Biochemistry and Regulation of Daunomycin Biosynthesis

点击翻译按钮获取中文摘要

9405730 Strohl Research is aimed at analyzing the structure and function of daunomycin biosynthesis polyketide synthase related genes and the enzymes of the daunomycin biosynthesis polyketide synthase complex. Daunomyucin is synthesized by several different species of Streptomyces. In studies, we found that the structure of the daunomycin polyketide synthase (DPS) gene cluster of Streptomyces sp. C5 contains differences from previously sequenced Type II polyketide gene clusters. Unique features of this st. C5 DPS gene cluster include the presence of a third open reading frame directly downstream of the polyketide synthase gene, an additional gene encoding a putative acytransferase, and the absence of the acyl carrier protein gene downstream from the other polyketide synthase genes. Instead, in the C5 DPS gene cluster, there is an acyl carrier protein gene "upstream" from the polyketide synthase genes. In this work, we will determine the DNA sequence between the acyl carrier protein and daunomycin polyketide synthase genes and analyze its function by complementation of mutants blocked in the early stages of daunomycin biosynthesis. We will examine the structure of DPS using antibodies against the putative enzyme components to determine if DPS contains a third subunit suggested by the sequence data. We also will probe protein-protein interactions between polyketide synthases and other daunomycin biosynthesis proteins using the two-hybrid genetic system as well as by ordinary methods of co-elution by gel filtration chromatography, co-immunoprecipitation, and chemical crosslinking. Finally, we will work to isolate and characterize the putative acyltransferase encoded by the gene associated with the DPS gene cluster. Our studies will provide new insights into structure and functional differences between DPS and Type II polyketide antibiotic synthases. Data from these studies will aid our understanding about the enzymology of secondary metabolite biosynthesi s, provide rationale for tailoring pathways for formation of novel hybrid secondary metabolites, and contribute to understanding the physiological and molecular regulation of antibiotic biosynthesis. %%% Daunomycin is used as a chemotherapeutic agent. We have been studying the biochemistry of its formation in Streptomyces, a type of bacterium, for the purpose of getting the microbe to make more of the compound or to make more potent analogues of it. Recently we isolated a fragment of DNA that contains more than thirty genes involved in the formation of the compound. Our goals are to analyze this DNA fragment further, to examine the structure- function of enzymes encoded by the DNA for daunomycin production, and test the ability of these enzymes to form clusters involving protein-protein interactions. Our studies will help predict how new molecules can be made through cell engineering. ***