CN103911390B - 一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法 - Google Patents
一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法 Download PDFInfo
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Abstract
本发明公开了一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法，属于遗传工程领域。本发明以可用于黄酮生产的大肠杆菌生产菌株为受体，将其OmpA基因进行沉默获得，能显著获得一株能耐受黄酮类化合物的工程菌，外源添加黄酮类化合物于E.coli生长环境中，相较于原始菌，工程菌的最大比生长速率相较于原始菌提高了2.35倍。
Description
技术领域
本发明涉及一种提高大肠杆菌黄酮类物质耐受性的方法发现一种蛋白的新功能，特别是一种通过调节膜蛋白OmpA表达量，以提高从而提高了E.coli对黄酮类化合物的耐受能力。
背景技术
黄酮类化合物(Flavonoids)是自然界种类最多的多酚类植物次级代谢产物，广泛存在于植物界中，目前已经发现的种类已超过8000多种。黄酮类化合物具有广泛的药学价值，具有较强的抑菌、抗炎、抗氧化、抗肿瘤、清除自由基、免疫调节、抗癌、抗病毒、降血糖、抗辐射和降血脂等多种生物学作用。随着对黄酮类化合物生物活性研究的逐步深入，对其的开发利用也越来越多，对其的需求量也越来越大，近年来，黄酮类化合物的市场每年增长30％以上，在药品和营养化学品领域上具有广阔的应用前景。但天然黄酮类物质的获得受到时间和区域的限制，而且在植物中含量低，不利于迅速研究和临床检验，并且化学结构很复杂，用化学合成法很复杂，不利于大规模的生产。因此用微生物生产黄酮类化合物越来越受到人们的重视。
随着黄酮类化合物的应用范围越来越广，其需求量也在不断的增大，从植物中提取有各种各样的问题，因此，通过微生物发酵生产黄酮类化合物具有很大的应用前景。目前，黄酮类化合物生物合成的代谢途径已经非常清楚，黄酮类化合物的基本骨架是由3个丙二酰辅酶A(malonylCoA)和1个香豆酰辅酶A(coumaroylCoA)在查尔酮合成酶(CHS)的催化下合成的，然后其他种类的黄酮是在母核的基础上通过羟基、甲氧基取代或烷基化等化学反应生成的。大肠杆菌遗传背景清晰，分子操作技术完善，是生物技术生产平台的首选，因此很早就有人通过改造E.coli相关代谢途径生产黄酮类化合物。目前很多研究成功通过基因工程等技术手段实现了多种黄酮类化合物在E.coli中的合成，如柚皮素、槲皮素、白藜芦醇等。但是黄酮类化合物本身对E.coli生长具有一定的抑制作用，主要是通过以下几种机制：①破坏细胞壁及细胞膜的完整性，改变细胞膜渗透性，进而破坏细胞膜的功能，影响细菌的生长；②抑制核酸的合成，槲皮素，芹黄素，五羟基黄酮等能够抑制E.coli的DNA旋转酶的活性进而抑制DNA的合成。芦丁能够促进E.coli的DNA裂解，导致SOS反应发生，从而抑制菌体的生长；③抑制能量代谢，通过抑制ATP合酶的活力进而抑制胞内ATP的含量，影响菌体的生长。因此利用E.coli生产黄酮类化合物必须要考虑黄酮类化合物对E.coli的毒性作用。
目前在国内未有关于增加E.coli对黄酮类化合物耐受性的相关蛋白的报道。
发明内容
本发明要解决的技术问题是找出能提高E.coli对黄酮类化合物耐受性的相关蛋白并提供一种能提高大肠杆菌黄酮类化合物的方法，以可用于黄酮生产的大肠杆菌生产菌株为受体，过量表达ompA基因。
所述ompA基因核苷酸序列如SEQ ID NO.1所示。
所述ompA基因克隆于E.coli BL21(DE3)菌株，并构建重组质粒pACYC-ompA并转入E.coli BL21(DE3)中。
所述黄酮类化合物为芦丁，柚皮素或白藜芦醇。
本发明的提出的基础及前期试验：
1)在E.coli稳定期时添加黄酮类化合物刺激后收集菌体细胞，提取膜蛋白，通过蛋白质组学的技术手段，发现OmpA蛋白的表达量有显著的提高。
2)提高qPCR技术验证该蛋白在这一生理过程中的mRNA水平。
3)通过Ncbi查出E.coli中该基因的核苷酸序列设计引物并克隆。
4)将基因与载体连接得到重组表达载体；
5)将得到的重组表达载体转化E.coli BL21(DE3)后得到重组菌株；
6)重组菌株进行IPTG诱导后进行SDS-PAGE验证蛋白是否表达；
7)通过对在黄酮类化合物存在的环境下生长曲线的测定，验证该蛋白的过量表达是否提高了E.coli对黄酮类化合物的耐受能力。
下面是本发明技术方案的具体描述：
OmpA蛋白的发现
挑取E.coli BL21(DE3)单菌落用LB培养基活化后，按1％接种量接种于25mL LB培养基，培养到稳定期(10h)添加1g/L黄酮类化合物(芦丁，柚皮素，白藜芦醇)刺激3h后收集菌体细胞。按照FOCUSTM Global Fractionation说明书提取膜蛋白，并用2-D Clean-Up试剂盒除去蛋白样品中的离子，最后用非干扰性蛋白质浓度测定试剂盒测定样品中的浓度，最后按照450μL含有800ng的浓度水化上样，使用24cm的pH4-7的胶条进行IEF，然后平衡两次后用13.5％的SDS-PAGE进行第二向，结束后用R250进行染色12h，脱色后用ImageScannerIII仪器对胶进行拍照，获得清楚图像后用PDQuest进行图谱分析，挖出差异较大的点进行脱色，酶解等步骤，然后利用MALDI-TOF/MS质谱对肽段样品进行蛋白质鉴定，通过检索Matrix Science数据库，确定蛋白种类。
qPCR
收集菌体的方法同上，利用天根公司的RNAprep Pure Cell/Bacteria试剂盒提取菌株细胞的总RNA，利用TaKaRa公司的RT reagent试剂盒将RNA反转录成cDNA构建cDNA文库，利用TaKaRa公司的SYBR Premix ExTaq试剂盒和LightCycler480II RealTime PCR仪器进行qPCR反应，具体程序如下：95℃预变性30s；95℃5s→55℃20s，40次循环(PCR反应)；95℃10s→65℃1min→95℃10s(溶解曲线分析)。为保证试验的重现性，每个样品3个平行，然后取平均值作为计算的基础。
质粒及重组菌的构建
将从Ncbi上查到的ompA的基因序列设计引物进行扩增，分别连接到克隆载体pMD19-T测序，获得正确的转化子后进行双酶切连接到质粒pACYC-Duet1上，构建得到重组质粒pACYC-ompA，将构建好的重组表达载体转化E.coli JM109，菌落PCR验证阳性转化子，提取构建好的重组质粒转入E.coli BL21(DE3)菌中获得过量表达ompA的重组菌株。
生长曲线验证
用M9培养基(13.3g/L M9CA Broth,0.2％葡萄糖,1mM硫酸镁,0.5μg/mLthiamine-HCl)活化工程菌(加入1％的氯霉素)和原始菌，按照1％的接种量接种入分别含有1g/L(芦丁，柚皮素或者白藜芦醇)，IPTG(0,100,200,400mM)M9培养基中，30℃200r/min培养，每隔1h测OD600，直至菌株生长到稳定期。获得菌株的生长曲线图，然后根据公式μ＝dx/x*dt算出菌株的比生长速率图，然后得出最大比生长速率μmax。
使用本发明提供的方法能显著提高工程菌耐黄酮类化合物能力，外源添加黄酮类化合物刺激E.coli生长，相较于原始菌，工程菌的最大比生长速率相较于原始菌提高了2.35倍。
具体实施方式
实施例1OmpA的发现
用接种针在E.coli BL21(DE3)平板上挑取单菌落接种于25mL LB培养基，活化12h。按照1％的接种量分别接种于4瓶25mL LB培养基，置于37℃摇床(200rpm)培养至稳定期后，其中三瓶分别添加25μL用DMSO溶解的浓度为0.1g/L的芦丁，柚皮素和白藜芦醇(终浓度为1g/L)，另外一瓶添加25μL DMSO作为对照，培养3h后8000rpm低温离心收集菌体，用去离子水洗3次后按照FOCUSTM Global Fractionation说明书提取膜蛋白，并用2-D Clean-Up试剂盒除去蛋白样品中的离子，最后用非干扰性蛋白质浓度测定试剂盒测定样品中的浓度，最后按照450μL含有800ng的浓度水化上样，使用24cm的pH4-7的胶条进行IEF，然后平衡两次后用13.5％的SDS-PAGE进行第二向，结束后用R250进行染色12h，脱色后用ImageScanner III仪器对胶进行拍照，获得清楚图像后用PDQuest进行图谱分析，其中一个胶点在有芦丁，柚皮素和白藜芦醇的样品中相较于对照组均有2倍以上的上调，将此点挖出进行脱色，脱水，酶解等步骤，然后利用MALDI-TOF/MS质谱对肽段样品进行蛋白质鉴定，通过检索Matrix Science数据库得出此点是E.coli属的OmpA蛋白。
实施例2耐黄酮类化合物工程菌的构建
将从NCBI上查到的ompA的基因序列设计引物进行扩增，分别连接到克隆载体pMD19-T测序，获得正确的转化子后进行双酶切连接到质粒pACYC-Duet1上，构建得到重组质粒pACYC-ompA，将构建好的表达载体转化到E.coli JM109，涂布到含有氯霉素的LB培养基上(酵母膏5g/L，蛋白胨10g/L，NaCl10g/L，固体培养基加20g/L琼脂，121℃灭菌15min)，挑取转化后平板上的转化子进行PCR验证，得到正确的单菌落进行发酵提取质粒，将质粒转入E.coli BL21(DE3)感受态，涂布到含有氯霉素的LB培养基上，挑取转化后平板上的转化子进行PCR验证，得到正确的单菌落即为重组菌株，也即获得高耐受黄酮类化合物的E.coliBL21(DE3)工程菌。
实施例2高耐受黄酮类化合物E.coli BL21(DE3)工程菌生长曲线测定
挑取工程菌和含有空质粒pACYC-Duet1的E.coli BL21(DE3)单菌落接种到含有氯霉素的M9培养基(13.3g/L M9CA Broth,0.2％葡萄糖,1mM硫酸镁,0.5μg/mL thiamine-HCl)中活化10h后，按1％接种量接入含有1％氯霉素，1g/L黄酮类化合物(芦丁，柚皮素，白藜芦醇)，以及不同浓度的IPTG(0,100,200,400μM)的M9培养基中，30℃200rpm培养，每隔1h测一次OD600直至菌株生长到稳定期。根据公式μ＝dx/x*dt算出菌株的比生长速率图，然后得出最大比生长速率μmax，发现在有1g/L芦丁的环境中工程菌的μmax为0.43，而原始菌株的为0.24，生长速率提升了1.8倍；在1g/L的柚皮素环境中工程菌的μmax为0.53，而原始菌株的为0.27，生长速率提升了1.96倍；在1g/L的白藜芦醇环境中工程菌的μmax为0.33，而原始菌株的为0.14，生长速率提升了2.35倍。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (4)
1.一种通过调节OmpA表达提高大肠杆菌黄酮类物质耐受性的方法，其特征在于以能够用于黄酮生产的大肠杆菌生产菌株为受体，对其OmpA基因进行过量表达，以提高对黄酮类物质的耐受性；所述OmpA基因的核苷酸序列如SEQ ID NO.1所示；所述黄酮类物质为芦丁，柚皮素或白藜芦醇。
2.一种对黄酮类物质耐受性提高的大肠杆菌，其特征在于所述大肠杆菌是以E.coliBL21(DE3)为宿主构建的过量表达核苷酸序列如SEQ ID NO.1所示的OmpA基因的重组菌株；所述黄酮类物质为芦丁，柚皮素或白藜芦醇。
3.权利要求2所述大肠杆菌的构建方法，其特征在于包括如下步骤：1)根据所查E.coliBL21(DE3)中OmpA基因序列设计引物进行克隆；2)将OmpA基因与载体连接得到重组表达载体；3)将得到的重组表达载体转化E.coli后得到重组菌株。
4.权利要求1获得的大肠杆菌在黄酮类化合物生产中的应用。
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