Advancing bio-recycling of nylon monomers through CRISPR-assisted engineering

Plastic waste is a global environmental crisis, and nylon—a widely used polyamide—contributing significantly due to its extensive applications in textiles, automotive components, and packaging. Post-lifecycle degradation of nylon releases monomers like 1,6-hexamethylenediamine (HD) and 6-aminocaproic acid (ACA), which persist in ecosystems, posing toxicity and bioaccumulation risks. In this study, we employed a CRISPR-assisted directed evolution (CDE) to engineer Pseudomonas putida KT2440 for efficient utilization of HD as the sole nitrogen source, coupling its degradation to bacterial growth. Genomic and transcriptomic analyses prioritized potential enzymes involved in HD degradation. Using CRISPR interference (CRISPRi) and expert-guided screening, we identified three key enzymes including KgtP transporter, AlaC transaminase, and FrmA dehydrogenase that are critical to the KAF pathway. The functionality of these enzymes was confirmed in P. putida and further validated through heterologous expression in Escherichia coli . The CDE and growth-coupled strategy, together with the KAF pathway we discovered, is essential for our future efforts to engineer synthetic bacterial consortia capable of degrading mixed plastic monomers. In the long term, we envision integrating these consortia with synthetic biology tools to degrade complex plastic polymers and convert them into valuable chemicals, advancing circular economic efforts for sustainable plastic waste management and environmental protection. CRISPR systems engineered Pseudomonas putida for efficient nylon monomer degradation, unveiling a novel pathway and advancing plastic waste recycling and environmental mitigation strategies. • CRISPR-evolution enables efficient bio-recycling of nylon monomers into adipic acid. • Engineered strain shows enhanced growth on nylon monomers as nitrogen sources. • Genomic and transcriptomic analyses revealed key genes for adipic acid production. • CRISPR interference validated enzymes critical for nylon monomer bio-conversion. • The bio-recycling pathway offers a sustainable solution for managing nylon waste.