| In the first part of this dissertation, an effort was made to define the pathways by which the NADPH is regenerated in E. coli cells under non-growing conditions. We used the reduction of ethyl acetoacetate by a Saccharomyces cerevisiae reductase (Gre2) overexpressed in E. coli as a model system and specifically deuterated glucose (C1-d1, C3- d1 or C6-d2) as isotopomer carbohydrate sources. Deuterium atoms originating at C1, C3 or C 6 of glucose are first transferred to NADP+ and then appeared at the C3 atom of ethyl-3-hydroxybutyrate, allowing us to measure the isotopic composition of the intracellular NADPH pool by GC/MS. In addition, we perturbed the NADP(H/+) levels of the host cell by environmental and genetic manipulation means and evaluated the effects of those perturbations on the productivity of the system under study. Our data showed that the NADPH sources present in E. coli cells under growing conditions are also active in non-dividing cells and most likely are not the limited factor for the maximum productivity of this particular biotransformation.;In the second part, a set of 15 genes encoding known and putative alkene reductases were successfully cloned by a rapid recombination technique and overexpressed as GST-fused proteins in E. coli. Their ability to reduce ca. 40 simple enones and enals was evaluated. This study uncovered subtle differences in substrate specificity among the distinct en-reductase subfamilies, as well as the function of a previously uncharacterized in S. cerevisiae gene product. |
