CN116144559A - 一种生产胞磷胆碱的基因工程菌及其构建方法与应用 - Google Patents
一种生产胞磷胆碱的基因工程菌及其构建方法与应用 Download PDFInfo
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- CN116144559A CN116144559A CN202211173656.4A CN202211173656A CN116144559A CN 116144559 A CN116144559 A CN 116144559A CN 202211173656 A CN202211173656 A CN 202211173656A CN 116144559 A CN116144559 A CN 116144559A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种生产胞磷胆碱的基因工程菌及其构建方法与应用,所述基因工程菌为重组菌株Escherichiacoli CKI‑CCT‑CMK‑NDK‑UDK,该基因工程菌遗传背景清晰、代谢背景低、异源表达能力强,可利用胞苷、氯化胆碱作为底物催化生产胞磷胆碱,具有操作简便、催化周期短、反应条件温和、成本低、摩尔转化率高等优点,为胞磷胆碱工业化生产提供新思路,具有重要的应用价值。
Description
技术领域
本发明涉及生物技术生产领域,尤其是一种生产胞磷胆碱的基因工程菌及其构建方法与应用。
背景技术
胞磷胆碱又称为胞嘧啶核苷二磷酸胆碱,能够改善脑组织代谢,降低血脂和血小板粘稠度,促进脑血液循环,还有助于脑功能恢复正常。临床上可应用于治疗脑损伤和外伤性脑休克,治疗由脑组织代谢受阻引起的神经性病和意识障碍,还对提高记忆力和延缓衰老有一定作用。胞磷胆碱市场需求量巨大,国内药品市场年需求量超过60吨,近年来胞磷胆碱在保健食品和功能饮料领域的应用非常活跃,国外需求迅猛增长。
目前,胞磷胆碱的生产方法主要有化学合成法和生物合成法。化学合成法使用多种工业有毒试剂,且底物转化率低,因此不适于大规模生产应用。生物合成法早期以酵母菌泥为生物酶源,以葡萄糖为底物进行从头发酵生产胞磷胆碱,但此方法耗时太久且酵母菌体使用量较大。
综上所述,传统的化学合成法与生物合成法已经无法满足市场上胞磷胆碱工业化生产需求,因此急需一种更加高效、方便的胞磷胆碱的生产方法。而酶催化法因其具有催化时间短、过程简单、成本低廉、易于工业化等优势,成为替代目前生产方法的首选。大肠杆菌因其遗传背景清晰、基因编辑方法完善、表达蛋白能力强优点,成为酶催化法生产胞磷胆碱的最好选择。
发明内容
本发明所要解决的技术问题在于提供一种生产胞磷胆碱的基因工程菌。
本发明所要解决的技术问题在于提供上述生产胞磷胆碱的基因工程菌的构建方法。
本发明所要解决的技术问题在于提供上述生产胞磷胆碱的基因工程菌的应用。
为解决上述技术问题,本发明的技术方案是:
一种生产胞磷胆碱的基因工程菌,为重组菌株Escherichia coliCKI-CCT-CMK-NDK-UDK,是由下述方法构建得到的:首先,构建工程菌E.coli CKI-CTT表达来源于酿酒酵母的胆碱激酶和磷酸胆碱胞苷酸转移酶,胆碱激酶能够催化氯化胆碱与磷酸盐合成磷酸胆碱,磷酸胆碱胞苷酸转移酶能够催化磷酸胆碱与三磷酸胞苷合成胞磷胆碱;构建工程菌Ecoli CMK-NDK表达来源于大肠杆菌自身的胞苷酸激酶和多功能核苷二磷酸激酶;构建工程菌Ecoli-UDK表达来源于大肠杆菌的胞苷激酶,胞苷激酶能够催化胞苷生成CMP,胞苷酸激酶能够催化CMP生成CDP,多功能核苷二磷酸激酶能够催化CDP生成CTP。
上述生产胞磷胆碱的基因工程菌的构建方法,利用合成生物学技术在野生型大肠杆菌中表达来源于酿酒酵母的胆碱激酶基因和磷酸胆碱胞苷酸转移酶基因,过表达内源的胞苷激酶、胞苷酸激酶以及多功能核苷二磷酸激酶基因获得可生产胞磷胆碱的大肠杆菌工程菌,步骤包括:
(1)构建工程菌E.coli CKI-CTT表达来源于酿酒酵母的胆碱激酶和磷酸胆碱胞苷酸转移酶,胆碱激酶能够催化氯化胆碱与磷酸盐合成磷酸胆碱,磷酸胆碱胞苷酸转移酶能够催化磷酸胆碱与三磷酸胞苷合成胞磷胆碱;
(2)构建工程菌E.coli CMK-NDK表达来源于大肠杆菌自身的胞苷酸激酶和多功能核苷二磷酸激酶;
(3)构建工程菌E.coli-UDK表达来源于大肠杆菌的胞苷激酶,胞苷激酶能够用于催化胞苷形成CMP,胞苷酸激酶能够催化CMP生成CDP,多功能核苷二磷酸激酶能够催化CDP生成CTP。
优选的,上述生产胞磷胆碱的基因工程菌的构建方法,所述野生型大肠杆菌为大肠杆菌E.coli BL21。
优选的,上述生产胞磷胆碱的基因工程菌的构建方法,以Pstv28质粒为载体在大肠杆菌E.coli BL21中表达异源的胆碱激酶和磷酸胆碱胞苷酸转移酶编码基因,构建出具有胆碱激酶活性和磷酸胆碱胞苷酸转移酶活性的菌株Escherichia coli CKI-CCT;其中,所述胆碱激酶基因来源于酿酒酵母,其扩增引物具有SEQ ID NO.1(EcoR I)、SEQ ID NO.2所示序列(Hind III);所述磷酸胆碱胞苷酸转移酶基因来源于酿酒酵母,其扩增引物具有SEQ ID NO.3(EcoR I)、SEQ ID NO.4所示序列(Hind III);所述质粒Pstv28具有SEQ IDNO.11所示核苷酸序列。
优选的,上述生产胞磷胆碱的基因工程菌的构建方法,以pTRC99a质粒为载体在大肠杆菌E.coli BL21中表达内源的胞苷酸激酶和多功能核苷二磷酸激酶编码基因,构建出具有胞苷酸激酶和多功能核苷二磷酸激酶活性的菌株Escherichia coli CKI-CCT-CMK-NDK;其中,所述胞苷酸激酶基因和多功能核苷二磷酸激酶编码基因均来自于大肠杆菌,胞苷酸激酶扩增引物具有SEQ ID NO.5(EcoRI)、SEQ ID NO.6所示序列(XbaI),多功能核苷二磷酸激酶扩增引物具有SEQ ID NO.7(EcoRI)、SEQ ID NO.8所示序列(XbaI);所述质粒pTRC99a具有SEQ ID NO.12所示核苷酸序列。
优选的,上述生产胞磷胆碱的基因工程菌的构建方法,以pTRC99a质粒为载体在大肠杆菌E.coli BL21中表达内源的胞苷激酶,构建出具有胞苷激酶活性的菌株EscherichiacoliCKI-CCT-CMK-NDK-UDK;其中,所述胞苷激酶编码基因来自于大肠杆菌,其扩增引物具有SEQ ID NO.9(SbfI)、SEQ ID NO.10所示序列(HindIII);所述质粒pTRC99a具有SEQ IDNO.12所示核苷酸序列。
上述生产胞磷胆碱的基因工程菌在制备胞磷胆碱方面的应用。
优选的,上述生产胞磷胆碱的基因工程菌的应用,胞磷胆碱的制备方法为:将所述基因工程菌(重组菌株Escherichia coli CKI-CCT-CMK-NDK-UDK)在培养基中培养,诱导产生高酶活性的胆碱激酶、胞苷激酶、多功能核苷二磷酸激酶和胞苷酸激酶之后,在发酵罐内随罐流加底物,进行全细胞催化,其中,以胆碱激酶、胞苷激酶、多功能核苷二磷酸激酶和胞苷酸激酶为催化剂,以胞苷、磷酸二氢钾、磷酸二氢钠、氯化胆碱为底物,在每30L发酵罐中随罐进行酶催化反应,反应体系中的胞苷的浓度为40mM,磷酸二氢钾的浓度为80mM,磷酸二氢钠的浓度为80mM,氯化胆碱的浓度为40mM,温度控制在24℃,初始转速为200r/min,后期调整转速使溶氧控制在30%-50%,反应过程中通过自动流加氨水维持PH值在7.0.合成胞磷胆碱。
优选的,上述生产胞磷胆碱的基因工程菌的应用,胞磷胆碱的制备包括菌种活化(步骤(1)、(2))和全细胞催化(步骤(3)-(5)),具体方法为:
(1)试管斜面培养:用接种环从甘油保菌管中挑取3-4环划线接种于带有壮观霉素和氨苄青霉素抗性的斜面培养基,于37℃培养箱中培养10-12h,转接1代;
(2)茄形瓶斜面培养:用接种环从试管斜面培养基中挑取全部菌体划线接种于带有壮观霉素和氨苄青霉素抗性的茄形瓶培养基中,于37℃培养箱中培养10-12h,转接2代;
(3)将茄形瓶中的菌体全部接种至含有壮观霉素和氨苄青霉素抗性的发酵培养基中,初始通气量2L/min,溶氧控制在30%-50%,通过自动流加氨水控制PH在7.0,培养温度为35℃;
(4)培养OD600=10-12时,添加最终浓度为0.1mMoL/L的IPTG诱导目的蛋白表达添加量为发酵体系的千分之一,调节诱导温度至24℃,诱导培养2-4h,表达关键酶的活性;
(5)开始以恒定速率向发酵罐中流加底物,当培养基中的葡萄糖消耗完之后,流加80%(m/v)的葡萄糖溶液,维持发酵培养基中的葡萄糖浓度在0.1-1g/L,直至流加结束,催化周期大约28-30h。
优选的,上述生产胞磷胆碱的基因工程菌的应用,所述斜面培养基为:葡萄糖2g/L,氯化钠2.5g/L,蛋白胨10g/L,酵母浸粉5g/L,磷酸二氢钾1g/L,硫酸镁0.2g/L,琼脂粉25g/L,PH7.0-7.2;灭菌条件:0.1MPa 30min,灭菌后加壮观霉素和氨苄青霉素各20mg/L。
优选的,上述生产胞磷胆碱的基因工程菌的应用,所述发酵培养基为:葡萄糖30g/L,酵母浸粉8g/L,MgSO4·7H2O 0.5g/L,KH2PO4 3g/L,蛋白胨2g/L,(NH4)2SO43 g/L,VB1.3.5.12各1mg/L,VH 1mg/L,微量元素混合液1mg/L,壮观霉素20mg/L,氨苄青霉素20mg/L,消泡剂2滴,其余为水;所述微量元素混合液组分含量为:钼酸铵0.28mg/L,硼酸5mg/L,CoCl2·6H2O1.4mg/L,MnSO4·H2O 0.5mg/L,CuSO4·7H2O 0.5mg/L,ZnSO4·7H2O 0.6mg/L。
上述成分称量固体后溶解于1L水中,在4℃保存。
反应液中上述胞磷胆碱的检测方法为:取反应液13000r/min离心2min,取上清液用去离子水稀释100倍,用0.22μm的无菌膜过滤至液相瓶待分析;使用高效液相色谱测定胞磷胆碱的含量,进样量为20μL,色谱柱为JADE-PAK(250×4.6mm)色谱柱,流动相为磷酸盐缓冲液:乙腈=97:3,柱温30℃,流速1mL/min,检测波长271nm,胞磷胆碱保留时间为2.8min全细胞催化反应周期30h。
有益效果:
本发明所述生产胞磷胆碱的基因工程菌,遗传背景清晰、代谢背景低、异源表达能力强,可利用胞苷、氯化胆碱作为底物催化生产胞磷胆碱,具有操作简便、催化周期短、反应条件温和、成本低、摩尔转化率高等优点,为胞磷胆碱工业化生产提供新思路,具有重要的应用价值。
1、使用全细胞催化法用于胞磷胆碱的合成,与现有报道的传统化学合成法、生物转化法和细胞破碎催化法相比具有生产成本低廉、反应温和、生产周期短、操作简单等特点;
2、采用多酶体系进行催化,以胞苷、氯化胆碱为主要底物制备胞磷胆碱,底物成本低廉,均为市售产品,很好的解决了传统化学合成法、生物转化法使用磷酸胆碱、CMP为底物成本居高不下的问题;
3、使用pTRC99a质粒和Pstv28质粒载体对多个酶进行串联表达,而且使用单株工程菌进行催化,简化了产酶菌株的数量,使胞磷胆碱的摩尔转化率高达80%;
4、催化过程反应温度、PH恒定,特定的催化反应条件最终获得较高的摩尔转化率,为胞磷胆碱的工业生产创造了良好的前景。
附图说明
图1为制备胞磷胆碱工程菌的构建方法图解;
图2为本发明中各时间段胞磷胆碱的产量与OD600图;
图3为反应液中胞磷胆碱的高效液相色谱图。
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。除非特别说明,本发明中所用的技术手段均为本领域技术人员所公知的方法。
本发明中所使用的原料,如无特殊说明,均为常规市售产品,本发明中所使用的方法。如无特殊说明,均为本领域常规方法,本发明所用各物质质量均为常规使用质量。
实施例1
全细胞催化法生产胞磷胆碱的基因工程菌的构建方法,如图1所示,具体构建步骤如下:
一、Pstv28-CKI-CCT质粒的构建
(1)以酿酒酵母(Saccharomyces cerevisiae S288c)基因组为模板,使用软件CEDesign单片段克隆中的PCR扩增线性化,根据胆碱激酶基因cki、磷酸胆碱胞苷酸转移酶基因cct以及Pstv28质粒载体序列(SEQ ID NO.11)设计扩增引物,所述两对引物分别包含了酶切位点EcoR I和Hind III;其中,
酿酒酵母的胆碱激酶编码基因扩增引物及酶切位点
5‘TATGACCATGATTACGAATTCTTACAAATAACTAGTATCGAGGAACTTGAA3‘EcoR I
5‘AGCGAACTGAATGGTACAAGAATCACGTCCAGG 3’Hind III
酿酒酵母的磷酸胆碱胞苷酸转移酶编码基因扩增引物及酶切位点
5‘CTTGTACCATTCAGTTCGCTGATTGTTTCTTCTT 3‘EcoR I
5‘ACGACGGCCAGTGCCAAGCTTATGGCAAACTCAACAACAGGGA 3’Hind III
(2)扩增获取目的基因片段cki和cct,将目的基因片段cki与cct进行重叠PCR,得到目的基因片段cki-cct。
(3)扩增获得Pstv28线性化载体片段,获得与目的基因片段相同粘性末端的Pstv28线性片段。
(4)使用Exnase II重组酶连接步骤(2)和步骤(3)中的基因片段和线性片段,得到重组表达载体Pstv28-cki-cct。
二、pTRC99a-cmk-ndk-udk质粒的构建
(1)以大肠杆菌(Escherichia coli K12,ATCC10798)基因组为模板,使用软件CEDesign单片段克隆中的PCR扩增线性化,根据胞苷酸激酶cmk、多功能核苷二磷酸激酶基因ndk以及pTRC99a质粒载体序列(SEQ ID NO.12)设计扩增引物,所述两对引物分别包含了酶切位点EcoRI和XbaI;其中,
大肠杆菌的胞苷酸激酶编码基因扩增引物及酶切位点
5‘AGGAAACAGACCATGGAATTCATGACGGCAATTGCCCCG 3‘EcoRI
5‘TAGCCATTTATGCGAGAGCCAATTTCTGG 3’XbaI
大肠杆菌的多功能核苷二磷酸激酶编码基因扩增引物及酶切位点
5‘GGCTCTCGCATAAATGGCTATTGAACGTACTTTTTCCA 3‘EcoRI
5‘TGCCTGCAGGTCGACTCTAGATTAACGGGTGCGCGGGCA 3’XbaI
(2)扩增获取目的基因片段cmk和ndk,将目的基因片段cmk与ndk进行重叠PCR,得到目的基因片段cmk-ndk。
(3)扩增获得pTRC99a载体片段,获得与目的基因片段相同粘性末端的pTRC99a线性片段。
(4)使用Exnase II重组酶连接步骤(2)和步骤(3)中的基因片段和线性片段,得到重组表达载体pTRC99a-cmk-ndk。
(5)以大肠杆菌(Escherichia coli K12,ATCC10798)基因组为模板,使用软件CEDesign单片段克隆中的PCR扩增线性化,根据胞苷激酶udk以及pTRC99a-cmk-ndk质粒载体序列设计扩增引物,所述两对引物分别包括了酶切位点Sbf I和Hind III;其中,
大肠杆菌的胞苷激酶编码基因扩增引物及酶切位点
5‘TAATCTAGAGTCGACCTGCAATGACTGATCAGTCTCATCAGTGCG 3‘SbfI
5‘TCCGCCAAAACAGCCAAGCTTTATTCAAAGAACTGACTTATTTTCGC 3’Hind III
(6)扩增获取目的基因片段udk。
(7)扩增获得pTRC99a-cmk-ndk线性化片段,获得与目的基因片段相同粘性末端的pTRC99a-cmk-ndk线性片段。
(8)使用Exnase II重组酶连接步骤(6)和步骤(7)中的目的基因片段和线性片段,得到重组表达载体pTRC99a-cmk-ndk-udk。
三、重组表达载体转入底盘细胞
分别将上述步骤一和步骤二得到的重组表达载体Pstv28-cki-cct与pTRC99a-cmk-ndk-udk化转至E.coli BL21(ACCC11171)中,得到具有胆碱激酶、磷酸胆碱胞苷酸转移酶、胞苷激酶、胞苷酸激酶、多功能核苷二磷酸激酶活性的菌株Escherichia coli CKI-CCT-CMK-NDK-UDK。
实施例2
应用实施例1所述重组菌株制备胞磷胆碱的方法,所述方法以酶为催化剂,以胞苷、氯化胆碱、磷酸二氢钾、磷酸二氢钠为底物,在30L发酵罐中随罐进行酶催化反应,系中的胞苷的浓度为40mM,磷酸二氢钾的浓度为80mM,磷酸二氢钠的浓度为80mM,氯化胆碱的浓度为40mM,温度控制在24℃,搅拌转速根据溶氧而定,反应过程中能通过自动流加氨水维持PH值在7.0.合成胞磷胆碱。
一、菌种活化
1.试管斜面培养:用接种环从甘油保菌管中挑取3-4环划线接种于带有壮观霉素和氨苄青霉素抗性的斜面培养基,于37℃培养箱中培养10-12h,转接1代;
2.茄形瓶斜面培养:用接种环从试管斜面培养基中挑取全部菌体划线接种于带有壮观霉素和氨苄青霉素抗性的茄形瓶培养基中,于37℃培养箱中培养10-12h,转接2代;
二、全细胞催化
1.将茄形瓶中的菌体全部接种至含有壮观霉素和氨苄青霉素抗性的发酵培养基中,初始通气量2L/min,溶氧控制在30%-50%,通过自动流加氨水控制PH在7.0左右,培养温度为35℃;
2.培养OD600=10-12时,添加最终浓度为0.1mMoL/L的IPTG诱导目的蛋白表达添加量为发酵体系的千分之一,调节诱导温度至24℃,诱导培养2-4h,表达关键酶的活性;
3.开始以恒定速率向发酵罐中流加底物,当培养基中的葡萄糖消耗完之后,流加80%(m/v)的葡萄糖溶液,维持发酵培养基中的葡萄糖浓度在0.1-1g/L,直至流加结束,催化周期大约28-30h。
所述斜面培养基为:葡萄糖2g/L,氯化钠2.5g/L,蛋白胨10g/L,酵母浸粉5g/L,磷酸二氢钾1g/L,硫酸镁0.2g/L,琼脂粉25g/L,PH 7.0-7.2;灭菌条件:0.1MPa 30min,灭菌后加壮观霉素和氨苄青霉素20mg/L。
所述发酵培养基为:葡萄糖30g/L,酵母浸粉8g/L,MgSO4·7H2O 0.5g/L,KH2PO43g/L,蛋白胨2g/L,(NH4)2SO4 3g/L,VB1.3.5.12各1mg/L,VH 1mg/L,微量元素混合液1mg/L,壮观霉素20mg/L,氨苄青霉素20mg/L,消泡剂2滴,其余为水。所述微量元素混合液组分含量为:钼酸铵0.28mg/L,硼酸5mg/L,CoCl2·6H2O 1.4mg/L,MnSO4·H2O 0.5mg/L,CuSO4·7H2O0.5mg/L,ZnSO4·7H2O 0.6mg/L,上述成分称量固体后溶解于1L水中,在4℃保存。
实施例3
实施例2中胞磷胆碱的测定
取反应液13000r/min离心2min,取上清液用去离子水稀释100倍,用0.22μm的无菌膜过滤至液相瓶待分析;使用高效液相色谱测定胞磷胆碱的含量,进样量为20μL,色谱柱为JADE-PAK(250×4.6mm)色谱柱,流动相为磷酸盐缓冲液:乙腈=97:3,柱温30℃,流速1mL/min,检测波长271nm,胞磷胆碱保留时间为2.8min。如图2、图3所示,经高效液相色谱检测,全细胞催化反应周期30h,胞磷胆碱产量达到了15g/L,摩尔转化率达到了80%。
以上所述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,本发明菌株的构建步骤不分先后顺序,本技术领域技术人员以本发明的方法或以本方法为基础进行的菌种改造等改进和润饰均视为本发明的保护范围。
Claims (10)
1.一种生产胞磷胆碱的基因工程菌,其特征在于:为重组菌株Escherichia coli CKI-CCT-CMK-NDK-UDK,是由下述方法构建得到的:首先,构建工程菌E.coli CKI-CTT表达来源于酿酒酵母的胆碱激酶和磷酸胆碱胞苷酸转移酶,胆碱激酶能够催化氯化胆碱与磷酸盐合成磷酸胆碱,磷酸胆碱胞苷酸转移酶能够催化磷酸胆碱与三磷酸胞苷合成胞磷胆碱;构建工程菌E coli CMK-NDK表达来源于大肠杆菌自身的胞苷酸激酶和多功能核苷二磷酸激酶;构建工程菌E coli-UDK表达来源于大肠杆菌的胞苷激酶,胞苷激酶能够催化胞苷生成CMP,胞苷酸激酶能够催化CMP生成CDP,多功能核苷二磷酸激酶能够催化CDP生成CTP。
2.权利要求1所述生产胞磷胆碱的基因工程菌的构建方法,其特征在于:利用合成生物学技术在野生型大肠杆菌中表达来源于酿酒酵母的胆碱激酶基因和磷酸胆碱胞苷酸转移酶基因,过表达内源的胞苷激酶、胞苷酸激酶以及多功能核苷二磷酸激酶基因获得可生产胞磷胆碱的大肠杆菌工程菌,步骤包括:
(1)构建工程菌E.coli CKI-CTT表达来源于酿酒酵母的胆碱激酶和磷酸胆碱胞苷酸转移酶,胆碱激酶能够催化氯化胆碱与磷酸盐合成磷酸胆碱,磷酸胆碱胞苷酸转移酶能够催化磷酸胆碱与三磷酸胞苷合成胞磷胆碱;
(2)构建工程菌E.coli CMK-NDK表达来源于大肠杆菌自身的胞苷酸激酶和多功能核苷二磷酸激酶;
(3)构建工程菌E.coli-UDK表达来源于大肠杆菌的胞苷激酶,胞苷激酶能够用于催化胞苷形成CMP,胞苷酸激酶能够催化CMP生成CDP,多功能核苷二磷酸激酶能够催化CDP生成CTP。
3.根据权利要求2所述的生产胞磷胆碱的基因工程菌的构建方法,其特征在于:所述野生型大肠杆菌为大肠杆菌E.coli BL21。
4.根据权利要求2所述的生产胞磷胆碱的基因工程菌的构建方法,其特征在于:以Pstv28质粒为载体在大肠杆菌E.coli BL21中表达异源的胆碱激酶和磷酸胆碱胞苷酸转移酶编码基因,构建出具有胆碱激酶活性和磷酸胆碱胞苷酸转移酶活性的菌株Escherichiacoli CKI-CCT;其中,所述胆碱激酶基因来源于酿酒酵母,其扩增引物具有SEQ ID NO.1、SEQ ID NO.2所示序列;所述磷酸胆碱胞苷酸转移酶基因来源于酿酒酵母,其扩增引物具有SEQ ID NO.3、SEQ ID NO.4所示序列;所述质粒Pstv28具有SEQ ID NO.11所示核苷酸序列。
5.根据权利要求2所述的生产胞磷胆碱的基因工程菌的构建方法,其特征在于:以pTRC99a质粒为载体在大肠杆菌E.coliBL21中表达内源的胞苷酸激酶和多功能核苷二磷酸激酶编码基因,构建出具有胞苷酸激酶和多功能核苷二磷酸激酶活性的菌株Escherichiacoli CKI-CCT-CMK-NDK;其中,所述胞苷酸激酶基因和多功能核苷二磷酸激酶编码基因均来自于大肠杆菌,胞苷酸激酶扩增引物具有SEQ ID NO.5、SEQ ID NO.6所示序列,多功能核苷二磷酸激酶扩增引物具有SEQ ID NO.7、SEQ ID NO.8所示序列;所述质粒pTRC99a具有SEQ ID NO.12所示核苷酸序列。
6.根据权利要求2所述的生产胞磷胆碱的基因工程菌的构建方法,其特征在于:以pTRC99a质粒为载体在大肠杆菌E.coli BL21中表达内源的胞苷激酶,构建出具有胞苷激酶活性的菌株Escherichia coliCKI-CCT-CMK-NDK-UDK;其中,所述胞苷激酶编码基因来自于大肠杆菌,其扩增引物具有SEQ ID NO.9、SEQ ID NO.10所示序列;所述质粒pTRC99a具有SEQ ID NO.12所示核苷酸序列。
7.权利要求1所述生产胞磷胆碱的基因工程菌在制备胞磷胆碱方面的应用。
8.根据权利要求7所述的应用,其特征在于:胞磷胆碱的制备方法为:将所述基因工程菌在培养基中培养,诱导产生高酶活性的胆碱激酶、胞苷激酶、多功能核苷二磷酸激酶和胞苷酸激酶之后,在发酵罐内随罐流加底物,进行全细胞催化,其中,以胆碱激酶、胞苷激酶、多功能核苷二磷酸激酶和胞苷酸激酶为催化剂,以胞苷、磷酸二氢钾、磷酸二氢钠、氯化胆碱为底物,在每30L发酵罐中随罐进行酶催化反应,反应体系中的胞苷的浓度为40mM,磷酸二氢钾的浓度为80mM,磷酸二氢钠的浓度为80mM,氯化胆碱的浓度为40mM,温度控制在24℃,初始转速为200r/min,后期调整转速使溶氧控制在30%-50%,反应过程中通过自动流加氨水维持PH值在7.0.合成胞磷胆碱。
9.根据权利要求7所述的应用,其特征在于:胞磷胆碱的制备包括菌种活化和全细胞催化,具体方法为:
(1)试管斜面培养:用接种环从甘油保菌管中挑取3-4环划线接种于带有壮观霉素和氨苄青霉素抗性的斜面培养基,于37℃培养箱中培养10-12h,转接1代;
(2)茄形瓶斜面培养:用接种环从试管斜面培养基中挑取全部菌体划线接种于带有壮观霉素和氨苄青霉素抗性的茄形瓶培养基中,于37℃培养箱中培养10-12h,转接2代;
(3)将茄形瓶中的菌体全部接种至含有壮观霉素和氨苄青霉素抗性的发酵培养基中,初始通气量2L/min,溶氧控制在30%-50%,通过自动流加氨水控制PH在7.0,培养温度为35℃;
(4)培养OD600=10-12时,添加最终浓度为0.1mMoL/L的IPTG诱导目的蛋白表达添加量为发酵体系的千分之一,调节诱导温度至24℃,诱导培养2-4h,表达关键酶的活性;
(5)开始以恒定速率向发酵罐中流加底物,当培养基中的葡萄糖消耗完之后,流加80%(m/v)的葡萄糖溶液,维持发酵培养基中的葡萄糖浓度在0.1-1g/L,直至流加结束,催化周期大约28-30h。
10.根据权利要求7所述的应用,其特征在于:
所述斜面培养基为:葡萄糖2g/L,氯化钠2.5g/L,蛋白胨10g/L,酵母浸粉5g/L,磷酸二氢钾1g/L,硫酸镁0.2g/L,琼脂粉25g/L,PH7.0-7.2;灭菌条件:0.1MPa 30min,灭菌后加壮观霉素和氨苄青霉素各20mg/L;
所述发酵培养基为:葡萄糖30g/L,酵母浸粉8g/L,MgSO4·7H2O 0.5g/L,KH2PO4 3g/L,蛋白胨2g/L,(NH4)2SO43 g/L,VB1.3.5.12各1mg/L,VH 1mg/L,微量元素混合液1mg/L,壮观霉素20mg/L,氨苄青霉素20mg/L,消泡剂2滴,其余为水;所述微量元素混合液组分含量为:钼酸铵0.28mg/L,硼酸5mg/L,CoCl2·6H2O 1.4mg/L,MnSO4·H2O 0.5mg/L,CuSO4·7H2O 0.5mg/L,ZnSO4·7H2O 0.6mg/L。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104774799A (zh) * | 2015-04-17 | 2015-07-15 | 南京工业大学 | 一株表达胆碱激酶及磷酰胆碱胞苷转移酶的基因工程菌及其构建方法与应用 |
AU2019101117A4 (en) * | 2019-09-26 | 2020-02-20 | Tianjin University Of Science And Technology | Method for the enzymatic production of uridine monophosphate and cytidine monophosphate |
US20200140910A1 (en) * | 2017-07-07 | 2020-05-07 | Suzhou Biosynthetica Co., Ltd | Recombinant microorganism for producing citicoline and method for producing citicoline |
CN111269870A (zh) * | 2020-03-06 | 2020-06-12 | 南京工业大学 | 一种高产胞苷酸的重组大肠杆菌及其应用 |
CN112481233A (zh) * | 2020-10-23 | 2021-03-12 | 天津科技大学 | 制备胞二磷胆碱用酶制剂以及酶催化制备胞二磷胆碱的方法 |
CN114262726A (zh) * | 2022-01-11 | 2022-04-01 | 深圳华酶生物科技有限公司 | 一种利用胞苷酶法合成胞磷胆碱钠的方法 |
-
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Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104774799A (zh) * | 2015-04-17 | 2015-07-15 | 南京工业大学 | 一株表达胆碱激酶及磷酰胆碱胞苷转移酶的基因工程菌及其构建方法与应用 |
US20200140910A1 (en) * | 2017-07-07 | 2020-05-07 | Suzhou Biosynthetica Co., Ltd | Recombinant microorganism for producing citicoline and method for producing citicoline |
AU2019101117A4 (en) * | 2019-09-26 | 2020-02-20 | Tianjin University Of Science And Technology | Method for the enzymatic production of uridine monophosphate and cytidine monophosphate |
CN111269870A (zh) * | 2020-03-06 | 2020-06-12 | 南京工业大学 | 一种高产胞苷酸的重组大肠杆菌及其应用 |
CN112481233A (zh) * | 2020-10-23 | 2021-03-12 | 天津科技大学 | 制备胞二磷胆碱用酶制剂以及酶催化制备胞二磷胆碱的方法 |
CN114262726A (zh) * | 2022-01-11 | 2022-04-01 | 深圳华酶生物科技有限公司 | 一种利用胞苷酶法合成胞磷胆碱钠的方法 |
Non-Patent Citations (1)
Title |
---|
王骏之等: "胞磷胆碱生产过程中的关键酶—胆碱激酶的表达优化", 《中国生物工程学会第六次全国会员代表大会暨第九届学术年会论文集》, 7 November 2015 (2015-11-07), pages 43 - 51 * |
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