Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy
Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy
复制标题
DOI：
10.1021/acssynbio.0c00290
复制
发表时间：
2020-07-17
影响因子：
4.7
通讯作者：
Niu, Wei
中科院分区：
文献类型：
--
作者：
Kramer, Levi;Le, Xuan;Niu, Wei
由于缺乏有效且负担得起的方法来识别有益突变体，酶活性的快速进化往往受到阻碍。我们报告了一种新的生长耦合选择方法的开发，用于基于这种氧化还原辅因子的回收来进化 NADPH 消耗酶。该方法依赖于转基因大肠杆菌菌株，该菌株会过度积累 NADPH。该方法应用于羧酸还原酶（CAR）的工程设计，以提高对 2-甲氧基苯甲酸和己二酸的催化活性。从单位点饱和诱变文库中鉴定出催化效率提高高达 17 倍的突变酶。获得的突变体成功应用于己二酸全细胞转化为1,6-己二醇（聚合物工业中常用的C-6单体）。
Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. We report the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified Escherichia coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on 2-methoxybenzoate and adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries. Obtained mutants were successfully applied to whole-cell conversions of adipate into 1,6-hexanediol, a C-6 monomer commonly used in polymer industry.