BIOSYNTHESIS OF TAXOL

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 从红豆杉属植物中分离出来的药物。这种药物的供应，以及它的 来自自然来源的半合成的前体非常有限，并且 完全综合是不现实的。任何试图改善生物多样性的尝试 紫杉醇及其同系物的生产需要了解 这一天然产物的生物合成和对 路径。在此基础上，提出了一种多步骤生物遗传方案。 确定的紫杉类代谢物的存在及其与生物合成的类比 较简单的萜类化合物的转化；然而，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 其半合成前体基因Slow过表达的研究 在完整的红豆杉植物或衍生细胞培养中该途径的步骤。这 分子方法为紫杉醇供应问题提供了一个可行的解决方案 因为它涉及到将相对较少的基因工程成 紫杉醇生产的现有背景，其中至少后者 这条路径的步骤似乎相当有效。这个目标将是 通过确定酶步骤的数量、类型和顺序而达到的 在无处不在的类异戊二烯分支点的转化中 中间体香叶基香叶基二磷酸，天然二萜类化合物 产品，并通过评估每一步对途径通量的贡献 以评价其作为基因克隆靶标的重要性。定义这一点 复杂的、多步骤的途径将主要通过 使用来自红豆杉(Taxus)茎或培养细胞的无细胞酶系统， 结合体内标记前体的研究，以确定 从简单的代谢物到复杂的代谢物的顺序过程。这 系统化的方法应该确定最合适的目标步骤， 为基因分离提供了必要的信息和工具。这个 紫杉醇途径的前两个步骤已经确定。这个 首先，香叶基香叶基二磷酸环化成4(5)，11(12)- Diene，是非常慢的，如果不是在路径中的速率限制，第二， 细胞色素P450催化的烯丙基重排羟化反应 4(5)，11(12)-二烯至-4(20)，11(12)-二烯-5α-醇 相对于随后的通路步骤来说非常慢。基于聚合酶链式反应的基因克隆 环化酶和P450羟基酶基因的策略已经被设计出来 这些努力构成了前两个具体目标。第三 特定目标是一种系统的方法来确定序列 从分类-4(20)，11(12)-二烯-5α-醇到 五醇水平，并评估每一步对 途径通量及其作为克隆靶标的重要性。第四个目标 重点介绍了逐步氧化中的酰化步骤的顺序 紫杉烷原子核，C9氧化的时间，以及破译 酶法合成氧杂环己烷。在最终目标中，转基因 红豆杉系统将被设计为过度表达慢途径步骤 利用现有技术，以及对产量的影响 紫杉醇和相关紫杉醇类化合物的含量将被确定。

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.