Reprogramming Native Cryptic Plasmids Enables Antibiotic-Selection-Free Urate Degradation by Escherichia coli Nissle 1917

Hyperuricemia is associated with renal injury and disturbances in gut microbial ecology. Intestinal urate clearance by engineered probiotics is limited by antibiotic-free functional maintenance and the oxygen requirement of urate oxidase (UOX). We reprogrammed the native cryptic plasmids of Escherichia coli Nissle 1917 (EcN) to construct M12U, a marker-free dual-plasmid strain. pMUT2 carried an HucR-regulated urate-responsive UOX module, while pMUT1 encoded a low-oxygen-responsive UOX module and Tat-targeted Vitreoscilla haemoglobin (tat-VHB). M12U degraded urate in vitro and retained activity under low-oxygen conditions. In hyperuricemic mice (n = 5 per group), oral M12U reduced serum urate, creatinine, blood urea nitrogen, inflammatory cytokines and renal histopathological injury. 16S rRNA profiling showed increased microbial richness and a partial shift in community composition toward healthy controls. Shotgun metagenomics revealed enrichment of Lactobacillus johnsonii and Bacteroides acidifaciens, together with increased metabolic potential related to the pentose phosphate pathway and glutathione metabolism. Faecal metabolomics showed reduced uric acid abundance and increased thiamine monophosphate and argininosuccinic acid. These results support further evaluation of dual-native-plasmid architectures for gut-directed urate clearance.