Integrative Omics and Gene Knockout Analyses Suggest a Possible Gossypol Detoxification Mechanism and Potential Key Regulatory Genes of a Ruminal Lactobacillus rhamnosus Strain

(WK331) newly isolated from the rumen. Biodegradation assays showed that WK331 removes over 80% of free gossypol, of which 50% was biodegraded and 30% was converted into bound gossypol. Mass spectrometry identified eight novel degradation products of gossypol and proposed two previously unrecognized metabolic pathways: removal of the aldehyde group and cleavage of the naphthalene ring. Transcriptomic analysis revealed that an antioxidant defense system comprising NADH oxidase, thioredoxin peroxidase, and glutathione peroxidase is pivotal for enhancing gossypol tolerance. Subsequent gene knockout analysis found that bifunctional acetaldehyde-CoA/alcohol dehydrogenase and catechol 2,3-dioxygenase play important regulatory roles in gossypol biodegradation. Collectively, our findings unmask a novel detoxification mechanism of gossypol in ruminal bacteria, which may contribute to the further development of gossypol-degrading enzymes.