CN108531501B - 尼古丁选择性降解菌的构建方法及其在废烟叶水提液中的应用 - Google Patents
尼古丁选择性降解菌的构建方法及其在废烟叶水提液中的应用 Download PDFInfo
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Abstract
本发明公开了一种尼古丁选择性降解菌的构建方法及其在废烟叶水提液中的应用，以Pseudomonas sp.JY‑Q为原始菌株，通过该菌全基因组信息分析，确定以下五个目的基因，构建目的基因的敲除载体，利用热转法将敲除载体转化到大肠杆菌缺失菌株WM3064中，利用载体上的Kanamycin抗性基因，筛选出第一次交换的重组子；将该菌株过夜扩培后，利用载体上携带的反筛选标记SacB基因，发生第二次交换的重组子——即目的基因随整合入基因组的质粒载体脱落，以实现基因无缝敲除，直至敲除全部目的基因。本发明所得的菌株与野生型JY‑Q的表型差异，主要包括生长差异、尼古丁和葡萄糖降解差异。
Description
技术领域
本发明属于代谢工程技术领域，通过构建假单胞菌JY-Q(Pseudomonas sp.JY-Q)基因缺失菌株，使其在尼古丁和葡萄糖等共存环境中(如废烟叶水提液,TWE)选择性降解尼古丁。
背景技术
尼古丁(Nicotine),又名烟碱，由C、H、N三种元素组成，是一种含氮杂环有机化合物，其分子量为162.23Da，分子式为C10H14N2，化学命名为1-甲基-2-(3-吡啶基)吡咯烷。室温下，尼古丁为一种淡黄色的油状液体，在光下久置易被氧化成暗灰色，有强烈的辛辣味，味苦，易挥发，腐蚀性强。常温常压下，尼古丁的密度为1.01g/mL，沸点为247℃，熔点为-79℃，易溶于乙醇、乙醚和氯仿等有机溶剂中，能溶于水，在紫外波长259nm处有特征吸收峰。尼古丁是一种环境有毒物质，同时也是一种精神毒物。它对人类能产生致毒、致癌等多种不良效应，由于其可以透过生物膜和血脑屏障等膜系统，造成血液粘稠，流速降低，引发心绞痛，气管炎等疾病，严重时可引发心脏病导致死亡。并且，大量尼古丁的存在可以影响土壤生态结构和地下水，对土壤和环境造成严重污染，扰乱生态平衡。烟草及其废弃物是自然环境和人类生活环境中尼古丁主要污染来源。目前，利用微生物降解环境中各种有机污染物发挥着举足轻重的作用。
Pseudomonas sp.JY-Q分离自废烟叶水提液(TWE)，并显示出较好的尼古丁降解活性和TWE耐受适应性(详情见专利申请号：201310320679.8，中国典型培养物保藏中心保藏编号:M2013236)。然而,在实际利用假单胞菌JY-Q降解一定稀释度TWE中的尼古丁时，由于样品中存在另其它碳源(如葡萄糖),JY-Q会优先利用葡萄糖，从而对JY-Q尼古丁降解起到竞争性抑制作用。为避免TWE中其他营养物质的影响，同时在基本无机盐培养基(BSM)中添加尼古丁和葡萄糖进行降解尼古丁实验，结果也发现与葡萄糖共存时尼古丁完全降解所需时间比以尼古丁为唯一碳源时要多，证明了葡萄糖的存在对JY-Q降解尼古丁有抑制作用。迄今，国内外均未见关于葡萄糖与特定污染物(如尼古丁)共存时选择性降解污染物的报道。
发明内容
本发明的目的是提供一种有效可行的方法，能够通过基因编辑技术对Pseudomonas sp.JY-Q菌株进行改造，使其成为在混合碳源环境中的尼古丁选择性降解菌。
本发明的另一目的是将前述的尼古丁选择性降解菌应用于复杂环境(如TWE)中高效降解尼古丁。
为实现发明目的，本发明采用的技术方案如下：
尼古丁选择性降解菌的构建方法，以Pseudomonas sp.JY-Q为原始菌株，包括下述步骤：
1)通过该菌全基因组信息分析，确定以下五个目的基因：一个葡萄糖激酶基因(AA098_22370)和四个葡萄糖脱氢酶基因(AA098_12490，AA098_22860，AA098_11910和AA098_05800)；
2)构建目的基因的敲除载体：设计待敲除目的基因上下游同源臂扩增引物(含约40bp敲除质粒部分序列)，在上游同源臂的正向引物5’端加入EcoRⅠ酶切位点，在下游同源臂的反向引物5’端加入BamHⅠ酶切位点，以融合PCR扩增出目的基因的上下游同源臂后，利用一步克隆法技术在体外将其与酶切后的线性载体pK18mobSacB重组；
3)利用热转法将敲除载体转化到大肠杆菌缺失菌株WM3064中，然后加入2,6-二氨基庚二酸,以此为供体菌，假单胞菌JY-Q为受体菌进行双亲本接合；之后，由于自杀质粒载体pK18mobSacB无法在假单胞菌中独立复制，其接合转化到宿主菌株后会插入到JY-Q的基因组上；利用载体上的Kanamycin抗性基因，可筛选出第一次交换的重组子；
4)将该菌株过夜扩培后，利用载体上携带的反筛选标记SacB基因，在反筛选平板，即15％蔗糖平板上，可将发生第二次交换的重组子——即目的基因随整合入基因组的质粒载体脱落，以实现基因无缝敲除；
5)重复步骤2)至4)，直至敲除全部目的基因。
通过以上步骤，可得到选择性降解菌株。并应用于废烟叶水提液(TWE)尼古丁的降解中。
经验证，本发明所得的菌株与野生型JY-Q的表型差异，主要包括生长差异、尼古丁和葡萄糖降解差异。
本发明所得的菌株，通过以下方法进行尼古丁和葡萄糖降解测定：
①尼古丁含量检测：取培养液适量，12,000r/min离心10min，上清液经0.25μm滤膜过滤后，使用高效液相色谱(HPLC)测定尼古丁浓度。色谱条件：色谱柱为Agilent SB-C18(4.6mm×150mm)；流动相采用水相-有机相双流动相，色谱甲醇和0.1mol·L-1KH2PO4(使用超纯水配置，pH＝3.0)，体积比为10:90；设定流速为1mL·min-1；设定检测波长为254nm，检测时间为5min。出峰时间在2.5min左右，峰面积与尼古丁浓度换算公式如下：
Y＝0.0003X-0.0223R2＝0.9991X:峰面积；Y:尼古丁浓度(g/L)
②葡萄糖含量检测：取培养液适量，12,000r/min离心10min，上清液经0.25μm滤膜过滤后，用SBA-40C生物传感器测定葡萄糖浓度。
附图说明
以下结合附图和本发明的实施方式来作进一步详细说明
图1为q-PCR分析假单胞菌JY-Q中葡萄糖代谢起始酶基因的表达水平；
图2为双交换同源重组示意图；
图3为菌落PCR验证第一次重组子；
图4为菌落PCR验证第二次重组子；
图5为缺失菌株gdh-05800基因上下游同源臂与JY-Q上下游同源臂序列对比图；
图6为JY-Q与缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800在2g/L葡萄糖固体培养基中的生长情况；
图7为JY-Q和缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800休止细胞在5％TWE中的尼古丁和葡萄糖降解曲线。
具体实施方式
实时荧光定量聚合酶链反应(RT-PCR)：本实验以Pseudomonas sp.JY-Q的全基因组信息为基础，通过已有的基因注释信息分析葡萄糖代谢相关途径，我们将关注点聚集在五个葡萄糖代谢起始酶基因，包括一个葡萄糖激酶基因(AA098_22370)和四个葡萄糖脱氢酶基因(AA098_12490，AA098_22860，AA098_11910和AA098_05800)。在基本无机盐培养基(BSM)中添加葡萄糖作为唯一碳源培养假单胞菌JY-Q至对数生长期，提取总RNA进行荧光定量实验，结果如图1所示。
从图1中显示五个基因都有所表达，其中gdh-22860(AA098_22860)基因表达量最高，约是gdh-12490基因的3倍。其次是gdh-05800基因，gdh-11910基因和gck-22370表达量相当，gdh-12490基因表达量最低。依据上述实验结果，依次对这五个基因进行敲除。
基因敲除：本发明采用双交换同源重组法实现基因的无缝敲除。首先，构建目的基因的敲除载体。设计引物，在目的基因上游同源臂的正向引物5’端加入EcoRⅠ酶切位点，在目的基因下游同源臂的反向引物5’端加入BamHⅠ酶切位点，以融合PCR扩增出目的基因的上下游同源臂(约1000bp)后，利用一步克隆法技术在体外将其与酶切后的线性载体pK18mobSacB重组；其次，利用热转法将敲除载体转化到大肠杆菌缺失菌株WM3064中，以此为供体菌，假单胞菌JY-Q为受体菌进行双亲本接合。之后，由于自杀质粒载体pK18mobSacB无法在假单胞菌中独立复制，其接合转化到宿主菌株后会插入到JY-Q的基因组上。利用载体上的Kanamycin抗性基因，可筛选出第一次交换的重组子；将该菌株过夜扩培后，利用载体上携带的反筛选标记(SacB基因)，在反筛选平板(15％蔗糖平板)上，可将发生第二次交换的重组子——即目的基因随整合入基因组的质粒载体脱落，以实现基因无缝敲除，但该方法仍有一定几率会恢复成野生型(如图2)。以gdh-05800基因(2412bp)为例，对缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910进行敲除实验。结果如图2所示。
将构建成功的WM3064/pk18mobSacB-(gdh-05800)-UD(供体菌)和缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910(受体菌)接合后，涂布于LB+Kan固体平板上，筛选得到第一次交换重组子。
以gdh-05800基因上游同源臂的正向引物A1和下游同源臂的反向引物B2为一对引物，进行菌落PCR验证。当敲除载体进行第一次交换插入到基因组后，其扩增产物有两条条带：一是gdh-05800基因的上下游同源臂(996bp)，二是gdh-05800基因加上上下游同源臂(3408bp)。由图3可知，3#等6个重组子条带正确。
挑取3#重组子过夜扩培后，取100μl培养液稀释100倍，取适量菌液涂布于15％蔗糖平板，筛选得到第二次交换重组子。
以gdh-05800基因上游同源臂的正向引物A1和下游同源臂的反向引物B2为一对引物，进行菌落PCR验证。第二次交换时敲除载体从基因组上脱落，发生两种情况：一是gdh-05800基因随敲除载体一起脱落，扩增产物为上下游同源臂(996bp)；二是敲除载体脱落但目的基因仍存在，其扩增产物为gdh-05800基因加上下游同源臂(3408bp)。从图4可知，只有3#重组子条带正确。为进一步确定敲除成功，以gdh-05800基因的A1B2为引物，3#菌株的基因组为模板进行扩增，产物交由杭州擎科梓熙生物技术有限公司完成测序。从图5所知，该菌株已缺失gdh-05800基因，缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800敲除完成。
表型差异：验证缺失菌株与野生型JY-Q的表型差异，主要包括生长差异、尼古丁和葡萄糖降解差异。如图6所示，试验结果表明：缺失菌株
JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800在2g/L葡萄糖固体培养基中无法生长，初步确定该缺失菌株已无法利用葡萄糖。
将种子液按100％接种量(细胞干重17.7mg)接种于PBS缓冲液(pH＝7.4)中，4℃饥饿处理20h后，添加5mL灭菌处理的TWE液体，使其最终体积为100mL。30℃、180rpm摇床培养，每隔6h连续取样，检测其尼古丁及葡萄糖含量，绘制曲线。结果如图7所示，曲线S1为野生型JY-Q的葡萄糖含量，曲线S2为缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800的葡萄糖含量，线S3为野生型JY-Q的尼古丁含量，线S4为缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800的尼古丁含量；30h后葡萄糖浓度没有变化，说明该缺失菌株JY-Q/ΔGckΔ12490Δ22860Δ11910Δ05800已无法利用葡萄糖作为营养来源，且其完全降解尼古丁所需时间比野生型JY-Q短，表明了缺失菌株的尼古丁降解效率更高。因此，葡萄糖共存时尼古丁选择性降解菌构建完成，且应用于废烟叶水提液中的尼古丁降解效果更优。
Claims (2)
1.尼古丁选择性降解菌的构建方法，以Pseudomonas sp.JY-Q为原始菌株，包括下述步骤：
1)通过该菌全基因组信息分析，确定以下五个目的基因：一个葡萄糖激酶基因AA098_22370和四个葡萄糖脱氢酶基因AA098_12490，AA098_22860，AA098_11910和AA098_05800；
2)构建目的基因的敲除载体：设计待敲除目的基因上下游同源臂扩增引物，在上游同源臂的正向引物5’端加入EcoRⅠ酶切位点，在下游同源臂的反向引物5’端加入BamHⅠ酶切位点，以融合PCR扩增出目的基因的上下游同源臂后，利用一步克隆法技术在体外将其与酶切后的线性载体pK18mobSacB重组；
3)利用热转法将敲除载体转化到大肠杆菌缺失菌株WM3064中，然后加入2,6-二氨基庚二酸,以此为供体菌，假单胞菌JY-Q为受体菌进行双亲本接合；之后，由于自杀质粒载体pK18mobSacB无法在假单胞菌中独立复制，其接合转化到宿主菌株后会插入到JY-Q的基因组上；利用载体上的Kanamycin抗性基因，可筛选出第一次交换的重组子；
4)将该菌株过夜扩培后，利用载体上携带的反筛选标记SacB基因，在反筛选平板，即15％蔗糖平板上，可将发生第二次交换的重组子——即目的基因随整合入基因组的质粒载体脱落，以实现基因无缝敲除；
5)重复步骤2)至4)，直至敲除全部目的基因。
2.权利要求1所得的尼古丁选择性降解菌应用于废烟叶水提液中以降解尼古丁的用途。
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