Optimizing Escherichia coli for Carbonyl Reductions

优化大肠杆菌的羰基还原

基本信息

批准号：
7185817
负责人：
JAMES DAVID ROZZELL
金额：
$ 42.68万
依托单位：
BIOCATALYTICS, INC.
依托单位国家：
美国
项目类别：
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

项目摘要

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（由申请人提供）：手性药物构成了新的药物活性物质的稳定增长，其合成需要可以产生具有高立体化学纯度的化合物的方法。手性酒精是手性药物合成中无处不在的中间体，因此，酮的立体选择性还原是在制备手性药物生产产品中的重要性反应。在第1阶段中，开发了一个大肠杆菌宿主菌株，该菌株的基因编码了被删除的天然酮基团酶。该突变菌株被证明是表达异源酮基督教基因基因的绝佳宿主，因为消除了宿主中产生背景酮激酶活性的基因，因此可以在不存在其他酮还原酶的情况下产生新的酮核酸酶，从而产生新的酮酮酶。宿主被证明可以有效地进行全细胞减少，以产生高立体化学纯度的手性醇。在第2阶段，我们计划创建一系列生产菌株，其中纳入了至少12种不同的酮类药物基因和其他生物体作为单个酮核酶活性。在项目过程中发现的新酮基因基因也可以用于产生新的生产菌株。最终结果将是一组宿主大肠杆菌菌株，每个大肠杆菌菌株含有单个酮果酶活性。这组新产生的生产菌株将构成第一组开发的宿主菌株，即仅包含一个用于生物转化的单个酮还原酶活性。辅因子回收酶也将被克隆到宿主菌株中，从而创建一组有机体，可用于经济生产大规模的多种手性醇。