Elucidation of a novel skatole degradation pathway and key genes in Rhodococcus rhodochrous S3

Skatole is a major component of the objectionable smell of manure and has an extremely low odor threshold, leading to severe odor problems and potential threats to animal and human health. Yet the molecular mechanisms underlying microbial skatole degradation remain poorly understood. To date, only a limited number of skatole monooxygenases have been reported; these enzymes belong to the Group E flavin-dependent monooxygenases and catalyze the initial oxidation of skatole, but the subsequent degradation pathways and associated genes remain unclear. In this study, we isolated a skatole-degrading strain Rhodococcus rhodochrous S3 from pig manure, elucidated a novel skatole degradation pathway and identified its key enzymes involved: SkoA is a Group B flavin-dependent monooxygenase responsible for oxidizing skatole to 3-methyloxindole (3-MOI); SkoB is a Baeyer-Villiger monooxygenase that catalyzes the conversion of 3-MOI to (4 R)-4-methyl-1,4-dihydro-3,1-benzoxazin-2-one (4-MDHBO); the esterase SkoC, encoded by a gene adjacent to skoB, hydrolyzes 4-MDHBO to form 2-carbamic acid-α-methylbenzyl alcohol (2-CMA), which is then completely degraded via decarboxylation and a series of oxygenation reactions through the catechol degradation pathway. Additionally, microbial distribution analysis revealed that SkoA-like skatole monooxygenases are widely distributed across Proteobacteria and Actinobacteria, indicating that these two phyla are the primary drivers of skatole degradation in nature. Together, these findings greatly improve our comprehension of microbial skatole degradation mechanisms and offers insights into the ecological distribution of skatole-degrading genes.