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

PLoS One. 2015 Dec 7;10(12):e0144519. doi: 10.1371/journal.pone.0144519. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Cells, Cultured
  • Forsythia / genetics
  • Forsythia / growth & development
  • Forsythia / metabolism*
  • Lignans / biosynthesis*
  • Molecular Structure
  • Plant Proteins / antagonists & inhibitors
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / growth & development
  • Plants, Genetically Modified / metabolism*
  • RNA, Small Interfering / genetics

Substances

  • Lignans
  • Plant Proteins
  • RNA, Small Interfering

Grants and funding

This work is supported by the Plant Factory project of the Ministry of Economy, Trade and Industry, Japan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.