Biosensor-driven adaptive laboratory evolution and modular pathway engineering for high-level production of nicotinamide mononucleotide in yeast.

Nicotinamide mononucleotide (NMN) has notable physiological effects, but its microbial production is constrained by the limited intracellular nicotinamide (NAM) pool. Here, the transcription factor NadR was semi-rationally reprogrammed to preferentially recognize NAM over nicotinic acid (NA), generating the NAM-specific biosensor NadR-M6. A growth-coupled selection platform was established by integrating NadR-M6 with a URA3 reporter and systematically optimizing genetic components, enabling robust growth-based discrimination across an extracellular NAM range of 0.05-60 mM. When combined with adaptive laboratory evolution (ALE), this platform facilitated a 7.6-fold increase in intracellular NAM content, reaching 6.3 mg/g DCW. This result indicates that biosensor-guided selection effectively enriched cells with enhanced nicotinamide accumulation. Subsequently, modular pathway engineering was applied to convert the expanded NAM pool into NMN while attenuating downstream NAD+ consumption to balance product accumulation with cell viability. The final strain, N35, reached an intracellular NMN titer of 2.6 g/L with a content of 118.5 mg/g DCW in a 5-L bioreactor. This study establishes a biosensor-driven evolutionary high-throughput screening platform for enhancing the intracellular precursor pools and enabling the biosynthesis of high-value nicotinamide-derived metabolites in yeast.