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 ΔnicR2A ΔnicR2B1 ΔnicR2B2 (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-ΔP_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.

中文翻译：

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同源转录调节因子NicR2A，NicR2B1和NicR2B2和内源性异位强启动子对假单胞菌烟碱代谢的差异作用。应变JY-Q

尼古丁是一种有毒的环境污染物，广泛存在于烟草废料中。作为天然尼古丁降解菌株，假单胞菌属（Pseudomonas sp。）。JY-Q菌株仍然难以降解高浓度的尼古丁。在这项研究中，我们调查了两个同源的转录调节因子和内源性异位强启动子对尼古丁降解效率的影响。比较基因组学分析表明，两个同源的转录调节因子，即NicR2A和NicR2Bs（NicR2B1加NicR2B2），可以抑制尼古丁降解基因的表达。当两个nicR2A和nicR2Bs被删除，所得到的突变体JY-QΔ nicR2A Δ nicR2B1 Δ nicR2B2（QΔABs）的烟碱降解效率比野生型JY-Q高17％。转录组测序（RNA-seq）分析显示，六个基因的转录水平（每千碱基片段的片段数[FPKM]值）高于JY-Q中其他基因的转录水平。基于这些基因的遗传组织，确定了三个推定的启动子P _RS28250，P _RS09985和P _RS24685。通过使用逆转录酶定量PCR（RT-qPCR）比较其表达水平来评估其启动子活性。我们发现RS28250，RS09985和RS24685的转录水平分别比编码6-羟基-3-琥珀酰吡啶吡啶羟化酶的hspB2高16.8、2.6和1.6倍，后者与烟碱降解有关。因此，选择了两个强大的内源启动子，即P _RS28250和P _RS09985，以替代nic2基因簇的原始启动子。内源性异位启动子的作用也与靶基因簇的位置有关。当启动子P _RS28250替换了_hspB2的启动子时，得到的突变体QΔABs- ΔP _hspB2 :: P _RS28250烟碱降解效率比JY-Q高69％。这项研究提出了一种可行的策略，可以通过去除抑制性调节蛋白并用强内源性异位启动子替代靶启动子来增强菌株尼古丁降解的能力。