Characterization of a novel nornicotine-degrading strain Mycolicibacterium sp. SMGY-1XX from a nornicotine-degrading consortium and preliminary elucidation of its biodegradation pathway by multi-omics analysis

Nicotine and nornicotine are all toxic alkaloids involved in the formation of carcinogenic tobacco-specific nitrosamines. Microbes play an important role in removing these toxic alkaloids and their derivatives from tobacco-polluted environments. By now, microbial degradation of nicotine has been well studied. However, limited information is available on the microbial catabolism of nornicotine. In the present study, a nornicotinedegrading consortium was enriched from a river sediment sample and characterized by metagenomic sequencing using a combination of Illumina and Nanopore technologies. The metagenomic sequencing analysis demonstrated that Achromobacter, Azospirillum, Mycolicibacterium, Terrimonas, and Mycobacterium were the dominant genera in the nornicotine-degrading consortium. A total of 7 morphologically distinct bacterial strains were isolated from the nornicotine-degrading consortium. These 7 bacterial strains were characterized by whole genome sequencing and examined for their ability to degrade nornicotine. Based on a combination of 16 S rRNA gene similarity comparisons, 16 S rRNA gene-based phylogenetic analysis, and ANI analysis, the accurate taxonomies of these 7 isolated strains were identified. These 7 strains were identified as Mycolicibacterium sp. strain SMGY-1XX, Shinella yambaruensis strain SMGY-2XX, Sphingobacterium soli strain SMGY-3XX, Runella sp. strain SMGY-4XX, Chitinophagaceae sp. strain SMGY-5XX, Terrimonas sp. strain SMGY-6XX, Achromobacter sp. strain SMGY-8XX. Among these 7 strains, Mycolicibacterium sp. strain SMGY-1XX, which has not been reported previously to have the ability to degrade nornicotine or nicotine, was found to be capable of degrading nornicotine, nicotine as well as myosmine. The degradation intermediates of nornicotine and myosmine by Mycolicibacterium sp. strain SMGY-1XX were determined and the nornicotine degradation pathway in strain SMGY-1XX was proposed. Three novel intermediates, myosmine, pseudooxy-nornicotine, and & gamma;-aminobutyrate, were identified during the nornicotine degradation process. Further, the most likely candidate genes responsible for nornicotine degradation in Mycolicibacterium sp. strain SMGY-1XX were identified by integrating genomic analysis, transcriptomic analysis, and proteomic analysis. The findings in this study will help to expand our understanding on the microbial catabolism of nornicotine and nicotine and provide new insights into the nornicotine degradation mechanism by consortia and pure culture, laying a foundation for the application of strain SMGY-1XX for the removal, biotransformation, or detoxification of nornicotine.