CN113755414B - 一种生产尿苷的重组微生物及生产尿苷的方法 - Google Patents
一种生产尿苷的重组微生物及生产尿苷的方法 Download PDFInfo
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- CN113755414B CN113755414B CN202010498760.5A CN202010498760A CN113755414B CN 113755414 B CN113755414 B CN 113755414B CN 202010498760 A CN202010498760 A CN 202010498760A CN 113755414 B CN113755414 B CN 113755414B
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Abstract
本发明提供了一种生产尿苷的重组微生物以及利用该重组微生物生产尿苷的方法。其中,对尿苷的降解及利用基因进行了敲除,这些基因编码核糖核苷水解酶,尿苷磷酸化酶,核苷磷酸化酶,胞苷/尿苷激酶以及核苷转运蛋白。同时,对尿苷生物合成途径中的关键酶进行了过表达,包括降解胞苷三磷酸到胞苷单磷酸的胞苷三磷酸焦磷酸化酶,以及催化尿苷单磷酸到尿苷的尿苷单磷酸磷酸化酶。此外,对嘧啶核苷途径进行了基因工程改造,解除合成途径的反馈抑制。通过生物发酵方法将重组菌株在5升发酵罐可达到20g/L以上的尿苷产量,可以实现工业化的量产,同时尿苷的生产成本低和减少污染少,绿色环保,具有较高的推广应用价值。
Description
技术领域
本发明属于生物技术领域,具体地,涉及一种生产尿苷的重组微生物,利用重组微生物生产尿苷以及利用该重组微生物通过生物方法生产尿苷。
背景技术
尿苷,又名尿嘧啶核苷,属于核苷的一种为白色针状结晶或粉末,无气味,味稍甜而微辛。能溶于水,微溶于稀醇,不溶于无水醇。
分子式C9H12N2O6,相对分子质量224.2,熔点163-167℃,比旋8.4°。它主要用于用于巨型红血球贫血,也可与其他核苷、碱基合用于治疗肝、脑血管及心血管等疾病,也是制造氟尿嘧啶(S-FC)、脱氧核苷、碘苷(IDUR)、溴苷(BUDR)、氟苷(FUDR)等药物的主要原料。
目前尿苷的发酵生产主要集中在日本,但其供应的产品数量较少,且售价偏高。国内仅上海太平洋生物高科技公司一家曾采用RNA水解法生产尿苷,由于工艺落后,成本高,现已停产。尿苷生产主要有化学法和生物法两种。
传统的嘧啶核苷的生产方法是使用核糖核酸(RNA)经化学方法水解,然后制备两种嘧啶核苷,这不仅需要大量的优质RNA原料,而且两种嘌呤核苷(腺苷和鸟苷)及两种嘧啶核苷(胞苷和尿苷)都必须平衡产销,否则成本价会很高。浙江先锋化工科技有限公司利用尿嘧啶和D-核糖在磷钨酸催化下进行缩合反应获得(CN101717420A)。反应成本高,且使用的有机溶剂存在环境污染。
而发酵法则是借助于微生物,通过诱变扩大其合成核苷的能力,从而能够大规模地单一地根据市场需求生产尿苷,降低此类药物的生产成本。20世纪80年代至90年代中期,日本科学家开始发表发酵法生产尿苷的成果,武田药品株式会社利用诱变筛选获得的杆菌发酵生产尿苷,但产量较低5g/L,且存在发酵不稳定等问题(CN85107905)。国内的研究尚未见放大生产的报道。
因此,需要开发一种能替代化学合成、降低污染的尿苷合成方法。人类已经利用微生物来发酵生产核苷类的产品,如beta-胸苷、鸟苷、肌苷和腺苷。而尿苷和上述核苷一样,作为补救合成途径中的一种嘧啶核苷酸中间体,在生物中普遍存在着尿苷的代谢途径,但它很少积累在细胞中或细胞生物的基质中。改造生物,特别是微生物,使其增强尿苷的合成并积累在其生长的基质中,就可以有效地实现尿苷大规模工业化生产,从而降低尿苷的生产成本和减少污染。
预计国内市场年需求量约30吨,国际市场150吨,而世界目前年产量还不足50吨,所以发酵法生产尿苷市场前景看好,出口创汇可观。
发明内容
发明目的:设计和生产具有高产量尿苷生产合成和积累能力的重组微生物,并利用该重组微生物实现尿苷的高产量生物法生产。
技术方案:
1.去除利用和降解尿苷的酶的编码基因使尿苷不再参与细胞中的代谢
首先为了使微生物能够积累尿苷,1)对细胞中利用尿苷合成其它嘧啶核苷酸的第一个酶(胞苷/尿苷激酶)的编码基因进行阻断或敲除,这样尿苷就不能被磷酸化成UMP,UMP能够进一步被用来合成UDP,UTP等等。2)在微生物还存在着一系列的酶,它们具有降解尿苷的能力。比如尿苷磷酸化酶(EC 2.4.2.3),催化尿苷到尿嘧啶的转化反应,核糖核苷水解酶(EC 3.2.2.3)将尿苷水解成尿嘧啶和核糖。对这些基因进行敲除或阻断,使得尿苷能够积累。此外,位于微生物细胞膜上的核苷转运蛋白,如大肠杆菌的NupG/NupC,(Craig JE1994,Mol Microbiol.11(6):1159-68.),可将胞外的核苷转运至胞内。进一步对编码这些转运蛋白的基因进行敲除,就可使尿苷在胞外积累,同时也方便产品的纯化。
2.从UMP和CMP的去磷酸化来合成尿苷
大多野生型的生物都能生产多种磷酸化酶或核苷酶,如酸性磷酸化酶、碱性磷酸化酶和核苷酶,例如大肠杆菌产生酸性磷酸化酶(AphA)、碱性磷酸化酶(PhoA)、和核苷酶(UshA,YjjG,UmpH,UmpG,SurE,YfbR)(see http://ecocyc.org/)。无论是在胞内还是在周质空间,这些酶对核苷的特异性都不强,但是这些酶在鸟苷、腺苷和肌苷的微生物发酵生产中担任着非常重要的角色。同样的,在重组细胞中这些酶也可以用来生产尿苷的。
另一方面,自然界生物中存在着对特定的核苷酸具有特异性的磷酸化酶或核苷酶,比如工业上在重组大肠杆菌中生产Beta-胸苷就是利用对dTMP(脱氧胸苷单磷酸)具有特异性的dTMP磷酸化酶(TMPase,US专利5213972)将dTMP去磷酸化来合成beta-胸苷的。在自然界中同样也存在对UMP有特异性的磷酸化酶或核苷酶(在这里统称为UMPase),同时这些酶也可以催化CMP生成CdR,进而在胞苷脱氨酶的作用下生成UdR。例如链球菌来源的S5NA(Zheng 2015JBC 290,31126-31137),酿酒酵母来源的PHM8(Kuznetsov 2015JBC 290,18678-18698),人体白细胞来源的核苷酸酶PN-1(Chiarelli 2004,Red cells 105,3340-3345),果蝇来源的CG3362(Buschmann J2013,J Biol Chem.,288(4):2441-51.),PBS2噬菌体来源的TMPase(Price AR1973,J Biol Chem.248(4):1372-80.),家鼠(murine)来源的mdNT1(Rampazzo C2000,J Biol Chem.Feb 25;275(8):5409-15)以及mdNT2(MoothaVK2003,Cell.115(5):629-40.),大肠杆菌来源的YjjG,YfdR,SurF,UmpG(ProudfootM2004,J Biol Chem.279(52):54687-94.),UshA(Innes D2001,J Basic Microbiol.41(6):329-37.),UmpH(Tremblay LW 2006,Biochemistry.45(4):1183-93),AphA(Passariello C2006,Biochim Biophys Acta.2006Jan;1764(1):13-9.),PhoA(Engstrom1961,Biochim Biophys Acta 52;36-48.)等等。
这些对UMP具有特异性的UMPase或/和它们同源的酶的编码基因可以用DNA合成的方法获得,也可以直接相应的生物中提取的mRNA为模板,通过PCR的方法获得。因此,将上述获得的任何一个基因在重组细胞中进行表达,成功表达的UMPase就可以在重组细胞中以UMP和CMP为底物来产生UdR,显著增尿苷的产量。
为了进一步获得特异性更高的某一UMPase,可以通过蛋白质工程的技术,也可以获得具有对UMP高特异性的UMPase突变体,用来在重组细胞中产生大量的UdR(如,通过蛋白质工程来提高芽孢杆菌中支链淀粉酶的热稳定性(Chang 2016,MPLoS One.11(10):e0165006.))。
3.增强细胞中CMP的供给
在生物中,胞苷单磷酸并不是嘧啶核苷酸从头合成途径中的中间体,它只是嘧啶核苷酸的补救合成中的中间体。它可能是RNA降解后的产物,细胞降解RNA后,所产生的CMP,被CMP激酶(Cmk)催化生成胞苷二磷酸(EC2.7.4.14,EC2.7.4.25),后者可以进一步磷酸化合成CTP,或经核糖核酸还原酶用来合成脱氧胞苷二磷酸(dCDP)。dCDP是脱氧核糖核苷酸(DNA)合成的原料,脱氧胞苷三磷酸(dCTP)和脱氧胸苷三磷酸(dTTP)的前体物。
为了增加尿苷的合成,重组细胞具有持续的胞苷单磷酸的供给就很重要。为此1)首先就必须使细胞中缺少CMP激酶(Cmk),使CMP专门用来合成胞苷,进而转化为尿苷。2)其次是在重组细胞中表达或超量表达CTP和CDP磷酸化酶或相应的核苷酸酶,从而增加CMP在胞内的浓度。有关的CTP和CDP磷酸化酶或相应的核苷酶可能并不存在于所有的、非基因改造过的细胞中。目前已知的能够催化CTP水解成CMP的磷酸化酶或核苷酶报道的有,大肠杆菌来源的NudG(O’Handley,et al 2000JBC 276,5421-5426),分枝杆菌来源的Mut2(Sang2013ASM 195,1552-1560),枯草杆菌来源的Maf蛋白-BSU28050(Tchigvintsev2013Chemistry&Biology 20,1386-1398),Deinococcus radiodurans来源的DR_0079(Buchko GW2008,Biochemistry.47(25):6571-82.),大肠杆菌来源的NudI(Xu W2006,JBiol Chem.281(32):22794-8.),大肠杆菌来源的MazG(Zhang J2002,J Bacteriol.184(19):5323-9.),大肠杆菌来源的NudB(Suzuki Y1974,J Biol Chem.249(8):2405-10.),大肠杆菌来源的PhnM(Kamat SS2011,Nature.480(7378):570-3.),大肠杆菌来源的RppH(Deana A2008,Nature.451(7176):355-8.)等等。
4.解除嘧啶核苷合成的反馈抑制,增加嘧啶核苷合成的代谢流和UdR的产量
嘧啶核苷酸合成途径催化第一步由氨基甲酰磷酸合成酶催化合成氨基甲酰磷酸。该酶的编码基因carAB分别受到代谢终产物嘌呤、嘧啶以及精氨酸的反馈阻遏作用(Devroede N2006,J Bacteriol.188(9):3236-45.),相应阻遏蛋白分别为PurR,PepA,ArgR(Kim2015.Microb Cell Fact 14:98),本发明通过对阻遏蛋白编码的基因purR,pepA,argR的敲除,进而解除了CarAB的转录的抑制,加快了氨基甲酰磷酸合成。同时氨基甲酰磷酸合成酶本身也会受到UMP的反馈抑制,根据文献报道(Delannay S1999,J Mol Biol.286(4):1217-28),将该酶的一个亚单位(CarB)的第948位氨基酸-丝氨酸突变成苯丙氨酸(S948F)就可有效解除UMP的抑制作用,本发明通过在染色体上对carB基因进行突变,获得携带编码CarB(S948F)突变基因的重组菌株。
嘧啶核苷酸合成途径催化第二步是由天冬氨酸氨甲酰基转移酶催化合成氨基甲酰天冬氨酸。该酶由一个起调控作用的亚基(PyrI)和一个起催化亚基(PyrB)组成,该酶在CTP浓度高的时候,CTP结合PyrI从而降低酶的活性。然而如果将编码天冬氨酸氨甲酰基转移酶调控亚基破坏掉,如将其编码基因的敲除,就解除终产物CTP对对其的反馈阻遏作用(Coudray L 2009,Bioorg Med Chem.17(22):7680-9)。
嘧啶核苷酸合成途径中pyrE编码乳清酸核糖磷酸转移酶,催化乳清酸合成乳清酸单磷酸,大肠杆菌W3110宿主菌中pyrE基因上游rph基因终止密码子附近存在移码突变(KajFrank Jensen1993,Journal Of Bacteriology,3401-3407.),导致部分rph基因转录无法终止,进而影响下游pyrE基因的表达,导致宿主菌中乳清酸的积累。pyrH编码的UMP激酶,催化UMP磷酸化合成UDP,但该酶受到其产物UDP的反馈抑制。对该酶第93位的天冬氨酸到丙氨酸的突变(D93A)能够有效解除相应的反馈抑制作用(Meyer P2008,J Biol Chem.283(51):36011-8.)。pyrG编码CTP合酶,催化UTP合成CTP,但该酶受到其产物CDP的反馈抑制,对该酶第160位天冬氨酸到谷氨酸(D160E),或第162位谷氨酸到丙氨酸(E162A)或第168位谷氨酸到赖氨酸(E168K)的突变可有效解除CDP对其反馈抑制作用(Zhu2014,ProteinEngineering,Design&Selection 27(7).225–233)。
另外在嘧啶核苷酸合成中,核糖是通过核糖磷酸焦磷酸为前体物合成到核苷上的,核糖磷酸焦磷酸激酶(Prs)催化5-磷酸核糖与ATP反应合成核糖磷酸焦磷酸PRPP。但是大肠杆菌来源的核糖磷酸焦磷酸激酶受到ADP的反馈抑制,而Prs上的第128位的天冬氨酸到丙氨酸的突变(D128A)可有效解除ADP对其反馈抑制作用(Shimaoka 2007J BiosciBioeng 103(3):255-61.)。
有益效果:本发明具有以下优点:(化学合成)与现有技术相比,本发明所述的生产尿苷的重组微生物以及应用该重组微生物利用生物法生产实现尿苷的高产量生产方法,并具有以下有益效果:通过利用遗传操作的生物材料的方法建立重组微生物,改造微生物,并利用微生物发酵的方法,使其增强尿苷的合成并积累在其生长的基质中,就可以有效地实现尿苷的大规模工业化生产,从而降低尿苷的生产成本和减少污染,绿色环保,具有较高的推广应用价值。
附图说明:
图1为大肠杆菌尿苷的代谢路径示意图;
图2为尿苷检测的HPLC图谱。
其中:carbamoyl phosphate:氨甲酰磷酸;carbamoyl aspartate:氨甲酰天冬氨酸;orotate:乳清酸盐;OMP:乳清酸-5'-单磷酸;UMP:尿苷-5'-单磷酸;UDP:尿苷-5'-二磷酸;UTP:尿苷-5'-三磷酸;UdR:尿苷;CdR:胞苷;CTP:胞苷-5'-三磷酸;CDP:胞苷-5'-二磷酸;CMP:胞苷-5'-单磷酸;Cytosine:胞嘧啶;PyrE:乳清酸磷酸核糖转移酶;PyrH:尿苷-5'-单磷酸激酶;PyrG:胞苷-5'-三磷酸合成酶;CTPase:胞苷-5'-三磷酸焦磷酸化酶;UMPase:尿苷-5'-单磷酸磷酸化酶;Cmk:胞苷-5'-单磷酸激酶;Udk:胞苷/尿苷激酶;Cdd:胞苷脱氨酶;RihA、B、C:嘧啶特异性核糖核苷水解酶1,2,3;XTPase:胞苷-5'-三磷酸磷酸化酶;PND:胞苷-5'-二磷酸磷酸化酶;Ndk:核苷二磷酸激酶;Udp:尿苷磷酸化酶;PpnP:核苷磷酸化酶;Upp:尿嘧啶磷酸核糖转移酶;CodA:胞嘧啶/异鸟嘌呤脱氨酶。
具体实施方式
下面将通过几个具体实施例,进一步阐明本发明,这些实施例只是为了说明问题,并不是一种限制。
本发明的技术方案可以应用大肠杆菌,枯草杆菌,谷氨酸棒杆菌,乳杆菌或者其他的微生物,下以大肠杆菌为例,对技术方案进行进一步说明。
实施例1在大肠杆菌中基因敲除的方法
本发明采用Datsenko的方法在大肠杆菌中进行基因敲除(Datsenko KA2000.Proc Natl Acad Sci USA,97(12):6640~6645),相应的基因敲除引物见Baba T2006Mol Syst Biol 2(1),0008。
实施例2摇瓶发酵验证重组菌株的方法
验证重组菌株在摇瓶发酵中生产尿苷的发酵培养基,具体为每升培养基中YC溶液100ml,甘油20g,5倍的盐溶液200ml,TM2溶液1ml,柠檬酸铁10mg,无水硫酸镁120mg,氯化钙111mg,硫胺素1ug,以去离子水定容。其中5倍的盐溶液为磷酸氢二钠每升30g,磷酸二氢钾每升15g,氯化钠每升2.5g,氯化铵5.0g,以离子水定容;TM2溶液为四水氯化锌每升2.0g,六水氯化钙每升2.0g,两水钼酸钠每升2.0g,五水硫酸铜每升1.9g,硼酸每升0.5g,盐酸每升100ml。发酵培养基M9中YC溶液:去离子水100ml;发酵培养基M10中YC溶液为蛋白胨0.3g,酵母粉0.15g,氯化钠0.3g,去离子水100ml;发酵培养基M11中YC溶液为蛋白胨0.5g,酵母粉0.25g,氯化钠0.5g,去离子水100ml;发酵培养基M12中YC溶液为蛋白胨0.1g,酵母粉0.05g,氯化钠0.1g,去离子水100ml;发酵培养基M13中YC溶液为大豆蛋白胨0.1g,酵母粉0.05g,去离子水100ml;上面溶液灭菌为121C,20-30分钟。
摇瓶发酵过程如下:首先接种重组菌株至含有抗生素的一定量的LB培养基(著[美]J.莎姆布鲁克黄培堂译,子克隆指南2002,1595)中,置34℃摇床中,250rpm培养过夜;取过夜种子100μl转移到至2ml含有抗生素的LB,于34℃摇床中,250rpm培养4-6h至OD 600值为1.5左右;随后将2ml二级种子全部转入装有18ml发酵培养基M11的摇瓶中,置34℃摇床中,250rpm培养OD600值为1左右时,添加IPTG至终浓度0.1mM后,继续培养20小时左右后,取1毫升的发酵液离心后(7000rmp,4分钟),取上清检测,检测方法见实施例4。
实施例3 5L发酵罐利用重组菌株发酵生产尿苷的方法
验证重组菌株在发酵罐中发酵生产尿苷的发酵培养基为MF1.16,具体为每升培养基中含硫酸铵10g,氯化钠2g,磷酸二氢钾2g,七水硫酸镁2g,甘油25g,氯化钙105mg,氯化锌10mg,TM2微量元素溶液1mL,柠檬酸铁9.4mg,蛋白胨0-10g,酵母粉0-10g,以去离子水定容。其中TM2微量元素溶液为氯化锌1.31g,氯化钙1.01g,四水合钼酸铵1.46g,硫酸铜1.9g,硼酸0.5g,盐酸10mL,以去离子水定容。补料培养基为每升含甘油500g,蛋白胨0-10g,酵母粉0-10g。
发酵如下:首先准备种子,从LB平板上挑取单克隆到含有抗生素的LB试管34℃过夜培养,按接种量1%接入装100mL LB的500mL摇瓶,34℃培养4小时,OD至1.5~2;以接种量5%接种至装有2L发酵培养基MF1.16的5L发酵罐,37℃培养,用氨水调节pH为6.9,溶氧转速偶联,维持溶氧在30%,当溶氧高于40%时,开始偶联补料,使溶氧维持在30%-45%。发酵8小时,OD600至16~25时,降低温度至35℃,加入IPTG,使终浓度为0.5mmol/L进行诱导,发酵27小时后开始取样检测,检测方法见实施例4。
实施例4 HPLC测定发酵液中的尿苷和相关副产物
吸取1ml发酵液于2ml离心管中,100℃加热5分钟,冷却至室温,稀释一定的倍数,离心过0.22um的滤膜,用高效液相色谱(HPLC)检测,HPLC的参数如下:采用Agilent SB C184.6*150mm 5um,流动相为甲醇和10mM PBS(pH4.0),流动相比例0.01-2.80分钟甲醇比例为2%,2.80-3.50分钟甲醇比例由2%上升到10%,3.50-3.60分钟甲醇比例由10%降至2%,3.60-8.5分钟甲醇比例为2%,利用紫外检测器检,测波长260nm;初始流动相的流速为1.0mL/min,发酵液的上样量5μL,柱温30℃。尿苷出峰时间为5.289分钟,胞苷出峰时间为3.580分钟,尿嘧啶出峰时间为2.581分钟,乳清酸出峰时间为1.780分钟,HPLC图谱如图2所示。
实施例5构建不能降解和利用尿苷的重组大肠杆菌
微生物有两种途径来合成尿苷(CdR)和尿苷的衍生物(如CTP,CDP,CMP),一种是从头合成途径(利用葡萄糖等碳源和氮源,以5′-磷酸核糖为出发物质),一种是补救合成途径(由嘧啶碱基通过核糖基化及磷酸化而合成)。
如图1所示,在大肠杆菌中,细胞中尿苷的流向有两个,一个是尿苷被用在补救合成途径中,经胞苷/尿苷激酶(Udk)合成UMP;另一个途径尿苷被核苷酸水解酶(RihA,RihB,RihC)或核苷磷酸化酶(PpnP)或尿苷磷酸化酶(Udp)水解为尿嘧啶(uracil)。本专利以大肠杆菌W3110(ATCC27325)为出发菌株,在W3110中敲除Udk、Udp、PpnP、RihA/RihB/RihC的编码基因,获得了重组菌(表1),这些菌株利用和降解尿苷的能力逐渐减弱,其中重组菌HX1在摇瓶发酵中完全不能利用和降解尿苷(摇瓶中添加1g/L尿苷,发酵24h检测,剩余尿苷1g/L)。
表1菌株改造
菌株 | 基因型 |
W3110 | F-mcrA mcrB IN(rrnD-rrnE)1lambda- |
HC2 | W3110ΔrihA |
HC3 | W3110ΔrihAΔudk |
HL5 | W3110ΔrihAΔudkΔudpΔrihC |
HX1 | W3110ΔrihAΔudkΔudpΔrihCΔppnP |
实施例6过表达核苷酸磷酸水解酶能提高尿苷产量
降解和利用尿苷的基因敲除之后尿苷的产量很低,说明代谢路径中生成尿苷的关键酶活性不足。大肠杆菌中的尿苷酸UMP经核苷磷酸水解酶(UMPase)水解生成尿苷和一分子磷酸,同时UMPase还会催化胞苷酸CMP水解为胞苷和一分子磷酸,胞苷进一步被胞苷脱氨酶脱氨生成尿苷。通过在重组菌种过表达UMPase,可以获得更多的尿苷。将表达不同携带编码UMPase基因的质粒转化大肠杆菌表达宿主BL21(DE3),通过随后在LB培养基中生长并IPTG诱导UMPase的表达后,用收集的菌体,超声破壁后用作粗酶溶液,以胞苷酸为底物进行体外转化实验,在Kuznetsova 2015(The Journal Of Biological Chemistry Vol.29(30),18678–18698)的条件及体系下,BL21(DE3)/pET30a-PHM8的细胞可转化CMP生成胞苷,反应1.5小时转化率可达31.3%。
以HL5为宿主,分别表达pHS01、pX01(pHS01-PHM8)后,在发酵培养基M9中进行摇瓶发酵后,HL5/pHS01尿苷产量只有60mg/L,而HL5/pX01为110mg/L,乳清酸的产量分别为100mg/L、145mg/L。实验结果表明过表达核苷酸磷酸水解酶能增加尿苷的生产。
实施例7过表达胞苷三磷酸水解酶增加尿苷的产量
如图1所示,大肠杆菌中的胞苷三磷酸CTP可以经三磷酸核苷焦磷酸水解酶(CTPase)水解生成胞苷酸CMP和焦磷酸,也会由核苷磷酸水解酶两步反应及经二磷酸胞苷CDP生成CMP。本专利通过过表达CTPase,来获得更多的胞苷酸(CMP),从而增加生产胞苷的前提物,进而提高尿苷产量。例如以HL4(HC3Δudp)为宿主,表达pHS01-PHM8后,在发酵培养基M10中摇瓶发酵,无尿苷生成,尿嘧啶120mg/L,乳清酸410mg/L;而表达pHS01-PHM8-nudG后尿苷产量增至325mg/L,同时尿嘧啶的产量降低为57mg/L,乳清酸409mg/L。说明菌体自身的胞苷三磷酸焦磷酸水解酶的活性低,而CTPase的过表达能够大幅度的提高尿苷的产量。
实施例8胞苷酸激酶的缺失可增加重组菌中尿苷的产量
细胞内的CTP、CDP及CMP三者之间可以互相转化,而CMP的回流一定程度上限制了胞苷的大量积累,进而减少了尿苷的生成。胞苷酸激酶(EC2.7.4.14,EC2.7.4.25)能够催化CMP生成CDP同时消耗一份子ATP。将回流路径中编码胞苷酸激酶的基因阻断,从而促使CMP积累后,经CMP水解酶等酶促反应生成胞苷,提高尿苷的产量。例如在HL7(HL5ΔpurR)、HL8(HL7△cmk)宿主中,表达pX04(pHS01-PHM8-nudG-pyrE-prs128)后,在发酵培养基M10中摇瓶发酵,尿苷产量HL7/pX04为1.5g/L,而cmk基因敲除后HL8/pX04为1.52g/L。
重组菌株pHS01-PHM8-nudG-pyrE-prs128简写为菌株HL7/pX04,其分类命名为大肠埃希氏菌(Escherichia coli.),该菌株已于2020年03月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编:100101,保藏编号为CGMCC No.19447。
实施例9过表达prs128提高尿苷的产量
在大肠杆菌嘧啶合成途径中,核糖磷酸焦磷酸(PRPP)会在乳清酸核糖磷酸转移酶(PyrE)的作用下将磷酸基转移至乳清酸生成乳清酸单磷酸(OMP)。而PRPP的不足会成为嘧啶合成途径中的限速步骤,导致胞苷产量较低且造成副产物乳清酸的积累。核糖磷酸焦磷酸激酶(Prs)催化5-磷酸核糖与ATP反应合成PRPP。但Prs受到ADP的反馈抑制,将Prs的第128位的天冬氨酸突变为丙氨酸获得的Prs128,就可以解除ADP的反馈抑制作用。例如以HL4为宿主,分别表达pX03、pX04(pHS01-PHM8-nudG-pyrE-prs128)后,在发酵培养基M12中摇瓶发酵尿苷产量从1.39g/L提高到1.51g/L。
实施例10敲除编码阻遏蛋白PurR的基因可以提高胞苷产量
嘧啶核苷合成途径第一步由氨基甲酰磷酸合成酶催化合成氨基甲酰磷酸。该酶的编码基因carAB受到代谢终产物嘌呤、嘧啶以及精氨酸的反馈阻遏作用。而嘧啶嘌呤核苷酸受由purR基因编码的阻遏蛋白的抑制。本发明通过对purR的敲除,进而解除了carAB的转录的抑制,加快了氨基甲酰磷酸的合成。例如在HL5、HL7(HL5 purR)为宿主中表达pX04,在发酵培养基M13中摇瓶发酵尿苷的产量从1.65g/L提高到1.74g/L。说明purR基因的敲除可以嘌呤对解除carAB的反馈阻遏,进而嘧啶的合成,提高尿苷的产量。HL7/pX04在5L发酵罐上放大,发酵44.6小时的产量为28.74g/L。
以上所述仅是发明的几个实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离发明原理的前提下,还可以做出若干改进,这些改进也应视为本发明的保护范围。
Claims (2)
1. 一种生产尿苷的重组微生物,其特征在于:所述重组微生物其分类命名为大肠埃希氏菌(Escherichia coli),该菌株已于2020年03月18日保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编:100101,保藏编号为CGMCC No. 19447。
2.一种利用重组微生物生产尿苷的方法,其特征在于:所述尿苷通过权利要求1所述的重组微生物经发酵,得到所述尿苷。
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