Cloning, overexpression and characterization of novel alkene reductases

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DESCRIPTION (provided by applicant): Reduction of carbon-carbon double bonds (C=C) is a fundamental reaction in synthetic organic chemistry. The ability to control the regio-, and stereo-, and chemo selectivity of reduction is of particular importance when designing new hydrogenation catalysts. For example, the chemo-selective reduction of C=C bonds that are conjugated to carbonyl groups has been a particular problem, as over-reduction leading to partial reduction of the carbonyl group occurs in most cases. The only promising methods reported this far use microwave irradiation to carry out the reaction, and this method, while usable at the laboratory scale, is not readily scalable for industrial application. A small number of enzyme activities that are capable of performing chemo-selective C=C reductions have been reported; however, limited characterization of these enzymes has been reported to date. Based on our experience in the cloning, over-expressing, and characterizing new enzymes for chemical synthesis applications, we have brought to the market more than 150 different enzymes formulated as active lyophilized powders, including transaminases, ketoreductases, nitrilases, amino acid dehydrogenases, aryl alcohol oxidases, human P450 oxidases, and many others. Processes based on these enzymes have proven to be practical and scalable. In 2005 alone, our enzymes were used for the commercial production of six different drugs in clinical trials. In this Phase 1 SBIR proposal we plan to investigate 12 ene reductases. The gene encoding each enzyme will be cloned and conditions for high expression in a bacterial host will be investigated. A thorough examination of the substrate range, chemo-selectivity (particularly a,¿-unsaturated carbonyl compounds), and stereoselectivity of each enzyme will be carried out. These results will establish the foundation for dramatically expanding the number of cloned ene reductase enzymes by genome mining and directed evolution in Phase II continuation of this project, along with a demonstration of the practical use of ene reductases in the synthesis of otherwise difficult to synthesize pharmaceutical intermediates. This research involves the development of new enzymatic catalysts for the manufacturing of drugs and drug intermediates. These new catalysts offer many advantages compared to the existing chemical ones. They are very selective in the reactions they perform, are recyclable and biodegradable, and operate under green environmentally friendly conditions.