[Metabolic engineering of Escherichia coli for efficient production of nicotinamide riboside]

Nicotinamide riboside (NR), an essential precursor for nicotinamide adenine dinucleotide (NAD⁺), has garnered increasing attention for its anti-aging properties and diverse pharmacological activities. Developing an efficient, sustainable, and environmentally friendly synthesis approach is of great significance for the industrial production of NR. In this study, we developed a modular metabolic engineering strategy to construct an engineered strain capable of synthesizing NR from nicotinamide efficiently. First, six genes, including rihA, were deleted to block the degradation pathways of NR and its key intermediates. Second, the key enzyme BaPrs^L135I and the NR transporter MdtL were introduced, significantly enhancing NR accumulation and extracellular export. Subsequently, pncA was deleted to improve the nicotinamide utilization efficiency. Furthermore, precursor supply was substantially increased by relieving purR-mediated feedback regulation and strengthening the pentose phosphate pathway. Through the coordinated optimization of these modules and introduction of the toxin-antitoxin system, the engineered strain NK22 produced 3.84 g/L NR after 36 h of shake-flask fermentation. Moreover, the strain achieved a final NR titer of 27.23 g/L in a 5 L fed-batch bioreactor under antibiotic-free conditions. This study establishes a next-generation NR-producing strain capable of antibiotic-free fermentation and provides a robust framework for the development of efficient and sustainable microbial cell factories for NR and its high-value derivatives.