BIOSYNTHESIS of Taxol

描述：(申请人提供)紫杉醇，一种高度官能化的二萜类化合物， 是从红豆杉属植物中分离得到的一种重要的抗癌药物。总计 合成紫杉醇是不现实的，在可预见的未来，供应 这种药物及其半合成的前体必须依赖于生物 生产方式。改进的生物工艺的发展必须 基于对紫杉醇生物合成途径的详细了解， 催化反应序列的酶，以及编码这些反应的基因 酶，尤其是那些负责缓慢步伐的酶，因为分子 可以预期，对该途径的遗传操作将导致 以合理的价格大量购买这种药物。的长期目标是 提高紫杉醇产量是通过确定紫杉醇的数量、类型 和类异戊二烯转化中的酶步骤序列 分支点中间体香叶基二磷酸为天然二萜类化合物 产品，并通过评估每一步对途径通量的贡献 为了评估作为cdna克隆(和基因过度表达)的重要性。 目标。定义这条多步骤路径是通过使用 诱导红豆杉(Taxus)培养细胞的无细胞酶系统 通过体内喂养研究，确定从简单到 复杂的代谢物。这种系统化的方法已经确定了前四个 紫杉醇途径的具体步骤和几个下游的转化， 为分离7个途径基因提供了工具。 已经获得了。这个正在进行的项目的具体目标是：1.确定 从紫杉醇的烯烃前体开始的早期氧化步骤， 紫杉二烯，到五醇的水平，来表征负责任的 细胞色素P450酶，并通过 基于同源性的克隆策略；2.确定后期氧化步骤 完成了紫杉醇核心的修饰，以表征 负责细胞色素P450酶，并通过以下方式获得相应的基因 类似的克隆策略；3.完成三者的比较研究 重组酰基转移酶现已面世(用于C2、C5和C10的酰化)， 并定义参与C13侧链组装的酰基转移酶和 分离相应的cdna；4.阐明其余的修改 紫杉烷核(C9氧化和氧杂环形成)及其成因 3-苯基异丝氨酸侧链的克隆，并设计合适的cDNA克隆 策略；以及6.改造红豆杉细胞以过表达慢通路 提高紫杉类药物产量的措施，并同时利用“感觉”和 “反义”技术帮助确定途径序列和通量 控制。

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.