BIOSYNTHESIS of Taxol

紫杉醇的生物合成

基本信息

批准号：
6633056
负责人：
RODNEY B CROTEAU
金额：
$ 28.74万
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 2006-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6633056
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

项目摘要

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，一种高度功能化的二萜类化合物，是从红豆杉 (Taxus) 物种中分离出来的一种重要的抗癌药物。全部的紫杉醇的合成是不切实际的，并且在可预见的将来，供应这种药物及其半合成前体的制备必须依赖于生物生产方法。改进生物过程的开发必须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，尤其是那些负责缓慢步伐的酶，因为分子该途径的基因操作有望导致生产以合理的成本大量生产药物。长期目标是通过确定紫杉醇的数量、类型来提高紫杉醇的产量以及类异戊二烯转化中的酶促步骤的顺序香叶基香叶基二磷酸到天然二萜的分支点中间体产物，并通过评估每个步骤对路径通量的贡献为了评估 cDNA 克隆（和基因过度表达）的重要性目标。定义这个多步骤路径是通过使用来完成的来自诱导红豆杉（红豆杉）培养细胞的无细胞酶系统，结合通过体内喂养研究，确定从简单到复杂的代谢物。这种系统方法确定了前四个紫杉醇途径的具体步骤和几个下游的转化，并提供了 cDNA 分离工具，其中七个途径基因已获得。这个正在进行的项目的具体目标是： 1. 定义由紫杉醇的烯烃前体引起的早期氧化步骤，紫杉二烯，达到五元醇的水平，表征负责任的细胞色素P450酶，并通过基于同源性的克隆策略； 2. 定义后期氧合步骤完成紫杉烷核心的修饰，以表征负责细胞色素P450酶，并通过以下方式获得相应的基因类似的克隆策略； 3.完成三者的比较研究现有重组酰基转移酶（用于 C2、C5 和 C 10 处的酰化），并定义参与 C13 侧链组装的酰基转移酶分离相应的cDNA； 4. 阐明其余修改紫杉烷核心（C9 氧化和氧杂环丁烷 D 环形成）和起源 3-苯基异丝氨酸侧链，并设计合适的 cDNA 克隆策略； 6. 改造红豆杉细胞以过度表达慢通路提高紫杉类产量的步骤，并利用“意义”和 “反义”技术协助定义途径序列和通量控制。