Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t‐resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β‐cyclodextrins

Abstract

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t-resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β-cyclodextrinsThe growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.

Tailoring tobacco hairy root metabolism for the production of stilbenes

“…Elicitation of the engineered HR with 50 mM MBCD and 100 µM of MeJA significantly increased the release of t -R and its derivatives t -Pn and t -Pt to the liquid medium, whereas piceid remained mainly inside the roots. In contrast, the positive effects of the combined elicitor treatment (MBCD + MeJA) on the t -R production has been previously reported in non-transgenic and transgenic grapevine cell cultures 14 , 60 , and also in transgenic S. marianum cell cultures carrying the VvSTS gene and supplemented with MBCD 17 . The ineffectiveness of elicitation in the HR culture system to improve the total stilbenoid contents was perhaps due to the fast metabolization of stilbenoids in the culture medium, as reported previously 15 , and an increased bioconversion of t -R into the more bioactive derivatives, t -Pn and t -Pt.…”

“…In addition, biotechnological studies have shown that Vitis vinifera cell cultures need elicitation treatments for an efficient t -R production, the best results being achieved by combining methyl jasmonate with the expensive cyclodextrins 16 . Silybum marianum transgenic cell suspensions carrying the V.vinifera stilbene synthase3 ( Vv STS) gene treated only with cyclodextrins have also yielded t -R 17 , as have HR of peanut 18 , 19 and V. rotundifolia 20 .…”

Biotechnological Production of Flavonoids: An Update on Plant Metabolic Engineering, Microbial Host Selection, and Genetically Encoded Biosensors

“…In this report, production of silymarin was less affected in the transgenic cultures, possibly due to an intrinsic precursor bottleneck from the monolignol branch limits silymarin synthesis. The fact is that overexpressed STS gene will react with the excessively produced precursors of non-bioactive compounds such as coniferyl alcohol, while the metabolic flux toward the silymarin production was kept unchanged, this may provide a route to extend the applications of plant cell cultures for both constitutive and foreign valuable metabolites production (Hidalgo, Martínez-Márquez et al 2017).…”

Transgenesis as a Tool for the Efficient Production of Selected Secondary Metabolites from Plant in Vitro Cultures

“…One example is the stable transformation of Silybum marianum (L.) Gaertn. cell suspension cultures with the Vitis vinifera L. stilbene synthase gene, allowing increased accumulation of t-resveratrol [56]. In addition, overexpression of the neutral / alkaline invertase (NINV) gene in Taxus chinensis (Rehder & E.H.Wilson)…”