BIOSYNTHESIS OF TAXOL

紫杉醇是一种高度功能化的二萜类化合物， 一种从紫杉（红豆杉属）中分离出来的药物。 这种药物的供应， 天然来源的半合成前体非常有限， 全合成是不切实际的。 任何试图改善生物学的尝试 生产紫杉醇及其同系物需要了解 这种天然产物的生物合成和调节 通路 提出了一种多步生物遗传方案， 确定的紫杉烷代谢物的出现和生物合成的类比 简单的萜类化合物的转化;然而，目前很少有 紫杉醇生物合成的实验支持信息， 这是未来供应所必须依赖的。 长期目标 这项研究的目的是增加这种有价值的药物的产量和/或 其半合成前体通过工程化过表达缓慢的 完整紫杉植物或衍生细胞培养物中的途径步骤。 这 分子方法为紫杉醇的供应问题提供了一个可行的解决方案 因为它涉及到相对较少的基因工程， 紫杉醇生产的现有背景，其中至少后者 这条途径的步骤似乎相当有效。 这一目标将 通过确定酶促步骤的数量、类型和顺序来实现 在普遍存在的类异戊二烯分支点的转变中， 天然二萜的中间体，香叶基香叶基二磷酸 产品，并通过评估每一步的贡献，途径通量 以评价作为cDNA克隆靶的重要性。 定义此 复杂的，多步骤的途径将主要通过 使用来自紫杉（Taxus）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， 从简单代谢物到复杂代谢物的顺序进展。这 系统性办法应确定最适当的目标步骤， 为cDNA的分离提供必要的信息和工具。 的 已经确定了紫杉醇途径的前两个关键步骤。 的 首先，香叶基香叶基二磷酸环化为taxa-4（5），11（12）- 二烯，在该途径中是非常缓慢的，如果不是速率限制的话，第二， 细胞色素P450催化羟化反应及烯丙基重排 紫杉-4（5），11（12）-二烯转化为紫杉-4（20），11（12）-二烯-5 α-醇， 相对于随后的路径步骤非常慢。 基于PCR的cDNA克隆 针对环化酶和P450羟化酶基因的策略已经被设计出来 而这些努力构成了前两个具体目标。 第三 具体目标是一个系统的方法来确定顺序， 氧合步骤导致从紫杉-4（20），11（12）-二烯-5 α-醇到 五醇的水平，并评估每个步骤对 途径通量及其作为克隆靶点的重要性。 第四个目标 重点是在逐步氧化酰化步骤的顺序 紫杉烷核，C9氧化的时间，并破译 氧杂环丁烷环形成的酶促途径。 最终，转基因 红豆杉系统将被改造为过表达慢途径步骤 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。

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.