Improving the production of NAD via multi-strategy metabolic engineering in Escherichia coli.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales. Here, we describe multi-strategy metabolic engineering to address the NAD-production bottleneck in E. coli. First, blocking the degradation pathway of NAD(H) increased the accumulation of NAD by 39%. Second, key enzymes involved in the Preiss-Handler pathway of NAD synthesis were overexpressed and led to a 221% increase in the NAD concentration. Third, the PRPP synthesis module and Preiss-Handler pathway were combined to strengthen the precursors supply, which resulted in enhancement of NAD content by 520%. Fourth, increasing the ATP content led to an increase in the concentration of NAD by 170%. Finally, with the combination of all above strategies, a strain with a high yield of NAD was constructed, with the intracellular NAD concentration reaching 26.9 μmol/g DCW, which was 834% that of the parent strain. This study presents an efficient design of an NAD-producing strain through global regulation metabolic engineering.