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.
Highlights
The consistent and appropriate intake of low-cost healthy diets and clinical drugs are the most promising and effective ways to improve the quality of life including a healthy life expectancy and to prevent lifestyle-related diseases
We present the development of a triple-transgenic F. koreana cell line, U18i-CPi-Fk, which exhibits a marked increase in production of pinoresinol aglycone and sesamin over that of CPi-Fk and the establishment of an optimized procedure for the long-term cryopreservation and recovery of U18i-CPi-Fk
We attempted to simultaneously introduce CYP81Q1, pinoresinol/lariciresinol reductase (PLR)-RNA interference (RNAi), and UGT71A18-RNAi cDNA sequences into F. koreana wildtype cells using an expression vector bearing these three sequences. This approach failed to obtain triple-transgenic cell cultures, because the expression of either PLR or UGT71A18 was not suppressed by RNAi in the resultant transgenic cells
Summary
The consistent and appropriate intake of low-cost healthy diets and clinical drugs are the most promising and effective ways to improve the quality of life including a healthy life expectancy and to prevent lifestyle-related diseases. Sesamin was shown to exert diverse beneficial effects on mammals including human [1,2,3,4,5,6,7], including an anti-hypertensive effect [9], the reduction of breast tumor growth [10], and recovery of liver damage caused by ethanol and lipid oxidation [11, 12]. These findings indicate that the demand for sesamin will rapidly increase in the near future. These shortcomings, combined with elevating demands on sesamin, indicate that a novel strategy for systematic sesamin production is clearly required
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