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.

De Novo Transcriptomes of Forsythia koreana Using a Novel Assembly Method: Insight into Tissue- and Species-Specific Expression of Lignan Biosynthesis-Related Gene

“…Subsequently, cell suspension culture lines of F . koreana were established from the calli as described previously [ 9 ] and were maintained at 22°C in Gamborg’s B-5 liquid medium supplemented with 6% sucrose and 0.05 mg l-1 2,4-D (Cell Culture Medium; CCM). All suspensions were cultured on a rotary shaker at 110 rpm in the dark and were subcultured every 2 weeks with an inoculum of 5 ml of saturated suspension cells.…”

“…There is a growing body of evidence for the diverse biological effects of lignans on mammals, including antioxidative, antiobesity, antitumor, antiviral activities, and the reduction of the risk of various cancers and cardiovascular diseases [ 6 – 8 ]. Quite recently, we also demonstrated the marked usefulness of triple-transgenic Forsythia callus suspensions as efficient, stable, and sustainable platforms for the production of a beneficial non- Forsythia lignan, sesamin [ 9 ].…”

Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists

“…In particular, these effects are due to the presence of a specific class of secondary metabolites, the lignans (phenylpropane dimers originating from the phenylpropanoid pathway [ 100 ]), which bind to the human sex hormone globulin and act as hormone balancer [ 101 ]. The production of lignans is usually achieved via plant cell suspension and hairy root cultures [ 102 , 103 , 104 , 105 , 106 ]. Their production in heterologous systems is not yet a viable option, since not all the players partaking in the pathway leading to specific lignin formation are known [ 107 ].…”

Bioproduction of Anticancer Podophyllotoxin and Related Aryltretralin-Lignans in Hairy Root Cultures of Linum Flavum L.

“…In HR-type systems, several studies have demonstrated the feasibility of engineering lignans biosynthesis in various flax species such as L. album, L. perenne, or L. corymbulosum [57,58,67]. In cell culture systems of F. koreana, it was possible to redirect the lignan biosynthetic pathway to the accumulation of new lignan compounds not naturally produced by this plant by a metabolic engineering approach [158,159]. Although yields were very low, the transient expression of several biosynthetic genes in tobacco leaves resulted in the production of an Etoposide precursor [83].…”