BIOSYNTHESIS of Taxol

紫杉醇的生物合成

基本信息

批准号：
7269986
负责人：
RODNEY B CROTEAU
金额：
$ 32.07万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7269986
关键词：
10p 14p Abbreviations Acetates Acetylation Acetyltransferase Acids Acylation Acyltransferase Address Affinity Chromatography Agreement Alkenes Ammonia Anabolism Anastomosis - action Antineoplastic Agents Area Attention Baccatin III Baculoviruses Benzoates Biochemical Biochemistry Biological Biological Assay Biological Factors Biological Models Biological Process Boxing C-terminal Candidate Disease Gene Cell Culture System Cell Line Cell physiology Cells Cephalomannine Chemistry Class Classification Clear Cell Cloning Coenzyme A Colorado Commit Complement 5a Complementary DNA Complex Condition Coupled Cryopreservation Cultured Cells Cyclization Cytochrome P450 Cytochromes DNA Sequence Rearrangement Databases Depth Development Diacetyl Dimethylallyltranstransferase Diphosphates Distal Diterpenes EST Library Electron Transport Engineering Enzymatic Biochemistry Enzyme Gene Enzymes Epoxy Compounds Erythritol Escherichia coli Esters Ethylene Oxide Evaluation Expressed Sequence Tags Facility Construction Funding Category Family Family member Felis catus Figs - dietary Foundations Frequencies Future Gene Duplication Gene Expression Generations Generic Drugs Genes Genetic Engineering Genomics Glyceraldehyde Glyceraldehyde 3-Phosphate Glycol Goals Gymnosperms Hand Homologous Gene Hydrogen Hydroxyl Radical Hydroxylation Imidazole Immunoblotting Isomerase Japanese Population Ketones Kinetics Knock-out Label Lead Legal patent Length Libraries Licensing Ligase Ligation Localized Mediating Messenger RNA Metabolic Metabolism Methods Microsomes Mixed Function Oxygenases Modification Molecular Molecular Cloning Molecular Genetics Monitor Monoterpenes NADPH-Ferrihemoprotein Reductase Nature Not Defined Nucleic Acids Numbers Object Attachment Organism Oxidases Oxidoreductase Oxygen Oxygenases Paclitaxel Pathway interactions Pattern Personal Satisfaction Pharmaceutical Preparations Pharmacy (field)Phenylalanine Phylogenetic Analysis Physical condensation Placement Plant Genes Plant Resins Plant Roots Plants Plasmids Plastids Polymerase Chain Reaction Positioning Attribute Preparation Principal Investigator Procedures Process Production Property Prosthesis Protein Overexpression Protocols documentation Protons Publications Purpose Pyruvate Pyruvates RNA Interference Radio Radiolabeled Range Rate Reaction Recombinants Recovery Regulation Relative (related person)Research Rest Reverse Transcription Route Science Screening procedure Series Side Source Spectrometry Spodoptera Staging Standards of Weights and Measures Steroid 21-Monooxygenase Structure Surveys Suspension substance Suspensions System Taxane Compound Taxoids Taxon Taxus Techniques Technology Terpenes Testing Time Time Study Tissues Transacylase Transcript Transcriptional Activation Transferase Transferase Gene Transgenic Organisms Trees Tritium Uncertainty Universities Up-Regulation Variant Walkers Withdrawal Work Yeasts acyl group assay development base benzoate benzoyl-coenzyme A cDNA Library catalyst cofactor cost deoxyxylulose phosphate design diene drug production enzyme biosynthesis epoxidase expression cloning farnesyltranstransferase feeding functional group gene cloning genetic manipulation geranylgeranyl diphosphate hexanoyl-coenzyme A his6 tag improved in vivo inhibitor/antagonist inorganic phosphate insight interest isopentenyl pyrophosphate isoprenoid kartocid mRNA Differential Displays member metabolomics methyl jasmonate microbial microbial host migration novel oxetane oxidation phenylisoserine polyol positional cloning pressure progenitor programs promoter protein expression prototype pyridine nucleotide radiotracer reconstitution stereochemistry taxadiene taxadiene synthase taxane taxusin thioester tool

项目摘要

Total synthesis of the anticancer agent Taxol is not practical and, for the foreseeable future, the supply of this diterpenoid drug, and its precursors for semisynthesis, must continue to rely on biological processes involving the isolation from yew (Taxus) species or cell cultures derived therefrom. Improvement of these biological methods of production must be based upon a detailed understanding of the complex pathway for Taxol biosynthesis, the enzymes which catalyze the sequence of reactions, and the genes for these enzymes, especially those responsible for slow steps, since the molecular genetic manipulation of the pathway can be expected to lead the production of the drug in high yield at more reasonable cost. The goal of improving Taxol production will be reached by determining the types and order of enzymatic steps from the diterpenoid branch-point intermediate geranylgeranyl diphosphate to the end- product, by cDNA cloning of the responsible genes, and by assessing the flux contribution of each step (and of diversionary side routes) by gene overexpression and suppression. Defining this multistep pathway is accomplished through the use of cell-free enzyme systems from induced yew (Taxus) cultured cells, combined with in vivo feeding studies, to determine the progression from simple to complex metabolites. This systematic approach has identified several early, intermediate and late steps of the Taxol pathway, and provided the tools for cDNA isolation with which thirteen pathway genes have been obtained and characterized by a broad range of cloning and expression strategies. The specific aims of this project are: 1. to clone and characterize the remaining five genes corresponding to intermediate enzymatic steps that complete modification of the taxoid core (Clp-hydroxylase, C9a-hydroxylase and C9 oxidase, C4,C20-epoxidase and oxomutase catalyzing formation the oxetane ring); 2. to clone and characterize the remaining two genes corresponding to the aroyl CoA ligase and C2'-hydroxylase needed to complete assembly of the C13 Af-benzoyl phenylisoserine side-chain; 3. to clone the corresponding genes and characterize the taxoid C7-0-, C9-O-, and C13-0-acetyltransferases which, along with taxoid 14p-hydroxylase, constitute major diversions of intermediate taxoids away from Taxol; and 4. to engineer Taxus cells for overexpression and suppression of each pathway (and side-route) gene, and to assess the influence on metabolic profile and yield as a means of ordering the sequence of reactions and defining flux controls at each step. Completion of these objectives will provide an understanding of Taxol biosynthesis and the foundation for multigene transgenic approaches to improve theproduction of Taxol, and of related intermediates for semisynthesis of Taxol and second generation taxoid drugs.

抗癌剂紫杉醇的全合成并不实用，并且在可预见的将来，这种药物的供应二萜类药物及其半合成前体必须继续依赖涉及以下生物过程从红豆杉（红豆杉）物种或源自其的细胞培养物中分离。这些生物学方法的改进生产必须基于对紫杉醇生物合成复杂途径的详细了解，即酶催化反应序列以及这些酶的基因，尤其是那些负责缓慢步骤的基因，因为该途径的分子遗传操作有望导致药物的高产量以更合理的成本获得收益。通过确定紫杉醇的种类和品种，达到提高紫杉醇产量的目的。从二萜类分支点中间体香叶基香叶基二磷酸到末端的酶促步骤顺序产物，通过负责基因的 cDNA 克隆，并通过评估每个步骤（以及通过基因过度表达和抑制来转移注意力）。定义此多步骤路径已完成通过使用来自诱导红豆杉（红豆杉）培养细胞的无细胞酶系统，并结合体内喂养研究，以确定从简单代谢物到复杂代谢物的进展。这种系统性方法已确定紫杉醇途径的几个早期、中期和晚期步骤，并提供了用于 cDNA 分离的工具已经获得了十三个途径基因，并通过广泛的克隆和表达策略对其进行了表征。该项目的具体目标是： 1. 克隆并表征其余五个基因对应的完成紫杉烷核心修饰的中间酶步骤（Clp-羟化酶、C9a-羟化酶和 C9氧化酶、C4,C20-环氧酶和氧化变位酶催化形成氧杂环丁烷环)； 2. 克隆和表征其余两个基因对应于完成组装所需的芳酰辅酶A连接酶和C2'-羟化酶 C13 Af-苯甲酰基苯基异丝氨酸侧链； 3.克隆相应基因并表征紫杉烷C7-0-， C9-O-和C13-0-乙酰转移酶与紫杉烷14β-羟化酶一起构成了主要的转移酶远离紫杉醇的中间紫杉烷； 4. 改造红豆杉细胞以过度表达和抑制每种途径（和旁路）基因，并评估对代谢谱和产量的影响，作为排序的一种手段反应顺序并定义每一步的通量控制。完成这些目标将提供了解紫杉醇生物合成和多基因转基因方法提高产量的基础紫杉醇，以及用于半合成紫杉醇和第二代紫杉醇药物的相关中间体。