Investigations of Coronafacic Acid Biosynthesis

冠发酸生物合成的研究

• 批准号: 9807774
• 负责人: Ronald Parry
• 金额: --
• 依托单位: William Marsh Rice University
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9807774&HistoricalAwards=false
• 关键词: Investigations; Coronafacic; Acid; Biosynthesis

Parry Coronatine is a novel phytotoxin produced by the phytopathogenic bacterium Pseudomonas syringae. Previous investigations of the biosynthesis of coronatine have revealed that the hydrindanone portion of the toxin, coronafacic acid, is a novel polyketide derived from two acetate units, one butyrate unit, and a four or five-carbon starter unit derived from (-ketoglutaric acid. The cyclopropyl amino acid moiety of the toxin, coronamic acid, has been shown to be biosynthesized from L-alloisoleucine by an unusual oxidative cyclization reaction. Genetic investigations of coronaline biosynthesis have identified a DNA region of ca. 6.9 kb that appears to be associated with coronamic acid formation and a region spanning ca. 21 kb that is associated with coronafacic acid biosynthesis. Sequencing of the coronafacic acid region has disclosed the presence of ten open reading frames which are part of a single transcript. The transcript begins with cfl and the remaining genes are designated as cfal-cfa9, numbering from the 5' end of the transcript. The cfl and cfa5 open reading frames encode proteins that are similar to enzymes that activate carboxylic acids as acyl adenylates. Two of the open reading frames, cfa6 and cfa7, are unusually large and appear to encode proteins that bear a close resemblance to known Type I polyketide synthases (PKSs). The remaining open reading frames encode proteins that are similar to components of Type II PKSs or fatty acid synthases. The organization of the coronafacic acid PKS is unlike that of any other PKS that has been reported. The research project has two major, long-term objectives. The first objective is to understand the nature of the steps involved in coronafacic acid biosynthesis at the biochemical level. Because of the complexity of coronafacic acid biosynthesis, the project will focus upon an analysis of the function of the first six genes in the pathway. The second objective is to utilize parts of the coronafacic acid PKS genes to engineer other PKSs in order to produce novel polyketide products. The experiments will focus upon the engineering of portions of the PKS that codes for the production of 6-deoxyerythronolide B (DEB), which is the polyketide portion of the antibiotic erythromycin. The PKS engineering experiments will be carried out in collaboration with Professor Chaitan Khosla of Stanford University.The investigation of polyketide synthases is currently an extremely active field of research. The reasons for this are a), that there is presently little understanding of the structural and mechanistic principles by which these enzymes are able to assemble highly reactive intermediates and catalyze the formation of specific products, and b), that the engineering of polyketide synthases has been remarkably successful in the creation of new polyketides. Because of the novel features of the coronafacic acid polyketide synthase, this project should lead to important new insights into polyketide biosynthesis and also provide new tools with which to engineer polyketide synthases. Since many biologically active compounds are polyketides, the engineering of polyketide synthases has great potential for the production of new substances with useful biological activity.

Parry Coronatine是一种由植物病原细菌Pseudomonasseringae产生的新型植物毒素。先前对冠菌素生物合成的研究表明，该毒素的去氢茚酮部分，冠孢酸，是一种新的聚酮化合物，由两个乙酸酯单元、一个丁酸酯单元和一个衍生自β-酮基谷氨酸的四碳或五碳起始单元衍生而来。环丙基氨基酸部分的毒素，冠氨酸，已被证明是生物合成的L-别异亮氨酸通过一个不寻常的氧化环化反应。对Coronaline生物合成的遗传学研究已经确定了ca. 6.9 kb，似乎与冠酰胺酸的形成和一个区域跨越约。21 kb，与coronafacic酸生物合成有关。冠状酸区的测序揭示了存在10个开放阅读框，它们是单个转录物的一部分。转录物以cfl开始，剩余的基因被指定为cfal-cfa 9，从转录物的5'端开始编号。cfl和cfa 5开放阅读框编码的蛋白质类似于将羧酸激活为酰基腺苷酸的酶。其中两个开放阅读框cfa 6和cfa 7非常大，似乎编码与已知的I型聚酮酶（PKS）非常相似的蛋白质。剩余的开放阅读框编码与II型PKS或脂肪酸脱氢酶的组分相似的蛋白质。冠状酸PKS的组织不同于已报道的任何其他PKS。该研究项目有两个主要的长期目标。第一个目标是了解在生物化学水平上冠状酸生物合成所涉及的步骤的性质。由于冠状酸生物合成的复杂性，该项目将集中在前六个基因的功能分析的途径。第二个目标是利用冠醚酸PKS基因的一部分来工程化其他PKS，以产生新的聚酮化合物产品。实验将集中于PKS编码产生6-脱氧红霉素B（DE B）部分的工程改造，DE B是抗生素红霉素的聚酮部分。PKS工程实验将与斯坦福大学的Chaitan Khosla教授合作进行。聚酮酶的研究是目前极为活跃的研究领域。其原因是a）目前对这些酶能够组装高活性中间体并催化形成特定产物的结构和机理原理了解甚少，和B）聚酮化合物脱氢酶的工程改造在产生新的聚酮化合物方面已经非常成功。由于冠状酸聚酮合酶的新功能，该项目应导致重要的聚酮生物合成的新见解，也提供了新的工具，工程聚酮脱氢酶。由于许多生物活性化合物是聚酮化合物，因此聚酮化合物脱氢酶的工程改造对于生产具有有用生物活性的新物质具有巨大潜力。