The present study revealed that the Bacillus sp. 05–5402 strain was effective in the degradation of TSNAs in flue-cured tobacco (Table 2). This result was consistent with the findings of previous studies[9, 19, 20], all of which suggested that microbial fermentation is effective in the degradation of TSNAs. Therefore, further optimization of the fermentation conditions was important to enhance the degradation rate of TSNAs for improving the industrial production of tobacco and tobacco products. Temperature and humidity during the fermentation process have been demonstrated to influence the degradation of TSNAs by microorganisms[10]. In a previous study, a lower TSNA content was detected in a high-humidity fermentation environment, while a higher TSNA content was detected under high-temperature conditions[21], which is consistent with the results of the present study. This effect could be related to the fact that the nitrate nitrogen present in tobacco leaves at a high water content is soluble in water and less likely to produce the precursors of TSNAs, compared to the levels produced in the presence of nitrite, thereby decreasing the levels of TSNAs in the leaves. Another study reported that increasing water content could promote the growth of microorganisms on tobacco leaves, facilitating the action of these microbes on TSNAs[22]. In summary, these studies demonstrated that fermentation conditions play a key role in determining the levels of TSNAs in tobacco.
The transcriptome responds to all RNAs transcribed within a tissue or a cell in a given environment and is, therefore, utilized to explain the effect of the environment on the subject[23]. The proteomics technology screens for functionally important proteins and has thereby provided novel research ideas to resolve and decipher various complex molecular mechanisms[24]. A combined transcriptomics and proteomics analysis is, consequently, a popular strategy in systems research to obtain information on both genes and proteins, along with gaining insights into the expressions, regulations, and interactions of these biomolecules in an organism[25, 26]. Both transcriptomics and proteomics have been applied widely for studying the degradation of harmful substances in tobacco and revealing the underlying mechanisms[27–29].
The studies on the degradation of harmful substances in tobacco have, however, focused mainly on nicotine, with few studies reported on the degradation mechanism of TSNAs. Nonetheless, the degradation mechanism of nicotine could be used as a reference for understanding the degradation mechanism of TSNAs, as TSNAs are derived from nicotine[30]. The degradation mechanisms of the two substances are indeed correlated. For instance, according to the KEGG analysis conducted in a previous study, ABC transporter could have a key role in the degradation of nicotine by Pseudomonas sp. JY-Q[27], and this ABC transporter was also enriched in the present study (Fig. 4). In addition, studies have reported structural similarities between TSNAs and nicotine, due to which the two may be catalyzed using the same enzymes, such as cytochrome P450 enzymes, for metabolism[31–33]. However, the transcriptome sequencing conducted in the present study did not reveal the enrichment of cytochrome P450 enzymes. This was attributable to a different strain used in the present study compared to those used in the previous studies[34] or to the possible inhibition of the P450 enzyme activity by NAT or NAB[33]. The precise mechanism, however, has to be investigated further.
Both transcriptomics and proteomics revealed the enrichment of carbon metabolism and glycolysis/gluconeogenesis pathways (Fig. 5), which have been previously linked to the degradation of TSNAs[4, 16, 35, 36]. In flue-cured tobacco, the glycolysis pathway produces ATP and energy to synthesize NADPH and the equivalent for the degradation of the precursors of N-nitrosamides[35, 37], which results in a lower content of nitrosamines and consequently the reduced biosynthesis of TSNAs. In addition, carbon metabolism is highly correlated with nitrogen metabolism in plants[38, 39]. A reduction in the nitrogen level was reportedly effective in decreasing the nitrate content[40]. Therefore, carbon metabolism could have significant roles in the degradation of TSNAs.
Furthermore, several pathways related to the metabolism of amino acids, such as lysine metabolism, tryptophan metabolism, arginine, and proline metabolism, etc., were enriched in the present study (Fig. 5). The pathways related to the biosynthesis of amino acids and the metabolism of various amino acids have been indicated to be responsible for the degradation of TSNAs[4, 7]. In addition, the metabolic process of amino acids could reportedly reduce the production of TSNAs by reducing the levels of nitrite[4]. These findings suggested that the metabolism of amino acids in the cells of the bacterial strain 05–5402 might exert a significant effect on the content of TSNAs.
In addition, nucleotide metabolism, oxidative phosphorylation, thiamine metabolism, and other pathways were enriched in the KEGG pathway analysis (Fig. 4) and were, therefore, inferred to have important roles in the degradation of TSNAs by strain 05–5402. However, no study to date has indicated the relation of these pathways to the degradation of alkaloids. Therefore, it was speculated that these pathways provide the necessary ATP for the biological activities of Bacillus pumilus and thereby promote the consumption and utilization of TSNAs, although the precise mechanism remains to be explored.
Using the combined transcriptomic and proteomic analysis, the present study revealed that the degradation of TSNAs by strain 05–5402 is associated with multiple biological response pathways, among which carbon metabolism, glycolysis/gluconeogenesis, amino acid biosynthesis, and metabolism of various amino acids have important roles. These results provide a feasible and effective approach to reducing the levels of harmful substances in flue-cured tobacco and would also serve as a reference for subsequent studies exploring the TSNA degradation pathways. However, the precise mechanism of degradation could not be deciphered in the present study as information regarding metabolites was not included and analyzed. Therefore, to reveal the precise mechanism of degradation, studies based on the integration of metabolomics and multi-omics are warranted.