Optimizing Escherichia coli for Carbonyl Reductions

优化大肠杆菌的羰基还原

• 批准号: 7050673
• 负责人: JAMES DAVID ROZZELL
• 金额: $ 41.88万
• 依托单位: BIOCATALYTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7050673
• 关键词: Escherichia coli; Saccharomyces; carbonyl compound; combinatorial chemistry; drug design /synthesis /production; drug discovery /isolation; enzyme activity; genetic strain; ketones; reduction; stereoisomer

DESCRIPTION (provided by applicant): Chiral drugs comprise a steadily increasing percentage of new pharmaceutically-active substances, and their synthesis demands methods that can produce compounds with high stereochemical purity. Chiral alcohols are ubiquitous intermediates in the synthesis of chiral drugs, and the stereoselective reduction of ketones is therefore a reaction of central importance in the preparation of chiral pharmaeutical products. In Phase 1 an Escherichia coli host strain was developed that had genes encoding native ketoreductase enzymes deleted. This mutant strain was demonstrated to be an excellent host for expressing heterologous ketoreductase genes because the genes producing background ketoereductase activities in the host were eliminated, thereby allowing new ketoreductase enzymes to be produced without the presence of other ketone-reducing enzymes that could lead to side reactions or reduced stereoselectivity. The host was shown to be effective in carrying out whole cell reductions to produce chiral alcohols with high stereochemical purity. In Phase 2 we plan create an array of production strains incorporating at least 12 different ketoreductase genes from Saccharomyces and other organisms as single ketoreductase activities. New ketoreductase genes discovered during the course of the project may also be used to produce new production strains. The end result will be a set of host Escherichia coli strains, each containing a single ketoreductase activity. This set of newly created production strains will constitute the first set of host strains developed to contain only a single ketone-reducing enzyme activity for biotransformation. A cofactor recycling enzyme will also be cloned into the host strains, creating a set of organisms useful for the economical production of a broad range of chiral alcohols at large scale.

描述（由申报人提供）：手性药物包括新的药物活性物质的比例稳步增加，它们的合成需要能够产生具有高立体化学纯度的化合物的方法。手性醇是手性药物合成中普遍存在的中间体，因此，酮的立体选择性还原是制备手性药物产品的一个重要反应。在第一阶段，培养了一株缺失了天然酮还原酶编码基因的大肠杆菌宿主菌株。该突变菌株被证明是表达外源酮还原酶基因的良好宿主，因为在宿主中产生酮还原酶活性的背景基因被消除，从而允许新的酮还原酶产生，而不存在可能导致副反应或降低立体选择性的其他酮还原酶。宿主被证明在进行全细胞还原以产生具有高立体化学纯度的手性醇方面是有效的。在第二阶段，我们计划创建一系列生产菌株，其中包含来自酵母菌和其他生物的至少12种不同的酮还原酶基因，作为单个酮还原酶活性。在项目过程中发现的新的酮还原酶基因也可用于生产新的生产菌株。最终的结果将是一组宿主大肠杆菌菌株，每个菌株都含有单一的酮还原酶活性。这组新创建的生产菌株将构成第一组宿主菌株开发仅含有单一酮还原酶活性用于生物转化。一种辅助因子回收酶也将被克隆到宿主菌株中，从而创造出一组可用于大规模经济生产各种手性醇的生物。