Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a ...single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism-an alcohol dehydrogenase and a cytochrome P450-produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-β-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products.The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine.
The composition of bioactive polyphenols from grape canes, an important viticultural byproduct, was shown to be varietal-dependent; however, the influence of soil-related terroir factors remains ...unexplored. Using spatial metabolomics and correlation-based networks, we investigated how continuous changes in soil features and topography may impact the polyphenol composition in grape canes. Soil properties, topography, and grape cane extracts were analyzed at georeferenced points over 3 consecutive years, followed by UPLC-DAD-MS-based metabolomic analysis targeting 42 metabolites. Principal component analyses on intra-vintage metabolomic data presented a good reproducibility in relation to geographic coordinates. A correlation-driven approach was used to explore the combined influence of soil and topographic variables on metabolomic responses. As a result, a metabolic cluster including flavonoids was correlated with elevation and curvature. Spatial metabolomics driven by correlation-based networks represents a powerful approach to spatialize field-omics data and may serve as new field-phenotyping tool in precision agriculture.
Many of the plant-derived compounds used in chemotherapies are currently produced by semisynthesis, which results in limited supplies at exorbitant market prices. However, the synthetic biology era, ...which began ca 15 years ago, has progressively yielded encouraging advances by using engineered microbes for the practical production of cheaper plant anticancer drugs.
Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. ...Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.
Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that
act as key components of the plant defense system against diseases. Considerable advances have ...been made in the chemical characterization of wine metabolites particularly volatile and polyphenolic compounds. However, the metabotyping (metabolite-phenotype characterization) of grape varieties, from polyphenolic-rich vineyard by-product is unprecedented. As this composition might result from the complex interaction between genotype, environment and viticultural practices, a field experiment was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. As a result, UPLC-MS-based targeted metabolomic analyses of grape stems from 8
L. cultivars allowed the determination of 42 polyphenols related to phenolic acids, flavonoids, procyanidins, and stilbenoids as resveratrol oligomers (degree of oligomerization 1-4). Using a partial least-square discriminant analysis approach, grape stem chemical profiles were discriminated according to their genotypic origin showing that polyphenol profile express a varietal signature. Furthermore, hierarchical clustering highlights various degree of polyphenol similarity between grape varieties that were in agreement with the genetic distance using clustering analyses of 22 microsatellite DNA markers. Metabolite correlation network suggested that several polyphenol subclasses were differently controlled. The present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with both health-benefit potential and plant defense traits.
Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of ...tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.
Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic ...intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.
Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole ...alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.
The monoterpene indole alkaloids (MIA) synthesized in
Catharanthus roseus
are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a ...complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within
C. roseus
cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of
C. roseus
cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring.
Summary
In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary ...metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1‐deoxy‐d‐xylulose 5‐phosphate synthase (DXS), 1‐deoxy‐d‐xylulose 5‐phosphate reductoisomerase (DXR) and 2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10‐hydroxylase (G10H), a cytochrome P450 monooxygenase involved in the first committed step in the formation of iridoid monoterpenoids display identical cell‐specific expression patterns. The co‐localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.
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