ENZYMATIC DETERMINANTS OF ERYTHROMYCIN STEREOCHEMISTRY

红霉素立体化学的酶决定因素

• 批准号: 6091814
• 负责人: RICHARD G SUMMERS
• 金额: $ 3.94万
• 依托单位: LAWRENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6091814
• 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。是 该项目的长期目标是生产新型红霉素衍生物， 这是传统的化学合成方法所不能达到的。新的抗生素 像这样的衍生品是迫切需要的，特别是鉴于 新出现的抗生素耐药性细菌带来的威胁增加。 具体而言，这项工作旨在确定决定酶结构域， 红霉素大环内酯环的立体化学， 基因工程抗生素生产细菌的知识， 以生产新的红霉素衍生物。 目前，红霉素合酶的基因已经被克隆， 关于这种化学复杂抗生素的生物合成是已知的。然而 酶结构域负责十个立体化学构型 红霉素大环内酯环中的不同位点是未知的。因为它有 已经表明，红霉素合酶可以被改变以产生新的 红霉素衍生物通过基因工程，知识， 红霉素立体化学的决定因素应该能够产生 一系列全新的抗生素衍生物，其中许多可能是 生物活性。事实上，仅仅通过立体化学的改变， 理论上可以获得一百种新的红霉素。 这里要采取的方法最初集中在体外构建 使用标准的编码改变的红霉素酶的遗传嵌合体 重组DNA技术。这些改变的酶将以酶促 领域交流，重点是那些最有可能涉及的领域， 红霉素立体化学的测定（即被认为产生红霉素的结构域 一个立体化学产物将被类似的结构域所取代， 产生相反的立体化学结果）。一旦基因嵌合体 在体外构建的，野生型基因的自然 产生红霉素的微生物将被替换（通过两步基因 替换方案）和由突变体产生的红霉素衍生物 将通过NMR分离和表征生物体。重要的是，大多数 这里提出的工作将由本科生化学和生物进行 因此，本研究项目也将提供一个理想的培训 对医学生物技术和遗传学感兴趣的学生的机会 工程.