Synergistic engineering of a GRAS-certified strain: Transforming Bacillus subtilis 168 into a high-performance...

Nicotine poses a significant threat to human health owing to its complex environmental exposure. Microbial detoxification presents a promising strategy for environmental remediation and in vivo intervention, but its application is limited by the scarcity of safe platforms, a challenge rooted in the trade-off between heterologous enzyme activity and host fitness. Here, we engineered the GRAS-certified Bacillus subtilis 168 into a nicotine-scavenging probiotic strain by introducing two FAD-dependent enzymes, nicotine oxidoreductase variant (NicA2v) and pseudooxynicotine amine oxidase (Pnao), which sequentially convert nicotine into 3-succinoylsemialdehyde-pyridine (SAP) by cleaving the toxic methylamine group. To overcome this limitation, we adopted a differentiated regulatory strategy: for NicA2v, balanced and sustained expression was achieved using a moderately strong promoter (P₄₃); for Pnao, conventional temperature and transcriptional regulation proved ineffective due to its narrow functional window. We therefore developed a novel upstream open reading frame (uORF)-based translational attenuator that modulates protein output without altering transcription. This enabled functional expression of a high-activity Pnao homolog (Pppnao-S16) from Pseudomonas putida S16, which was further optimized via protein engineering to generate the enhanced variant Pppnao-S16-R54Q. The final strain, engineered via CRISPR-Cas9-mediated genome integration of dual P₄₃-NicA2v copies and one uORF-mediated Pppnao-S16-R54Q cassette, efficiently degraded 20 μM nicotine to SAP within 24 h. Furthermore, this strain demonstrated strong tolerance to 2.5 mM nicotine, underscoring its potential to reduce health risks associated with nicotine exposure. Hence, this work delivers a safe, high-performance probiotic chassis for nicotine detoxification and provides a novel perspective for constructing microbial platforms against other hazardous xenobiotics.