Optimizing Escherichia Coli for Carbonyl reduction

手性醇是多种医药、农用化学品和大宗化工产品的关键中间体。利用酶合成高产率和立体化学纯度的手性醇的优化平台将在药物生产中具有很高的实用价值。工程菌全细胞特别适合于通过不对称酮还原生产手性醇。这些细胞以简单易用的包装提供酶和还原的烟酰胺辅因子(NADH和NADPH)。然而，内源性β-酮酯还原酶在大肠杆菌中的存在使利用大肠杆菌细胞作为异源羰基还原酶过表达宿主的努力复杂化，特别是当异源酶的立体选择性与大肠杆菌活性不匹配的时候。 我们已经分离了主要的大肠杆菌β-酮酯还原酶，并证明它是由yqhE基因编码的。YQHE被击倒的菌株将被创造出来。该工程菌将被用作表达酮基还原酶的宿主，该酶具有10个基团，底物范围很广。在该工程菌宿主中表达的酮还原酶之一将被用作全细胞催化剂，通过生产100g(S)-4-氯-3-羟基丁酸乙酯来展示该菌株的商业潜力。(Ju)-4-氯-3-羟丁酸乙酯是降胆固醇药物立普妥的关键中间体。

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.