CN104726467B - 一种核苷酸序列及其在提高菌株耐酸性中的应用 - Google Patents
一种核苷酸序列及其在提高菌株耐酸性中的应用 Download PDFInfo
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Abstract
本发明公开了一种核苷酸序列，该核苷酸为全局转录调控因子的基因序列，本发明还公开了该核苷酸序列在提高菌株耐酸性中的应用。
Description
技术领域
本发明属于生物工程技术领域，特别涉及一种核苷酸序列及其在提高菌株耐酸性中的应用。
背景技术
细胞对酸胁迫的抗性差是传统发酵工业节能减排的关键共性问题。有机酸、氨基酸产业作为我国发酵产业的重要组成部分，其目的产物导致了不利于细胞生长与维持正常代谢活性的酸性微环境，并严重影响了生产条件稳定性的维持。迄今为止，绝大多数工业微生物的耐酸研究都集中在大规模的诱变和筛选，研究周期长、工作量大，且属于暗箱操作，不能根据目标表型获得相应的基因型，即不清楚突变发生的位置和机理。因此，研究微生物酸耐受性机制，提高微生物对酸耐受性十分重要。目前研究表明，工业微生物的耐酸机制同时涉及有机酸的跨膜运输、体内协同转运、以及与能量因子偶联等多种作用形式，是个复杂的科学问题。
大肠杆菌基因组共含有4000多个基因，这些基因的转录特异性受到两步调控：第一步就是RNA聚合酶σ因子的转录识别，第二步是约240-260个其他特异性转录因子的识别。在大肠杆菌的不同生长环境中，其RNA聚合酶是通过转录特异基因的选择性σ因子改变基因组的转录来调控整个生长过程。目前大肠杆菌中发现的σ因子有7种，分别为：σD（σ70）、σN（σ54）、σS（σ38）、σH（σ32）、σF（σ28）、σE（σ24）和σfecI。其中，σD是最主要的σ因子，负责与细胞生长相关的1000多个基因的转录控制，是提高大肠杆菌耐酸性的最佳对象。
随机片段交换法是一种新的突变文库构建方法，它汇集了多种DNA序列的突变形式，各种形式可以单独发生，也可以同时发生。2006年，Fujii等利用此方法对TEM-1型β-内酰胺酶进行进化，经过三轮突变，使其对头孢他啶（ceftazidime）的最小抑制浓度提高了5000倍。因此，将随机片段交换法替代易错PCR构建基因突变文库方法，不仅可以简化实验操作步骤，同时又能增强突变文库的多样性，将大大提高工业微生物耐酸表型定向筛选的效率。
发明内容
本发明的目的是通过突变全局转录调控因子σD，筛选得到能够提高大肠杆菌耐酸性的突变菌株，并获得该转录调控因子的多核苷酸序列。
为了达到本发明的技术目的，发明人以携带rpoD基因(gene ID 947567)完整序列（含上游天然启动子区域和下游终止子区域）的大肠杆菌E. coli DH5 alpha基因组为模板，构建rpoD基因的突变文库。将以随机片段交换法获得的rpoD突变基因为目的片段，以低拷贝表达质粒pKSC为载体，通过T4DNA连接酶连接，得到带有rpoD突变基因的重组质粒，经热激法转化E. coli DH5 alpha感受态细胞，涂布含有30 μg/mL卡那霉素的LB平板，37℃培养过夜。洗脱单菌落，利用平板试管交替筛选的模式，最后筛选得到显著提高大肠杆菌酸耐酸性的trpoD。
在本发明中的大肠杆菌基因工程菌中的σD突变基因trpoD携带12个突变位点，分别为Pro60Ser, Thr95Ile, Phe221Leu, Ile249Phe, Ile287Val, Ile511Val,His518Leu, Glu538Val, Ala542Val, Asp566Asn, Glu605Gly, Leu607Gln，其中，碱基序列1-194位为天然启动子区域，2051-2075位为终止密码子区域；携带该sigma D突变基因trpoD的大肠杆菌E.coli DH5 alpha无需添加IPTG诱导表达，且能耐低pH，而普通大肠杆菌在低pH条件下就停止生长。
基于此，本发明提供的技术方案包括：
一种多核苷酸，包含由SEQ ID NO：1代表的核苷酸序列，或由SEQ ID NO：1代表的核苷酸序列中第1至1842位的核苷酸残基组成的核苷酸序列。
来源于大肠杆菌的如权1所述的多核甘酸。
一种蛋白质，包含由SEQ ID NO：2代表的全长氨基酸序列，或由SEQ ID NO：2代表的全长氨基酸序列中第1至614位氨基酸残基组成的氨基酸序列。
本发明所述的蛋白质其含有，Pro60Ser, Thr95Ile, Phe221Leu, Ile249Phe,Ile287Val, Ile511Val, His518Leu, Glu538Val, Ala542Val, Asp566Asn, Glu605Gly,Leu607Gln，12个突变位点。
含有如权利要求1所述多核苷酸序列，或如权利要求2或3所述蛋白质的多核苷酸序列表达载体。
含有如权利要求5所述表达载体的菌株；该菌株优选为大肠杆菌。
本发明所述的多核苷酸在提高菌株耐酸性中的应用。
构建耐酸性菌株的方法，包含如下步骤：
（1）以携带rpoD基因完整序列的大肠杆菌E. coli DH5 alpha基因组为模板进行随机突变，获得rpoD突变基因；
（2）以rpoD突变基因为目的片段，以低拷贝质粒pKSC为表达载体，构建σD基因突变库；
（3）将载体转入大肠杆菌，并进行高通量筛选，以低pH为筛选压力，进行液体和琼脂平板交替筛选。
通过本发明的技术方案，可以带来以下有益技术效果：
（1）通过对全局转录调控因子σD连续突变，显著提高了大肠杆菌对低pH的耐受性，为解决大肠杆菌等工业微生物细胞对酸胁迫响应调控过程提供了方法，使人们能更有效地控制和设计微生物细胞抗逆元器件，有助于提高工业微生物的应用效益。
（2）提供了新的抗酸性多核苷酸序列，该序列可用于提高工业微生物的耐酸性。
附图说明
图1 携带sigma D突变基因trpoD的质粒pKSC电泳图。
图2 大肠杆菌E. coli DH5 alpha-pKSC-trpoD和大肠杆菌E. coli DH5 alpha-pKSC-rpoD在pH4.0时的耐酸情况。
图3 大肠杆菌E. coli DH5 alpha-pKSC-trpoD和大肠杆菌E. coli DH5 alpha-pKSC-rpoD在pH3.5时的耐酸情况。
具体实施方式
下面结合实施例对本发明做进一步说明。所列的实施例仅作阐示
实施例1本实施例说明利用随机片段交换法定向突变出发菌株大肠杆菌E. coliDH5 alpha中编码sigma D的基因rpoD，并构建σD突变基因文库的方法。
（1）利用LB培养基，于37℃，有氧条件下过夜活化大肠杆菌E. coli DH5 alpha，利用Takara细菌基因组抽提试剂盒提取基因组。
（2）以大肠杆菌E. coli DH5 alpha基因组为模板，PCR扩增rpoD基因片段，所用引物，单下划线同源片段：
rpoD-F: CCGAATTCTGATTTAACGGCTTAAGTGCCGAAG；
rpoD-R: TACGGCTTGCCGGGTGCGGCGTAAC。
PCR反应条件：94℃变性4 min，经94℃30 sec，56℃30 sec，72℃1 min，30个循环，再经过72℃延伸10 min，降温至16℃。获得的PCR产物经电泳分析确认。
（3）对目的基因rpoD进行纯化；用DNase I消化，获得一系列大小在100-300 bp之间的DNA碎片，再用末端脱氧核苷酸转移酶（TdT）在小片段的3'端随机添加/删除5个左右的核苷酸；以带3'尾巴的短片段互为引物进行PCR重新组装，加入两端引物扩增出全长突变基因rpoD。获得的PCR产物经电泳分析确认。
（4）对随机片段交换法获得的突变基因片段进行纯化；以低拷贝质粒pKSC为表达载体，经过HindIII和EcoRI限制性内切酶消化，通过T4DNA连接酶16℃金属浴连接过夜，构建σD突变基因文库。其中附图1为携带σD突变基因trpoD的质粒pKSC电泳图。其中pKSC表达质粒的构建参见文献Chong H, Geng H, Zhang H, et al. Enhancing E. coli isobutanoltolerance through engineering its global transcription factor cAMP receptorprotein (CRP)[J]. Biotechnology and bioengineering, 2014, 111(4): 700-708.。
（5）热激法转化σD突变基因（含有卡那霉素基因）至大肠杆菌E. coli DH5 alpha感受态，并涂布于带有卡那霉素的LB平板筛选阳性重组子，通过PCR鉴定，获得含有σD突变基因的菌株大肠杆菌E. coli DH5 alpha。
实施例2 本实施例说明以低pH为筛选压力，利用液体和琼脂平板交替筛选的方式获得耐酸性显著提高的大肠杆菌基因工程菌的方法。
（1）利用LB培养基，于37℃，有氧条件下培养含有σD突变基因的菌株大肠杆菌E. coli DH5 alpha至OD600=0.2，以菌株大肠杆菌E. coli DH5 alpha-pKSC-rpoD为对照组，加入8 mol/L稀盐酸，培养基pH分别调至4.5，5，6，7，37℃培养24 h后，将菌液稀释106后，吸取700 μL菌液涂布于LB/Kana平板上，37℃培养至单菌落产生。
（2）以pH4.5为初筛起点，挑取平板上的100个单菌落。将菌株接种至10 mL试管中，检测100株菌株的OD600值，其中101号为对照菌株（WT）。当菌株生长到OD600为0.2时，试管中pH调至4.5，37℃培养12 h，其中有10株菌株生长OD600=0.6~0.8。挑取该10株突变菌株进行进一步筛选。
（3）将10株突变菌株接种至10 mL试管中，检测10株菌株的OD600值，其中11号为对照菌株（WT）。当菌株生长到OD600为0.2时，试管中pH调至4，37℃培养12h。根据培养结果显示，G1生长情况最好，OD600=0.868，而对照菌株停止生长。
（4）提取G1携带σD突变基因的质粒pKSC，导入大肠杆菌E. coli DH5 alpha感受态，并涂布于带有卡那霉素的LB平板筛选阳性重组子，通过PCR鉴定，获得含有σD突变基因trpoD的菌株大肠杆菌E. coli DH5 alpha。
实施例3 本实例说明新构建的大肠杆菌基因工程菌株E. coli DH5 alpha-pKSC-trpoD和携带野生型σD基因rpoD的大肠杆菌基因工程菌株E .coli DH5 alpha-pKSC-rpoD氨基酸序列和耐低pH能力的对比。
（1）利用LB培养基，于37℃，有氧条件下过夜培养大肠杆菌E. coli DH5 alpha-pKSC-trpoD。收集菌体，提取质粒，送至南京金斯瑞生物科技公司进行测序。
（2）根据测序结果，以携带野生型σD基因rpoD的大肠杆菌E. coli DH5 alpha-pKSC-rpoD为对照，大肠杆菌E. coli DH5 alpha-pKSC-trpoD共有12个突变位点，分别为Pro60Ser, Thr95Ile, Phe221Leu, Ile249Phe, Ile287Val, Ile511Val, His518Leu,Glu538Val, Ala542Val, Asp566Asn, Glu605Gly, Leu607Gln，其中，突变位点主要集中于rpoD片段的Region 4区域，影响RNA聚合酶的启动子区域识别。
（3）以携带野生型σD基因rpoD的大肠杆菌E. coli DH5 alpha-pKSC-rpoD为对照，将突变菌株大肠杆菌E. coli DH5 alpha-pKSC-trpoD大肠杆菌E. coli DH5 alpha-pKSC-rpoD接种至10mL试管中。当菌株生长到OD600为0.2时，试管中pH调至3.5，4，37℃培养10h，见图3。
序列表
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Claims (1)
1.由SEQ ID NO:1代表的核苷酸序列在提高大肠杆菌E. coli DH5菌株耐酸性中的应用。
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