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.

紫杉醇是一种高度官能化的二萜，是重要的抗癌 从紫杉物种中分离出的药物。 该药物的供应及其 从天然来源的半合成前体非常有限，并且 完全合成是不切实际的。 任何改善生物学的尝试 生产紫杉醇及其同类物需要了解 这种天然产物的生物合成和调节 路径。 已经提出了一种多步生物遗传方案 出现定义的紫类代谢物，类似于生物合成 更简单的萜类化合物的转换；但是，目前几乎没有 关于紫杉醇生物合成的实验支持的信息， 未来供应必须取决于的过程。 长期目标 这项研究的是增加这种有价值的药物和/或 通过工程慢速表达，其半合成前体 完整的紫杉植物或衍生细胞培养的途径步骤。 这 分子方法为紫杉醇供应问题提供了可行的解决方案 因为它涉及相对较少的基因的工程 紫杉醇生产的现有背景，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 在无处不在的类异戊二烯分支的转化中 中间体，黄烷基凝酰二磷酸，到二萜天然 产品，并通过评估每个步骤对通路通量的贡献 为了评估重要性作为cDNA克隆靶标。 定义这个 复杂的多步途径将主要通过 使用紫杉（出租车）茎或培养细胞的无细胞酶系统， 与标记的前体的体内研究结合在一起，以确定 顺序从简单到复杂的代谢产物进展。这 系统的方法应确定最合适的目标步骤， 并提供必要的信息和工具进行cDNA隔离。 这 已经定义了紫杉醇途径的前两个投入步骤。 这 首先，黄烷基凝酰二磷酸到分类群-4（5），11（12） - 狄内（Diene 细胞色素P450催化的羟基化，烯丙基重排 在Taxa-4（5），11（12）-Diene到Taxa-4（20），11（12）-dien-5alpha-Ol也是 相对于随后的途径步骤非常慢。 基于PCR的cDNA克隆 已经设计了环化酶和P450羟化酶基因的策略 这些努力构成了前两个具体目标。 第三 具体目的是一种系统的方法来确定序列 从Taxa-4（20），11（12）-dien-5alpha-Ol到达的氧合步骤 五角星的水平并评估每个步骤的贡献 途径通量及其作为克隆目标的重要性。 第四目标 着重于进行性氧合中的酰基化步骤的序列 紫杉烷核，C9氧化的时机和破译 氧烷环形成的酶促途径。 在最终目标中，转基因 出租车系统将用于慢速步骤的过表达 使用现有技术以及对生产产量的影响 将确定紫杉醇和相关的型号。