Investigations of Coronafacic Acid Biosynthesis

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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.