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，一种高度功能化的二萜， 是一种从紫杉（Taxus）物种中分离出来的重要抗癌药。全部的 紫杉醇的综合是不切实际的，在可预见的将来是供应 该药物及其半合成的前体必须依靠生物学 生产方法。改进的生物过程的发展必须是 基于对紫杉醇生物合成途径的详细理解， 催化反应序列的酶，以及编码这些反应的基因 酶，尤其是负责缓慢步骤的酶，因为分子 可以预期该途径的遗传操纵会导致生产 以合理的成本以更大数量的药物进行。长期目标 通过确定数量，类型，可以提高紫杉醇生产 以及类异丙因的转化中的酶促步骤序列 分支点中间的黄烷基凝血酶二磷酸到二萜天然 产品，并通过评估每个步骤对途径通量的贡献 为了评估重要性为cDNA克隆（和基因过表达） 目标。通过使用来定义这一多步途径 来自诱导紫杉（Taxus）培养细胞的无细胞酶系统的组合 通过体内喂养研究，确定从简单到 复杂的代谢物。这种系统的方法已经确定了前四个 紫杉醇途径的特定步骤和几个下游的转换， 并提供了七个途径基因的cDNA隔离工具 已获得。这个正在进行的项目的具体目的是：1。定义 从紫杉醇的烯烃前体导致的早期充氧步骤， 义务义务，达到五角星的水平，以表征负责任的 细胞色素P450酶，并通过A获得相应的基因 基于同源的克隆策略； 2。定义晚期氧合步骤 完成了符号核心的修改，以表征 负责的细胞色素P450酶，并通过 类似的克隆策略； 3。完成三个的比较研究 重组酰基转移酶现已携手携带（在C2，C5和C 10处进行酰化）， 并定义涉及C13侧链组件的酰基转移酶和 隔离相应的cDNA； 4。阐明其余的修改 紫杉烷核（C9和Oxetane D形成的氧化）和原点 3-苯基硅烷侧链，并设计合适的cDNA克隆 策略；和6。要设计出慢速途径过表达的出租车电池 提高紫类生产产量的步骤，并利用“意义”和 “反义”技术以帮助定义途径序列和通量 控件。