CN110343728B - 一种生物转化合成六氢哒嗪-3-羧酸的方法 - Google Patents
一种生物转化合成六氢哒嗪-3-羧酸的方法 Download PDFInfo
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- CN110343728B CN110343728B CN201910482492.5A CN201910482492A CN110343728B CN 110343728 B CN110343728 B CN 110343728B CN 201910482492 A CN201910482492 A CN 201910482492A CN 110343728 B CN110343728 B CN 110343728B
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Abstract
本发明公开了一种生物转化合成六氢哒嗪‑3‑羧酸的方法，属于生物技术领域。所述方法包括：(1)构建含有L‑鸟氨酸羟化酶编码基因和L‑哌嗪酸合成酶编码基因的重组质粒；(2)通过基因敲除构建鸟氨酸转氨甲酰酶编码基因argI缺失的大肠杆菌基因工程菌；(3)将步骤(1)构建的重组质粒转化入步骤(2)改造后的大肠杆菌基因工程菌中，得到重组基因工程菌；(4)诱导培养重组基因工程菌，收集菌体，以菌体全细胞或细胞破碎后的粗酶液作为催化剂，以L‑鸟氨酸为底物，经生物转化反应，制备得到六氢哒嗪‑3‑羧酸。生物催化方法立体选择特异性高，底物转化率高，反应条件温和易控，符合绿色环保的要求。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种生物转化合成六氢哒嗪-3-羧酸的方法。
背景技术
六氢哒嗪-3-羧酸(hexahydropyridazine-3-carboxylic acid，L-piperazicacid)及其结构衍生类化合物是一类独特的含有氮-氮(N-N)键连接的非蛋白质氨基酸。六氢哒嗪-3-羧酸存在于很多具有显著生物活性的天然产物中，例如天然免疫抑制剂萨菲菌素(sanglifehrin A)和抗肿瘤活性肽吕宋肽菌素(luzopeptins)中都含有此结构单元。此外，六氢哒嗪-3-羧酸结构上具有的刚性构象，使得它能够作为结构单元在肽类合成中引入β-转角(β-turns)结构。这些独特的性质激发了合成化学家致力于开发六氢哒嗪-3-羧酸的化学合成途径，将其作为重要的药物和化学品合成中间体。因此研究此化合物的高效合成具有重要及广泛的意义。
在过去的几年中，六氢哒嗪-3-羧酸的纯立体选择特异性合成以及在大分子化学结构单元中的六氢哒嗪-3-羧酸结构单元受到了越来越多的关注。比如Oelke等总结了含有六氢哒嗪-3-羧酸的天然产物分离，生物学相关性以及化学合成的信息。
六氢哒嗪-3-羧酸可以通过两种方法合成，其一以手性化合物为起始原料，比如L-(+)谷氨酸，经九步合成得到此化合物，这种方法线路长，成本高，工艺复杂，反应难度大，且总收率较低(不足30％)；第二种方法是用非手性的化合物为原料，经Diels-Alder反应，虽然此过程相对比较简单，收率也高，但是所得的产物为外消旋，需要经过化学拆分，且一般化学拆分剂都比较昂贵。
专利WO2001/083458中报道了六氢哒嗪-3-羧酸化学合成的另外一个方法。首先将D-谷氨酸转化为(R)-2,5-二羟基戊酸酯，再将羟基转化为合适的离去基团如甲磺酸酯后，用双保护的肼处理酯，得到所需的(3S)-哌嗪酸衍生物，再通过精制得到六氢哒嗪-3-羧酸。
生物转化的方法可以克服上述缺点，通常生物催化反应立体选择特异性高，底物转化率高，反应速率快，且反应条件温和易控，成本也更低。因此，如何利用生物方法制备六氢哒嗪-3-羧酸是本领域技术人员需要解决的问题。
发明内容
本发明的目的在于提供一种生物转化合成六氢哒嗪-3-羧酸的方法，利用含有胞内表达L-鸟氨酸羟化酶和L-哌嗪酸合成酶的大肠杆菌基因工程菌全细胞或细胞破碎后的粗酶液催化L-鸟氨酸制备六氢哒嗪-3-羧酸，该方法立体选择特异性高、工艺简单、成本低廉且操作简便，以解决化学生产方法带来的立体选择性低、污染严重、工艺复杂、成本高、收率低等问题。
为实现上述目的，本发明采用如下技术方案：
一种生物转化合成六氢哒嗪-3-羧酸的方法，包括以下步骤：
(1)构建含有L-鸟氨酸羟化酶编码基因和L-哌嗪酸合成酶编码基因的重组质粒；
(2)通过基因敲除构建鸟氨酸转氨甲酰酶编码基因argI缺失的大肠杆菌基因工程菌；
(3)将步骤(1)构建的重组质粒转化入步骤(2)改造后的大肠杆菌基因工程菌中，得到重组基因工程菌；
(4)诱导培养重组基因工程菌，收集菌体，以菌体全细胞或细胞破碎后的粗酶液作为催化剂，以L-鸟氨酸为底物，经生物转化反应，制备得到六氢哒嗪-3-羧酸。
步骤(1)中，构建包含催化L-鸟氨酸到六氢哒嗪-3-羧酸的关键酶(L-鸟氨酸羟化酶和L-哌嗪酸合成酶)编码基因的重组表达质粒。
所述的L-鸟氨酸羟化酶，以黄素腺嘌呤二核苷酸(FAD)为辅因子，利用氧气和还原型烟酰胺腺嘌呤二核苷酸(NADH)或还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)催化L-鸟氨酸转化为L-N5-羟化鸟氨酸。所述L-鸟氨酸羟化酶的氨基酸序列为SEQ ID NO.1所示。
任何对SEQ ID NO.1所示氨基酸序列中的氨基酸进行缺失、插入或替换的处理获得的多肽片段或其变体，只要其与SEQ ID NO.1所示氨基酸序列具有50％以上同源性且具有L-鸟氨酸羟化酶功能的，均属于本发明的保护范围。
所述的L-哌嗪酸合成酶，以血红素(heme b)为辅因子，可催化L-N5-羟基鸟氨酸生成六氢哒嗪-3-羧酸。所述L-哌嗪酸合成酶的氨基酸序列为SEQ ID NO.2所示。任何对SEQID NO.2所示氨基酸序列中的氨基酸进行缺失、插入或替换的处理获得的多肽片段或其变体，只要其与SEQ ID NO.2所示氨基酸序列具有50％以上同源性且具有L-哌嗪酸合成酶功能的，均属于本发明的保护范围。
将编码上述L-鸟氨酸羟化酶和L-哌嗪酸合成酶的基因片段连接到表达质粒中制得重组质粒。表达质粒可采用在大肠杆菌中诱导表达外源基因的表达质粒。
作为优选，步骤(1)中，所述重组质粒的原始载体为表达质粒pETDuet-1。
步骤(2)中，采用基因敲除技术敲除宿主大肠杆菌基因组中鸟氨酸转氨甲酰酶编码基因argI，使其自身不消耗底物L-鸟氨酸。
作为优选，所述基因敲除的方法采用red重组技术。
所述宿主菌为大肠杆菌E.coli BL21。
步骤(3)中，将上述重组质粒导入鸟氨酸转氨甲酰酶编码基因argI缺失的宿主细胞中，构建含有L-鸟氨酸羟化酶和L-哌嗪酸合成酶编码基因的重组基因工程菌。
步骤(4)中，利用诱导表达了L-鸟氨酸羟化酶和L-哌嗪酸合成酶的工程菌全细胞或细胞破碎液作为催化剂，催化L-鸟氨酸生成六氢哒嗪-3-羧酸。
所述诱导培养包括：将重组基因工程菌接种至含有抗生素的LB液体培养基中，37℃振荡培养8-12h，再以1％接种量接种至新鲜的含有抗生素的LB液体培养基中，37℃振荡培养至菌体浓度OD600＝0.6-0.8，然后向培养液中加入终浓度0.1mM的IPTG，30℃诱导培养8-10h。
所述生物转化反应包括：以LB培养基为反应介质，每200mL中，加入1-10g湿菌体全细胞或全细胞破碎后的粗酶液，底物L-鸟氨酸的质量百分比浓度为0.2-1％，在15～50℃、150rpm～250rpm条件下反应3-15h。
作为优选，所述生物转化反应包括：以LB培养基为反应介质，每200mL中，加入10g湿菌体全细胞或全细胞破碎后的粗酶液，底物L-鸟氨酸的质量百分比浓度为0.2％，在30℃、200rpm条件下反应10h。
本发明的另一个目的是提供一种用于生物转化合成六氢哒嗪-3-羧酸的重组基因工程菌，所述重组基因工程菌中含有L-鸟氨酸羟化酶编码基因和L-哌嗪酸合成酶编码基因。
所述L-鸟氨酸羟化酶的氨基酸序列如SEQ ID NO.1所示，所述L-哌嗪酸合成酶的氨基酸序列如SEQ ID NO.2所示。
与现有技术相比，本发明具备的有益效果：
本发明提供一种生物转化合成六氢哒嗪-3-羧酸的方法，利用全细胞或全细胞破碎后的粗酶液催化L-鸟氨酸制备六氢哒嗪-3-羧酸，生物催化方法立体选择特异性高，底物转化率高，反应过程中也不需要用到贵重金属或易燃易爆的试剂，反应条件温和易控，解决了六氢哒嗪-3-羧酸现有化学生产方法带来的污染严重、工艺复杂、成本高、得率低等问题，生物法生产具有良好的技术应用和产业化前景，符合绿色环保的要求。
附图说明
图1为pETDuet-KtzI-KtzT重组质粒构建示意图。
图2为大肠杆菌E.coli BL21-ΔargI构建示意图。
图3为转化产物高效液相色谱检测结果。
图4为转化产物液质联用检测结果。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
以下实施例所用主要实验材料来源为：
大肠杆菌宿主菌BL21(DE3)、大肠杆菌表达质粒pETDuet-1、pIJ773质粒、pKD46质粒均为本实验室保存，也可以购买得到。
T4DNA连接酶、限制性内切酶(NcoI、HindIII、NdeI和XhoI)、DNA聚合酶购自大连宝生物有限公司。
质粒DNA抽提试剂盒、PCR纯化试剂盒购自TSINGKE公司。
异丙基-β-D-硫代吡喃半乳糖苷(IPTG)、氨苄青霉素、阿普拉霉素购自Sangon公司。其他所有试剂均为国产或进口分析纯试剂。
实施例中LB平板培养基组成为(g/L)：蛋白胨10，酵母粉5，氯化钠10，琼脂20，溶于水。
LB液体培养基组成为(g/L)：蛋白胨10，酵母粉5，氯化钠10，溶于水。
实施例1
构建含有L-鸟氨酸羟化酶编码基因ktzI和L-哌嗪酸合成酶编码基因ktzT的重组质粒
根据L-鸟氨酸羟化酶和L-哌嗪酸合成酶的氨基酸序列，分别如SEQ ID NO.1和SEQID NO.2所示，并依据大肠杆菌偏好的密码子进行密码子优化，根据表达载体pETDuet-1的特点，设计PCR引物，在L-鸟氨酸羟化酶编码基因ktzI两端设计酶切位点NcoI和HindIII，在L-哌嗪酸合成酶编码基因ktzT两端设计酶切位点NdeI和XhoI，通过基因工程的常规操作以全合成的方式合成KtzI和KtzT基因DNA片段。
用NcoI和HindIII限制性内切酶酶切ktzI基因DNA片段和表达质粒pETDuet-1，经PCR纯化试剂盒纯化回收后，用T4DNA连接酶连接，构建得到重组表达质粒pETDuet-ktzI。然后用NdeI和XhoI限制性内切酶酶切ktzT基因DNA片段和重组表达质粒pETDuet-ktzI，经PCR纯化试剂盒纯化回收后，用T4DNA连接酶连接，从而构建得到胞内重组表达质粒pETDuet-ktzI-ktzT。
实施例2
构建自身不消耗底物L-鸟氨酸的宿主大肠杆菌E.coli BL21-ΔargI
根据鸟氨酸氨甲酰转移酶编码基因argI(NCBI数据库编号:AJH11494)在大肠杆菌E.coli BL21(DE3)基因组上的位置，设计argI两端同源臂的片段，利用含有阿普拉霉素抗性标记的pIJ773质粒为模板，PCR获得含有阿普拉霉素抗性标记的同源重组片段，通过常规的大肠杆菌red重组方法，将鸟氨酸氨甲酰转移酶编码基因argI替换成阿普拉霉素抗性标记，涂布于含有阿普拉霉素(终浓度为50μg/ml)的LB固体培养基平板上，于37℃培养过夜，第二天平板上长出的菌落中随机挑取克隆并抽提质粒，用鉴定引物进行测序，
argI1:5’-GGCACACTTATTGTTAGTCCCAG-3’
argI2:5’-ATCCTATCCTTTTGGCCTCTGGA-3’。
筛选获得敲除成功的宿主大肠杆菌E.coli BL21-ΔargI。
实施例3
构建含有L-鸟氨酸羟化酶和L-哌嗪酸合成酶编码基因的基因工程菌
将实施例1中构建的重组质粒pETDuet-ktzI-ktzT转化至实施例2中构建的E.coliBL21-ΔargI宿主菌中，涂布于含有氨苄青霉素(终浓度为50μg/ml)的LB固体培养基平板上，于37℃培养过夜，构建基因工程菌E.coli BL21-ΔargI/pETDuet-ktzI-ktzT。
实施例4
含有L-鸟氨酸羟化酶和L-哌嗪酸合成酶的基因工程菌全细胞的制备
将实施例3中构建的重组基因工程菌E.coli BL21-ΔargI/pETDuet-ktzI-ktzT接种至含有50μg/ml氨苄青霉素的LB液体培养基中，37℃振荡培养8-12h，再以1％接种量(v/v)接种至新鲜的含有50μg/ml氨苄青霉素的LB液体培养基中，37℃振荡培养至菌体浓度OD600约0.6左右，再向LB液体培养基中加入终浓度0.1mM的IPTG，30℃诱导培养8-10h后，将培养液在4℃，10000rpm离心10min，弃去上清液，收集湿菌体，即为含有胞内表达重组L-鸟氨酸羟化酶和L-哌嗪酸合成酶的大肠杆菌E.coli BL21-ΔargI/pETDuet-ktzI-ktzT湿菌体。
实施例5
生物转化制备六氢哒嗪-3-羧酸
以实施例4中获得的含有L-鸟氨酸羟化酶和L-哌嗪酸合成酶的大肠杆菌E.coliBL21-ΔargI/pETDuet-ktzI-ktzT湿菌体全细胞或全细胞破碎后的粗酶液为催化剂，以L-鸟氨酸为底物，进行生物转化制备六氢哒嗪-3-羧酸。
转化体系及转化操作如下：在200ml LB中，加入10g湿菌体全细胞或全细胞破碎后的粗酶液，底物L-鸟氨酸0.2％(w/v)，30℃、200rpm条件下反应10h，取样离心(12000rpm，5min)，吸取上清，进行芴酰氯(Fmoc-Cl)衍生后供HPLC分析检测或直接用液质联用检测，如图3和图4所示，证实催化产物为六氢哒嗪-3-羧酸(质谱信号:m/z 131)。
总之，以上所述仅作为本发明的较佳实施例，凡依据本发明专利范围所做的均等变化和修饰，皆应属于本发明专利的涵盖范围。
序列表
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<120> 一种生物转化合成六氢哒嗪-3-羧酸的方法
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Claims (3)
1.一种生物转化合成六氢哒嗪-3-羧酸的方法，其特征在于，包括以下步骤：
(1)构建含有L-鸟氨酸羟化酶编码基因和L-哌嗪酸合成酶编码基因的重组质粒，所述重组质粒的原始载体为表达质粒pETDuet-1，所述L-鸟氨酸羟化酶的氨基酸序列如SEQ IDNO.1所示，所述L-哌嗪酸合成酶的氨基酸序列如SEQ ID NO.2所示；
(2)通过基因敲除构建鸟氨酸转氨甲酰酶编码基因argI缺失的大肠杆菌基因工程菌，宿主菌为大肠杆菌E.coliBL21；
(3)将步骤(1)构建的重组质粒转化入步骤(2)改造后的大肠杆菌基因工程菌中，得到重组基因工程菌；
(4)诱导培养重组基因工程菌，收集菌体，以LB培养基为反应介质，每200mL中，加入10g湿菌体全细胞作为催化剂，以L-鸟氨酸为底物，质量百分比浓度为0.2％，30℃、200rpm条件下反应10h，制备得到六氢哒嗪-3-羧酸。
2.如权利要求1所述的生物转化合成六氢哒嗪-3-羧酸的方法，其特征在于，步骤(2)中，所述基因敲除的方法采用red重组技术。
3.如权利要求1所述的生物转化合成六氢哒嗪-3-羧酸的方法，其特征在于，步骤(4)中，所述诱导培养包括：将重组基因工程菌接种至含有抗生素的LB液体培养基中，37℃振荡培养8-12h，再以1％接种量接种至新鲜的含有抗生素的LB液体培养基中，37℃振荡培养至菌体浓度OD600＝0.6-0.8，然后向培养液中加入终浓度0.1mM的IPTG，30℃诱导培养8-10h。
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