Transgenic Forsythia plants expressing sesame cytochrome P450 produce beneficial lignans

Lignans are widely distributed plant secondary metabolites that have received attention for their benefits to human health. Sesamin is a furofran lignan that is conventionally extracted from Sesamum seeds and shows anti-oxidant and anti-inflammatory activities in the human liver. Sesamin is biosynthesized by the Sesamum-specific enzyme CYP81Q1, and the natural sources of sesamin are annual plants that are at risk from climate change. In contrast, Forsythia species are widely distributed perennial woody plants that highly accumulate the precursor lignan pinoresinol. To sustainably supply sesamin, we developed a transformation method for Forsythia leaf explants and generated transgenic Forsythia plants that heterologously expressed the CYP81Q1 gene. High-performance liquid chromatography (HPLC) and LC-mass spectrometry analyses detected sesamin and its intermediate piperitol in the leaves of two independent transgenic lines of F. intermedia and F. koreana. We also detected the accumulation of sesamin and piperitol in their vegetatively propagated descendants, demonstrating the stable and efficient production of these lignans. These results indicate that CYP81Q1-transgenic Forsythia plants are promising prototypes to produce diverse lignans and provide an important strategy for the cost-effective and scalable production of lignans.

The aging of world populations highlights the importance of plant secondary metabolites such as alkaloids, flavonoids, terpenoids, and lignans with benefits for human health 1,2 . Lignans are phenylpropanoid dimers with diverse functions, and dietary lignans have attracted attention as food nutrients 3,4 . (+)-Sesamin is a furofuran lignan that is commercially available as a health-promoting supplement 5 . In mammals, (+)-Sesamin metabolites attenuate oxidation and inflammation for the protection of the liver 6,7 . (+)-Sesamin also shows anti-cancer properties 8 . (+)-Sesamin is commercially available via extraction at concentrations (4-6 mg/g) from Sesamum indicum (sesame) seed oil 5,9,10 . Sesame plants, the strongest known synthesizers of (+)-sesamin, are annuals that are threatened by climate change 11,12 . Thus, new plant sources are required for the efficient and stable production of (+)-sesamin.
Previously, we demonstrated the ectopic accumulation of (+)-sesamin in cultured CYP81Q1-transgenic Fk cells 26,27 and showed their ability to produce (+)-sesamin. However, the mass production of (+)-sesamin using transgenic cells is not practical in light of the cost of large-scale cell culture. In contrast, given the large biomass generated by Forsythia leaves, CYP81Q-transgenic Forsythia plants could efficiently and stably produce (+)-sesamin. In this study, we generated CYP81Q1-transgenic Forsythia plants that stably produce the intermediate (+)-piperitol and the product (+)-sesamin.

Results
Initially, we established a practical method for the transformation of Forsythia plants ( Fig. 2A). Fi leaf explants (n = 451) were soaked in a suspension of Agrobacterium tumefaciens cells harboring the Pro35S:nGFP 28 plasmid and regenerated shoots and roots during culture for two years (see "Materials and methods", Fig. 2B-G, Table 1). Eight of ten independent kanamycin-resistant lines exhibited signals for nGFP presence and expression when examined by fluorescence microscopy (Fig. 2H) and genomic (Fig. 2I) and RT-PCR analyses (Fig. 2J); no nGFP presence or expression was seen in wild-type (WT) plants. Two kanamycin-resistant plants did not show GFP presence or expression, probably due to somaclonal variation that eliminated the transgene (Fig. 2I,J). Even after vegetative propagation for four years, newly generated FiPro35S:nGFP leaves maintained GFP fluorescence, demonstrating stable Pro35S:nGFP transformation of Forsythia plants.
We also introduced the 35S promoter-regulated sesame CYP81Q1 gene (Pro35S:CYP81Q1) 29 into Fi and Fk. After co-culture of 956 Fi and 273 Fk leaf explants with Agrobacterium cells harboring the Pro35S:CYP81Q1 plasmid, the resulting transgenic Forsythia plants were propagated vegetatively in soil through repeated rounds of cutting and growth in our plant culture room-conditions under which Forsythia plants continuously developed their leaves without flowering (Table 1). Eventually, two independent transgenic lines developed normally on soil (Fig. 3A) and showed CYP81Q1 gene presence (Fig. 3B) and expression (Fig. 3C).

Discussion
In this study, we provide evidence that transgenic Forsythia plants produce ectopic lignans, (+)-piperitol and (+)-sesamin. These results suggest that sesamolin and sesaminol, which are antioxidant sesame lignans metabolized from (+)-sesamin by CYP92B14 30 , is expected to be produced via additional introduction of the CYP92B14 gene into Pro35S:CYP81Q1 plants. Another lignan, podophyllotoxin, may also be produced in transgenic Forsythia plants. Podophyllotoxin is at present extracted from the rhizomes of Podophyllum species and clinically  www.nature.com/scientificreports/ utilized in cancer therapy 31 . In Podophyllum podophyllotoxin biosynthesis, matairesinol, which Forsythia species also accumulate in the pathway downstream of pinoresinol, is metabolized to pluviatolide by CYP719A23 32 . Additional enzymes (CYP71CU1, 2-oxoglutarate/Fe(II)-dependent dioxygenase, and O-methyltransferases) convert pluviatolide to the proposed precursor deoxypodophyllotoxin 33 . Thus, methods for introducing multiple genes into Forsythia plants will pave the way for the generation of podophyllotoxin and its related compounds by transgenic plants. In a previous study, we generated triple-transgenic Forsythia cultured cells 26 , suggesting the possibility of multigene transformation of Forsythia plants.
The production of various specialized plant metabolites has been attempted in transgenic and synthetic biology-based microorganisms 1,34-40 . However, most microorganisms appear to lack (+)-sesamin and its precursor (+)-pinoresinol 9,41 . Moreover, tremendous bioinformatic and screening processes are often required for the generation of genetically-engineered microorganisms that produce plant lignans. Additionally, mass production using such engineered microorganisms could be limited by genetic instability, infectious contamination, unexpected product toxicity, and low fermentation performance 38,40 . In contrast, Forsythia species are perennial shrubs, and thus Pro35S:CYP81Q1 plants are easily cultivated via explant, a marked advantage for stable growth and mass propagation in a plant factory. Moreover, a plant factory provides plasticity in place and time of the production of sesamin in plants 42 , unlike agricultural production that is limited by climates, seasons, and farmlands. Thus, we will be able to produce sesamin using our transgenic Forsythia plants in a plant factory.
The transgenic Forsythia plants have limitation in the content of sesamin as compared with sesame seeds. To increase the content of sesamin in the transgenic Forsythia plants, we will be able to apply multigene transformation strategy. Previously, triple-transgenic Forsythia cells with the RNAi construct for endogenous pinoresinollariciresinol reductase, and the overexpression-construct of pinoresinol glucosylating enzyme, which increase the level of the precursor pinoresinol, as well as CYP81Q1 gene, produced higher level of sesamin than the single-transgenic CYP81Q1 cells 26 . The same strategy of multigene transformation of Forsythia plants is expected to increase the content of sesamin. Moreover, overexpression of enzymes upstream of pinoresinol stimulated accumulation of podophyllotoxin-related lignans 60 . Thus, overexpression of the upstream enzymes may increase the content of sesamin in transgenic Forsythia plants. Because the content of pinoresinol was not changed in the control and the Pro35S:CYP81Q1 plants (Supplemental Fig. S1), the moderate activity of CYP81Q1 seems www.nature.com/scientificreports/ to limit the rate of production of sesamin from pinoresinol. As many cytochrome P450 enzymes function in complex with their native oxidoreductases 30 , co-expression of CYP81Q1 and S. indicum oxidoreductase genes in transgenic Forsythia plants is a future option to strengthen the activity of CYP81Q1 for higher production of sesamin. In addition, the red light condition increased the content of sesamin in the transgenic Forsythia cells 26 , suggesting that irradiation of red light to transgenic Forsythia plant may increase the content of sesamin.
In conclusion, we have generated Forsythia plants as promising prototypes for the efficient and sustainable heterologous production of beneficial lignans.   Microscopy. GFP fluorescence was observed using a M205 fluorescence stereomicroscope (Leica Microsystems, Germany) using the GFP3 filter and recorded by LAS AF software (Leica Microsystems, Germany).  Fig. S2).

Measurement of lignans. Cultured transgenic and control
Forsythia plants were transferred into soil in pots, acclimatized for three weeks until rooting, and grown for two months until the plants reached 15 to 20 cm height. The third to fifth leaves from the top of each plant were pooled, frozen in liquid nitrogen, lyophilized to permit measurement of dry weight (DW) using an FDU-2110 device (EYELA, Japan), extracted with 50% methanol (v/v) containing 2.25 µM (final concentration) 2′-ethoxysesamin (Supplementary Fig. S3 and Table S2) as the internal standard, and processed as described previously 29 . The leaf extracts were subjected into reversephase HPLC (Alliance 2960, Waters Corporation, MA) using a Develosil C30-UG-5 column (4.6 × 150 mm; Nomura Chemical, Japan) under conditions described previously 26  www.nature.com/scientificreports/ chromatograms while referencing standard curves of authentic piperitol and sesamin with technical duplicates or triplicates. For LC-MS analysis, the leaf extracts were subjected to LC-MS-IT-TOF (Shimazu, Japan) and analyzed as described previously 30 . Lignans were detected using a photodiode array detector and and analyzed by LabSolutions LCMS version 3.8.1 (https:// www. an. shima dzu. co. jp/ lcms/ suppo rt/ downl oad/ index. htm, Shimazu, Japan).

Statistical analysis.
The lignan contents were measured using five or six biological replicates and statistically analyzed by two-tailed Student's t tests (Table 2 and Supplemental Fig. S1).