CN103952417B - 枯草芽孢杆菌双组分调控系统突变基因yvrH及应用 - Google Patents
枯草芽孢杆菌双组分调控系统突变基因yvrH及应用 Download PDFInfo
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Abstract
本发明公开了一种枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)及应用,枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A),是SEQ?ID?No.1所示。本发明所构建的包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌生物安全,遗传背景清晰、包含的突变基因yvrH(G665A)可以大幅提高枯草芽孢杆菌合成核黄素的能力,可在摇瓶发酵条件下,提高核黄素积累水平15%以上。
Description
技术领域
本发明属于生物技术与分子生物学领域,具体地涉及一种枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)及该基因编码的氨基酸序列及应用。
背景技术
以细菌为宿主菌的基因工程菌具有发酵周期短、原料要求简单、成熟的基因工程技术等优点。在芽孢杆菌属中,包括枯草芽孢杆菌(Bacillussubtilis)在内的许多菌株具有可靠的安全性。传统菌株选育发现,枯草芽孢杆菌的突变株能够过量合成叶酸、腺苷、肌苷、鸟苷、核黄素等一系列嘌呤途径代谢中间产物或该途径的衍生代谢产物,目前成为选育高产核苷类代谢产物重要出发菌株。作为模式菌株,目前对其生理生化特性及遗传背景已经有了比较深入的了解,相关的分子生物学方法和基因操作技术都比较成熟,也有利于通过理性代谢工程及系统生物学手段来选育核苷类代谢产物高产菌种。如核黄素。核黄素(分子式C17H20O6N4,IUPAC中文名:7,8-二甲基-10-(1'-D-核糖基)-异咯嗪)是人体必需的13种维生素之一,为黄素酶类的辅酶组成部分,在生物体内主要以黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)的形式存在。它作为黄素蛋白的辅酶参与机体组织呼吸链电子传递及氧化还原反应,在呼吸和生物氧化中起着重要的作用。
双组分信号转导系统(two-componentsignal-transductionsystem)普遍存在于细菌,是细胞感应各种各样的环境刺激和做出相应反馈的重要机制。该机制同样存在于低等真核生物和低等植物中。典型的双组分系统通常包括两个部分,组氨酸蛋白激酶(Sensorkinase)和反应调节蛋白(responseregulator)。其中组氨酸蛋白激酶感应不同类型的信号(环境变化),并通过磷酸转移反应调控反应调节蛋白的功能。通常,磷酸化后的反应调节蛋白可以做为转录增强子或抑制子直接结合至受其调节的基因的启动子区域,影响这些被调节基因的表达水平,进而使细胞对外界的信号做出正确的生理反应。该系统可以调节各种各样的细胞过程,比如适应性和渗透性等行为,是允许细胞的基因网络对于不同的外界环境做出适应和维持细胞生存能力的重要机制。
在枯草芽孢杆菌中,参与细胞膜和细胞壁合成调控的yvrGH系统在细胞生理活动中发挥重要作用。按照组氨酸磷酸化基序(motif)周围的氨基酸序列进行分类,yvrG属于类别ClassIIIA,该激酶N-端包含了两个跨膜区域,为膜蛋白。而yvrH属于ClassOmpR类别。YvrGH系统正调控7个转录单位(wprA,wapA-yxxG,dltABCDE,sunA,sunT-bdbA-yolJ-bdbB,yvrI-yvrHa和sigX-risX),负调控lytABC操纵子。敲除实验发现,该双组分系统的失活可导致细胞对抗生素和抗菌剂敏感性的提高。
目前,尚未有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)用于核黄素高产菌种的选育的报道。
发明内容
本发明的目的是提供一种能够提高枯草芽孢杆菌核黄素合成能力的枯草芽孢杆菌双组分调控系统突变基因yvrH。
本发明的第二个目的是提供一种枯草芽孢杆菌双组分调控系统突变基因yvrH所编码的氨基酸序列。
本发明的第三个目的是提供一种包含有枯草芽孢杆菌双组分调控系统突变基因yvrH的工程菌。
本发明的第四个目的是提供包含有枯草芽孢杆菌双组分调控系统突变基因yvrH的工程菌的应用。
本发明的技术方案概述如下:
枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A),所述突变基因yvrH(G665A)序列是用SEQIDNo.1所示。
枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)所编码的氨基酸序列,所述氨基酸序列是用SEQIDNo.2所示。
包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌。
包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌在产核黄素的应用。
本发明所构建的包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌生物安全,遗传背景清晰、包含的突变基因yvrH(G665A)可以大幅提高枯草芽孢杆菌合成核黄素的能力,可在摇瓶发酵条件下,提高核黄素积累水平15%以上。
附图说明
图1为工程菌株B.subtilisRC1、B.subtilisRC2和B.subtilisRC3核黄素合成水平发酵验证。
具体实施方式
下面结合实施例对本发明做进一步说明,下述实施例是为了使本领域的技术人员能够更好地理解本发明,但对本发明不作任何限制。
本发明所用到的原始菌株B.subtilis168来源为BGSC(BacillusGeneticStockCenter,http://www.bgsc.org/)。本发明所用到的原始质粒pUC18购买于生工生物工程(上海)股份有限公司(http://www.sangon.com/)。
本发明所用到的核黄素标准品从Sigma公司(http://www.sigmaaldrich.com/sigma-aldrich)购买,所用限制性内切酶、去磷酸化酶、DNA连接酶等分子生物学试剂从Thermo公司购买(http://www.thermoscientificbio.com/fermentas)。所用其他生化试剂从生工生物工程(上海)股份有限公司购买(http://www.sangon.com/)。
实施例1:无抗生素标记筛选枯草芽孢杆菌系统出发菌株B.subtilisBUK和基础操作质粒pSS的构建
本发明中所用到的枯草芽孢杆菌出发菌株B.subtilisBUK来源于B.subtilis168,基础操作质粒pSS来源于pUC18,二者详细构建过程可参考公开发表文献1。
本发明对该基础操作质粒pSS的构建方法以及抗性基因的选择不做限定。
实施例2:工程菌株B.subtilisRC1和B.subtilisRC2构建过程
(1)向菌株B.subtilisBUK基因组上无痕引入基因突变ribC*(G596A)操作流程如下:
利用ribC*-F-U、ribC*-F-L和ribC*-B-U、ribC*-B-L两对引物,以B.subtilis168基因组为模版,使用KOD-plus高保真DNA聚合酶扩增分别得到大小为894bp和928bp的上下游同源臂ribC*-F和ribC*-B。ribC*-F的PCR片段经切胶回收后,使用ThermoFastdigestAatII和XhoI双酶切,连接、转化质粒pSS后得到质粒pSS-ribC*-F。ribC*-B的PCR片段经切胶回收后,使用ThermoFastdigestSalI和ScaI双酶切,连接、转化质粒pSS-ribC*-F后得到这质粒pSS-ribC*-FB。将测序结果正确的质粒通过Spizizen转化导入枯草芽孢杆B.subtilisBUK中,用含氯霉素LB固体培养基中筛选重组成功的阳性克隆,并用菌落PCR验证。挑出的转化子接种于5mlLB液体培养基中,200rpm震荡培养6h(OD约为2),并在五氟尿嘧啶基本培养基(添加终浓度为5μmol/LFMN)上挑出菌落,使用引物ribC*-F-U、ribC*-B-L进行PCR和测序验证,得到ribC*(G596A)正确引入的阳性菌株B.subtilisRC1。
(2)向菌株B.subtilisRC1基因组上无痕引入基因突变ribO*(G+39A)具体操作如下:
利用ribO*-F-U、ribO*-F-L和ribO*-B-U、ribO*-B-L两对引物,以B.subtilis168基因组为模版,使用KOD-plus高保真DNA聚合酶扩增分别得到大小为922bp和1234bp的上下游同源臂ribO*-F和ribO*-B。ribO*-F的PCR片段经切胶回收后,使用ThermoFastdigestAatII和XhoI双酶切,连接、转化质粒pSS后得到质粒pSS-ribO*-F。ribO*-B的PCR片段经切胶回收后,使用ThermoFastdigestSalI和KpnI双酶切,连接、转化质粒pSS-ribO*-F后得到这质粒pSS-ribO*-FB。将测序结果正确的质粒通过Spizizen转化导入枯草芽孢杆B.subtilisRC1中,用含氯霉素LB固体培养基中筛选重组成功的阳性克隆,并用菌落PCR验证。挑出的转化子接种于5mlLB液体培养基中,200rpm震荡培养6h(OD约为2),并在五氟尿嘧啶基本培养基(添加终浓度为5μmol/LFMN)上挑出菌落,使用引物ribO*-F-U、ribO*-B-L进行PCR和测序验证,得到ribO*正确引入的阳性菌株B.subtilisRC2。
实施例3:工程菌株B.subtilisRC3构建(引入枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A))
利用yvrH*-F-U、yvrH*-F-L和yvrH*-B-U、yvrH*-B-L两对引物,以B.subtilis168基因组为模版,使用KOD-plus高保真DNA聚合酶扩增分别得到大小为1067bp和849bp的上下游同源臂yvrH*-F和yvrH*-B。yvrH*-F的PCR片段经切胶回收后,使用ThermoFastdigestAatII和XhoI双酶切,连接、转化质粒pSS后得到质粒pSS-yvrH*-F。yvrH*-B的PCR片段经切胶回收后,使用ThermoFastdigestSalI和KpnI双酶切,连接、转化质粒pSS-yvrH*-F后得到质粒pSS-yvrH*-FB。将测序结果正确的质粒通过Spizizen转化导入枯草芽孢杆B.subtilisRC2中,用含氯霉素LB固体培养基中筛选重组成功的阳性克隆。将正确的阳性克隆接种于5mlLB液体培养基中,200rpm震荡培养6h,并在五氟尿嘧啶基本培养基上挑出菌落使用引物yvrH*-F-U、yvrH*-B-L进行PCR和测序验证,得到突变基因yvrH(G665A)正确引入的阳性菌株,即包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌,命名为B.subtilisRC3,其中枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)和所编码的氨基酸序列分别用SEQIDNo.1和SEQIDNo.2所示。
LB液体培养基配方为:10g/L蛋白胨,5g/L酵母提取物,10g/LNaCl,调节pH至7.5。0.1Mpa压力下灭菌20min。LB固体培养基配方为:在LB液体培养基中加入琼脂粉(终浓度15g/L),0.1Mpa压力下灭菌20min。五氟尿嘧啶基本培养基配方见表1:
表1五氟尿嘧啶基本培养基配方
母液 | 添加量 |
葡萄糖(40%) | 2ml |
1000×微量元素 | 100μL |
Trp(0.5%) | 1ml |
谷氨酰胺(4%) | 5ml |
10×Spizizen基本盐 | 10ml |
VB1(0.1%) | 100μL |
5FU(20mM) | 50μL |
琼脂 | 1.5g |
加水至 | 100ml |
表1中成分的百分比浓度均为质量体积百分比。
表1中的10×Spizizen基本盐:2g/L(NH4)2SO4,18.3g/LK2HPO4,6g/LKH2PO4,12g/LC6H5Na3O7·2H2O,pH7.2,0.1Mpa压力下灭菌20min。
表1中的1000×微量元素:27g/LFeCl3·6H2O,2g/LZnCl2·4H2O,2g/LCaCl2·2H2O,2g/LNa2MoO4·2H2O,1.9g/LCuSO4·5H2O,0.5g/LH3BO3pH7.2,0.1Mpa压力下灭菌20min。
实施例4:双组分调控系统突变基因yvrH(G665A)引入后细胞转录调控变化
1)采用LB培养基,37℃,240rpm条件下,将菌体培养至对数生长期中期。
2)4℃条件下,6000rpm离心5min,收集1.5ml菌体。弃上清。
3)向菌体沉淀添加终浓度为3mg/ml溶菌酶,混匀。室温放置15min。
4)采用InvitrogenTMTRIzol试剂盒提取totalRNA。
5)采用FastQuantRTKit试剂盒逆转录totalRNA模板,合成cDNA。
6)以rrnA为参照基因,采用FastFireqPCRPreMix试剂盒进行RT-PCR荧光定量,RT-PCR所用引物见表3。上机RocheLightcyler480进行RT-PCR分析,分析结果汇总见表2。
表2RT-PCR分析突变基因yvrH(G665A)对所调控基因表达水平的影响
基因 | rrnA | wapA | wprA | dltA | lytA | lytB | lytC |
上游引物 | RP-16sRNA-F | RP-wapA-F | RP-wprA-F | RP-dltA-F | RP-lytA-F | RP-lytB-F | RP-lytC-F |
下游引物 | RP-16sRNA-B | RP-wapA-B | RP-wprA-B | RP-dltA-B | RP-lytA-B | RP-lytB-B | RP-lytC-B |
B.subtilis RC2 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
B.subtilis RC3 | 1.00 | 0.06 | 0.05 | 0.32 | 2.37 | 2.56 | 2.60 |
7)结果发现,枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)在一定程度上解除了对其调控基因的表达,如负向调控表达的lytA、lytB和lytC基因的转录水平均上调2倍以上,而正向调控的基因wapA、wprA、dltA等均下调至很低的水平。
实施例5:工程菌株B.subtilisRC2和B.subtilisRC3核黄素合成能力对比
1)分别划线菌株B.subtilisRC1、B.subtilisRC2和B.subtilisRC3于LB平板,37℃过夜培养。
2)挑单菌落接种5mLLB液体培养基的摇管,培养至对数生长期中期。。
3)按初始OD=0.02接种量转接培养菌液至50mlYE培养基。摇床转速240rpm,41℃培养60h。
其中,YE培养基配方:100g/L葡萄糖,20g/L酵母粉,0.5g/LMgSO4,0.5g/LKH2PO4,
0.5g/LK2HPO4,调节pH至7.0,0.1Mpa压力下灭菌20min。
发酵结果见图1。从发酵结果可以看出,相对于菌株B.subtilisRC2,包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌(B.subtilisRC3)提高了核黄素产量15%以上。说明这种遗传修饰对于核黄素等核苷类高产菌株选育具有较好的应用前景。
本发明的菌株的构建,其步骤的前后顺序不限定,本领域的技术人员按本发明公开的内容达到本发明的目的均属于本发明的保护范围。
本发明中的菌株代号如B.subtilisRC1、B.subtilisRC2和B.subtilisRC3等是为了方便描述,但不应理解为对本发明的限定。
上述方法构建的包含有枯草芽孢杆菌双组分调控系统突变基因yvrH(G665A)的工程菌的用途,包括但是不局限于核黄素。
参考文献1:Shi,T.,Wang,G.,Wang,Z.,Fu,J.,Chen,T.,Zhao,X.,2013.EstablishmentofaMarkerlessMutationDeliverySysteminBacillussubtilisStimulatedbyaDouble-StrandBreakintheChromosome.PLoSone.8,e81370.
表3菌株构建所用引物序列
Claims (4)
1.枯草芽孢杆菌双组分调控系统突变基因yvrH,其特征是所述突变基因是SEQIDNo.1所示。
2.权利要求1所述的枯草芽孢杆菌双组分调控系统突变基因yvrH所编码的蛋白质,其特征是所述蛋白质的氨基酸序列用SEQIDNo.2所示。
3.包含有权利要求1所述的枯草芽孢杆菌双组分调控系统突变基因yvrH的工程菌。
4.权利要求3所述的工程菌在产核黄素的应用。
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