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Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD + , suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. strain JY-Q still has difficulties degrading 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, namely, NicR2A and NicR2Bs (NicR2B1 plus NicR2B2), can repress nicotine degradation gene expression. When both nicR2A and nicR2Bs were deleted, the resulting mutant JY-Q Δ nicR2A Δ nicR2B1 Δ nicR2B2 (QΔABs) exhibits a 17% higher nicotine degradation efficiency than wild-type JY-Q. Transcriptome sequencing (RNA-seq) analysis showed that the transcription levels (fragments per kilobase per million [FPKM] value) of six genes were higher than those of the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, P RS28250 , P RS09985 , and P RS24685 , were identified. Their promoter activities were evaluated by comparing their expression levels using reverse transcriptase quantitative PCR (RT-qPCR). We found that the transcription levels of RS28250 , RS09985 , and RS24685 were respectively 16.8, 2.6, and 1.6 times higher than that of hspB2 , encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely, P RS28250 and P RS09985 , were selected to replace the original promoters of nic2 gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter P RS28250 replaced the promoter of hspB2 , the resultant mutant QΔABs-Δ P hspB2 :: P RS28250 exhibited nicotine-degrading efficiency 69% higher than that of JY-Q. This research suggests a feasible strategy to enhance strains' capacity for nicotine degradation by removal of repressing regulatory proteins and replacing the target promoter with strong endogenous ectopic promoters. IMPORTANCE This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild-type (WT) strain JY-Q by removing repressing regulatory proteins NicR2A and 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 the pollutant-degrading capacity of natural strains through genomic modification., (Copyright © 2021 American Society for Microbiology.)

Pseudomonas sp. JY-Q was isolated from nicotine-rich environment and could degrade and tolerate high-content nicotine. Its specific genetic architecture comprised duplicated homologous nicotine-degrading clusters for different functional modules on the whole pathway. Its adaptive and genomic properties caused our concern whether the duplicated homologous gene clusters confer additive effects on nicotine degradation and result in strain JY-Q strong capability. After deletion of representative genes from duplicated homologous gene clusters of upstream module Nic1, midstream module Spm, and downstream module Nic2, the nicotine degradation efficiency of the wild type and mutant strains were examined. As the first genes of clusters Nic1-1 and Nic1-2, nicA2 and nox are both involved in nicotine degradation, but nox exhibited more contribution to nicotine metabolism due to the higher transcriptional amount of nox than that of nicA2. Likewise, the sub-clusters spm1 and spm2 showed additive effect on nicotine metabolism. As two hpo-like genes of clusters Nic2-1 and Nic2-2, hpo1, and hpo2 also showed additive effect on the nicotine degrading, but hpo1 provided more contribution than hpo2. The third hpo-like gene in cluster NA (nicotinic acid degrading), nicX is not necessary for 2,5-dihydroxypyridine transformation when hpo1 and hpo2 exist. A variety of transposases and integrases observed around Nic1 and Nic2 cluster genes suggests that the duplicated genes could evolve from horizontal gene transfer (HGT)-related dissemination. This study provide an insight into a novel adaptability mechanism of strains in extreme environment such as high nicotine concentration, and potential novel targets to enhance strain synthesis/degradation ability for future applications.