CN116396876A - 一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法 - Google Patents
一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法 Download PDFInfo
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Abstract
本发明涉及一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法,属于分子生物学和生物工程技术领域。所述的生产人参皂苷Rd的酿酒酵母工程菌是在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β‑葡萄糖苷酶EGH1,过表达葡萄糖磷酸变位酶1、葡萄糖磷酸变位酶2和UDP‑葡萄糖焦磷酸化酶,糖基转移酶Pn1‑31、糖基转移酶PnUGT53、糖基转移酶PnUGT50和原人参二醇合成酶PPDS。本发明获得的酿酒酵母工程菌能够利用葡萄糖发酵生产人参皂苷Rd,产量为56.68±16.21mg/L;酵母生长繁殖较快,仅需发酵罐就可以生产,为人工细胞高效合成人参皂苷Rd奠定了基础。
Description
技术领域
本发明属于分子生物学和生物工程技术领域,涉及异源合成人参皂苷Rd的重组酿酒工程菌及其构建方法,具体涉及一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法。
背景技术
人参皂苷Rd具有保护心血管系统、保护神经系统、抗衰老、抗肿瘤、免疫调节、镇痛等多种药理活性,药用价值较高。人参皂苷Rd可用于肿瘤、心血管系统、肾脏衰竭及炎症等疾病治疗。目前人参皂苷Rd已经成为治疗脑卒中的国家一类候选新药,其作为新型神经保护剂的临床应用具有良好的发展前景。人参皂苷Rd的分子式如式(Ⅰ)所示。
人参皂苷Rd主要的获取方式主要是从人参属植物中提取,但人参皂苷Rd在人参属植物中的含量都比较低,而人参属植物种植存在栽培周期长、连作障碍、农药及重金属残留等问题,有限的天然资源及人工栽培技术极大的限制了人参皂苷Rd的推广和应用。另外,化学合成因使用昂贵的起始原料和繁琐的合成程序而黯然失色。因此,利用合成生物学技术改造微生物生产人参皂苷Rd提供了一种最有潜力的替代方法。
发明内容
本发明的目的是为了解决现有技术的不足,提供一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法。通过ZW04BY酿酒酵母工程菌中异源表达人参皂苷Rd通路合成的3个关键糖基转移酶实现了人参皂苷Rd在酿酒酵母中的从头生产,其合成途径如图1所示,并且此工程菌人参皂苷Rd产量较高。本发明的方法为人工细胞高效合成人参皂苷Rd奠定了基础。
为实现上述目的,本发明采用的技术方案如下:
一种生产人参皂苷Rd的酿酒酵母工程菌,在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β-葡萄糖苷酶EGH1,过表达葡萄糖磷酸变位酶1、葡萄糖磷酸变位酶2和UDP-葡萄糖焦磷酸化酶,糖基转移酶Pn1-31、糖基转移酶PnUGT53、糖基转移酶PnUGT50和原人参二醇合成酶PPDS;所述糖基转移酶PnUGT50的核苷酸序列如SEQ ID NO.1所示;所述葡萄糖磷酸变位酶1的核苷酸序列如SEQ ID NO.2所示;所述葡萄糖磷酸变位酶2的核苷酸序列如SEQID NO.3所示;所述UDP-葡萄糖焦磷酸化酶如SEQ ID NO.4所示;所述糖基转移酶Pn1-31的核苷酸序列如SEQ ID NO.5所示;所述优化后的糖基转移酶PnUGT53的核苷酸序列如SEQ IDNO.6所示;原人参二醇合成酶PPDS的核苷酸序列如SEQ ID NO.7所示。
进一步,优选的是,所述酿酒酵母基因组中整合过表达原人参二醇至人参皂苷Rd途径中的所有基因,包括原人参二醇合成酶PPDS、糖基转移酶PnUGT50和糖基转移酶PnUGT53。
本发明还提供上述生产人参皂苷Rd的酿酒酵母工程菌的构建方法,包括以下步骤:
(1)通过在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β-葡萄糖苷酶EGH1,通过PCR扩增,将葡萄糖磷酸变位酶1、葡萄糖磷酸变位酶2和UDP-葡萄糖焦磷酸化酶、糖基转移酶Pn1-31和糖基转移酶PnUGT53导入起始菌株ZW04BY,获得重组菌株1;
(2)在重组菌株1的基础上,分别在组合型启动子TDH3+UASTEF1-CIT1-CLB2和ADH1启动子的控制之下,过表达密码子优化后的糖基转移酶PnUGT50和糖基转移酶PnUGT53,获得重组菌株2;
(3)将来自人参的原人参二醇合成酶基因PPDS进行酵母密码子优化,串联组合型启动子TDH3+UASTEF1-CIT1-CLB2和G418抗性筛选标签,一起导入起始酿酒酵母重组菌株2的δ序列位点,获得重组菌株3;
(4)获得的重组菌株3在摇瓶条件下进行发酵测产,获得的生产人参皂苷Rd的酿酒酵母工程菌。
进一步,优选的是,步骤(1)的具体方法为:
(1.1)Y1-LKG-1基因盒重组载体构建:
(1.1.1)以酵母菌株ZW04BY的基因组为模板,采用引物LEU(Dn)+pADH1-F和LEU2-R扩增获得同源臂下游片段;
(1.1.2)以质粒pHDE-Cas9为模板,采用引物KANMX+LEU(up)-F和KANMX+HindIII-R进行PCR扩增,获得G418片段;
(1.1.3)以UASTEF1+CIT1+CLB2为模板,采用引物UAS+PTDH3-R和UAS+KANMX-F进行PCR扩增,获得UAS片段;
(1.1.4)将获得的同源臂下游片段、G418片段、UAS片段为模板,采用引物Leu2-up-F和UAS+pTDH3-R经融合PCR扩增,获得-LKG-1基因盒;
(1.1.5)LKG-1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-1基因盒重组载体;
(1.2)Y1-LKG-2基因盒重组载体构建:
(1.2.1)以Y2-EGH-3基因盒重组载体为模板,采用引物pTDH3+UAS-F和pTDH3+sPn50-R进行PCR扩增,获得启动子TDH3片段;
(1.2.2)以pESC-SnyPnUGT50为模板,采用引物sPn50+EGFP-R和sPn50+pTDH3-F进行PCR扩增,获得SnyPn50片段;
(1.2.3)以质粒pT4-CMV-GFP为模板,采用引物EGFP+sPnUGT50-F和EGFP+tCYC 1-R进行PCR扩增,获得EGFP序列;
(1.2.4)将获得的TDH3、SnyPn50、EGFP为模板,采用引物pTDH3+UAS-F和EGFP+tCYC1-R经融合PCR扩增,获得LKG-2基因盒;
(1.2.5)LKG-2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-2基因盒重组载体;
(1.3)Y1-LKG-3基因盒重组载体构建:
(1.3.1)以酵母菌株ZW04BY的基因组为模板,采用引物Leu2-up-F和LEU2(up)+KANMX-R扩增同源臂上游片段Leu2-UP片段;
(1.3.2)以酵母菌株BY4742基因组为模板,采用引物ADH1+PNUGT53-F和pADH1+LEU2(Dn)-R进行PCR扩增,获得ADH1片段;
(1.3.3)以Y2-EGH-2基因盒重组载体为模板,采用引物PNUGT53+pADH1-R和tCYC1+GFP-F进行PCR扩增,获得PNUGT53片段;
(1.3.4)将获得的PNUGT53、ADH1、Leu2-UP片段为模板,采用引物tCYC1+GFP-F和LEU2-R经融合PCR扩增,获得LKG-3基因盒;
(1.3.5)LKG-3基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-3基因盒重组载体;
(1.4)将所得的Y1-LKG-1、Y1-LKG-2和Y1-LKG-3基因盒重组载体质粒线性化一起转入起始菌株ZW04BY,获得重组菌株1;
步骤(1.1.1)~(1.1.3)、(1.2.1)~(1.2.3)、(1.3.1)~(1.3.3)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min;
步骤(1.1.4)、(1.2.4)、(1.3.4)中PCR反应体系均为50μL:模板1μL,上游引物10mM2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
进一步,优选的是,步骤(2)的具体方法为:
(2.1)Y2-EGH-1基因盒重组载体构建:
(2.1.1)以酵母菌株BY4742基因组为模板,采用引物EGH1-UP-F和
EGH1-UP+pLYS2-R进行PCR扩增,获得EGH1-UP片段;
(2.1.2)以酵母菌株W303基因组为模板,采用引物LYS2+pADH1-R和pLYS2+EGH1-UP-F进行PCR扩增,获得pLYS2片段;
(2.1.3)将获得的EGH1-UP和pLYS2片段为模板,采用引物EGH1-UP-F和LYS2+pADH1-R经融合PCR扩增,获得EGH-1基因盒;
(2.1.4)EGH-1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-1基因盒重组载体;
(2.2)Y2-EGH-2基因盒重组载体构建:
(2.2.1)以酵母菌株W303基因组为模板,采用引物ADH1+Lys2-F和ADH1+Pn1-31-R进行PCR扩增,获得pADH1片段;
(2.2.2)以三七基因组为模板,采用引物pn1-31+ADH1-F和Pn1-31+tPGI-R进行PCR扩增,获得Pn1-31片段;
(2.2.3)以得酵母菌株W303基因组为模板,采用引物tPGI+Pn1-31-F和tPGI+pTEF1-R进行PCR扩增,获得tPGI片段;
(2.2.4)以得酵母菌株W303基因组为模板,采用引物pTEF1+PNUGT53-F和pTEF1+tPGI-F扩增获得pTEF1片段;
(2.2.5)以质粒YCplac22为模板,采用引物tCYC1+PNUGT53-F和tCYC1+tPFK1-R进行PCR扩增,获得tCYC1片段;
(2.2.6)以三七基因组为模板,采用引物PNUGT53+tCYC-R和PNUGT53+pTEF1-F进行PCR扩增,获得PnUGT53;
(2.2.7)将获得的pADH1、Pn1-31、tPGI、PnUGT53、pTEF1、tCYC1片段为模板,采用引物ADH1+Lys2-F和tCYC1+tPFK1-R经融合PCR扩增,获得EGH-2基因盒;
(2.2.8)EGH-2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-2基因盒重组载体;
(2.3)Y2-EGH-3基因盒重组载体构建:
(2.3.1)以酵母菌株W303为模板,采用引物ScUGP1+tPFK1-F和ScUGP1+pTDH3-R进行PCR扩增获得ScUGP1片段;
(2.3.2)以酵母菌株W303为模板,采用引物tTDH2+pTDH3-F和tTDH2+ScPGM2-R进行PCR扩增获得pTDH2片段;
(2.3.3)以酵母菌株W303为模板,采用引物tPFK1+tCYC1-F和tPFK1+ScUGP1-R进行PCR扩增获得PFK1片段;
(2.3.4)以酵母菌株W303为模板,采用引物pTDH3+ScUGP1-F和pTDH3+tTDH2-R进行PCR扩增获得pTDH3片段;
(2.3.5)将获得的ScUGP1、PFK1、pTDH3、pTDH2片段为模板,采用引物tPFK1+tCYC1-F和tTDH2+ScPGM2-R经融合PCR扩增,获得EGH-3基因盒;
(2.3.6)EGH-3基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-3基因盒重组载体;
(2.4)Y2-EGH-4基因盒重组载体构建:
(2.4.1)以酵母菌株BY4742基因组为模板,采用引物ScPGM2+tTDH2-F和ScPGM2+pEBA1-R进行PCR扩增获得ScPGM2片段,采用引物pEBA1+ScPGM2-F和pFBA+tADH1-R进行PCR扩增获得pEBA1片段;
(2.4.2)以酵母菌株W303基因组为模板,采用引物tADH+pFBA1-F和tADH1+ScPGM1-R进行PCR扩增获得tADH片段;
(2.4.3)将获得的ScPGM2、pEBA1、tADH片段为模板,采用引物ScPGM2+tTDH2-F和tADH1+ScPGM1-R经融合PCR扩增,获得EGH-4基因盒;
(2.4.4)EGH-4基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-4基因盒重组载体;
(2.5)Y2-EGH-5基因盒重组载体构建:
(2.5.1)以质粒YCplac22为模板,采用引物pPGK1+ScPGM1-R和pPGK1+EGH1-F进行PCR扩增获得pPGK1片段;
(2.5.2)以酿酒酵母BY4742基因组为模板,采用引物ScPGM1+tADH1-R和ScPGM1+pPGK1-F进行PCR扩增获得ScPGM1片段;
(2.5.3)以酵母菌株BY4742基因组为模板,采用引物EGH1+pPGK1-F和EGH1-Dn-R进行PCR扩增获得EGH1-Dn片段;
(2.5.4)将获得的ScPGM1、PGK1、EGH1-Dn片段为模板,采用引物ScPGM1+tADH1-R和EGH1-Dn-R经融合PCR扩增,获得EGH-P5基因盒;
(2.5.5)EGH-5基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-5基因盒重组载体;
(2.6)将所得的Y2-EGH-1、Y2-EGH-2、Y2-EGH-3、Y2-EGH-4和Y2-EGH-5基因盒重组载体质粒线性化一起转入重组菌株1,获得重组菌株2;
步骤(2.1.1)~(2.1.2)、(2.2.1)~(2.2.6)、(2.3.1)~(2.3.4)、(2.4.1)~(2.4.2)、(2.5.1)~(2.5.3)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min;
步骤(2.1.3)、(2.2.7)、(2.3.5)、(2.4.3)、(2.5.4)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
进一步,优选的是,步骤(3)的具体方法为:
(3.1)Y3-PPDS-1基因盒重组载体构建:
(3.1.1)以酵母菌株BY4742基因组为模板,采用引物UP-F和UP+KAMX-R扩增获得片段上游同源臂片段;
(3.1.2)以UASTEF1+CIT1+CLB2为模板,采用引物UAS+pTDH3-R和UAS+KANMX-F进行PCR扩增获得UAS片段;
(3.1.3)以pHDE-Cas9质粒为模板,采用引物KANMX+HindIII-R和KANMX+UP-F进行PCR扩增获得KANMX片段;
(3.1.4)将获得的UAS、KANMX、上游同源臂片段为模板,采用引物UP-F和UAS+pTDH3-R经融合PCR扩增,获得L-F1基因盒;
(3.1.5)L-F1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y3-PPDS-1基因盒重组载体;
(3.2)Y3-PPDS-2基因盒重组载体构建:
(3.2.1)以酵母菌株BY4742基因组为模板,采用引物Dn-R和03Dn+PRM9-F进行PCR扩增获得片段下游同源臂Dn片段;
(3.2.2)以经酵母密码子优化PPDS序列为模板,采用引物PPDS+PRM9-R和PPDS+pTDH3-F进行PCR扩增获得SnyPPDS片段;
(3.2.3)以酵母菌株W303基因组为模板,引物pTDH3+PPDS-R和pTDH3+UAS-F扩增获得启动子pTDH3片段,采用引物PRM9+03Dn-F和PRM9+PPDS-R扩增获得终止子PRM9片段;
(3.2.4)将获得的Dn、PRM9、SnyPPDS、pTDH3为模板,引物Dn-R和pTDH3+UAS-F经融合PCR扩增,获得L-F2基因盒;
(3.2.5)L-F2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y3-PPDS-2基因盒重组载体;
(3.3)将所得的Y3-PPDS-1、Y3-PPDS-2和Y3-PPDS-3基因盒重组载体质粒线性化一起转入重组菌株2,获得重组菌株3;
步骤(3.1.1)~(3.1.3)、(3.2.1)~(3.2.3)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min;
步骤(3.1.4)、(3.2.4)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
本发明同时提供一种人参皂苷Rd的制备方法,将如权利要求1-2任一项所述的生产人参皂苷Rd的酿酒酵母工程菌发酵,从发酵液中获得人参皂苷Rd。
进一步,优选的是,发酵培养基的配方为20g/L葡萄糖、20g/L蛋白胨,10g/L酵母浸粉,余量为水,发酵温度条件为30℃。
本发明在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β-葡萄糖苷酶EGH1,过表达葡萄糖磷酸变位酶1(ScPGM1)、葡萄糖磷酸变位酶2(ScPGM2)和UDP-葡萄糖焦磷酸化酶(ScUGP1),以及3个糖基转移酶——Pn1-31、PnUGT53、PnUGT50和原人参二醇合成酶PPDS。所述优化后的糖基转移酶PnUGT50的核苷酸序列以SEQ ID NO.1所示;所述葡萄糖磷酸变位酶1(ScPGM1)的核苷酸序列以SEQ ID NO.2所示;所述葡萄糖磷酸变位酶2(ScPGM2)的核苷酸序列以SEQ ID NO.3所示;所述UDP-葡萄糖焦磷酸化酶(ScUGP1)以SEQ ID NO.4所示;所述优化后的糖基转移酶Pn1-31的核苷酸序列以SEQ ID NO.5所示;所述优化后的糖基转移酶PnUGT53的核苷酸序列以SEQ ID NO.6所示;原人参二醇合成酶PPDS以SEQ ID NO.7所示。
本发明通过在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β-葡萄糖苷酶EGH1,过表达葡萄糖磷酸变位酶1(ScPGM1)、葡萄糖磷酸变位酶2(ScPGM2)和UDP-葡萄糖焦磷酸化酶(ScUGP1),以及2个糖基转移酶——Pn1-31和PnUGT53,获得重组菌株1;在菌株1的基础上,过表达三七的糖基转移酶PnUGT50和PnUGT31,获得重组菌株2;在其基础上异源过表达原人参二醇合成酶PPDS获得菌株3;
本发明发明人考虑在合成生物学中,酿酒酵母是较为常用的平台生物。酿酒酵母具有高效产生异戊二烯起始合成单位异戊二烯焦磷酸,丙二烯甲基焦磷酸以及关键中间代谢物香叶基二磷酸,法尼基二磷酸的能力,因此广泛用于甾醇、类固醇和其他萜类化合物的生产。另外,在萜类化合物生物合成中,真核生物的细胞膜对植物萜类转化酶如细胞色素P450等比原核生物更加适合。基于此,研究出一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法。
本发明与现有技术相比,其有益效果为:
本发明构建的生产人参皂苷Rd的酿酒酵母工程菌能够利用葡萄糖发酵生产人参皂苷Rd,产量为56.68±16.21mg/L;酵母生长繁殖较快,仅需发酵罐就可以生产,无论是生态保护、土地利用面积或生产周期等均比植物生产提取高效,并可降低生产成本,同时也比化学合成具有更高效、绿色、无毒污染等优点,易于推广应用。
附图说明
图1为人参皂苷Rd在酿酒酵母中的生物合成路径示意图;其中实线代表本次改造插入的糖基转移酶;
图2为重组菌株1的插入片段图谱(L-F1至L-F3基因盒);
图3为重组菌株2的插入片段图谱(Y1-EGH-1至Y1-EGH-5基因盒);
图4为重组菌株3的插入片段图谱(Y2-LKG-1至Y2-LKG-3基因盒);
图5为重组菌株3生产人参皂苷Rd的HPLC检测图。其中标准品峰1代表人参皂苷Rb1,峰2代表人参皂苷Rg3,峰3代表人参皂苷Rh2,峰4代表原人参二醇;
图6为重组菌株生产的人参皂苷Rd的LC-MS检测图;
图7为与起始菌株相比,重组菌株4在摇瓶发酵中人参皂苷Rd产量图。
具体实施方式
下面结合实施例对本发明作进一步的详细描述。
本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品。
一、本发明涉及的菌株和质粒来源如下:
1.出发菌株为ZW04BY(BY4742,HXT7p-tHMG1-ADH1t,TEF2p-synPgCPR1-TDH2t,TPIIp-ERG1-ENO2t,GPM1p-ERG20-CYC1t,PGK1p-ERG9-FBA1t,TDH3p-synDDS-PGT1t,TEF1p-synPPDS-PGK1t,ENO2p-ERG12-CPS1t,TEF2p-ERG13-IDP1t,TPIIp-ERG8-PRM5t,GPM1p-ERG19-HIS5t,PGK1p-IDI-PRM9t,TDH3p-ERG10-SPG5t,TEF1p-tHMG1-ADH1t,TDH3p-synPPDS-CPS1t),该菌株由本课题组按照参考文献(Wang P,Wei W,Ye W,Li X,Zhao W,Yang C,Li C,Yan X,Zhou Z.Synthesizing ginsenoside Rh2 in Saccharomycescerevisiae cell factory at high-efficiency.Cell Discov.2019Jan 15;5:5.doi:10.1038/s41421-018-0075-5)中的方法构建。
2.质粒pHDE-Cas9、YCplac22由中国科学院天津工业生物技术研究所江会锋研究员赠予,pT4-CMV-GFP质粒为商业购买获得。
3.所用引物均由北京擎科生物科技有限公司(昆明分公司)合成,引物序列见表1。
表1
二、本发明所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
三、本发明涉及的基因如下:
1.优化后的糖基转移酶PnUGT50的核苷酸序列以SEQ ID NO.1所示;
2.葡萄糖磷酸变位酶1(ScPGM1)的核苷酸序列以SEQ ID NO.2所示;
3.葡萄糖磷酸变位酶2(ScPGM2)的核苷酸序列以SEQ ID NO.3所示;
4.UDP-葡萄糖焦磷酸化酶(ScUGP1)以SEQ ID NO.4所示;
5.优化后的糖基转移酶Pn1-31的核苷酸序列以SEQ ID NO.5所示;
6.优化后的糖基转移酶PnUGT53的核苷酸序列以SEQ ID NO.6所示。
7.优化后的原人参二醇合成酶PPDS以SEQ ID NO.7所示。
四、本发明涉及的培养基及配制
YPD液体培养基:10g/L酵母浸粉,20g/L蛋白胨,20g/L葡萄糖,溶剂为去离子水;配制:将各成分溶于去离子水,搅拌溶解,115℃灭菌25min即得。
Sc-his-lys平板:无水葡萄糖20g/L;Yeast Nitrogen Base 6.7g/L;精氨酸、半胱氨酸、苏氨酸、色氨酸、亮氨酸、腺嘌呤各0.1g/L;天冬氨酸、异亮氨酸、苯丙氨酸、脯氨酸、丝氨酸、酪氨酸、缬氨酸、甲硫氨酸、尿嘧啶各0.05g/L;配制:将各成分溶于去离子水,搅拌溶解,115℃灭菌25min即得。
YPD+G418平板:无水葡萄糖20g/L,蛋白胨20g/L,酵母浸粉10g/L,1.5%琼脂粉,去离子水配制为YPD固体培养基,115℃灭菌25min即得;G418用去离子水配制为100mg/ml母液,过滤除菌后加入2ml至1L的未凝固的YPD固体培养基中使G418终浓度为200mg/L,倒入平板获得YPD+G418平板。
实施例1:重组质粒的构建
(一)Y3-PPDS-1基因盒重组载体构建
(1)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株BY4742基因组,以此基因组为模板,采用引物UP-F和UP+KANMX-R扩增获得片段上游同源臂片段;
(2)通过北京擎科生物有限公司合成UASTEF1+CIT1+CLB2序列,以此为模板(Blazeck J,Garg R,Reed B,et al.Controlling promoter strength and regulation inSaccharomyces cerevisiae using synthetic hybrid promoters.Biotechnology andbioengineering[J].2012.109:2884-2895),采用引物UAS+pTDH3-R和UAS+KANMX-F扩增获得UAS片段;
(3)以pHDE-Cas9质粒为模板,采用引物KANMX+HindIII-R和KANMX+UP-F扩增获得KANMX片段;
(4)将获得的UAS、KANMX、上游同源臂片段为模板,采用引物UP-F和UAS+pTDH3-R融合PCR扩增获得L-F1基因盒;
(5)L-F 1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y3-PPDS-1基因盒重组载体。
(二)Y3-PPDS-2基因盒重组载体构建
(1)以酵母菌株BY4742基因组为模板,采用引物Dn-R和03Dn+PRM9-F扩增获得片段下游同源臂Dn片段;
(2)以经酵母密码子优化PPDS序列为模板,采用引物PPDS+PRM9-R和PPDS+pTDH3-F扩增获得SnyPPDS片段;
(3)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,引物pTDH3+PPDS-R和pTDH3+UAS-F扩增获得启动子pTDH3片段,采用引物PRM9+03Dn-F和PRM9+PPDS-R扩增获得终止子PRM9片段;
(4)将获得的Dn、PRM9、SnyPPDS、pTDH3为模板,引物Dn-R和pTDH3+UAS-F融合PCR扩增获得L-F2基因盒;
(5)L-F2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y3-PPDS-2基因盒重组载体。
(三)Y2-EGH-1基因盒重组载体构建
(1)以酵母菌株BY4742基因组为模板,采用引物EGH1-UP-F和EGH1-UP+pLYS2-R扩增获得EGH1-UP片段;
(2)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物LYS2+pADH1-R和pLYS2+EGH1-UP-F扩增获得pLYS2片段;
(3)将获得的EGH1-UP和pLYS2片段为模板,采用引物EGH1-UP-F和LYS2+pADH1-R经融合PCR扩增获得EGH-1基因盒;
(6)EGH-1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-1基因盒重组载体。
(四)Y2-EGH-2基因盒重组载体构建
(1)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物ADH1+Lys2-F和ADH1+Pn1-31-R扩增获得pADH1片段;
(2)使用Solarbio酵母基因组DNA提取试剂盒获得三七基因组,以此为模板,采用引物pn1-31+ADH1-F和Pn1-31+tPGI-R扩增获得Pn1-31片段;
(3)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物tPGI+Pn1-31-F和tPGI+pTEF1-R扩增获得tPGI片段;
(4)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物pTEF1+PNUGT53-F和pTEF1+tPGI-F扩增获得pTEF1片段;
(5)以质粒YCplac22为模板,采用引物tCYC1+PNUGT53-F和tCYC1+tPFK1-R扩增获得tCYC1片段;
(6)使用Solarbio酵母基因组DNA提取试剂盒获得三七基因组,采用引物PNUGT53+tCYC-R和PNUGT53+pTEF1-F扩增获得PnUGT53;
(7)将获得的pADH1、Pn1-31、tPGI、PnUGT53、pTEF1、tCYC1片段为模板,采用引物ADH1+Lys2-F和tCYC1+tPFK1-R为扩增获得EGH-2基因盒;
(8)EGH-2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-2基因盒重组载体。
(五)Y2-EGH-3基因盒重组载体构建
(1)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物ScUGP1+tPFK1-F和ScUGP1+pTDH3-R扩增获得ScUGP1片段;
(2)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物tTDH2+pTDH3-F和tTDH2+ScPGM2-R扩增获得pTDH2片段;
(3)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物tPFK1+tCYC1-F和tPFK1+ScUGP1-R扩增获得PFK1片段;
(4)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物pTDH3+ScUGP1-F和pTDH3+tTDH2-R扩增获得pTDH3片段;
(5)将获得的ScUGP1、PFK1、pTDH3、pTDH2片段为模板,采用引物tPFK1+tCYC1-F和tTDH2+ScPGM2-R经融合PCR扩增获得EGH-3基因盒片段;
(6)EGH-3基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-3基因盒重组载体。
(六)Y2-EGH-4基因盒重组载体构建
(1)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株BY4742基因组,以此为模板,采用引物ScPGM2+tTDH2-F和ScPGM2+pEBA1-R扩增获得ScPGM2片段,采用引物pEBA1+ScPGM2-F和pFBA+tADH1-R扩增获得pEBA1片段;
(2)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株W303基因组,以此为模板,采用引物tADH+pFBA1-F和tADH1+ScPGM1-R扩增获得tADH片段;
(3)将获得的ScPGM2、pEBA1、tADH片段为模板,采用引物ScPGM2+tTDH2-F和tADH1+ScPGM1-R经融合PCR扩增获得EGH-4基因盒片段;
(4)EGH-4基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-4基因盒重组载体。
(七)Y2-EGH-5基因盒重组载体构建
(1)以质粒YCplac22为模板,采用引物pPGK1+ScPGM1-R和pPGK1+EGH1-F扩增获得pPGK1片段;
(2)以酿酒酵母BY4742基因组为模板,采用引物ScPGM1+tADH1-R和ScPGM1+pPGK1-F扩增获得ScPGM1片段;
(3)以酵母菌株BY4742基因组为模板,采用引物EGH1+pPGK1-F和EGH1-Dn-R扩增获得EGH1-Dn片段;
(4)将获得的ScPGM1、PGK1、EGH1-Dn片段为模板,采用引物ScPGM1+tADH1-R和EGH1-Dn-R经融合PCR扩增获得EGH-P5基因盒片段。
(5)EGH-P5基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-5基因盒重组载体。
(八)Y1-LKG-1基因盒重组载体构建
(1)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株ZW04BY的基因组,以此为模板,采用引物LEU(Dn)+pADH1-F和LEU2-R扩增获得同源臂下游片段;
(2)以质粒pHDE-Cas9为模板,采用引物KANMX+LEU(up)-F和KANMX+HindIII-R扩增获得G418片段;
(3)以合成的UASTEF1+CIT1+CLB2为模板,采用引物UAS+PTDH3-R和UAS+KANMX-F扩增获得UAS片段;
(4)将获得的同源臂下游片段、G418片段、UAS片段为模板,采用引物Leu2-up-F和UAS+pTDH3-R融合PCR扩增获得LKG-1基因盒;
(5)LKG-1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-1基因盒重组载体。
(九)Y1-LKG-2基因盒重组载体构建
(1)以Y2-EGH-3基因盒重组载体为模板,采用引物pTDH3+UAS-F和pTDH3+sPn50-R为扩增获得启动子TDH3片段;
(2)以合成序列pESC-SnyPnUGT50为模板,采用引物sPn50+EGFP-R和sPn50+pTDH3-F扩增获得SnyPn50片段;
(3)以质粒pT4-CMV-GFP为模板,采用引物EGFP+sPnUGT50-F和EGFP+tCYC1-R扩增获得EGFP序列;
(4)将获得的TDH3、SnyPn50、EGFP为模板,采用引物pTDH3+UAS-F和EGFP+tCYC1-R融合PCR扩增获得LKG-2基因盒。
(5)LKG-2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,测序正确,得到Y1-LKG-2基因盒重组载体。
(十)Y1-LKG-3基因盒重组载体构建
(1)使用Solarbio酵母基因组DNA提取试剂盒获得酵母菌株ZW04BY的基因组,以此为模板,采用引物Leu2-up-F和LEU2(up)+KANMX-R扩增同源臂上游片段Leu2-UP片段;
(2)以酵母菌株BY4742基因组为模板,采用引物ADH1+PNUGT53-F和pADH1+LEU2(Dn)-R扩增获得ADH1片段;
(3)以Y2-EGH-2基因盒重组载体为模板,采用引物PNUGT53+pADH1-R和tCYC1+GFP-F扩增获得PNUGT53片段;
(4)将获得的PNUGT53、ADH1、Leu2-UP片段为模板,采用引物tCYC1+GFP-F和LEU2-R融合PCR扩增获得LKG-3基因盒;
(5)LKG-3基因盒通过pEASY-Blunt Cloning Kit(北京全式金生物技术有限公司)连接pEASY载体构建重组载体,得到Y1-LKG-3基因盒重组载体。
实施例中,所有用到的试剂盒均按照试剂盒说明书进行操作。
(一)Y3-PPDS-1基因盒重组载体构建中(1)~(3)、(二)Y3-PPDS-2基因盒重组载体构建中(1)~(3)、(三)Y2-EGH-1基因盒重组载体构建中(1)~(2)、(四)Y2-EGH-2基因盒重组载体构建中(1)~(6)、(五)Y2-EGH-3基因盒重组载体构建中(1)~(4)、(六)Y2-EGH-4基因盒重组载体构建中(1)~(2)、(七)Y2-EGH-5基因盒重组载体构建中(1)~(3)、(八)Y1-LKG-1基因盒重组载体构建中(1)~(3)、(九)Y1-LKG-2基因盒重组载体构建中(1)~(3)、(十)Y1-LKG-3基因盒重组载体构建中(1)~(3)中的PCR采用Q5 High-Fidelity DNAPolymerases或Phusion Plus DNA聚合酶克隆试剂盒进行;PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;酶混合物为Q5 High-Fidelity DNA Polymerases或Phusion Plus DNA聚合酶克隆试剂盒中的相应试剂。PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
(一)Y3-PPDS-1基因盒重组载体构建中(4)、(二)Y3-PPDS-2基因盒重组载体构建(4)、(三)Y2-EGH-1基因盒重组载体构建中(3)、(四)Y2-EGH-2基因盒重组载体构建中(7)、(五)Y2-EGH-3基因盒重组载体构建中(5)、(六)Y2-EGH-4基因盒重组载体构建中(3)、(七)Y2-EGH-5基因盒重组载体构建中(4)、(八)Y1-LKG-1基因盒重组载体构建中(4)、(九)Y1-LKG-2基因盒重组载体构建中(4)、(十)Y1-LKG-3基因盒重组载体构建中(4)中的PCR采用Q5High-Fidelity DNA Polymerases克隆试剂盒进行;PCR反应体系均为50μL:每个模板的量均为25ng-100ng,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;酶混合物为采用Q5High-Fidelity DNA Polymerases克隆试剂盒中的相应试剂。PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
总的来说,基因表达盒的建立需经过2轮PCR克隆,第一轮片段克隆采用Q5High-Fidelity DNA Polymerases或Phusion Plus DNA聚合酶克隆试剂盒获得基础片段。PCR反应体系均为50μL:由1μL模板,10mM的上游引物和10mM的下引物各2μL,酶混合物25μL,去离子水补足50μL组成。PCR反应程序为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。PCR结束后,进行跑胶,确认扩增成功后进行目的条带回收。基因切胶回收使用北京全式金生物技术有限公司的EasyPure Quick Gel Extraction Kit试剂盒进行目的基因回收。回收后在NanoReady超微量紫外可见分光光度计上测定其回收浓度,-20℃冰箱中保存;第二轮融合PCR采用Q5High-Fidelity DNA Polymerases克隆试剂盒获得基础片段相连的基因表达盒,参与融合PCR的各个片段模板需相同浓度(25ng-100ng),PCR反应程序及其余步骤均同第一轮PCR。最后获得的基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体。
以上获得的重组载体转化大肠杆菌TransT1保存,重组单菌落送测序公司测序。经测序确认无误后进行保种。方法为50%(W/V)甘油与菌液按1:1加入保种管,充分混匀后放入-80℃超低温冰箱中保存。
实施例3:重组菌株构建
(一)载体的线性化
(1)以所得的Y1-LKG-1基因盒重组载体为模板,采用引物Leu2-up-F和UAS+pTDH3-R经PCR扩增获得Y1-LKG-1线性化片段;
(2)以所得的Y1-LKG-2基因盒重组载体为模板,采用引物pTDH3+UAS-F和EGFP+tCYC1-R进行PCR扩增获得Y1-LKG-2线性化片段;
(3)以所得的Y1-LKG-3基因盒重组载体为模板,采用引物tCYC1+GFP-F和LEU2-R进行PCR扩增获得Y1-LKG-3线性化片段;
(4)以所得的Y2-EGH-1基因盒重组载体为模板,采用引物EGH1-UP-F和LYS2+pADH1-R经PCR扩增获得Y2-EGH-1线性化片段;
(5)以所得的Y2-EGH-2基因盒重组载体为模板,采用引物ADH1+Lys2-F和tCYC1+tPFK1-R经PCR扩增获得Y2-EGH-2线性化片段;
(6)以所得的Y2-EGH-3基因盒重组载体为模板,采用引物pTDH3+ScUGP1-F和pTDH3+tTDH2-R经PCR扩增获得Y2-EGH-3线性化片段;
(7)以所得的Y2-EGH-4基因盒重组载体为模板,采用引物ScPGM2+tTDH2-F和tADH1+ScPGM1-R经PCR扩增获得Y2-EGH-4线性化片段;
(8)以所得的Y2-EGH-5基因盒重组载体为模板,采用引物ScPGM1+tADH1-R和EGH1-Dn-R经PCR扩增获得Y2-EGH-5线性化片段;
(9)以所得的Y3-PPDS-1基因盒重组载体为模板,采用引物UP-F和UAS+pTDH3-R融合PCR扩增获得Y3-L-F1线性化片段。
(10)以所得的Y3-PPDS-2基因盒重组载体为模板,采用引物Dn-R和pTDH3+UAS-F融合PCR扩增获得Y3-L-F2线性化片段。
以上PCR克隆采用Q5 High-Fidelity DNA Polymerases克隆试剂盒获取线性化片段。PCR反应体系均为50μL:由1μL模板,10mM的上游引物和10mM的下引物各2μL,酶混合物25μL,去离子水补足50μL组成。酶混合物为采用Q5 High-Fidelity DNA Polymerases克隆试剂盒中的相应试剂。PCR反应程序为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。PCR结束后,进行跑胶,确认扩增成功后进行目的条带回收。
(二)酵母转化
取10ul酵母菌株甘油菌或活化后平板上的单菌落于2mL-3mL的YPD液体培养基中,放在30℃,200rpm摇床中过夜培养;将培养的菌液转至20mL YPD液体培养基中,使其初始OD值为0.2,在30℃,200rpm的摇床中培养3~4h至OD为0.6~0.9;无菌50mL离心管收菌,5000rpm离心5min,倒掉上清液,加入20mL无菌水洗涤一次,5000rpm离心5min之后倒掉上清液;加入1mL 100mM LiAc洗涤,转移至无菌1.5mL管,8000rpm离心30s,弃上清液;加入200ul100mM LiAc重悬,分装50ul到新无菌1.5mL cp管中,8000rpm离心30s,吸出上清液;分别加入无菌240ul 50%(W/V)PEG4000,36ul 1M LiAc,10ul 10mg/mL鲑鱼精DNA,(400-800ng)线性化片段(即制备重组菌株1时,加入400-800ng Y1-LKG-1线性化片段、400-800ng Y1-LKG-2线性化片段和400-800ng Y1-LKG-3线性化片段,三者浓度相同;制备重组菌株2时,加入400-800ng Y2-EGH-1线性化片段、400-800ng Y2-EGH-2线性化片段、400-800ng Y2-EGH-3、400-800ng Y2-EGH-4线性化片段和400-800ng Y2-EGH-5线性化片段,五者浓度相同;制备重组菌株3时,加入400-800ng Y3-L-P1线性化片段、400-800ng Y3-L-P2线性化片段,二者浓度相同;)和ddH2O补足至360ul,吹吸混匀。在30℃孵育30min,42℃孵育30min,涂于Sc-his-lys或YPD+G418平板,在30℃培养箱中倒置培养2-3天。
将所得的线性化片段Y1-LKG-1、Y1-LKG-2和Y1-LKG-3,一起转入起始菌株ZW04BY,其线性化后在酵母基因组中组合的插入片段如图2所示,获得重组菌株1;将所得的线性化片段Y2-EGH-1、Y2-EGH-2、Y2-EGH-3、Y2-EGH-4和Y2-EGH-5一起转入重组菌株1,在酵母基因组中组合的插入片段如图3所示,获得重组菌株2;将所得的线性化片段Y3-L-P1和Y3-L-P2一起转入重组菌株2,在酵母基因组中组合的插入片段如图4所示,获得重组菌株3,即生产人参皂苷Rd的酿酒酵母工程菌。
实施例3:产人参皂苷Rd的酿酒酵母基因工程菌的应用
1.工程菌的培养及产物提取
分别采用实施例2中重组菌生产人参皂苷Rd。具体方法如下:活化重组菌,于YPD液体培养基中30℃、220rpm条件下培养48h,得到种子液。将种子液以1%的接种量接种于30mlYPD液体培养基中,在30℃、220rpm震荡培养4天。发酵结束后,取500微升发酵液与500微升甲醇充分混匀,超声30分钟,12000rpm离心20分钟。取200微升进行产物检测。
2.HPLC、LC-MS检测条件
HPLC分析:仪器:安捷伦超高效液相色谱仪1200;色谱柱:Agilent Poroshell120EC-C18(100mm×3.0mm,2.7μm),紫外检测器,检测波长203nm;流动相:A相为纯水;B相为乙腈;起始浓度:A:85%、B:15%,流速:0.9mL/min。进样体积5μL。
柱温:30℃,检测器:PDA检测器。梯度洗脱程序:采用线性梯度洗脱,(浓度为B相百分比)0-7min 15%B线性变化至30%B,7-11min 30%B线性变化至40%B,11-17min40%B线性变化至42%B,17-25min 42%B线性变化至100%B,25-27min保持100%B。重组菌株3的发酵产物的HPLC检测图如图6所示。
LC-MS测量使用Micro ToF MS(Bruker Daltonics)配备HP1100系列LC系统(安捷伦科技),质谱参数:采用Dual AJS ESI离子源,离子扫描模式为ESI负离子或正离子扫描;扫描范围:m/z 100~1700。重组菌株3产生人参皂苷Rd的LC-MS检测图如图5所示。
3.结果
重组菌3摇瓶发酵生产了56.68±16.21mg/L人参皂苷Rd,其中其它人参皂苷的副产物为1313.75±87.33mg/L原人参二醇,55.67±1.44mg/L人参皂苷Rh2。重组菌株3的发酵产物HPLC检测图如图6所示,其发酵产量如图7所示。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (8)
1.一种生产人参皂苷Rd的酿酒酵母工程菌,其特征在于,在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β-葡萄糖苷酶EG H 1,过表达葡萄糖磷酸变位酶1、葡萄糖磷酸变位酶2和UDP-葡萄糖焦磷酸化酶,糖基转移酶Pn 1-31、糖基转移酶PnUGT53、糖基转移酶PnUGT50和原人参二醇合成酶PPDS;所述糖基转移酶PnUGT50的核苷酸序列如SEQ ID NO.1所示;所述葡萄糖磷酸变位酶1的核苷酸序列如SEQ ID NO.2所示;所述葡萄糖磷酸变位酶2的核苷酸序列如SEQ ID NO.3所示;所述UDP-葡萄糖焦磷酸化酶如SEQ ID NO.4所示;所述糖基转移酶Pn 1-31的核苷酸序列如SEQ ID NO.5所示;所述优化后的糖基转移酶PnUGT53的核苷酸序列如SEQ ID NO.6所示;原人参二醇合成酶PPDS的核苷酸序列如SEQ ID NO.7所示。
2.根据权利要求1所述的生产人参皂苷Rd的酿酒酵母工程菌,其特征在于,所述酿酒酵母基因组中整合过表达原人参二醇至人参皂苷Rd途径中的所有基因,包括原人参二醇合成酶PPDS、糖基转移酶PnUGT50和糖基转移酶Pn UGT53。
3.权利要求1所述的生产人参皂苷Rd的酿酒酵母工程菌的构建方法,其特征在于,包括以下步骤:
(1)通过在产原人参二醇的起始菌株ZW04BY酿酒酵母中敲除β-葡萄糖苷酶EG H 1,通过PCR扩增,将葡萄糖磷酸变位酶1、葡萄糖磷酸变位酶2和UDP-葡萄糖焦磷酸化酶、糖基转移酶Pn 1-31和糖基转移酶PnUGT53导入起始菌株ZW04BY,获得重组菌株1;
(2)在重组菌株1的基础上,分别在组合型启动子TDH3+UASTEF1-CIT1-CLB2和ADH 1启动子的控制之下,过表达密码子优化后的糖基转移酶PnUGT50和糖基转移酶PnUGT53,获得重组菌株2;
(3)将来自人参的原人参二醇合成酶基因PPDS进行酵母密码子优化,串联组合型启动子TDH3+UASTEF1-CIT1-CLB2和G418抗性筛选标签,一起导入起始酿酒酵母重组菌株2的δ序列位点,获得重组菌株3;
(4)获得的重组菌株3在摇瓶条件下进行发酵测产,获得的生产人参皂苷Rd的酿酒酵母工程菌。
4.根据权利要求3所述的生产人参皂苷Rd的酿酒酵母工程菌的构建方法,其特征在于,步骤(1)的具体方法为:
(1.1)Y1-LKG-1基因盒重组载体构建:
(1.1.1)以酵母菌株ZW04BY的基因组为模板,采用引物LEU(Dn)+pADH1-F和LEU2-R扩增获得同源臂下游片段;
(1.1.2)以质粒pHDE-Cas9为模板,采用引物KANMX+LEU(up)-F和KA NMX+HindIII-R进行PCR扩增,获得G418片段;
(1.1.3)以UASTEF1+CIT1+CLB2为模板,采用引物UAS+PTDH3-R和UAS+KANMX-F进行PCR扩增,获得UAS片段;
(1.1.4)将获得的同源臂下游片段、G418片段、UAS片段为模板,采用引物Leu2-up-F和UAS+pTDH3-R经融合PCR扩增,获得-LKG-1基因盒;
(1.1.5)LKG-1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-1基因盒重组载体;
(1.2)Y1-LKG-2基因盒重组载体构建:
(1.2.1)以Y2-EGH-3基因盒重组载体为模板,采用引物pTDH3+UAS-F和pTDH3+sPn50-R进行PCR扩增,获得启动子TDH3片段;
(1.2.2)以pESC-SnyPnUGT50为模板,采用引物sPn50+EGFP-R和sPn50+pTDH3-F进行PCR扩增,获得SnyPn50片段;
(1.2.3)以质粒pT4-CMV-GFP为模板,采用引物EGFP+sPnUGT50-F和EGFP+tCYC1-R进行PCR扩增,获得EGFP序列;
(1.2.4)将获得的TDH3、SnyPn50、EGFP为模板,采用引物pTDH3+UAS-F和EGFP+tCYC1-R经融合PCR扩增,获得LKG-2基因盒;
(1.2.5)LKG-2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-2基因盒重组载体;
(1.3)Y1-LKG-3基因盒重组载体构建:
(1.3.1)以酵母菌株ZW04BY的基因组为模板,采用引物Leu2-up-F和LE U2(up)+KANMX-R扩增同源臂上游片段Leu2-UP片段;
(1.3.2)以酵母菌株BY4742基因组为模板,采用引物ADH1+PNUGT53-F和pADH1+LEU2(Dn)-R进行PCR扩增,获得ADH1片段;
(1.3.3)以Y2-EGH-2基因盒重组载体为模板,采用引物PNUGT53+pADH1-R和tCYC1+GFP-F进行PCR扩增,获得PNUGT53片段;
(1.3.4)将获得的PNUGT53、ADH1、Leu2-UP片段为模板,采用引物tC YC1+GFP-F和LEU2-R经融合PCR扩增,获得LKG-3基因盒;
(1.3.5)LKG-3基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y1-LKG-3基因盒重组载体;
(1.4)将所得的Y1-LKG-1、Y1-LKG-2和Y1-LKG-3基因盒重组载体质粒线性化一起转入起始菌株ZW04BY,获得重组菌株1;
步骤(1.1.1)~(1.1.3)、(1.2.1)~(1.2.3)、(1.3.1)~(1.3.3)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min;
步骤(1.1.4)、(1.2.4)、(1.3.4)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
5.根据权利要求3所述的生产人参皂苷Rd的酿酒酵母工程菌的构建方法,其特征在于,步骤(2)的具体方法为:
(2.1)Y2-EGH-1基因盒重组载体构建:
(2.1.1)以酵母菌株BY4742基因组为模板,采用引物EGH1-UP-F和EGH1-UP+pLYS2-R进行PCR扩增,获得EGH1-UP片段;
(2.1.2)以酵母菌株W303基因组为模板,采用引物LYS2+pADH1-R和pL YS2+EGH1-UP-F进行PCR扩增,获得pLYS2片段;
(2.1.3)将获得的EGH1-UP和pLYS2片段为模板,采用引物EGH1-UP-F和LYS2+pADH1-R经融合PCR扩增,获得EGH-1基因盒;
(2.1.4)EGH-1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-1基因盒重组载体;
(2.2)Y2-EGH-2基因盒重组载体构建:
(2.2.1)以酵母菌株W303基因组为模板,采用引物ADH1+Lys2-F和ADH1+Pn1-31-R进行PCR扩增,获得pADH1片段;
(2.2.2)以三七基因组为模板,采用引物pn1-31+ADH1-F和Pn1-31+tPGI-R进行PCR扩增,获得Pn1-31片段;
(2.2.3)以得酵母菌株W303基因组为模板,采用引物tPGI+Pn1-31-F和tPGI+pTEF1-R进行PCR扩增,获得tPGI片段;
(2.2.4)以得酵母菌株W303基因组为模板,采用引物pTEF1+PNUGT53-F和pTEF1+tPGI-F扩增获得pTEF1片段;
(2.2.5)以质粒YCplac22为模板,采用引物tCYC1+PNUGT53-F和tCYC1+tPFK1-R进行PCR扩增,获得tCYC1片段;
(2.2.6)以三七基因组为模板,采用引物PNUGT53+tCYC-R和PNUGT53+pTEF1-F进行PCR扩增,获得PnUGT53;
(2.2.7)将获得的pADH1、Pn1-31、tPGI、PnUGT53、pTEF1、tCYC1片段为模板,采用引物ADH1+Lys2-F和tCYC1+tPFK1-R经融合PCR扩增,获得EGH-2基因盒;
(2.2.8)EGH-2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-2基因盒重组载体;
(2.3)Y2-EGH-3基因盒重组载体构建:
(2.3.1)以酵母菌株W303为模板,采用引物ScUGP1+tPFK1-F和ScUGP1+pTDH3-R进行PCR扩增获得ScUGP1片段;
(2.3.2)以酵母菌株W303为模板,采用引物tTDH2+pTDH3-F和tTDH2+ScPGM2-R进行PCR扩增获得pTDH2片段;
(2.3.3)以酵母菌株W303为模板,采用引物tPFK1+tCYC1-F和tPFK1+ScUGP1-R进行PCR扩增获得PFK1片段;
(2.3.4)以酵母菌株W303为模板,采用引物pTDH3+ScUGP1-F和pTDH3+tTDH2-R进行PCR扩增获得pTDH3片段;
(2.3.5)将获得的ScUGP1、PFK1、pTDH3、pTDH2片段为模板,采用引物tPFK1+tCYC1-F和tTDH2+ScPGM2-R经融合PCR扩增,获得EGH-3基因盒;
(2.3.6)EGH-3基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-3基因盒重组载体;
(2.4)Y2-EGH-4基因盒重组载体构建:
(2.4.1)以酵母菌株BY4742基因组为模板,采用引物ScPGM2+tTDH2-F和ScPGM2+pEBA1-R进行PCR扩增获得ScPGM2片段,采用引物pEBA1+ScP GM2-F和pFBA+tADH1-R进行PCR扩增获得pEBA1片段;
(2.4.2)以酵母菌株W303基因组为模板,采用引物tADH+pFBA1-F和tA DH1+ScPGM1-R进行PCR扩增获得tADH片段;
(2.4.3)将获得的ScPGM2、pEBA1、tADH片段为模板,采用引物ScPG M2+tTDH2-F和tADH1+ScPGM1-R经融合PCR扩增,获得EGH-4基因盒;
(2.4.4)EGH-4基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-4基因盒重组载体;
(2.5)Y2-EGH-5基因盒重组载体构建:
(2.5.1)以质粒YCplac22为模板,采用引物pPGK1+ScPGM1-R和pPGK1+EGH1-F进行PCR扩增获得pPGK1片段;
(2.5.2)以酿酒酵母BY4742基因组为模板,采用引物ScPGM1+tADH1-R和ScPGM1+pPGK1-F进行PCR扩增获得ScPGM1片段;
(2.5.3)以酵母菌株BY4742基因组为模板,采用引物EGH1+pPGK1-F和EGH1-Dn-R进行PCR扩增获得EGH1-Dn片段;
(2.5.4)将获得的ScPGM1、PGK1、EGH1-Dn片段为模板,采用引物ScP GM1+tADH1-R和EGH1-Dn-R经融合PCR扩增,获得EGH-P5基因盒;
(2.5.5)EGH-5基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y2-EGH-5基因盒重组载体;
(2.6)将所得的Y2-EGH-1、Y2-EGH-2、Y2-EGH-3、Y2-EGH-4和Y2-EG H-5基因盒重组载体质粒线性化一起转入重组菌株1,获得重组菌株2;
步骤(2.1.1)~(2.1.2)、(2.2.1)~(2.2.6)、(2.3.1)~(2.3.4)、(2.4.1)~(2.4.2)、(2.5.1)~(2.5.3)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min;
步骤(2.1.3)、(2.2.7)、(2.3.5)、(2.4.3)、(2.5.4)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
6.根据权利要求3所述的生产人参皂苷Rd的酿酒酵母工程菌的构建方法,其特征在于,步骤(3)的具体方法为:
(3.1)Y3-PPDS-1基因盒重组载体构建:
(3.1.1)以酵母菌株BY4742基因组为模板,采用引物UP-F和UP+KAMX-R扩增获得片段上游同源臂片段;
(3.1.2)以UASTEF1+CIT1+CLB2为模板,采用引物UAS+pTDH3-R和UAS+KANMX-F进行PCR扩增获得UAS片段;
(3.1.3)以pHDE-Cas9质粒为模板,采用引物KANMX+HindIII-R和KAN MX+UP-F进行PCR扩增获得KANMX片段;
(3.1.4)将获得的UAS、KANMX、上游同源臂片段为模板,采用引物UP-F和UAS+pTDH3-R经融合PCR扩增,获得L-F1基因盒;
(3.1.5)L-F1基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y3-PPDS-1基因盒重组载体;
(3.2)Y3-PPDS-2基因盒重组载体构建:
(3.2.1)以酵母菌株BY4742基因组为模板,采用引物Dn-R和03Dn+PRM9-F进行PCR扩增获得片段下游同源臂Dn片段;
(3.2.2)以经酵母密码子优化PPDS序列为模板,采用引物PPDS+PRM9-R和PPDS+pTDH3-F进行PCR扩增获得SnyPPDS片段;
(3.2.3)以酵母菌株W303基因组为模板,引物pTDH3+PPDS-R和pTDH3+UAS-F扩增获得启动子pTDH3片段,采用引物PRM9+03Dn-F和PRM9+PPDS-R扩增获得终止子PRM9片段;
(3.2.4)将获得的Dn、PRM9、SnyPPDS、pTDH3为模板,引物Dn-R和pTDH3+UAS-F经融合PCR扩增,获得L-F2基因盒;
(3.2.5)L-F2基因盒通过pEASY-Blunt Cloning Kit连接pEASY载体构建重组载体,得到Y3-PPDS-2基因盒重组载体;
(3.3)将所得的Y3-PPDS-1、Y3-PPDS-2和Y3-PPDS-3基因盒重组载体质粒线性化一起转入重组菌株2,获得重组菌株3;
步骤(3.1.1)~(3.1.3)、(3.2.1)~(3.2.3)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PCR反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min;
步骤(3.1.4)、(3.2.4)中PCR反应体系均为50μL:模板1μL,上游引物10mM 2μL,下游引物10mM 2μL,酶混合物25μL,去离子水补足50μL;PC R反应程序均为:94℃、5min;94℃、30S,56℃、1.5min,72℃、1min,35个循环;72℃、7min。
7.一种人参皂苷Rd的制备方法,其特征在于,将如权利要求1-2任一项所述的生产人参皂苷Rd的酿酒酵母工程菌发酵,从发酵液中获得人参皂苷Rd。
8.根据权利要求7所述的人参皂苷Rd的制备方法,其特征在于,发酵培养基的配方为20g/L葡萄糖、20g/L蛋白胨,10g/L酵母浸粉,余量为水,发酵温度条件为30℃。
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