ENZYMATIC DETERMINANTS OF ERYTHROMYCIN STEREOCHEMISTRY

红霉素立体化学的酶决定因素

• 批准号: 6588377
• 负责人: RICHARD G SUMMERS
• 金额: $ 8.92万
• 依托单位: UNIVERSITY OF THE SOUTH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6588377
• 关键词: analog; biosynthesis; biotechnology; enzyme activity; enzymes; erythromycin; fusion gene; mutant; nuclear magnetic resonance spectroscopy; polymerase chain reaction; recombinant DNA; stereochemistry

DESCRIPTION (adapted from applicant's abstract): The proposed research focuses on the biosynthesis of the clinically important antibiotic erythromycin. It is the long-term goal of this project to produce novel erythromycin derivatives that cannot be prepared by traditional chemical syntheses. New antibiotic derivatives such as these are urgently needed, particularly in light of the increased threat posed by newly emergent antibiotic resistant bacteria. In specific, this work seeks to determine the enzymatic domains that dictate the stereochemistry of the erythromycin macrolactone ring and then use this knowledge to genetically engineer the antibiotic producing bacteria, Saccharopolyspora erythraea, to produce new erythromycin derivatives. Currently, the genes for the erythromycin synthase have been cloned, and much is known about the biosynthesis of this chemically complex antibiotic. Yet, the enzymatic domains responsible for the stereochemical configuration of ten distinct sites in the erythromycin macrolactone ring are unknown. Since it has already been shown that the erythromycin synthase can be altered to produce new erythromycin derivatives through genetic engineering, knowledge of the determinants of erythromycin stereochemistry should enable the production of entirely new series of antibiotic derivatives, many of which may be biologically active. Indeed, just through alterations in stereochemistry, over a hundred new erythromycins are theoretically accessible. The approach to be taken here centers initially on the in vitro construction of genetic chimeras encoding altered erythromycin synthases using standard recombinant DNA techniques. These altered synthases will feature enzymatic domain interchanges focusing on those domains most likely involved in the determination of erythromycin stereochemistry (i.e. a domain thought to produce one stereochemical outcome will be replaced with an analogous domain thought to produce the opposite stereochemical outcome). Once the genetic chimeras have been constructed in vitro, the wild type genes of the natural erythromycin-producing organism will be replaced (via a two step gene replacement protocol) and the erythromycin derivatives produced by the mutant organisms will be isolated and characterized by NMR. Importantly, most of the work proposed here will be conducted by undergraduate chemistry and biology majors, consequently this research project will also provide an ideal training opportunity for students interested in medical biotechnology and genetic engineering.

描述（改编自申请人的摘要）：拟议的研究重点 临床上重要的抗生素红霉素的生物合成。这是 该项目的长期目标是生产新型红霉素衍生物 传统化学合成方法无法制备。新抗生素 迫切需要诸如此类的衍生物，特别是考虑到 新出现的抗生素耐药细菌造成的威胁增加。 具体来说，这项工作旨在确定决定的酶域 红霉素大环内酯环的立体化学，然后使用它 对产生抗生素的细菌进行基因改造的知识， 红霉素糖多孢菌，用于生产新的红霉素衍生物。 目前，红霉素合酶基因已被克隆，许多 这种化学复杂的抗生素的生物合成是已知的。然而， 负责十个立体化学构型的酶结构域 红霉素大环内酯环中的不同位点尚不清楚。既然它有 已经表明红霉素合酶可以被改变以产生新的 通过基因工程的红霉素衍生物，知识 红霉素立体化学的决定因素应该能够生产 全新系列的抗生素衍生物，其中许多可能是 具有生物活性。事实上，仅仅通过立体化学的改变， 理论上可以使用一百种新的红霉素。 这里采取的方法最初集中在体外构建 使用标准编码改变的红霉素合酶的遗传嵌合体 重组DNA技术。这些改变的合酶将具有酶促作用 域交换专注于那些最有可能涉及的域 红霉素立体化学的测定（即被认为产生的域 一种立体化学结果将被认为是的类似域所取代 产生相反的立体化学结果）。一旦基因嵌合体具有 体外构建的天然野生型基因 产生红霉素的生物体将被取代（通过两步基因 替换方案）和突变体产生的红霉素衍生物 生物体将被分离并通过核磁共振表征。重要的是，大多数 这里提出的工作将由化学和生物学本科生进行 专业，因此该研究项目也将提供理想的培训 为对医学生物技术和遗传学感兴趣的学生提供机会 工程。