1. Honoo Satake Metabolic engineering of lignan biosynthesis in Forsythia

2. OH OCH 3 OH HO O O HH OH OCH 3 OCH 3 OH O OH CH 3 coniferyl alcohol x 2 (+)-pinoresinol HO O HH OH OCH 3 OCH 3 OH (+)-lariciresinol (-)-matairesinol HO OH H 3 CO H H OCH 3 O O Forsythia (+)-pinoresinol glucoside HO O O HH O-Glc OCH 3 OCH 3 (-)-podophyllotoxin Linum (-)-matairesinol HO OH H 3 CO H H OCH 3 O O O O HH O O O O O O HH OH OCH 3 O O O O HH O O O O (+)-piperitol (+)-sesamin OH (+)-sesaminol Sesamum Lignans are species-unique plant secondary compounds

3. 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 HH O O O O (+)-sesamin O O HH OH OCH 3 O O (+)-piperitol HO O O HH OH OCH 3 OCH 3 (+)-pinoresinol CYP81Q 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.

4. 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?

5. 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. Construction of “sesamin-producing Forsythia” by metabolic engineering of the lignan biosynthesis pathways

6. HO O O HH OH OCH 3 OCH 3 (+)-pinoresinol O O HH O O O O (+)-sesamin HO O HH OH OCH 3 OCH 3 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

7. CPi-Fk WT PLR CYP81Q1 npt Ⅱ rRNA #1 #2 #1 Generation of F. koreana transgenic cell, CPi-Fk Callus formation Suspension culture 30 days selection CPi-Fk agrobacterium-based transformation CYP81Q (and nptII) is expressed in CPi-Fk. PLR expression is suppressed in CPi-Fk.

8. 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 ND Sesamin (mg g -1 DW) WT CPi-Fk Production of sesamin by CPi-Fk MASS quantification

9. 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

10. 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 CPi-Fk Wildtype 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 -2 s- 1 PPFD (photosynthetic photon flux density)

11. Pinoresinol aglycone (mg -1 g DW) Total pinoresinol (mg -1 g DW) ND Pinoresinol aglycone and glucosides Sesamin Pinoresinol aglycone Aglycon and glucosides Pinoresinol 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

12. CPi-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 plant More efficient sesamin production using Forsythia transgenic plants

13. F0 medium F medium FM0 mediumF medium Callus Shoot formation and elongation Rooting Days 0 10 20 30 45 60 90 120 Rooting Callus Shoot 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.

14. Rooting shoots 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.

15. Elucidation of transgenic Forsythia from the callus

16. rRNA npt Ⅱ hpt Ⅱ F. koreana F. intermedia #1 wt #1 #2 wt rRNA npt Ⅱ hpt Ⅱ Genomic PCR RT-PCR F. koreana F. intermedia Transgenic #1 #1 #2 ×25 ×30 ×25 ×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.

17. Conclusion and perspectives We are now attempting to generate CYP81Q1 and PLR-RNAi-introduced transgenic 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.

18. This project has been financially supported by the Ministry of Economy, 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.