RESUMEN
The monoterpene indole alkaloids vindoline and catharanthine, which are exclusively synthesized in the medicinal plant Catharanthus roseus, are the two important precursors for the production of pharmaceutically important anti-cancer medicines vinblastine and vincristine. Hairy root culture is an ideal platform for alkaloids production due to its industrial scalability, genetic and chemical stability, and availability of genetic engineering tools. However, C. roseus hairy roots do not produce vindoline due to the lack of expression of the seven-step pathway from tabersonine to vindoline [Murata & De Luca (2015) Plant Journal, 44, 581-594]. The present study describes the genetic engineering of the first two genes tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT) in the missing vindoline pathway under the control of a glucocorticoid-inducible promoter to direct tabersonine toward vindoline biosynthesis in C. roseus hairy roots. In two transgenic hairy roots, the induced overexpression of T16H and 16OMT resulted in the accumulation of vindoline pathway metabolites 16-hydroxytabersonine and 16-methoxytabersonine. The levels of root-specific alkaloids, including lochnericine, 19-hydroxytabersonine and hörhammericine, significantly decreased in the induced hairy roots in comparison to the uninduced control lines. This suggests tabersonine was successfully channeled to the vindoline pathway away from the roots competing pathway based on the overexpression. Interestingly, another two new metabolites were detected in the induced hairy roots and proposed to be the epoxidized-16-hydroxytabersonine and lochnerinine. Thus, the introduction of vindoline pathway genes in hairy roots can cause unexpected terpenoid indole alkaloids (TIA) profile alterations. Furthermore, we observed complex transcriptional changes in TIA genes and regulators detected by RT-qPCR which highlight the tight regulation of the TIA pathway in response to T16H and 16OMT engineering in C. roseus hairy roots.
Asunto(s)
Catharanthus/enzimología , Sistema Enzimático del Citocromo P-450/biosíntesis , Expresión Génica , Alcaloides Indólicos/metabolismo , Proteínas de Plantas/biosíntesis , Raíces de Plantas/enzimología , Plantas Modificadas Genéticamente/enzimología , Quinolinas/metabolismo , Catharanthus/genética , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Plantas/genética , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/genéticaRESUMEN
The aromatic amino acid biosynthesis pathway is a source to a plethora of commercially relevant chemicals with very diverse industrial applications. Tremendous efforts in microbial engineering have led to the production of compounds ranging from small aromatic molecular building blocks all the way to intricate plant secondary metabolites. Particularly, the yeast Saccharomyces cerevisiae has been a great model organism given its superior capability to heterologously express long metabolic pathways, especially the ones containing cytochrome P450 enzymes. This review contains a collection of state-of-the-art metabolic engineering work devoted towards unraveling the mechanisms for enhancing the flux of carbon into the aromatic pathway. Some of the molecules discussed include the polymer precursor muconic acid, as well as important nutraceuticals (flavonoids and stilbenoids), and opium-derived drugs (benzylisoquinoline alkaloids).