CN100432216C - 代谢控制的工程化 - Google Patents
代谢控制的工程化 Download PDFInfo
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- CN100432216C CN100432216C CNB2005100659541A CN200510065954A CN100432216C CN 100432216 C CN100432216 C CN 100432216C CN B2005100659541 A CNB2005100659541 A CN B2005100659541A CN 200510065954 A CN200510065954 A CN 200510065954A CN 100432216 C CN100432216 C CN 100432216C
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Abstract
本发明特征在于在代谢物和代谢通量的调控下在细胞中生产异源分子的方法,所述方法可增加异源多肽和代谢物的合成。
Description
本申请是申请日为2002年9月20日,申请号为00810878.1,发明名称为“代谢控制的工程化”的中国专利申请的分案申请。
技术领域
本发明涉及代谢控制的工程化,更具体地涉及工程化的宿主细胞,其可以增加异源多肽和代谢物的产量,本发明还涉及在宿主细胞中生产异源多肽和代谢物的方法。
背景技术
重组DNA技术的应用使得可工程化宿主细胞,以产生所需的化合物,如多肽和次生代谢物。在工程细胞中大规模生产多肽使得可生产具有药学用途的蛋白质和具有工业用途的酶。次生代谢物是来自自然界的产物,长久以来已知其生物学和医学重要性。由于这种分子固有的结构复杂性,传统的化学合成通常需要大量人力并使用昂贵的前体和辅因子以制备该化合物。近年来,在细胞中表达异源蛋白已经促进在生物中异源生物合成途径的工程化,以从非昂贵的起始材料生产代谢物。以此方式,已产生了许多化合物,包括聚酮化合物,β-内酰胺抗生素,单萜,类固醇,和芳香化合物。
发明内容
本发明部分基于如下发现,即可通过使用受次生代谢物如乙酰磷酸浓度调节的启动子调节多肽(如生物合成酶)的表达,而增加异源多肽和代谢物的产生。术语“异源”是指多肽或代谢物是通过人为导入的。异源多肽或代谢物可与天然存在的内源性物质相同。
术语“代谢物”指是一或多个生物化学反应的产物的有机化合物。
代谢物其自身可以是其它反应的前体。次生代谢物是产生自另一代谢物的代谢物。
因此本发明一方面涉及一种含有一核酸序列的细菌宿主细胞,该核酸序列包含一个启动子和一个编码异源多肽的核酸序列。细菌宿主细胞例如包括大肠杆菌,枯草芽孢杆菌,鼠伤寒沙门氏菌,根瘤土壤杆菌,嗜热水生菌,和豌豆根瘤菌细胞。此核酸序列可操纵地与由应答调节蛋白控制的启动子连接。换而言之,核酸序列与启动子的连接方式是使核苷酸序列在体外或体内表达。“启动子”是指导遗传物质转录的任何DNA片段。启动子是由应答调节蛋白控制的,例如大肠杆菌的ntrC,phoB,phoP,ompR,cheY,creB,或torB,或其它细菌菌种的同源物。另外,应答调节蛋白可以是簇合直向同源组(cluster orthologous group,COG)COG0745的另一成员,其由http://www.ncbi.nlm.nih.gov/COG/所定义(Tatusov等,核酸研究(2000);28:33-36)。在一实施方案中,启动子是与大肠杆菌ntrC结合的。术语“ntrC”是指大肠杆菌ntrC蛋白(SWISSPROT:P06713,http://www.expasy.ch/)及在其它适当细菌中的同源物。本文所用“结合”是指平衡结合常数(KD)小于100nM,优选小于1nM的物理缔合作用。一适当的启动子例如是大肠杆菌glnAp2启动子,如以GenBank登记号No.M10421公布的DNA序列的大约93-323位之间的区域(Reitzer & Magasanik(1985)美国科学院院报82:1979-1983)。此区域包括glnA基因的未翻译序列。另外,可在glnA编码序列和异源多肽的编码序列之间构建一翻译融合体。
宿主细胞被遗传修饰从而启动子由乙酰磷酸在无氮饥饿的情况下调节。例如,宿主细胞可通过缺失或突变编码组氨酸蛋白激酶的基因而遗传修饰,例如,该组氨酸蛋白激酶是COG0642的成员(如由http://www.ncbi.nlm.nih.gov/COG/所述;Tatusov等,如前),例如glnL,phoR,phoQ,creC,或envZ。在另一实施例中,组氨酸蛋白激酶对控制启动子的应答调节蛋白有特异性。组氨酸蛋白激酶可由glnL,如大肠杆菌glnL(SWISSPROT P06712,http://www.expasy.ch/)编码。
尽管宿主细胞被遗传修饰从而启动子由乙酰磷酸在无氮饥饿的情况下调节,以表达异源多肽或代谢物,但宿主细胞可在任何所需条件下繁殖,例如在氮饥饿条件,氮缺乏条件,或氮富足条件下繁殖。
由此核酸序列编码的异源多肽可以是产生代谢物所需的生物合成酶,其可以是哺乳动物蛋白,例如分泌的生长因子,单克隆抗体,或胞外基质组分。在另一实施例中,异源多肽可以是用于疫苗中的所需抗原,例如来自病毒,细菌,真菌或原生动物病原体的表面蛋白。
本发明的另一方面涉及一种试剂盒,其含有一种核酸序列,该核酸序列包括由应答调节蛋白控制的启动子。此试剂盒还任选的含有已经遗传修饰的细菌宿主细胞,从而启动子在不存在氮饥饿情况下由乙酰磷酸调节。此试剂盒还可提供使用说明。所述核酸序列可含有位于启动子3’端的限制酶多接头,这样插入多接头的序列与由应答调节蛋白控制的启动子可操纵地连接。在该试剂盒的一实施方案中,启动子是大肠杆菌glnAp2启动子,细菌宿主细胞是含有glnL基因缺失或突变的大肠杆菌细胞。
本发明另一方面涉及含有第一个表达盒的宿主细胞。第一个表达盒包括启动子,如上述那些启动子,以及编码异源代谢物生物合成所需的酶的核酸序列。本文所用术语“酶”是指具有催化一个化学反应或多个反应的能力的多肽。所述核酸序列可操纵地与启动子连接,该启动子在无氮饥饿情况下由乙酰磷酸调节。宿主细胞还含有表达代谢物生物合成所需的其它酶的另外的核酸序列。这种另外的核酸序列可以是表达内源酶的内源序列,或表达异源酶的导入的序列。
在一实施例中,异源代谢物是类异戊二烯,多羟基链烷酸酯,聚酮化合物,β-内酰胺抗生素,芳香化合物,或其前体,例如上游代谢物,或其衍生物,例如下游代谢物。例如,类异戊二烯可以是类胡萝卜素,固醇,紫杉醇,二萜,赤霉素,和醌。类异戊二烯的特别例子包括二磷酸异戊酯,二磷酸二甲基烯丙酯,二磷酸香叶酯,二磷酸法呢酯,二磷酸香叶基香叶酯,和八氢番茄红素。类胡萝卜素特别例子包括β-胡萝卜素,ζ-胡萝卜素,虾青素,玉米黄质,玉米黄质-β-葡糖苷,六氢番茄红素,链孢红素,叶黄素和torulene。当所需的异源代谢物是类异戊二烯时,异源酶可以是异戊烯二磷酸异构酶,二磷酸香叶基香叶酯合酶,或1-脱氧木酮糖-5-磷酸合酶。当所需的异源代谢物是多羟基链烷酸酯时,异源酶可以是3-酮酯酰还原酶,或聚-3-羟基链烷酸酯聚合酶。
宿主细胞可以是细菌细胞,例如大肠杆菌细胞。此宿主细胞任选地通过缺失或突变一个基因而遗传修饰,该基因例如是上述编码组氨酸蛋白激酶的基因。在一特异的实施例中,宿主细胞还含有第二个表达盒,其含有与受乙酰磷酸浓度调节的启动子如glnAp2可操纵连接的编码烯醇丙酮酸磷酸合酶的核酸序列。
本发明另一方面涉及一种在宿主细胞中生产异源类异戊二烯的方法。该方法包括过表达烯醇丙酮酸磷酸合酶,和表达异源类异戊二烯合成所需的生物合成酶。在一实施方案中,在宿主细胞中编码丙酮酸激酶或烯醇丙酮酸磷酸羧化酶的基因经遗传缺失或弱化。在另一实施方案中,编码烯醇丙酮酸磷酸羧激酶的基因在宿主细胞中过表达。本发明的另一方面涉及一种在宿主细胞中生产番茄红素的方法,此方法包括表达以下异源酶:1-脱氧-D-木酮糖-5-磷酸合酶,二磷酸香叶基香叶酯合酶,八氢番茄红素合酶,和八氢番茄红素饱和酶。在此方法的一实施方案中,异戊烯二磷酸异构酶例如使用glnAp2启动子而过表达。
本发明的另一方面涉及一种核酸序列,其含有一个启动子和编码产生第一种代谢物所需的生物合成酶的序列。该启动子可操纵地与该序列连接,并由第二种代谢物调节,该第二种代谢物的浓度是生物合成第一种代谢物的前体的可用性的指示。在一实施例中,第二种代谢物是从生物合成第一种代谢物的前体中产生的废物。
在一实施方案中,第一种代谢物是多羟基链烷酸酯,例如多羟基丁酸酯,所述核酸序列编码生物合成酶,例如3-酮酯酰辅酶A(coA)还原酶,或聚-3-羟基辛酰辅酶A聚合酶。在另一种情况中,第一种代谢物是聚酮化合物,β-内酰胺抗生素,或芳香化合物。在另一种情况中,第一种代谢物是类异戊二烯,例如本文提及的类异戊二烯。所述核酸序列可编码类异戊二烯生成所需的生物合成酶,例如异戊烯二磷酸异构酶,二磷酸香叶基香叶酯合酶,1-脱氧木酮糖-5-磷酸合酶,烯醇丙酮酸磷酸合酶,二磷酸法呢酯合酶,二磷酸香叶基香叶酯合酶,八氢番茄红素合酶,八氢番茄红素脱饱和酶,或番茄红素环化酶。类异戊二烯的一个前体可以是丙酮酸。丙酮酸浓度与乙酸和乙酰磷酸浓度相关。因此,在这种情况下,第二种代谢物是乙酰磷酸。对乙酰磷酸的应答的启动子可通过应答调节蛋白控制,如以上提及的应答调节蛋白。这种启动子在特异的宿主细胞中可只应答乙酰磷酸。在一特定的实施例中,应答乙酰磷酸浓度的启动子与大肠杆菌ntrC结合,例如大肠杆菌glnAp2启动子。
启动子可以由cAMP调节。启动子可以是结合CAP(分解代谢物激活蛋白)的细菌启动子。在哺乳动物中,启动子可以是含有cAMP应答元件(CRE)的启动子,其结合CREB,CREM,或ATF-1蛋白。在酵母细胞中,启动子可以是由cAMP调节的启动子,或结合Gis1,Msn2或Msn4蛋白的启动子。启动子的其它可能的调节信号可以是果糖-1-磷酸,或果糖-6-磷酸。大肠杆菌FruR蛋白调节这种启动子。
所述核酸序列可包含在质粒中,其还可含有一个细菌复制源点,和一个选择标记。此序列还可包含酵母或其它真核生物复制源点,和适当的选择标记,并可整合入基因组中。
在宿主细胞中对生物合成异源化合物的优化,依赖于细胞生理学感应参数,并依赖于利用这些参数调节生物合成。本领域的一种标准方法是培养细胞,使用者外源性加入试剂如诱导物,以激活所需产物的生物合成所需的基因。已广泛观测到高水平诱导重组蛋白或途径导致生长延迟及降低代谢活性。(Kurland和Dong(1996)分子微生物学21:1-4)。外源性应用诱导物根据经验进行,并不监测细胞中生物合成资源的可用性。相反,天然途径依赖于反馈机制控制这种过程。在本发明中一些启动子与特异的遗传限定的宿主细胞和异源多肽的组合,令人预料到地产生高度精确的通用控制线路,其应答细胞代谢状态而调节异源多肽或代谢物合成的通量。实际上,动态控制的重组途径使产量增加,生长延迟最小化,并降低副产物形成所产生的毒性。对生理状态应答而调节基因表达也有益于其它应用,如基因治疗。
本发明的一或多种实施方案详见下述。通过以下阐述和权利要求可清楚本发明的其它特征,目的和优点。
具体实施方式
本发明提供了代谢控制工程方法,例如利用特异宿主细胞中的启动子优化蛋白质表达以生产蛋白质或合成代谢物的方法。
本发明的中心成分是一表达盒,其包含一个启动子和编码希望表达的异源多肽的核酸序列。此表达盒用本领域的标准方法构建,从而编码核酸序列可操纵地与启动子相连,例如由启动子调节。选择受细胞生理学或细胞代谢状态参数调节的启动子。有许多启动子可以使用。在一些应用中,所述表达盒包含于质粒中,如具有细菌复制源点和选择标记的细菌质粒。表达盒可用本领域的标准方法整合入细胞的基因组中。
如果表达盒用于工程异源多肽在晚期对数生长期或稳定期的调控产生,则可由此选择启动子。例如,可选择应答其水平在对数生长期或稳定期积累的小分子信号如第二信使的启动子。第二信使可以是作为生物化学途径的前体,中间体,或废物而积累的分子。在细菌中,小分子信号可以是糖酵解的中间体,如果糖-1-磷酸或果糖-6-磷酸,或糖酵解的废物,如乙酸或乙酰磷酸。在真核细胞中,cAMP浓度是熟知的营养状态信号。
表达盒中的启动子可基于大规模表达分析试验的结果选择,例如基因芯片试验。由乙酰磷酸诱导的基因可通过与从在乙酸中生长的细胞中制备的微阵列标记cDNA杂交,并将信号与参照信号对比而鉴别,参照信号例如是用从早期对数生长的细胞中制备的cDNA所得的信号。此试验可在原核或真核细胞,例如细菌,酵母,植物和哺乳动物细胞中进行。在原核生物中进行的这种试验例如见Talaat等,(2000)自然生物技术18:679-82和Oh&Liao(2000)生物技术进展16:278-86所述。一旦鉴别出在所需条件下表达的基因,其启动子可用在表达盒中。或者,可通过外源性加入所需分子(如代谢途径中的前体),或通过人工操纵试验条件(如生长至晚期对数期,或在生物合成酶过量产生同时仍生长),进行此试验。可基于诱导的基因鉴别启动子。
在一种情况下,使用表达盒在细菌细胞中工程代谢物的调控产生。可选择由第二种代谢物调节的启动子,该第二种代谢物的浓度是生物合成第一种代谢物的前体的可用性的指标。例如,如果第一种代谢物是合成自前体丙酮酸和甘油醛-3-磷酸的类异戊二烯,则第二种代谢物可以是乙酰磷酸。在富集环境中,细胞产生过量的乙酰辅酶A,这是丙酮酸的一种产物。过量的乙酰辅酶A用于产生ATP和乙酸,乙酸作为废物分泌。乙酸浓度随细胞密度提高。乙酸,乙酰辅酶A和乙酰磷酸浓度通过以下生物化学反应而相关:
因此,高乙酰磷酸浓度表明过量的乙酰辅酶A和过量的丙酮酸。通过缺失或突变例如glnL而遗传修饰的宿主细胞,使ntrC功能成为乙酰磷酸依赖性的(Feng等,(1992)细菌学杂志174:6061-6070)。在此方式中,由ntrC调节的启动子,例如glnAp2启动子,可用于控制对乙酰磷酸应答的基因表达。glnAp2启动子可使用本领域标准方法获得。例如,可设计并合成与glnAp2启动子退火的引物。可使用聚合酶链反应(PCR)扩增含有glnAp2启动子的核酸片段,此片段可用于进一步构建过程。类似地,可容易地制备含有组氨酸蛋白激酶基因例如glnL缺失的大肠杆菌菌株。见Link等,(1997)细菌学杂志179:6228-6237详细阐述了一种可能的方法。编码所需异源多肽的序列可克隆入glnAp2启动子的下游,以便其可操纵地与启动子相连。然后可将具有失活的glnL基因的宿主细胞用此序列转化。培养转化的菌株,并在生长期间监测多肽的产生。在高细胞密度可观测到强的蛋白质表达,如Farmer和Liao(2000)自然生物技术18:533-537所述,其内容在此并入参考。
哺乳动物细胞可用作生产多肽或代谢物的宿主细胞。可选择例如对cAMP应答的启动子。这种启动子可含有cAMP应答元件(CRE),其结合CREB,CREM或ATF-1蛋白。使用本领域标准方法,可将所需编码序列置于启动子的控制下,并转化入哺乳动物细胞中。在一些情况中,可将此构建体插入病毒中,如失活的病毒。这种应用使得在基因治疗方案中可实现由异源生物合成酶产生的蛋白质或代谢物的调控产生。植物细胞也可用作宿主细胞。同样,可选择适当的启动子,例如应答植物激素,代谢物,或产生所需代谢物的前体的启动子。启动子可通过微阵列试验鉴别。在将所需启动子与所需编码序列在适当载体中融合后,可将此构建体电穿孔入根瘤农杆菌中,然后使用本领域标准方法用于转化植物细胞。在另一实施例中,可操作酵母细胞使之在代谢控制下表达异源多肽或代谢物。例如,酿酒酵母启动子可以是由cAMP调节的启动子,例如与Gis1,Msn2,或Msn4蛋白结合的启动子。应答各种代谢条件的所有酵母基因的调控正在日益增加地充分研究。例如,DeRisi等,(1997)科学278:680-686阐述了在各种代谢条件下,近于全部的根瘤农杆菌基因的转录模式的试验。可基于这些数据选择由所需代谢物调节的启动子。本领域熟知产生酵母质粒和转化酵母的方法。
可使用上述表达方法重建各种代谢途径。例如,通过构建以下基因的表达载体,可将产生番茄红素的途径导入大肠杆菌中,所述基因为:来自大肠杆菌的dxs(编码1-脱氧-D-木酮糖-5-磷酸合酶),来自Archaeoglobus fulgidus的gps(编码二磷酸香叶基香叶酯(GGPP)合酶),和来自Erwinia uredovora的crtBI(分别编码番茄红素合酶和脱饱和酶)。这些基因可位于单个或多个质粒上,或可整合入大肠杆菌染色体中。另外,可使用任何方法过表达烯醇丙酮酸磷酸合酶,例如通过与glnAp2启动子融合而进行。异戊基二磷酸异构酶可使用任何方法过表达,例如通过与glnAp2启动子融合而进行。
在另一实施例中,可将产生多羟基链烷酸酯(PHA),例如多羟基丁酸酯的途径导入大肠杆菌中。PHA是一类具有各种热塑性和商业用途的羟酸线性聚酯。编码3-酮酯酰辅酶A还原酶和聚-3-羟基链烷酸酯聚合酶的铜绿假单胞菌基因可置于所需启动子例如glnAp2的调节下,因为乙酰辅酶A水平可表明合成PHA的前体的可用性。
不用进一步详细阐述,确信以上阐述已充分说明了本发明。以下实施例只是举例说明本发明,而无任何限制之意。文中所引用的文献均以全文并入参考。
方法
生长条件:将所有大肠杆菌全部生长于含有指定的培养基的摇瓶中,此摇瓶置于37℃水浴摇床(Model G76;New Brunswick Scientific,Edison,NJ)中。将培养物生长于基本培养基中,该培养基由含有0.5%(wt/vol)葡萄糖的M9限定盐34,或含有1.5%(wt/vol)葡萄糖的YE限定盐组成。YE限定盐(每升)由14g K2HPO4,16g KH2PO4,5g(NH4)2SO4,1g MgSO4,和1mg硫胺素组成。在550nm经分光光度法监测细胞浊度。
代谢物测定:用HPLC(Constametric 3500溶剂输送系统和分光监测仪3100可变波长检测仪;LDC Analytical,Riviera Beach,FL)经保持在65℃的有机酸层析柱(Aminex HPX-87H,Bio-Rad实验室,Hercules,CA),测定乙酸,丙酮酸和其它有机酸。流动相由0.01NH2SO4组成,流速保持在0.6ml/分钟。在210nm检测层析柱峰。用Sigma试剂盒No.315-100测定葡萄糖。为定量番茄红素,将1ml细菌培养物用丙酮提取,离心,并在474nm测定上清吸光度。通过与标准曲线吸光度对比计算番茄红素浓度。
SDS-PAGE和酶分析:SDS-PAGE方法见于Laemmli(1970)自然227:680-685所述。测定β-半乳糖苷酶活性基本如Miller(1992)细菌遗传学简要课程,冷泉港实验室出版社,冷泉港NY所述。
结果
在大肠杆菌中使用glnAp2启动子与lacZ的异源融合体
乙酰磷酸水平提高可表明过量葡萄糖通量。本发明特征是宿主细胞,核酸序列,和利用乙酰磷酸作为信号调节所需代谢途径中控速酶的表达,以充分利用过量碳通量并使通量不产生毒性产物,乙酸。
为测试glnAp2作为产物表达的动态控制物的潜力,构建含有可操纵地与glnAp2启动子连接的异源lacZ基因的核酸序列。含有启动子和两个ntrC结合位点的glnAp2启动子区域,通过本领域的标准方法可易于获得。此glnAp2启动子经PCR从大肠杆菌基因组DNA中扩增出,使用正向引物5’-CAGCTGCAAAGGTCATTGCACCAAC(含有工程PvuII位点),和反向引物5’-GGTACCAGTACGT-GTTCAGCGGACATAC(含有工程KpnI位点)。这两个引物扩增公布的DNA序列16(GenBank登记号No.M10421)的93-343位之间的区域。
将glnAp2PCR片段也克隆入pRS551的EcoRI位点,因此产生p2GFPuv,其含有在无启动子的lacZ基因之前的glnAp2。通过同源重组(Simons等,(1987)基因53:85-96),将glnAp2-lacZ区域转移至λRS45中,产生噬菌体λp2GFPuv。通过用λp2GFPuv噬菌体感染(Silhavy等,(1984),基因融合试验,冷泉港实验室出版社,冷泉港NY),将glnAp2-lacZ融合体分别整合入BW13711(lacX74)和BW18302(lacX glnL2001;Feng等,如前)的染色体中而构建JCL1595和JCL1596。此菌株含有glnL2001等位基因,其由glnL编码序列的23和182密码子之间的内部缺失组成,并预计产生无效突变(Feng等,如前)。
β-半乳糖苷酶活性的时程是在野生型和glnL突变体中测定的。glnAp2-β-半乳糖苷酶活性以时间依赖型方式增加,这类似于从glnL宿主(JCL1596)中分泌的乙酸浓度,而在等基因的野生型对照(JCL1595)中未发现启动子活性被诱导。
表1:glnAp2-lacZ的β-半乳糖苷酶活性
因此,在没有glnL的情况下,glnAp2能应答由乙酸分泌代表的过量碳通量。随着细胞接近晚指数期,生物合成需求降低,细胞开始呈现过量碳通量,这由乙酸的产生增加而表明。在这点上,在大约6小时,令人意外的glnAp2-β-半乳糖苷酶活性开始提高(~700nmol/分钟/mg蛋白,见表1),而野生型菌株(JCL1595)中glnAp2-β-半乳糖苷酶活性相对较低,并在整个期间保持恒定(~100nmol/分钟/mg蛋白,见表1)。在10小时以后,在无glnL情况下,glnAp2-β-半乳糖苷酶活性达到~1500nmol/分钟/mg蛋白,见表1。glnASp2的诱导分布图也完全与lac启动子(Plac)的分布图形成鲜明对照。菌株VJS632(lac+)中染色体Plac活性在用IPTG(异丙基硫代-β-D-半乳糖苷)诱导后迅速提高,并在细胞中达到恒定水平表达(~550nmol/分钟/mg蛋白,见表1),其不依赖于生长时期。
在大肠杆菌中使用glnAp2启动子与pps和aroG的异源融合体
将两个不同的代谢酶,烯醇丙酮酸磷酸合酶(pps)和3-脱氧-D-阿拉伯庚酮糖酸(arabinoheptulosonate)-7-磷酸(DAHP)合酶(aroG)的表达置于glnAp2启动子的控制下。作为对照,将同样的这两个蛋白也用呈静置对照的tac启动子(Ptac),在相同遗传背景和环境条件下过表达。表达pps的标准方法导致生长延迟(Patnaik等,细菌学杂志174:7527-7532)。
通过将含有aroG pRW5tkt的PCR片段克隆入pJF118EH的EcoRI-BamHI位点中,构建质粒pAROG。质粒pPS706已由Patnaik等(如前)阐述。这两个质粒表达在Ptac启动子下的相应基因。将含有glnAp2启动子的PCR片段分别克隆入质粒pAROG和pPS706的EcoRV-EcoRI位点中,产生含有在glnAp2启动子控制下的相应基因的质粒p2AROG3和pPSG706。
用所有4个质粒转化宿主菌株BW18302(lacXglnL2001)。将具有相应质粒的菌株培养于M9盐-葡萄糖培养基中。在5小时后对比生长情况。
表2:过表达菌株的生长
在生长5小时后的OD<sub>550</sub> | |
无质粒 | ~0.5 |
P<sub>tac</sub>-aroG | ~0.5 |
glnAp2-aroG | ~0.5 |
P<sub>tac</sub>-pps | ~0.12 |
glnAp2-pps | ~0.4 |
如前所述,用Ptac-pps过表达pps导致生长明显延迟。然而,使用glnAp2意外地产生接近正常的生长(见表2)。在15小时后,从每个菌株中分离蛋白质,并在10%SDS-PAGE凝胶上进行分析。当与Ptac启动子控制的pps基因相对比时,由glnAp2启动子控制的pps基因有至少多500%的pps蛋白质表达。另一令人惊奇的发现是,利用glnAp2启动子进行表达与利用Ptac启动子进行表达相比,在细胞的提取物中AroG蛋白也至少多300%,aroG蛋白的过表达没有公开的害处。
通过idi过表达在大肠杆菌中生产番茄红素
通过表达来自大肠杆菌的dxs基因(编码1-脱氧-D-木酮糖5-磷酸合酶),来自Archaeoglobus fulgidus的gps基因(编码二磷酸香叶基香叶酯(GGPP)合酶),和来自Erwinia uredovora的crtBI基因(分别编码八氢番茄红素合酶和脱饱和酶),在大肠杆菌中重建重组番茄红素途径。将这些基因插入低拷贝数质粒pCL1920中形成pCW9,并同时过表达。
使用glnAp2启动子控制idi(异戊烯二磷酸异构酶)的表达。含有idi基因的构建体衍生自无启动子载体pJF118。插入glnAp2启动子形成p2IDI。作为对照,插入Ptac启动子形成pTacIDI。将这些质粒分别导入含有pCW9的glnL菌株(BW18302)中。含有p2IDI的菌株(glnAp2-idi)在含有葡萄糖的规定培养基中培养26小时后,产生100mg/L的番茄红素。与此相反,含有Ptac-idi的菌株在相同条件下,只产生少量的番茄红素(<5mg/L)。另外,p2IDI菌株比pTacIDI产生几乎少3倍的乙酸,这表明为流向乙酸的碳通量已更改为流向番茄红素。
表3在大肠杆菌分批培养物中番茄红素形成的碳收率
在葡萄糖上番茄红素碳收率(mol C/mol C) | |
宿主(BW18302) | 0.0000 |
+pTacIDI(Ptac-idi) | 0.0003 |
+pTacIDI(Ptac-idi)/pPS184(Ptac-pps) | 0.0012 |
+p2IDI(glnAp2-idi) | 0.014 |
+p2IDI(glnAp2-idi)/pPSG184(glnAp2-pps) | 0.022 |
使用pps增加番茄红素产量
从另一相容质粒pPSG18中经glnAp2过表达pps,同时用pCL1920表达番茄红素途径其余基因(dxs,gps,crtBI)。pps和idi与番茄红素途径共表达使番茄红素的终滴度提高50%,并使产量提高3倍,从0.05mg/ml/小时提高至0.16mg/ml/小时(表3)。这与含有pTacIDI和pPS184的对比菌株(Ptac-idi+Ptac-pps)相反,它们的产量没有明显改进,并发生实际生长抑制。
生产番茄红素的其它宿主细胞
将pykF::cat和pykA::kan等位基因导入野生型菌株中,以产生两个单突变体菌株(JCL1610(pykF)和JCL1612(pykA)),和一个双突变体菌株(JCL1613(pykF pykA))(Ponce等,(1995)细菌杂志177:5719-5722)。双突变体菌株能达到终番茄红素滴度为大约14mg番茄红素/g干细胞,而每个单突变体菌株所得番茄红素滴度为大约2.5mg番茄红素/g干细胞。单pyk突变体产生的番茄红素水平与野生型菌株相似,大约为4mg番茄红素/g干细胞。另外,过表达Pck(烯醇丙酮酸磷酸羧激酶)使番茄红素的终滴度提高大约3倍。过表达Ppc(烯醇丙酮酸磷酸羧化酶)使番茄红素的产量降低大约30%。
其它实施方案
本发明的许多实施方案已经阐述。另外应知在不违背本发明精神和范围之下,可对本发明加以各种修改。因此,其它实施方案在所附权利要求的范围内。例如,提及的多肽和基因的所有同源物均在本发明范围内。
Claims (6)
1.一种包含一核酸序列的细菌宿主细胞,该核酸序列包含一个启动子和一编码异源多肽的核酸序列;所述核酸序列可操纵地与由应答调节蛋白控制的启动子相连;其中所述应答调节蛋白是ntrC;所述宿主细胞经遗传修饰从而所述启动子在不存在氮饥饿的情况下由乙酰磷酸诱导/激活,且其中所述启动子与ntrC结合。
2.权利要求1的宿主细胞,其中启动子是glnAp2。
3.一种包含一核酸序列的细菌宿主细胞,该核酸序列包含一个启动子和一编码异源多肽的核酸序列;所述核酸序列可操纵地与由应答调节蛋白控制的启动子相连;所述宿主细胞通过缺失或者突变组氨酸蛋白激酶基因glnL被遗传修饰,从而所述启动子在不存在氮饥饿的情况下由乙酰磷酸诱导/激活。
4.一种包含一核酸序列的细菌宿主细胞,该核酸序列包含一个启动子和一编码异源多肽的核酸序列;所述异源多肽是催化生产代谢物的代谢途径中的反应的生物合成酶,所述核酸序列可操纵地与由应答调节蛋白控制的启动子相连;所述宿主细胞经遗传修饰从而所述启动子在不存在氮饥饿的情况下由乙酰磷酸诱导/激活。
5.权利要求4的宿主细胞,其中所述异源多肽催化产生类异戊二烯的代谢途径中的反应。
6.权利要求5的宿主细胞,其中所述应答调节蛋白选自ntrC、phoB、phoP、ompR、cheY、creB和torR,且所述宿主细胞通过缺失或突变编码组氨酸蛋白激酶的基因而被遗传修饰。
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