LED, 600-700 nm, 630 nm- peak;

white light (white fluorescent tubes)

Light intensity: 100 μmol m-2s-1 PPFD (photosynthetic photon flux density)

0.0

0.5

1.0

1.5

Dark Dark White Blue

Pin

ore

sin

ol a

gly

con

e

(mg

-1 g

DW

)

WT CPi-Fk

0

1

2

3

4

Dark Dark White Blue

To

tal p

ino

resi

no

l

(mg

-1 g

DW

)

WT CPi-Fk

Pin

ore

sino

l agl

ycon

e(m

g-1 g

DW

)T

ota

l pin

ores

inol

(mg-1

g D

W)

0

1

2

3

Dark Dark White BlueS

esa

min

(mg

-1 g

DW

)

WT CPi-Fk

NDND

Pin

ore

sin

ol ag

lycon

ean

d g

lucosid

es

Sesam

in

Pin

ore

sin

ol

ag

lycon

ePercent ( % )

Aglycone / TotalPinoresinol

Wild type Dark 29±6.6Dark 23±5.1

CPi-Fk White 30±3.3Blue 40±7.7

Cell strain Light Aglycon and glucosidesPinoresinol aglycone

• Pinoresinol production is increased under blue or white light.

• Sesamin production is also approx. 3-fold higher under blue or white light (2.5 mg/g DW) than under dark conditions (0.8 mg/g DW).

Effects of light on lignan production by CPi-Fk

CPi-Fk may provide stable and sustainable sesamin productionCPi-Fk may provide stable and sustainable sesamin production

Insight into sesamin production efficiency

CPi-Fk

・ 0.8 ～ 2.65 mg/g DW

・ 10-fold proliferation for two weeks

・ Cultivation anytime

Sesamum seeds

・ 1 ～ 5 mg/g of sesame oil

・ Cultivation once a year

・ 10-fold greater lignan than suspension culture

・ Much larger biomass with lower cost

・ Propagation from a cut explant (without the requirement of flowering or seed formation)

Forsythia plantForsythia plant

More efficient sesamin production usingMore efficient sesamin production using Forsythia Forsythia transgenic plantstransgenic plants

F0 medium F medium

FM0 medium F mediumCallus

Shoot formation and elongation

Rooting

Days 0 10 　　　　　 　 　　 　 20 　　　　　 　　　　　　 30 　　　 45 60 　　　　　　　　　　　　 90 120

Rooting

CallusShoot formation and elongation

F. koreana

F.intermedia

Days 0 10 　　　　　 　 　　 　 14 　　　　　　　 　　　 30 　　　 55 60 　　　　　　　　　　 90 120

Elucidation of regeneration condition of Forsythia plants from calli

• Optimal media are different between F. koreana and F. intermedia.

• F. koreana and F. intermedia grow to 10-cm plants in 120 days.

0

20

40

60

80

100

Re

ge

ne

ratio

n (

%)

F. koreana

F. intermedia

Medium only Kanamycin (mg l-1) Hygromycin (mg l-1) 5 25 50 0.5 2.5 5

Rooting shoots

0

5

10

15

20

25

30

35

Day

s fo

r roo

ting

F. koreana

F. intermedia

Medium only Kanamycin (mg l-1) Hygromycin (mg l-1) 5 25 50 0.5 2.5 5Rooting

( X / 3 ) 3, 3 2, 1 3, 0 0, 0 1, 0 1, 0 0, 0

Elucidation of hygromycin resistance of Forsythia

Regenerating shoots

• 5 mg/L hygromycin completely eliminates non-transgenic F. koreana and F. intermedia at regeneration and rooting stages.

Elucidation of transgenic Forsythia from the callus

Day 0 3 10 60 120

F. koreana FM0F. intermedia F0

Ticarcillin 0 300 200 200Hygromycin 0 0 5 5

F

rRNA

nptⅡ

hptⅡ

F. koreana F. intermedia

　　　　　　　　　 #1 wt #1 #2 wt

rRNA

nptⅡ

hptⅡ

Genomic PCR

RT-PCR

F. koreana F. intermedia　 Transgenic #1 #1 　 #2×25

×30

×30

×25

×30

×30

　　 Wild type

Construction of hygromycin-resistant Forsythia

• Hygromycin-resistant transgenic F. koreana and F. intermedia have been generated.

• These transgenic Forsythia plants still grow and propagate.

Conclusion and perspectives

We are now attempting to generate CYP81Q1 and PLR-RNAi-introducedtransgenic Forsythia plants.

• Transgenic Forsythia can produce sesamin.

• CPi-Fk is a promising platform for industrial production of sesamin.

• Transgenic Forsythia plants are expected to be sustainable sesamin producers in plant factories.

• CPi-Fk, a CYP81Q1 and PLR-RNAi-introduced Forsythia suspension cell, produces an exogenous lignan, sesamin.

• Blue LED and white fluorescent light increase sesamin production by CPi-Fk.

• Basal procedures for transgenic Forsythia plants have been established.

This project has been financially supported by the Ministry ofEconomy, Technology, and Industry (METI), Japan, since 2006

SUNBOR

Kim H.-J. Morimoto K.

Yamagaki T.Ono E.

Osaka University

Suntory Holdings

Co-workers and acknowledgements

Kobayashi A.

Murata J.

Okazawa A.