BIOSYNTHESIS OF TAXOL

紫杉醇的生物合成

• 批准号: 2894902
• 负责人: RODNEY B CROTEAU
• 金额: $ 17.51万
• 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 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2894902
• 关键词: Conifera; acylation; bioreactors; biosynthesis; cell free system; chemical kinetics; chemical structure; cyclization; cytochrome P450; diterpenes; enzyme mechanism; geranyl compound; mass spectrometry; medicinal plants; molecular cloning; paclitaxel; plant extracts; plant physiology; radiotracer; tissue /cell culture; transfection

Taxol, a highly functionalized diterpenoid, is an important anticancer drug isolated from yew (Taxus) species. The supply of this drug, and its precursors for semisynthesis, from natural sources is very limited, and total synthesis is not practical. Any attempt to improve the biological production of taxol and its congeners requires an understanding of the biosynthesis of this natural product and of the regulation of the pathway. A multi-step biogenetic scheme has been proposed based on the occurrence of defined taxoid metabolites and on analogy to biosynthetic transformations of simpler terpenoids; however, there is presently little experimentally-supported information on the biosynthesis of taxol, a process upon which future supply must depend. The long-term objective of this research is to increase the yields of this valuable drug and/or its semisynthetic precursors by engineering the overexpression of slow steps of the pathway in intact yew plants or derived cell cultures. This molecular approach offers a feasible solution to the taxol supply problem in that it involves the engineering of relatively few genes into an existing background for taxol production in which at least the latter steps of the pathway seem reasonably efficient. This goal will be reached by determining the number, types and sequence of enzymatic steps in the transformation of the ubiquitous 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 target. Defining this complex, multi-step pathway will be accomplished primarily through the use of cell-free enzyme systems from yew (Taxus) stem or cultured cells, combined with in vivo studies with labeled precursors, to determine the sequential progression from simple to complex metabolites. This systematic approach should identify the most appropriate target steps, and provide the necessary information and tools for cDNA isolation. The first two committed steps of the taxol pathway have been defined. The first, the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)- diene, is very slow if not rate limiting in the pathway, and the second, the cytochrome P450-catalyzed hydroxylation with allylic rearrangement of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol, is also very slow relative to subsequent pathway steps. PCR-based cDNA cloning strategies for both cyclase and P450 hydroxylase genes have been devised and these efforts constitute the first two specific aims. The third specific aim is a systematic approach to determining the sequence of oxygenation steps leading from taxa-4(20),11(12)-dien-5alpha-ol to the level of a pentaol and evaluating the contribution of each step to pathway flux and its importance as a cloning target. The forth aim focuses on the sequence of acylation steps in the progressive oxygenation of the taxane nucleus, the timing of C9-oxidation, and deciphering the enzymatic route to oxetane ring formation. In the final aim, transgenic Taxus systems will be engineered for overexpression of slow pathway steps using existing technologies, and the influence on the production yields of taxol, and related taxoids, will be determined.

紫杉醇是一种高度功能化的二萜类化合物，是一种重要的抗癌药物 从红豆杉（红豆杉）物种中分离出的药物。 该药物的供应及其 来自天然来源的半合成前体非常有限，并且 全合成不实用。 任何改善生物学的尝试 紫杉醇及其同系物的生产需要了解 这种天然产物的生物合成和调节 途径。 在此基础上提出了多步生物遗传方案 确定的紫杉烷代谢物的出现以及与生物合成的类比 更简单的萜类化合物的转化；但目前还很少有 关于紫杉醇生物合成的实验支持信息，紫杉醇是一种 未来供应必须依赖的过程。 长期目标 这项研究的目的是提高这种有价值的药物的产量和/或 它的半合成前体通过设计慢的过度表达 完整红豆杉植物或衍生细胞培养物中该途径的步骤。 这 分子方法为紫杉醇供应问题提供了可行的解决方案 因为它涉及将相对较少的基因改造为 紫杉醇生产的现有背景，其中至少后者 该路径的步骤似乎相当有效。 这个目标将是 通过确定酶促步骤的数量、类型和顺序来达到 在普遍存在的类异戊二烯分支点的转化中 香叶基香叶基二磷酸的中间体，是天然二萜的中间体 产品，并通过评估每个步骤对路径通量的贡献 为了评估作为 cDNA 克隆靶标的重要性。 定义这个 复杂的、多步骤的途径将主要通过 使用来自红豆杉（红豆杉）干细胞或培养细胞的无细胞酶系统， 结合标记前体的体内研究，以确定 从简单到复杂代谢物的顺序进展。这 系统方法应确定最合适的目标步骤， 并提供cDNA分离所需的信息和工具。 这 紫杉醇途径的前两个关键步骤已经确定。 这 首先，香叶基香叶基二磷酸环化为taxa-4(5),11(12)- 二烯，即使不是路径中的速率限制，也是非常慢的，第二个， 细胞色素 P450 催化的羟基化和烯丙基重排 Taxa-4(5),11(12)-diene 变为taxa-4(20),11(12)-dien-5alpha-ol，也是 相对于后续途径步骤非常慢。 基于 PCR 的 cDNA 克隆 针对环化酶和 P450 羟化酶基因的策略已被设计出来 这些努力构成了前两个具体目标。 第三个 具体目标是确定顺序的系统方法 从taxa-4(20),11(12)-dien-5alpha-ol到 五醇的水平并评估每个步骤的贡献 途径通量及其作为克隆靶点的重要性。 第四个目标 重点关注渐进氧化中酰化步骤的顺序 紫杉烷核的形成、C9 氧化的时间以及破译 氧杂环丁烷环形成的酶促途径。 最终目标是转基因 红豆杉系统将被设计用于慢通路步骤的过度表达 使用现有技术，以及对产量的影响 紫杉醇和相关紫杉烷的含量将被确定。