CN116411013B - 一种代谢工程改造酿酒酵母从头合成δ-生育三烯醇的方法 - Google Patents
一种代谢工程改造酿酒酵母从头合成δ-生育三烯醇的方法 Download PDFInfo
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- CN116411013B CN116411013B CN202310158802.4A CN202310158802A CN116411013B CN 116411013 B CN116411013 B CN 116411013B CN 202310158802 A CN202310158802 A CN 202310158802A CN 116411013 B CN116411013 B CN 116411013B
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Abstract
本发明公开了一种代谢工程改造酿酒酵母从头合成δ‑生育三烯醇的方法，本发明在酿酒酵母中构建并优化了δ‑生育三烯酚合成途径，再通过构建底物传输通道并过表达转运体PDR11，获得了胞外分泌高效生产δ‑生育三烯酚的基因工程菌株，为代谢工程改造酿酒酵母从头合成特定构型的天然维生素E奠定了基础。本发明提供的底物传输通道的构建方法简单有效，可解决多种外源酶组装不当导致的催化效率低问题。
Description
技术领域
本发明涉及一种代谢工程改造酿酒酵母从头合成δ-生育三烯醇的方法，属于代谢工程技术领域。
背景技术
δ-生育三烯醇，作为维生素E中的一个同分异构体，除了具有抗氧化作用，还因具有对人类健康更丰富的生物活性，特别是抗癌活性而受到关注。维生素E是生育酚和生育三烯酚的总称，包括α、β、γ、δ-生育酚和α、β、γ、δ-生育三烯酚这8种化合物，作为最主要的抗氧化剂之一，是维持机体代谢的必需维生素，功效广泛，市场需求量日益增长。虽然自1938年已经实现了化学法全合成维生素E，主要利用2,3,5-三甲基氢醌(主环)和异植物醇(支链)两种中间体以“一步缩合法”合成，当前技术较为成熟，但由于其无法合成特定立体构型的维生素单体从而影响维生素E的活性，而被广泛用于动物饲料添加剂。2018年国内实现了将生物与化学法结合来合成维生素E，但仅能利用微生物发酵法合成异植物醇(C20H40)的前体物质法尼烯(C15H24)，合成过程仍较复杂且主要依赖于化学合成。目前在保健、医疗、美容等方面，人们更趋向于选择天然合成的维生素E。现有的天然维生素E仍主要依赖植物提取，受限于植物资源的有限性和成本过高。因此利用基因工程法在微生物中人为地构建维生素E的生物合成途径来实现特定构型的天然维生素E从头合成是一种更方便、更经济的方式。
作为GRAS(Generally Regard as Safe)菌株，酿酒酵母已被广泛用于萜类和芳香族化合物等天然产物的生物合成。相比大肠杆菌，酿酒酵母具有高安全性，低致病性，高抗逆性，受噬菌体污染的概率较低等优点，此外，酿酒酵母对几种异戊二烯类化合物，如法尼基二磷酸(FPP)和牻牛儿基二磷酸盐(GGPP)的耐受性高于大肠杆菌，更适合用于高水平生产异戊二烯类化合物的底盘细胞。由于GGPP是δ-生育三烯酚生物合成途径中的关键前体之一，因此酿酒酵母是更有利于δ-生育三烯酚生产的宿主菌株。酿酒酵母中不具有δ-生育三烯酚合成途径，所以需要利用代谢工程将δ-生育三烯酚合成途径引入酿酒酵母中，构建δ-生育三烯酚生产工程菌株。近年来，已有学者将光合作用生物的δ-生育三烯酚合成途径引入到酿酒酵母中，实现了δ-生育三烯酚在酿酒酵母中的从头合成。
目前，虽已经实现了δ-生育三烯酚在酿酒酵母中的从头合成，但将光合作用生物来源的基因导入酿酒酵母后，其表达、定位和组装模式存在不确定性，进而会引起一系列问题：异源酶的错误定位导致酶与底物分隔，合成产物所需前体被内源竞争性途径消耗，上下游酶之间的表达不平衡以及多种外源酶的组装不当导致的难以实现协同催化作用等。这些问题极大地限制了外源基因在细胞工厂中的功能性，从而导致δ-生育三烯酚生物合成效率低。
发明内容
为了解决目前δ-生育三烯酚生物合成效率低的问题，本发明提供一种高效合成δ-生育三烯酚的酿酒酵母菌，通过整合来自光合作用生物来源的δ-生育三烯酚合成通路，强化前体途径，突变δ-生育三烯酚合成模块中的关键酶，并构建底物传输通道对δ-生育三烯酚合成模块中的关键酶进行组装，以及过表达转运体PDR11，使得酿酒酵母可以胞外高效分泌生产δ-生育三烯酚。
本发明的第一个目的是提供一种代谢工程改造酿酒酵母从头合成δ-生育三烯醇的方法，所述方法是在酿酒酵母基因组上整合对羟基苯丙酮酸双加氧酶HPPD、尿黑酸牻牛儿基牻牛儿基转移酶HGGT和截短后的生育酚环化酶tTC，并过表达GGPP合成酶CrtE和FPP合成酶突变体FPSF112A。
进一步地，对羟基苯丙酮酸双加氧酶HPPD的NCBI编号为NP_172144.3；尿黑酸牻牛儿基牻牛儿基转移酶HGGT的NCBI编号为BAA17774；截短后的生育酚环化酶tTC的氨基酸序列如SEQ ID NO.1所示；GGPP合成酶CrtE的NCBI编号为AAS49033.1；FPP合成酶突变体FPSF112A的氨基酸序列如SEQ ID NO.2所示。
进一步地，对羟基苯丙酮酸双加氧酶HPPD的编码基因插入至酿酒酵母基因组HO位点；尿黑酸牻牛儿基牻牛儿基转移酶HGGT和截短后的生育酚环化酶tTC的编码基因插入酿酒酵母基因组DPP1位点；GGPP合成酶CrtE和FPP合成酶突变体FPSF112A的编码基因插入酿酒酵母基因组GAL1～7位点。
进一步地，所述方法还包括对截短后的生育酚环化酶tTC进行突变，将氨基酸序列如SEQ ID NO.1所示的亲本序列的第331位点天冬酰胺(N)突变为脯氨酸(P)。
进一步地，所述方法还包括过表达采用蛋白支架SH3组装的HPPD和HGGT-tTCN331P复合体。
进一步地，所述复合体插入酿酒酵母基因组的416d位点。
进一步地，HGGT和tTCN331P之间采用短蛋白连接体(GGGGS)3融合表达。
进一步地，所述蛋白支架SH3的核苷酸序列如SEQ ID NO.4所示。
进一步地，所述方法还包括采用过表达内源转运蛋白PDR11，获得分泌生产δ-生育三烯酚的酿酒酵母。
进一步地，所述方法中，是以酿酒酵母S.cerevisiae CEN PK2-1C为宿主。
本发明的第二个目的是提供一种从头合成δ-生育三烯醇的酿酒酵母，所述酿酒酵母是在酿酒酵母宿主的基因组上整合表达对羟基苯丙酮酸双加氧酶HPPD、尿黑酸牻牛儿基牻牛儿基转移酶HGGT和截短后的生育酚环化酶tTC，并过表达GGPP合成酶CrtE和FPP合成酶突变体FPSF112A。
进一步地，对羟基苯丙酮酸双加氧酶HPPD的NCBI编号为NP_172144.3；尿黑酸牻牛儿基牻牛儿基转移酶HGGT的NCBI编号为BAA17774；截短后的生育酚环化酶tTC的氨基酸序列如SEQ ID NO.1所示；GGPP合成酶CrtE的NCBI编号为AAS49033.1；FPP合成酶突变体FPSF112A的氨基酸序列如SEQ ID NO.2所示。
进一步地，以启动子PADH1和PPGK1控制δ-生育三烯酚合成途径中的基因表达，PADH1的核苷酸序列如SEQ ID NO.5所示，PPGK1的核苷酸序列如SEQ ID NO.6所示。
进一步地，对羟基苯丙酮酸双加氧酶HPPD的编码基因插入至酿酒酵母基因组HO位点；尿黑酸牻牛儿基牻牛儿基转移酶HGGT和截短后的生育酚环化酶tTC的编码基因插入酿酒酵母基因组DPP1位点；GGPP合成酶CrtE和FPP合成酶突变体FPSF112A的编码基因插入酿酒酵母基因组GAL1～7位点。
进一步地，所述酿酒酵母中，截短后的生育酚环化酶tTC为将氨基酸序列如SEQIDNO.1所示的亲本序列的第331位点天冬酰胺(N)突变为脯氨酸(P)的截短后的生育酚环化酶tTC突变体。
进一步地，所述酿酒酵母中，过表达了采用蛋白支架SH3组装的HPPD和HGGT-tTCN331P复合体。
进一步地，HGGT和tTCN331P之间采用短蛋白连接体(GGGGS)3融合表达。
进一步地，所述蛋白支架SH3的核苷酸序列如SEQ ID NO.4所示。
进一步地，所述酿酒酵母中，过表达了内源转运蛋白PDR11。
进一步地，所述酿酒酵母宿主为酿酒酵母S.cerevisiae CEN PK2-1C。
本发明的第三个目的是提供所述酿酒酵母在发酵生产δ-生育三烯醇中的应用。
本发明的有益效果是：
本发明在酿酒酵母中构建并优化了δ-生育三烯酚合成途径，再通过构建底物传输通道并过表达转运体PDR11，获得了胞外分泌高效生产δ-生育三烯酚的酿酒酵母菌株，为代谢工程改造酿酒酵母从头合成特定构型的天然维生素E奠定了基础。本发明提供的底物传输通道的构建方法简单有效，可解决多种外源酶组装不当导致的催化效率低问题。
附图说明
图1是VE-2与VE-3菌株的δ-生育三烯酚产量；
图2是VE-3与VE-4菌株的δ-生育三烯酚产量；
图3是VE-4与VE-5菌株的δ-生育三烯酚产量；
图4是VE-5与VE-6菌株的δ-生育三烯酚产量。
具体实施方式
下面结合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。
涉及的检测方法：三重四级杆复合线性离子阱液质联用仪，QTRAP 5500，ChromCore C8柱，流动相A相为水(含0.1％FA)、流动相B相为乙腈，柱温50℃，进样量2μL，流速0.35mL/min，梯度洗脱程序为80％流动相B相(0-1min)；99％流动相B相(1-8min)；80％流动相B相(8-10.5min)，阳离子模式，离子对为137/397和177/397。
实施例1：δ-生育三烯酚从头合成重组酿酒酵母菌株的构建
根据NCBI上公布的拟南芥来源的对羟基苯丙酮酸双加氧酶HPPD(NCBI ID：NP_172144.3)、集胞藻来源的尿黑酸牻牛儿基牻牛儿基转移酶HGGT(NCBI ID：BAA17774)、拟南芥来源的截短后的生育酚环化酶tTC(在NCBI ID：NP_567906.1的基础上截短了47个氨基酸，氨基酸序列如SEQ ID NO.1所示)、红豆杉来源的GGPP合成酶CrtE(NCBI ID：AAS49033.1)和家鸡来源的FPP合成酶突变体FPSF112A(未突变的FPP合成酶的NCBI ID：P08836.2，在此基础上，将112位苯丙氨酸突变为丙氨酸，突变后氨基酸序列如SEQ ID NO.2所示)，按照酿酒酵母密码子偏好性进行密码子优化并进行全基因合成。
由于酿酒酵母具有同源重组能力，按照基因整合框的每个相邻片段都具有45-50bp的重叠部分设计引物。在酿酒酵母基因组HO，DPP1和GAL1～7整合位点序列两侧设计基因整合框的上下游同源臂扩增引物，分别以酿酒酵母基因组DNA为模板，通过PCR将上下游同源臂扩增下来。以质粒pML104-HIS和pML104-TRP为模板设计引物，扩增获得相应的氨基酸筛选标签。再结合重叠延伸PCR引物设计方法，设计引物扩增上述δ-生育三烯酚合成途径酶、启动子和终止子。以全基因合成的HPPD、HGGT、tTC、CrtE和FPSF112A质粒为模板，扩增获得相应的基因片段。以酿酒酵母基因组DNA为模板，扩增获得启动子PADH1和PPGK1，终止子TCYC1和TADH1。通过重叠延伸PCR获得相应基因表达框。
按照Cre-loxp系统，将扩增的上下游同源臂、氨基酸筛选标签与基因整合框转化至酿酒酵母感受态细胞，挑取菌落PCR正确的转化子进行测序验证，获得具有δ-生育三烯酚从头合成通路的重组酿酒酵母菌株VE-2。
为了加强前体GGPP的供应，在VE-2菌株的基础上过表达红豆杉来源的GGPP合成酶CrtE和家鸡来源的FPP合成酶突变体FPSF112A。在酿酒酵母基因组GAL1～7整合位点序列两侧设计基因整合框的上下游同源臂扩增引物，以酿酒酵母基因组DNA为模板，通过PCR将上下游同源臂扩增下来。以质粒pML104-LEU为模板设计引物，扩增获得亮氨酸筛选标签。以全基因合成的CrtE和FPSF112A质粒为模板，扩增获得相应的基因片段。以酿酒酵母基因组DNA为模板，扩增获得启动子PADH1和PPGK1，终止子TCYC1和TADH1。通过重叠延伸PCR获得相应基因表达框。按照上述Cre-loxp系统基因编辑方法，获得优化前体途径后的合成δ-生育三烯酚的基因工程菌株VE-3。
构建tTCN331P突变体质粒，以全基因合成的tTC质粒为模板，用引物N331-F和N331-R进行环状PCR，经测序获得tTCN331P突变体质粒。
按照上述基因整合方法，以VE-3为出发菌株，在DPP1整合表达tTCN331P突变体，替代未突变的tTC，最终获得从头合成δ-生育三烯酚的基因工程菌株VE-4。
引物序列：
N331-F：CTGAAAACGAACCACATGTTGTTGAATTAGAAGCTAGAACCAAC
N331-R：TCAACAACATGTGGTTCGTTTTCAGCAGTGATGTACCA。
实施例2：δ-生育三烯酚合成途径中底物传输通道的构建
构建HGGT和tTCN331P融合表达质粒pY16-HGGT-(GGGGS)3-tTCN331P，分别以全基因合成HGGT质粒和已构建的tTCN331P突变体质粒为模板，通过引物HGGT-F和引物HGGT-R扩增HGGT、引物tTCN331P-F和引物tTCN331P-R扩增tTCN331P，以pY16-URA质粒为模板，通过引物pY16-F1和pY16-R1扩增载体框架。将获得的DNA片段纯化后，通过Gibson组装法插入pY16-URA载体，经测序获得质粒pY16-HGGT-(GGGGS)3-tTCN331P。
构建HPPD和HGGT-tTCN331P复合体组装表达质粒pY16-HPPD-HGGT-tTCN331P-SH3，以全基因合成HPPD质粒为模板，通过引物HPPD-F和引物HPPD-R扩增HPPD，以质粒pY16-HGGT-(GGGGS)3-tTCN331P为模板，通过含有SH3-ligand序列引物HGGT-tTCN331P-F和引物HGGT-tTCN331P-R扩增HGGT-tTCN331P复合体(SH3-ligand的核苷酸序列如SEQ IDNO.3所示)，以合成序列SH3-domain为模板(SH3-domain的核苷酸序列如SEQ ID NO.4所示)，通过引物SH3-F和SH3-R扩增SH3-domain，以酿酒酵母基因组DNA为模板，通过引物P-F和P-R扩增获得启动子PADH1和PPGK1，以pY16-URA质粒为模板，通过引物pY16-F2和pY16-R2扩增载体框架。将获得的DNA片段纯化后，通过Gibson组装法插入pY16-URA载体，经测序获得质粒pY16-HPPD-HGGT-tTCN331P-SH3。
按照实施例1中所述基因整合方法，以VE-4为出发菌株，在416d位点整合使用SH3组装后的HPPD和HGGT-tTCN331P复合体，构建底物传输通道，获得高效合成δ-生育三烯酚的基因工程菌株VE-5。
引物序列：
HGGT-F：CAAATATAAAACAATGGCTACTATTCAAGCTTTTTGGAG
HGGT-R：GCCACCGCCGCTTCCACCGCCACCAAAAATAGTATTAGAAAAATTTGGC AACCACAAAG
tTCN331P-F：GCGGTGGAAGCGGCGGTGGCGGAAGCATGGCTTCTATTAGTACTCCAA ACTCTG
tTCN331P-R：CATAAGAAATTCGCTCATAATCCAGGTGGCTTGAAGAATG
pY16-F1：CTGGATTATGAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAG
pY16-R1：TAGTAGCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTG A
HPPD-F：GTACGGTGGAGGAGGAAGCGGCGGTGGCGGATCCGGTCACCAAAATGC TGCCG
HPPD-R：TAAGAAATTCGCTTAACCGACCAATTGCTTGGC
HGGT-tTCN331P-F：GAGGTAAGGCTGGTGGGGGGCTTCCGCCACCGCCGCTTCCACCGCCACCTAATCCAGGTGGCTTGAAGAATG
HGGT-tTCN331P-R：CATACAATCAACTATGGCTACTATTCAAGCTTTTTGGAG
SH3-F：ATATAAAACAATGGCAGAGTATGTGCGTGCT
SH3-R：ACCGGATCCGCCACCGCCGCTTCCTCCTCCACCGTACTTCTCCACATAAGG AACG
P-F：GAATAGTAGCCATAGTTGATTGTATGCTTGGTATAGCTTG
P-R：TACTCTGCCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTC
pY16-F2：TGGTCGGTTAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGpY16-R2：AAGCCCCCCACCAGCCTTACCTCCTAAGCGTAGACGTTAATCATGTAATT AGTTATGTCACGCTTACATTC。
实施例3：胞外分泌生产δ-生育三烯酚
构建pY16-PDR11质粒，以酵母基因组为模板，通过引物PDR11-F和引物PDR11-R扩增PDR11，以pY16-URA质粒为模板，通过引物pY16-F3和pY16-R3扩增载体框架。将获得的DNA片段纯化后，通过Gibson组装法插入pY16-URA载体，经测序获得质粒pY16-PDR11。
将质粒pY16-PDR11转化至VE-5菌株，获得胞外分泌生产δ-生育三烯酚菌株VE-6。
摇瓶发酵方法：
(1)将重组菌株VE-2、VE-3、VE-4、VE-5和VE-6划线于YPD平板，30℃下培养直至长出大量菌落。
(2)接种一环单菌落至种子培养基，30℃下220rpm培养至20h。
(3)按起始2％的接种量将种子培养液接种至发酵培养基中，30℃，220rpm培养48h后添加50％(v/v)十二烷，继续在30℃，220rpm条件下培养至144h。测量OD600后，将菌液转至50mL离心管，常温6000rpm离心10min。取1mL有机相在真空旋转蒸发器中旋蒸后，再用1mL乙腈复溶后经0.22μm的有机系滤膜过滤后使用三重四级杆复合线性离子阱液质联用仪进行定量分析。
引物序列：
PDR11-F：TAGAACTAGTGGATCCCCCGGCGGATGTCTCTTTCCAAATAT
PDR11-R：GACGGTATCGATAAGCTTGATTATACGCTTTGTTCGTTTGGAT
pY16-F3：CGGGGGATCCACTAGTTCTA
pY16-R3：TCAAGCTTATCGATACCGTCG。
菌株VE-2和VE-3摇瓶发酵结果对比如图1所示，菌株VE-2和VE-3的δ-生育三烯酚产量分别为59.2μg/L和384.5μg/L，与菌株VE-2相比，菌株VE-3的δ-生育三烯酚产量提高了5.5倍，以验证过表达红豆杉来源的GGPP合成酶CrtE和家鸡来源的FPP合成酶突变体FPSF112A，加强GGPP供应后对提高δ-生育三烯酚合成的效果。
菌株VE-3和VE-4摇瓶发酵结果对比如图2所示，菌株VE-4的δ-生育三烯酚产量是菌株VE-3的1.8倍，达703.7μg/L，以验证定点突变生育酚环化酶tTC后对提高δ-生育三烯酚产量的效果。
菌株VE-4和VE-5摇瓶发酵结果对比如图3所示，菌株VE-5的δ-生育三烯酚产量为1801.5μg/L，与菌株VE-4相比，菌株VE-5的δ-生育三烯酚产量提高了1.6倍，以验证构建底物传输通道对提高δ-生育三烯酚产量的效果。
菌株VE-5和VE-6摇瓶发酵结果对比如图4所示，菌株VE-6的δ-生育三烯酚产量是菌株VE-5的1.7倍，达3062.6μg/L，以验证胞外分泌高效生产δ-生育三烯酚的效果。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (9)
1.一种代谢工程改造酿酒酵母从头合成δ-生育三烯醇的方法，其特征在于，所述方法是在酿酒酵母基因组上整合对羟基苯丙酮酸双加氧酶HPPD、尿黑酸牻牛儿基牻牛儿基转移酶HGGT和截短后的生育酚环化酶tTC，并过表达GGPP合成酶CrtE和FPP合成酶突变体FPSF112A；
所述对羟基苯丙酮酸双加氧酶HPPD的NCBI编号为NP_172144.3；所述尿黑酸牻牛儿基牻牛儿基转移酶HGGT的NCBI编号为BAA17774；所述截短后的生育酚环化酶tTC的氨基酸序列如SEQ ID NO.1所示；所述GGPP合成酶CrtE的NCBI编号为AAS49033.1；所述FPP合成酶突变体FPSF112A的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的方法，其特征在于，所述方法还包括对截短后的生育酚环化酶tTC进行突变，将氨基酸序列如SEQ ID NO.1所示的亲本序列的第331位点天冬酰胺突变为脯氨酸。
3.根据权利要求1或2所述的方法，其特征在于，所述方法还包括过表达采用蛋白支架SH3组装的HPPD和HGGT-tTCN331P复合体，其中，所述蛋白支架SH3的核苷酸序列如SEQ IDNO.4所示。
4.根据权利要求3所述的方法，其特征在于，HGGT和tTCN331P之间采用短蛋白连接体（GGGGS）3融合表达。
5.根据权利要求1所述的方法，其特征在于，所述方法还包括采用过表达内源转运蛋白PDR11，获得分泌生产δ-生育三烯酚的酿酒酵母。
6.一种从头合成δ-生育三烯醇的酿酒酵母，其特征在于，所述酿酒酵母是在酿酒酵母宿主的基因组上整合表达对羟基苯丙酮酸双加氧酶HPPD、尿黑酸牻牛儿基牻牛儿基转移酶HGGT和截短后的生育酚环化酶tTC，并过表达GGPP合成酶CrtE和FPP合成酶突变体FPSF112A；
所述对羟基苯丙酮酸双加氧酶HPPD的NCBI 编号为NP_172144.3；所述尿黑酸牻牛儿基牻牛儿基转移酶HGGT的NCBI 编号为BAA17774；所述截短后的生育酚环化酶tTC的氨基酸序列如SEQ ID NO.1所示，或将氨基酸序列如SEQ ID NO.1所示的亲本序列的第331位点天冬酰胺突变为脯氨酸；所述GGPP合成酶CrtE的NCBI编号为AAS49033.1；所述FPP合成酶突变体FPSF112A的氨基酸序列如SEQ ID NO.2所示。
7.根据权利要求6所述的酿酒酵母，其特征在于，所述酿酒酵母中，过表达了采用蛋白支架SH3组装的HPPD和HGGT-tTCN331P复合体；其中，所述蛋白支架SH3的核苷酸序列如SEQ IDNO.4所示，HGGT和tTCN331P之间采用短蛋白连接体（GGGGS）3融合表达。
8.根据权利要求6所述的酿酒酵母，其特征在于，所述酿酒酵母中，过表达了内源转运蛋白PDR11。
9.权利要求6~8任一项所述酿酒酵母在发酵生产δ-生育三烯醇中的应用。
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