Recent advances in the cell, developmental, and molecular biology of alkaloid biosynthesis have heightened our appreciation for the complexity and importance of plant secondary pathways. Several biosynthetic genes involved in the formation of tropane, benzylisoquinoline, and terpenoid indole alkaloids have now been isolated. The early events of signal perception, the pathways of signal transduction, and the function of gene promoters have been studied in relation to the regulation of alkaloid metabolism. Enzymes involved in alkaloid biosynthesis are associated with diverse subcellular compartments including the cytosol, vacuole, tonoplast membrane, endoplasmic reticulum, chloroplast stroma, thylakoid membranes, and perhaps unique "biosynthetic" or transport vesicles. Localization studies have shown that sequential alkaloid biosynthetic enzymes can also occur in distinct cell types, suggesting the intercellular transport of pathway intermediates. Isolated genes have also been used to genetically alter the accumulation of specific alkaloids and other plant secondary metabolites. Metabolic modifications include increased indole alkaloid levels, altered tropane alkaloid accumulation, elevated serotonin synthesis, reduced indole glucosinolate production, redirected shikimate metabolism, and increased cell wall-bound tyramine formation. This review discusses the biochemistry, cell biology, molecular regulation, and metabolic engineering of alkaloid biosynthesis in plants.

ALKALOID BIOSYNTHESIS IN PLANTS: Biochemistry, Cell Biology, Molecular Regulation, and Metabolic Engineering Applications.

In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed.

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Multicellular Compartmentation of Catharanthus roseus Alkaloid Biosynthesis Predicts Intercellular Translocation of a Pathway Intermediate

Full details of the development of a direct coupling of catharanthine with vindoline to provide vinblastine are described along with key mechanistic and labeling studies. Following an Fe(III)-promoted coupling reaction initiated by generation of a presumed catharanthine radical cation that undergoes a subsequent oxidative fragmentation and diastereoselective coupling with vindoline, addition of the resulting reaction mixture to an Fe(III)−NaBH4/air solution leads to oxidation of the C15′−C20′ double bond and reduction of the intermediate iminium ion directly providing vinblastine (40−43%) and leurosidine (20−23%), its naturally occurring C20′ alcohol isomer. The yield of coupled products, which exclusively possess the natural C16′ stereochemistry, approaches or exceeds 80% and the combined yield of the isomeric C20′ alcohols is >60%. Preliminary studies of Fe(III)−NaBH4/air oxidation reaction illustrate a generalizable trisubstituted olefin scope, identify alternatives to O2 trap at the oxidized carbon, p...

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Total synthesis of vinblastine, vincristine, related natural products, and key structural analogues.

The literature concerning the regulation and the biosynthesis of secondary metabolites in cell and tissue cultures of Catharanthus roseus is reviewed. The aim of this review is to summarise the progress achieved since the previous review of this subject from 1988 to December 1993. Several factors influencing the production of indole alkaloids are discussed. Special attention is given to large-scale cultivation methods. Some economic considerations on the production of ajmalicine are also discussed.

Cell and tissue cultures of Catharanthus roseus: A literature survey

Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.

https://link.springer.com/content/pdf/10.1007%2Fs11101-006-9047-8.pdf

Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Altered alkaloid pattern in dark grown seedlings of Catharanthus roseus: the isolation and characterization of 4-desacetoxyvindoline: a novel indole alkaloid and proposed precursor of vindoline

Studies involving selective functionalization of the indole unit in a number of bisindole alkaloids and related synthetic derivatives are described. These investigations include such reagents as te...

Total synthesis of indole and dihydroindole alkaloids. XII. Selective functionalization of various bisindoles. Efficient syntheses of leurosine and related bisindole alkaloid derivatives

Studies describing the preparation of various derivatives of vinblastine and vincristine are presented. Vindoline (3) is transformed into a series of derivatives and the latter via Polonovski-type ...

Total synthesis of indole and dihydroindole alkaloids. XVI. Derivatives of vinblastine and vincristine: change of functionality in the vindoline unit

Oxidation of 16,18-dicarbomethoxycleavamine gave the epoxide (6) and the enamide (9). Similar oxidation of 3′,4′-dehydrovinblastine (8) or leurosine (7) gave the natural product catharine (3). Pota...

Total synthesis of indole and dihydroindole alkaloids. XVII. The total synthesis of catharine and vinamidine (catharinine)

Robinson annelation of thujone (1) with methyl and ethyl vinyl ketones proceeds stereospecifically to afford the tricyclic enones 6 and 4 respectively. Enone 4 was converted to the glycol 13 which ...

John Balsevich

Papers

Altered alkaloid pattern in dark grown seedlings of Catharanthus roseus: the isolation and characterization of 4-desacetoxyvindoline: a novel indole alkaloid and proposed precursor of vindoline

Total synthesis of indole and dihydroindole alkaloids. XII. Selective functionalization of various bisindoles. Efficient syntheses of leurosine and related bisindole alkaloid derivatives

Total synthesis of indole and dihydroindole alkaloids. XVI. Derivatives of vinblastine and vincristine: change of functionality in the vindoline unit

Total synthesis of indole and dihydroindole alkaloids. XVII. The total synthesis of catharine and vinamidine (catharinine)

The chemistry of thujone. III. Thujone as a chiral synthon for the preparation of sesquiterpenes. Synthesis of (+)-β-cyperone