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. • A multi-strategy engineering method was developed for NAD + overproduction in E. coli . • Synergy of metabolic pathways to achieve global regulation of NAD + production. • Enhance the supply of co-substrate PRPP in NAD + synthesis network. • Regulate the ATP level to provide the driving force for NAD + overproducing.