Optimizing Escherichia Coli for Carbonyl reduction

优化大肠杆菌的羰基还原

• 批准号: 6788947
• 负责人: JAMES DAVID ROZZELL
• 金额: $ 10万
• 依托单位: BIOCATALYTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2004-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6788947
• 关键词: Escherichia coli; aldehyde /ketone oxidoreductase; bacterial genetics; bacterial proteins; biotechnology; catalyst; chemical synthesis; enzyme biosynthesis; esters; genetic manipulation; nicotinamide; reduction; technology /technique development

Chiral alcohols are key intermediates in a wide variety of pharmaceutical, agrochemical and bulk chemical products. An optimized platform for using enzymes to synthesize chiral alcohols in high yield and stereochemical purity would have high utility for the production of pharmaceuticals. Whole cells of engineered Escherichia coli are particularly convenient for producing chiral alcohols by asymmetric ketone reduction. The cells provide both enzyme and reduced nicotinamide cofactors (NADH and NADPH) in a simple-to-use package. However, the presence of the endogenous beta-keto ester reductase in E. coli complicates efforts to use E. coli cells as overexpression hosts for heterologous carbonyl reductases, especially when the stereoselectivity of the heterologous enzymes doesn't match that of the E. coli activity. We have isolated the major E. coli beta-keto ester reductase and shown that it is encoded by the yqhE gene. A strain in which the yqhE gone is knocked out will be created. The engineered E. coli will be used as host to express ketoreductases, which BioCatalytics has a group of 10 with broad substrate range. One of the ketoreductase expressed in this engineered E. coli host will be used as whole cell catalyst to demonstrate the commercial potential of this strain by producing a 100 g of (S)-ethyl 4-chloro-3-hydroxybutyrate, a key intermediate for anti-cholesterol drug Lipitor.

手性醇是医药、农药和大宗化学品的重要中间体。使用酶以高产率和立体化学纯度合成手性醇的优化平台对于药物生产具有高实用性。工程化大肠杆菌的全细胞特别便于通过不对称酮还原来生产手性醇。该细胞在一个简单易用的包装中提供酶和还原型烟酰胺辅因子（NADH和NADPH）。然而，在E.大肠杆菌使使用大肠杆菌的努力复杂化。大肠杆菌细胞作为异源羰基还原酶的过表达宿主，特别是当异源酶的立体选择性与大肠杆菌的立体选择性不匹配时。coli活性。 我们已经分离出了E大调。大肠杆菌β-酮酯还原酶，并表明它是由yqhE基因编码。一个菌株，其中yqhE消失敲除将被创建。工程菌E.大肠杆菌作为宿主表达酮还原酶，BioCatalytics有一组10种底物，底物范围广。在该工程菌中表达的一种酮还原酶。大肠杆菌宿主将被用作全细胞催化剂，以证明该菌株的商业潜力，通过生产100克的（S）-乙基4-氯-3-羟基丁酸酯，抗胆固醇药物立普妥的关键中间体。