Unraveling and overcoming ammonium toxicity in haloalkaliphilic methanotrophs for economic biomanufacturing and methane mitigation

Methane, a potent greenhouse gas, contributes significantly to global warming. Aerobic methanotrophs represent promising microbial platforms for methane mitigation and sustainable methane-based biomanufacturing. However, their reliance on costly nitrate and sensitivity to ammonium toxicity limits industrial scalability, while ammonia-driven N₂O emissions exacerbate their environmental footprint. Here, we resolve these dual challenges by uncovering the genetic basis of ammonium toxicity in methanotrophs and obtaining an engineered strain with enhanced ammonium detoxification and reduced N₂O emissions. Using an industrially promising methanotroph Methylotuvimicrobium buryatense 5GB1C as a model, we identified an out membrane porin PorA that was involved in ammonium uptake. Inactivation of porA remarkably alleviates ammonium toxicity and suppressed N₂O generation. Furthermore, we dissected the ammonium detoxification pathway, linking cytochrome P460 to N₂O via hydroxylamine conversion. Through combinatorial mutagenesis, we engineered a strain with a 2-fold improvement in growth under ammonium and 83 % lower N₂O emissions. Fed-batch fermentation achieved a dry cell weight up to 8.57 g/L using ammonium and the·biomass yield on methane was 0.69 g/g, matching nitrate-based performance. Additionally, the function of PorA was demonstrated in another methanotroph Methylotuvimicrobium alcaliphilum 20Z. This work provides new insights about ammonium toxicity in haloalkaliphilic methanotrophs and offers a sustainable chassis for methane conversion, thereby advancing both circular biomanufacturing and climate change mitigation.