CN117264858A - 一种高效生产胞苷酸的方法及应用 - Google Patents
一种高效生产胞苷酸的方法及应用 Download PDFInfo
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Abstract
本发明公开了一种高效生产胞苷酸的方法及应用,属于生物技术领域。本发明通过在基因位点pta整合枯草芽孢杆菌的嘧啶操纵子基因簇pyr_Bs,在大肠杆菌中重构UMP合成途径,提高5’‑胞苷酸合成途径的代谢通量,实现高效生产5’‑胞苷酸(5’‑CMP)。在60L发酵罐中发酵60h的产量达到49g/L。
Description
技术领域
本发明涉及一种高效生产胞苷酸的方法及应用,属于生物技术领域。
背景技术
胞苷5’-单磷酸(简称5’-CMP)属于胞嘧啶核苷酸的一种,是RNA的结构成分之一。5’-CMP应用于不同的领域,它可作为药物合成的前体或中间体应用于医药领域,也可作为食品营养强化剂应用于食品领域,是一种重要的核苷酸。在食品领域,5’-CMP是一种重要的增鲜剂,可以添加到味精等调味品中,提高食品的鲜味,在节约粮食方面具有重大意义;在医药领域,5’-CMP在生物医药行业中是合成核苷酸药物的关键中间体,在核苷酸药物中,例如三磷酸胞苷、胞嘧啶β-D-呋喃阿拉伯糖苷、二磷酸胞嘧啶胆碱和聚肌苷酸-聚胞苷酸等的生产都需要5’-CMP作中间体。在人体和哺乳动物的免疫系统中,5’-CMP发挥重要作用。研究表明,添加5’-CMP的婴幼儿奶粉的功效更接近母乳,与未添加5’-CMP婴幼儿奶粉相比,能够明显提高婴幼儿的免疫力。
目前,生产5’-CMP的方法主要包括:酵母核酸水解法、化学合成法、酶合成法和生物发酵法。酵母核酸水解法传统方法利用RNA制备5’-CMP,包括核酸化学水解法和利用核酸水解酶水解法。常用的核酸水解酶是5’-磷酸二酯酶,利用5’-磷酸二酯酶(5’-PDE)水解酵母RNA制备风味核苷酸和其他具有医用价值的核苷酸,再经离子交换法分离获得相应的核苷单磷酸盐。化学水解法是将酵母RNA在强酸溶液(硫酸或硝酸)中脱氨制备胞苷酸或尿苷酸。核酸水解法在原材料来源和反应条件等方面具有较大优势,但该方法存在反应时间长,水解后得到的四种核苷酸混合物分离工序复杂,且成本高等问题。化学合成法主要是利用甲基化的核苷与磷酸或者焦磷酸发生磷酸化反应,如利用2’,3’-异丙基亚甲基核苷与磷酰氯发生磷酸化反应,合成5’-核苷酸,在反应前加入少量水,可大大提高核苷酸的产率。另外,用三烷基磷酸酯替代磷酰氯,和核苷发生磷酸化反应也可以获得相应的核苷酸产物。化学合成法所需试剂成本高,反应后的废水对环境存在污染。尿苷-胞苷激酶是生物体嘧啶核苷酸代谢补救途径中的重要催化剂,它可以使胞苷/尿苷磷酸化生成5’-CMP,但需要NTP为该反应提供磷酸基团。已报道的在大肠杆菌(Escherichia coli)、保加利亚乳杆菌(Lactobacillus bulgaricus)和枯草芽孢杆菌(Bacillus subtilis)中尿苷和胞苷进行磷酸化反应时,需要GTP为该反应提供磷酸基团,但是GTP的采购成本过高。
生物发酵法是依靠微生物的发酵作用合成所需要的物质,微生物发酵法合成具有条件简单、生长周期短、生产产品成本高、发酵条件易于控制、产量高、生产原料成本低等优点,适合大规模工业化生产。前期调研中,关于5’-CMP下游代谢产物胞苷的微生物生产方面的研究较多,但对于5’-CMP的微生物生产研究较少。微生物中有两种途径来合成单磷酸胞苷,从头合成途径和嘧啶补救途径。嘧啶补救途径是细胞直接从外部环境吸收胞嘧啶或者脱氧胞苷三磷酸经转化得到胞苷,胞苷再经尿苷胞苷激酶生成5’-CMP。5’-CMP从头合成途径是以谷氨酰胺、碳酸和ATP为原料,在一系列酶的作用下,经过一系列反应,生成UMP。在嘧啶核苷酸代谢途径中,UMP是合成所有嘧啶核苷酸的前体物质,经过一系列胺化水解反应最终合成5’-CMP。在野生菌株中,5’-CMP的积累量非常低,这是因为5’-CMP属于嘧啶核苷酸代谢产物,嘧啶核苷酸代谢还包括UMP、UTP、CTP、CDP等,这些物质均是DNA或RNA的组成物质,具有复杂的调控机制。随着微生物学技术的发展,微生物的嘧啶核苷酸代谢机制逐渐清晰,利用微生物从头合成尿苷和胞苷的研究逐渐增多,清晰的代谢路径以及调控机制为研究微生物通过代谢工程生产5’-CMP提供了理论依据。
CN 111269870 A披露了一种高产胞苷酸的重组大肠杆菌及其应用,从胞苷酸生产菌株中克隆得到的胞苷激酶基因,重组菌经破碎后得到的粗酶液有着良好的催化活性和稳定性,加入胞苷、三磷酸腺苷(ATP)以及Mg2+,反应得到胞苷酸。但,该方法需要对重组菌进行破碎、再对酶进行分离,且产量不高。CN 202111280840.4披露了一种生产5’-胞苷酸的方法,基因敲除技术对大肠杆菌的胞苷单磷酸焦磷酸化酶基因ushA和核苷酸5'-单磷酸核苷酶基因ppnN进行敲除,并通过过量表达尿苷激酶基因udk,获得可积累5’-胞苷酸(5’-CMP)的大肠杆菌突变体。但,上述重组菌株积累5’-CMP时需要诱导剂IPTG和抗生素的补加,该诱导剂和抗生素价格昂贵且对细胞有毒性,同时如果5’-胞苷酸(5’-CMP)的积累浓度进一步获得提高,将进一步促进其工业化生产。
因此,目前研究目标在于利用代谢工程和合成生物学手段对E.coli出发菌株进行改造,以实现胞苷酸的高效生产。
发明内容
[技术问题]
本发明要解决的技术问题是现有技术中缺少能够高产5’-胞苷酸的重组菌株。
[技术方案]
本发明采用基因整合技术在大肠杆菌基因组上整合枯草芽孢杆菌的嘧啶操纵子pyr_Bs,在大肠杆菌中重构UMP合成途径,提高5’-CMP的合成途径的代谢通量,增强5’-胞苷酸的合成通量,可以高效生产5’-胞苷酸(5’-CMP),促进其在医药等领域中的应用。
本发明提供了一种生产胞苷酸的重组大肠杆菌,在出发菌株中过表达天门冬氨酸氨基甲酰转移酶pyrB、二氢乳清酸酶pyrC、嘧啶特异性氨甲酰磷酸合成酶pyrAA、嘧啶特异性氨甲酰磷酸合成酶pyrAB-E949、二氢乳清酸脱氢酶基因pyrK、二氢乳清酸脱氢酶基因pyrD、乳清苷酸脱羧酶基因pyrF和乳清酸磷酸核糖基转移酶基因pyrE,所述嘧啶特异性氨甲酰磷酸合成酶pyrAB-E949是在pyrAB的基础上,缺失第949位谷氨酸得到的。
在一种实施方式中,在出发菌株的基因组上整合枯草芽孢杆菌的嘧啶操纵子pyr_Bs,所述嘧啶操纵子pyr_Bs的基因簇顺序为pyrB、pyrC、pyrAA、pyrAB-E949、pyrK、pyrD、pyrF和pyrE;所述pyrB的核苷酸序列如SEQ ID NO.1所示,所述pyrC的核苷酸序列如SEQ IDNO.2所示,所述pyrAA的核苷酸序列如SEQ ID NO.3所示,所述pyrAB-E949的核苷酸序列如SEQ ID NO.4所示,所述pyrK的核苷酸序列如SEQ ID NO.5所示,所述pyrD的核苷酸序列如SEQ ID NO.6所示,所述pyrF的核苷酸序列如SEQ ID NO.7所示,所述pyrE的核苷酸序列如SEQ ID NO.8所示。
在一种实施方式中,所述嘧啶操纵子pyr_Bs的整合位点为基因yghX或pta。
在一种实施方式中,所述出发菌株为E.coli CMP12或E.coli CMP13。
在一种实施方式中,所述E.coli CMP12及其构建方法已公开于专利CN116463273A,所述E.coli CMP12以E.coli MG1655为底盘细胞,敲除了基因组上的核苷酸5'-单磷酸核苷酶基因ppnN,胞苷单磷酸焦磷酸化酶基因ushA、嘌呤核苷酸酶基因yrfG、嘧啶5’-核苷酸酶基因yjjG、UMP磷酸酶基因umpH、5’-核苷酸酶基因umpG和胞苷脱氨酶基因cdd;
在一种实施方式中,在umpH位点整合5’-CTP二磷酸水解酶基因nudG;所述基因nudG的核苷酸序列如SEQ ID NO.20所示;在umpG位点整合乳清酸磷酸核糖转移酶基因pyrE,所述基因pyrE的核苷酸序列如SEQ ID NO.15所示;
在一种实施方式中,还整合表达尿苷-胞苷激酶基因udk、磷酸核糖焦磷酸合成酶基因prs、葡萄糖-6-磷酸脱氢酶基因zwf和6-磷酸葡糖酸脱氢酶基因gnd,所述基因udk的核苷酸序列如SEQ ID NO.16所示,所述基因prs的核苷酸序列如SEQ ID NO.19所示,所述基因zwf的核苷酸序列如SEQ ID NO.18所示,所述基因gnd的核苷酸序列如SEQ ID NO.17所示。
在一种实施方式中,在所述出发菌株poxB位点整合基因pyrH(R92G/D93G)或pyrH(D93A);所述基因pyrH(R92G/D93G)的核苷酸序列如SEQ ID NO.21所示,所述基因pyrH(D93A)的核苷酸序列如SEQ ID NO.22所示。
在一种实施方式中,所述基因ppnN的核苷酸序列如SEQ ID NO.9所示,所述基因ushA的核苷酸序列如SEQ ID NO.10所示,所述基因yrfG的核苷酸序列如SEQ ID NO.11所示,所述基因yjjG的核苷酸序列如SEQ ID NO.12所示,所述基因umpH的核苷酸序列如SEQID NO.13所示,所述基因umpG的核苷酸序列如SEQ ID NO.14所示;所述基因cdd的核苷酸序列如SEQ ID NO.23所示。
在一种实施方式中,E.coli CMP12-M4为以E.coli MG1655为出发菌株,分别敲除7个基因ppnN、ushA、yrfG、yjjG、umpH、umpG、cdd、yghX,同时过表达基因nudG、pyrE、pyrH(R92G/D93G)、udk、PPP。
在一种实施方式中,E.coli CMP12-B4为以E.coli MG1655为出发菌株,分别敲除7个基因ppnN、ushA、yrfG、yjjG、umpH、umpG、cdd、pta,同时过表达基因nudG、pyrE、pyrH(R92G/D93G)、udk、PPP。
在一种实施方式中,E.coli CMP13-M4为以E.coli MG1655为出发菌株,分别敲除7个基因ppnN、ushA、yrfG、yjjG、umpH、umpG、cdd、yghX,同时过表达基因nudG、pyrE、pyrH(D93A)、udk、PPP。
在一种实施方式中,E.coli CMP13-B4为以E.coli MG1655为出发菌株,分别敲除7个基因ppnN、ushA、yrfG、yjjG、umpH、umpG、cdd、pta,同时过表达基因nudG、pyrE、pyrH(D93A)、udk、PPP。
本发明还提供了一种发酵生产5’-胞苷酸的方法,是将所述重组大肠杆菌在含葡萄糖的培养基中发酵。
在一种实施方式中,发酵过程不添加任何抗生素和诱导剂(TPTG)。
在一种实施方式中,所述发酵是将菌株接种至LB培养基中,37℃和200rpm,培养6-16h;对于摇瓶发酵,按照0.2-10%接种量接种至含有30-100mL LB培养基的250mL三角瓶中,37℃和200rpm,培养50-80h。
在一种实施方式中,所述发酵是将LB培养基中的种子液按照1-12%接种量,转接至发酵培养基中,在37℃和200rpm条件下进行发酵培养,通过测定发酵液中葡萄糖控制葡萄糖浓度为10±2g/L;控制溶氧为30%,当溶氧低于30%,提高搅拌转速、通气量和罐压。采用氨水控制pH为6-8。
在一种实施方式中,用于发酵的发酵培养基含有:葡萄糖20-70g/L、氯化铁2-15mg/L、MgSO4 0.1-3g/L、柠檬酸钠1-20mg/L、氯化钙10-500mg/L、磷酸氢二钠1-6g/L、磷酸二氢钠1-9g/L、氯化锌1-60mg/L、酵母粉0.5-12g/L、蛋白胨0.2-15g/L、微量元素1-30mL(含有氯化铜2-51g/L、硫酸锌3-28g/L、钼酸钠3-50g/L)。
在一种实施方式中,发酵过程还进行补料;所述补料培养基含有:葡萄糖400-800g/L、蛋白胨1-16g/L、酵母粉1-15g/L。
本发明还提供了所述重组大肠杆菌在医药领域生产含5’-胞苷酸的产品中的应用。
有益效果
本发明通过整合枯草芽孢杆菌的嘧啶操纵子基因簇pyr_Bs,在大肠杆菌中重构UMP合成途径,提高5’-胞苷酸合成途径的代谢通量,实现高效生产5’-胞苷酸(5’-CMP)。在60L发酵罐中发酵60h的产量达到49g/L,基本不积累中间代谢产物,有利于5’-胞苷酸的分离纯化。
本发明提供的重组大肠杆菌,能够以葡萄糖为底物,在不补加诱导剂IPTG和抗生素的条件下,一步发酵得到5’-胞苷酸,发酵上清液中5’-胞苷酸浓度高,便于分离纯化,工艺简单。
附图说明
图1为枯草芽孢杆菌嘧啶操纵子示意图;
图2为枯草芽孢杆菌嘧啶操纵子迭代整合方法示意图;
图3为基因整合验证结果图;A:pyrB-pyrC基因整合验证;B:pyrAA基因整合验证;
图4为5’-胞苷酸产量图(摇瓶);
图5为5’-胞苷酸产量图(60L发酵罐)。
具体实施方式
胞苷酸的HPLC检测条件:液相色谱仪岛津10A,色谱柱:INERTSIL ODS-SP 5μm4.6*250mm,流动相:缓冲液配制:0.1mol/L磷酸二氢钾水溶液:0.01mol/L的四丁基氢氧化铵:甲醇=95:95:10,然后磷酸调节pH至4.5,波长:276nm,流速:1.0ml/min。
本发明所用pEtac质粒的构建方法已公开于论文《代谢工程改造大肠杆菌合成羟基酪醇》。
以下实施方式中的基因序列如下:pyrB的核苷酸序列如SEQ ID NO.1所示,pyrC的核苷酸序列如SEQ ID NO.2所示,pyrAA的核苷酸序列如SEQ ID NO.3所示,pyrAB-E949的核苷酸序列如SEQ ID NO.4所示,pyrK的核苷酸序列如SEQ ID NO.5所示,pyrD的核苷酸序列如SEQ ID NO.6所示,pyrF的核苷酸序列如SEQ ID NO.7所示,pyrE的核苷酸序列如SEQ IDNO.8所示,ppnN的核苷酸序列如SEQ ID NO.9所示,ushA的核苷酸序列如SEQ ID NO.10所示,yrfG的核苷酸序列如SEQ ID NO.11所示,yjjG的核苷酸序列如SEQ ID NO.12所示,umpH的核苷酸序列如SEQ ID NO.13所示,umpG的核苷酸序列如SEQ ID NO.14所示,pyrE的核苷酸序列如SEQ ID NO.15所示,udk的核苷酸序列如SEQ ID NO.16所示,gnd的核苷酸序列如SEQ ID NO.17所示,zwf的核苷酸序列如SEQ ID NO.18所示,prs的核苷酸序列如SEQ IDNO.19所示,nudG的核苷酸序列如SEQ ID NO.20所示,pyrH(R92G/D93G)的核苷酸序列如SEQIDNO.21所示,pyrH(D93A)的核苷酸序列如SEQ ID NO.22所示,cdd的核苷酸序列如SEQ IDNO.23所示。其中核苷酸序列SEQ ID NO.1~8、20~23来源于枯草芽孢杆菌,核苷酸序列SEQID NO.9~19来源于大肠杆菌。
构建重组菌株所需要的引物序列:
表1所用的引物序列
所构建的重组菌株:
表2具体实施方式中涉及的菌株:
实施例1出发菌株的构建
1、E.coli CMP12菌株的构建:
菌株构建方法公开于专利CN116463273A,以E.coli MG1655为底盘细胞,敲除了基因组上的核苷酸5'-单磷酸核苷酶基因ppnN(核苷酸序列如SEQ ID NO.9所示),胞苷单磷酸焦磷酸化酶基因ushA(核苷酸序列如SEQ ID NO.10所示)、嘌呤核苷酸酶基因yrfG(核苷酸序列如SEQ ID NO.11所示)、嘧啶5’-核苷酸酶基因yjjG(核苷酸序列如SEQ ID NO.12所示)、UMP磷酸酶基因umpH(核苷酸序列如SEQ ID NO.13所示)、5’-核苷酸酶基因umpG(核苷酸序列如SEQ ID NO.14所示)和胞苷脱氨酶基因cdd(核苷酸序列如SEQ ID NO.23所示);在umpH位点整合5’-CTP二磷酸水解酶基因nudG(核苷酸序列如SEQ ID NO.20所示);在umpG位点整合乳清酸磷酸核糖转移酶基因pyrE(核苷酸序列如SEQ ID NO.8所示);还整合表达尿苷-胞苷激酶基因udk(核苷酸序列如SEQ ID NO.16所示)、磷酸核糖焦磷酸合成酶基因prs(核苷酸序列如SEQ ID NO.19所示)、葡萄糖-6-磷酸脱氢酶基因zwf(核苷酸序列如SEQ IDNO.18所示)和6-磷酸葡糖酸脱氢酶基因gnd(核苷酸序列如SEQ ID NO.17所示);在poxB位点整合基因pyrH(R92G/D93G)(核苷酸序列如SEQ ID NO.21所示)。
2、E.coli CMP13菌株的构建
以E.coli CMP12为出发菌株,采用引物pyrH-D93A-RS和pyrH-D93A-FW,以pEtac-pyrH质粒为模板构建获得整合表达框pyrH(D93A),采用基因编辑技术利用pyrH(D93A)替换E.coli CMP12基因组上的基因pyrH(R92G/D93G)。利用引物△poxB::pyrH-up-FW和△poxB::pyrH-up-RS、△poxB::pyrH-down-FW和△poxB::pyrH-down-RS分别获得敲除基因pyrH上下游同源臂,然后利用引物△pyrH-up-FW和△pyrH-down-RS构建获得基因pyrH(R92G/D93G)敲除框。采用引物sg-pyrH-FW和pTargetF-RS,以质粒pTargetF为模板,进行全质粒PCR构建质粒pTargetF-pyrH。将pTargetF-pyrH和Donor DNA pyrH(R92G/D93G)敲除框导入含有pCas质粒的重组菌株E.coli CMP12的电转感受态中,筛选获得E.coli CMP12基因组上敲除基因pyrH(R92G/D93G)的重组菌株,之后通过设计引物与表达框,将基因pyrH(D93A)整合到基因yjiP-yjiR的基因间隔区,采用引物sg-SCD-FW和pTargetF-RS,以质粒pTargetF为模板,进行全质粒PCR构建质粒pTargetF-SCD。利用引物CD-zh-pyrH(D93A)-up-FW、CD-zh-pyrH(D93A)-up-RS、CD-zh-pyrH(D93A)-down-FW、CD-zh-pyrH(D93A)-down-RS,以E.coli MG1655基因组为模板,PCR扩增整合基因pyrH(D93A)的上下游同源臂,利用引物CD-zh-pyrH(D93A)-mid-FW、CD-zh-pyrH(D93A)-mid-RS,以pEtac-pyrH为模板获得基因pyrH(D93A)的表达框,再利用CD-zh-pyrH(D93A)-up-FW和CD-zh-pyrH(D93A)-down-RS,以上下游同源臂和表达框为模板进行融合PCR,将构建的重组质粒pTargetF-SCD和整合表达框导入到敲除基因pyrH(R92G/D93G)的重组菌株中,整合基因pyrH(D93A)构建获得E.coliCMP13。
实施例2用于生产5’胞苷酸的重组大肠杆菌的构建
(1)构建用于过表达尿苷激酶基因的重组质粒
以E.coli CMP13为出发菌株,分别利用引物:
pEtac-pyrB-pyrC-FW:CGGAATTCATGAAGCATTTAACGACGATGAGTG;
pEtac-pyrB-pyrC-RS:CCGCTCGAGTCATTTGACAAGTCTCCCCTCT;
pEtac-pyrAA-FW:CGGAATTCATGAAGAGACGATTAGTACT;
pEtac-pyrAA-RS:CCGCTCGAGCTACGCGTTTTGGCATACCG;
pEtac-pyrAB-FW:AAGGAAAAAAGCGGCCGCATGCCAAAACGCGTAGACAT;
pEtac-pyrAB-RS:CCGCTCGAGTCATATAGTGACTGCCGCCTCC;
pEtac-pyrK-pyrD-pyrF-pyrE-FW:TCACACAGGAAACAGAATTCATGAAAAAAGCGTATTTGAC;
pEtac-pyrK-pyrD-pyrF-pyrE-RS:CAAGCTTGTCGACGGAGCTCTTATTTTTTAAACCAATCTT。
以枯草芽孢杆菌168基因组为模板,进行PCR扩增,获得pyrB-pyrC、pyrAA、pyrAB、pyrK-pyrD-pyrF-pyrE目的片段。核苷酸序列PCR扩增的条件:预变性98℃,5min;变性95℃,30s;退火55℃,30s;延伸72℃,1min;设置循环29次;再延伸72℃,10min;最后16℃保温。PCR扩增的体系:5×PS buffer 20μL,dNTP 10μL,上游/下游引物(10μmol·L1)2μL,模板1μL,酶1μL,水66μL。采用限制性内切酶Xho I和EcoR I酶切PCR获得的pyrB-pyrC、pyrAA、pyrAB基因片段和pEtac质粒(pEtac质粒的构建方法已公开于论文《代谢工程改造大肠杆菌合成羟基酪醇》)。利用连接酶将酶切后的pyrB-pyrC、pyrAA、pyrAB、pyrK-pyrD-pyrF-pyrE基因片段分别和pEtac质粒进行连接,采用C112一步克隆酶构建重组质粒pEtac-pyrB-pyrC、pEtac-pyrAA、pEtac-pyrAB和pEtac-pyrK-pyrD-pyrF-pyrE,将连接产物采用化学转化的方式导入E.coli JM109感受态中,挑取转化子提质粒酶切验证。构建获得重组质粒pEtac-pyrB-pyrC、pEtac-pyrAA、pEtac-pyrAB和pEtac-pyrK-pyrD-pyrF-pyrE。
(2)构建pyrAB(E949*)突变体。
利用引物pEtac-pyrAB-949-FW和pEtac-pyrAB-949-RS,以构建好的质粒pEtac-pyrAB为模板进行全质粒PCR,获得表达pyrAB第949位谷氨酸缺失的突变体的重组质粒,将重组质粒通过化学转化导入E.coli JM109感受态中,挑取转化子提质粒酶切验证,获得表达pyrAB-E949突变体的重组质粒pEtac-pyrAB-E949。
(3)在yghX基因位点或pta基因位点整合枯草芽孢杆菌嘧啶操纵子pyr_Bs
利用(Wu H,Li Y,Qian M,et al.Metabolic engineering of Escherichia colifor high-yield uridine production[J].Metabolic Engineering,2018,49:S1096717618302507-)研究中的大片段基因整合的迭代整合方法,在大肠杆菌E.coliMG1655(ΔppnNΔushAΔyrfGΔyjjGΔumpHΔumpG nudG pyrE pyrH(D93A)Δcdd udkPPP)整合枯草芽孢杆菌的嘧啶操纵子pyr_Bs,所述嘧啶操纵子pyr_Bs的基因簇顺序为pyrB、pyrC、pyrAA、pyrAB-E949、pyrK、pyrD、pyrF和pyrE。
经benchling网站设计yghX基因位点的sgRNA识别序列(https://benchling.com/),设计引物sg-yghX-FW和pTarget-RS、sg-pta-FW和pTarget-RS,分别利用sg-yghX-FW和pTarget-RS、sg-pta-FW和pTarget-RS,以pTarget质粒为模板进行全质粒PCR,将PCR产物用产物清洁试剂盒进行纯化,将纯化后的产物利用DpnⅠ进行消化,将消化产物通过化学转化导入E.coli JM109感受态中。挑转化子提质粒送测序验证,序列比对正确即pTarget-yghX、pTarget-pta质粒构建成功。然后根据(Wu H,Li Y,Qian M,etal.Metabolic engineering of Escherichia coli for high-yield uridineproduction[J].Metabolic Engineering,2018,49:S1096717618302507)研究中提到的两个效率高的PS+PAM序列设计sgRNA识别序列,分别用引物sg-yghX-1-FW和pTarget-RS、sg-yghX-2-FW和pTarget-RS,以pTarget质粒为模板进行全质粒PCR,将PCR产物进行产物清洁试剂盒进行纯化,将纯化后的产物利用DpnⅠ进行消化,将消化产物通过化学转化导入E.coli JM109感受态中,挑转化子提质粒送测序验证,序列比对正确即质粒构建pTarget-yghX-1和pTarget-yghX-2成功。
首先以E.coli MG1655基因组为模板,利用zh-pyrBC-up-FW和zh-pyrBC-up-RS、zh-pyrBC-down-FW和zh-pyrBC-down-RS、PCR扩增yghX基因位点的上下游同源臂,利用pEtac-pyrB-pyrC为模板,利用引物zh-pyrBC-mid-FW和zh-pyrBC-mid-RS,PCR扩增基因pyrB和pyrC表达盒片段pyrB-pyrC,设计获得基因pyrB-pyrC表达盒片段的引物时,要在3’端引入外源的1号PS+PAM序列P1(核苷酸序列为TGCGCTGGTTGATTTCTTCTGTT),即pTarget-yghX-1识别位点。PS+PAM序列就是在基因编辑中sgRNA特异性识别的序列,我们通过查阅相关资料获得两个效率高的sgRNA特异性识别的序列,将其加在整合表达框的末端作为下一个基因整合的特异性识别位点。
将获得的yghX基因位点上下游同源臂分别和基因pyrB-pyrC表达盒片段组合作为模板,利用引物zh-pyrBC-up-FW和zh-pyrBC-down-RS进行融合PCR,获得yghX基因位点整合框。将pTarget-yghX重组质粒和yghX基因位点的整合框,通过电转化分别导入E.coliCMP12的感受态细胞中。挑取转化子进行菌落PCR验证,未整合pyrB-pyrC基因的对照菌株条带大小为1728bp,正确转化子的条带大小为4051bp。琼脂糖凝胶电泳结果如图3A所示,从图可以看出基因pyrB-pyrC已被整合在E.coli基因组上,获得yghX基因位点整合pyrB-pyrC的重组菌株命名为E.coli CMP13-M1和E.coli CMP12-M1。
以E.coli CMP13-M1基因组为模板PCR扩增,分别利用引物zh-pyrAA-up-FW、zh-pyrAA-up-RS、zh-pyrAA-down-FW、zh-pyrBC-down-RS扩增在yghX基因位点整合pyrAA基因所需的上下游同源臂;以pEtac-pyrAA为模板,利用引物pta-zh-pyrAA-mid-FW、pta-zh-pyrAA-mid-RS分别PCR扩增基因pyrAA表达盒片段,设计基因pyrAA表达盒片段的引物时,在3’端引入外源的2号PS+PAM序列P2(核苷酸序列为AGGAATATCCGCAATAATTAGTT)即pTarget-yghX-2识别位点。以获得的上下游同源臂和基因pyrAA表达盒片段为模板,利用引物zh-pyrAA-up-FW、zh-pyrBC-down-RS进行融合PCR,获得yghX基因位点pyrAA的整合框。将pTarget-yghX-1重组质粒和yghX基因位点的pyrAA整合框,通过电转化导入E.coli CMP13-M1和E.coli CMP12-M1感受态细胞中。挑取转化子进行菌落PCR验证,未整合pyrAA基因的对照菌株条带大小为1111bp,正确转化子的条带大小为2630bp。琼脂糖凝胶电泳结果如图3B所示。从图可以看出基因pyrAA已被整合在E.coli基因组上,获得重组菌株E.coli CMP13-M2和E.coli CMP12-M2。
剩余的基因pyrAB、pyrK-pyrD-pyrF-pyrE按照图2的方式,依次进行整合,利用引物zh-pyrAB-up-FW和zh-pyrAB-up-RS、zh-pyrAB-mid-FW和zh-pyrAB-mid-RS、zh-pyrAB-down-FW和zh-pyrBC-down-RS分别构建整合基因pyrAB的上游同源臂、表达盒和下游同源臂,设计基因pyrAA表达盒片段的引物时,在3’端引入外源的1号PS+PAM序列P1即与pTarget-yghX-1识别位点。再利用引物zh-pyrAB-up-FW和zh-pyrBC-down-RS,以上游同源臂、表达盒和下游同源臂基因片段为模板,进行融合PCR,获得基因pyrAB-E949的整合框,将基因pyrAB-E949的整合框和pTarget-yghX-2导入E.coli CMP13-M2和E.coli CMP12-M2感受态细胞中,获得重组菌株E.coli CMP13-M3和E.coli CMP12-M3。
利用引物zh-pyrKDFE-up-FW和zh-pyrKDFE-up-RS、zh-pyrKDFE-mid-FW和zh-pyrKDFE-mid-RS、zh-pyrKDFE-down-FW和zh-pyrBC-down-RS,利用E.coli CMP13-M3基因组为模板,分别获得整合pyrKDFE基因的上游同源臂、表达盒和下游同源臂,利用引物zh-pyrKDFE-up-FW和zh-pyrBC-down-RS,以pyrK-pyrD-pyrF-pyrE基因的上游同源臂、表达盒和下游同源臂为模板,进行融合PCR,获得整合pyrK-pyrD-pyrF-pyrE的整合表达框,将pyrK-pyrD-pyrF-pyrE的整合表达框和pTarget-yghX-1导入E.coli CMP13-M3和E.coliCMP12-M3的感受态中,获得重组菌株E.coli CMP13-M4和E.coli CMP12-M4。
以E.coli MG1655基因组为模板,利用pta-zh-pyrBC-up-FW和pta-zh-pyrBC-up-RS、pta-zh-pyrBC-down-FW和zh-pyrBC-down-RS分别PCR扩增pta基因位点的上下游同源臂,利用pEtac-pyrB-pyrC为模板,利用引物pta-zh-pyrBC-mid-FW和pta-zh-pyrBC-mid-RS分别PCR扩增基因pyrB和pyrC表达盒片段pyrB-pyrC,设计获得基因pyrB-pyrC表达盒片段的引物时,要在3’端引入外源的1号PS+PAM序列P1(核苷酸序列为TGCGCTGGTTGATTTCTTCTGTT),即pTarget-pta-1识别位点。
将获得的pta基因位点的上下游同源臂分别和基因pyrB-pyrC表达盒片段组合作为模板,利用引物pta-zh-pyrBC-up-FW和zh-pyrBC-down-RS进行融合PCR,获得pta基因位点整合框。将pTarget-pta重组质粒和pta基因位点的整合框,通过电转化分别导入E.coliCMP12的感受态细胞中。挑取转化子进行菌落PCR验证,获得pta基因位点整合pyrB-pyrC的重组菌株命名为E.coli CMP13-B1和E.coli CMP12-B1。
以E.coli CMP13-B1和E.coli CMP12-B1基因组为模板PCR扩增,分别利用引物pta-zh-pyrAA-up-FW、pta-zh-pyrAA-up-RS、pta-zh-pyrAA-down-FW、zh-pyrBC-down-RS扩增在pta基因位点整合pyrAA基因所需的上下游同源臂;以pEtac-pyrAA为模板,利用引物pta-zh-pyrAA-mid-FW、pta-zh-pyrAA-mid-RS分别PCR扩增基因pyrAA表达盒片段,设计基因pyrAA表达盒片段的引物时,在3’端引入外源的2号PS+PAM序列P2(核苷酸序列为AGGAATATCCGCAATAATTAGTT)即pTarget-yghX-2识别位点。以获得的上下游同源臂和基因pyrAA表达盒片段为模板,利用引物pta-zh-pyrAA-up-FW、zh-pyrBC-down-RS分别进行融合PCR,获得pta基因位点pyrAA的整合框。将pTarget-pta-1重组质粒和pta基因位点的pyrAA整合框,通过电转化导入E.coli CMP13-B1和E.coli CMP12-B1感受态细胞中。挑取转化子进行菌落PCR验证,获得重组菌株E.coli CMP13-B2和E.coli CMP12-B2。
剩余的基因pyrAB、pyrK-pyrD-pyrF-pyrE按照图2的方式,依次进行整合,利用引物pta-zh-pyrAB-up-FW和pta-zh-pyrAB-up-RS、pta-zh-pyrAB-mid-FW和pta-zh-pyrAB-mid-RS、pta-zh-pyrAB-down-FW和zh-pyrBC-down-RS分别构建整合基因pyrAB的上游同源臂、表达盒和下游同源臂,设计基因pyrAA表达盒片段的引物时,在3’端引入外源的1号PS+PAM序列P1即与pTarget-pta-1识别位点。再利用引物pta-zh-pyrAB-up-FW和zh-pyrBC-down-RS,以上游同源臂、表达盒和下游同源臂基因片段为模板,进行融合PCR,获得基因pyrAB-E949的整合框,将基因pyrAB-E949的整合框和pTarget-pta-2导入E.coli CMP13-B2和E.coli CMP12-B2感受态细胞中,获得重组菌株E.coli CMP13-B3和E.coli CMP12-B3。
利用引物pta-zh-pyrKDFE-up-FW和pta-zh-pyrKDFE-up-RS、pta-zh-pyrKDFE-mid-FW和pta-zh-pyrKDFE-mid-RS、pta-zh-pyrKDFE-down-FW和zh-pyrBC-down-RS,利用E.coli CMP13-B3基因组为模板,分别获得整合pyrKDFE基因的上游同源臂、表达盒和下游同源臂,利用引物pta-zh-pyrKDFE-up-FW和zh-pyrBC-down-RS,以pyrK-pyrD-pyrF-pyrE基因的上游同源臂、表达盒和下游同源臂为模板,进行融合PCR,获得整合pyrK-pyrD-pyrF-pyrE的整合表达框,将pyrK-pyrD-pyrF-pyrE的整合表达框和pTarget-pta-1导入E.coliCMP13-B3和E.coli CMP12-B3的感受态中,获得重组菌株E.coli CMP13-B4和E.coliCMP12-B4。
实施例3 5’-CMP在摇瓶中发酵生产
摇瓶发酵:将菌株接种至LB培养基中,37℃和200rpm,培养6-16h;对于摇瓶发酵,按照1%接种量接种至含有50mL LB培养基的250mL三角瓶中,37℃和200rpm,培养72h。
整合位点为yghX时,在摇瓶水平,在发酵至72h时,E.coli CMP13-M4的5’-CMP产量可达468.0mg·L-1,较对照菌株E.coli CMP13的5’-CMP产量(431.5mg·L-1)提高了1.08倍;在摇瓶水平,在发酵至72h时,E.coli CMP12-M4的5’-CMP产量可达503.7mg·L-1,较对照菌株E.coli CMP12的5’-CMP产量(456mg·L-1)提高了1.1倍。同样的,整合位点为pta时,在摇瓶水平,在发酵至72h时,E.coli CMP13-B4的5’-CMP产量可达485.2mg·L-1,较对照菌株E.coli CMP13的5’-CMP产量(431.5mg·L-1)提高了1.12倍。在摇瓶水平,在发酵至72h时,E.coli CMP12-B4的5’-CMP产量可达536.9mg·L-1,较对照菌株E.coli CMP12的5’-CMP产量(456mg·L-1)提高了1.18倍。
实施例4 5’-CMP在60L发酵罐中发酵生产
将菌株E.coli CMP12-M4和E.coli CMP13-M4分别在60L发酵罐体系下进行高密度发酵,发酵条件为:将LB培养基中的种子液按照6%接种量,转接至发酵培养基中(60L发酵罐,初始装液量为28L,菌株初始OD值为0.12),在37℃和200rpm条件下进行发酵培养,通过测定发酵液中葡萄糖控制葡萄糖浓度为10±2g/L;控制溶氧为30%,当低于30%,提高搅拌转速、通气量和罐压。采用氨水控制pH为6.8。发酵结束后,离心除菌体,取上清液,测定其中的5’-CMP的含量。发酵60h时可以高效生产5’-CMP,菌株E.coli CMP12的产量达到39.6g/L,菌株E.coli CMP13的产量达到36.3g/L,菌株E.coli CMP12-M4和E.coli CMP13-M4的产量分别达到43.6g/L和41.5g/L。同样的,发酵60h时可以高效生产5’-CMP,菌株E.coli CMP12-B4和E.coli CMP13-B4的产量分别达到49.0g/L和42.8g/L。
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 (10)
1.一种生产胞苷酸的重组大肠杆菌,其特征在于,在出发菌株中过表达天门冬氨酸氨基甲酰转移酶pyrB、二氢乳清酸酶pyrC、嘧啶特异性氨甲酰磷酸合成酶pyrAA、嘧啶特异性氨甲酰磷酸合成酶pyrAB-E949、二氢乳清酸脱氢酶pyrK、二氢乳清酸脱氢酶pyrD、乳清苷酸脱羧酶pyrF和乳清酸磷酸核糖基转移酶pyrE。
2.如权利要求1所述的重组大肠杆菌,其特征在于,在出发菌株的基因组上整合枯草芽孢杆菌的嘧啶操纵子pyr_Bs,所述嘧啶操纵子pyr_Bs的基因簇依次包括pyrB、pyrC、pyrAA、pyrAB-E949、pyrK、pyrD、pyrF和pyrE。
3.如权利要求1所述的重组大肠杆菌,其特征在于,所述pyrB的核苷酸序列如SEQ IDNO.1所示,所述pyrC的核苷酸序列如SEQ ID NO.2所示,所述pyrAA的核苷酸序列如SEQ IDNO.3所示,所述pyrAB-E949的核苷酸序列如SEQ ID NO.4所示,所述pyrK的核苷酸序列如SEQ ID NO.5所示,所述pyrD的核苷酸序列如SEQ ID NO.6所示,所述pyrF的核苷酸序列如SEQ ID NO.7所示,所述pyrE的核苷酸序列如SEQ ID NO.8所示。
4.如权利要求1~3任一所述的重组大肠杆菌,其特征在于,所述嘧啶操纵子pyr_Bs整合在基因yghX或pta所在位置。
5.如权利要求4所述的重组大肠杆菌,其特征在于,所述出发菌株以E.coli MG1655为底盘细胞,敲除了基因组上的核苷酸5'-单磷酸核苷酶基因ppnN,胞苷单磷酸焦磷酸化酶基因ushA、嘌呤核苷酸酶基因yrfG、嘧啶5’-核苷酸酶基因yjjG、UMP磷酸酶基因umpH、5’-核苷酸酶基因umpG和胞苷脱氨酶基因cdd;
在umpH位点整合5’-CTP二磷酸水解酶基因nudG;所述基因nudG的核苷酸序列如SEQ IDNO.20所示;在umpG位点整合乳清酸磷酸核糖转移酶基因pyrE,所述基因pyrE的核苷酸序列如SEQ ID NO.15所示;
还整合表达尿苷-胞苷激酶基因udk、磷酸核糖焦磷酸合成酶基因prs、葡萄糖-6-磷酸脱氢酶基因zwf和6-磷酸葡糖酸脱氢酶基因gnd,所述基因udk的核苷酸序列如SEQ IDNO.16所示,所述基因prs的核苷酸序列如SEQ ID NO.19所示,所述基因zwf的核苷酸序列如SEQ ID NO.18所示,所述基因gnd的核苷酸序列如SEQ ID NO.17所示。
6.如权利要求5所述的重组大肠杆菌,其特征在于,在所述出发菌株poxB位点整合基因pyrH(R92G/D93G)或pyrH(D93A);所述基因pyrH(R92G/D93G)的核苷酸序列如SEQ ID NO.21所示,所述基因pyrH(D93A)的核苷酸序列如SEQ ID NO.22所示。
7.一种生产5’-胞苷酸的方法,其特征在于,将权利要求1~6任一所述的重组大肠杆菌在含葡萄糖的培养基中发酵。
8.如权利要求7所述的方法,其特征在于,所述葡萄糖的浓度为8~12g/L。
9.如权利要求8所述的方法,其特征在于,在35~40℃,发酵至少72h。
10.权利要求1~6任一所述的重组大肠杆菌在医药领域生产含5’-胞苷酸的产品中的应用。
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