Metabolic engineering of lignan
biosynthesis in Forsythia
Honoo Satake
Lignans are species-unique plant secondary compounds
HO
H
H
H3CO
O
O
O
O
HO
O
H
O
H
CH 3O
OCH3
OCH 3
OCH 3
OH
(-)-matairesinol
Linum
(-)-podophyllotoxin
OH
O
O
OCH3
OCH3
O
CH3
OH
OH
O
O
OH
O
O
H
OH
coniferyl alcohol x 2
H
H
OCH3
O
O
O
O
(+)-pinoresinol
OH
H
O
O
HO
H
H
O
O
OH
OCH3
H
H
O
O
O
O
(+)-piperitol
Sesamum
(+)-sesaminol
(+)-sesamin
OCH3
OCH3
H
OH
O-Glc
H3CO
O
O
O
HO
H
H
H
H
H
O
HO
HO
OCH3
(+)-pinoresinol glucoside
O
OH
OCH3
OCH3
(+)-lariciresinol
OH
(-)-matairesinol
Forsythia
Sesamin: A sesame lignan exerting beneficial actions on humans
•A furofuran lignan
• Most abundantly contained in sesame seeds (~0.5% of sesame oil)
•Sesamin is biosynthesized from pinoresinol via the formation of
two methylenedioxy bridges by a unique enzyme, CYP81Q.
OCH3
OCH3
OH
O
OH
O
CYP81Q
O
H
CYP81Q
O
H
H
O
H
H
O
H
O
O
HO
O
OCH3
O
O
O
(+)-pinoresinol
(+)-piperitol
(+)-sesamin
•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.
Low efficiency in acquisition of sesamin via extraction
from sesame seeds
・ 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.
Metabolic engineering of lignan biosynthesis
(=Generation of sesamin-producing transgenic plants)
Convert “agricultural production” into industrial production
using a transgenic plant in a “plant factory”
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
Strategy for production of sesamin by metabolic engineering
of Forsythia spp.
OCH3
OH
OCH3
OH
O
H
O
H
PLR
O
H
H
OH
HO
OCH3
HO
(+)-pinoresinol
CYP81Q
OCH3
(+)-lariciresinol
Engineering:
O
O
Suppression of PLR by RNAi
O
H
H
Introduction of Sesamum CYP81Q
O
O
O
(+)-sesamin
Examination of sesamin production using
Forsythia suspension cell cultures
Generation of F. koreana transgenic cell, CPi-Fk
agrobacterium-based transformation
30 days
30 days
selection
Callus formation
CPi-Fk
#1
#2
Suspension culture
CPi-Fk
WT
#1
PLR
CYP81Q1
nptⅡ
rRNA
• CYP81Q (and nptII) is expressed in CPi-Fk.
• PLR expression is suppressed in CPi-Fk.
Production of sesamin by CPi-Fk
quantification
Sesamin
(mg g-1 DW)
1.0
0.8
0.6
0.4
MASS
0.2
0.0
ND
WT
WT
CPi-Fk
CPi-Fk
CPi-Fk produces approx. 0.8 mg/g DW of sesamin
• Transgenic Forysthia can produce sesamin.
• This is the first report of metabolic engineering of a lignan.
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
Effects of light on the growth of CPi-Fk
150
150
Growth rate (%)
Growth rate (%)
Wildtype
WT
100
50
0
CPi-Fk
CPi-Fk
100
50
0
Dark
White
Blue
Red
Dark
White
Blue
Red
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)
• CPi-Fk grows under blue LED or white fluorescent light as well as in the dark.
• Red light moderately reduces CPi-Fk growth.
4
3
2
1
0
Dark
WT
White
Blue
CPi-Fk
1.5
Sesamin
Sesamin
aglycone
Pinoresinol
aglycone
Pinoresinol
Pinoresinol
aglycone
-1
-1
(mg
g DW)
(mg
g DW)
Dark
Percent ( % )
Pinoresinol
Aglyconeaglycone
/ Total
Cell strain Light Aglycon and glucosides
Pinoresinol
Wild type Dark
29±6.6
Dark
23±5.1
CPi-Fk
White
30±3.3
Blue
40±7.7
1.0
0.5
0.0
3
(mg-1 g DW)
-1-1ggDW)
(mg
(mg
DW)
Pinoresinol aglycone
glucosides
andTotal
Total
pinoresinol
pinoresinol
Effects of light on lignan production by CPi-Fk
2
1
0
Dark
WT
Dark
White
CPi-Fk
Blue
ND
Dark
WT
Dark
White
Blue
CPi-Fk
• 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).
Insight into sesamin production efficiency
Sesamum seeds
CPi-Fk
・ 1～5 mg/g of sesame oil
・ 0.8～2.65 mg/g DW
・ Cultivation once a year
・ 10-fold proliferation for two weeks
・ Cultivation anytime
CPi-Fk may provide stable and sustainable sesamin production
Forsythia plant
・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)
More efficient sesamin production using Forsythia transgenic plants
Elucidation of regeneration condition of Forsythia plants
from calli
F.
koreana
Days
0
10
20
FM0 medium
Callus
30
45
60
90
F medium
120
Shoot formation and elongation
Rooting
F.
intermedia
Days
0
10
14
F0 medium
Callus
30
55
60
90
F medium
Shoot formation and elongation
Rooting
• Optimal media are different between F. koreana and F. intermedia.
• F. koreana and F. intermedia grow to 10-cm plants in 120 days.
120
Elucidation of hygromycin resistance of Forsythia
Rooting shoots
Regenerating shoots
35
100
F. koreana
30
80
F. intermedia
60
40
20
Days for rooting
Regeneration (%)
F. koreana
F. intermedia
25
20
15
10
5
0
Medium only
-1
Kanamycin (mg l )
5
25
50
-1
Hygromycin (mg l )
0.5
2.5
5
0
Medium only
Rooting
( X / 3 ) 3, 3
-1
-1
Kanamycin (mg l )
5
25
50
Hygromycin (mg l )
0.5
2.5
5
2, 1
1, 0
3, 0
0, 0
1, 0
• 5 mg/L hygromycin completely eliminates non-transgenic F. koreana and
F. intermedia at regeneration and rooting stages.
0, 0
Elucidation of transgenic Forsythia from the callus
Day
0
F. koreana FM0
F. intermedia F0
Ticarcillin
0
Hygromycin
0
3
10
60
120
F
300
0
200
5
200
5
Construction of hygromycin-resistant Forsythia
F. koreana F. intermedia
Genomic PCR #1 wt #1 #2 wt
F. koreana F. intermedia
rRNA
×25 Transgenic #1
nptⅡ
×30
hptⅡ
×30
RT-PCR
rRNA
×25
nptⅡ
×30
hptⅡ
×30
#1
• Hygromycin-resistant transgenic F. koreana and F. intermedia
have been generated.
• These transgenic Forsythia plants still grow and propagate.
#2
Wild type
Conclusion and perspectives
• 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.
• 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.
We are now attempting to generate CYP81Q1 and PLR-RNAi-introduced
transgenic Forsythia plants.
Co-workers and acknowledgements
SUNBOR
Osaka University
Kobayashi A.
Okazawa A.
Kim H.-J.
Morimoto K.
Suntory Holdings
Murata J.
Yamagaki T.
Ono E.
This project has been financially supported by the Ministry of
Economy, Technology, and Industry (METI), Japan, since 2006