CN114395497B - 一种以葡萄糖为底物微生物合成白皮杉醇的工程菌、构建及其应用 - Google Patents
一种以葡萄糖为底物微生物合成白皮杉醇的工程菌、构建及其应用 Download PDFInfo
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Abstract
本发明公开了一种以葡萄糖为底物微生物合成白皮杉醇的工程菌、构建和应用。本发明通过在工程菌株中过表达L‑酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶和羟化酶复合体基因,实现了白皮杉醇的从头合成策略,并显著提高了产物白皮杉醇的含量,为其安全生物转化提供了简单且有效的实施方法。本发明公开的以葡萄糖为底物微生物合成白皮杉醇的工程菌产量可达到7‑8g/L。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种以葡萄糖为底物生物合成白皮杉醇工程菌的构建及其应用。
背景技术
白皮杉醇(3,3',4,5'-四羟基反式二苯乙烯)是一种生物活性分子,为白藜芦醇的3'-羟基化产物,在葡萄、虎杖和百香果等多种天然植物中存在。它是一种极强的抗氧化剂,在自由基的清除过程中表现出比白藜芦醇更高的抗氧化活性;除此之外白皮杉醇也能抑制白血病、淋巴瘤和黑色素瘤等多种肿瘤细胞的增殖;有研究证实白皮杉醇具有提高免疫调节力、抗炎、抗菌、抑制皮肤黑色素生成和缓解动脉血管舒张多种积极作用。
白皮杉醇对人体的众多有益效果使得其在功能性食品、药品和化妆品领域中具有极高的潜在价值。然而,天然的白皮杉醇极难获取,1kg干的大黄藓根中仅能提取白皮杉醇0.3g,且白皮杉醇化学性质不稳定、易被氧化,使得天然提取的白皮杉醇极其昂贵。由于白皮杉醇容易被氧化、且顺反构型难以控制,所以制备比较困难。目前白皮杉醇的合成方法有化学合成、生物合成两种,其中化学合成存在成本高、反应条件苛刻、操作复杂、产率低等缺点。生物合成白皮杉醇属于绿色合成工艺,具有三废少且易处理等特点,应用前景广阔,但现有技术生物合成白皮杉醇产量较低,不能达到工业化生产的要求。
发明内容
本发明的目的之一是提供一种以葡萄糖为底物生物合成白皮杉醇的工程菌、构建及应用。该工程菌通过表达L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶和羟化酶复合体,实现以葡萄糖为底物生物合成白皮杉醇。
本发明的目的之二是提供一种以葡萄糖为底物微生物合成白皮杉醇的工程菌株的构建方法。本发明系统利用酶偶联法同时催化四个反应体系,四个酶催化体系分别为L-酪氨酸在L-酪氨酸氨解酶催化下生成对香豆酸、对香豆酰基辅酶A连接酶催化对香豆酸生成对香豆酰基辅酶A、然后在白藜芦醇合成酶催化下生成白藜芦醇,最后经羟化酶复合体催化生成白皮杉醇,在此同时利用过表达酪氨酸的反馈抑制调节相关基因DAHP合成酶和分支酸变位酶基因解除底物抑制作用,提高了催化反应的效率,最终提高白皮杉醇的产量。
本发明的目的之三在于提供一种以葡萄糖为底物微生物合成白皮杉醇的工程菌在以葡萄糖为底物生物合成白皮杉醇的生物转化方法。
上述工程菌中,所述过表达L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶和羟化酶复合体的方法是将所述L-酪氨酸氨解酶基因、对香豆酰基辅酶A连接酶基因、白藜芦醇合成酶基因、DAHP合成酶基因、分支酸变位酶基因、羟化酶复合体(羟化酶HpaB基因和氢化酶HpaC基因)全部置于表达质粒的启动子之后进行表达,通过hp4d启动子启动转录;优选地,所述DAHP合成酶、分支酸变位酶和羟化酶复合体在整合型质粒载体pINA1312中进行表达;所述L-酪氨酸氨解酶基因、对香豆酰基辅酶A连接酶基因、白藜芦醇合成酶通过整合型质粒载体pINA1269中进行表达;
提供一种以葡萄糖为底物生物合成白皮杉醇的工程菌,所述的工程菌含有L-酪氨酸氨解酶基因、对香豆酰基辅酶A连接酶基因、白藜芦醇合成酶、DAHP合成酶ARO4K221L基因、分支酸变位酶ARO7G139S基因、羟化酶HpaB和羟化酶HpaC基因,其核苷酸序列分别如SEQ IDNo.1,SEQ ID No.2,SEQ ID No.3,SEQ ID No.4、SEQ ID No.5、SEQ ID No.6和SEQ ID No.7所示,表达L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶、羟化酶HpaB和羟化酶HpaC。
按上述方案,所述L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶、羟化酶HpaB和羟化酶HpaC的氨基酸序列分别如SEQ ID No.8,SEQ ID No.9,SEQ ID No.10,SEQ ID No.11、SEQ ID No.12、SEQ ID No.13和SEQ ID No.14所示。
所述L-酪氨酸氨解酶基因、对香豆酰基辅酶A连接酶基因、白藜芦醇合成酶、羟化酶HpaB和羟化酶HpaC属于异源酶。所述L-酪氨酸氨解酶基因TAL来源于黄杆菌(Flavobacterium johnsoniae)(FjTAL);所述4-香豆跣辅酶A连接酶基因(对香豆酰基辅酶A连接酶基因)4CL来源于拟南芥(Arabidopsis thaliana)(At4CL);所述白藜芦醇合酶基因STS来源于葡萄(Vitis vinifera)(VvSTS);所述羟化酶HpaB来源于大肠杆菌(Escherichiacoli)(EcHpaB);所述羟化酶HpaC来源于大肠杆菌(Escherichia coli)(EcHpaC);所述DAHP合成酶ARO4K221L和分支酸变位酶ARO7G139S基因来源于解脂耶氏酵母;其中,ARO4K221L为ARO4基因的第221位的赖氨酸突变为亮氨酸;ARO7G139S为ARO7基因的第139位的甘氨酸突变为丝氨酸。
所述L-酪氨酸氨解酶基因、对香豆酰基辅酶A连接酶基因、白藜芦醇合成酶、羟化酶HpaB、羟化酶HpaC、DAHP合成酶和分支酸变位酶基因的核苷酸序列是根据解脂耶氏酵母密码子偏爱性进行密码子优化后的序列。
上述以葡萄糖为底物微生物合成白皮杉醇的工程菌的构建方法,包括以下步骤:
(1)利用PCR技术或酶切方法对载体进行线性化处理,回收产物为线性化载体片段;
利用特异性引物分别以人工合成的L-酪氨酸氨解酶基因FjTAL、对香豆酰基辅酶A连接酶基因At4CL、白藜芦醇合成酶基因VvSTS、羟化酶EcHpaB基因和羟化酶EcHpaC基因为模板进行PCR扩增,回收得到目的片段;
(2)使用无缝克隆或酶切酶连的方式将所述含有L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶和羟化酶复合体编码基因片段与所述线性化载体片段进行连接、转化,得到重组表达载体-FjTAL、重组表达载体-At4CL、重组表达载体-VvSTS、重组表达载体-EcHpaB、重组表达载体-EcHpaC;
(3)以人工合成的YIARO4K211L、YIARO7G139S基因为模板,使用Overlap PCR扩增得到融合基因:YIARO4K211L-YIARO7G139S酶切连接后构建得到重组质粒-YIARO4K211L-YIARO7G139S;
(4)分别使用特异性引物扩增hp4d-EcHpaB-XPR2、hp4d-EcHpaC-XPR2表达框,并使用Overlap PCR连接,扩增得到hp4d-EcHpaB-XPR2-hp4d-EcHpaC-XPR2双重表达框,通过无缝克隆插入重组质粒-YIARO4K211L-YIARO7G139S,最终得到重组质粒-YIARO4K211L-YIARO7G139S-EcHpaB-EcHpaC;
(5)将所得质粒-YIARO4K211L-YIARO7G139S-EcHpaB-EcHpaC经Not I酶切,线性化后转入解脂耶氏酵母Po1f中,并涂布到SC-Ura酵母缺陷型培养基上,培养至长出转化子,随机挑取若干阳性转化子,获得工程菌Ⅰ;
(7)分别使用特异性引物扩增hp4d-At4CL-XPR2、hp4d-VvSTS-XPR2表达框,并使用Overlap PCR连接,扩增得到hp4d-At4CL-XPR2-hp4d-VvSTS-XPR2双重表达框,通过无缝克隆插入重组质粒-FjTAL,最终得到重组质粒-FjTAL-At4CL-VvSTS;
(8)将所得的重组质粒-FjTAL-At4CL-VvSTS经限制性内切酶NotⅠ酶切,线性化后利用LiAC转化法转入步骤(5)的工程菌I中,并涂布到SC-Leu酵母缺陷型培养基上,培养至长出转化子,随机挑取若干阳性转化子,获得以葡萄糖为底物微生物合成白皮杉醇的工程菌Ⅱ。
白皮杉醇的生物转化方法也属于本发明的保护范围,包括以下步骤:
(1)将以葡萄糖为底物微生物合成白皮杉醇的工程菌培养,得到种子液;
(2)将所述的种子液接入到含有底物葡萄糖的发酵培养基中,进行发酵培养,生物转化合成白皮杉醇。
按上述方案,所述的生物转化时间为96-120h。
按上述方案,上述生物转化方法合成的白皮杉醇的产量为7-8g/L。
按上述方案,上述生物转化方法具体为:
将以葡萄糖为底物微生物合成白皮杉醇的工程菌划线到YNB固体培养基上培养至长出单菌落,挑取单菌落接种至YNB液体培养基中,得到种子液,所述种子液的OD600=10-12;
将种子液接入到含有底物葡萄糖的发酵培养基中,进行发酵培养,发酵过程中进行葡萄糖补料;
按上述方案,所述种子液的培养条件为28-30℃,200-225rpm。
按上述方案,所述发酵培养基组分为:底物葡萄糖8%,(NH4)2SO4 0.5%、KH2PO40.3%、MgSO4·7H2O 0.05%、微量金属溶液0.2%,维生素溶液0.1%、消泡剂204 0.05%。
按上述方案,所述发酵条件为28-30℃,200-225rpm,初始pH为5.9-6.2,发酵过程中加pH调节剂调控体系pH为5.9-6.2,具体地,每隔4.5-5.5h通过人工添加pH调节剂5M KOH调控pH为5.9-6.2。
按上述方案,在发酵阶段,每隔22-24小时添加18-22g/L葡萄糖。
按上述方案,所述YNB液体培养基为20-80g/L葡萄糖,6.7g/L无氨基酵母氮源,优选为20-60g/L葡萄糖为碳源,6.7g/L无氨基酵母氮源。
与现有技术相比,本发明具有如下有益效果:
本发明整合了L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、羟化酶复合体、DAHP合成酶和分支酸变位酶的基因工程菌株,通过过表达L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、羟化酶复合体、DAHP合成酶和分支酸变位酶的策略,显著地提高了产物白皮杉醇的含量,为白皮杉醇的安全生物转化提供了简单且有效的实施方法。该工程菌生物转化白皮杉醇的产量高达7-8g/L。
附图说明
图1为表达DAHP合成酶、分支酸变位酶、羟化酶HpaB和羟化酶HpaC的重组质粒pINA1312-YIARO4K211L-YIARO7G139S-EcHpaB-EcHpaC的构建示意图;
图2为表达L-酪氨酸氨解酶、4-香豆跣辅酶A连接酶和白藜芦醇合成酶的重组质粒pINA1269-FjTAL-At4CL-VvSTS的构建示意图。
具体实施方式
实施例1产白皮杉醇工程菌株的构建
1.pINA1312-YIARO4K211L-YIARO7G139S质粒的构建
(1)以人工合成的YIARO4K211L基因为模板,利用引物YIARO4K211L-YIARO7G139S-F1和YIARO4K211L-YIARO7G139-R1进行PCR扩增,所述扩增产物经回收后得到YIARO4K211L的目的片段,所述引物YIARO4K211L-YIARO7G139S-F1序列5'-CCCCACGTGATGCCGCCGAAAGTGGTGAT-3',所述引物YIARO4K211L-YIARO7G139S-R1序列5'-CGGGGTACCTTTCTGCGCGCGCATGCTAT-3';
(2)以人工合成的YIARO7G139S基因为模板,利用引物YIARO4K211L-YIARO7G139S-F2和YIARO4K211L-YIARO7G139S-R2进行PCR扩增,所述扩增产物经回收后得到YIARO7G139S的目的片段,所述引物YIARO4K211L-YIARO7G139S-F2序列5'-CCCCACGTGATGGATTTTACCAAAGCGGATAC-3',所述引物YIARO4K211L-YIARO7G139S-R2序列5'-CGGGGTACCTTCCAGGCGGCGCAGCAG-3';
(3)以上述回收的YIARO4K211L、YIARO7G139S目的片段同时作为模板,利用引物YIARO4K211L-YIARO7G139S-F1和YIARO4K211L-YIARO7G139S-R2进行PCR扩增,所述扩增产物经回收后得到YIARO4K211L-YIARO7G139S的融合基因片段;
(4)使用限制性内切酶Pml I、Kpn I同时对YIARO4K211L-YIARO7G139S回收产物与pINA1312质粒DNA进行双酶切,连接后得到重组质粒pINA1312-YIARO4K211L-YIARO7G139S。
2.pINA1312-YIARO4K211L-YIARO7G139S-EcHpaB-EcHpaC质粒的构建
(1)以人工合成的EcHpaB、EcHpaC基因为模板,分别利用引物EcHpaB-F、R和EcHpaC-F、R进行PCR扩增,所述扩增产物经回收后得到EcHpaB、EcHpaC的目的片段。所述引物EcHpaB-F序列5'-CCCCACGTGATGAAGCCAGAAGATTTTAGAGCTTCTACAC-3',所述引物EcHpaB-R序列5'-CGGGGTACCTCACTTTAATAACTTATCTAACATGTTAATGTCATCGTTGT-3';所述引物EcHpaC-F序列5'-CCCCACGTGATGCAATTGGACGAGCAAAGATTAAGATT-3',所述引物EcHpaC-R序列5'-CGGGGTACCTCAGATTGCAGCTTCCATTTCCAAC-3';
(2)使用限制性内切酶Pml I、Kpn I同时对EcHpaB、EcHpaC回收产物与pINA1269质粒DNA进行双酶切,连接后得到两种重组质粒pINA1269-EcHpaB和pINA1269-EcHpaC;
(3)以pINA1269-EcHpaB质粒DNA为模板,使用引物Expression Cassette-F1、R1进行PCR扩增,回收获得包含EcHpaB基因的hp4d-EcHpaB-XPR2 DNA片段;所述引物ExpressionCassette-F1序列5'-CGCGCGAGGCAGCAGATCCACTAGTAGAGACCGGGTTGGCGGC-3',所述引物Expression Cassette-R1序列5'-TCAGCATGCACGCGTATCGATAAGCTAGCTTGCAAATTAAAGCCTTCGAGCGTCCCAA-3';
(4)以pINA1269-EcHpaC质粒DNA为模板,使用引物Expression Cassette-F2、R2进行PCR扩增,回收获得包含EcHpaC基因的hp4d-EcHpaC-XPR2 DNA片段;所述引物ExpressionCassette-F2序列5'-AAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTAGCTTATCGATACGCGTGCA-3',所述引物Expression Cassette-R2序列5'-TTTACCGCAGCAGATCCGCGGCTATTTACAACAATATCTGGTCAAATTTCAGTTTCGT-3';
(5)以上述hp4d-EcHpaB-XPR2 DNA片段、hp4d-EcHpaC-XPR2 DNA片段同时作为模板,使用引物Expression Cassette-F1、R2进行PCR扩增,回收获得包含EcHpaB、EcHpaC基因的双重表达框,即:hp4d-EcHpaB-XPR2-hp4d-EcHpaB-XPR2;
(6)使用限制性内切酶Spe I对上述构建pINA1312-YIARO4K211L-YIARO7G139S质粒进行酶切,回收酶切产物;将上述hp4d-EcHpaB-XPR2-hp4d-EcHpaB-XPR2双重表达框与酶切产物进行无缝连接,获得重组质粒pINA1312-YIARO4K211L-YIARO7G139S-EcHpaB-EcHpaC。
3.解脂耶氏酵母工程菌Ⅰ的构建
将上述质粒pINA1312-YIARO4K211L-YIARO7G139S-EcHpaB-EcHpaC经限制性内切酶NotⅠ酶切,线性化后利用LiAC转化法转入解脂耶氏酵母Po1f中,并涂布到SC-Ura酵母缺陷型培养基上,于30℃培养至长出转化子,挑取阳性转化子,获得初始工程菌株Ⅰ。
4.pINA1269-FjTAL-At4CL-VvSTS质粒的构建
(1)以人工合成的FjTAL、At4CL、VvSTS基因为模板,分别利用引物FjTAL-F、R,At4CL-F、R和VvSTS-F、R进行PCR扩增,所述扩增产物经回收后得到FjTAL、At4CL和VvSTS的目的片段。所述引物FjTAL-F序列5'-CCCCACGTGATGAACACCATCAACGAGTACCTGT-3',所述引物FjTAL-R序列5'-CGGGGTACCCTAGTTGTTAATCAGATGGTCCTTGACCT-3';所述引物At4CL-F序列5'-CCCCACGTGATGGCGCCACAAGAACAAGC-3',所述引物At4CL-R序列5'-CCCCACGTGTTAATGCAGAACGGATTGTTGATTCTCTTG-3';所述引物VvSTS-F序列5'-CCCCACGTGATGGCTTCAGTTGAAGAGTTTCGTAAC-3',所述引物VvSTS-R序列5'-CGGGGTACCTTAATTGGTAACTGTAGGGATTGAGTGTAGTACT-3'。
(2)使用限制性内切酶Pml I、Kpn I同时对FjTAL、At4CL和VvSTS回收产物与pINA1269质粒DNA进行双酶切,连接后得到三种重组质粒pINA1269-FjTAL、pINA1269-At4CL和pINA1269-VvSTS;
(3)以pINA1269-At4CL质粒DNA为模板,使用引物Expression Cassette-F1、R1进行PCR扩增,回收获得包含At4CL基因的hp4d-At4CL-XPR2 DNA片段;
(4)以pINA1269-VvSTS质粒DNA为模板,使用引物Expression Cassette-F2、R2进行PCR扩增,回收获得包含VvSTS基因的hp4d-VvSTS-XPR2 DNA片段;
(5)以上述hp4d-At4CL-XPR2 DNA片段、hp4d-VvSTS-XPR2 DNA片段同时作为模板,使用引物Expression Cassette-F1、R2进行PCR扩增,回收获得包含At4CL、VvSTS基因的双重表达框,即:hp4d-At4CL-XPR2-hp4d-VvSTS-XPR2;
(6)使用限制性内切酶Spe I对上述构建pINA1269-FjTAL质粒进行酶切,回收酶切产物;将上述hp4d-At4CL-XPR2-hp4d-VvSTS-XPR2双重表达框与酶切产物进行无缝连接,获得重组质粒pINA1269-FjTAL-At4CL-VvSTS。
5.解脂耶氏酵母工程菌Ⅱ的构建
将上述质粒pINA1269-FjTAL-At4CL-VvSTS经限制性内切酶NotⅠ酶切,线性化后利用LiAC转化法转入工程菌Ⅰ中,并涂布到SC-Leu酵母缺陷型培养基上,于30℃培养至长出转化子,挑取阳性转化子,获得最终工程菌株Ⅱ。
实施例2解脂耶氏酵母工程菌分批补料发酵
1.将工程菌Ⅱ划线到YNB固体培养基上培养至长出单菌落,挑取单菌落,接种至YNB液体培养基中,培养15h,得到种子液,所述YNB培养基:20g/L葡萄糖为碳源,6.7g/L无氨基酵母氮源;所述种子液的培养条件为30℃,225rpm,所述种子液的OD600=10;
2.将2%所述的种子液接入到2L发酵培养基中,进行发酵培养;所述发酵培养基组分为:底物葡萄糖8%,(NH4)2SO4 0.5%、KH2PO4 0.3%、MgSO4·7H2O 0.05%、微量金属溶液0.2%,维生素溶液0.1%、消泡剂204 0.05%;所述发酵条件为30℃,225rpm,初始pH为6.0;在发酵阶段,每隔24小时添加20g/L葡萄糖;每隔5h通过人工添加5M KOH调控pH为6.0。
3.转化120h结束后利用高效液相色谱(HPLC)检测白皮杉醇的产量。
结果表明:
本发明整合了L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、羟化酶复合体、DAHP合成酶和分支酸变位酶的基因工程菌株,利用hp4d启动子使L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、羟化酶复合体、DAHP合成酶和分支酸变位酶在解脂耶氏酵母中表达,从而提高了白皮杉醇产量,为利用工程菌株生物转化生产白皮杉醇提供了可行性,最终白皮杉醇产量达到了7.5g/L。
高效液相色谱检测白皮杉醇:
(1)取上述发酵液1mL,5000rpm离心10min,取上清液并稀释一百倍;
(2)所述上清稀释液经0.22μm滤膜过滤于棕色液相瓶中,得到待测样品;
(3)高效液相色谱的色谱条件为:色谱柱为C18(250mm*4.6mm,5μm)或等效色谱柱,检测波长为280nm;流动相A为水(含0.05%三氟乙酸),B为乙腈;流速为1mL/min,进样量为10μL,柱温箱温度为30℃;梯度洗脱条件:0-1min,90%A和10%B;1-5min,80%A和20%B;5-8min,60%A和40%B;8-15min,100%B;15-20min,70%A和30%B;20-30min,90%A和10%B。
(5)根据白皮杉醇标准品的峰面积,最终计算白皮杉醇的含量,结果表明,发酵液中白皮杉醇含量达7.5g/L。
上述实施例中载体、基因和耗材均可从商业途径中得到。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
SEQUENCE LISTING
<110> 河北维达康生物科技有限公司
<120> 一种以葡萄糖为底物微生物合成白皮杉醇的工程菌、构建和应用
<130> 1
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 1521
<212> DNA
<213> 人工合成
<400> 1
atgaacacca tcaacgagta cctgtctctg gaagagttcg aggccatcat cttcggcaac 60
cagaaggtga ccatctctga cgtggtggtg aaccgagtga acgagtcttt caacttcctg 120
aaggaattct ctggcaacaa ggtgatctac ggcgtgaaca ccggcttcgg ccccatggct 180
cagtaccgaa tcaaggaatc tgaccagatc cagctgcagt acaacctgat ccgatctcac 240
tcttctggca ccggcaagcc tctgtctccc gtgtgcgcca aggccgccat tctggcccga 300
ctgaacaccc tgtcgctggg caactctggc gtgcacccct ctgtgatcaa cctgatgtct 360
gagctgatca acaaggacat tacccctctg atcttcgagc acggcggcgt gggcgcctct 420
ggcgacctgg tgcagctgtc tcacctggct ctggtgctga tcggcgaggg cgaagtgttc 480
tacaagggcg agcgacgacc cactcctgag gtgttcgaga tcgagggact gaagcccatc 540
caggtcgaga tccgagaggg actcgccctg atcaacggca cctccgtgat gaccggcatc 600
ggcgtggtga acgtgtacca cgccaagaag ctgctggact ggtccctgaa gtcctcttgc 660
gccattaacg agctggtgca ggcctacgac gaccacttct ctgccgagct gaaccagacc 720
aagcgacaca agggccagca agagatcgcc ctgaagatgc gacagaacct gtctgactct 780
accctgattc gaaagcgaga ggaccacctg tactctggcg agaacaccga ggaaatcttc 840
aaggaaaagg tgcaagagta ctactctctc cgatgcgtgc cccagattct gggccccgtg 900
ctggaaacca tcaacaacgt ggcctctatt ctcgaggacg agttcaactc tgccaacgac 960
aaccccatca tcgacgtgaa gaaccagcac gtctaccacg gcggcaactt ccacggcgac 1020
tacatctccc tcgagatgga caagctgaag atcgtgatca ccaagctgac catgctggcc 1080
gagcgacagc tgaactacct gctgaactct aagatcaacg agctgctgcc tcctttcgtg 1140
aacctgggca ccctgggctt caacttcggc atgcagggcg tgcagttcac cgccacctct 1200
accaccgccg agtctcagat gctgtctaac cccatgtacg tgcactctat ccccaacaac 1260
aacgataacc aggacatcgt gtctatgggc accaactccg ccgtgattac ctctaaggtg 1320
atcgagaacg ccttcgaggt gctggccatc gagatgatca ccatcgtgca ggccattgac 1380
tacctgggcc agaaggacaa gatctcttct gtgtctaaga agtggtacga cgagattcga 1440
aacatcatcc ccacctttaa ggaagatcag gtgatgtacc ccttcgtgca gaaggtcaag 1500
gaccatctga ttaacaacta g 1521
<210> 2
<211> 1620
<212> DNA
<213> 人工合成
<400> 2
atggcgccac aagaacaagc agtaagtcaa gttatggaaa agcaatcaaa taataataat 60
tccgacgtga tctttagaag taagctacct gacatctaca ttccaaatca cttgtcactg 120
catgactata tttttcaaaa catctctgaa ttcgccacaa aaccttgttt aatcaatgga 180
ccaaccggtc atgtgtacac ttattcagat gtccatgtta ttagtaggca aattgccgct 240
aattttcata aattgggagt taaccaaaat gatgttgtca tgttactgct accaaactgc 300
cctgagtttg tcttgagttt tctagccgca tcattcagag gcgcaacagc tacagcagcc 360
aatcctttct ttactccagc cgaaattgca aagcaggcca aagcctccaa tactaaatta 420
atcatcactg aagctagata cgtagataaa attaagcctc ttcaaaatga tgatggtgtc 480
gtgattgttt gtatagatga taacgagtct gtcccaattc cagaaggatg tttaaggttc 540
acggaattaa ctcaaagtac gacagaagca tctgaagtca tagactctgt tgaaatttct 600
ccggatgatg ttgtcgcttt accatattct tcagggacta cgggcctgcc taaaggcgtt 660
atgttaactc ataagggtct ggtaactagt gtggcgcaac aagttgatgg agaaaatcct 720
aatttatatt ttcattcaga tgacgtcatt ctgtgtgtgt tacctatgtt tcacatctat 780
gcactgaaca gtatcatgtt atgcggttta agagtcggcg ccgctatcct gatcatgcct 840
aaatttgaaa ttaatcttct tctggaacta attcagagat gtaaagtcac agttgcccca 900
atggttccac cgattgtcct tgctattgct aagtctagtg aaacagaaaa atacgattta 960
tcaagtatta gagttgttaa gagtggtgcc gcccctttag gcaaagaact agaagatgcc 1020
gttaatgcta agtttccgaa tgctaagtta ggtcaaggct acggtatgac ggaagccggt 1080
ccggtcttag ctatgtctct tggttttgcc aaggagccgt ttccggttaa atccggtgca 1140
tgcggtacag tggtcaggaa tgccgagatg aagatagtcg accctgatac cggcgattct 1200
ctttctagaa atcagcccgg cgaaatttgc ataagaggtc accaaattat gaagggttat 1260
cttaataacc cagcggctac tgcagagact attgataaag acggctggtt gcatactggt 1320
gacattggct taattgacga tgatgacgag ttgttcatag ttgacagatt aaaagagttg 1380
ataaaatata agggattcca agtggctcct gccgaattgg aagccctttt gattggtcat 1440
cccgacatta cagatgtggc cgtcgtcgcc atgaaggaag aagcagctgg tgaagtacca 1500
gttgcctttg tagttaaatc taaagattct gaactgtcag aagacgatgt gaaacaattc 1560
gtttccaagc aagttaaatc atgtgtttta caagagaatc aacaatccgt tctgcattaa 1620
<210> 3
<211> 1179
<212> DNA
<213> 人工合成
<400> 3
atggcttcag ttgaagagtt tcgtaacgct caacgtgcta agggcccagc cactattttg 60
gctataggaa ccgcaactcc tgatcattgt gtctaccaat ccgattacgc tgattattac 120
ttcagagtta ctaaatccga gcatatgacg gaattaaaga aaaaatttaa tagaatctgc 180
gacaaatcca tgatcaagaa gcgttatatc catttaactg aagaaatgct tgaagaacat 240
cctaatatcg gtgcttatat ggctccatca cttaatataa gacaagaaat tataacagcc 300
gaagtgccaa gattaggcag agatgccgca cttaaagctt tgaaggaatg ggggcagcca 360
aagtcaaaga ttacgcattt agtattttgt acaacatctg gcgttgaaat gcctggggca 420
gattacaaac tggctaatct tctaggcttg gaaacgagtg ttcgtagagt catgctatat 480
caccaaggtt gttacgcggg cggaacagtt ttacgtacgg ctaaggatct tgccgaaaat 540
aatgcaggtg cccgtgtctt agttgtatgt tcagaaatta cagtagtcac attcagaggc 600
ccatctgaag acgcattaga tagcttagtt ggtcaggcat tgtttggtga cggatcaagt 660
gctgtgattg tcgggtcaga tcccgatgta tcaatagaaa gaccattgtt tcagttggtc 720
tcagccgctc aaacttttat tcccaactca gcaggtgcaa tagccggtaa cctacgtgag 780
gtggggttaa ccttccattt atggcctaat gttccaactt tgatatctga aaatatagaa 840
aaatgcttaa cacaagcgtt cgaccccttg ggtatttcag actggaattc tttgttttgg 900
atagctcatc cgggtggtcc agctatctta gacgcagtag aggcaaagtt gaacctggaa 960
aaaaagaagc tagaagccac tagacatgtc ctatccgagt acggaaacat gagctccgca 1020
tgcgttttgt tcattttgga tgaaatgaga aaaaagagcc ttaaaggaga aaatgccact 1080
actggtgaag gtttggattg gggcgttttg ttcggttttg gtcctggatt gactattgag 1140
actgtagtac tacactcaat ccctacagtt accaattaa 1179
<210> 4
<211> 1074
<212> DNA
<213> 人工合成
<400> 4
atgtcccgtt cctcctctcc caacgcctcc tctgctgagg acgtgcgaat tctgggctac 60
gaccccctcc tcgctcccgc tcttctccag actgaggttg cctccaccaa aaacgcccga 120
gagaccgtct ccaagggccg aaaggactcc attgatgtca tcaccggcaa gtccgacaag 180
ttgctgtgca ttgtcggtcc ctgctccctc cacgacccca aggccgccat ggagtacgcc 240
cagcgactca aggagctgtc tgacaagctg tctggtgagc tcgtcatcgt tatgcgagcc 300
tacctcgaga agccccgaac caccgttggc tggaagggtc tgatcaacga ccccgacatg 360
gacgagtctt tcaacatcaa caagggtctg cgactctccc gaaaggtctt ctgcgacctt 420
accgatctgg gtctgcccat tgcctccgag atgctcgata ccatttctcc ccagttcctg 480
gccgacctgc tctccctggg tgccattggt gctcgaacca ccgagtccca gctgcaccga 540
gagctcgcct ccggtctgtc tttccccgtt ggtttcaaga acggaaccga cggtactctg 600
ggtgttgccg ttgatgctgt ccaggccgcc tctcaccctc accacttcat gggtgtcacc 660
ctgcagggtg ttgccgccat caccaccacc aagggtaacg agaactgctt catcattctg 720
cgaggaggta agaagggcac caactacgac gccgagtccg tcgccgagtg caagaaggcc 780
accgagtcca tgctcatggt tgactgctct cacggcaact ccaacaagga ctaccgaaac 840
cagcccaagg tttccaaggc cgttgctgag caggttgctg ctggcgagaa gaagatcatc 900
ggtgtcatga tcgagagtaa tatccacgag ggcaaccaga aggtccccaa ggagggcccc 960
tctgccctta aatacggtgt ctccatcacc gacgcctgtg tctcttggga gaccaccgtg 1020
gacatgctca ccgagctggc caacgccgtc aaggagcgac gaaacaagaa ctaa 1074
<210> 5
<211> 771
<212> DNA
<213> 人工合成
<400> 5
atggatttta ccaaagcgga taccgtgctg gatctggcga acattcgcga tagcctggtg 60
cgcatggaag ataccattgt gtttaacctg attgaacgcg cgcagttttg ccgcagcgaa 120
tttgtgtata aagcgggcaa cagcgatatt ccgggcttta aaggcagcta tctggattgg 180
tttctgcagg aaagcgaaaa agtgcatgcg aaactgcgcc gctatgcggc gccggatgaa 240
caggcgtttt ttccggatga tctgccggaa gcgattctgc cgccgattga ttatgcgccg 300
attctggcgc cgtatagcaa agaagtgagc gtgaacgatg aaattaaaaa aatttatacc 360
gatgatattg tgccgctggt gtgcgcgggc accggcgatc agccggaaaa ctatagcagc 420
gtgatggtgt gcgatattga aaccctgcag gcgctgagcc gccgcattca ttttggcaaa 480
tttgtggcgg aaagcaaatt tctgagcgaa accgaacgct ttaccgaact gattaaaaac 540
aaagatattg cgggcattga agcggcgatt accaacagca aagtggaaga aaccattctg 600
gcgcgcctgg gcgaaaaagc gctggcgtat ggcaccgatc cgaccctgcg ctggagccag 660
cgcacccagg gcaaagtgga tagcgaagtg gtgaaacgca tttataaaga atgggtgatt 720
ccgctgacca aaaaagtgga agtggattat ctgctgcgcc gcctggaata a 771
<210> 6
<211> 1563
<212> DNA
<213> 人工合成
<400> 6
atgaagccag aagattttag agcttctaca cagagaccat ttacaggtga agaatatctg 60
aagagtttgc aagatggcag ggaaatatac atctatggtg agagagttaa agacgttact 120
acgcatcccg cattcagaaa tgcagcagct agtgtagcac agctttatga tgctttacac 180
aaaccagaaa tgcaagattc tttatgttgg aataccgata caggttctgg aggctatacc 240
cataaatttt ttagagttgc aaagtccgcg gatgatctac gtcaacaaag agatgctatt 300
gctgagtggt cccgtttaag ttatggttgg atgggtagaa cgccagatta caaggctgca 360
tttggttgtg ctctgggtgc caacccaggt ttttacggac aattcgaaca gaacgcgaga 420
aactggtaca ccaggattca agaaactgga ttatacttta accacgccat agtgaaccct 480
cctattgata gacatttacc aactgacaag gttaaggacg tttacattaa attggaaaaa 540
gaaacagacg ctggtatcat cgtctctggt gctaaggtag ttgccacgaa ttctgctttg 600
acccattata acatgattgg tttcggctcc gcgcaagtga tgggtgaaaa tccggatttc 660
gctctaatgt tcgttgctcc aatggacgca gacggcgtga aattgatttc aagggcttct 720
tatgaaatgg tcgcaggagc tacgggctct ccatatgact accccttatc ttctaggttc 780
gatgaaaatg atgccatttt ggtgatggat aacgtgctta tcccatggga aaatgtttta 840
atctacagag atttcgatag atgtagaaga tggacaatgg aaggcgggtt tgcgagaatg 900
taccccttgc aagcatgtgt aaggttggca gttaaattgg atttcataac ggctttacta 960
aaaaagtctt tggaatgcac tggaacgttg gagttccgtg gtgtccaggc tgacttaggt 1020
gaagtagttg cttggaggaa tactttttgg gccttgagtg atagcatgtg ttccgaagct 1080
acaccgtggg taaacggtgc ttatttgcct gaccatgctg ccctacaaac gtacagagtc 1140
ttagctccaa tggcttatgc aaaaattaag aacattatcg agaggaacgt tacttctgga 1200
ttgatatact tgccttcaag tgcgagggat ttaaataatc cacaaattga tcaatacctt 1260
gctaagtacg ttaggggatc taatggaatg gatcatgtac aaagaattaa gattctgaag 1320
ctgatgtggg atgctatcgg tagtgagttc ggtggtaggc atgaattata tgagatcaat 1380
tacagtggtt ctcaggatga aatccgttta caatgtctga ggcaagctca aaatagcggt 1440
aatatggaca agatgatggc aatggtcgat agatgcctat ctgaatacga tcaagatggc 1500
tggaccgttc cacacttaca caacaacgat gacattaaca tgttagataa gttattaaag 1560
tga 1563
<210> 7
<211> 513
<212> DNA
<213> 人工合成
<400> 7
atgcaattgg acgagcaaag attaagattc agagatgcta tggcttcact ttcagctgca 60
gtcaatatta ttacaaccga aggtgacgct ggtcagtgtg gtattacagc aacggcagtt 120
tgttctgtta cagatactcc accaagtttg atggtgtgca ttaacgctaa ctccgcaatg 180
aatccagtct ttcaagggaa tgggaaattg tgtgtaaacg tgttgaatca tgaacaagag 240
ttaatggcaa gacacttcgc tgggatgact gggatggcaa tggaagagag attttcccta 300
tcttgttggc agaagggtcc tctggcacaa cctgtattga aaggttcttt ggcgtcctta 360
gagggtgaaa tcagggatgt ccaggctatt gggacacacc tggtttatct ggttgaaatt 420
aagaacatta ttctgtcagc cgaagggcac ggtcttattt acttcaaaag gcgttttcac 480
ccagttatgt tggaaatgga agctgcaatc tga 513
<210> 8
<211> 506
<212> PRT
<213> 人工合成
<400> 8
MNTINEYLSL EEFEAIIFGN QKVTISDVVV NRVNESFNFL KEFSGNKVIY GVNTGFGPMA 60
QYRIKESDQI QLQYNLIRSH SSGTGKPLSP VCAKAAILAR LNTLSLGNSG VHPSVINLMS 120
ELINKDITPL IFEHGGVGAS GDLVQLSHLA LVLIGEGEVF YKGERRPTPE VFEIEGLKPI 180
QVEIREGLAL INGTSVMTGI GVVNVYHAKK LLDWSLKSSC AINELVQAYD DHFSAELNQT 240
KRHKGQQEIA LKMRQNLSDS TLIRKREDHL YSGENTEEIF KEKVQEYYSL RCVPQILGPV 300
LETINNVASI LEDEFNSAND NPIIDVKNQH VYHGGNFHGD YISLEMDKLK IVITKLTMLA 360
ERQLNYLLNS KINELLPPFV NLGTLGFNFG MQGVQFTATS TTAESQMLSN PMYVHSIPNN 420
NDNQDIVSMG TNSAVITSKV IENAFEVLAI EMITIVQAID YLGQKDKISS VSKKWYDEIR 480
NIIPTFKEDQ VMYPFVQKVK DHLINN 506
<210> 9
<211> 539
<212> PRT
<213> 人工合成
<400> 9
MAPQEQAVSQ VMEKQSNNNN SDVIFRSKLP DIYIPNHLSL HDYIFQNISE FATKPCLING 60
PTGHVYTYSD VHVISRQIAA NFHKLGVNQN DVVMLLLPNC PEFVLSFLAA SFRGATATAA 120
NPFFTPAEIA KQAKASNTKL IITEARYVDK IKPLQNDDGV VIVCIDDNES VPIPEGCLRF 180
TELTQSTTEA SEVIDSVEIS PDDVVALPYS SGTTGLPKGV MLTHKGLVTS VAQQVDGENP 240
NLYFHSDDVI LCVLPMFHIY ALNSIMLCGL RVGAAILIMP KFEINLLLEL IQRCKVTVAP 300
MVPPIVLAIA KSSETEKYDL SSIRVVKSGA APLGKELEDA VNAKFPNAKL GQGYGMTEAG 360
PVLAMSLGFA KEPFPVKSGA CGTVVRNAEM KIVDPDTGDS LSRNQPGEIC IRGHQIMKGY 420
LNNPAATAET IDKDGWLHTG DIGLIDDDDE LFIVDRLKEL IKYKGFQVAP AELEALLIGH 480
PDITDVAVVA MKEEAAGEVP VAFVVKSKDS ELSEDDVKQF VSKQVKSCVL QENQQSVLH 539
<210> 10
<211> 392
<212> PRT
<213> 人工合成
<400> 10
MASVEEFRNA QRAKGPATIL AIGTATPDHC VYQSDYADYY FRVTKSEHMT ELKKKFNRIC 60
DKSMIKKRYI HLTEEMLEEH PNIGAYMAPS LNIRQEIITA EVPRLGRDAA LKALKEWGQP 120
KSKITHLVFC TTSGVEMPGA DYKLANLLGL ETSVRRVMLY HQGCYAGGTV LRTAKDLAEN 180
NAGARVLVVC SEITVVTFRG PSEDALDSLV GQALFGDGSS AVIVGSDPDV SIERPLFQLV 240
SAAQTFIPNS AGAIAGNLRE VGLTFHLWPN VPTLISENIE KCLTQAFDPL GISDWNSLFW 300
IAHPGGPAIL DAVEAKLNLE KKKLEATRHV LSEYGNMSSA CVLFILDEMR KKSLKGENAT 360
TGEGLDWGVL FGFGPGLTIE TVVLHSIPTV TN 392
<210> 11
<211> 357
<212> PRT
<213> 人工合成
<400> 11
MSRSSSPNAS SAEDVRILGY DPLLAPALLQ TEVASTKNAR ETVSKGRKDS IDVITGKSDK 60
LLCIVGPCSL HDPKAAMEYA QRLKELSDKL SGELVIVMRA YLEKPRTTVG WKGLINDPDM 120
DESFNINKGL RLSRKVFCDL TDLGLPIASE MLDTISPQFL ADLLSLGAIG ARTTESQLHR 180
ELASGLSFPV GFKNGTDGTL GVAVDAVQAA SHPHHFMGVT LQGVAAITTT KGNENCFIIL 240
RGGKKGTNYD AESVAECKKA TESMLMVDCS HGNSNKDYRN QPKVSKAVAE QVAAGEKKII 300
GVMIESNIHE GNQKVPKEGP SALKYGVSIT DACVSWETTV DMLTELANAV KERRNKN 357
<210> 12
<211> 256
<212> PRT
<213> 人工合成
<400> 12
MDFTKADTVL DLANIRDSLV RMEDTIVFNL IERAQFCRSE FVYKAGNSDI PGFKGSYLDW 60
FLQESEKVHA KLRRYAAPDE QAFFPDDLPE AILPPIDYAP ILAPYSKEVS VNDEIKKIYT 120
DDIVPLVCAG TGDQPENYSS VMVCDIETLQ ALSRRIHFGK FVAESKFLSE TERFTELIKN 180
KDIAGIEAAI TNSKVEETIL ARLGEKALAY GTDPTLRWSQ RTQGKVDSEV VKRIYKEWVI 240
PLTKKVEVDY LLRRLE 256
<210> 13
<211> 520
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<213> 人工合成
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MKPEDFRAST QRPFTGEEYL KSLQDGREIY IYGERVKDVT THPAFRNAAA SVAQLYDALH 60
KPEMQDSLCW NTDTGSGGYT HKFFRVAKSA DDLRQQRDAI AEWSRLSYGW MGRTPDYKAA 120
FGCALGANPG FYGQFEQNAR NWYTRIQETG LYFNHAIVNP PIDRHLPTDK VKDVYIKLEK 180
ETDAGIIVSG AKVVATNSAL THYNMIGFGS AQVMGENPDF ALMFVAPMDA DGVKLISRAS 240
YEMVAGATGS PYDYPLSSRF DENDAILVMD NVLIPWENVL IYRDFDRCRR WTMEGGFARM 300
YPLQACVRLA VKLDFITALL KKSLECTGTL EFRGVQADLG EVVAWRNTFW ALSDSMCSEA 360
TPWVNGAYLP DHAALQTYRV LAPMAYAKIK NIIERNVTSG LIYLPSSARD LNNPQIDQYL 420
AKYVRGSNGM DHVQRIKILK LMWDAIGSEF GGRHELYEIN YSGSQDEIRL QCLRQAQNSG 480
NMDKMMAMVD RCLSEYDQDG WTVPHLHNND DINMLDKLLK 520
<210> 14
<211> 170
<212> PRT
<213> 人工合成
<400> 14
MQLDEQRLRF RDAMASLSAA VNIITTEGDA GQCGITATAV CSVTDTPPSL MVCINANSAM 60
NPVFQGNGKL CVNVLNHEQE LMARHFAGMT GMAMEERFSL SCWQKGPLAQ PVLKGSLASL 120
EGEIRDVQAI GTHLVYLVEI KNIILSAEGH GLIYFKRRFH PVMLEMEAAI 170
Claims (10)
1.一种以葡萄糖为底物生物合成白皮杉醇的解脂耶氏酵母工程菌,其特征在于:所述的工程菌含有L-酪氨酸氨解酶基因、对香豆酰基辅酶A连接酶基因、白藜芦醇合成酶、DAHP合成酶ARO4K221L、分支酸变位酶ARO7G139S基因、羟化酶HpaB基因和羟化酶HpaC基因,其核苷酸序列分别如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQID No.6和SEQ ID No.7所示,表达L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶和羟化酶复合体。
2.根据权利要求1所述的工程菌,其特征在于:所述L-酪氨酸氨解酶基因来源于黄杆菌(Flavobacterium johnsoniae)(FjTAL) ;所述对香豆酰基辅酶A连接酶基因来源于拟南芥( Arabidopsis thaliana)(At4CL);所述白藜芦醇合酶基因来源于葡萄(Vitis vinifera)(VvSTS);所述DAHP合成酶ARO4K221L基因和分支酸变位酶ARO7G139S基因来源于解脂耶氏酵母,其中,ARO4 K221L为ARO4基因的第221位的赖氨酸突变为亮氨酸;ARO7 G139S为ARO7基因的第139位的甘氨酸突变为丝氨酸;所述羟化酶HpaB和羟化酶HpaC基因来源于大肠杆菌;
L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶、DAHP合成酶、分支酸变位酶、羟化酶HpaB和羟化酶HpaC的氨基酸序列分别如SEQ ID No.8、SEQ ID No.9、SEQ IDNo.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13和SEQ ID No.14所示。
3.权利要求1所述的以葡萄糖为底物微生物合成白皮杉醇的工程菌的构建方法,包括以下步骤:
(1)利用PCR技术或酶切方法对载体进行线性化处理,回收产物为线性化载体片段,利用特异性引物分别以人工合成的L-酪氨酸氨解酶基因FjTAL、对香豆酰基辅酶A连接酶基因At4CL、白藜芦醇合成酶基因VvSTS、羟化酶EcHpaB基因和羟化酶EcHpaC基因为模板进行PCR扩增,回收得到目的片段;
(2)使用无缝克隆或酶切酶连的方式将所述含有L-酪氨酸氨解酶、对香豆酰基辅酶A连接酶、白藜芦醇合成酶和羟化酶复合体编码基因片段与所述线性化载体片段进行连接、转化,得到重组表达载体-FjTAL、重组表达载体-At4CL、重组表达载体-VvSTS、重组表达载体-EcHpaB、重组表达载体-EcHpaC;
(3)以人工合成的YIARO4 K211L 、YIARO7 G139S基因为模板,使用Overlap PCR扩增得到融合基因:YIARO4 K211L -YIARO7 G139S酶切连接后构建得到重组质粒-YIARO4 K211L -YIARO7 G139S;
(4)分别使用特异性引物扩增hp4d-EcHpaB-XPR2、hp4d-EcHpaC-XPR2表达框,并使用Overlap PCR连接,扩增得到hp4d-EcHpaB-XPR2-hp4d-EcHpaC-XPR2双重表达框,通过无缝克隆插入重组质粒-YIARO4 K211L -YIARO7 G139S,最终得到重组质粒-YIARO4 K211L -YIARO7 G139S - EcHpaB-EcHpaC;
(5)将所得质粒-YIARO4 K211L -YIARO7 G139S -EcHpaB-EcHpaC经Not I酶切,线性化后转入解脂耶氏酵母Po1f中,并涂布到SC-Ura酵母缺陷型培养基上,于30℃培养至长出转化子,随机挑取若干阳性转化子,获得工程菌Ⅰ;
(7)分别使用特异性引物扩增hp4d-At4CL-XPR2、hp4d-VvSTS-XPR2表达框,并使用Overlap PCR连接,扩增得到hp4d-At4CL-XPR2-hp4d-VvSTS-XPR2双重表达框,通过无缝克隆插入重组质粒-FjTAL,最终得到重组质粒-FjTAL-At4CL-VvSTS;
(8)将所得的重组质粒-FjTAL-At4CL-VvSTS经限制性内切酶Not Ⅰ酶切,线性化后利用LiAC转化法转入步骤(5)的工程菌Ⅰ中,并涂布到SC-Leu酵母缺陷型培养基上,于30℃培养至长出转化子,随机挑取若干阳性转化子,获得工程菌Ⅱ。
4.白皮杉醇的生物转化方法,其特征在于:包括以下步骤:
将权利要求1所述的以葡萄糖为底物微生物合成白皮杉醇的工程菌培养,得到种子液;
将所述的种子液接入到含有底物葡萄糖的发酵培养基中,进行发酵培养,生物转化合成白皮杉醇。
5.根据权利要求4所述的白皮杉醇的生物转化方法,其特征在于:所述的生物转化时间为96h-120h。
6.根据权利要求4所述的白皮杉醇的生物转化方法,其特征在于:其合成的白皮杉醇的产量为7-8g/L。
7.根据权利要求4所述的白皮杉醇的生物转化方法,其特征在于:具体步骤为:
将以葡萄糖为底物微生物合成白皮杉醇的工程菌进行划线到YNB固体培养基上培养至长出单菌落,挑取单菌落,接种至YNB液体培养基中,得到种子液所述种子液的OD600=10-12;
将种子液接入到含有底物葡萄糖的发酵培养基中,进行发酵培养,发酵过程中进行葡萄糖补料;
8. 根据权利要求7所述的白皮杉醇的生物转化方法,其特征在于:所述YNB液体培养基为20-80 g/L葡萄糖,6.7 g/L YNB;
所述发酵培养基组分为:底物葡萄糖8%, (NH4)2SO4 0.5%、KH2PO4 0.3%、MgSO4·7H2O0.05%、微量金属溶液0.2%,维生素溶液0.1%、消泡剂204 0.05%。
9.根据权利要求4所述的白皮杉醇的生物转化方法,其特征在于:所述种子液的培养条件为28-30℃,200-225rpm;
所述发酵条件为28-30℃,200-225rpm,初始pH为5.9-6.2,发酵过程中加pH调节剂调控体系pH为5.9-6.2。
10.根据权利要求4所述的白皮杉醇的生物转化方法,其特征在于:在发酵阶段,每隔22-24小时添加18-22g/L葡萄糖。
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