Microbial cleavage and mineralization of acesulfame by Shinella sp. strain KJ01

Acesulfame (ACE), a widely used artificial sweetener, has long been regarded as a persistent marker compound in wastewater treatment systems. Although emerging evidence indicates that ACE can be microbially degraded, the mechanisms governing its initial cleavage and ultimate environmental fate remain poorly resolved. Here, we isolated an ACE-degrading bacterium, Shinella sp. strain KJ01, from activated sludge using D₂O-probed Raman-activated cell sorting. Integrated evidence from total organic carbon removal, CO₂ production, and transient accumulation of transformation products (TP_S) indicates substantial mineralization of ACE by strain KJ01. Comprehensive TP profiling further revealed that hydrolysis was the major initial transformation route of ACE in strain KJ01, while trace and transient intermediates suggested the possible occurrence of minor monooxygenation- and deoxygenation-related side reactions. Multi-omics analyses identified a formylglycine-dependent arylsulfonase (AtsA) as a key enzyme associated with the initial cleavage of ACE, which was further validated through in vivo heterologous expression and in vitro enzymatic assays. AtsA catalyzes the conversion of ACE to acetoacetamide-N-sulfonic acid, initiating structural destabilization and enabling downstream metabolism. A metagenomic survey of wastewater treatment plants revealed widespread occurrence of atsA, with its abundance positively associated with regional ACE loads, suggesting pollutant-driven functional enrichment. Together, these findings link enzyme-level mechanisms to system-scale microbial processes and provide a mechanistic framework for understanding the environmental fate of persistent anthropogenic contaminants in wastewater treatment systems.