Optimizing Escherichia coli for Carbonyl Reductions

描述(申请人提供)：手性药物包括稳步增加的新的药用活性物质的百分比，它们的合成需要能够产生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手性药物制备中的一个重要反应。在第一阶段中，开发了一种含有编码自然酮还原酶基因的大肠杆菌宿主菌株。该突变菌株被证明是表达异源酮还原酶基因的良好宿主，因为在宿主中产生本底酮还原酶活性的基因被消除，从而允许在不存在其他可能导致副反应或立体选择性降低的酮还原酶的情况下产生新的酮还原酶。结果表明，该载体能有效地进行全细胞还原反应，生成具有较高立体化学纯度的手性醇。在第二阶段，我们计划创建一系列生产菌株，将来自酵母菌和其他生物的至少12个不同的酮还原酶基因作为单一酮还原酶活性。在项目过程中发现的新的酮还原酶基因也可能被用来生产新的生产菌株。最终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。这组新产生的生产菌株将构成第一组宿主菌株，开发成只包含用于生物转化的单一酮类还原酶活性。一种辅因子循环酶也将被克隆到宿主菌株中，创造出一套有助于经济地大规模生产广泛的手性醇的微生物。

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.