High-Level Production of NMN in <i>Escherichia coli</i> Through Co-Utilization of Glucose and Glycerol.

Nicotinamide mononucleotide (NMN), a direct precursor of the essential coenzyme nicotinamide adenine dinucleotide (NAD⁺), confers anti-aging effects and multiple health benefits. Engineered microorganisms represent a promising platform for sustainable industrial production of NMN. 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 <i>pgi</i> and <i>pykA</i>/<i>pykF</i> were sequentially deleted to disrupt glucose catabolism through the glycolytic pathway, thereby potentially improving precursor availability for NMN biosynthesis. Second, a feedback-resistant glycerol kinase mutant (<i>glpK*</i>) was introduced to enhance glycerol utilization, aiming to compensate for the growth defects associated with impaired glycolysis. These modifications enabled glycerol to primarily support cell growth and energy metabolism, while improving glucose allocation toward NMN biosynthesis by reducing its competitive consumption through glycolysis. 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. Our research provides an effective strategy to achieve industrial-scale production of NMN, laying a foundation for the widespread application of NMN.