Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans

Abstract

Sesamin is a furofuran lignan biosynthesized from the precursor lignan pinoresinol specifically in sesame seeds. This lignan is shown to exhibit anti-hypertensive activity, protect the liver from damages by ethanol and lipid oxidation, and reduce lung tumor growth. Despite rapidly elevating demand, plant sources of lignans are frequently limited because of the high cost of locating and collecting plants. Indeed, the acquisition of sesamin exclusively depends on the conventional extraction of particular Sesamum seeds. In this study, we have created the efficient, stable and sustainable sesamin production system using triple-transgenic Forsythia koreana cell suspension cultures, U18i-CPi-Fk. These transgenic cell cultures were generated by stably introducing an RNAi sequence against the pinoresinol-glucosylating enzyme, UGT71A18, into existing CPi-Fk cells, which had been created by introducing Sesamum indicum sesamin synthase (CYP81Q1) and an RNA interference (RNAi) sequence against pinoresinol/lariciresinol reductase (PLR) into F. koreanna cells. Compared to its transgenic prototype, U18i-CPi-Fk displayed 5-fold higher production of pinoresinol aglycone and 1.4-fold higher production of sesamin, respectively, while the wildtype cannot produce sesamin due to a lack of any intrinsic sesamin synthase. Moreover, red LED irradiation of U18i-CPi-Fk specifically resulted in 3.0-fold greater production in both pinoresinol aglycone and sesamin than production of these lignans under the dark condition, whereas pinoresinol production was decreased in the wildtype under red LED. Moreover, we developed a procedure for sodium alginate-based long-term storage of U18i-CPi-Fk in liquid nitrogen. Production of sesamin in U18i-CPi-Fk re-thawed after six-month cryopreservation was equivalent to that of non-cryopreserved U18i-CPi-Fk. These data warrant on-demand production of sesamin anytime and anywhere. Collectively, the present study provides evidence that U18i-CP-Fk is an unprecedented platform for efficient, stable, and sustainable production of sesamin, and shows that a transgenic and specific light-regulated Forsythia cell-based metabolic engineering is a promising strategy for the acquisition of rare and beneficial lignans.

Fig 1. Biosynthesis pathways of major lignans in Forsythia and Sesamum.

Chemical conversions at each step are indicated in red. Solid and broken lines represent identified and unidentified enzyme-catalyzed reactions, respectively.

Fig 2. Generation of U18i-CPi-Fk.

Expression of CYP81Q, PLR, and UGT71A18 in F. koreana wildtype, CPi-Fk, and U8i-CPi-Fk.

Fig 3. Amounts of pinoresinol aglycone, total pinoresinol (aglycone and glucosides), and sesamin.

Comparison of the three lignans between CPi-Fk and U18-CPi-Fk (A). The ratio of pinoresinol aglycone to total aglycone was also calculated (B). Pinoresinol aglycone was quantified separately after a culture period of 15 d under the same conditions. Values (%) in (B) are presented as the ratio of pinoresinol aglycone (2.21 ± 0.72 μg/ g DW and 0.34 ± 0.09 μg/ g DW) to total pinoresinol (3.71 ± 0.40 μg/ g DW and 2.88 ± 0.47 μg/ g DW) in CPi-Fk and U18i-CPi-Fk, respectively. Data are obtained from three independent experiments as the mean ± SEM (P < 0.05).

Fig 4. Comparison of the production of (A) pinoresinol and (B) sesamin in U18i-CPi-Fk cells under the dark, white, blue, and red continuous light conditions.

Lignans were quantified separately for each sample after a culture period of two weeks in the same medium. Data are presented as the average of three independent experiments (mean ± S.E.M., P < 0.05).

Fig 5. Differential effects of red LED irradiation on the increase of lignan production in Forsythia wildtype and U18i-CP-Fk.

Each indicated lignan in wildtype (A) and U18i-CP-Fk (B) in the dark (black) and red LED (gray) was assessed. Each point represents the mean ± S.E.M (P < 0.05) of three preparations.

Fig 6. Evaluation of lignan production after cryopreservation of U18i-CPi-Fk.

Pinoresinol aglycone and sesamin were extracted from U18-CPi-Fk before cryopreservation (white) and after re-thawing following 6-month cryopreservation (gray). Each lignan was assessed separately after 55-day culture since the recovery under the same condition and expressed as the mean ± SEM of three independent experiments (P < 0.05).

Fig 7. Strategy for efficient, stable, and sustainable lignan production via metabolic engineering of Forsythia cells.