Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.
ACS Synth Biol. 2020 Jul 17;9(7):1632-1637. doi: 10.1021/acssynbio.0c00290. Epub 2020 Jul 6.
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 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 monomer commonly used in polymer industry.
酶活性的快速进化通常受到缺乏有效且经济实惠的方法来鉴定有益突变体的阻碍。我们报告了一种新的基于还原辅酶 NADPH 循环的生长偶联选择方法的开发，用于进化 NADPH 消耗酶。该方法依赖于一种遗传修饰的 菌株，该菌株过度积累 NADPH。该方法应用于羧酸还原酶 (CAR) 的工程改造，以提高对 2-甲氧基苯甲酸和己二酸的催化活性。通过单点饱和突变文库鉴定出催化效率提高高达 17 倍的突变酶。获得的突变体成功地应用于己二酸到 1,6-己二醇的全细胞转化，1,6-己二醇是聚合物工业中常用的 C 单体。
