Putative anaerobic transformation pathway of microcystin-RR inferred from 15N labeling and multi-omics in an enriched shrimp pond sediment microbial community.

The degradation mechanism of MC-RR by an anaerobic degrading microbial community (ADMC), enriched from shrimp pond sediment, was investigated using LC-MS/MS, metagenomic, and metatranscriptomic analyses. Three key degradation products of 15N-labeled MC-RR were tentatively identified: Adda-Glu-Mdha-Ala (m/z 618.3216), a deamination product (m/z 600.2965), and Glu-MeAsp-Ala-Arg-CO2 (m/z 466.2233). The pathway was inferred to involve hydrolytic ring-opening at Arg-Adda or Ala-Arg bonds, followed by deamination, decarboxylation, and stepwise degradation into short peptides and amino acids. Metagenomics revealed Citrobacter amalonaticus as the core dominant species and Shewanella as a low-abundance but transcriptionally active genus. Metatranscriptomic differential expression analysis (1,648 DEGs; 460 upregulated, 1,188 downregulated) showed significant upregulation of non-canonical peptidases including U32 family (YhbU, UbiU), M23 family (MepM), and S9 family serine peptidases, suggesting the involvement of a non-canonical, mlr-independent peptidase system in MC-RR transformation. Genes of the dissimilatory nitrate reduction to ammonium (DNRA) pathway (narG-nirB-nrfA) and nitric oxide reductase (norB) were concurrently upregulated, a transcriptional pattern consistent with DNRA-linked nitrogen turnover and NO detoxification during degradation, although the corresponding nitrogen fluxes were not directly measured. Among quorum sensing (QS) systems, the AI-2 system exhibited the most pronounced transcriptional response, with AHLs, DSF, and c-di-GMP genes also concurrently upregulated, suggesting a multi-signal transcriptional response during degradation of this complex substrate. These findings provide an important theoretical reference for revealing the mechanisms of anaerobic degradation of microcystins (MCs) by complex microbial communities in situ environments, while also offering scientific data to support the targeted development of efficient MCs-degrading microbial community or specific MCs-degrading enzymes.