Abstract
The cofactor regeneration system plays a crucial role in redox biocatalysis for organic synthesis and the pharmaceutical industry. The alcohol dehydrogenase (ADH)-based regeneration system offers a promising solution for the in situ regeneration of NAD(P)H. However, its widespread use is hindered by low activity and poor expression of ADH in Escherichia coli. Herein, the BioBricks (promoter, ribosome binding site [RBS], functional gene, and terminator) were assembled and engineered to constitute an efficient NADH regeneration system. The semi-rational design was employed to enhance the catalytic efficiency of GstADH (an ADH from Geobacillus stearothermophilus), resulting in a beneficial GstADH variant with a 2.1-fold increase in catalytic efficiency. Furthermore, the RBS optimization was used to increase the expression of ADH genes, leading to the identification of an RBS with a 3.2-fold increased translation rate. Using this developed system, the NADH generating velocity reached more than 2 s(-1) even toward 0.1 mM NAD(+), indicating that it is the most promising NADH regeneration so far. Finally, the engineered system was utilized for the asymmetric biosynthesis of l-phosphinothricin (a chiral herbicide), with a high yield (>95%). IMPORTANCE: 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 adh 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.