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.

手性醇是多种药物，农业化学和散装化学产品的关键中间体。一个优化的平台，用于使用酶以高产量和立体化学纯度合成手性醇的生产能力很高。通过不对称酮的减少，工程大肠杆菌的整个细胞对于产生手性醇特别方便。这些细胞在简单的包装中既提供酶和还原烟酰胺辅因子（NADH和NADPH）。然而，大肠杆菌中内源性β-酮酯还原酶的存在使使用大肠杆菌细胞作为异源羰基还原酶的过表达宿主的努力复杂化，尤其是当异源酶的立体选择性与大肠杆菌活性的立体选择性不匹配时。 我们已经分离了大肠杆菌β-酮酯还原酶，并表明它是由YQHE基因编码的。将产生YQHE消失的压力。工程大肠杆菌将用作表达酮糖酶的宿主，生物催化酶的基板范围为10组。在该工程大肠杆菌宿主中表达的酮中还原酶之一将用作全细胞催化剂，通过产生100 g的（S） - 乙基4-氯-3-羟基丁酸酯，这是抗胆固醇药物lipitor的关键中间体，以证明该菌株的商业潜力。