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

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.