CN116515869B - 一种2,4-二氨基丁酸转移酶突变体基因及其应用 - Google Patents
一种2,4-二氨基丁酸转移酶突变体基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种2,4‑二氨基丁酸转移酶突变体基因及其应用,所述基因编码氨基酸序列如SEQ ID NO.2所示的2,4‑二氨基丁酸转移酶突变体。该突变体基因应用于四氢嘧啶的合成,先通过构建2,4‑二氨基丁酸转移酶突变体基因EctBm、2,4‑二氨基丁酸乙酰转移酶EctA和四氢嘧啶合成酶EctC的表达框,共表达2,4‑二氨基丁酸转移酶突变体基因、2,4‑二氨基丁酸乙酰转移酶基因和四氢嘧啶合成酶基因,获得2,4‑二氨基丁酸转移酶突变体、2,4‑二氨基丁酸乙酰转移酶和四氢嘧啶合成酶,在此基础上,以葡萄糖或甘油为碳源,经一步法发酵大肠杆菌高效合成四氢嘧啶即可。该突变体与底物天冬氨酸‑β‑半醛具有较高的结合力,有效提高了四氢嘧啶的合成效率。
Description
技术领域
本发明属于四氢嘧啶的合成领域,尤其涉及一种用于四氢嘧啶合成中的2,4-二氨基丁酸转移酶突变体基因及其应用。
背景技术
四氢嘧啶,又称四氢甲基嘧啶羧酸,是一种氨基酸衍生物。在自然界中,它广泛存在于嗜盐细菌中,在高盐、高温、紫外线辐射等极端条件下,用于保护嗜盐菌免受伤害。鉴于四氢嘧啶具有修护皮肤屏障抵御颗粒污染物侵害、修护及抵抗UV损伤、修复敏感肌及抗刺激、减少光老化及抗衰老等功能,其被广泛应用于化妆品和药品等领域。
在微生物细胞中,四氢嘧啶通过2,4-二氨基丁酸转移酶、2,4-二氨基丁酸乙酰转移酶和四氢嘧啶合成酶等途径酶共同作用催化天冬氨酸--半醛而形成。然而上述3种酶的匹配性问题,尤其是2,4-二氨基丁酸转移酶的低底物亲和力问题是制约四氢嘧啶高效合成的关键因素,从而进一步限制了四氢嘧啶的工业化生产及其应用。
发明内容
发明目的:本发明的第一目的是提供一种2,4-二氨基丁酸转移酶突变体基因,其编码的2,4-二氨基丁酸转移酶突变体具有高底物亲和力;
本发明的第二目的是提供上述2,4-二氨基丁酸转移酶突变体基因的应用,该2,4-二氨基丁酸转移酶突变体基因应用于四氢嘧啶合成中,能够有效提高其与天冬氨酸--半醛的结合力,进而促进了四氢嘧啶的高效合成。
技术方案:
第一方面,本发明提供了一种2,4-二氨基丁酸转移酶突变体基因,所述基因编码氨基酸序列如SEQ ID NO.2所示的2,4-二氨基丁酸转移酶突变体。
作为一种实施方案,所述2,4-二氨基丁酸转移酶突变体基因的核苷酸序列如SEQID NO.1所示。
第二方面,本发明提供了一种2,4-二氨基丁酸转移酶突变体,所述突变体的氨基酸序列如SEQ ID NO.2所示。
第三方面,本发明提供了一种重组表达载体,其包括所述的2,4-二氨基丁酸转移酶突变体基因。
第四方面,本发明提供了一种重组微生物,所述微生物包含所述的2,4-二氨基丁酸转移酶突变体基因,或者表达所述的2,4-二氨基丁酸转移酶突变体。
第五方面,本发明提供了一种四氢嘧啶的制备方法,包括利用所述的2,4-二氨基丁酸转移酶突变体基因、所述的2,4-二氨基丁酸转移酶突变体、所述的重组表达载体、或所述的重组微生物生产四氢嘧啶。
作为一种实施方案,所述的四氢嘧啶的制备方法,包括以下步骤:
(1)在表达载体上串联2,4-二氨基丁酸转移酶突变体表达框,2,4-二氨基丁酸乙酰转移酶表达框及四氢嘧啶合成酶的表达框,构建四氢嘧啶的重组表达载体;
(2)采用宿主细胞转化步骤(1)中的重组表达载体,获得四氢嘧啶的重组菌株,诱导培养,合成四氢嘧啶。
优选的,步骤(1)中,所述重组表达载体的构建方法包括如下步骤:
(1-1)构建携带有2,4-二氨基丁酸转移酶突变体基因、T7启动子和T7终止子的表达框;
(1-2)构建携带有2,4-二氨基丁酸乙酰转移酶、T7启动子和T7终止子的表达框;
(1-3)构建携带有四氢嘧啶合成酶、T7启动子和T7终止子的表达框;
(1-4)在表达载体上,基于同尾酶串联2,4-二氨基丁酸转移酶突变体基因、2,4-二氨基丁酸乙酰转移酶和四氢嘧啶合成酶的表达框构建四氢嘧啶的重组表达载体。
优选的,所述表达载体为pET系列载体,所述宿主细胞包括大肠杆菌、枯草杆菌、酵母、昆虫细胞或杆状病毒。
优选的,步骤(2)中,所述诱导培养是将宿主细胞株活化后,转接到发酵培养基中,于20-37℃条件下进行发酵培养,培养至OD600 nm为0.6-0.8,加入终浓度为0.01-1mM的IPTG,诱导后离心收集上清。
第六方面,本发明提供了所述的2,4-二氨基丁酸转移酶突变体基因、所述的2,4-二氨基丁酸转移酶突变体、所述的重组表达载体、或所述的重组微生物在合成四氢嘧啶中的应用。
有益效果:与现有技术相比,本发明基因编码的2,4-二氨基丁酸转移酶突变体是基于2,4-二氨基丁酸转移酶的晶体结构及底物天冬氨酸-β-半醛的化学结构,利用生物信息学软件分子对接、虚拟突变后获得;该突变体与底物天冬氨酸-β-半醛具有较高的结合力,其应用于四氢嘧啶的合成中,有效提高了四氢嘧啶的合成效率。
附图说明
图1为2,4-二氨基丁酸转移酶突变体和原酶活性表征;
图2为本发明2,4-二氨基丁酸转移酶突变体基因的表达框的构建示意图T7-EctBm-T7ter;
图3为本发明2,4-二氨基丁酸乙酰转移酶基因的表达框的构建示意图T7-EctA-T7ter;
图4为本发明四氢嘧啶合成酶基因的表达框的构建示意图T7-EctC-T7ter;
图5为本发明四氢嘧啶重组表达载体pET28a(+)-T7-EctBm-T7ter-T7-EctA-T7ter-T7-EctC-T7ter的构建示意图;
图6为本发明四氢嘧啶产量检测结果;
图7为本发明不同方式合成四氢嘧啶的成本核算图。
具体实施方式
下面结合附图及实施例对本发明的技术方案做进一步详细说明。需说明的是本发明所采用的宿主细胞、报表的载体、酶类及其他试剂均可购自市售。其中,pET28a(+)的货号为69864-3(Novagen)。
实施例1 2,4-二氨基丁酸转移酶突变体的获得
根据备选基因2,4-二氨基丁酸转移酶的晶体结构(PDB:6RL5)及底物天冬氨酸-β-半醛的化学结构,利用生物信息学软件Discovery Studio分子对接、虚拟突变后,获得与底物天冬氨酸-β-半醛结合力提高的突变体(EctBm)的氨基酸序列如SEQ ID NO.2所示;经密码子优化合成EctBm的核苷酸序列如SEQ ID NO.1(上海生工生物工程有限公司合成)所示。
分别设计引物F1和R1,F2和R2,利用高保真PCR聚合酶Prime Star分别以SEQ IDNO.1和为模版扩增EctBm和EctB基因片段,经Gibbson组装整合到表达载体pET28a(+)质粒(Invitrogen)的BamHⅠ和SacI位点,获得EctBm和EctB的重组表达载体。将上述构建的重组表达载体pET28a(+)-EctBm和pET28a(+)-EctB分别与大肠杆菌E.coli BL21感受态冰上孵育30min后,于42℃热击90s转化E.coli BL21获得表达EctBm和EctB的重组大肠杆菌表达菌株E.coli BL21 pET28a(+)-EctBm和E.coli BL21 pET28a(+)-EctB。
分别挑取重组大肠杆菌表达菌株E.coli BL21 pET28a(+)-EctBm和E.coli BL21pET28a(+)-EctB单菌落,接种含有50mg/L卡那霉素的LB培养基,37℃过夜培养后,按10%(V/V)的比例转接至50mL含有50mg/L卡那霉素的LB培养基中,待菌体培养至OD600=0.6~0.8后,添加终浓度为1mM的IPTG诱导EctBm和EctB的表达。诱导培养5h后8000rpm,4℃离心5min,收集菌体。用预冷的Tris-HCl(pH 7.0)清洗、重悬菌体至50mL,冰浴超声,条件为:400W,工作2s,间歇3s,200个循环至菌液澄清,14000rpm,4℃离心20min,收集上清,即为粗酶液。
将获得的粗酶液经镍柱纯化后,获得EctBm和EctB的纯品。所用程序如下:用Buffer A(20mM Tris-HCl,0.5M NaCl,20mM咪唑,pH 7.4)平衡Ni-NTA Agarose Fast Flow柱子后,将水蛭酪氨酸磺酸转移酶粗酶液1、2分别透过0.22μm滤膜后,上清液以3mL/min的速度通过Ni-NTA Agarose Fast Flow柱子,结合10min后,Buffer B(20mM Tris-HCl,0.5MNaCl,500mM咪唑,pH 7.4)洗脱收集酶活性部分,浓缩,冷冻干燥。
将获得的1mg 2,4-二氨基丁酸转移酶突变体和原酶,加入10mg天冬氨酸-β-半醛和10mg谷氨酸的1mL Tris-HCl(pH7.0)缓冲液中,37℃,反应2min后,加入等体积的甲醇溶液终止反应,并利用HPLC测定其中的谷氨酸的残留量来进行进行2,4-二氨基丁酸转移酶突变体和原酶活性表征,结果表明突变体的比酶活为1.82U/mg,比原酶比酶活(1.47U/mg)高23.8%,其中U代表每分钟催化1mg的谷氨酸降解的酶量(图1)。
实施例2 2,4-二氨基丁酸转移酶突变体基因(EctBm)的表达框的构建
分别设计引物F3和R3,F4和R4,F5和R5,利用高保真PCR聚合酶Prime Star分别以SEQ ID NO.1、pET28a(+)质粒(Invitrogen)、pET28a(+)质粒(Invitrogen)为模版扩增EctBm、T7启动子、T7终止子的基因片段,经Gibbson组装,获得2,4-二氨基丁酸转移酶突变体基因的表达框T7-EctBm-T7ter,如图2所示。
实施例3 2,4-二氨基丁酸乙酰转移酶基因(EctA)的表达框的构建
分别设计引物F6和R6,F4和R4,F5和R5,利用高保真PCR聚合酶Prime Star分别以GenBank:CP032412.1、pET28a(+)质粒(Invitrogen)、pET28a(+)质粒(Invitrogen)为模版扩增EctA、T7启动子、T7终止子的基因片段,经Gibbson组装,获得2,4-二氨基丁酸乙酰转移酶基因的表达框T7-EctA-T7ter,如图3所示。
实施例4四氢嘧啶合成酶基因(EctC)的表达框的构建
分别设计引物F7和R7,F4和R4,F5和R5,利用高保真PCR聚合酶Prime Star分别以GenBank:CP032412.1、pET28a(+)质粒(Invitrogen)、pET28a(+)质粒(Invitrogen)为模版扩增EctC、T7启动子、T7终止子的基因片段,经Gibbson组装,获得四氢嘧啶合成酶基因的表达框T7-EctC-T7ter,如图4所示。
实施例5四氢嘧啶重组表达载体的构建
设计引物F8和R8,利用高保真PCR聚合酶Prime Star,以pET28a(+)质粒(Invitrogen)为模版扩增pET28a(+)质粒片段,pET28a(+),经Gibbson组装,获得重组表达载体pET28a(+)-T7-EctBm-T7ter;并以该载体为模版,经BamH I和Not I酶切后,与基因表达框T7-EctA-T7ter经Bgl II和Not I酶切后的片段,利用T4连接酶进行连接获得重组表达载体pET28a(+)-T7-EctBm-T7ter-T7-EctA-T7ter;并以该载体为模版,经BamH I和Not I酶切后,与基因表达框T7-EctC-T7ter经BglII和Not I酶切后的片段,利用T4连接酶进行连接获得重组表达载体pET28a(+)-T7-EctBm-T7ter-T7-EctA-T7ter-T7-EctC-T7ter(如SEQ ID NO.3所示),如图5所示。
本发明重组表达载体的构建中所采用的引物序列如下表1所示
表1引物序列
实施例6大肠杆菌细胞工厂的构建
将上述构建的重组表达载体pET28a(+)-T7-EctBm-T7ter-T7-EctA-T7ter-T7-EctC-T7ter与大肠杆菌E.coli BL21感受态冰上孵育30min后,于42℃热击90s转化E.coli BL21获得积累四氢嘧啶的重组大肠杆菌表达菌株E.coli BL21pET28a(+)-T7-EctBm-T7ter-T7-EctA-T7ter-T7-EctC-T7ter,已备后续四氢嘧啶的生产。
实施例7大肠杆菌细胞工厂的培养
挑取重组大肠杆菌表达菌株E.coli BL21 pET28a(+)-T7-EctBm-T7ter-T7-EctA-T7ter-T7-EctC-T7ter或对照菌株E.coli BL21 pET28a(+)-T7-EctB-T7ter-T7-EctA-T7ter-T7-EctC-T7ter单菌落,分别接种于3mL LB培养基(加入终浓度为50mg/L的卡那霉素)中,37℃,200rpm培养过夜。按1%(v/v)的比例转接50mL发酵培养基(加入终浓度为50mg/L的卡那霉素)中,37℃,培养至OD600nm为0.6-0.8,加入终浓度为0.1mM的IPTG,37℃,诱导24h后8000rpm,4℃离心5min,收集上清,备测四氢嘧啶含量。
上述所采用的发酵培养基的配方为:每升培养基中含23.6g酵母粉,11.8g蛋白胨,9.4g磷酸氢二钾,2.2g磷酸二氢钾和40g葡萄糖。
检测四氢嘧啶含量测定
利用高效液相色谱-质谱联用(HPLC-MS)检测发酵液上清中的四氢嘧啶含量。所用HPLC-MS的高效液相色谱条件为色谱柱:ZORBAX StableBond Aq4.6×150mm;流动相:2%的甲醇水溶液等度洗脱;流速:1mL/min。检测波长:230nm;其对应的所用质谱条件:离子源:ESI;雾化器流速:1.5L/min;干燥气流速:5L/min;离子源温度:200℃;传输线温度:250℃。
结果表明,以目前的色谱条件,四氢嘧啶的出峰时间为3.5min,实施例7中四氢嘧啶的浓度为72.33±1.85g/L,对照样品中四氢嘧啶的浓度为55.67±2.34g/L(图6)。据此得到大肠杆菌发酵生产四氢嘧啶的成本约为182元/kg,对照样品的成本为791.30元/kg,比对照样品低23%(图7)。
除上述实施例外,本发明所采用的发酵培养基的配方可为:每升培养基中含5-40g酵母粉,5-20g蛋白胨,2-15g磷酸氢二钾,0.5-6g磷酸二氢钾和1-8mL甘油。也可选用:每升培养基中含5-40g酵母粉,5-20g蛋白胨,2-15g磷酸氢二钾,0.5-6g磷酸二氢钾和20-100g葡萄糖。
此外,在诱导培养时,将大肠杆菌工程菌株活化后,转接到发酵培养基中,可于20-37℃条件下进行发酵培养,培养至OD600 nm为0.6-0.8,加入终浓度为0.01-1mM的IPTG,诱导后离心收集菌体。
Claims (9)
1.一种2,4-二氨基丁酸转移酶突变体基因,其特征在于,所述基因编码氨基酸序列如SEQ ID NO. 2所示的2,4-二氨基丁酸转移酶突变体。
2.根据权利要求1所述的2,4-二氨基丁酸转移酶突变体基因,其特征在于,所述2,4-二氨基丁酸转移酶突变体基因的核苷酸序列如SEQ ID NO. 1所示。
3.一种2,4-二氨基丁酸转移酶突变体,其特征在于,所述突变体的氨基酸序列如SEQID NO. 2所示。
4.一种重组表达载体,其特征在于,包括权利要求1或2所述的2,4-二氨基丁酸转移酶突变体基因。
5.一种重组微生物,其特征在于,所述微生物包含权利要求1或2所述的2,4-二氨基丁酸转移酶突变体基因,或者表达权利要求3所述的2,4-二氨基丁酸转移酶突变体。
6.一种四氢嘧啶的制备方法,其特征在于,包括利用权利要求1或2所述的2,4-二氨基丁酸转移酶突变体基因、权利要求3所述的2,4-二氨基丁酸转移酶突变体、权利要求4所述的重组表达载体、或权利要求5所述的重组微生物生产四氢嘧啶;所述制备方法包括以下步骤:
(1)在表达载体上串联2,4-二氨基丁酸转移酶突变体表达框,2,4-二氨基丁酸乙酰转移酶表达框及四氢嘧啶合成酶的表达框,构建四氢嘧啶的重组表达载体;所述2,4-二氨基丁酸转移酶突变体表达框为携带有2,4-二氨基丁酸转移酶突变体基因、启动子和终止子的表达框;所述2,4-二氨基丁酸乙酰转移酶表达框为携带有2,4-二氨基丁酸乙酰转移酶基因、启动子和终止子的表达框,所述2,4-二氨基丁酸乙酰转移酶基因来源于GenBank:CP032412.1;所述四氢嘧啶合成酶的表达框为携带有四氢嘧啶合成酶基因、启动子和终止子的表达框,所述四氢嘧啶合成酶基因来源于GenBank: CP032412.1;
(2)采用宿主细胞转化步骤(1)中的重组表达载体,获得四氢嘧啶的重组菌株,诱导培养,合成四氢嘧啶。
7.根据权利要求6所述的四氢嘧啶的制备方法,其特征在于,步骤(1)中,所述重组表达载体的构建方法包括如下步骤:
(1-1)构建携带有2,4-二氨基丁酸转移酶突变体基因、T7启动子和T7终止子的表达框;
(1-2)构建携带有2,4-二氨基丁酸乙酰转移酶基因、T7启动子和T7终止子的表达框;
(1-3)构建携带有四氢嘧啶合成酶基因、T7启动子和T7终止子的表达框;
(1-4)在表达载体上,基于同尾酶串联2,4-二氨基丁酸转移酶突变体基因、2,4-二氨基丁酸乙酰转移酶和四氢嘧啶合成酶的表达框构建四氢嘧啶的重组表达载体。
8.根据权利要求6所述的四氢嘧啶的制备方法,其特征在于,所述表达载体为pET系列载体,所述宿主细胞包括大肠杆菌或枯草杆菌;步骤(2)中,所述诱导培养是将宿主细胞株活化后,转接到发酵培养基中,于20-37℃条件下进行发酵培养,培养至OD600 nm为0.6-0.8,加入终浓度为0.01-1 mM的IPTG,诱导后离心收集上清。
9.权利要求1或2所述的2,4-二氨基丁酸转移酶突变体基因、权利要求3所述的2,4-二氨基丁酸转移酶突变体、权利要求4所述的重组表达载体、或权利要求5所述的重组微生物在合成四氢嘧啶中的应用。
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