CN112501099A - 一种脱氮基因工程菌的构建方法及应用 - Google Patents
一种脱氮基因工程菌的构建方法及应用 Download PDFInfo
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Abstract
本发明涉及一种脱氮基因工程菌的构建方法及应用,包括根据枯草芽孢杆菌宿主对硝化途径的氨单加氧酶编码基因和羟胺氧化酶编码基因的密码子进行优化合成,通过DNA同源重组组装进入枯草芽孢杆菌游离质粒置于P43启动子下进行串联表达,获得脱氮基因工程菌。本发明提高微生物菌株对恶劣环境的影响,这对于微生物菌株开发及生物强化技术在污水处理中的推广应用具有现实而深广的意义。
Description
技术领域
本发明涉及生活污水处理技术领域,具体涉及一种脱氮基因工程菌的构建方法及应用。
背景技术
水是生命生存进化的基础,全球可供人类利用的水资源仅占全球总水量的十万分之七。然而,人类社会的快速发展,水环境的污染日趋严重。其中来源于生活污水、工业废水、养殖废水、垃圾渗滤液和农田氮肥等的高浓度水污染因子-氮引起水体富营养化,使水体中硝酸盐和亚硝酸盐浓度过高,人畜若长期饮用会引起中毒,而一些有毒藻类的生长与大量繁殖会排放大量毒素于水体中,导致水生动物的大量死亡,从而严重破坏水体的生态平衡。
生物脱氮技术是目前应用广泛且经济效益较高的脱氮方法,传统的生物脱氮由好氧自养硝化作用和厌氧异养反硝化作用共同完成,而近些年来异养硝化和好氧反硝化的发现打破了传统的生物脱氮理论,与传统生物脱氮相比,不仅可以使硝化和反硝化在同一反应器中完成,加快反应过程,减小反应器容积,缩短水力停留时间,降低运行成本,提高系统抗冲击能力,处理高浓度的含氮废水,还可以在反硝化过程中同步除磷。因而逐渐成为目前生物脱氮研究的热点。
为了提高污水生物处理的效率,近些年来国内外学者主要集中在降解环境污染物的高活性菌株的筛选和降解特性研究等方面,大量功能环境微生物被分离和鉴别,具潜力和经济价值的污水处理微生物菌种被发现。尽管当前国内外已分离鉴定和商品化的功能微生物菌种数量众多,根据近十几年的应用观察发现,由于微生物菌种其遗传特性,在条件恶劣的环境中往往收到环境因子胁迫而导致难以发挥其原有(实验条件下)的生物处理活性,难以在分散式污水处理设备上形成有效常态应用。
运用生物工程技术,采用细胞融合、基因重组技术等遗传工程手段,可以将某种降解污染能力强的微生物的降解基因,转入繁殖能力强、适应性好的受体微生物中,构建出高效的具有广谱降解能力的基因工程菌。
发明内容
本发明为解决上述问题,通过分子生物学技术对菌种进行鉴定、代谢分析改造,进行高效脱氮菌种构建,提供了一种脱氮基因工程菌的构建方法。
本发明第二方面是为了提供通过该构建方法得到的脱氮基因工程菌的应用。
为了实现上述目的,本发明采用以下技术方案:
一种脱氮基因工程菌的构建方法,所述构建方法包括根据枯草芽孢杆菌宿主对硝化途径的氨单加氧酶AMO编码基因和羟胺氧化酶HAO编码基因的密码子进行优化合成;通过DNA同源重组组装进入枯草芽孢杆菌游离质粒pP43NMK,置于P43启动子下进行串联表达,产生重组质粒pP43NMK-AMO-HAO;将重组质粒转化枯草芽孢杆菌Bacillus subtilis 168,获得重组菌株B.subtilis 168DN。
作为本发明的一种优选方案,所述重组菌株B.subtilis 168DN的菌落形态:30℃在LB平板培养12h,菌落表面粗糙不透明,边缘不规则,呈淡黄色。
作为本发明的一种优选方案,所述重组菌株B.subtilis 168DN的细胞形态:16h培养物的细胞具有圆端的直杆菌或细胞接近球状,芽孢椭圆到柱状。
作为本发明的一种优选方案,所述重组菌株B.subtilis 168DN的生理生化特征:革兰氏阳性,需氧型菌,化能异养。
作为本发明的一种优选方案,所述羟胺氧化酶HAO编码基因含有如SEQ ID NO.1所示的基因序列。
作为本发明的一种优选方案,所述氨单加氧酶AMO编码基因含有如SEQ ID NO.2所示的基因序列。
作为本发明的一种优选方案,所述构建方法包括以下步骤:通过质粒重组,AMO和NeHAO基因串联重组进入pP43NMK质粒,重组至枯草芽孢杆菌基因组上,获得重组菌株B.subtilis 168DN。
本发明提供了脱氮基因工程菌的应用,上述的构建得到的脱氮基因工程菌在脱氮中的应用。
作为本发明的一种优选方案,所述的脱氮基因工程菌在生活污水中应用。
作为本发明的一种优选方案,取污水处理站的进水,将氨氮与总氮浓度均控制到50mg/L-60mg/L,然后将脱氮基因工程构建菌菌剂按1%接入调配后的生活污水中。
与现有技术相比,本发明具有以下有益效果:
本发明借助现代基因工程技术来改造底盘微生物,在分子水平上对菌种氮代谢途径基因进行改造,强化目的菌株在恶劣环境下氮移除途径基因的表达水平,提供脱氮能力,进而在全局水平上优化影响细胞的代谢功能;通过代谢工程技术或过程工程调控等手段对关键节点进行扰动,提高微生物菌株对恶劣环境的影响,这对于微生物菌株开发及生物强化技术在污水处理中的推广应用具有现实而深广的意义。
附图说明
图1是实施例2中脱氮基因工程菌的生长曲线图;
图2是实施例2中脱氮基因工程菌的脱氮效果图;
图3是实施例3中脱氮基因工程菌在不同碳氮比条件下氨氮去除效果图;
图4是实施例4中脱氮基因工程菌在不同底物浓度条件下氨氮的去除效果图。
具体实施方式
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或者按照各制造商所建议的条件。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1
重组枯草芽孢杆菌硝化途径的构建
根据枯草芽孢杆菌宿主对硝化途径的氨单加氧酶(Ammonia monoxygenase,AMO)编码基因和羟胺氧化酶(Hydroxylamine oxidase,NeHAO)编码基因的密码子进行优化合成。通过DNA同源重组组装进入枯草芽孢杆菌游离质粒pP43NMK,置于P43启动子下进行串联表达,产生重组质粒pP43NMK-AMO-HAO。将重组质粒转化枯草芽孢杆菌(Bacillus subtilis168),获重组菌株B.subtilis 168DN。对重组菌株的单克隆进行DNA测序分析,确定在重组菌株中成功构建了硝化途径。
羟胺氧化酶(Hydroxylamine oxidase,NeHAO)的测序结果SEQ ID NO.1:
gaattcaaaggaggaaggatcaatgagaatcggcgaatggatgcgcggactgcttttatgcgctggccttatgatgtgtggagttgtgcatgccgatatttcaacagttccggatgaaacgtatgatgcccttaaactggatagaggcaaagcaacaccgaaagaaacgtatgaagcactggttaaacgctataaagatccggcgcatggagctggcaaaggaacaatgggagattattgggaaccgattgctatctcaatctacatggacccgaacacattttacaaaccgccggttagcccgaaagaagtggccgaaagaaaagattgcgttgaatgtcatagcgatgaaacaccggtctgggttagagcgtggaaacgctctacacatgctaatttagataaaatcagaaacctgaaatcagatgatccgctttactacaagaaaggcaaactggaagaagttgaaaataacttacgcagcatgggcaaactgggagaaaaagaaacacttaaagaagtgggatgcatcgattgtcatgtggatgtcaacaagaaagataaagctgatcatacaaaagatattagaatgccgacagccgatacatgcggcacatgtcatttacgcgaatttgctgaaagagaatctgaacgcgatacaatggtgtggcctaatggacaatggccggccggaagaccgtcacatgcgctggattatacagctaacattgaaacaacagtgtgggcaacgatgccgcaacgcgaagtcgcagaaggatgcacaatgtgtcatacaaatcagaacaaatgcgataactgtcatacaagacatgaattttctgctgccgaatcacgcaaaccggaagcatgcgccacatgtcatagcggcgtcgatcataataactgggaagcatatacaatgtctaaacatggaaaacttgcggaaatgaatagagataaatggaactgggaagttcgcttaaaagatgcttttagcaaaggcggacaaaatgccccgacatgcgcagcgtgtcacatggaatacgaaggcgaatacacacataacatcacaagaaaaacacgctgggcaaactatccgtttgtgccgggaatcgcggaaaacattacatcagattggagcgaagctagactggattcttgggtccttacatgcacacagtgtcatagcgaaagatttgcacgctcttatctggatcttatggataaaggcacactggaaggacttgcgaaatatcaagaagcaaatgcgatcgtgcataaaatgtatgaagatggcacactgacaggacagaaaacaaatcgcccgaacccgccggaaccggaaaaaccgggctttggaatttttacacaactgttttggtcaaaaggcaataacccggcgagcttagaactgaaagtcttagaaatgggcgaaaataacctggcaaaaatgcatgtcggacttgcacatgttaatccgggcggatggacatatacagaaggctggggaccgatgaacagagcgtatgtcgaaattcaagatgaatacacaaaaatgcaagaattatctgccctgcaagcacgcgttaataaattagaaggcaaacagacatctctgcttgatctgaaaggcacaggagaaaaaatctcacttggcggattaggcggaggcatgttactggctggcgcccttgcattaattggatggagaaaacgcaaacagacaagagcgtaactgcag
氨单加氧酶(Ammonia monoxygenase,AMO)编码基因的测序结果SEQ ID NO.2:
ggtaccaagagaggaatgtacacatgtctatctttcgcacagaagaaatcctgaaagcagcgaaaatgcctccggaagcagtccacatgtcaagactgattgatgcggtttattttccgattcttatcattctgcttgtgggcacatatcacatgcattttatgttactggctggagattgggatttttggatggattggaaagatcgccaatggtggccggttgtgacaccgattgtgggcatcacatattgctcagctatcatgtactacctgtgggtcaattatcgccagccgtttggagccacattatgcgtcgtttgtcttttaatcggcgaatggctgacaagatattggggattttattggtggagccattatccgattaactttgttacaccgggaatcatgcttccgggcgcacttatgctggattttacactgtaccttacaagaaactggcttgtcacagcactggttggcggcggctttttcggactgctgttttatccgggcaactggccgatttttggaccgacacatcttccgatcgtggtcgaaggcacattactgagcatggcagattatatgggccacctgtatgttagaacaggaacaccggaatatgtgcgccatatcgaacaaggatctttaagaacatttggaggccatacaacagttatcgctgcatttttctcagcgtttgttagcatgcttatgtttacagtgtggtggtatttaggcaaagtctattgtacagcatttttctatgtgaaaggaaaaagaggccgcattgtgcatcgcaatgatgtcacagcctttggagaagaaggctttccggaaggaatcaaataagaattccggctcgag。
本发明中的筛选方法包括:
a.筛选分离:取活性污泥样品于无菌生理盐水中,制备菌悬液;吸取菌悬液移至驯化培养基中,振荡培养,为第一个驯化周期,以此类推,进行多次的驯化;
b.脱氮验证:取步骤a驯化完成、培养的菌液,按梯度稀释法分别将样品稀释成10-1~10-8的8种不同梯度的菌悬液,并用各自的固体选择培养基进行平板涂布,放入振荡器培养;从平板中挑取脱氮基因工程菌,在96孔板中培养后,进行验证,从96孔板中各取菌液至NH4 +与NO2 -验证板中,加入氨氮,亚硝酸盐显色剂,观察颜色变化,选取优质菌株;
c.迭代培养:从96孔板中挑选显色明显的菌株,克隆混合后以氨氮为唯一氮源,进行迭代培养,定时期取样测定培养液中的氨氮、硝酸盐和亚硝酸盐的浓度,进一步筛选高效脱氮基因工程菌;
d.复筛:将上述初筛菌株的菌液接入含氯化铵的LB培养基中培养,测氨氮浓度,选取氨氮去除率最高的一株,将该菌接种至发酵培养基中进行扩培,得到脱氮基因工程菌;所述培养基按下述比例配制:1L水中,蛋白胨10g,酵母粉5g,氯化钠10g,pH 7.2,121℃湿热灭菌30min后加入1‰抗生素。
实施例2
生长曲线
控制生活污水中氨氮初始浓度为50-60mg/L,碳氮比为5:1;将基因工程构建菌在添加1‰抗生素的LB培养基中活化培养24h,菌液以1%接种量接种于生活污水中,于200r/min,30℃摇床中震荡培养。由实验结果可以看出,脱氮基因工程菌在24h时OD600达到最大值,之后趋于平稳,因此本发明的菌株生长状态稳定,见图1所示;参见图2所示,本发明的菌株具有脱氮效果。
实施例3
不同碳氮比条件下的脱氮性能
参见图3,保持生活污水中初始氨氮浓度50-60mg/L不变,调整污水中碳源浓度,使得C/N分别为2、3、4、5、8、12。将基因工程构建菌在添加1‰抗生素的LB培养基中活化培养24h,菌液以1%接种量接种于不同碳氮比的生活污水中,于200r/min,30℃摇床中震荡培养。结果如图所示,氨氮及总氮降解效率随碳氮比的增加而上升,在碳氮比为5、8、12时降解率可达到25%以上,氨氮降解率分别为25.95%、26.49%,26.04%,总氮降解率分别为20.74%、24.28%、29.68%。这是由于当碳氮比较高时可为微生物提供充足的碳源,因此本发明菌株可提高其脱氮效果。
实施例4
不同底物浓度条件下的脱氮性能
参见图4,设置生活污水氨氮浓度分别为20-30、30-40、40-50、50-60、60-70mg·L-1,将基因工程构建菌在添加1‰抗生素的LB培养基中活化培养24h,菌液以1%接种量接种于碳氮比为5:1的生活污水中,于200r/min,30℃摇床中震荡培养。在不同底物浓度条件下,NH4+-N浓度越高,细胞生长速度越快,而氨氮与总氮的降解率也随之升高。当底物浓度从20mg/L-30mg/L范围上升至60mg/L-70mg/L范围时,氨氮降解率也从24.97%增加至38.24%,总氮降解率从23.46%上升至38.59%。因此本发明的菌株可提高污水的脱氮效果。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员,在不脱离本发明的精神和范围的情况下,当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化,均为本发明的等效实施例;同时,凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变,均仍属于本发明的技术方案的范围内。
序列表
<110> 浙江双良商达环保有限公司
<120> 一种脱氮基因工程菌的构建方法及应用
<141> 2020-12-14
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1741
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaattcaaag gaggaaggat caatgagaat cggcgaatgg atgcgcggac tgcttttatg 60
cgctggcctt atgatgtgtg gagttgtgca tgccgatatt tcaacagttc cggatgaaac 120
gtatgatgcc cttaaactgg atagaggcaa agcaacaccg aaagaaacgt atgaagcact 180
ggttaaacgc tataaagatc cggcgcatgg agctggcaaa ggaacaatgg gagattattg 240
ggaaccgatt gctatctcaa tctacatgga cccgaacaca ttttacaaac cgccggttag 300
cccgaaagaa gtggccgaaa gaaaagattg cgttgaatgt catagcgatg aaacaccggt 360
ctgggttaga gcgtggaaac gctctacaca tgctaattta gataaaatca gaaacctgaa 420
atcagatgat ccgctttact acaagaaagg caaactggaa gaagttgaaa ataacttacg 480
cagcatgggc aaactgggag aaaaagaaac acttaaagaa gtgggatgca tcgattgtca 540
tgtggatgtc aacaagaaag ataaagctga tcatacaaaa gatattagaa tgccgacagc 600
cgatacatgc ggcacatgtc atttacgcga atttgctgaa agagaatctg aacgcgatac 660
aatggtgtgg cctaatggac aatggccggc cggaagaccg tcacatgcgc tggattatac 720
agctaacatt gaaacaacag tgtgggcaac gatgccgcaa cgcgaagtcg cagaaggatg 780
cacaatgtgt catacaaatc agaacaaatg cgataactgt catacaagac atgaattttc 840
tgctgccgaa tcacgcaaac cggaagcatg cgccacatgt catagcggcg tcgatcataa 900
taactgggaa gcatatacaa tgtctaaaca tggaaaactt gcggaaatga atagagataa 960
atggaactgg gaagttcgct taaaagatgc ttttagcaaa ggcggacaaa atgccccgac 1020
atgcgcagcg tgtcacatgg aatacgaagg cgaatacaca cataacatca caagaaaaac 1080
acgctgggca aactatccgt ttgtgccggg aatcgcggaa aacattacat cagattggag 1140
cgaagctaga ctggattctt gggtccttac atgcacacag tgtcatagcg aaagatttgc 1200
acgctcttat ctggatctta tggataaagg cacactggaa ggacttgcga aatatcaaga 1260
agcaaatgcg atcgtgcata aaatgtatga agatggcaca ctgacaggac agaaaacaaa 1320
tcgcccgaac ccgccggaac cggaaaaacc gggctttgga atttttacac aactgttttg 1380
gtcaaaaggc aataacccgg cgagcttaga actgaaagtc ttagaaatgg gcgaaaataa 1440
cctggcaaaa atgcatgtcg gacttgcaca tgttaatccg ggcggatgga catatacaga 1500
aggctgggga ccgatgaaca gagcgtatgt cgaaattcaa gatgaataca caaaaatgca 1560
agaattatct gccctgcaag cacgcgttaa taaattagaa ggcaaacaga catctctgct 1620
tgatctgaaa ggcacaggag aaaaaatctc acttggcgga ttaggcggag gcatgttact 1680
ggctggcgcc cttgcattaa ttggatggag aaaacgcaaa cagacaagag cgtaactgca 1740
g 1741
<210> 2
<211> 869
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtaccaaga gaggaatgta cacatgtcta tctttcgcac agaagaaatc ctgaaagcag 60
cgaaaatgcc tccggaagca gtccacatgt caagactgat tgatgcggtt tattttccga 120
ttcttatcat tctgcttgtg ggcacatatc acatgcattt tatgttactg gctggagatt 180
gggatttttg gatggattgg aaagatcgcc aatggtggcc ggttgtgaca ccgattgtgg 240
gcatcacata ttgctcagct atcatgtact acctgtgggt caattatcgc cagccgtttg 300
gagccacatt atgcgtcgtt tgtcttttaa tcggcgaatg gctgacaaga tattggggat 360
tttattggtg gagccattat ccgattaact ttgttacacc gggaatcatg cttccgggcg 420
cacttatgct ggattttaca ctgtacctta caagaaactg gcttgtcaca gcactggttg 480
gcggcggctt tttcggactg ctgttttatc cgggcaactg gccgattttt ggaccgacac 540
atcttccgat cgtggtcgaa ggcacattac tgagcatggc agattatatg ggccacctgt 600
atgttagaac aggaacaccg gaatatgtgc gccatatcga acaaggatct ttaagaacat 660
ttggaggcca tacaacagtt atcgctgcat ttttctcagc gtttgttagc atgcttatgt 720
ttacagtgtg gtggtattta ggcaaagtct attgtacagc atttttctat gtgaaaggaa 780
aaagaggccg cattgtgcat cgcaatgatg tcacagcctt tggagaagaa ggctttccgg 840
aaggaatcaa ataagaattc cggctcgag 869
Claims (10)
1.一种脱氮基因工程菌的构建方法,其特征在于,所述构建方法包括根据枯草芽孢杆菌宿主对硝化途径的氨单加氧酶AMO编码基因和羟胺氧化酶HAO编码基因的密码子进行优化合成;通过DNA同源重组组装进入枯草芽孢杆菌游离质粒pP43NMK,置于P43启动子下进行串联表达,产生重组质粒pP43NMK-AMO-HAO;将重组质粒转化枯草芽孢杆菌Bacillussubtilis168,获得重组菌株B.subtilis 168DN。
2.根据权利要求1所述的一种脱氮基因工程菌的构建方法,其特征在于,所述重组菌株B.subtilis 168DN的菌落形态:30℃在LB平板培养12h,菌落表面粗糙不透明,边缘不规则,呈淡黄色。
3.根据权利要求1所述的一种脱氮基因工程菌的构建方法,其特征在于,所述重组菌株B.subtilis 168DN的细胞形态:16h培养物的细胞具有圆端的直杆菌或细胞接近球状,芽孢椭圆到柱状。
4.根据权利要求1所述的一种脱氮基因工程菌的构建方法,其特征在于,所述重组菌株B.subtilis 168DN的生理生化特征:革兰氏阳性,需氧型菌,化能异养。
5.根据权利要求1所述的一种脱氮基因工程菌的构建方法,其特征在于,所述羟胺氧化酶HAO编码基因含有如SEQ ID NO.1所示的基因序列。
6.根据权利要求1所述的一种脱氮基因工程菌的构建方法,其特征在于,所述氨单加氧酶AMO编码基因含有如SEQ ID NO.2所示的基因序列。
7.根据权利要求1所述的一种脱氮基因工程菌的构建方法,其特征在于,所述构建方法包括以下步骤:通过质粒重组,AMO和NeHAO基因串联重组进入pP43NMK质粒,重组至枯草芽孢杆菌基因组上,获得重组菌株B.subtilis 168DN。
8.一种脱氮基因工程菌的应用,其特征在于,权利要求1-7任一项所述的构建得到的脱氮基因工程菌在脱氮中的应用。
9.根据权利要求8所述的一种脱氮基因工程菌的应用,其特征在于,所述的脱氮基因工程菌在生活污水中应用。
10.根据权利要求8所述的一种脱氮基因工程菌的应用,其特征在于,取污水处理站的进水,将氨氮与总氮浓度均控制到50mg/L-60mg/L,然后将脱氮基因工程构建菌菌剂按1%接入调配后的生活污水中。
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