Investigations of Coronafacic Acid Biosynthesis

Parry Coronatine是由植物病原菌紫丁香假单胞菌产生的一种新的植物毒素。以往对冠醚生物合成的研究表明，该毒素的氢化部分冠醚是一种新的聚酮，由两个乙酸酯单元、一个丁酸酯单元和一个由酮戊二酸衍生的四碳或五碳起始单元组成。该毒素的环丙基氨基酸部分，冠花酸，已被证明是由L-异亮氨酸通过一个不寻常的氧化环化反应生物合成的。冠状病毒生物合成的遗传学研究已经确定了一个约6.9kb的DNA区域和一个约21kb的区域，该区域似乎与冠花酸的形成有关。冠状表面酸区的测序揭示了作为单个转录本的一部分的10个开放阅读框的存在。转录本以CFL开头，其余的基因被指定为CFAL-Cfa9，从转录本的5‘端开始编号。Cfl和cfa5开放阅读框编码的蛋白质类似于激活羧酸的酶，如酰基腺苷。其中两个开放阅读框，cfa6和cfa7，异常大，似乎编码的蛋白质与已知的I型聚酮合成酶(PKS)非常相似。其余的开放阅读框编码的蛋白质类似于II型PKSS或脂肪酸合成酶的成分。冠麻酸PKS的组织结构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。第一个目标是在生化水平上了解冠麻酸生物合成所涉及的步骤的性质。由于冠麻酸生物合成的复杂性，该项目将重点分析该途径中前六个基因的功能。第二个目标是利用冠状表面酸PKS基因的一部分来改造其他PKS，以生产新的聚酮产品。实验将集中在对PKS的部分编码的工程上，该PKS编码生产6-脱氧赤藓内酯B(DEB)，这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。聚酮合成酶的研究目前是一个非常活跃的研究领域。其原因是，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制原理知之甚少，以及b)聚酮合成酶工程在创造新的聚酮方面取得了显著的成功。由于冠状表面酸性聚酮合成酶的新特性，该项目将为聚酮生物合成带来重要的新见解，并为设计聚酮合成酶提供新的工具。由于许多具有生物活性的化合物都是聚酮类化合物，因此聚酮合成酶工程在生产具有有用生物活性的新物质方面具有巨大的潜力。

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.