Assembly and engineering of BioBricks to develop an efficient NADH regeneration system

The alcohol dehydrogenase (ADH)-based coenzyme regeneration system serves as a useful tool in redox biocatalysis. This system effectively replenishes NAD(P)H by utilizing isopropanol as a substrate, with the added advantage of easily separable acetone as a by-product. Previous studies focused on discovering new <i>adh</i> genes and engineering the ADH protein for higher catalytic efficiency, neglecting the optimization of other gene components. In this study, a remarkably efficient NADH regeneration system was developed using BioBricks assembly for system initialization. The ADH engineering was used to enhance catalytic efficiency, and RBS optimization for elevated ADH expression, which resulted in not only a 2.1-fold increase in catalytic efficiency but also a 3.2-fold increase in translation rate. Together, these improvements resulted in an overall 6.7-fold enhancement in performance. This system finds application in a wide range of NADH-dependent biocatalysis processes and is particularly advantageous for the biosynthesis of fine chemicals.