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.

手性醇是多种医药、农化和大宗化工产品的关键中间体。利用酶合成高收率、高立体化学纯度的手性醇的优化平台对药物生产具有重要的实用价值。工程大肠杆菌的全细胞特别便于通过不对称酮还原产生手性醇。细胞在一个简单易用的包装中提供酶和还原性烟酰胺辅助因子（NADH和NADPH）。然而，大肠杆菌中内源性β -酮酯还原酶的存在使利用大肠杆菌细胞作为外源羰基还原酶过表达宿主的努力变得复杂，特别是当外源酶的立体选择性与大肠杆菌活性不匹配时。