Malic enzyme-based system for transhydrogenation between nicotinamide cofactors

The nicotinamide cofactors including nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) play important roles in facilitating redox reactions for energy metabolism and biosynthesis. To expand the cofactor menu, a non-natural cofactor nicotinamide cytosine dinucleotide (NCD) has been introduced recently. The reduced forms of these cofactors carry reducing equivalents that are essential for cellular metabolism. However, there is a long standing challenge to rationally transfer reducing equivalent from one cofactor to another, albeit such process is highly demanding in metabolic engineering. This study develops a new approach based on malic enzyme (ME)-mediated transhydrogenation to enable reducing equivalents exchange among different cofactors. We used wild-type ME, MaeB and an engineered ME* that favors NAD, NADP and NCD, respectively, to demonstrate such conversions. When an in vitro system initiated with equal amount of NADH and NCD in the presence of ME, ME* and excess amount of pyruvate was held for 2 h, up to 65% NADH was consumed and 57% NCDH was generated. When implemented into NCD self-sufficient Escherichia coli cells, the system directed reducing equivalents toward NCDH-linked formation of lactate. Overall, this work offers an effective strategy to regulate intracellular reducing equivalents that may serve as a novel tool for metabolic engineering and synthetic biology.