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

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%).