A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories.

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.

GATA and Phytochrome Interacting Factor Transcription Factors Regulate Light-Induced Vindoline Biosynthesis in Catharanthus roseus.

Catharanthus roseus is the exclusive source of an array of terpenoid indole alkaloids including the anticancer drugs vincristine and vinblastine, derived from the coupling of catharanthine and vindoline. Leaf-synthesized vindoline is regulated by light. A seven-step enzymatic process is involved in the sequential conversion of tabersonine to vindoline; however, the regulatory mechanism controlling the expression of genes encoding these enzymes has not been elucidated. Here, we identified CrGATA1, an Leu-Leu-Met domain GATA transcription factor that regulates light-induced vindoline biosynthesis in C. roseus seedlings. Expression of CrGATA1 and the vindoline pathway genes T16H2, T3O, T3R, D4H, and DAT was significantly induced by light. In addition, CrGATA1 activated the promoters of five light-responsive vindoline pathway genes in plant cells. Two GATC motifs in the D4H promoter were critical for CrGATA1-mediated transactivation. Transient overexpression of CrGATA1 in C. roseus seedlings resulted in up-regulation of vindoline pathway genes and increased vindoline accumulation. Conversely, virus-induced gene silencing of CrGATA1 in young C. roseus leaves significantly repressed key vindoline pathway genes and reduced vindoline accumulation. Furthermore, we showed that a C. roseus Phytochrome Interacting Factor, CrPIF1, is a repressor of CrGATA1 and vindoline biosynthesis. Transient overexpression or virus-induced gene silencing of CrPIF1 in C. roseus seedlings altered CrGATA1 and vindoline pathway gene expression in the dark. CrPIF1 repressed CrGATA1 and DAT promoter activity by binding to G/E-box/PBE elements. Our findings reveal a regulatory module involving Phytochrome Interacting Factor -GATA that governs light-mediated biosynthesis of specialized metabolites.

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

Vinblastine, 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.

Structural and functional characterization of the Vindoline biosynthesis pathway enzymes of Catharanthus roseus.

Vinblastine and its related compound vincristine are important mono terpenoid indole alkaloids accumulated in the leaves of Catharanthus roseus (Madagascar periwinkle). They serve as major anticancer drugs. Vinblastine is formed by the condensation of vindoline and catharanthine. The vindoline moiety is derived from tabersonine via vindoline biosynthesis pathway. The reaction sequence from tabersonine to vindoline is now well established and the enzymes involved in this pathway are identified. However, to date, the structures of the enzymes involved in the vindoline biosynthesis pathway are not known, leading to limited mechanistic understanding of the substrate binding and catalysis. The purpose of this work is to provide structural insight regarding all the steps of the vindoline pathway via rigorous homology modeling, molecular docking, and molecular dynamics analyses. Substrate and cofactors required for each step were docked onto the computationally built and validated three-dimensional (3D) model of the corresponding enzyme, and the catalytic reaction was analyzed from the structural point of view. Possible binding modes of the substrates and cofactors were generated and corresponding binding residues were identified. Enzyme-substrate models were verified based on structure evaluation methods and molecular dynamics based approaches. Findings of our analysis would be useful in rational designing of these important enzymes aimed toward bio-production of vindoline.

Discovery of a P450-catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids.

Here we report the discovery of a cytochrome P450 that is required for the biosynthesis of vindoline, a plant-derived natural product used for semi-synthesis of several anti-cancer drugs. This enzyme catalyzes the formation of an epoxide that can undergo rearrangement to yield the vincamine-eburnamine backbone, thereby providing evidence for the long-standing hypothesis that the aspidosperma- and eburnamine-type alkaloids are biosynthetically related.

Effects of Adding Vindoline and MeJA on Production of Vincristine and Vinblastine, and Transcription of their Biosynthetic Genes in the Cultured CMCs of Catharanthus roseus.

Vincristine and vinblastine were found by Liquid Chromatography-Mass Spectrometry (LC-MS) in Catharanthus roseuscambial meristem cells (CMCs) jointly treated with 0.25 mM vindoline and methyl jasmonate (MeJA), suggesting that C. roseus CMCs contain a complete set of the enzymes which are in response to convert vindoline into vincristine and vinblastine. Based on the facts that the transcript levels of vindoline-biosynthetic genes (STR, SGD and D4H) were up-regulated instead of being down-regulated by adding itself to the culture, and that the transcriptional factor ORCA3 was up-regulated simultaneously, we further confirmed that the transcription of STR, SGD, D4H was manipulated by ORCA3.

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus.

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003-0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.

The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots.

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

Progress in the development of early diagnosis and a drug with unique pharmacology to improve cancer therapy.

Cancer continues to be one of the major health and socio-economic problems worldwide, despite considerable efforts to improve its early diagnosis and treatment. The identification of new constituents as biomarkers for early diagnosis of neoplastic cells and the discovery of new type of drugs with their mechanistic actions are crucial to improve cancer therapy. New drugs have entered the market, thanks to industrial and legislative efforts ensuring continuity of pharmaceutical development. New targets have been identified, but cancer therapy and the anti-cancer drug market still partly depend on anti-mitotic agents. The objective of this paper is to show the effects of KAR-2, a potent anti-mitotic compound, and TPPP/p25, a new unstructured protein, on the structural and functional characteristics of the microtubule system. Understanding the actions of these two potential effectors on the microtubule system could be the clue for early diagnosis and improvement of cancer therapy.

The leaf epidermome of Catharanthus roseus reveals its biochemical specialization.

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis-enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus.

The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ss-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.

Monitoring the production yields of vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis. Semiquantitative determination by flow-injection electrospray ionization mass spectrometry.

A semiquantitative determination of two bis-indole antitumor alkaloids, vincristine and vinblastine, has been performed by flow-injection electrospray ionization mass spectrometric analysis of the extracts of Catharanthus roseus. Leaves and flowers of two different phenotypes (pink flower and white flower) obtained from somatic embryogenesis were thus examined and compared with the field-grown mother plant. Different amounts of vincristine and vinblastine were detected depending on the examined samples.

The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer.

The gene encoding acetyl CoA:deacetylvindoline 4-O-acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross-reacting with anti-DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light-treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of approximately 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit-ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O-acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.

Enzymology of indole alkaloid biosynthesis in Catharanthus roseus.

Indole alkaloids in Catharanthus roseus have been in focus because of their medicinal value. These alkaloids consist of an indole moiety provided by tryptamine and a terpenoid portion provided by the secologanin. The most important catharanthus alkaloids are vinblastine (VLB), vincristine (VCR) and ajmalicine. VLB and VCR are clinically useful anticancer agents whereas ajmalicine is used for the treatment of circulatory diseases. VCR and VLB are the most expensive because of their low abundance in the plant, and are formed by the coupling of monomeric indole alkaloids vindoline and catharanthine, catalysed by peroxidases. The pathway that lead to monomeric indole alkaloids involves more than 20 enzymes of which 16 enzymes have been isolated and characterized biochemically, and only three at the molecular level. The present state of knowledge on enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their regulation has been discussed.