While CtXR (Candida tenuis Xylose Reductase) exhibits no C-C double bond reductase activity, other AKRs (Aldo-Keto Reductase) and short chain dehydrogenases/reductases (SDRs) accept enoates as substrates. In this project, we will engineer C-C double bond activity into CtXR by mimicking the active site of AKR 1D in which active-site histidine is replaced by a glutamate and the particular activity is present. Likewise, an SDR-type catalytic triad will be realized in a Tyr51Phe mutant. AKRs, and reductases in general, can be used in either of two ways to catalyze the reduction of ketones: As whole cell systems, or as purified enzymes. In both cases, the coenzyme (NADH or NADPH) is present in substoichiometric amounts and has to be recycled during the course of the reaction. Baker's yeast mediated reductions offer the advantage that the supply of NADH or NADPH is accomplished by the metabolism of a co-substrate. Unfortunately, bioreductions often go along with poor stereoselectivities, which can be traced to the presence of multiple NADPH-dependent reductases with divergent enantio- and diastereomeric preferences in the cell. We introduce an integrated approach of process development to overcome these limitations, which combines (i) structure based enzyme engineering, (ii) cell engineering and (iii) process engineering.
|Tatsächlicher Beginn/ -es Ende||1/01/08 → 1/01/11|
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