CN117384813A - 依克多因合成菌株及其构建方法和应用 - Google Patents
依克多因合成菌株及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及依克多因合成菌株及其构建方法和应用，该菌株通过在宿主菌中表达ectABC基因簇获得；所述宿主菌为大肠杆菌（Escherichia coli）MG1655；所述ectABC基因簇的核苷酸序列如SEQ ID NO：1所示。本发明的菌株能够使用低价氮源高效合成依克多因，依克多因产量高并且所得发酵液中杂质较少。
Description
技术领域
本发明属于微生物技术领域，涉及依克多因合成菌株及其构建方法和应用。
背景技术
依克多因，又称四氢嘧啶，是环状氨基酸的衍生物，是一种重要的相容性溶质。依克多因是耐盐微生物中最广泛的相容性溶质，可以帮助生物抵抗高渗透压的胁迫。在已知的相容溶质，例如海藻糖、谷氨酸和甜菜碱中，四氢嘧啶具有很强的细胞保护能力。
因其具有长效保湿作用，被广泛应用于化妆品、医药等领域。依克多因是一种比甘油更有效的长效保湿剂，可以通过多种方式减少紫外线和可见光对细胞的伤害，它能够保护皮肤免受氧化损伤，对抗皮肤老化。它也是一种优良的生物功能稳定剂、皮肤保护剂和潜在药物。由于四氢嘧啶在化妆品、医药等领域的广泛应用，每年依克多因的需求量约为1.5万吨，市场规模达数十亿美元，零售价约为1000美元/公斤。
目前依克多因的发酵工艺中，有使用野生菌发酵和工程改造菌两种路径。野生菌发酵一般需要高盐环境，对发酵设备的腐蚀较严重，而且产量较低。以大肠杆菌和谷氨酸棒杆菌为主的工程改造菌已经可以达到较高的产量。对于依克多因发酵，发酵的成本中培养基成本和分离成本都占有不小的比例，而且分离的难易程度会影响得率。目前，大多数报道的培养基需要添加酵母粉、天冬氨酸等高价或者复杂的成分作为氮源，较高的培养基价格和分离成本限制了工业生产的成本。因此目前需要一种能够用成本较低的原料高产依克多因，且杂质较少利于分离的依克多因合成菌株及发酵方法。
发明内容
本发明的目的第一目的在于提供一株依克多因合成菌株。
为实现上述技术目的，本发明采用如下技术方案：
一株依克多因合成菌株，通过在宿主菌中表达ectABC基因簇获得；所述宿主菌为大肠杆菌(Escherichia coli)MG1655；所述ectABC基因簇的核苷酸序列如SEQ ID NO：1所示。
本发明的第二目的在于提供上述述菌株的构建方法，包括：
扩增ectABC基因簇基因序列，以酶切一步克隆方式插入载体质粒，获得表达质粒；
将表达质粒导入宿主菌中，获得所述依克多因合成菌株。
作为一种优选的实施方式，所述载体质粒选用pTrc99a质粒。
作为一种优选的实施方式，使用如下引物扩增ectABC基因簇基因序列：
ectAF：5’-CATGGAATTCGAGCTCGGTACCATGAGCACGCCAATAATACC-3’
ectCR：5’-CAGGTCGACTCTAGAGGATCCCTCACCAGTAGGTGCGGCG-3’。
本发明的第三目的在于提供上述菌株在发酵生产依克多因中的应用。
作为一种优选的实施方式，所述菌株以氯化铵为唯一氮源发酵生产依克多因。
作为一种优选的实施方式，菌株发酵的碳源为葡萄糖、果糖或甘油。
作为一种优选的实施方式，菌株发酵的发酵培养基组成为：碳源20-30g/L、氮源6g/L、柠檬酸钠二水合物1g/L，磷酸二氢钾2g/L，硫酸镁1.2g/L，硫酸铁0.1g/L，硫酸锰0.1g/L。
作为一种优选的实施方式，使用发酵罐发酵时，通过分批补料补加碳源，保持碳源浓度不超过20g/L；或通过流加补料补加碳源，保持碳源浓度在1-5g/L。
作为一种优选的实施方式，使用该菌株发酵产依克多因的方式如下：
将所述依克多因合成菌株划线至固体LB培养基，在35-39℃下培养8-16h；
挑取培养的菌种，接种至液体LB培养基，在35-39℃下培养8-16h，获得种子液；
将种子液接种至发酵培养基，加入氨苄青霉素和IPTG诱导剂，在35-39℃下发酵培养36-60h。
本发明具有如下有益效果：
(1)本发明构建了一株可以使用廉价无机氮源生产依克多因的大肠杆菌，该大肠杆菌在以廉价无机氮源氯化铵为唯一氮源的培养基上可获得最高产量，且发酵液杂质含量较低，提高了发酵效率，降低了分离的难度，能够为工业化生产带来巨大的经济效益。
(2)本发明中使用优化的发酵培养基进行分批补料或流加补料发酵，最高可以达到64.66g/L的依克多因产量，产率约为0.20-0.24g/g葡萄糖，且发酵总用时52h，发酵时间短，为菌株工业化合成依克多因提供有利条件。
附图说明
图1是实施例3、8的液相色谱检测结果对比。
图2是实施例31的发酵结果。
具体实施方式
下面结合附图说明和具体实施方式对本发明的技术方案作进一步阐述。
实施例中依克多因检测方法如下：
每隔4-12h取2mL样品，取一部分并稀释至合适倍数，使用分光光度计检测600nm处的吸光度以体现菌体量。其余样品在12000rpm下离心2min，吸取上清液，一部分使用生物传感器分析仪检测剩余糖浓度；一部分稀释到合适的倍数后用0.22微米孔径的滤膜过滤，并加入液相样品瓶中，使用高效液相色谱法(HPLC)进行检测：液相色谱使用C18色谱柱，流动相为20％乙腈，80％水。通过与标准样品的峰面积对比算得依克多因浓度。
实施例1
本发明所述的依克多因合成菌株的构建方法为：
选择酶切位点KpnI和BamHI，使用KpnI和BamHI核酸内切酶对pTrc99a质粒按照说明书进行酶切线性化。使用如下引物，对菌株Halomonas sp.或者基因合成所得的序列(序列SEQ ID NO：2)进行PCR扩增，获得依克多因合成基因簇ectABC：
ectAF：5’-CATGGAATTCGAGCTCGGTACCATGAGCACGCCAATAATACC-3’
ectCR：5’-CAGGTCGACTCTAGAGGATCCCTCACCAGTAGGTGCGGCG-3’
使用诺维赞公司的ClonExpress IIOne Step Cloning Kit一步克隆试剂盒，按照说明书通过一步克隆的方法连接线性化所得的质粒与PCR扩增所得的片段，得到质粒pTrc99a-ectABC。
在冰上将10μL所得pTrc99a-ectABC质粒加入自制或购买的100μL大肠杆菌(Escherichia coli)MG1655的化学感受态细胞中混匀，静置30min后放入42℃水浴锅热激45秒，然后冰上冷却2min，加入800μL LB培养基(酵母粉5g/L、蛋白胨10g/L、氯化钠10g/L。115℃灭菌20min)，放入37℃摇床复苏1h。取100μL涂至在37℃培养箱预热的有氨苄青霉素的LB固体平板中(酵母粉5g/L、蛋白胨10g/L、氯化钠10g/L、15g/L琼脂。115℃灭菌20min)，放入37℃培养箱过夜生长，筛选并测序得到的阳性菌株，确认基因序列正确后得到发酵使用的工程菌株，命名为Fect1。
实施例2
本实施例中对菌株进行发酵培养，步骤如下：
(1)平板培养：将菌株Fect1使用划线接种至LB固体培养基(酵母粉5g/L、蛋白胨10g/L、氯化钠10g/L、15g/L琼脂。115℃灭菌20min)培养，培养温度37℃，培养时间12-18h；
(2)种子培养：从保藏有菌种的平板中挑取或者从保种管取菌种，接种于5mL LB培养基的试管中，放入37℃摇床中，180rpm培养12h作为种子液；
(3)摇瓶发酵：将活化好的种子液接入摇瓶中，接种量为1％(v/v)。发酵培养基为葡萄糖20g/L，酵母粉2g/L，蛋白胨4g/L，柠檬酸钠二水合物1g/L，磷酸二氢钾2g/L，硫酸镁0.7g/L，硫酸铁0.1g/L，硫酸锰0.1g/L，其中，酵母粉和蛋白胨作为氮源。放入37℃摇床中，180rpm发酵48h。4h时加入IPTG 0.1mmol/L。每12h加入氨水调整pH至7。
实施例3-9
实施例3-9研究了不同氮源对发酵结果的影响(实施例3-9为同批次发酵)，结果如表1所示：
表1实施例2-9的发酵结果
其中，实施例8、9为纯无机氮源，实施例3、8的液相色谱检测结果对比如图1所示，可以看出，使用纯无机氮源的情况下，产物中杂质非常少。
实施例10-15
实施例10-15研究了不同浓度氯化铵氮源对依克多因发酵的影响，除发酵培养基中氮源成分外(实施例10-15选用氯化铵作为唯一氮源)，其余步骤与实施例2相同(实施例10-15为同批次发酵，与实施例3-9为不同发酵批次)。结果如表2所示：
表2实施例10-15的发酵结果
可以看出，氯化铵在6g/L时依多克因达到最大产量。
实施例16-22
实施例16-22研究了不同碳源对依克多因发酵的影响，除发酵培养基中碳源成分外，其余步骤与实施例8相同(实施例16-22为同批次发酵)。碳源种类对依克多因发酵的影响如表3所示：
表3实施例16-22的发酵结果
考虑到葡萄糖与甘油结果差距较小以及甘油具有较高的成本，选择葡萄糖为碳源。
实施例23-28
实施例23-28与实施例16的不同之处仅在于，使用了不同浓度的硫酸镁(实施例23-28为同批次发酵)，表4列出了硫酸镁浓度对依克多因发酵的影响：
表4实施例23-28的发酵结果
实施例29-31
实施例29-31研究了不同碳源补料策略对依克多因发酵的影响，使用之前优化所得条件，进行发酵罐实验，以验证条件的产量。
(1)平板培养：将菌株Fect1划线至LB固体培养基培养，培养温度37℃，培养时间12-18h；
(2)种子培养：从保藏有菌种的平板中挑点或者从保种管取菌种，接种于5mL LB培养基的试管中，放入37℃摇床中，180rpm培养12h作为种子液；
(3)发酵罐培养：将活化好的种子液接入发酵罐中，接种量为10％(v/v)，搅拌速度为600-800rpm，控制溶氧在40％，37℃发酵培养，用氨水全程控pH为7.0。发酵培养基为氯化铵6g/L，柠檬酸钠二水合物1g/L，磷酸二氢钾2g/L，硫酸镁1.2g/L，硫酸铁0.1g/L，硫酸锰0.1g/L。开始发酵时加入0.1mg/L氨苄青霉素。初始葡萄糖20g/L，葡萄糖耗完时开始补糖。OD600达到5时加入IPTG 0.1mmol/L。一共发酵58h，从图中可以看出发酵52h后已达到最大产量。
表5实施例29-31的发酵结果
实施例31的结果如图2所示，在发酵52h时已基本达到最大产量，大肠杆菌OD600可达107.1，依克多因产量可达64.66g/L。此结果远高于现有使用低成本培养基的发酵结果，进一步提高了生产效率，可以为工程改造的大肠杆菌生产依克多因的工业化生产带来可观的经济效益。
Claims (10)
1.一株依克多因合成菌株，其特征在于，通过在宿主菌中表达ectABC基因簇获得；所述宿主菌为大肠杆菌（Escherichia coli）MG1655；所述ectABC基因簇的核苷酸序列如SEQ IDNO：1所示。
2.权利要求1所述菌株的构建方法，其特征在于，包括：
扩增ectABC基因簇基因序列，以酶切一步克隆方式插入载体质粒，获得表达质粒；
将表达质粒导入宿主菌中，获得所述依克多因合成菌株。
3.根据权利要求2所述的构建方法，其特征在于，所述载体质粒选用pTrc99a质粒。
4. 根据权利要求2所述的构建方法，其特征在于，使用如下引物扩增ectABC基因簇基因序列：
ectAF：5’- CATGGAATTCGAGCTCGGTACCATGAGCACGCCAATAATACC-3’
ectCR：5’- CAGGTCGACTCTAGAGGATCCCTCACCAGTAGGTGCGGCG-3’。
5.权利要求1所述菌株在发酵生产依克多因中的应用。
6.根据权利要求5所述的应用，其特征在于，所述菌株以氯化铵为唯一氮源发酵生产依克多因。
7.根据权利要求5所述的应用，其特征在于，菌株发酵的碳源为葡萄糖、果糖或甘油。
8. 根据权利要求5所述的应用，其特征在于，菌株发酵的发酵培养基组成为：碳源20-30g/L、氮源6g/L、柠檬酸钠二水合物1 g/L，磷酸二氢钾2 g/L，硫酸镁1.2 g/L，硫酸铁0.1g/L，硫酸锰0.1 g/L。
9.根据权利要求5所述的应用，其特征在于，使用发酵罐发酵时，通过分批补料补加碳源，保持碳源浓度不超过20g/L；或通过流加补料补加碳源，保持碳源浓度在1-5g/L。
10.根据权利要求5所述的应用，其特征在于，包括：
将所述依克多因合成菌株划线至固体LB培养基，在35-39℃下培养8-16h；
挑取培养的菌种，接种至液体LB培养基，在35-39℃下培养8-16h，获得种子液；
将种子液接种至发酵培养基，加入氨苄青霉素和IPTG诱导剂，在35-39℃下发酵培养36-60h。
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