Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants

The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.1) Fundo Europeu de Desenvolvimento Regional funds through the Operational Competitiveness Programme COMPETE and by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the projects FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013) and FCOMP-01-0124-FEDER-019664 (PEst-C/SAU/LA0002/2010); (2) by the FCT scholarships co-supported by FCT and POPH-QREN (European Social Fund), SFRH/BD/41907/2007 (IC) and SFRH/BD/74257/2010 (HN); (3) by a Postdoctoral fellowship financed by national funds through FCT under the project Incentivo/SAU/LA0002/2014 (VM); (4) by a Scientific Mecenate Grant from Grupo Jerónimo Martins.info:eu-repo/semantics/publishedVersio

A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate

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Exploiting Spermidine N -Hydroxycinnamoyltransferase Diversity and Substrate Promiscuity to Produce Various Trihydroxycinnamoyl Spermidines and Analogues in Engineered Yeast

International audienceTrihydroxycinnamoyl spermidines (THCSpd) are plant specialized metabolites with promising pharmacological activities as antifungals, antibacterial, antiviral, and antidepressant drugs. However, their characterization and potential pharmaceutical exploitation are greatly impaired by the sourcing of these compounds, restricted to the pollen of core Eudicot plant species. In this work, we developed a precursor-directed biosynthesis of THCSpd in yeast using a dual enzymatic system based on 4-coumarate-CoA ligases (4CL) and spermidine N-hydroxycinnamoyltransferases (SHT). The system relies on the yeast endogenous spermidine pool and only requires hydroxycinnamic acids as exogenous precursors. By exploring 4CL isoforms and SHT diversity among plants, we have driven the production of 8 natural THCSpd, using single or mixed hydroxycinnamic acid precursors. Substrate promiscuities of 4CL and SHT were genuinely exploited to produce 8 new-to-nature THCSpd from exotic hydroxycinnamic and dihydrohydroxycinnamic acids, together with 3 new-to-nature THCSpd containing halogenated hydroxycinnamoyl moieties. In this work, we established a versatile and modular biotechnological production platform allowing the tailor-made THCSpd synthesis, constituting pioneer metabolic engineering for access to these valuable natural products

Identifying Genes Involved in Alkaloid Biosynthesis in Vinca minor through Transcriptomics and Gene Co-Expression Analysis

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Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle

International audienceVinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds

A chromosome-scale genome assembly of Rauvolfia tetraphylla facilitates identification of the complete ajmaline biosynthetic pathway

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Alternative splicing creates a pseudo-strictosidine β- d -glucosidase modulating alkaloid synthesis in Catharanthus roseus

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