| Summary: | <i>β</i>-Nicotinamide mononucleotide (NMN), as a key precursor of an essential coenzyme nicotinamide adenine dinucleotide (NAD<sup>+</sup>), is most recognized for its pathological treatment effects and anti-aging functions. Here, the biosynthesis of NMN from the inexpensive feedstock substrate nicotinamide (Nam) using previously isolated <i>Saccharomyces boulardii</i>-YS01 was investigated. Ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UPLC-ESI-QqQ-MS/MS) was established for the determination and targeted analysis of NMN, nicotinamide riboside (NR), nicotinic acid (NA), Nam, and NAD<sup>+</sup> in YS01 cells. Satisfactory precision and accuracy values were achieved with recoveries above 70% for five analytes. A 5~100 times higher content of NMN in YS01 (0.24~103.40 mg/kg) than in some common foods (0.0~18.8 mg/kg) was found. Combined with genome sequencing and enzyme function annotation, target-acting enzymes, including nudC, ISN1, URH1, PNP, and SIR2, were identified, and the biosynthetic pathway of NMN via Nam was suggested. The initial addition of 3 g/L Nam in the culture medium effectively promoted the generation of NMN, which raised the content of NMN by 39%. This work supplements an alternative resource for NMN production and lays the theoretical foundation for the further construction of NMN transgenic synthesis hosts.
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