honoo satake metabolic engineering of lignan biosynthesis in forsythia
TRANSCRIPT
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.