Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans.
Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans.
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DOI：
10.1371/journal.pone.0144519
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Satake H
中科院分区：
文献类型：
--
作者：
Murata J;Matsumoto E;Morimoto K;Koyama T;Satake H
芝麻素是一种呋喃类木脂素，由芝麻中的前体木脂素松脂醇生物合成。这种木脂素被证明具有抗高血压活性，保护肝脏免受乙醇和脂质氧化的损害，并减少肺癌的生长。尽管需求迅速上升，但由于定位和收集植物的成本很高，植物木脂素的来源往往有限。事实上，芝麻素的获取完全依赖于对特定芝麻种子的传统提取。本研究利用三转基因朝鲜连翘悬浮细胞U18I-CPI-FK建立了高效、稳定、可持续的芝麻素生产体系。这些转基因细胞培养物是通过将针对松脂醇-葡萄糖化酶UGT71A18的RNAi序列稳定地导入现有的CPI-FK细胞中而产生的，而CPI-FK细胞是通过将芝麻芝麻素合成酶(CYP81Q1)和针对松脂醇/落叶松脂醇还原酶(PLR)的RNA干扰序列(RNAi)导入朝鲜落叶松脂醇还原酶(PLR)而产生的。与其转基因原型相比，U18I-CPI-FK的松脂醇苷元产量和芝麻素产量分别增加了5倍和1.4倍，而野生型由于缺乏任何内在的芝麻素合成酶而不能产生芝麻素。此外，红色LED照射U18I-CPI-FK后，在黑暗条件下，松脂醇苷元和芝麻素的产量比这些木脂素的产量高出3.0倍，而在红色LED照射下，野生型松脂醇的产量下降。此外，我们还开发了一种基于海藻酸钠的U18I-CPI-FK在液氮中长期保存的方法。冻存6个月后复温的U18I-CPI-FK的芝麻素产量与未冻存的U18I-CPI-FK相当。这些数据保证了随时随地按需生产芝麻素。综上所述，本研究提供了证据表明U18i-CP-FK是一个前所未有的高效、稳定和可持续生产芝麻素的平台，并表明基于转基因和特定光调控的连翘细胞代谢工程是获得稀有和有益木脂素的一种有前途的策略。
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.