Tailoring tobacco hairy root metabolism for the production of stilbenes

Abstract

Tobacco hairy root (HR) cultures, which have been widely used for the heterologous production of target compounds, have an innate capacity to bioconvert exogenous t-resveratrol (t-R) into t-piceatannol (t-Pn) and t-pterostilbene (t-Pt). We established genetically engineered HR carrying the gene encoding stilbene synthase (STS) from Vitis vinifera and/or the transcription factor (TF) AtMYB12 from Arabidopsis thaliana, in order to generate a holistic response in the phenylpropanoid pathway and coordinate the up-regulation of multiple metabolic steps. Additionally, an artificial microRNA for chalcone synthase (amiRNA CHS) was utilized to arrest the normal flux through the endogenous chalcone synthase (CHS) enzyme, which would otherwise compete for precursors with the STS enzyme imported for the flux deviation. The transgenic HR were able to biosynthesize the target stilbenes, achieving a production of 40 μg L−1 of t-R, which was partially metabolized into t-Pn and t-Pt (up to 2.2 μg L−1 and 86.4 μg L−1, respectively), as well as its glucoside piceid (up to 339.7 μg L−1). Major metabolic perturbations were caused by the TF AtMYB12, affecting both primary and secondary metabolism, which confirms the complexity of biotechnological systems based on seed plant in vitro cultures for the heterologous production of high-value molecules.

Engineering Metabolism in Nicotiana Species: A Promising Future

“…BY-2 cells are characterized by a fast growth rate relative to other plant cell suspension lines, and the ease of cell cycle synchronization and transformation with Agrobacterium. Several compounds, such as the betalain amaranthin [80], stilbenes [81], and more than 20 pharmaceutical proteins and peptides have already been successfully produced in BY-2 cell suspension cultures, including interleukins and antibodies [82].…”

“…HRCs are appropriated for the production of specialized metabolites due to characteristics such as genetic stability, high biomass production and efficient biosynthetic capacity, and are able to produce specialized metabolites for a long period of time [84]. Some examples of specialized metabolites produced using Nicotiana transgenic HRCs are tropane alkaloids such as scopolamine and hyoscyamine [85], stilbenes [81] and geraniol [86].…”

Green (cell) factories for advanced production of plant secondary metabolites

“…The possibility of exploiting plant in vitro systems as a "green cell factory" has significantly increased over the last decade. Metabolic engineering through transformation with Agrobacterium species followed by manipulation of plant metabolic signaling pathway has been intensively used to enhance the production of pharmaceutically valuable SMs in plants and plant in vitro systems [16]. The biosynthesis of the SMs is regulated by complex networks.…”

“…Investigating the differences in MYB14 and MYB15 expression and/or transcriptional activity in different grapevine cultivars might lead to the development of molecular markers for the breeding of grapevines with optimized stilbene content and composition [26]. To increase the amount of trans-resveratrol (up-to 40 lg/L), Hidalgo et al [16] established a transgenic HRs from tobacco harboring the gene encoding STS from V. vinifera and/or the TF AtMYB12 from Arabidopsis thaliana aiming to activate and coordinate the up-regulation of resveratrol through the phenylpropanoid pathway. Additionally, an artificial microRNA for chalcone synthase (amiRNA CHS) was used to suppress the metabolite flux through CHS inhibition, which might be a competitive pathway that utilize the precursors of the STS enzyme.…”

“…On the other hand in the TF/S/Inh (contain T-DNA, VvSTS, AtMB12 TF, amiRNA-CHS) lines the interference between amiRNA-CHS and the natural CHS gene expression resulted in decreased levels of flavonoids and the lowest levels of stilbene production. This might be related with the simultaneously low expression of VvSTS and AtMYB12 TF genes [16]. Other TFs, such as VvWRKY8 can be used to suppress the resveratrol biosynthesis, e.g.…”