CN117603896A - 合成酪氨酸及其衍生物的基因工程菌及其构建方法和应用 - Google Patents
合成酪氨酸及其衍生物的基因工程菌及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及合成酪氨酸及其衍生物的基因工程菌及其构建方法和应用。本发明提供了以葡萄糖为起始原料,通过生物发酵方式生产酪氨酸及其衍生物的底盘菌株及方法,并提供了基因改造菌株,从而合成酪氨酸、白藜芦醇、柚皮素、阿魏酸和香兰素的方法。本发明提供的微生物全合成酪氨酸及其衍生物的方法以葡萄糖为起始底物很大程度的降低了原料的成本,同时微生物全合成是一种可控性强、可定制性、结构优化的合成方法,本发明提供的基因工程改造的菌株生产酪氨酸的效率更高。
Description
技术领域
本发明涉及生物技术领域,具体涉及合成酪氨酸及其衍生物的基因工程菌及其构建方法和应用。
背景技术
酪氨酸(Tyrosine)是芳香族氨基酸,在人及动物的新陈代谢和生长发育中起着重要的作用,其衍生物如白藜芦醇、柚皮素、阿魏酸和香兰素等可维持人体健康代谢,广泛应用在食品、饲料、医药和化工等行业。
白藜芦醇(Resveratrol)是一种天然存在于一些植物中的化合物,特别是葡萄皮、红葡萄酒、花生和一些草药中含有较高的浓度。它是一种多酚类化合物,具有抗氧化和抗炎症的作用,有潜在的健康益处,常被用于护肤或健康补充剂使用。
阿魏酸(Ferulic Acid)是一种天然存在于多种植物中的有机化合物,属于羟基肉桂酸的一种形式。它在植物界中相当常见,主要存在于谷物、蔬菜、水果和一些草本植物中。具有抗氧化和抗炎症的作用,通常作为饮食中的天然成分存在,也可作为药物和护肤品的成分使用。
柚皮素(Naringenin)是一种天然存在于柚子、橙子、柠檬、葡萄柚以及某些其他水果和蔬菜中的生物活性化合物,属于黄酮类化合物,也具有抗氧化、抗炎的作用,除此之外还能够改善胰岛素敏感性,有助于控制血糖水平,因此对于糖尿病的管理具有潜在益处。
香兰素(Vanillin)是一种常见的香料化合物,常以游离态和葡萄糖苷的形式存在于植物中,如香荚兰豆、丁香油、甜菜根、秘鲁香脂、吐鲁香脂和天冬门等,其中较集中存在于香荚兰豆中。纯净的香兰素具有浓郁的奶香气,有“香料之王”的美誉,被广泛应用于食品、饮料、香料和医药等领域中。
白藜芦醇、柚皮素、阿魏酸、香兰素均属于"羟基肉桂酸类化合物"(hydroxycinnamic acids)的多酚化合物,均具有苯环结构和一个或多个羟基(OH)基团和酚基团,由于具有一个或多个酚基团,使上述物质具有强大的抗氧化性质。
目前,市场上酪氨酸及其衍生物的生产方式主要包括植物提取法、微生物转化法和化学合成法。植物提取法主要是从植物中提取,但植物的种植受气候的影响,其产量有限,价格昂贵,不能满足市场需求。化学合成法生产的酪氨酸及其衍生物价格低廉,但其生产过程对环境造成严重污染,而且香型单一,安全性也备受质疑,不符合下游应用市场对天然原料的消费趋势,并且欧盟(European Union,EU)和美国食品药品监督管理局(Food andDrug Administration,FDA)不允许将其用于食品。现有技术目前较为常用的是以阿魏酸作为底物,通过微生物发酵制备酪氨酸及其衍生物,但阿魏酸作为原料的生产成本较高,寻求更为廉价的底物成为酪氨酸及其衍生物生物法制备的一个重要研究方向。
以葡萄糖为起始原料,生物全合成制备白藜芦醇、柚皮素、阿魏酸、香兰素很大程度的降低了原料的成本,但反应路径关键酶的活性和表达强度受限,存在抑制和阻遏,需要进行精确调控和优化。在上述物质的生物全合成路径中,酪氨酸均为重要的中间物质,因此构建用于合成酪氨酸衍生物的酪氨酸底盘是核心的关键技术之一。
发明内容
有鉴于此,本发明要解决的技术问题在于提供合成酪氨酸及其衍生物的基因工程菌及其构建方法和应用,本发明提供了以葡萄糖为起始原料,通过生物发酵方式生产酪氨酸及其衍生物的菌种及方法,并提供了基因改造菌株,从而产生酪氨酸及其衍生物的方法。
本发明提供了合成酪氨酸的基因工程菌,包括对底盘菌进行如下基因改造:
抑制和/或敲除tyrR基因、pheA基因、metJ基因、dkgB基因、yeaE基因、lacZ基因和yjgB基因中的至少一种;
整合表达解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种,所述解除反馈抑制基因包括aroGfbr基因和/或tyrAfbr基因,所述整合的位点包括dkgB位点、yeaE位点、lacZ位点和yjgB位点中的至少一种;
过表达tktA基因、ppsA基因和aroE基因中的至少一种。
在一些实施例中,所述aroGfbr基因于所述dkgB位点表达;
所述tyrAfbr基因于所述yeaE位点表达;
所述GroEL基因于所述lacZ位点表达;
所述DnaJK基因于所述yjgB位点表达。
在一些实施例中,所述整合表达还包括将所述解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种的启动子更换为较原启动子启动效果更强的启动子。
在一些具体实施例中,所述aroGfbr基因、tyrAfbr基因、tktA基因、ppsA基因和aroE基因由T7启动子调控,所述GroEL基因由J23119启动子调控,所述DnaJK基因由HCE启动子调控。
在一些实施例中,所述底盘菌包括酵母菌、大肠杆菌、枯草杆菌或微藻中的任意一种。
优选地,本发明采用大肠杆菌作为底盘菌进行改造。
本发明提供了所述的基因工程菌的构建方法,所述构建方法包括对所述底盘菌进行如下的改造:
抑制和/或敲除tyrR基因、pheA基因、metJ基因、dkgB基因、yeaE基因、lacZ基因和yjgB基因中的至少一种;
整合表达解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种,所述解除反馈抑制基因包括aroGfbr基因和/或tyrAfbr基因,所述整合的位点包括dkgB位点、yeaE位点、lacZ位点和yjgB位点中的至少一种;
过表达tktA基因、ppsA基因和aroE基因中的至少一种。
在一些实施例中,所述aroGfbr基因于所述dkgB位点表达;
所述tyrAfbr基因于所述yeaE位点表达;
所述GroEL基因于所述lacZ位点表达;
所述DnaJK基因于所述yjgB位点表达;
所述整合表达还包括将所述解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种的启动子更换为较原启动子启动效果更强的启动子。
在一些具体实施例中,所述aroGfbr基因、tyrAfbr基因、tktA基因、ppsA基因和aroE基因由T7启动子调控,所述GroEL基因由J23119启动子调控,所述DnaJK基因由HCE启动子调控。
本发明提供了所述的基因工程菌或所述的构建方法制备的基因工程菌在合成酪氨酸和/或酪氨酸衍生物中的应用。
在一些具体实施例中,所述酪氨酸衍生物包括白藜芦醇、柚皮素、阿魏酸和香兰素中的至少一种。
本发明提供了合成酪氨酸和/或酪氨酸衍生物的方法,包括:采用所述的基因工程菌或所述的构建方法制备的基因工程菌进行发酵。
在一些实施例中,所述酪氨酸衍生物为阿魏酸,合成所述阿魏酸的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括整合顺天黄杆菌来源的FsTAL、西班牙糖丝菌来源的SeSAM5和拟南芥来源的突变体AtCOMTQ310G至yahK位点获得合成阿魏酸的改造菌株,采用所述改造菌株进行发酵。
在一些实施例中,所述酪氨酸衍生物为香兰素,合成所述香兰素的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括整合顺天黄杆菌来源的FsTAL、西班牙糖丝菌来源的SeSAM5和拟南芥来源的突变体AtCOMTQ310G至yahK位点,过表达链霉菌来源的SsFcs和SsEch获得合成香兰素的改造菌株,采用所述改造菌株进行发酵。
在一些具体实施例中,所述整合表达还包括将所述顺天黄杆菌来源的FsTAL、西班牙糖丝菌来源的SeSAM5和拟南芥来源的突变体AtCOMTQ310G中的至少一种的启动子更换为较原启动子启动效果更强的启动子。
在一些实施例中,所述酪氨酸衍生物为白藜芦醇,合成所述白藜芦醇的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括顺天黄杆菌来源的FsTAL、拟南芥来源的At4CL和葡萄来源的VvSTS1获得合成白藜芦醇的改造菌株,采用所述改造菌株进行发酵。
在一些实施例中,所述酪氨酸衍生物为柚皮素,合成所述柚皮素的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括顺天黄杆菌来源的FsTAL、拟南芥来源的At4CL、矮牵牛来源的PhCHS和苜蓿来源的MsCHI获得合成柚皮素的改造菌株,采用所述改造菌株进行发酵。
在一些具体实施例中,所述发酵的底物包括葡萄糖。
在一些具体实施例中,所述发酵的底物包括葡萄糖。
本发明提供了以葡萄糖为起始原料,通过生物发酵方式生产酪氨酸及其衍生物的底盘菌株及方法,并提供了基因改造菌株,从而合成酪氨酸、白藜芦醇、柚皮素、阿魏酸和香兰素的方法。与现有技术相比:
(1)、本发明以葡萄糖为起始底物进行酪氨酸及其衍生物的合成,很大程度的降低了原料的成本;
(2)、本发明通过对菌株进行优化构建,平衡了代谢通量,扩大代谢过程中可利用的碳源,提高了底物利用率;
(3)、提供了可控性强、可定制性、结构优化的酪氨酸及其衍生物的合成方法,提供的基因改造菌株,可通过调节反应路径关键酶的活性和表达强度,进行精确调控和优化;
(4)、与其他改造相比,本发明提供的基因工程改造的菌株生产酪氨酸及其衍生物的效率更高。
具体实施方式
本发明提供了香兰素的基因工程菌及其构建方法、应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。下面结合实施例,进一步阐述本发明。
实施例1构建合成酪氨酸合成菌株
以E.coli BL21(DE3)为初始底盘菌,对其进行改造,构建以葡萄糖为底物合成酪氨酸的菌株TYR001-TYR022,其中具体采用的培养基及菌株构建方法如下。
采用的培养基包括:
(1)M9培养基配方:
氯化铵1g/L,氯化钠0.5g/L,12水磷酸氢二钠17.9g/L,磷酸二氢钾3g/L,硫酸镁2mM,氯化钙0.1mM,葡萄糖20g/L
(2)LB培养基配方:
胰蛋白胨10g/L,NaCl 10g/L,酵母提取物5g/L,10M NaOH溶液调节培养基pH在7.0±0.5。
(3)TB培养基配方:
胰蛋白胨12g/L,酵母提取物24g/L,甘油4‰(v/v),10MNaOH溶液调节培养基pH在7.0±0.5;17mM KH2PO4,72mM K2HPO4·3H2O。
具体构建方法(均采用CRISPR/Cas9基因编辑技术):
一、敲除阻遏基因tyrR
1)在E.coli BL21(DE3)的基因组中寻找到tyrR基因,然后用在线网站Zhang Lab的CRISPOR(http://crispor.tefor.net/)预测tyrR基因的gRNA序列,选择效率高、无脱靶的gRNA序列;设计部分互补的带有gRNA序列的正反向引物tyrR-gRNA-F和tyrR-gRNA-R(引物见表1),以质粒pEcgRNA为模板进行PCR扩增带有靶向目的基因gRNA序列的pEcgRNAtyrR,PCR产物用限制性内切酶Dpn I消化2h去除模板;琼脂糖凝胶电泳后回收目的条带,回收时用ddH2O洗脱,转化E.coli DH5α感受态细胞,涂Spe抗性平板于37℃过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Spe的LB培养基,测序验证,提取测序正确的质粒pEcgRNA-tyrR备用。
2)在E.coli BL21(DE3)的基因组中寻找到tyrR基因,找出tyrR基因上下游各500bp的序列作为同源臂序列tyrR-up、tyrR-down,设计引物扩增(引物见表1),再以之为模板进行OE-PCR得到tyrR-donor DNA,回收后测序验证,备用。
3)将pEcCas9质粒转化入E.coli BL21(DE3)底盘感受态细胞中,涂Kan平板;挑取Kan平板上的阳性克隆于10mL LB(Kan+2g/L L-araB)培养基中,37℃培养约5~6h至OD~1.0;将正确的100ng pEcgRNA-tyrR和400ng tyrR-donor DNA电转到带有pEcCas9质粒的E.coli细胞(~OD 1.0),涂平板(Kan+Spe);同时,将正确的100ng gRNA电转到带有pEcCas9质粒的阳性克隆中,涂板(Kan+Spe),作为阴性对照;37℃过夜培养;
4)挑取同时电转gRNA和donor DNA平板上的单克隆于300uL含有Kan+2g/Lrhamnose的LB培养基中,37℃,200rpm摇至对数期(~5h左右),取1~2uL菌液进行菌液PCR,将菌液PCR验证正确的菌液划线于Kan平板中,同时正确的PCR产物送测序验证;
5)挑取Kan平板上的阳性克隆,分别挑于300uL含有Kan、Kan+Spe和20g/L蔗糖的LB培养基,37℃、200rpm培养至对数期(~5h左右);将在含Kan+Spe培养基不长、但在含蔗糖培养基长的菌于蔗糖平板上划线(终浓度20g/L),37℃培养;挑取蔗糖平板上单菌落分别于300uL含有Kan、不含抗性的LB培养基,37℃、200rpm培养至对数期(约5h左右);其中在Kan不长而在不含抗性LB上长的菌即为敲除tyrR基因的工程菌(编号TYR001),保存菌株备用(20%甘油)。
二、敲除苯丙氨酸竞争途径基因pheA
1、构建pheA基因单独敲除菌株(编号TYR002):以E.coliBL21(DE3)为出发菌株,采用与“一、敲除阻遏基因tyrR”步骤1)-5)相同的方法进行pheA基因敲除,涉及到的引物列于表1中,最后得到在Kan不长而在不含抗性的LB上长的菌即为敲除pheA基因的工程菌,保存菌株备用(20%甘油)。
2、构建tyrR基因和pheA基因同时敲除菌株(编号TYR003):以TYR001为出发菌株,采用与“一、敲除阻遏基因tyrR”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“一”制备得到的TYR001菌株中。最后得到在Kan不长而在不含抗性的LB上长的菌即为同时敲除tyrR、pheA基因的工程菌,保存菌株备用(20%甘油)。
三、敲除甲基供体合成途径的全局调控基因metJ
1、构建metJ基因单独敲除菌株(编号TYR004):以E.coliBL21(DE3)为出发菌株,采用与“一、敲除阻遏基因tyrR”步骤1)-5)相同的方法进行,涉及到的引物列于表1中,最后得到在Kan不长而在不含抗性的LB上长的菌即为敲除metJ基因的工程菌,保存菌株备用(20%甘油)。
2、构建tyrR基因和metJ基因同时敲除菌株(编号TYR005):采用与“一、敲除阻遏基因tyrR”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“二”制备得到的TYR001菌株中,最后得到同时敲除tyrR、metJ基因的工程菌,保存菌株备用(20%甘油)。
3、构建pheA基因和metJ基因同时敲除菌株(编号TYR006):采用与“一、敲除阻遏基因tyrR”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“二”制备得到的TYR002菌株中。最后得到同时敲除pheA、metJ基因的工程菌,保存菌株备用(20%甘油)。
4、tyrR基因、pheA基因和metJ基因同时敲除菌株(编号TYR007):采用与“一、敲除阻遏基因tyrR”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“二”制备得到的TYR003菌株中。最后得到同时敲除tyrR、pheA、metJ基因的工程菌,保存菌株备用(20%甘油)。
四、将基因aroGfbr整合至dkgB位点或LacZ位点(aroGfbr基因由T7启动子调控)
1、构建tyrR基因、pheA基因和metJ基因同时敲除并将基因aroGfbr整合至dkgB位点的基因工程菌(编号TYR008),具体构建方法如下:
1)dkgB基因敲除方法采用与“一、敲除阻遏基因tyrR”步骤1)相同的方法进行;
2)在E.coliBL21(DE3)的基因组中寻找到dkgB基因,找出dkgB基因上下游各500bp的序列作为同源臂序列dkgB-up、dkgB-down,设计引物扩增携带同源臂序列的dkgB-up、aroGfbr、dkgB-down(引物见表1),再以之为模板进行OE-PCR得到aroGfbr-donorDNA,回收后测序验证,备用。
3)将pEcCas9质粒转化入前述“三”制备得到的TYR007菌株中,涂Kan平板。后续筛选步骤与“一”步骤3)-5)相同。最后得到编号TYR008(ΔtyrRΔpheAΔmetJΔdkgB::aroGfbr)的菌株,保存菌株备用(20%甘油)。
2、构建tyrR基因、pheA基因和metJ基因同时敲除并将基因aroGfbr整合至LacZ位点的基因工程菌(编号TYR009),具体构建方法与菌株TYR008构建方法相同。
最后得到编号TYR009(ΔtyrRΔpheAΔmetJΔlacZ::aroGfbr)的菌株,保存菌株备用(20%甘油)。
五、将基因tyrAfbr整合至yeaE位点(tyrAfbr基因由T7启动子调控)
构建tyrR基因、pheA基因和metJ基因同时敲除并将基因tyrAfbr整合至yeaE位点的基因工程菌,构建方法同编号TYR008菌株的构建方法涉及到的引物列于表1中。最后得到编号TYR010的菌株,保存菌株备用(20%甘油)。
六、将基因tyrAfbr整合至LacZ位点(tyrAfbr基因由T7启动子调控)
1、构建tyrR基因、pheA基因和metJ基因同时敲除并将基因tyrAfbr整合至LacZ位点的基因工程菌(编号TYR011),构建方法同编号TYR008菌株的构建方法。最后得到编号TYR011(ΔtyrRΔpheAΔmetJΔlacZ::tyrAfbr)的菌株,保存菌株备用(20%甘油)。
七、将基因tyrAfbr整合至yeaE位点(tyrAfbr基因由T7启动子调控)
构建tyrR基因、pheA基因和metJ基因同时敲除,将基因aroGfbr整合至dkgB位点,将基因tyrAfbr整合至yeaE位点的基因工程菌,构建方法同编号TYR008菌株的构建方法,区别在于,步骤3)是将pEcCas9质粒转化入前述“四”制备得到的TYR008菌株中。最后得到编号TYR012的菌株,保存菌株备用(20%甘油)。
八、将基因tktA、ppsA以及aroE分别单独过表达(替换为T7强启动子);
同样采用CRISPR/Cas9技术进行,与前述“四”相同的方法进行,引物见表1。区别在于,步骤3)是将pEcCas9质粒转化入前述“七”制备得到的TYR012菌株,分别得到编号TYR013、TYR014、TYR015的菌株,保存菌株备用(20%甘油)。
九、将基因tktA、ppsA以及aroE组合过表达(替换为T7强启动子)
tktA、ppsA过表达:同样采用CRISPR/Cas9技术进行,与前述“四”相同的方法进行,引物见表1。区别在于,步骤2)步骤3)是将pEcCas9质粒转化入前述“八”制备得到的TYR013菌株,得到编号TYR016的菌株,保存菌株备用(20%甘油);
tktA、aroE过表达:同样采用CRISPR/Cas9技术进行,与前述“四”相同的方法进行,引物见表1。区别在于,步骤2)步骤3)是将pEcCas9质粒转化入前述“八”制备得到的TYR013菌株,得到编号TYR017的菌株,保存菌株备用(20%甘油);
ppsA、aroE过表达:同样采用CRISPR/Cas9技术进行,与前述“四”相同的方法进行,引物见表1。区别在于,步骤2)步骤3)是将pEcCas9质粒转化入前述“八”制备得到的TYR014菌株,得到编号TYR018的菌株,保存菌株备用(20%甘油);
tktA、ppsA、aroE过表达:同样采用CRISPR/Cas9技术进行,与前述“四”相同的方法进行,引物见表1。区别在于,步骤2)步骤3)是将pEcCas9质粒转化入前述“八”制备得到的TYR016菌株,得到编号TYR019的菌株,保存菌株备用(20%甘油)。
十、将GroEL整合至lacZ位点(GroEL基因由J23119启动子调控)
采用与前述“四”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“四”制备得到的TYR019菌株中。最后得到编号TYR020的菌株,保存菌株备用(20%甘油)。
十一、将DnaJK整合至yjgB位点(DnaJK基因由HCE启动子调控)
采用与前述“四”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“四”制备得到的TYR019菌株中。最后得到编号TYR021的菌株,保存菌株备用(20%甘油)。
十二、采用与前述“四”步骤1)-5)相同的方法进行,涉及到的引物列于表1中。区别在于,步骤3)是将pEcCas9质粒转化入前述“四”制备得到的TYR020菌株中。最后得到编号TYR022的菌株,保存菌株备用(20%甘油)。
表1
上述实施例1中涉及基因及其登录号信息如下:tyrR基因登录号:CP055259.1;
pheA基因登录号:CP054224.1;
metJ基因登录号:CP082100.1;
dkgB基因登录号:CP042892.1;
aroGfbr基因登录号:CP082100.1;
lacZ基因登录号:CP053602.1;
yeaE基因登录号:CP127119.1;
tyrAfbr基因登录号:CP082087.1;
tktA基因登录号:CP053602.1;
ppsA基因登录号:CP053597.1;
aroE基因登录号:CP082100.1;
yjgB基因登录号:CP054457.1。
采用本实施例中制备的酪氨酸菌株进行发酵并统计酪氨酸产量,具体试验流程如下:
将实施例1构建的TYR001-TYR022菌株划线于无抗性的LB平板,37℃过夜培养后挑取单克隆于5mLLB培养基,37℃、200rpm过夜培养。按照1:100接种量接种于100mL TB培养基,37℃、200rpm培养48h,至少3个平行组。发酵结束后,取1mL发酵液直接经等体积甲醇淬灭,离心过滤后上清于HPLC检测酪氨酸含量,具体如表2所示。
表2
结果表明,对底盘进行改造,敲除阻遏基因tyrR、苯丙氨酸竞争途径基因pheA、甲基供体合成途径的全局调控基因metJ;调整解除反馈抑制基因aroGfbr、tyrAfbr的位点和拷贝数;调整GroEL、DnaJK的位点,增加前体碳代谢基因tktA、ppsA以及aroE的表达,均能在一定程度上提高酪氨酸的产量。
效果例1构建合成阿魏酸菌株
以实施例1构建的工程菌株为底盘菌进行改造,构建阿魏酸高产菌株FA001-FA009。
具体改造流程如下:
一:咖啡酸-O-甲基转移酶(AtCOMT)突变体构建
以AtCOMT为模板,以AtCOMT-F、AtCOMT-Q310G-R2和AtCOMT-Q310G-F2、AtCOMT-R为引物进行PCR扩增,回收DNA片段;然后再以AtCOMT-F和AtCOMT-R为引物,DNA片段AtCOMT(Q310G)-P1/P2为模板进行PCR扩增,得到AtCOMT(Q310G)突变体DNA。
上述咖啡酸-O-甲基转移酶(AtCOMT)来源于:Arabidopsis thaliana拟南芥。
二:质粒及菌株构建
采用pET21d为表达质粒,利用In-Fusion策略构建重组表达质粒;
具体步骤如下:
2.1、全基因合成针对大肠杆菌密码子优化后的FsTAL、SeSAM5、AtCOMT,通过引物扩增携带T7启动子的FsTAL(酪氨酸解氨酶TAL,来源于:Flavobacterium suncheonense顺天黄杆菌)、SeSAM5(对香豆酸羟化酶C3H,来源于:Saccharothrix espanaensis西班牙糖丝菌)和AtCOMT基因片段(t7-FsTAL-t7-SeSAM5-t7-AtCOMT)和pET21d线性化载体片段(引物见表3);按照试剂盒说明,将DNA片段和线性化载体片段进行同源重组反应,50℃反应1h后,转化E.coli DH5α感受态细胞,涂Amp抗性平板于37度过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Amp的LB培养基,测序验证,提取测序正确的质粒FAP001(pET21d-t7-FsTAL-t7-SeSAM5-t7-AtCOMT)备用;
将质粒FAP001转入partⅠ部分的TYR020、TYR021、TYR022中,用含有Amp抗性的平板进行培养,挑取阳性克隆,提取质粒,测序验证正确后,获得菌株FA001、FA002、FA003。
2.2、以FAP001为模板,设计AtCOMT突变体引物(引物见表3),进行PCR扩增,PCR产物用限制性内切酶Dpn I消化2h去除模板;琼脂糖凝胶电泳后回收目的条带,回收时用ddH2O洗脱,然后转化E.coli DH5α感受态细胞,涂Amp抗性平板于37度过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Amp的LB培养基,测序验证,提取测序正确的质粒FAP002(pET21d-t7-FsTAL-t7-SeSAM5-t7-AtCOMT(Q310G))备用。
将质粒FAP002转入partⅠ部分的TYR020、TYR021、TYR022中,用含有Amp抗性的平板进行培养,挑取阳性克隆,提取质粒,测序验证正确后,获得菌株FA004、FA005、FA006。
三、基因整合及菌株制备
1.构建表达框并整合至基因组的yahK位点
1)在E.coli BL21(DE3)的基因组中寻找到yahK基因,然后用在线网站Zhang Lab的CRISPOR(http://crispor.tefor.net/)预测yahK基因的gRNA序列,选择效率高、无脱靶的gRNA序列;设计部分互补的带有gRNA序列的正反向引物yahK-gRNA-F和yahK-gRNA-R(引物见表3),以质粒pEcgRNA为模板进行PCR扩增带有靶向目的基因gRNA序列的pEcgRNAyahK,PCR产物用限制性内切酶Dpn I消化2h去除模板;琼脂糖凝胶电泳后回收目的条带,回收时用ddH2O洗脱,转化E.coli DH5α感受态细胞,涂Spe抗性平板于37℃过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Spe的LB培养基,测序验证,提取测序正确的质粒pEcgRNA-yahK备用。
2)在E.coliBL21(DE3)的基因组中寻找到yahK基因,找出yahK基因上下游各500bp的序列作为同源臂序列yahK-up、yahK-down,设计引物扩增携带同源臂序列的yahK-up、t7-FsTAL-t7-SeSAM5-t7-AtCOMT(Q310G)、yahK-down,再以之为模板进行OE-PCR得到t7-FsTAL-t7-SeSAM5-t7-AtCOMT(Q310G)-donorDNA,回收后测序验证,备用。
3)将pEcCas9质粒转化入partⅠ部分的TYR020中,涂Kan平板;挑取Kan平板上的阳性克隆于10mLLB(Kan+2g/LL-araB)培养基中,37℃培养约5~6h至OD~1.0;将正确的100ngpEcgRNA-yahK和400ng t7-FsTAL-t7-SeSAM5-t7-AtCOMT(Q310G)-donorDNA电转到带有pEcCas9质粒的细胞(~OD 1.0),涂板(Kan+Spe);同时,将正确的100ng gRNA电转到带有pEcCas9质粒的阳性克隆中,涂板(Kan+Spe),作为阴性对照;37℃过夜培养;
4)挑取电转gRNA和donorDNA平板的单克隆于300uL含有Kan+2g/Lrhamnose的LB培养基中,37℃,200rpm摇至对数期(~5h左右),取1~2uL菌液进行菌液PCR,将菌液PCR验证正确的菌液划线于Kan平板中;
5)挑取Kan平板上的阳性克隆,分别挑于300uL含有Kan、Kan+Spe和20g/L蔗糖的LB培养基,37℃、200rpm培养至对数期(~5h左右);将在含Kan+Spe培养基不长、但在含蔗糖培养基长的菌于蔗糖平板上划线(终浓度20g/L);挑取蔗糖平板上单菌落分别于300uL含有Kan、空的LB培养基,37℃、200rpm培养至对数期(约5h左右);其中在Kan不长而在LB空上长的菌即为敲除yahK基因的工程菌(编号FA007),保存菌株备用(20%甘油),得到FA007菌株。
2.构建表达框并整合至基因组的ybfE位点
采用与前述“1”步骤1)-5)相同的方法进行,涉及到的引物列于表3中。区别在于整合位点是,步骤3)是将pEcCas9质粒转化入partI制备得到的TYR020菌株中。最后得到编号FA008的菌株,保存菌株备用(20%甘油)。
3.构建表达框并整合至基因组的cspH位点
采用与前述“1”步骤1)-5)相同的方法进行,涉及到的引物列于表3中。区别在于整合位点是,步骤3)是将pEcCas9质粒转化入partI制备得到的TYR020菌株中。最后得到编号FA009的菌株,保存菌株备用(20%甘油)。
实施例2中涉及基因序列信息或登录号信息如下:
yahK基因登录号:CP133854.1;
ybfE基因登录号:CP133913.1;
cspH基因登录号:CP133871.1;
FsTAL序列(SEQ ID NO:84)
atgagcaccattcatgaatttctgagcattgcggaatttacgagcattgtgtttaaaaacaccaaagtggaagtgagcgatgcggtggtgaaacgcgtggaagcgagctttaactttctgaaagaatttagcgaaaacaaagtgatttatggcgtgaacaccggctttggcccgatggcgcagtatcgcattaaagatgaagatcgcctgcagctgcagtataacctgattcgcagccatagcagcggcaccggcaaaccgctgagcccggtgtgcgtgaaaagcgcgattctggcgcgcctgaacaccctgagcctgggcaacagcggcgtgcatccgagcgtgatttatctgatgcaagaatttattaaccgcgatattaccccgctgatttttgaacatggcggcgtgggcgcgagcggcgatctggtgcagctggcgcatctggcgctgaccctgattggcgaaggcgaagtgttttataaaggcgaacgccgcgcgaccaaagaagtgtttgaactggaaggcctgcagccgattaacgtggaaattcgcgaaggcctggcgctgatgaacggcacgagcgtgatgaccggcattggcattgtgaacgtgtataacgcgaaaaaagtgctggattgggcgattaaactgagctgcgcgattaacgaaattgtgaaagcgtatgatgatcatctgagcgcggaactgaacaacaccaaacagcattttggtcagcaagaagtggcgaaacgcatgcgcgaaaacctggcggatagcaccctggtgcgcaaacgcgaagatcatctgtataccggcgaaaacaccgaagaagtgtttaaagaaaaagtgcaagaatattatagcctgcgctgcgtgccgcagattctgggcccggtgctggataccattcagaacgtggcggaagtgctggaaaacgaaattaacagcgcgaacgataacccgattgtggatgtggaaaatcagcatgtgtatcatggcggcaactttcatggcgattatattagcctggaaatggataaactgaaactggtggtgaccaaactgaccatgctggcggaacgtcagctgaactatctgctgaacagcaaactgaacgaaattctgccgccgtttgtgaacctgggcaccctgggctttaactttggcatgcaaggcgtgcagtttaccgcgacgagcaccaccgcggaatgtcagaccctgagcagtagcatgtatattcatagcattccgaacaataacgataaccaagatattgtgagcatgggcaccaacgcggcggtgattaccggcaaagtgattgaaaacgcgtttgaagtgctggcgattgaactgattaccattgtgcaagcgattgatgcgctgaactataaaaacgatattagcagcgtgacccgcaaaatgtatgatgaagtgcgcgaaattgtgccggaatttaaacaagatatgattatgtatccgtatgtgcagaaagtgaaagaatatctgatgatttaa
SeSAM5序列(SEQ ID NO:85)
atgaccatcacctctccggcgccggcgggtcgtctgaacaacgttcgtccgatgaccggtgaagaatacctggaatctctgcgtgacggtcgtgaagtttacatctacggtgaacgtgttgacgacgttaccacccacctggcgttccgtaactctgttcgttctatcgcgcgtctgtacgacgttctgcacgacccggcgtctgaaggtgttctgcgtgttccgaccgacaccggtaacggtggtttcacccacccgttcttcaaaaccgcgcgttcttctgaagacctggttgcggcgcgtgaagcgatcgttggttggcagcgtctggtttacggttggatgggtcgtaccccggactacaaagcggcgttcttcggtaccctggacgcgaacgcggaattctacggtccgttcgaagcgaacgcgcgtcgttggtaccgtgacgcgcaggaacgtgttctgtacttcaaccacgcgatcgttcacccgccggttgaccgtgaccgtccggcggaccgtaccgcggacatctgcgttcacgttgaagaagaaaccgactctggtctgatcgtttctggtgcgaaagttgttgcgaccggttctgcgatgaccaacgcgaacctgatcgcgcactacggtctgccggttcgtgacaaaaaattcggtctggttttcaccgttccgatgaactctccgggtctgaaactgatctgccgtacctcttacgaactgatggttgcgacccagggttctccgttcgactacccgctgtcttctcgtctggacgaaaacgactctatcatgatcttcgaccgtgttctggttccgtgggaaaacgttttcatgtacgacgcgggtgcggcgaactctttcgcgaccggttctggtttcctggaacgtttcaccttccacggttgcacccgtctggcggttaaactggacttcatcgcgggttgcgttatgaaagcggttgaagttaccggtaccacccacttccgtggtgttcaggcgcaggttggtgaagttctgaactggcgtgacgttttctggggtctgtctgacgcgatggcgaaatctccgaactcttgggttggtggttctgttcagccgaacctgaactacggtctggcgtaccgtaccttcatgggtgttggttacccgcgtatcaaagaaatcatccagcagaccctgggttctggtctgatctacctgaactcttctgcggcggactggaaaaacccggacgttcgtccgtacctggaccgttacctgcgtggttctcgtggtatccaggcgatcgaccgtgttaaactgctgaaactgctgtgggacgcggttggtaccgaattcgcgggtcgtcacgaactgtacgaacgtaactacggtggtgaccacgaaggtatccgtgttcagaccctgcaggcgtaccaggcgaacggtcaggcggcggcgctgaaaggtttcgcggaacagtgcatgtctgaatacgacctggacggttggacccgtccggacctgatcaacccgggtacctaa
AtCOMT序列(SEQ ID NO:86)
atgggttcaacggcagagacacaattaactccggtgcaagtcaccgacgacgaagctgccctcttcgccatgcaactagccagtgcttccgttcttccgatggctttaaaatccgcctta8gagcttgaccttcttgagattatggccaagaatggttctcccatgtctcctaccgagatcgcttctaaacttccgaccaaaaatcctgaagctccggtcatgctcgaccgtatcctccgtcttcttacgtcttactccgtcttaacctgctccaaccgtaaactttccggtgatggcgttgaacggatttacgggcttggtccggtttgcaagtatttgaccaagaacgaagatggtgtttccattgctgctctttgtcttatgaaccaagacaaggttctcatggaaagctggtaccatttgaaggatgcaattcttgatggtgggattccattcaacaaggcttatggaatgagcgcgttcgagtaccacgggactgaccctagattcaacaaggtctttaacaatggaatgtctaaccattccacaatcaccatgaagaagattcttgagacctataagggttttgaaggattgacttctttggttgatgttggtggtggcattggtgctacactcaaaatgattgtctccaagtaccctaatcttaaaggcatcaactttgatctcccacatgtcatcgaagatgctccttctcatcctggtattgagcatgttggaggagatatgtttgtaagtgtccctaaaggtgatgccatattcatgaagtggatatgtcatgactggagtgacgaacattgcgtgaaattcttgaagaactgctacgagtcacttccagaggatggaaaagtgatattagcagagtgtatacttccagagacaccagactcaagcctctcaaccaaacaagtagtccatgtcgattgcattatgttggctcacaatcccggaggcaaagaacgaaccgagaaagagtttgaggcattagccaaagcatcaggcttcaagggcatcaaagttgtctgcgacgcttttggtgttaaccttattgagttactcaagaagctctaa
AtCOMT(Q310G)序列具体为:替换SEQ ID NO:86中第928~930位碱基为ggc。
表3引物设计
采用本实施例中制备的阿魏酸菌株发酵并统计阿魏酸产量,具体试验流程如下:
将实施例2构建的FA001-FA006菌株划线于含Amp的LB平板(FA007-FA009菌株划线于无抗性的LB平板),37℃过夜培养后挑取单克隆于5mLLB培养基,37℃、200rpm过夜培养。按照1:100接种量接种于100mLTB培养基,37℃、200rpm,培养至OD600~0.7,加入0.1mMIPTG,25℃,160rpm诱导培养48h,至少3个平行组(FA007-FA009无需诱导,培养48h,至少3个平行组)。发酵结束后,取1mL发酵液直接经等体积甲醇淬灭,离心过滤后上清于HPLC检测阿魏酸含量,具体结果如表4所示。
表4
结果表明,表达咖啡酸-O-甲基转移酶(COMT)采用突变体AtCOMT(Q310G)的菌株相对于表达传统咖啡酸-O-甲基转移酶(AtCOMT)的菌株,阿魏酸产量明显得到了提高。同时,相对于外源基因以质粒型式表达,将外源基因整合在基因组上能够提高菌株的稳定性,但会比引入外源质粒表达水平低。本申请通过构建基因表达框并整合至基因组的高效表达位点,使阿魏酸的产量仍然能维持在同一水平。
效果例2构建合成香兰素的菌株
以效果例1构建的工程菌株为底盘菌进行改造,构建生产香兰素的工程菌。
具体试验流程如下:
一:质粒构建
以pET28a为表达载体,利用In-Fusion策略构建重组表达质粒;具体步骤:全基因合成针对大肠杆菌密码子优化后的SsFcs(反式阿魏酰辅酶A合成酶,trans-Feruloyl-CoAsynthetase,来源于:Streptomyces sp.V-1链霉菌)、SsEch(烯酰辅酶A水合酶/醛缩酶,Enoyl-CoAhydratase/aldolase来源于:Streptomyces sp.V-1链霉菌),通过引物扩增含有同源臂的SsFcs-RBS-SsEch和线性化pET28a载体(引物见表5);
按照试剂盒说明,将DNA片段和线性化载体片段进行同源重组反应,50度反应1h后,转化E.coli DH5α感受态细胞,涂Kan抗性平板于37度过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Kan的LB培养基,测序验证,提取测序正确的质粒VANP101(pET28a-t7-SsFcs-SsEch)。
二:菌株构建
将质粒VANP101电转入FA007菌株,得到菌株VAN10。具体操作步骤是:将冻存的FA007菌株接种于5~10mL LB培养基,37℃培养至OD600~1.0;将质粒VANP101电转入FA007菌株,涂Kan抗性平板于37度过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Kan的LB培养基,37度过夜培养,得到菌株VAN10,保存菌株于-80℃(诱导表达方法:接种于50mLTB培养基,200rpm,37℃培养至OD600~0.7,加入0.1mM IPTG,20℃诱导表达)。
将VAN10菌株接种于50mL含有Kan的M9培养基,37℃、200rpm,培养至OD600~0.7,加入0.1mM IPTG,20℃,160rpm诱导培养72h,至少3个平行组。发酵结束后,取1mL发酵液直接经等体积甲醇淬灭,离心过滤后上清于HPLC测定香兰素的含量。测得香兰素的含量为1056mg/L,结果表明,制备获得的底盘菌能够有效用于生产香兰素中。
效果例2中涉及基因序列信息如下:
SsFcs基因登录号:KC847405.1,SsEch基因登录号:KC847406.1。
表5
引物名称 | 序列5-3 | SEQ ID NO |
Fcs-F | gtgcgcaaccagggtctgggctc | 121 |
Fcs-R1 | tcagccgaagcggcggcggac | 122 |
Ech-F1 | tccgccgccgcttcggctgactagaaataattttgtttaactttaagaaggagatat | 123 |
Ech-R | ctacttctccgggtcgaaggcgctcag | 124 |
28a-over-F2 | ccttcgacccggagaagtagaagcttgcggccgcactcgagc | 125 |
28a-over-R2 | cccagaccctggttgcgcacatggctgccgcgcggcaccaggc | 126 |
效果例3构建合成白藜芦醇的菌株
以实施例1构建的工程菌株为底盘菌进行改造,构建生产白芦藜醇的工程菌。
具体构建及测试方法如下:
全基因合成At4CL、VvSTS1,通过引物扩增携带T7启动子的FsTAL(酪氨酸解氨酶,来源于:Flavobacterium suncheonense顺天黄杆菌)、At4CL(对香豆酰辅酶A连接酶,来源于:Arabidopsis thaliana拟南芥,GenBank登录号:QJD21997)、VvSTS1(芪合酶,来源于:Vitis vinifera葡萄,登录号:NP_001267939)基因片段(T7-FsTAL-T7-At4CL-T7-VvSTS1)和pET21d线性化载体片段(引物见表6);按照试剂盒说明,将DNA片段和线性化载体片段进行同源重组反应,50℃反应1h后,转化E.coli DH5α感受态细胞,涂Amp抗性平板于37度过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Amp的LB培养基,测序验证,提取测序正确的质粒RESP001(pET21d-T7-FsTAL-T7-At4CL-T7-VvSTS1)备用;
表6
将质粒RESP001转入实施例1部分的TYR020、TYR021、TYR022中,用含有Amp抗性的平板进行培养,挑取阳性克隆,获得菌株RES001、RES002、RES003,20%甘油浓度保存于-80℃冰箱备用;
将重组菌RES001、RES002、RES003划线于含Amp抗性平板,37度过夜培养后挑取单菌落接种于5mL含Amp的LB培养基,37度过夜后按照(1:100)接种于100mLTB培养基,200rpm、37℃培养至OD600=0.6~0.8,加入0.1mM IPTG,20℃、160rpm诱导培养48h,至少3个平行组。发酵结束后,取1mL发酵液直接经等体积甲醇淬灭,离心过滤后上清于HPLC检测白藜芦醇含量如下表7所示。
表7
白芦藜醇菌株 | 白芦藜醇产量(mg/L) |
RES001-1 | 290 |
RES001-2 | 287 |
RES001-3 | 293 |
RES002-1 | 303 |
RES002-2 | 305 |
RES002-3 | 308 |
RES003-1 | 278 |
RES003-2 | 265 |
RES003-3 | 266 |
结果表明,制备获得的底盘菌能够有效用于生产白芦藜醇中。
效果例4构建合成柚皮素的菌株
以实施例1构建的工程菌株为底盘菌进行改造,构建生产柚皮素的工程菌。
具体构建及测试方法如下:
全基因合成PhCHS、MsCHI,通过引物扩增携带T7启动子的FsTAL(酪氨酸解氨酶,来源于:Flavobacterium suncheonense顺天黄杆菌)、At4CL(对香豆酰辅酶A连接酶,来源于:Arabidopsis thaliana拟南芥,GenBank登录号:QJD21997)、PhCHS(查尔酮合成酶,来源于:Petuniaxhybrida矮牵牛,:GenBank登录号:AB678720)和MsCHI(查尔酮异构酶,来源于:Medicago sativa苜蓿,GenBank登录号:OQ504213)基因片段(T7-FsTAL-At4CL-T7-PhCHS-MsCHI)和pET21d线性化载体片段(引物见表8);按照试剂盒说明,将DNA片段和线性化载体片段进行同源重组反应,50℃反应1h后,转化E.coli DH5α感受态细胞,涂Amp抗性平板于37度过夜培养;菌落PCR筛选阳性克隆,挑取阳性克隆于5mL含Amp的LB培养基,测序验证,提取测序正确的质粒NARP001(pET21d-T7-FsTAL-At4CL-T7-PhCHS-MsCHI)备用;
表8
将质粒NARP001转入实施例1部分的TYR020、TYR021、TYR022中,用含有Amp抗性的平板进行培养,挑取阳性克隆,获得菌株NAR001、NAR002、NAR003,20%甘油浓度保存于-80℃冰箱备用;
将重组菌NAR001、NAR002、NAR003划线于含Amp抗性平板,37度过夜培养后挑取单菌落接种于5mL含Amp的LB培养基,37度过夜后按照(1:100)接种于100mLTB培养基,200rpm、37℃培养至OD600=0.6~0.8,加入0.1mM IPTG,20℃、160rpm诱导培养48h,至少3个平行组。发酵结束后,取1mL发酵液直接经等体积甲醇淬灭,离心过滤后上清于HPLC检测(2S)-柚皮素含量如下表9所示。
表9
柚皮素菌株 | 柚皮素产量(mg/L) |
NAR001-1 | 180 |
NAR001-2 | 178 |
NAR001-3 | 182 |
NAR002-1 | 200 |
NAR002-2 | 192 |
NAR002-3 | 201 |
NAR003-1 | 150 |
NAR003-2 | 158 |
NAR003-3 | 151 |
结果表明,制备获得的底盘菌能够有效用于生产柚皮素中。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (14)
1.合成酪氨酸的基因工程菌,其特征在于,包括对底盘菌进行如下基因改造:
抑制和/或敲除tyrR基因、pheA基因、metJ基因、dkgB基因、yeaE基因、lacZ基因和yjgB基因中的至少一种;
整合表达解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种,所述解除反馈抑制基因包括aroGfbr基因和/或tyrAfbr基因,所述整合的位点包括dkgB位点、yeaE位点、lacZ位点和yjgB位点中的至少一种;
过表达tktA基因、ppsA基因和aroE基因中的至少一种。
2.根据权利要求1所述的基因工程菌,其特征在于,
所述aroGfbr基因于所述dkgB位点表达;
所述tyrAfbr基因于所述yeaE位点表达;
所述GroEL基因于所述lacZ位点表达;
所述DnaJK基因于所述yjgB位点表达。
3.根据权利要求1或2所述的基因工程菌,其特征在于,所述整合表达还包括将所述解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种的启动子更换为较原启动子启动效果更强的启动子。
4.根据权利要求1所述的基因工程菌,其特征在于,所述底盘菌包括酵母菌、大肠杆菌、枯草杆菌或微藻中的任意一种。
5.权利要求1~4任一项所述的基因工程菌的构建方法,其特征在于,所述构建方法包括对所述底盘菌进行如下的改造:
抑制和/或敲除tyrR基因、pheA基因、metJ基因、dkgB基因、yeaE基因、lacZ基因和yjgB基因中的至少一种;
整合表达解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种,所述解除反馈抑制基因包括aroGfbr基因和/或tyrAfbr基因,所述整合的位点包括dkgB位点、yeaE位点、lacZ位点和yjgB位点中的至少一种;
过表达tktA基因、ppsA基因和aroE基因中的至少一种。
6.根据权利要求5所述的构建方法,其特征在于,
所述aroGfbr基因于所述dkgB位点表达;
所述tyrAfbr基因于所述yeaE位点表达;
所述GroEL基因于所述lacZ位点表达;
所述DnaJK基因于所述yjgB位点表达;
所述整合表达还包括将所述解除反馈抑制基因、GroEL基因和DnaJK基因中的至少一种的启动子更换为较原启动子启动效果更强的启动子。
7.权利要求1~4任一项所述的基因工程菌或权利要求5或6所述的构建方法制备的基因工程菌在合成酪氨酸和/或酪氨酸衍生物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述酪氨酸衍生物包括白藜芦醇、柚皮素、阿魏酸和香兰素中的至少一种。
9.合成酪氨酸和/或酪氨酸衍生物的方法,其特征在于,包括:采用权利要求1~4任一项所述的基因工程菌或权利要求5或6所述的构建方法制备的基因工程菌进行发酵。
10.根据权利要求9所述的方法,其特征在于,所述酪氨酸衍生物为阿魏酸,合成所述阿魏酸的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括整合顺天黄杆菌来源的FsTAL、西班牙糖丝菌来源的SeSAM5和拟南芥来源的突变体AtCOMTQ310G至yahK位点获得合成阿魏酸的改造菌株,采用所述改造菌株进行发酵。
11.根据权利要求9所述的方法,其特征在于,所述酪氨酸衍生物为香兰素,合成所述香兰素的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括整合顺天黄杆菌来源的FsTAL、西班牙糖丝菌来源的SeSAM5和拟南芥来源的突变体AtCOMTQ310G至yahK位点,过表达链霉菌来源的SsFcs和SsEch获得合成香兰素的改造菌株,采用所述改造菌株进行发酵。
12.根据权利要求9所述的方法,其特征在于,所述酪氨酸衍生物为白藜芦醇,合成所述白藜芦醇的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括顺天黄杆菌来源的FsTAL、拟南芥来源的At4CL和葡萄来源的VvSTS1获得合成白藜芦醇的改造菌株,采用所述改造菌株进行发酵。
13.根据权利要求9所述的方法,其特征在于,所述酪氨酸衍生物为柚皮素,合成所述柚皮素的方法包括:以所述基因工程菌为底盘菌进行改造,所述改造包括顺天黄杆菌来源的FsTAL、拟南芥来源的At4CL、矮牵牛来源的PhCHS和苜蓿来源的MsCHI获得合成柚皮素的改造菌株,采用所述改造菌株进行发酵。
14.根据权利要求9~13任一项所述的方法,其特征在于,所述发酵的底物包括葡萄糖。
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