FUNGAL POLYKETIDE SYNTHASES

真菌聚酮化合物合成酶

• 批准号: 6163945
• 负责人: CHARLES J SIH
• 金额: $ 14.32万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6163945
• 关键词: Aspergillus; Moniliales; active sites; bioreactors; brefeldin A; enzyme complex; enzyme mechanism; enzyme structure; fatty acid synthase; fungal genetics; fusion gene; lovastatin; microorganism culture; microorganism metabolism; transfection

As our long-term goal, we will attempt to provide the understanding needed to design new polyketide synthases (PKSs) with novel properties, by characterizing the physical and functional nature of the fungal PKSs involved in the biosynthesis of the biologically active compounds mevinolin (lovastatin) and brefeldin A. We will strive to establish how these two multifunctional proteins, which are likely to contain sets of largely identical or very similar active sites and utilize the same two substrates, acetyl-and malonyl-Coenzyme A, are nonetheless able to catalyze the assembly of two distinct products with different carbon chain lengths, functionalization and cyclization patterns. Although we and others are studying different PKSs from bacteria as well as fungi for similar reasons, none of the mechanistic hypotheses emerging from these other studies are suited to the unique demands placed on the PKSs that are the focus of this proposal. The parallels between mevinolin and brefeldin A biosynthesis are expected to facilitate the understanding of the two PKSs by the construction of hybrid genes to make novel protein catalysts. In due course, we hope that the resulting knowledge will be instrumental in the design of novel protein catalysts that can make biotechnologically valuable compounds, including herbicides, insecticides, antiparasiticides, pharmaceuticals and biodegradable polymers, for many industrial uses. The research will be pursued in the following two laboratories. University of Wisconsin-Madison personnel will: 1) purify and physically characterize the mevinolin PKS, obtained by overexpression of the gene for this enzyme in a microbial host, and determine the structure of the product produced from acetyl-and malonyl-CoA in vivo and in vitro, through a collaboration with John Vederas at the University of Alberta; 2) extend the sequence analysis of the mevinolin gene cluster to include all of the genes for mevinolin biosynthesis; 3) clone and sequence the brefeldin A PKS gene(s) from P. brefeldianum DNA; and 4) initiate studies of the properties of mutuant and hybrid forms of the mevinolin PKS. University of Alberta, Canada, personnel will: 1) study the incorporation of various compounds into mevinolin or its precursor in vivo using a recombinant strain provided by UW-Madison; 2) if possible, extend this work in vitro using a cell-free system made from such a strain or the purified mevinolin PKS provided by UW-Madison; and 3) isolate and characterize any new intermediates of mevinolin biosynthesis uncovered.

作为我们的长期目标，我们将努力提供理解 需要设计具有新性质的新聚酮酶（PKS）， 通过表征真菌PKS的物理和功能性质， 参与生物活性化合物的生物合成 mevinolin（洛伐他汀）和brefeldin A。 我们将努力确定如何 这两种多功能蛋白质，可能含有一组 很大程度上相同或非常相似的活性位点，并利用相同的两个 底物，乙酰辅酶A和丙二酰辅酶A，仍然能够 催化具有不同碳的两种不同产物的组装 链长、官能化和环化模式。 虽然我们 其他人正在研究来自细菌和真菌的不同PKS 出于类似的原因，没有一个机械假说出现在 这些其他研究适合于对PKS提出的独特要求 这是本提案的重点。 mevinolin与 和布雷菲德菌素A的生物合成，预计将促进 通过构建杂交基因来理解这两个PKS， 制造新型蛋白质催化剂。 在适当的时候，我们希望， 知识将有助于设计新的蛋白质催化剂 可以制造具有生物技术价值的化合物， 除草剂、杀虫剂、杀寄生虫剂、药物和 可生物降解的聚合物，用于许多工业用途。 研究将在以下两个实验室进行。 威斯康星大学麦迪逊分校的人员将：1）净化和身体 表征mevinolin PKS，通过基因的过表达获得 这种酶在微生物宿主，并确定结构的 在体内和体外由乙酰-和丙二酰-CoA产生的产物， 通过与阿尔伯塔大学的John Vederas合作; 2)扩展mevinolin基因簇的序列分析， 用于mevinolin生物合成的所有基因; 3）克隆并测序 来自布雷菲德青霉DNA的布雷菲德菌素A PKS基因;和4）启动 mevinolin突变体和杂交体性质的研究 PKS 加拿大阿尔伯塔大学，人员将：1）研究 将各种化合物掺入到美维诺林或其前体中， 体内使用由UW-Madison提供的重组菌株; 2）如果可能， 在体外使用无细胞系统扩展这项工作， 菌株或由UW-Madison提供的纯化的mevinolin PKS;和3） 分离和表征mevinolin生物合成任何新中间体 发现了