CN116426455B - 一种重组大肠杆菌及其构建方法与其在生产3-脱氢莽草酸中的应用 - Google Patents
一种重组大肠杆菌及其构建方法与其在生产3-脱氢莽草酸中的应用 Download PDFInfo
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Abstract
本发明涉及一种重组大肠杆菌及其构建方法与其在生产3‑脱氢莽草酸中的应用。本发明所述重组大肠杆菌在大肠杆菌宿主菌中,敲除3‑脱氢莽草酸脱氢酶基因aroE、负调控转录抑制因子tyrR、乙酸激酶基因ackA、丙酮酸脱氢酶基因poxB和奎尼酸脱氢酶基因YdiB,并过量表达磷酸烯醇式丙酮酸合酶ppsA、转酮醇酶tktA和转醛缩酶talA、DHQ合酶aroB、3‑脱氢奎尼酸脱水酶aroD和抗反馈抑制的3‑脱氧‑D‑阿拉伯庚酮糖‑7‑磷酸(DAHP)合成酶基因aroGFBR。本发明发酵生产3‑脱氢莽草酸的工艺操作简单易行、培养基成本低廉,适用于工业化生产。
Description
技术领域
本发明涉及生物工程技术领域,尤其是指一种重组大肠杆菌及其构建方法与其在生产3-脱氢莽草酸中的应用。
背景技术
3-脱氢莽草酸(3-dehydroshikimic acid,DHS)分子式为C7H8O5,分子量172.14,DHS是微生物体内芳香族氨基酸生物合成代谢途径中的一种重要中间产物,是莽草酸(SA)、己二烯二酸(MA)和原儿茶酸(PCA)生物合成的关键中间体,该类化学品作为食品、药品和日用化学品的主要原料而备受关注。DHS有一个六元碳环和两个不对称碳,化学合成既困难又昂贵。另一方面,3-脱氢莽草酸是在植物,真菌和细菌的莽草酸途径中发现的天然代谢物,使其通过微生物发酵生产可行,因此3-脱氢莽草酸作为生物基生产是一种很有前途的替代目前基于石化的生产路线,这可以缓解环境污染和能源短缺等问题。因此,研究3-脱氢莽草酸的生产具有重要的应用前景。
大肠杆菌中的3-脱氢莽草酸生物合成从葡萄糖开始,通过中心碳代谢两种中间体磷酸烯醇式丙酮酸(PEP)和赤藓糖-4-磷酸(E4P)通过3-脱氧-D-阿拉伯庚糖酸-7-磷酸合酶(DAHP),DHQ合酶(aroB)和DHQ脱水酶(aroD)转化为3-脱氢莽草酸。几十年来,已经应用了许多方法来增加莽草酸盐生物合成途径的碳通量,以生产几种芳香族化合物。但由于大肠杆菌中莽草酸路径的代谢流小,关键限速靶点不清楚,导致目前大肠杆菌生产3-脱氢莽草酸的产量,得率,以及生产强度都较低。因此,需要理性筛选改造靶点,以构建一株高产3-脱氢莽草酸的大肠杆菌。
发明内容
为解决上述技术问题,本发明提供了一种重组大肠杆菌及其构建方法与其在生产3-脱氢莽草酸中的应用,采用CRISPR/Cas9系统工具重编程大肠杆菌代谢途径,包括敲除3-脱氢莽草酸脱氢酶基因aroE,敲除负调控转录抑制因子tyrR并在该位点过表达DHQ合酶aroB和3-脱氢奎尼酸脱水酶aroD,敲除乙酸激酶基因ackA并在该位点过表达抗反馈抑制的DAHP合成酶基因aroGFBR,敲除丙酮酸脱氢酶基因poxB并在该位点过表达抗反馈抑制的DAHP合成酶基因aroGFBR,以及敲除奎尼酸脱氢酶基因YdiB并在该位点过表达磷酸烯醇式丙酮酸合酶ppsA、转酮醇酶tktA和转醛缩酶talA。最终,获得了一株高产莽草酸的大肠杆菌,结合发酵条件的优化实现3-脱氢莽草酸的高效生产。
本发明的第一个目的在于提供一种重组大肠杆菌,所述重组大肠杆菌在大肠杆菌宿主菌中,敲除3-脱氢莽草酸脱氢酶基因aroE,敲除负调控转录抑制因子tyrR并在该位点过表达DHQ合酶aroB和3-脱氢奎尼酸脱水酶aroD,敲除乙酸激酶基因ackA并在该位点过表达抗反馈抑制的DAHP合成酶基因aroGFBR,敲除丙酮酸脱氢酶基因poxB并在该位点过表达抗反馈抑制的DAHP合成酶基因aroGFBR,以及敲除奎尼酸脱氢酶基因YdiB并在该位点过表达磷酸烯醇式丙酮酸合酶ppsA、转酮醇酶tktA和转醛缩酶talA。。
在本发明的一个实施例中,3-脱氢莽草酸脱氢酶编码基因aroE的NCBI编号为NP_417740.1、负调控转录抑制因子编码基因tyrR的NCBI编号为NP_415839.1、乙酸激酶编码基因ackA的NCBI编号为NP_416799.1、丙酮酸脱氢酶编码基因poxB的NCBI编号为NP_415392.1、奎尼酸脱氢酶编码基因YdiB的NCBI编号为NP_416207.1、磷酸烯醇式丙酮酸合酶编码基因ppsA的NCBI编号为NP_416217.1、转酮醇酶编码基因tktA的NCBI编号为YP_026188.1、转醛醇酶编码基因talA的NCBI编号为NP_414549.1、DHQ合酶的编码基因aroB的NCBI编号为NP_417848.1、3-脱氢奎尼酸脱水酶的编码基因aroD的NCBI编号为NP_416208.1、3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶编码基因aroGFBR的核苷酸序列如SEQ IDNO.1所示。
SEQ ID NO.1:atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccgctactgaaaatgccgcgaatacggttgcccatgcccgaaaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggttgtgattggcccatgctcaattcatgatcctgtcgcggcaaaagagtatgccactcgcttgctggcgctgcgtgaagagctgaaagatgagctggaaatcgtaatgcgcgtctattttgaaaagccgcgtaccacggtgggctggaaagggctgattaacgatccgcatatggataatagcttccagatcaacgacggtctgcgtatagcccgtaaattgctgcttgatattaacgacagcggtctgccagcggcaggtgagtttctcAatatgatcaccccacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcatcagggctttcttgtccggtcggcttcaaaaatggcaccgacggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgccagcacaggtgatgatcgatttcagccatgctaactcgtccaaacaattcaaaaagcagatggatgtttgtgctgacgtttgccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggagccgctggcctacggtaagagcatcaccgatgcctgcatcggctgggaagataccgatgctctgttacgtcaactggcgaatgcagtaaaagcgcgtcgcgggtaa。
在本发明的一个实施例中,过表达DAHP合成酶编码基因aroGFBR、DHQ合酶的编码基因aroB、3-脱氢奎尼酸脱水酶的编码基因aroD、转醛醇酶编码基因talA、转酮醇酶编码基因tktA和磷酸烯醇式丙酮酸合酶编码基因ppsA是通过使用强启动子PJ23119分别控制表达,其中DAHP合成酶编码基因aroGFBR过表达了两个拷贝。
在本发明的一个实施例中,所述大肠杆菌宿主菌为MG1655(MG1655,Gao C,Guo L,Ding Q,Hu GP,Ye C,Liu J,Chen XL,Liu LM.Dynamic consolidated bioprocessing fordirect production ofxylonate and shikimate from xylan by Escherichiacoli.Metabolic Engineering,2020,60:128-137.),获得的重组菌株依次为FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05。
本发明的第二个目的在于提供一种重组大肠杆菌的构建方法,包括以下步骤:
(1)扩增获得ppsA、tktA、talA、aroB、aroD和aroGFBR基因片段;将基因片段连接到pJ01表达载体上,从表达载体上扩增得到表达框;
(2)以MG1655基因组为模板,扩增得到aroE、tyrR、ackA、poxB和YdiB基因的上下游同源臂,分别将上下游同源臂与(1)中的表达框构建成敲除框或整合框;
(3)将(2)中的敲除框或整合框转化至含有pcas9质粒的宿主菌株MG1655中,得到一系列重组菌株FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05。
进一步的,构建方法具体为:
(1)扩增获得ppsA、tktA、talA、aroB、aroD和aroGFBR基因片段;将基因片段连接到pJ01表达载体上,所有基因均以强启动子PJ23119控制表达。再以构建完成的表达质粒为模板,扩增得到PJ23119-aroB-PJ23119-aroD、PJ23119-aroGFBR、PJ23119-ppsA-PJ23119-tktA表达框;
(2)以MG1655基因组为模板,扩增得到aroE、tyrR、ackA、poxB和YdiB基因的上下游同源臂,将aroE基因的上下游同源臂构建成敲除框,将tyrR基因的上下游同源臂与PJ23119-aroB-PJ23119-aroD构建成tyrR-PJ23119-aroB-PJ23119-aroD整合框,将ackA基因的上下游同源臂与PJ23119-aroGFBR构建成ackA-PJ23119-aroGFBR整合框,将poxB基因的上下游同源臂与PJ23119-aroGFBR构建成poxB-PJ23119-aroGFBR整合框,将YdiB基因的上下游同源臂与PJ23119-ppsA-PJ23119-tktA构建成YdiB-PJ23119-ppsA-PJ23119-tktA整合框;
(3)依次将aroE基因敲除框与gRNA-N20-aroE,tyrR-PJ23119-aroB-PJ23119-aroD整合框与gRNA-N20-tyrR,ackA-PJ23119-aroGFBR整合框与gRNA-N20-ackA、poxB-PJ23119-aroGFBR整合框与gRNA-N20-poxB和YdiB-PJ23119-ppsA-PJ23119-tktA整合框与gRNA-N20-YdiB转化至含有pcas9质粒的宿主菌株MG1655中,得到一系列重组菌株FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05。
本发明的第三个目的在于提供所述重组大肠杆菌在生产3-脱氢莽草酸中的应用。
在本发明的一个实施例中,所述3-脱氢莽草酸生产是通过将重组大肠杆菌在发酵培养基中进行全有氧发酵,得到含有3-脱氢莽草酸的发酵液。
在本发明的一个实施例中,在所述的全有氧发酵的过程中,初始培养基中葡萄糖耗尽时,流加500~800g/L葡萄糖,并控制葡萄糖浓度在0~5g/L。
在本发明的一个实施例中,在所述的全有氧发酵过程中,pH控制在6.8~7.1;发酵温度控制在36~38℃;溶氧维持在20~50%,进一步的溶氧维持为20%~40%。
在本发明的一个实施例中,所述的发酵培养基配方为:葡萄糖8~12g/L,酵母粉3~7g/L,一水柠檬酸1.5~2.5g/L,磷酸氢二钾5~9g/L,七水硫酸镁1.5~2.5g/L,硫酸铵4~6g/L,氯化钠0.5~1.5g/L,色氨酸0.3~0.5g/L,酪氨酸0.5~0.8g/L,苯丙氨酸0.5~0.8g/L,金属离子液0.8~1.2mL/L。每1L培养基含有1mL微量元素液;微量元素液:FeCl2·6H2O 2.4g/L、CoCl2·6H2O 0.3g/L、CuCl20.15 g/L、ZnCl2·4H2O 0.3g/L、NaMnO40.3 g/L、H3BO30.075 g/L、MnCl2·4H2O 0.495g/L,溶于0.1M HCl中配制。
本发明的上述技术方案相比现有技术具有以下优点:
本发明采用CRISPR/Cas9系统技术敲除发酵过程中主要副产物乙酸合成相关基因ackA和poxB,在发酵过程中,大幅减少了发酵过程中乙酸的积累;同时过表达tktA、talA、ppsA提高PEP和E4P的供应,以及过表达莽草酸途径相关基因如aroB、aroD和抗反馈抑制的aroGFBR以强化莽草酸途径通量,有效提高了3-脱氢莽草酸的产量,最终得到的工程菌株E.coli FMME-DHS05在发酵罐水平上发酵生产3-脱氢莽草酸产量最高可达73.6g/L。本发明发酵生产3-脱氢莽草酸的工艺操作简单易行、培养基成本低廉,适用于工业化生产。
大肠杆菌(Escherichia coli)E.coli FMME-DHS05,所述大肠杆菌已于2023年4月25日保藏于江南大学食品科学与技术国家重点实验室微生物制造研究室,保藏地址为中国无锡,江南大学。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中,
图1是本发明图1为基于靶点改造大肠杆菌生产3-脱氢莽草酸路径图;
图2为过表达DHQ合酶基因aroB和3-脱氢奎尼酸脱水酶基因aroD质粒pJ01-aroB-aroD结构示意图;
图3为过表达转酮醇酶基因tktA和转醛缩酶基因talA的质粒pJ01-tktA-talA结构示意图;
图4为本发明过表达抗反馈抑制的DHAP合成酶基因aroGFBR和磷酸烯醇式丙酮酸合酶基因ppsA的质粒pJ01-aroGFBR-ppsA结构示意图;
图5为本发明实施例重组菌株FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05的摇瓶发酵结果;
图6为构建工程菌株E.coliFMME-DHS05的发酵罐补料分批发酵结果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
1、本发明实施例中序列表中相关核苷酸序列信息:
SEQ ID NO.1序列信息为抗反馈抑制的DHAP合成酶基因aroGFBR的核苷酸序列。
2、本发明实施例所涉及材料和方法:
(1)培养基:
固体培养基:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L,琼脂粉20g/L。
一级种子培养基:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L。
二级种子培养基:蛋白胨32g/L,酵母提取物20g/L,氯化钠5g/L,甘油5.2g/L。
发酵培养基:葡萄糖10g/L,酵母粉5g/L,一水柠檬酸2g/L,磷酸氢二钾3g/L,硫酸铵5g/L,七水硫酸镁1g/L,氯化钠1g/L,色氨酸0.3g/L,酪氨酸0.3g/L,苯丙氨酸0.3g/L,金属离子液1mL/L。
(2)葡萄糖的测定:
发酵液预处理:取发酵液12000r/min离心5min取上清。稀释至适宜倍数,用M-100生物传感分析仪检测发酵液葡萄糖浓度。
(3)3-脱氢莽草酸的测定:
高效液相色谱法:配置1g/L浓度3-脱氢莽草酸溶液,稀释至0.1、0.2、0.3、0.4和0.5g/L,利用高效液相色谱仪(HPLC)进行检测,获得出峰时间以及不同浓度莽草酸所对应的峰面积,以3-脱氢莽草酸溶液的浓度为横坐标,以峰面积为纵坐标,绘制标准曲线,获得线性回归方程。线性回归方程的回归系数应该在0.990以上方可以使用。仪器为waters高效液相色谱仪,色谱柱采用ZORBAX SB-C18液相色谱柱;流动相为20%甲醇+0.07%高氯酸;流速设定为1mL/min;检测器为紫外检测器,检测波长为278nm,柱温为40℃。
发酵液预处理:取发酵液12000r/min离心10min取上清。稀释适宜倍数后,将样品过膜处理,使用HPLC进行检测,将所得峰面积代入线性回归方程,所得结果乘稀释倍数即为发酵液中莽草酸浓度。
实施例1:表达载体pJ01-aroB-aroD的构建
本发明所用的DHQ合酶基因aroB和3-脱氢奎尼酸脱水酶基因aroD来源于大肠杆菌,提取大肠杆菌的基因组DNA;根据已公布的基因组信息序列,分别设计验证引物对aroB-F/aroB-R和aroD-F/aroD-R(表1),以提取大肠杆菌的基因组DNA为模板,采用标准的PCR扩增体系和程序,分别扩增获取aroB和aroD基因。PCR扩增获取的roB和aroD,采用琼脂糖凝胶核酸电泳切胶回收,表达载体pJ01利用限制性内切酶ApaI和Sal I进行双酶切3h,酶切产物采用琼脂糖凝胶核酸电泳凝胶回收,aroB、aroD基因和线性化质粒大小分别为1089、759和2502bp。首先分别将aroB和aroD基因与线性化pJ01质粒采用ClonExpress II One StepCloning Kit连接酶37℃连接30min,转化至JM109感受态细胞中,挑取单菌落PCR使用YZ-pJ01-F/YZ-pJ01-R(表1)验证,阳性转化子进行测序,比对正确,证明表达载体构建成功,质粒命名为pJ01-aroB和pJ01-aroD。接下来以pJ01-aroD为模板,使用引物pJ-aroD-F/pJ-aroD-R扩增获取Pj23119-aroD,将表达载体pJ01-aroB利用限制性内切酶BamH I和Pst I进行双酶切3h,酶切产物采用琼脂糖凝胶核酸电泳凝胶回收,Pj23119-aroD和pJ01-aroB线性化质粒大小分别为932和3591bp,将Pj23119-aroD片段与线性化pJ01-aroB质粒采用ClonExpressII One Step Cloning Kit连接酶37℃连接30min,转化至JM109感受态细胞中,挑取单菌落PCR使用YZ-pJ01-F/YZ-pJ01-R验证,阳性转化子进行测序,比对正确,证明表达载体构建成功,质粒命名为pJ01-aroB-aroD。
表1实施例1中相关引物序列
实施例2:表达载体pJ01-tktA-talA的构建
本发明所用的转酮醇酶基因tktA和转醛缩酶基因talA来源于大肠杆菌,提取大肠杆菌的基因组DNA;根据已公布的基因组信息序列,分别设计验证引物对tktA-F/tktA-R和talA-F/talA-R(表2),以提取大肠杆菌的基因组DNA为模板,采用标准的PCR扩增体系和程序,分别扩增获取tktA和talA基因。PCR扩增获取的tktA和talA,采用琼脂糖凝胶核酸电泳切胶回收,表达载体pJ01利用限制性内切酶ApaI和Sal I进行双酶切3h,酶切产物采用琼脂糖凝胶核酸电泳凝胶回收,tktA和talA基因和线性化质粒大小分别为1992、951和2502bp。首先分别将tktA和talA基因与线性化pJ01质粒采用ClonExpress II One Step CloningKit连接酶37℃连接30min,转化至JM109感受态细胞中,挑取单菌落PCR使用YZ-pJ01-F/YZ-pJ01-R(表2)验证,阳性转化子进行测序,比对正确,证明表达载体构建成功,质粒命名为pJ01-tktA和pJ01-talA。接下来以pJ01-talA为模板,使用引物pJ-talA-F/pJ-talA-R扩增获取Pj23119-talA,将表达载体pJ01-tktA利用限制性内切酶BamH I和Pst I进行双酶切3h,酶切产物采用琼脂糖凝胶核酸电泳凝胶回收,Pj23119-talA和pJ01-tktA线性化质粒大小分别为1088和4490bp,将Pj23119-talA片段与线性化pJ01-tktA质粒采用ClonExpress II OneStep Cloning Kit连接酶37℃连接30min,转化至JM109感受态细胞中,挑取单菌落PCR使用YZ-pJ01-F/YZ-pJ01-R验证,阳性转化子进行测序,比对正确,证明表达载体构建成功,质粒命名为pJ01-tktA-talA。
表2实施例2中相关引物序列
引物序列 | 引物序列(5′-3′) |
tktA-F | TAAAGAGGAGAAAAAGCTTGGGCCCatgtcctcacgtaaagagcttgcc |
tktA-R | aatgatgatgatgatgatggtcgacttacagcagttcttttgctttcgc |
talA-F | CTAGTAAAGAGGAGAAAAAGCTTGGGCCCatgaacgagttagacggcatcaaac |
talA-R | aactcaatgatgatgatgatgatggtcgacttatagtttggcggcaagaagatc |
pJ-talA-F | ccttagctttcgctaaggatg |
pJ-talA-R | cctttctgcgTGGATCCgtttaaactcaat |
YZ-pJ01-F | ccttagctttcgctaaggatg |
YZ-pJ01-R | cgcctttgagtgagctgatacc |
实施例3:表达载体pJ01-aroGFBR-ppsA的构建
本发明所用的抗反馈抑制的DHAP合成酶基因aroGFBR来源于本实验前期构建(GaoC,Guo L,Ding Q,Hu GP,Ye C,Liu J,Chen XL,Liu LM.Dynamic consolidatedbioprocessing for direct production of xylonate and shikimate from xylan byEscherichia coli.Metabolic Engineering,2020,60:128-137.),磷酸烯醇式丙酮酸合酶基因ppsA来源于大肠杆菌,提取大肠杆菌的基因组DNA;根据已公布的基因组信息序列,分别设计验证引物对aroG-F/aroG-R和ppsA-F/ppsA-R(表3),以提取大肠杆菌的基因组DNA为模板,采用标准的PCR扩增体系和程序,分别扩增获取aroGFBR和ppsA基因。PCR扩增获取的aroGFBR和ppsA,采用琼脂糖凝胶核酸电泳切胶回收,表达载体pJ01利用限制性内切酶ApaI和Sal I进行双酶切3h,酶切产物采用琼脂糖凝胶核酸电泳凝胶回收,aroGFBR和ppsA基因和线性化质粒大小分别为2379、1053和2502bp。首先分别将aroGFBR和ppsA基因与线性化pJ01质粒采用ClonExpress II One Step Cloning Kit连接酶37℃连接30min,转化至JM109感受态细胞中,挑取单菌落PCR使用YZ-pJ01-F/YZ-pJ01-R(表3)验证,阳性转化子进行测序,比对正确,证明表达载体构建成功,质粒命名为pJ01-aroGFBR和pJ01-ppsA。接下来以pJ01-aroGFBR为模板,使用引物pJ-aroG-F/pJ-aroG-R扩增获取Pj23119-aroG,将表达载体pJ01-ppsA利用限制性内切酶BamH I和Pst I进行双酶切3h,酶切产物采用琼脂糖凝胶核酸电泳凝胶回收,Pj23119-aroG和pJ01-ppsA线性化质粒大小分别为1201和4875bp,将Pj23119-aroG片段与线性化pJ01-ppsA质粒采用ClonExpress II One Step Cloning Kit连接酶37℃连接30min,转化至JM109感受态细胞中,挑取单菌落PCR使用YZ-pJ01-F/YZ-pJ01-R验证,阳性转化子进行测序,比对正确,证明表达载体构建成功,质粒命名为pJ01-aroGFBR-ppsA。
表3实施例3中相关引物序列
实施例4:敲除3-脱氢莽草酸脱氢酶基因aroE
根据NCBI数据库中Escherichia coliMG1655的aroE基因序列设计aroE基因敲除框上下同源臂引物aroE-up-F/aroE-up-R和aroE-down-F/aroE-down-R(表4),以大肠杆菌MG1655基因组为模板扩增aroE的上下游同源臂并进行胶回收。使用引物aroE-up-F/aroE-down-R通过重叠PCR将上下游同源臂融合得到aroE基因敲除框。需要指出的是,aroE基因敲除框上下同源臂之间含有30-40bp的同源序列。使用CRISPR gRNADesigntool网站设计aroE基因的gRNA-N20序列,根据使用gRNA-N20序列使用引物N20-aroE-F/N20-aroE-R(表4)以商业化质粒TargetF-frdA-100为模板通过反向PCA构建aroE基因的gRNA-N20-aroE质粒。将aroE的敲除框以及gRNA-N20-aroE通过电转化的形式导入含有pCas9质粒的E.coli MG1655感受态细胞中(电转化电压和时间分别为2100V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)和壮观霉素(33g/mL)的LB固体培养基平板上,倒置培养12-24h。待平板长出单菌落后,使用验证引物YZ-aroE-F和YZ-aroE-R(表1)筛选阳性转化子。成功敲除的转化子电泳条带大小为1405bp,没有敲除的对照组电泳条带大小为2166bp。挑取筛选正确的转化子接种LB培养基中,并添加IPTG诱导以消去gRNA-N20-aroE质粒,再将已消除gRNA-N20-aroE质粒的转化子接种于LB培养基中培养,培养温度为42℃,以消除pCas9质粒,最终获得aroE基因成功敲除的菌株命名为FMME-DHS01。
表4实施例4相关引物序列
实施例5:敲除负调控转录抑制因子tyrR以及过表达DHQ合酶基因aroB和3-脱氢奎尼酸脱水酶基因aroD
根据NCBI数据库中Escherichia coli MG1655的tyrR基因序列设计tyrR基因敲除框上下同源臂引物tyrR-up-F/tyrR-up-R和tyrR-down-F/tyrR-down-R(表5),以Escherichia coli MG1655基因组为模板扩增tyrR的上下游同源臂并进行胶回收。使用引物KZ-ZHK-F/KZ-ZHK-R以pJ01-aroB-aroD质粒为模板扩增Pj23119-aroB-Pj23119-aroD表达框,使用引物tyrR-up-F/tyrR-down-R通过重叠PCR将上下游同源臂以及Pj23119-aroB-Pj23119-aroD表达框融合得到tyrR-Pj23119-aroB-Pj23119-aroD整合框。需要指出的是,tyrR基因敲除框上下同源臂与Pj23119-aroB-Pj23119-aroD表达框之间含有30-40bp的同源序列。使用CRISPRgRNA Design tool网站设计tyrR基因的gRNA-N20序列,根据使用gRNA-N20序列使用引物N20-tyrR-F/N20-tyrR-R(表5)以商业化质粒TargetF-frdA-100为模板通过反向PCA构建tyrR基因的gRNA-N20-tyrR质粒。将tyrR-Pj23119-aroB-Pj23119-aroD整合框以及gRNA-N20-tyrR通过电转化的形式导入含有pCas9质粒的FMME-DHS01感受态细胞中(电转化电压和时间分别为2100V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)和壮观霉素(33g/mL)的LB固体培养基平板上,倒置培养12-24h。待平板长出单菌落后,使用验证引物YZ-tyrR-F和YZ-tyrR-R(表5)筛选阳性转化子。成功整合的转化子电泳条带大小为3279bp,没有整合成功的对照组电泳条带大小为2173bp。接下来使用如实施例1中类似操作消除gRNA-N20-tyrR质粒和pCas9质粒,最终获得菌株命名为FMME-DHS02。
表5实施例5中相关引物序列
引物序列 | 引物序列(5v-3′) |
tyrR-up-F | TGCCGTGGATTGACGATGAC |
tyrR-up-R | gaaattctgcctcgtgatacgcctaGCACGAGTAGATCGAGTAAT |
tyrR-down-F | ttcctcgctcactgactcgctCGTTTCACATACCGCGATTG |
tyrR-down-R | GTTCGCCCAGTCTCGTTTCT |
N20-tyrR-F | TGCGTCTATACCGGAAGATGgttttagagctagaaatagcaagtt |
N20-tyrR-R | CATCTTCCGGTATAGACGCAactagtattatacctaggactgagc |
KZ-ZHK-F | taggcgtatcacgaggcaga |
KZ-ZHK-R | agcgagtcagtgagcgaggaa |
YZ-tyrR-F | CGACTCGGGATTAAAGCTATGGAGC |
YZ-tyrR-R | CTGGCAACTGAGATCGACAACACCG |
实施例6:敲除乙酸激酶基因ackA以及过表达抗反馈抑制的DHAP合成酶基因aroGFBR
根据NCBI数据库中Escherichia coli MG1655的ackA基因序列设计敲除引物ackA-up-F/ackA-up-R和ackA-down-F/ackA-down-R(表6),以Escherichia coli MG1655基因组为模板扩增ackA的上下游同源臂并进行胶回收。使用引物KZ-ZHK-F/KZ-ZHK-R以pJ01-aroGFBR质粒为模板扩增Pj23119-aroGFBR表达框,使用引物ackA-up-F/ackA-down-R通过重叠PCR将上下游同源臂以及Pj23119-aroGFBR表达框融合得到ackA-Pj23119-aroGFBR整合框。需要指出的是,ackA基因敲除框上下同源臂与Pj23119-aroGFBR表达框之间含有30-40bp的同源序列。使用CRISPR gRNADesigntool网站设计ackA基因的gRNA-N20序列,根据使用gRNA-N20序列使用引物N20-ackA-F/N20-ackA-R(表6)以商业化质粒TargetF-frdA-100为模板通过反向PCA构建ackA基因的gRNA-N20-ackA质粒。将ackA-Pj23119-aroGFBR整合框以及gRNA-N20-ackA通过电转化的形式导入含有pCas9质粒的FMME-DHS02感受态细胞中(电转化电压和时间分别为2100V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)和壮观霉素(33g/mL)的LB固体培养基平板上,倒置培养12-24h。待平板长出单菌落后,使用验证引物YZ-ackA-F和YZ-ackA-R(表6)筛选阳性转化子。成功整合的转化子电泳条带大小为2501bp,没有整合成功的对照组电泳条带大小为2185bp。接下来使用如实施例1中类似操作消除gRNA-N20-ackA质粒和pCas9质粒,最终获得菌株命名为FMME-DHS03。
表6实施例6中相关引物序列
实施例7:敲除丙酮酸脱氢酶基因poxB以及过表达抗反馈抑制的DHAP合成酶基因aroGFBR和磷酸烯醇式丙酮酸合酶基因ppsA
根据NCBI数据库中Escherichia coliMG1655的poxB基因序列设计敲除引物poxB-up-F/poxB-up-R和poxB-down-F/poxB-down-R(表7),以Escherichia coli MG1655基因组为模板扩增poxB的上下游同源臂并进行胶回收。使用引物KZ-ZHK-F/KZ-ZHK-R以pJ01-aroGFBR质粒为模板扩增Pj23119-aroGFBR表达框,使用引物poxB-up-F/poxB-down-R通过重叠PCR将上下游同源臂以及Pj23119-aroGFBR表达框融合得到poxB-Pj23119-aroGFBR整合框。需要指出的是,poxB基因敲除框上下同源臂与Pj23119-aroGFBR表达框之间含有30-40bp的同源序列。使用CRISPR gRNADesigntool网站设计poxB基因的gRNA-N20序列,根据使用gRNA-N20序列使用引物N20-poxB-F/N20-poxB-R(表7)以商业化质粒TargetF-frdA-100为模板通过反向PCA构建poxB基因的gRNA-N20-poxB质粒。将poxB-Pj23119-aroGFBR整合框以及gRNA-N20-poxB通过电转化的形式导入含有pCas9质粒的FMME-DHS03感受态细胞中(电转化电压和时间分别为2100V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)和壮观霉素(33g/mL)的LB固体培养基平板上,倒置培养12-24h。待平板长出单菌落后,使用验证引物YZ-poxB-F和YZ-poxB-R(表7)筛选阳性转化子。成功整合的转化子电泳条带大小为2496bp,没有整合成功的对照组电泳条带大小为2709bp。接下来使用如实例1中消除gRNA-N20-poxB质粒和pCas9质粒,最终获得菌株命名为FMME-DHS04。
表7实施例7中相关引物序列
实施例8:敲除奎尼酸脱氢酶基因YdiB以及过表达转酮醇酶基因tktA和转醛缩酶基因talA
根据NCBI数据库中Escherichia coli MG1655的YdiB基因序列设计敲除引物YdiB-up-F/YdiB-up-R和YdiB-down-F/YdiB-down-R(表8),以Escherichia coli MG1655基因组为模板扩增YdiB的上下游同源臂并进行胶回收。使用引物KZ-ZHK-F/KZ-ZHK-R以pJ01-tktA-talA质粒为模板扩增Pj23119-tktA-Pj23119-talA表达框,使用引物YdiB-up-F/YdiB-down-R通过重叠PCR将上下游同源臂以及Pj23119-tktA-Pj23119-talA表达框融合得到YdiB-Pj23119-tktA-Pj23119-talA整合框。需要指出的是,YdiB基因敲除框上下同源臂与Pj23119-tktA-Pj23119-talA表达框之间含有30-40bp的同源序列。使用CRISPR gRNADesign tool网站设计YdiB基因的gRNA-N20序列,根据使用gRNA-N20序列使用引物N20-YdiB-F/N20-YdiB-R(表8)以商业化质粒TargetF-frdA-100为模板通过反向PCA构建tyrR基因的gRNA-N20-YdiB质粒。将YdiB-Pj23119-tktA-Pj23119-talA整合框以及gRNA-N20-YdiB通过电转化的形式导入含有pCas9质粒的FMME-DHS04感受态细胞中(电转化电压和时间分别为2100V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)和壮观霉素(33g/mL)的LB固体培养基平板上,倒置培养12-24h。待平板长出单菌落后,使用验证引物YZ-YdiB-F和YZ-YdiB-R(表8)筛选阳性转化子。成功整合的转化子电泳条带大小为4428bp,没有整合成功的对照组电泳条带大小为1613bp。接下来使用如实施例1中类似操作消除gRNA-N20-YdiB质粒和pCas9质粒,最终获得菌株命名为FMME-DHS05。
表8实施例8中相关引物序列
引物序列 | 引物序列(5′′-3′) |
YdiB-up-F | CATTTTACAGCTCGGCGTTTCGGTC |
YdiB-up-R | gaaattctgcctcgtgatacgcctaCAATTCGTATTTTGCGGTAACATCC |
YdiB-down-F | ttcctcgctcactgactcgctTTAAACAGGTCATGGGGTTCGGTGC |
YdiB-down-R | CTTTGGCACTGCGGAAGGTAAACAG |
N20-YdiB-F | AAACACCGATTGTGTCGTCAgttttagagctagaaatagcaagtt |
N20-YdiB-R | TGACGACACAATCGGTGTTTactagtattatacctaggactgagc |
KZ-ZHK-F | taggcgtatcacgaggcaga |
KZ-ZHK-R | agcgagtcagtgagcgaggaa |
YZ-YdiB-F | ACAGCAGCCATTATCTACCTGTACC |
YZ-YdiB-R | GATGGCTGCACGATTGAGTGCAAT |
实施例9:重组菌株FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05摇瓶发酵
(1)种子活化及培养
一级种子培养基配置:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L;
二级种子培养基配置:蛋白胨32g/L,酵母提取物20g/L,氯化钠5g/L,甘油5.2g/L。
一级种子培养:挑取单菌落接种于LB培养基中,37℃摇床培养12-14h;
二级种子培养:取1mL一级种子液并接种至装有50mL培养基的500mL三角瓶,往复式摇床200rpm/min,37℃恒温培养约5-6h左右,OD600控制在12-18之间。
(2)发酵培养
在500mL摇瓶中对重组菌株FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05进行发酵培养,具体发酵条件为:
发酵培养基:葡萄糖10g/L,酵母粉5g/L,一水柠檬酸2g/L,磷酸氢二钾3g/L,硫酸铵5g/L,七水硫酸镁1g/L,氯化钠1g/L,色氨酸0.3g/L,酪氨酸0.3g/L,苯丙氨酸0.3g/L,金属离子液1mL/L,pH指示剂苯酚红8mg/L,调pH至7.0。
115℃灭菌15min。待温度冷却至37℃后,准备接种。
接种量:以接种量10%,将制备好的种子液接种至500mL摇瓶中;
发酵温度为37℃,水平摇床转速200rpm/min;
发酵pH:间隔4h使用氨水调一次pH,氨水的加入量根据pH指示剂显示的发酵培养基颜色进行微调;
发酵过程中,间隔4h测一次发酵液中残糖浓度,待发酵培养基中葡萄糖低于1g/L后,补加葡萄糖至终浓度为10g/L,发酵周期为48h。
采用高效液相色谱(HPLC)测得重组菌株FMME-DHS01、FMME-DHS02、FMME-DHS03、FMME-DHS04、FMME-DHS05发酵液上清中最终的3-脱氢莽草酸产量分别为0.35、2.12、4.37、9.57和15.23g/L(图5)。
实施例10:重组菌株E.coliFMME-DHS05发酵罐补料分批发酵
(1)种子活化及培养
一级种子培养基配置:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L;
二级种子培养基配置:蛋白胨32g/L,酵母提取物20g/L,氯化钠5g/L,甘油5.2g/L。
一级种子培养:挑取单菌落接种于LB培养基中,37℃摇床培养12-14h;
二级种子培养:取1mL一级种子液并接种至装有50mL培养基的500mL三角瓶,往复式摇床200rpm/min,37℃恒温培养约5-6h左右,OD600控制在12-18之间。
(2)发酵培养
在5L发酵罐中对重组菌株E.coli FMME-DHS05进行发酵培养,具体发酵条件为:
发酵培养基:葡萄糖10g/L,酵母粉5g/L,一水柠檬酸2g/L,磷酸氢二钾3g/L,硫酸铵5g/L,七水硫酸镁1g/L,氯化钠1g/L,色氨酸0.3g/L,酪氨酸0.3g/L,苯丙氨酸0.3g/L,金属离子液1mL/L。
115℃灭菌15min。灭菌后安装至控制台,开启温度自控,待温度冷却至37℃后,流加氨水调节pH至7.0,准备接种。
接种量:以接种量10%,将制备好的种子液接种至发酵罐中;
发酵温度:发酵过程中,开启温度自控,维持发酵温度在37℃;
发酵pH:使用氨水调节pH在6.95~6.75之间;
溶氧条件:初始条件:通气量2vvm,转速300rpm;
发酵过程中,通过调节通气量和搅拌桨转速将发酵过程中的溶氧维持在20%以上。
发酵过程中,待发酵培养基中初始葡萄糖低于1g/L后,通过脉冲式流加800g/L的葡萄糖溶液,控制葡萄糖浓度在5g/L以下,发酵周期为48h。
采用高效液相色谱(HPLC)测得发酵液上清中最终的3-脱氢莽草酸产量为73.6g/L(图6),对葡萄糖得率达到0.24g/g。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (9)
1.一种重组大肠杆菌,其特征在于,所述重组大肠杆菌在大肠杆菌宿主菌中,敲除3-脱氢莽草酸脱氢酶基因aroE、负调控转录抑制因子tyrR、乙酸激酶基因ackA、丙酮酸脱氢酶基因poxB和奎尼酸脱氢酶基因YdiB,并过量表达磷酸烯醇式丙酮酸合酶ppsA、转酮醇酶tktA和转醛醇酶talA、DHQ合酶aroB、3-脱氢奎尼酸脱水酶aroD和抗反馈抑制的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶基因aroGFBR;3-脱氢莽草酸脱氢酶编码基因aroE的NCBI编号为NP_417740.1、负调控转录抑制因子编码基因tyrR的NCBI编号为NP_415839.1、乙酸激酶编码基因ackA的NCBI编号为NP_416799.1、丙酮酸脱氢酶编码基因poxB的NCBI编号为NP_415392.1、奎尼酸脱氢酶编码基因YdiB的NCBI编号为NP_416207.1、磷酸烯醇式丙酮酸合酶编码基因ppsA的NCBI编号为NP_416217.1、转酮醇酶编码基因tktA的NCBI编号为YP_026188.1、转醛醇酶编码基因talA的NCBI编号为NP_414549.1、DHQ合酶的编码基因aroB的NCBI编号为NP_417848.1、3-脱氢奎尼酸脱水酶的编码基因aroD的NCBI编号为NP_416208.1、3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶编码基因aroGFBR的核苷酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的重组大肠杆菌,其特征在于,过表达3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶编码基因aroGFBR、DHQ合酶的编码基因aroB、3-脱氢奎尼酸脱水酶的编码基因aroD、转醛醇酶编码基因talA、转酮醇酶编码基因tktA和磷酸烯醇式丙酮酸合酶编码基因ppsA,都是通过使用强启动子PJ23119分别控制表达。
3.根据权利要求1所述的重组大肠杆菌,其特征在于,所述3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶编码基因aroGFBR过表达了两个拷贝。
4.一种如权利要求1-3中任一项所述的重组大肠杆菌的构建方法,其特征在于,包括以下步骤:
(1)构建aroE、tyrR、ackA、poxB和YdiB基因敲除框,依次将基因敲除框片段转入大肠杆菌宿主菌,筛选获得敲除目标基因的菌株;
(2)扩增获得ppsA、tktA、talA、aroB、aroD和aroGFBR基因片段,将基因片段连接到表达载体上,然后将连接有基因片段的表达载体转入步骤(1)的菌株中,得到所述的重组大肠杆菌。
5.权利要求1-3任一项所述重组大肠杆菌在生产3-脱氢莽草酸中的应用。
6.根据权利要求5所述的应用,其特征在于,所述3-脱氢莽草酸生产是通过将重组大肠杆菌在发酵培养基中进行全有氧发酵,得到含有3-脱氢莽草酸的发酵液。
7.根据权利要求6所述的应用,其特征在于,在所述的全有氧发酵的过程中,初始培养基中葡萄糖耗尽时,流加500~800 g/L葡萄糖,并控制葡萄糖浓度在5~10 g/L。
8.根据权利要求6所述的应用,其特征在于,在所述的全有氧发酵过程中,pH控制在6.8~7.1,发酵温度控制在36~38℃,溶氧维持在20~50%。
9.根据权利要求6所述的应用,其特征在于,所述的发酵培养基配方为:葡萄糖8~12 g/L,酵母粉3~7 g/L,一水柠檬酸1.5~2.5 g/L,磷酸氢二钾5~9 g/L,七水硫酸镁1.5~2.5 g/L,硫酸铵4~6 g/L,氯化钠0.5~1.5 g/L,色氨酸0.3~0.5 g/L,酪氨酸0.5~0.8 g/L,苯丙氨酸0.5~0.8 g/L,金属离子液0.8~1.2 mL/L。
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