Tailoring tobacco hairy root metabolism for the production of stilbenes

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.