High-level production of NMN in Escherichia coli through co-utilization of glucose and glycerol

Background: Nicotinamide mononucleotide (NMN), a direct precursor of the essential coenzyme nicotinamide adenine dinucleotide (NAD⁺), confers anti-aging effects and multiple health benefits. Engineered microorganism represents a promising platform for sustainable industrial production of NMN. Results: Here, the previously reported NMN-producing strain NMN008 was engineered to co-utilize glucose and glycerol for the biosynthesis of NMN from nicotinamide (NAM). First, the glycolytic genes pgi and pykA/F were sequentially deleted to disrupt glucose catabolism through the glycolytic pathway, thereby redirecting a greater proportion of glucose flux into the pentose phosphate (PP) pathway. Second, a feedback-resistant glycerol kinase mutant ( glpK* ) was introduced to enhance glycerol utilization, while facilitating glucose uptake by providing phosphoenolpyruvate (PEP) as the driving force. These modifications enabled glycerol to primarily support cell growth, whereas conserving glucose into the PP pathway to increase the availability of phosphoribosyl pyrophosphate (PRPP), an essential co-substrate for NMN biosynthesis. As a result, the final strain achieved an NMN titer of 32.92 g/L in a 2 L bioreactor, representing a 26.28% increase in NMN production and a substantial 34.48% improvement in carbon conversion efficiency. Conclusions: Our research provides an effective strategy to achieve industrial-scale production of NMN, laying a foundation for the widespread application of NMN.