BIOSYNTHESIS of Taxol

紫杉醇的生物合成

• 批准号: 6512723
• 负责人: RODNEY B CROTEAU
• 金额: $ 28.75万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6512723
• 关键词: Conifera; biosynthesis; cell free system; complementary DNA; cytochrome P450; enzyme activity; genetically modified plants; microsomes; molecular cloning; paclitaxel; recombinant proteins; serine; transferase

DESCRIPTION: (provide by applicant) Taxol, a highly functionalized diterpenoid, is an important anticancer drug isolated from yew (Taxus) species. Total synthesis of taxol is not practical and, for the foreseeable future, the supply of this drug, and its precursors for semisynthesis, must rely on biological methods of production. The development of improved biological processes must be based upon a detailed understanding of the pathway for taxol biosynthesis, the enzymes which catalyze the sequence of reactions, and the genes encoding these enzymes, especially those responsible for slow steps, since the molecular genetic manipulation of the pathway can be expected to lead to the production of the drug in larger quantities at reasonable cost. The long-term goal of improving taxol production is being reached by determining the number, types and sequence of enzymatic steps in the transformation of the isoprenoid branch-point intermediate geranylgeranyl diphosphate to the diterpenoid natural product, and by assessing the contribution of each step to pathway flux in order to evaluate importance as a cDNA cloning (and gene overexpression) target. Defining this multi-step pathway is being 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 the first four specific steps of the taxol pathway and several downstream' transformations, and has provided the tools for cDNA isolation with which seven pathway genes have been obtained. The specific aims of this ongoing project are: 1. to define the early oxygenation steps leading from the olefin precursor of taxol, taxadiene, to the level of a pentaol, to characterize the responsible cytochrome P450 enzymes, and to acquire the corresponding genes by a homology-based cloning strategy; 2. to define the late-stage oxygenation steps that complete the modification of the taxoid core, to characterize the responsible cytochrome P450 enzymes, and to acquire the corresponding genes by a similar cloning strategy; 3. to complete comparative studies on the three recombinant acyltransferases now in hand (for acylation at C2, C5 and C 10), and to define the acyltransferases involved in C13 side-chain assembly and to isolate the corresponding cDNAs; 4. to elucidate the remaining modifications to the taxane core (oxidation at C9 and oxetane D-ring formation) and the origin of the 3-phenylisoserine side chain, and to devise suitable cDNA cloning strategies; and 6. to engineer Taxus cells for overexpression of slow pathway steps to increase taxoid production yields, and to exploit both 'sense' and 'antisense' technology to assist in defining pathway sequence and flux controls.

描述：（由申请人提供）紫杉醇，一种高度官能化的二萜类化合物， 是一种重要的抗癌药物，分离自红豆杉（Taxus）物种。总 紫杉醇的合成是不实际的，并且在可预见的将来， 这种药物及其半合成前体的生产必须依赖于生物 生产方法。必须开发改进的生物过程 基于对紫杉醇生物合成途径的详细了解， 催化反应序列的酶，以及编码这些酶的基因 酶，特别是那些负责缓慢步骤，因为分子 对该途径的遗传操作可以预期导致产生 以合理的价格大量生产药物。的长期目标 提高紫杉醇的产量是通过确定紫杉醇的数量、类型 以及类异戊二烯转化中的酶促步骤的顺序 分支点中间体香叶基香叶基二磷酸到天然二萜 产品，并通过评估每一步的贡献，途径通量， 为了评估作为cDNA克隆（和基因过表达）的重要性， 目标定义这一多步骤途径是通过使用 诱导红豆杉（Taxus）培养细胞的无细胞酶系统，结合 通过体内喂养研究，以确定从简单到 复杂代谢物。这种系统的方法确定了前四个 紫杉醇途径的具体步骤和几个下游转化， 并提供了cDNA分离的工具， 已经获得。该项目的具体目标是：1.以限定 早期的氧化步骤由紫杉醇的烯烃前体引起， 紫杉二烯，达到五醇的水平，以表征负责任的 细胞色素P450酶，并获得相应的基因， 基于同源性的克隆策略; 2.来定义后期的氧合步骤 完成紫杉烷核心的修饰， 负责细胞色素P450酶，并获得相应的基因， 类似的克隆策略; 3.完成三者的比较研究 重组酰基转移酶现在在手（用于在C2、C5和C10的酰化）， 并定义参与C13侧链组装的酰基转移酶， 分离相应的cDNA; 4.为了阐明剩余的修改， 紫杉烷核心（C9氧化和氧杂环丁烷D环形成）和起源 的3-苯基异丝氨酸侧链，并设计合适的cDNA克隆 战略; 6。改造红豆杉细胞使其过表达慢信号通路 步骤，以增加紫杉烷生产产量，并利用"正义"和 "反义"技术，以帮助确定途径序列和流量 对照