Cloning, overexpression and characterization of novel alkene reductases

描述（申请人提供）：碳碳双键还原（C=C）是合成有机化学中的基本反应。在设计新的加氢催化剂时，控制区域、立体和化学选择性的能力是特别重要的。例如，共轭羰基的C=C键的化学选择性还原一直是一个特别的问题，因为在大多数情况下，过度还原会导致羰基的部分还原。迄今为止报道的唯一有前途的方法是使用微波辐射来进行反应，这种方法虽然在实验室规模上可用，但不容易扩展到工业应用。已经报道了少数能够进行化学选择性C=C还原的酶活性；然而，迄今为止对这些酶的描述有限。基于我们在克隆、过表达和表征化学合成应用新酶方面的经验，我们已经向市场推出了150多种不同的活性冻干粉末酶，包括转氨酶、酮还原酶、腈酶、氨基酸脱氢酶、芳基醇氧化酶、人类P450氧化酶等。基于这些酶的工艺已被证明是实用和可扩展的。仅在2005年，我们的酶就被用于临床试验中六种不同药物的商业生产。在这个1期SBIR提案中，我们计划研究12个烯还原酶。编码每种酶的基因将被克隆，并在细菌宿主中进行高表达的条件将被研究。对底物范围、化学选择性（特别是A -不饱和羰基化合物）和每种酶的立体选择性进行彻底的检查。这些结果将为本项目第二阶段继续进行的基因组挖掘和定向进化中显著增加克隆的烯还原酶的数量奠定基础，同时也将证明烯还原酶在合成其他难以合成的药物中间体中的实际应用。这项研究涉及开发用于制造药物和药物中间体的新酶催化剂。与现有的化学催化剂相比，这些新催化剂具有许多优点。它们在进行的反应中具有很强的选择性，可回收和可生物降解，并且在绿色环保条件下运行。

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.