Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. JY-Q still has difficulties to degrade high-concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators NicR2A and NicR2Bs can repress nicotine-degrading genes expression. When both of nicR2A and nicR2Bs were deleted, the resulting mutant QΔnicR2AΔnicR2B1ΔnicR2B2 exhibit 17% higher nicotine degradation efficiency than wide type JY-Q. The RNA-seq analysis showed that the transcription level (FPKM value) of six genes was particularly higher than the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250, PRS09985 and PRS24685, were identified. Their promoter activities were evaluated by comparing their expression levels using RT-qPCR. We found that the transcription levels of RS28250, RS09985 and RS24685 were 16.8-, 2.6-, and 1.6-times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase involved in nicotine degradation. Thus, two strong endogenous promoters PRS28250 and PRS09985 were selected to replace the original promoters of Nic2 gene clusters. The effect of endogenous ectopic promoter was also related to the replaced position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant, QΔABs-ΔPhspB2::PRS28250, exhibited 69% higher nicotine degrading efficiency than the JY-Q. This research suggests a feasible strategy to enhance strain ability by removal of repressing regulatory proteins and replacing target promoter with strong endogenous ectopic promoters. IMPORTANCE This study evaluated the differential effects of homologous NicR2A/NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild type strain JY-Q by removing repressing regulatory proteins, NicR2A/NicR2Bs, and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving pollutant-degrading capacity of naturally strains through genomic modification.
Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the co-occurrence of these two pathways is only observed in Pseudomonas sp. JY-Q. Nicotine pyrrolidine catabolism pathway of strain JY-Q consists of function modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenases (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm (namely Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 both were in-frame deleted, the mutant still grew well in the basic salt medium supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains re-organized domains similar to that of Spm. When JY-Q_nicAB gene was introduced into another Pseudomonas strain, which is unable to degrade NA, the resultant recombinant strain exhibited the ability transforming SP to HSP except the ability NA metabolism. Here, we ccan conclude that NicAB in strain JY-Q exhibits an additional role of SP transforming. The other genes in NA cluster, NicXDFE (Nic2 homolog), then also exhibit the role of HSP subsequent metabolism for energy yield. This finding also suggests that the co-occurrence of nicotine and NA degradation genes in strain JY-Q represents a contribution for JY-Q making it more effective and flexible for the degradation of nicotine. Importance 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) both are valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant kept the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.
Tobacco (Nicotiana tabacum L.) is a very important economic crop in China but lead to a large quantity of tobacco waste during processing. Nowadays, industrial tobacco waste was mainly treated via reconstituted tobacco process by papermaking method, during which liquid tobacco waste extract (TWE) is the key fermentation step to control the final product quality. As bioaugmentation with specific functional microorganisms was one effective method in the quality optimization of fermented products, some native strains were isolated from TWE and used as bioaugmentation agents. In this study, Zygosaccharomyces sp. MC-5K3 isolated from TWE was used as the additive to improve the quality of fermented TWE. The results proved that the addition of Zygosaccharomyces sp. MC-5K3 significantly influence the microbial diversity of fermented TWE especially for the fungal groups that Zygosaccharomyces become the only dominant fungal genus instead of some pathogenic bacterial genera. Then the metabolomics profiling of fermented TWE showed that the variation in the concentration of 3-Hydroxybenzoic acid was the key influence factor on fermented TWE quality after MC-5K3 was added. The correlation analysis showed that the change of microbial diversity of the fermented production due to the addition of MC-5K3 leaded to some important differential metabolites such as 3-Hydroxybenzoic acid and finally improved various qualities of tobacco products. In conclusion, this study lays a theoretical foundation and new microbiology methods for improving the quality of tobacco products by new microbiology methods.
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