Comprehensive Transformation of Escherichia coli for Nicotinamide Mononucleotide Production

Abstract

Nicotinamide mononucleotide (NMN) is a key precursor of nicotinamide adenine dinucleotide and an important source of cellular energy. It can prevent neuronal mitochondrial defects and alleviate heart fibrosis. Strategies to improve NMN production have important implications for human health. Through plasmid expression technology and CRISPR/Cas9 technology, we engineered Escherichia coli for efficient NMN production. First, we upregulated the expression of genes encoding key enzymes in the NMN synthesis pathway, enabling E. coli to directly produce NMN, and established the important role of the nicotinamide mononucleotide transporter in the transport of NMN from cells. The content of NMN was 0.24 g·L−1 at 24 h. Second, we strengthened the adenosine triphosphate (ATP) cycle, and the concentration of NMN was 0.49 g·L−1 at 24 h. Third, we increased the synthesis of the NMN precursor 5-phosphate ribose-1-phosphate and obtained an NMN content of 0.49 g·L−1 at 12 h and 1.11 g·L−1 at 24 h. Fourth, we introduced nicotinamide riboside kinase (NRK) and found that it was effective only for a period of time. The content of NMN was 0.54 g·L−1 at 12 h but only 1.05 g·L−1 at 24 h. Finally, we combined these strategies to regulate the whole metabolic flow, revealing that integrating multiple pathways promoted NMN production. During fermentation, we added 1 g·L−1 nicotinamide and 10 g·L−1 glucose, yielding an extracellular NMN concentration of 1.11 g·L−1.