CN113897325B - 一种生产红景天苷的重组大肠杆菌及其构建方法和应用 - Google Patents
一种生产红景天苷的重组大肠杆菌及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及一种重组大肠杆菌,以大肠杆菌CCTCC NO:M2019390为宿主细胞,表达来源于拟南芥的糖基转移酶基因UGT85A1,获得一株红景天苷的高产菌株E.coli YMGRS。本发明的重组大肠杆菌实现了从头合成红景天苷,底物为葡萄糖,价格低廉,且该菌株在装有2.5L M9Y培养基的5L发酵罐发酵后,红景天苷最高产量为9.3g/L,单位菌体浓度的产量为0.31g/(L·OD600),生产强度高。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种生产红景天苷的重组大肠杆菌及其构建方法和应用。
背景技术
红景天苷(Salidroside)是红景天中的主要药用成分。临床研究结果表明,红景天苷不但有抗缺氧、抗寒冷、抗疲劳、抗微波辐射、抗病毒、抗肿瘤等明显功能,而且还具有增强注意力、提高工作效率、延缓机体衰老、防止老年疾病等功效,尤其在军事医学、航天医学、运动医学和保健医学等方面具有十分重要的应用价值,是一种极具开发前景的环境适应药物,近年来备受关注。红景天苷无毒无害,可直接作为天然保健食品或药品,也可添加到焙烤制品、肉制品、面制品、乳制品、果冻、饮料等几乎大部分食品中;红景天苷在化妆品、饲料添加剂方面也有很好的市场前景。因此,红景天苷市场潜力不断扩大,具有很好的市场前景。
目前红景天苷的生产方法是从红景天植物中提取获得。红景天属于高寒多年生草本植物,天然资源有限,并且尚未实现大面积的人工栽培;红景天中红景天苷含量很低,随着开采力度不断加大,导致红景天属植物逐渐枯竭,同时提取法工艺复杂、成本高,并且产物中含有有毒物质百脉根苷。化学合成的方法有文献报道,但未实现工业化。化学合成方法大都存在需要进行选择性保护、活化或使用昂贵的金属催化剂等缺点,往往残留少量或痕量的其它有毒化学品,具有不安全性,生产成本仍然较高。
目前,已有以大肠杆菌为宿主实现以葡萄糖为原料利用微生物高效发酵合成红景天苷,并且已初步建立了提取工艺。与化学合成法相比,微生物合成红景天苷具有反应条件温和、目标产物可以发酵大量生产的特点,红景天苷主要存在于发酵液中,提取工艺简单;与传统生产方法相比,降低了生产成本和环境污染,实现可持续发展,具有很好的应用前景。但是,现有技术当中红景天苷的产量还有很大的提升空间。因此,价格低廉的生物法合成高产红景天苷成为必然的趋势。
发明内容
为解决上述技术问题,本发明以E.coliYMGR5A为出发菌构建了一种重组大肠杆菌,提供了一种高效的红景天苷合成方法。
本发明的第一个目的是提供一种生产红景天苷的重组大肠杆菌,以大肠杆菌CCTCC NO:M2019390为宿主细胞,表达来源于拟南芥的糖基转移酶基因UGT85A1(GenBank登录号:At1g22400)。本发明的大肠杆菌CCTCC NO:M2019390已记载于公开号为CN110452865A的中国专利中,命名为E.coliYMGR5A。
进一步地,重组大肠杆菌可以pEtac、pTrc99a、pKK223-3、pLac03等为表达载体,发明人首先采用与构建重组质粒pKK223-3-UGT85A1相同的引物和构建方法,以pEtac为表达载体构建重组质粒pEtac-UGT85A1(抗性标记为卡那霉素抗性),并进一步获得重组大肠杆菌E.coli YMGR5A/pEtac-UGT85A1,并利用大量其他表达载体构建,最终发现,以pKK223-3为表达载体构建的重组菌的红景天苷产量显著高于其他菌。
进一步地,糖基转移酶基因UGT85A1(GenBank登录号:At1g22400)的氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供上述重组大肠杆菌的构建方法,包括以下步骤:
将糖基转移酶基因UGT85A1插入到质粒pKK223-3中,得到重组质粒,将重组质粒转化入大肠杆菌CCTCC NO:M2019390中构建得到上述重组大肠杆菌E.coli YMGR5A/pKK223-3-UGT85A1,该大肠杆菌命名为E.coli YMGRS。
进一步地,以pKK223-3为表达载体构建重组质粒时,酶切位点为EcoR I与HindIII。
本发明的第三个目的是提供一种生产红景天苷的方法,采用上述重组大肠杆菌发酵生产红景天苷。
进一步地,将重组大肠杆菌接种至种子培养基中培养,得到种子液;将种子液接种至发酵培养基中,发酵制备红景天苷。
进一步地,将重组大肠杆菌接种至LB固体培养基上培养,得到单菌落,挑取单菌落接种于LB液体培养基中,于35-40℃、180-220r/min条件下培养10-14h,得到种子液;按体积比1%-10%的接种量将种子液接种至LB液体培养基中,在35-40℃、180-220r/min条件下培养8-12h后收集菌体;将得到的菌体接种至M9Y液体培养基中于25-35℃发酵35-120h得到红景天苷。
摇瓶发酵条件:按1%(v/v)接种量接入到装有LB液体培养基的锥形瓶中,于37℃、200r/min培养10h后收集菌体,并用生理盐水清洗菌体一次;转入装有M9Y液体培养基的锥形瓶中,于30℃、200r/min培养。
发酵罐发酵条件:按1%(v/v)接种量接入到装有LB液体培养基的锥形瓶中,于37℃、200r/min培养4~5h,按照10%接种量转入装有M9Y液体培养基的发酵罐中,通气量1VVM,于30℃培养。发酵过程中适量补加葡萄糖和酵母粉。
借由上述方案,本发明至少具有以下优点:
本发明以大肠杆菌CCTCC NO:M2019390为宿主细胞,表达来源于拟南芥的糖基转移酶基因UGT85A1,构建的重组大肠杆菌能够实现将微生物法从头合成转化为红景天苷,底物为葡萄糖,价格低廉,生产强度较高,单位菌体浓度的产量为0.31g/(L·OD600),比目前报道最高产量的菌株(酿酒酵母)的单位菌体浓度的产量0.24g/(L·OD600)高29%。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为本发明所构建的菌株E.coliYMGRS与出发菌株YMGR5A发酵的酪醇产量结果;
图2为本发明所构建的菌株E.coliYMGRS发酵的红景天苷产量结果;
图3为本发明所构建的菌株E.coliYMGRS发酵罐发酵的OD600及红景天苷产量结果;
图4为本发明所构建的菌株E.coliYMGRS主要代谢途径改造。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例中所涉及的培养基如下:
LB培养基配方(g/L):酵母粉5,蛋白胨10,NaCl 10,固体培养基另加1.5%-2.0%琼脂粉。
M9Y培养基配方(g/L):Na2HPO4·12H2O 17.1,KH2PO43,NaCl 0.5,NH4Cl 1,葡萄糖20,酵母粉0.25,补加终浓度MgSO45mmol·L-1。
下述实施例中所涉及的检测方法如下:
酪醇及红景天苷检测方法,采用高效液相色谱(HPLC)检测。色谱检测条件具体如下:Agela Innoval C18色谱柱(4.6×250mm,孔径为5μm);流动相为80%的0.1%甲酸水溶液,20%的甲醇;流速0.7mL/min;进样量10μL;紫外检测器,检测波长280nm;柱温为25℃。
下述实施例中所涉及的引物如表1所示。
表1引物序列
实施例1糖基转移酶基因UGT85A1游离表达大肠杆菌菌株的构建
1、重组质粒的构建
UGT85A1目的基因经金唯智公司合成获得,并将EcoR I与Hind III酶切位点设计在引物UGT85A1-L和UGT85A1-R中,将目的片段与pEtac、pTrc99a、pKK223-3、pLac03等质粒进行两个位点的酶切纯化后,用Solution I连接酶进行连接,经过化学转化法转入E.coliJM109中,涂布于含有氨苄或卡那青霉素抗性的LB固体培养基平板上,于37℃培养箱培养12h左右,将长出的单菌落提质粒进行酶切验证,将正确的菌株进行培养后重组质粒pKK223-3-UGT85A1、pEtac-UGT85A1、pTrc99a-UGT85A1、pLac03-UGT85A1的提取。我们还利用其他大量的表达质粒构建了表达UGT85A1基因的重组质粒,由于载体本身酶切位点的不同,为适应所用载体的特点,扩增UGT85A1基因的引物设计时酶切位点相应部分的序列按照引物设计的一般方法进行了调整。
2、糖基转移酶基因UGT85A1游离表达大肠杆菌菌株的构建
(1)制备E.coliYMGR5A感受态细胞:将E.coliYMGR5A菌株接种至LB固体培养基上划线培养,挑取单菌落接种于装有20mL LB液体培养基的100mL锥形瓶中,于37℃、200r/min进行活化培养12h左右,制备得到种子液。将制备得到的种子液以1%接种量接种至装有50mL LB液体培养基的250mL锥形瓶中,在37℃、200r/min条件下培养至OD600=0.6-0.8时,冰浴30min后于4℃下5000r/min离心5min收集菌体,将获得的菌体用预冷超纯水洗涤1次,用0.1mol/L的预冷的10%甘油溶液洗涤3次,向所收得的菌体中加入800ul的10%甘油。每份100μL分装与于1.5mL的EP管中于-70℃储存备用。
(2)将步骤(1)制备得到的重组质粒pKK223-3-UGT85A1、pEtac-UGT85A1等质粒采用电转导入E.coliYMGR5A感受态细胞中,涂布于含有氨苄或卡那青霉素的LB固体培养基中,于37℃培养至长出转化子。将转化子进行养菌提质粒,将质粒进行EcoRI与Hind III酶切验证,验证正确的菌株进行甘油管保藏,得到游离型重组大肠杆菌E.coliYMGR5A/pKK223-3-UGT85A1、E.coliYMGR5A/pEtac-UGT85A1、E.coli YMGR5A/pTrc99a-UGT85A1、E.coliYMGR5A/pLac03-UGT85A1等重组菌。
实施例2摇瓶发酵生产红景天苷
(1)将实施例1制备得到的重组大肠杆菌E.coliYMGR5A/pKK223-3-UGT85A1菌株在LB固体培养基上划线培养,得到单菌落,挑取单菌落接种于装有20mLLB液体培养基的100mL锥形瓶中,于37℃、200r/min培养12h;制备得到种子液。
(2)将步骤(1)制备得到的种子液按1%(v/v)的接种量接入到装有50mL LB液体培养基的250mL锥形瓶中,于37℃、200r/min培养10h后收集菌体;
(3)将步骤(2)得到的菌体采用生理盐水清洗菌体一次后,转入装有50mLM9Y液体培养基的250mL锥形瓶中,于30℃、200r/min培养48h。
每隔12h取样检测产物含量,结果如表2~3和图1~2所示。其中,酪醇产量结果如图1所示(数据见表2),红景天苷产量结果如图2所示(数据见表3)。
表2菌株YMGR5A和YMGRS不同发酵时间的酪醇产量
表3菌株YMGRS不同发酵时间的红景天苷产量
采用其他表达质粒所获得的重组大肠杆菌也采用同样的方法进行发酵及产量的检测,结果显示,其红景天苷的产量均在1.5g/L以下,低于E.coli YMGR5A/pKK223-3-UGT85A1的产量。
实施例3
(1)将实施例1制备得到的E.coliYMGRS菌株在LB固体培养基上划线培养,得到单菌落,挑取单菌落接种于装有20mL LB液体培养基的100mL锥形瓶中,于37℃、200r/min培养12h;制备得到种子液;
(2)将步骤(1)制备得到的种子液按1%(v/v)的接种量接入到装有50mL LB液体培养基的250mL锥形瓶中,于37℃、200r/min培养4~5h,得到发酵液;
(3)将步骤(2)制备得到的发酵液按照10%(v/v)的接种量转入装有2.5LM9Y液体培养基的5L发酵罐中,通气量1VVM,控制溶氧在40%左右,于30℃培养至120h;
发酵过程中适量补加葡萄糖和酵母粉。每隔4h左右取样检测OD600和红景天苷产量。结果如图3所示(数据见表4)。
表4菌株YMGRS不同发酵时间的红景天苷产量及OD600
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序列表
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Claims (6)
1.一种生产红景天苷的方法,其特征在于:采用重组大肠杆菌发酵生产红景天苷,所述重组大肠杆菌以大肠杆菌CCTCCNO:M2019390为宿主细胞,以pKK223-3为表达载体表达氨基酸序列如SEQ ID NO.1所示的来源于拟南芥的糖基转移酶基因UGT85A1;
发酵生产时,将活化后的菌体接种至M9Y液体培养基中进行发酵培养,并在发酵过程中补加葡萄糖和酵母粉。
2.根据权利要求1所述的方法,其特征在于:所述糖基转移酶基因UGT85A1的核苷酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的方法,其特征在于,所述重组大肠杆菌的构建方法包括以下步骤:将糖基转移酶基因UGT85A1插入到质粒pKK223-3中,得到重组质粒,将所述重组质粒转化入大肠杆菌CCTCCNO:M2019390中构建得到所述重组大肠杆菌。
4.根据权利要求1所述的方法,其特征在于:将所述重组大肠杆菌接种至种子培养基中培养,得到种子液;将种子液接种至发酵培养基中,发酵制备红景天苷。
5.根据权利要求4所述的方法,其特征在于:按体积比1%-10%的接种量将种子液接种至发酵培养基中,于25-35℃发酵35-120h得到红景天苷。
6.根据权利要求4所述的方法,其特征在于:将所述重组大肠杆菌接种至LB固体培养基上培养,得到单菌落,挑取单菌落接种于LB液体培养基中,于35-40℃、180-220r/min条件下培养10-14h,得到种子液;将得到的种子液接种至LB液体培养基中,在35-40℃、180-220r/min条件下培养8-12h后收集菌体;将得到的菌体接种至M9Y液体培养基中进行发酵培养制备红景天苷。
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