Biosynthesis of Maytansinoids and Analogs

美登木素生物碱及其类似物的生物合成

• 批准号: 6748448
• 负责人: Tin-Wein Yu
• 金额: $ 5.42万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-15 至 2004-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6748448
• 关键词: Streptomyces; antineoplastic antibiotics; bacterial genetics; biological products; bioreactors; biosynthesis; biotechnology; carbohydrate analog; drug design /synthesis /production; drug screening /evaluation; enzyme activity; macrolide antibiotics; microorganism mass culture; microorganism metabolism; plant extracts; polyketide synthase; recombinant proteins; site directed mutagenesis

DESCRIPTION (provided by applicant): The ansamitocin family of ansamycin-type microbial metabolites produced by the Actinomycete Actinosynnema pretiosum and the structurally almost identical plant-derived maytansine and its congeners are extraordinarily potent antitumor agents. Despite its toxicity, maytansine at one time was considered an outstanding candidate for clinical development, but it failed to show significant efficacy in phase II clinical trials, probably due to dose-limiting toxicity. However, interest in these compounds continues, e.g., as "warheads" for antibody-targeted delivery, and their high potency calls for further efforts to modify their structures with the goal of identifying analogs which retain high antitumor activity coupled with lower toxicity than the parent compounds. The structural complexity of these compounds limits chemical approaches to modified structures to those accessible by semi-synthesis from the natural product starting materials; more deep-seated backbone structural modifications require biochemical approaches based on genetic alteration of the biosynthetic machinery generating the parent compounds. With this rationale in mind we have cloned and sequenced the ansmitocin (asm) biosynthetic gene cluster from A. pretiosum and are in the process of analyzing the functions of its individual genes. In the next 4-year period of this grant we wish to continue this work by determining the functions of all the downstream modification genes/enzymes, expressing the polyketide synthase (PKS) assembling the backbone of ansamitocin in a heterologous host and studying its structure and mode of operation, and clarifying the structure and mode of formation of a rare polyketide chain extension unit required for the function of the asm PKS. Based on the insights gained from this work, we will then assemble a system, which allows the expression of all the asm biosynthetic genes to produce ansamitocins in a heterologous host, Streptomyces coelicolor, from a series of gene cassettes under the control of an external promoter. Once this system is established, we will demonstrate the feasibility of producing ansamitocin analogs by introducing genetic modifications into the expressed gene cluster. In addition we plan to investigate the genetic control of ansamitocin production in A. pretiosum with the aim to increase yields of the parent and engineered mutant compounds. This work will thus provide the tools for the preparation of structural analogs of the ansamitocins, which can be evaluated for an improved therapeutic ratio as anticancer agents.

描述（由申请人提供）：放线菌静脉阳离子pretiosum和结构上几乎相同的植物衍生的Maytansine产生的Ansamycin型微生物代谢产物的Ansamitocin家族，其共同体是非常有效的抗衰老者。尽管有毒性，但一次Maytansine被认为是临床发育的出色候选者，但在II期临床试验中未能显示出明显的疗效，这可能是由于剂量限制性毒性所致。但是，对这些化合物的兴趣仍在继续，例如，作为抗体靶向递送的“弹头”，他们的高效力要求采取进一步的努力来修饰其结构，目的是确定与母亲化合物相比，保留高抗毒剂与毒性低的毒性相结合的类似物。这些化合物的结构复杂性将化学方法限制为通过自然产物起始物质从半合成而获得的修改结构的方法。更深层的主链结构修饰需要基于生成父化合物的生物合成机制的遗传改变的生化方法。 考虑到这种理由，我们已经克隆并测序了来自A. petiosum的Ansmitocin（ASM）生物合成基因簇，并正在分析其各个基因的功能。在这笔赠款的下一个4年期间，我们希望通过确定所有下游修饰基因/酶的功能来继续这项工作，表达在异源宿主中组装Ansamitocin的主链并研究其操作的结构和模式，并阐明Ansamitocin的主体的结构和模式，并阐明了ANSAMITOCIN的结构和模式，并阐明了ANSAMITOS的主体的结构和模式。然后，根据这项工作从中获得的见解，我们将组装一个系统，该系统允许所有ASM生物合成基因的表达在异源宿主（链霉菌链霉菌）中产生Ansamitocin，这是由一系列基因cassettes在外部启动子控制下的一系列基因cassettes中产生的。建立该系统后，我们将通过将遗传修饰引入表达的基因簇中来证明产生ANSAMITOCIN类似物的可行性。此外，我们计划研究青春期曲霉中Ansamitocin产生的遗传控制，旨在提高父和工程突变体化合物的产量。因此，这项工作将为制备Ansamitocins的结构类似物提供工具，可以评估其作为抗癌药的改善治疗比例。