Biochemistry and Regulation of Daunomycin Biosynthesis

道诺霉素生物合成的生物化学和调控

• 批准号: 9405730
• 负责人: John Reeve
• 金额: --
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405730&HistoricalAwards=false
• 关键词: Biochemistry; Regulation; Daunomycin; Biosynthesis

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. ***

9405730 Strohl研究旨在分析柔红霉素生物合成聚酮合成酶相关基因的结构和功能，以及柔红霉素生物合成聚酮合成酶复合体的酶。柔红菌素是由几种不同的链霉菌合成的。在研究中，我们发现链霉菌柔红霉素聚酮合成酶(DPS)基因簇的结构。C5含有与之前测序的II型聚酮基因簇不同的基因。这是圣彼得堡的独特之处。C5DPS基因簇包括聚酮合成酶基因下游的第三个开放阅读框，编码一个可能的酰基转移酶的额外基因，以及其他聚酮合成酶基因下游不存在的酰基载体蛋白基因。相反，在C5DPS基因簇中，存在聚酮合成酶基因的上游酰基载体蛋白基因。在这项工作中，我们将测定酰基载体蛋白和柔红霉素聚酮合成酶基因之间的DNA序列，并通过对柔红霉素生物合成早期受阻的突变体进行互补来分析其功能。我们将使用针对假定的酶成分的抗体来检查DPS的结构，以确定DPS是否包含序列数据所建议的第三亚单位。我们还将使用双杂交遗传系统以及通过凝胶过滤层析、免疫共沉淀和化学交联法的普通共洗脱方法来探索聚酮合成酶和其他柔红霉素生物合成蛋白之间的蛋白质-蛋白质相互作用。最后，我们将致力于分离和鉴定与DPS基因簇相关的基因编码的推测的酰基转移酶。我们的研究将为DPS和II型聚酮抗生素合成酶的结构和功能差异提供新的见解。这些研究数据将有助于我们理解S次生代谢物生物合成的酶学，为定制新的杂交次生代谢物的形成途径提供理论基础，并有助于理解抗生素生物合成的生理和分子调控。使用%柔红霉素作为化疗药物。我们一直在研究它在链霉菌中形成的生物化学，这是一种细菌，目的是让微生物制造更多的化合物或制造更有效的类似物。最近，我们分离了一段DNA，它包含了30多个与化合物形成有关的基因。我们的目标是进一步分析这一DNA片段，检测DNA编码的柔红霉素产生酶的结构和功能，并测试这些酶形成涉及蛋白质-蛋白质相互作用的簇的能力。我们的研究将有助于预测如何通过细胞工程制造新分子。***