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Taxol Biosynthesis Molecular Cloning and Characterization of a ..

Taxol: biosynthesis, molecular genetics, and biotechnological applications[J].

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CroteauTaxol: biosynthesis, molecular genetics, ..

Next generation sequencing technologies have enabled metagenomic and metagenetic analysis of soil microorganism species and gene composition of microbiota [, ]. However, there are currently no studies characterizing species and gene composition of root associated microbiome of the roots from Taxus. In this study, we used bar-coded high-throughput sequencing with primers targeting the 16S and 18S rRNA genes to survey root associated bacterial and fungal diversity of Taxus root, in conjunction with a metagenome approach to survey microbial species and gene composition in its root associated microbiome. We also studied genes putatively associated with taxol biosynthesis in the Taxus root associated microbiome to estimate the prevalence of taxol biosynthetic genes in the root associated microbiome.

Taxol Biosynthesis: Molecular Cloning and Characterization of a Cytochrome P450 Taxoid 7β ..

Protein purification of cytochrome P450 enzymes from Taxus microsomes as the basis for cDNA cloning was eliminated as not feasible, because the number of P450 species involved and their likely similarity in physical properties would almost certainly preclude bringing the individual proteins to homogeneity for amino acid microsequencing. As an alternative approach to cytochrome P450 cloning by reverse genetics, a strategy () was used that was based on a differential display of mRNA-reverse transcription-PCR method for the isolation of transcriptionally active cytochrome P450s in Taxus cells induced (with methyl jasmonate) for Taxol production (). This strategy led to the acquisition of a family of unique but closely related cytochrome P450 sequences that were expressed in yeast (Saccharomyces cerevisiae) and yielded the taxane 10β-hydroxylase as a 1497-bp cDNA encoding a 498-residue cytochrome capable of transforming taxadienyl acetate to the 10β-hydroxy derivative. However, only about half of the full-length clones afforded a characteristic CO-difference spectrum with absorbance maximum near 450 nm (), indicating the presence of functional heme-containing and properly folded recombinant cytochrome P450 in the yeast host (). To further develop and validate this general approach to defining the oxygenation steps of the Taxol biosynthetic pathway, a more reliable functional expression system was required.

Taxol biosynthesis and molecular genetics

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acytransferase that functions in taxol biosynthesis[J].

Cytochrome P450 monooxygenases play a prominent role in the biosynthesis of the diterpenoid anticancer drug Taxol, as they appear to constitute about half of the 19 enzymatic steps of the pathway in yew (Taxus) species. A combination of classical biochemical and molecular methods, including cell-free enzyme studies and differential-display of mRNA-reverse transcription polymerase chain reaction (RT-PCR) combined with a homology-based searching and random sequencing of a cDNA library from induced T. cuspidata cells, led to the discovery of six novel cytochrome P450 taxoid (taxane diterpenoid) hydroxylases. These genes show unusually high sequence similarity with each other (>70%) but low similarity (α- and the 7β-hydroxylase. This general approach could accelerate the functional analysis of candidate cDNAs from the extant family of P450 genes to identify the remaining oxygenation steps of this complex pathway.

Only eight C–H oxygenation events separate the minimally oxidized hydrocarbon taxadiene (1) from Taxol (2). Known by enzymologists as the oxidase phase, the approximate order of oxidation is proposed to occur at C-5, C-10/C-13, C-9, C-7/C-2, and then C-1/C-4/C-20 (Figure A). Divergent oxidation pathways and acylation patterns lead to hundreds of members in the taxane family. Intriguingly, the first three of eight oxygenation events involve the formal activation of allylic C–H bonds. In an ongoing effort to replicate the two phases of Taxol biosynthesis in the laboratory, we focused on uncovering the innate reactivity of 1 so as to achieve a controlled oxidation of these positions (C-5, C-10, and C-13). In order to obtain material to launch these investigations, an artificial cyclase phase for taxanes was devised, culminating in the first enantioselective, scalable total synthesis of (+)-taxadiene (1). In fact, decagram-scale reproduction of that route was independently accomplished by Albany Molecular Research Inc. to provide “taxadieneone,” the precursor for 1. We present a systematic approach to mimicking the early stages of the Taxol oxidase phase and may form the basis of an eventual scalable total synthesis of 2. Extensive empirical studies into the fundamental reactivity of taxadiene (1), computational modeling, and reagent development were all enlisted to achieve the first total synthesis of (−)-taxuyunnanine D (3) in only five steps from 1.

Taxol: Biosynthesis, Molecular Genetics and ..

(2004) Taxol biosynthesis: molecular cloning and characterization of a cytochrome P450 ..

Sources of Drugs: Biological, marine, mineral and plant tissue cultures as sources of drugs;
Classification of Drugs: Morphological, taxonomical, chemical and pharmacological classification of drugs; Study of medicinally important plants belonging to the families with special reference to: Apocynacae, Solanaceae, Rutacease, Umbelliferae, Leguminosae, Rubiaceae, Liliaceae, Graminae, Labiatae, Cruciferae, Papaveraceae; Cultivation, Collection, Processing and Storage of Crude Drugs: Factors influencing cultivation of medicinal plants, Types of soils and fertilizers of common use. Pest management and natural pest control agents, Plant hormones and their applications, Polyploidy, mutation and hybridization with reference to medicinal plants. Quality Control of Crude Drugs: Adulteration of crude drugs and their detection by organoleptic, microscopic, physical, chemical and biological methods and properties. Introduction to Active Constituents of Drugs: Their isolation, classification and properties.
Systematic pharmacognostic study of the followings:
CARBOHYDRATES and derived products: agar, guar gum acacia, Honey, Isabagol, pectin, Starch, sterculia and Tragacanth; Lipids: Bees wax, Castor oil, Cocoa butter, Codliver oil, Hydnocarpus oil, Kokum butter, Lard, Linseed oil, Rice, Bran oil, Shark liver oil and Wool fat; RESINS: Study of Drugs Containing Resins and Resin Combinations like Colophony, podophyllum, jalap, cannabis, capsicum, myrrh, asafoetida, balsam of Tolu, balsam of Peru, benzoin, turmeric, ginger;
TANNINS: Study of tannins and tannin containing drugs like Gambier, black catechu, gall and myrobalan;
VOLATILE OILS: General methods of obtaining volatile oils from plants, Study of volatile oils of Mentha, Coriander, Cinnamon, Cassia, Lemon peel, Orange peel, Lemon grass, Citronella, Caraway, Dill, Spearmint, Clove, Fennel, Nutmeg, Eucalyptus, Chenopodium, Cardamom, Valerian, Musk, Palmarosa, Gaultheria, Sandal wood; Phytochemical Screening: Preparation of extracts, Screening of alkaloids, saponins, cardenolides and bufadienolides, flavonoids and leucoanthocyanidins, tannins and polyphenols, anthraquinones, cynogenetic glycosides, amino acids in plant extracts; FIBERS: Study of fibers used in pharmacy such as cotton, silk, wool, nylon, glass-wool, polyester and asbestos.
Study of the biological sources, cultivation, collection, commercial varieties, chemical constituents, substitutes, adulterants, uses, diagnostic macroscopic and microscopic features and specific chemical tests of following groups of drugs:
GLYCOSIDE CONTAINING DRUGS: Saponins : Liquorice, ginseng, dioscorea, sarsaparilla, and senega. Cardioactive glycosides: Digitalis, squill, strophanthus and thevetia, Anthraquinone cathartics: Aloe, senna, rhubarb and cascara, Others: Psoralea, Ammi majus, Ammi visnaga, gentian, saffron, chirata, quassia.
ALKALOID CONTAINING DRUGS: Pyridine-piperidine: Tobacco, areca and lobelia. Tropane: Belladonna, hyoscyamus, datura, duboisia, coca and withania. Quinoline and Isoquinoline: Cinchona, ipecac, opium. Indole: Ergot, rauwolfia, catharanthus, nux-vomica and physostigma. Imidazole: Pilocarpus. Steroidal: Veratrum and kurchi. Alkaloidal Amine: Ephedra and colchicum. Glycoalkaloid: Solanum. Purines: Coffee, tea and cola. Biological sources, preparation, identification tests and uses of the following enzymes: Diastase, papain, pepsin, trypsin, pancreatin. Studies of Traditional Drugs: Common vernacular names, botanical sources, morphology, chemical nature of chief constituents, pharmacology, categories and common uses and marketed formulations of following indigenous drugs: Amla, Kantkari, Satavari, Tylophora, Bhilawa, Kalijiri, Bach, Rasna, Punamava, Chitrack, Apamarg, Gokhru, Shankhapushpi, Brahmi, Adusa, Atjuna, Ashoka, Methi, Lahsun, Palash, Guggal, Gymnema, Shilajit, Nagarmotha and Neem. The holistic concept of drug administration in traditional systems of medicine. Introduction to ayurvedic preparations like Arishtas, Asvas, Gutikas, Tailas, Chumas, Lehyas and Bhasmas.
General Techniques of Biosynthetic Studies and Basic Metabolic Pathways/Biogenesis: Brief introduction to biogenesis of secondary metabolites of pharmaceutical importance. Terpenes: monoterpenes, sesquiterpenes, diterpenes, and triterpenoids. Carotenoids: a-carotenoids, ß-carotenes, vitamin A, Xanthophylls of medicinal importance. Glycosides: Digitoxin, digoxin, hecogenin, sennosides, diosgenin and sarasapogenin. Alkaloids: Atropine and related compounds, Quinine, Reserpine, Morphine, Papaverine, Ephedrine, Ergot and Vinca alkaloids. Lignans, quassanoids and flavonoids. Role of plant-based drugs on National economy: A brief account of plant based industries and institutions involved in work on medicinal and aromatic plants in India. Utilization and production of phyto-constituents such as quinine, calcium sennosides, podophyllotoxin, diosgenin, solasodine, and tropane alkaloids. Utilization of aromatic plants and derived products with special reference to sandalwood oil, mentha oil, lemon grass oil, vetiver oil, geranium oil and eucalyptus oil. World-wide trade in medicinal plants and derived products with special reference to diosgenin (disocorea), taxol (Taxus sps) digitalis, tropane alkaloid containing plants, Papain, cinchona, Ipecac, Liquorice, Ginseng, Aloe, Valerian, Rauwolfia and plants containing laxatives. Plant bitters and sweeteners. Plant Tissue Culture: Historical development of plant tissue culture, types of cultures, nutritional requirements, growth and their maintenance. Applications of plant tissue culture in pharmacognosy. Marine pharmacognosy: Novel medicinal agents from marine sources. Natural allergens and photosensitizing agents and fungal toxins. Herbs as health foods. Herbal cosmetics. Standardization and quality control of herbal drugs, WHO guidelines for the standardization of herbal drugs.

Rheb is downregulated by Tsc1/Tsc2 complex that acts as a GTPase activating protein (GAP) for Rheb. Mutations in the Tsc1 or Tsc2 gene leads to genetic disorder called tuberous sclerosis that is associated with the appearance of benign tumors at multiple sites in the body. Our current effort is aimed at defining proteins involved in the Rheb signaling pathway.Nanodelivery of anticancer drugs: Another research interest of our lab is utilization of silica nanoparticles for controllable drug delivery system for cancer therapy. One of the major problems in clinical use of anti-cancer drugs is that many of them are hydrophobic, which poses a critical obstacle for cancer therapy. We have used mesoporous silica nanoparticles prepared in the presence of surfactants. These nanoparticles have the diameter of approximately 130 nm and contain thousands of pores whose diameter is about 3 nm. We incorporated different hydrophobic anticancer drugs, such as camptothecin (CPT) and taxol, into the pores of the mesoporous silica nanoparticles and delivered the drug to a variety of human cancer cells. This caused cell death. We are also exploring ways to use molecular valves to carry out controlled release of anti-cancer drugs with the mesoporous silica nanoparticles. One approach is to use molecules that change conformation by light exposure to accomplishcontrolled delivery. Targeting to cancer by attaching ligands specific to cancer cells is currently being investigatedin our lab.Protein lipidation and prenyltransferase inhibitors: Protein prenylation is aposttranslational modification of proteins involving the addition of isoprenoids, intermediates in cholesterol biosynthesis. Two types of modification, farnesylation and geranylgeranylation, occur with a variety of proteins. Farnesylation is of particular interest, since many of these farnesylated proteins are involved in signal transduction. Farnesylated proteins include Ras superfamily G-proteins as well as tyrosine phosphatases. Farnesylation is catalyzed by protein farnesyltransferase which recognizes the CysAAX motif at the C-termini of substrate proteins and transfers a farnesyl group forming a thioether bond.

Wildung MR (2006) Taxol biosynthesis and molecular genetics.
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  • Taxol Biosynthesis and Molecular Genetics

    Kaspera R, Wildung MR: Taxol biosynthesis and molecular genetics.

  • (2006) Taxol biosynthesis and molecular genetics

    (2006) Taxol biosynthesis and molecular genetics ..

  • as have the biosynthesis and molecular genetics of ..

    Croteau R, Ketchum REB, Kaspera R, Wildung MR (2006) Taxol biosynthesis and molecular genetics. Phytochem Rev 5: 75–97.

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