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. ? ?
