CN110129245B - 一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌及其构建方法与应用 - Google Patents
一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌,该菌株中胞外核酸酶基因ExeP失活,所述的谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13032,所述胞外核酸酶ExeP的基因序列如SEQ ID NO.1所示,失活后的胞外核酸酶ExeP的基因序列如SEQ ID NO.2所示。本发明通过敲除敲除胞外核酸酶ExeP,减少胞外DNA的降解,加强了谷氨酸棒杆菌的成膜能力,利用本发明构建的敲除胞外核酸酶ExeP的谷氨酸棒杆菌发酵产脯氨酸,提高了脯氨酸连续化发酵的产量,缩短了发酵周期。
Description
技术领域
本发明属于生物工程技术领域,具体涉及到一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌及其构建方法与应用。
背景技术
生物膜广泛存在于自然界,在生物膜形成过程中,细菌自身分泌产生的的胞外聚合物(EPS)是生物膜形成的物质基础,具有分层分布的特点,对细菌的粘附及聚集特性起到了关键作用。从化学组成而言,胞外聚合物主要由蛋白质、多聚糖、胞外DNA等构成。新研究表明eDNA对于EPS结构的完整性及生物膜的三维结构起到关键作用,不仅影响EPS关键组分的空间分布,而且直接决定生物膜的最初形成过程。eDNA作为 EPS的关键组分,对于维系生物膜的空间稳定性具有十分重要的作用,eDNA在生物膜形成过程中强化了细胞聚集,与蛋白质、多聚糖等相互结合缠绕,起到了“脚手架”和“蜘蛛网”的作用,进一步促进了生物膜的结构完整性。
谷氨酸棒状杆菌作为重要的工业菌株,其成膜能力非常弱,难以连续化发酵,了解和阐明eDNA对生物膜的重要作用有可以运用到谷氨酸棒状杆菌上,弄清eDNA与生物膜中其他胞外分泌物的关系可以为我们筛选改造谷氨酸棒状杆菌的基因,可以促进谷氨酸棒状杆菌生物膜的形成。
发明内容
本发明所要解决的技术问题是提供一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌,以促进谷氨酸棒杆菌生物膜的形成。
本发明还要解决的技术问题是提供谷氨酸棒杆菌的构建方法。
本发明最后要解决的技术问题是提供上述谷氨酸棒杆菌的应用。
为了解决上述问题,本发明采用的技术方案如下:
一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌,该菌株中胞外核酸酶基因ExeP失活,所述的胞外核酸酶基因ExeP失活是指胞外核酸酶基因ExeP不能正常表达,或者胞外核酸酶基因ExeP不能表达得到具有胞外核酸酶功能的蛋白质。
胞外核酸酶基因ExeP被为细胞外内切核酸酶,编码细胞外内切核酸酶的产生,可以分解胞外DNA。
其中,所述的谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13032。
其中,所述胞外核酸酶ExeP的基因序列如SEQ ID NO.1所示。
其中,失活后的胞外核酸酶ExeP的基因序列如SEQ ID NO.2所示。
上述敲除胞外核酸酶ExeP的谷氨酸棒杆菌的构建方法,包括如下步骤:
(1)构建ExeP基因敲除片段,所述基因敲除片段的核苷酸序列如SEQ ID NO.3 所示;
(2)将ExeP基因敲除片段克隆到敲除质粒上,得到重组质粒;
(3)将重组质粒转化谷氨酸棒杆菌,筛选得到胞外核酸酶基因ExeP失活的基因工程菌。
步骤(2)中,所述的敲除质粒为pk18mobsacB。
步骤(3)中,所述的谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13032。
上述敲除胞外核酸酶ExeP的谷氨酸棒杆菌在微生物发酵中的应用在本发明的保护范围之内。
上述敲除胞外核酸酶ExeP的谷氨酸棒杆菌在制备脯氨酸中的应用在本发明的保护范围之内。
有益效果:
本发明构建了一株敲除胞外核酸酶ExeP的谷氨酸棒状杆菌,通过敲除胞外核酸酶ExeP,减少谷氨酸棒杆菌胞外DNA的降解,加强了谷氨酸棒杆菌的成膜能力,提高了谷氨酸棒杆菌连续化发酵的产量,缩短了发酵周期。
附图说明
图1:左右同源臂PCR琼脂糖凝电泳图(第一泳道为左同源臂,第二泳道为2000DNAMarker,第三泳道为右同源臂)。
图2:左右同源臂Cross-over PCR琼脂糖凝电泳图(第一泳道为2000DNAMarker,第二泳道为左右同源臂Cross-over PCR条带)。
图3:ExeP基因敲除转化子基因组PCR琼脂糖凝电泳图(第一泳道为重组菌基因组PCR条带,第二、三泳道为原始菌基因组PCR条带,第四泳道为10000DNAMarker)。
图4:原始菌和重组菌生物膜CLSM电镜图。
图5:脯氨酸连续固定化发酵产量示意图。
图6:脯氨酸连续固定化发酵周期示意图。
具体实施方式
实施例1:构建ExeP基因敲除菌。
用于ExeP基因敲除的重组质粒pk18mobsacB/△ExeP是通过Cross-over PCR获得的。左同源臂(726bp)引物ExeP-L-F、ExeP-L-R和右同源臂(750bp)引物ExeP-R-F、 ExeP-R-R分别以原始菌株谷氨酸棒状杆菌ATCC13032基因组为模板进行常规PCR分别获得左右同源臂。纯化后的左右同源臂等量混合为模板,ExeP-L-F、ExeP-R-R为引物进行Cross-overPCR,获得将左右同源臂连接到一起的ExeP内部有缺失的片段。将该片段与经BamH1单酶切后的pk18mobsacB质粒一步克隆,获得用于基因敲除的质粒 pk18mobsacB/△ExeP。
谷氨酸棒状杆菌ATCC13032中ExeP基因敲除方法:谷氨酸棒状杆菌ATCC13032 中ExeP基因敲除是通过两次同源重组的原理,其大意为,通过电转化先将pk18mobsacB/ △ExeP重组质粒转入谷氨酸棒状杆菌ATCC13032的感受态中进行基因打靶。在含卡那霉素的LB平板上筛选经过第一次同源重组的卡那霉素抗性克隆,即pk18mobsacB/△ ExeP质粒通过同源重组整合到了谷氨酸棒状杆菌ATCC13032的基因组上。将转化子转接到LB液体培养基中,30℃、220r/min振荡培养过夜后涂布于含10%蔗糖的LB平板上,通过蔗糖致死基因(sacB基因)负筛选出经过第2次同源重组的克隆。将转化子分别转接到含有30mg/L卡那霉素的LB平板和LB平板上生长,能在LB平板上生长,但对卡那霉素敏感的菌株即是经过第2次同源重组的克隆。经过第2次同源重组的不一定是基因缺失的突变,也可能是恢复突变,需要对基因组进一步的PCR验证。
表1引物序列
基因ExeP(777bp)+前后各1000bp:这一共是2777bp,其中前726bp作为上同源臂,后750bp作为下同源臂,上下同源臂PCR如下图1所示,上下同源臂通过cross-over PCR如图2所示,质粒敲除结果如图3所示。ExeP基因组序列和敲除ExeP基因序列如下所示。
实施例2:CLSM检测生物膜
改造菌株构造成功后进行96孔板以及12孔板实验,以及CLSM电镜实验。电镜照片可以直观具体的看出生物膜多少及形态。
为更深入详细的观察生物膜结构,以及重组菌和原始菌生物膜的量以及在微观结构上的区别,我们用DAPI荧光染色剂染细胞核再使用CLSM电镜20倍目镜条件下观察生物膜微观结构。结果如图4所示,敲除胞外核酸酶基因ExeP的生物膜量明显多于原始菌。
经过验证改造后的菌株确实比原始菌株生物膜形成量多,这为以后做连续化发酵提供了基础。
实施例3:基因重组菌发酵产脯氨酸。
活化培养基成分如下:蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L。
种子培养基成分如下:葡萄糖30g/L、玉米浆20g/L、硫酸铵7g/L、七水硫酸镁0.7g/L、磷酸二氢钾1.5g/L、尿素3g、生物素0.2g/L。
发酵培养基成分如下:葡萄糖110g/L、玉米浆25g/L、硫酸铵25g/L、七水硫酸镁0.5g/L、磷酸二氢钾1.5g/L、尿素3g/L、生物素0.3g/L、Ca2+5g/L。
菌株活化:在超净台中,将-80℃下装有谷氨酸棒杆菌的甘油冻存管置于冰上,用微量移液器分别吸取200μL原始菌(谷氨酸棒杆菌ATCC13032)和改造菌(谷氨酸棒杆菌ATCC13032-△ExeP)加入装有5ml活化培养基的50ml离心管中,在30℃,220rpm 条件下活化18h。
摇瓶种子培养:在超净台中,将上述活化完成后的菌液以5%(v/v)的接种量分别接入装有50ml种子培养液的500ml摇瓶中,瓶口包裹八层纱布,在30℃,220rpm/min 条件下培养12h。
摇瓶发酵培养:每500ml的摇瓶加入50ml的发酵培养液,将上述培养好的种子液以10%(v/v)的接种量接入到发酵培养基中,在30℃,220rpm/min,去牛皮纸,八层纱布包裹条件下发酵72,每12h加入250μL尿素。
固定化发酵:在发酵过程中,第一批时菌体已经吸附到固定化载体上,此时摇瓶培养已经过72h,第二批时倒掉发酵液,留下吸附着菌的固定化载体,再倒入新的50ml 发酵液,培养至糖耗尽,约60h。随后批次固定化发酵均采用此法,游离发酵过程中不添加固定化载体。
聚氨酯载体材料的预处理:将聚氨酯载体剪成0.5cm×0.5cm×0.5cm的立方体,用纯水洗净烘干后于乙醇中浸泡1h,再用纯水清洗2遍后沸水浴20min后放入烘箱烘干,称重1.5g,然后放入倒有发酵液的摇瓶当中一起灭菌,115℃,15min。
实施例4:还原糖的测定
运用DNS(二硝基水杨酸)在碱性条件下,与还原糖发生氧化还原反应,生成3- 氨基-5-硝基水杨酸,该产物在煮沸条件下显棕红色,且在一定浓度范围内颜色深浅与还原糖含量成比例,用紫外分光光度计(OD540)测定还原糖含量。
样品适当稀释后,使预计含糖量在0.1-1.0mg/mL之间,取稀释后的0.5mL样品于10mL刻度试管中,再加入0.5mL DNS,沸水煮沸5min,立即冷却5min,加8mL纯水混匀,另设0.5mL纯水和0.5mL DNS样品为空白样。事先做好本次实验使用的DNS 还原糖含量标准曲线,用紫外分光光度计在540nm波长下检测吸光值,作为还原糖含量的量度。
实施例5:脯氨酸含量的测定
每瓶发酵液取样100μl用0.1M盐酸溶液稀释60倍,再将每个样品分别取400μl稀释液,接着进行PITC柱前衍生,用RP-HPLC法进行脯氨酸含量检测,计算出每个样品的脯氨酸浓度。
利用出发菌和本发明构建的重组菌进行连续发酵实验,通过10批次发酵实验,发酵结果见表1。由表中数据可以看出,在原始菌连续化发酵到第七批的时候发酵产量趋于稳定,改造菌连续化发酵到第十批时产量达到最高,出发菌和重组菌第十批次产量分别为13.16g/L和16.01g/L,均比出发菌的初始产量高。从图5可以看出,改造后的菌株固定化产量比原始菌高出60%。从图6可以看出,改造后的菌株固定化发酵周期比原始菌缩短32%。
表2固定化菌株连续发酵实验
序列表
<110> 南京工业大学
<120> 一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌及其构建方法与应用
<160> 7
<170> SIPOSequenceListing 1.0
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<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
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atggctcgat cacaaaaaag aacacctgct atcaggtcaa ccaaaaaagt taaaagtgta 60
atatccagca tcatcacgat tgccgtggct gcagtcgctt ttgcagctta cgttatagat 120
ggtggggtag aagaggcgtc tggaacaccg acgtcttcgg aaagctcggt agcggcaact 180
gctccagcgg catctagcga gactgcggct gaataccgtg cgatgctcgc ttcccttgac 240
gttaaaggtc gtgcgccagg aacaggatat gaccgcgaat tattcggacc agcatggacc 300
gacactgttt ccgtggaata tggacacaat ggctgcgata cccgcaacga catcctgcaa 360
cgcgacctgg atgacatcca acttcgcgaa ggcaccaagg attgtatcgt cacgagcggc 420
ctgctcagcg atccattttc tggcgaactt attgatttcg ttcgcggtga acgttccggc 480
gacgtgcaga tcgatcacct ggtcccatta catgacgcat gggtcaaggg agcacagcag 540
tgggatgagc aaactcgaaa gaactttgcc aacgatcccg acaaccttct cgccgttaaa 600
ggtacgctta accagcaaaa aggtgcaggc gatgcagcaa cctggcttcc accaaacaca 660
gcttttaggt gcgattacgc aaagaaaatc atcaccgtta aagatcgcta caacgtgtgg 720
gtgactgagg ctgaagcaag cgccctggaa cgccaattag atacgtgtgc tgcataa 777
<210> 2
<211> 300
<212> DNA
<213> 人工序列(Artificial Sequence)
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atggctcgat cacaaaaaag aacacctgct atcaggtcaa ccaaaaaagt taaaagtgta 60
atatccagca tcatcacgat tgccgtggct gcagtcgctt ttgcagctta cgttatagat 120
ggtggggtag aagaggcgtc tggaacaccg ggcgatgcag caacctggct tccaccaaac 180
acagctttta ggtgcgatta cgcaaagaaa atcatcaccg ttaaagatcg ctacaacgtg 240
tgggtgactg aggctgaagc aagcgccctg gaacgccaat tagatacgtg tgctgcataa 300
<210> 3
<211> 1452
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aattcgagct cggtacccgg ggatccacga gcgctgggac agtaatgccg cgcgggtaat 60
cggggaagcg gttgaaatac gcgtcctgta gacgaggaat ctgcagcacg ggatcaagat 120
ggtttggatc taaatcgaaa gtccaggaac gaacgtcgtg agtcgggccg gtcagaccaa 180
gcgagatcac gttctccagg ccgagaagac gacgggtgat aacagtgcgg tgtgcccatg 240
gacatgcgcg ggcagcgact aaacggtagc gaccagcctc gacaggccaa tggaaagtgc 300
catcttcctg agcaattggt tcggatcccg ctggaacgtc tgcgacgatg cggtcatcga 360
tgtagttggt atcgcgaacg aactcgccgt ctgctgatgc attttgtggg gcgcctgccc 420
aatcggatga cgtgttagcc actgtgtggt gtcctctctt ggaaatttag atgatatgtc 480
aacaatatta cgaagtggaa tgaaaatcaa cgattgaaca aatgttcggg gtgattgcag 540
acgtgtcctg cacagttgat atggtgtgaa accatcatgg ctcgatcaca aaaaagaaca 600
cctgctatca ggtcaaccaa aaaagttaaa agtgtaatat ccagcatcat cacgattgcc 660
gtggctgcag tcgcttttgc agcttacgtt atagatggtg gggtagaaga ggcgtctgga 720
acaccgggcg atgcagcaac ctggcttcca ccaaacacag cttttaggtg cgattacgca 780
aagaaaatca tcaccgttaa agatcgctac aacgtgtggg tgactgaggc tgaagcaagc 840
gccctggaac gccaattaga tacgtgtgct gcataacagt cacataagca tttggggcgc 900
gccgaagtta tatccacatt gcggaagtta attcgcatat tctggggcac atgactgata 960
actcgcgaaa ttcaaatcag aattttgatc aaccagacct gaacgatctc gatgatgatt 1020
cggccatccc tacttacaag ggaccatcgc cttccgcgaa ttcctccaac gctaccgagc 1080
gcctcggcag tgtttacgac cgaactggcc gtgcagcgcc ccagaacatt ccgccggcat 1140
caaatgctgc tgaaaccaca gcatttgagc gtccagataa tcaatctgcg aagccagtgg 1200
cttccgaagc tccaacaact attacgccgg caagctctgg tgcattagca tccgatgctc 1260
cgacgtctta tgttcaggcg cagccttccc agcagcagac tcctcagcaa gttccgccag 1320
ctccgccgac acaagttact cgtcagatta gccgtcctga agagcctggt taccagccag 1380
aaccgtctta ttcggagcct tacactgact ctgatttcgc gccagcggat cctctagagt 1440
cgacctgcag gc 1452
<210> 4
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aattcgagct cggtacccgg ggatccacga gcgctgggac agtaatgc 48
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cggtgttcca gacgcctctt c 21
<210> 6
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaagaggcgt ctggaacacc gggcgatgca gcaacctggc tt 42
<210> 7
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
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gcctgcaggt cgactctaga ggatccgctg gcgcgaaatc agagtcag 48
Claims (6)
1.一株敲除胞外核酸酶ExeP的谷氨酸棒杆菌,其特征在于,该菌株中胞外核酸酶基因ExeP失活;
所述的谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13032;
所述胞外核酸酶ExeP的基因序列如SEQ ID NO.1所示;
失活后的胞外核酸酶ExeP的基因序列如SEQ ID NO.2所示。
2.权利要求1所述敲除胞外核酸酶ExeP的谷氨酸棒杆菌的构建方法,其特征在于,包括如下步骤:
(1)构建ExeP基因敲除片段,所述基因敲除片段的核苷酸序列如SEQ ID NO.3所示;
(2)将ExeP基因敲除片段克隆到敲除质粒上,得到重组质粒;
(3)将重组质粒转化谷氨酸棒杆菌,筛选得到胞外核酸酶基因ExeP失活的基因工程菌。
3.根据权利要求2所述的敲除胞外核酸酶ExeP的谷氨酸棒杆菌的构建方法,其特征在于,步骤(2)中,所述的敲除质粒为pk18mobsacB。
4.根据权利要求2所述的敲除胞外核酸酶ExeP的谷氨酸棒杆菌的构建方法,其特征在于,步骤(3)中,所述的谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13032。
5.权利要求1所述敲除胞外核酸酶ExeP的谷氨酸棒杆菌在微生物发酵中的应用。
6.权利要求1所述敲除胞外核酸酶ExeP的谷氨酸棒杆菌在制备脯氨酸中的应用。
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