CN110903993A - 一种产生菜籽甾醇的酿酒酵母工程菌及其构建方法和应用 - Google Patents
一种产生菜籽甾醇的酿酒酵母工程菌及其构建方法和应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种产生菜籽甾醇的酿酒酵母工程菌及其构建方法和应用,属于代谢工程技术领域,所述酿酒酵母工程菌含有甾醇Δ7‑还原酶基因和葡萄糖脱氢酶基因。本发明提供的酿酒酵母工程菌能够将麦角甾醇还原成菜籽甾醇。在本发明中,所述酿酒酵母工程菌中共表达甾醇Δ7‑还原酶和葡萄糖脱氢酶,就可以葡萄糖脱氢酶形成的NADPH作为辅因子,利用甾醇Δ7‑还原酶将酿酒酵母固有的麦角甾醇还原成菜籽甾醇,从而达到生物制备菜籽甾醇的目的。
Description
技术领域
本发明属于代谢工程技术领域,尤其涉及一种产生菜籽甾醇酿酒酵母工程菌及其构建方法和应用。
背景技术
菜籽甾醇(Brassicasterol,CAS号474-67-9)是一种重要的甾体化合物,它可以合成芸苔素内酯等农药,具有很重要的价值。菜籽甾醇主要通过植物提取获得,但由于菜籽甾醇在植物中的含量较低,分离纯化比较困难。化学合成菜籽甾醇已经获得成功,尚没有工业化生产的报道。菜籽甾醇来源稀缺导致了市售的菜籽甾醇价格非常昂贵。
发明内容
有鉴于此,本发明的目的在于提供一种产生菜籽甾醇酿酒酵母工程菌及其构建方法和应用,本发明提供的酿酒酵母工程菌能够将麦角甾醇还原成菜籽甾醇。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明提供了一种产生菜籽甾醇的酿酒酵母工程菌,所述酿酒酵母工程菌含有甾醇Δ7-还原酶基因和葡萄糖脱氢酶基因。
优选的,所述甾醇Δ7-还原酶基因的核苷酸序列如SEQ ID No.1所示。
优选的,所述甾醇Δ7-还原酶基因编码的甾醇Δ7-还原酶的氨基酸序列如 SEQID No.2所示。
优选的,所述葡萄糖脱氢酶基因的核苷酸序列如SEQ ID No.3所示。
优选的,所述葡萄糖脱氢酶基因编码的葡萄糖脱氢酶的氨基酸序列如 SEQ IDNo.4所示。
本发明还提供了上述技术方案所述的酿酒酵母工程菌的构建方法,包括以下步骤:
1)将甾醇Δ7-还原酶基因插入到pESC-Trp质粒中,得到pESCTrp-Δ7Red2 质粒;
2)将葡萄糖脱氢酶基因插入到步骤1)得到的pESCTrp-Δ7Red2质粒中,得到pESCTrp-Δ7Red2-GDH质粒;
3)将所述步骤2)得到的pESCTrp-Δ7Red2-GDH质粒导入酿酒酵母中,得到酿酒酵母工程菌。
优选的,扩增甾醇Δ7-还原酶基因使用的引物包括Fhis49398引物和 Rhis49398引物;
所述Fhis49398引物的核苷酸序列如SEQ ID No.5所示,所述Rhis49398 引物的核苷酸序列如SEQ ID No.6所示。
优选的,扩增甾醇Δ7-还原酶基因使用的程序为:94℃2min;94℃20s, 55℃30s,72℃1min,30个循环;72℃7min。
优选的,扩增葡萄糖脱氢酶基因使用的引物包括FBamH12276引物和 RSal12276引物;
所述FBamH12276引物的核苷酸序列如SEQ ID No.7所示,所述 RSal12276引物的核苷酸序列如SEQ ID No.8所示。
本发明还提供了上述技术方案所述的酿酒酵母工程菌在制备菜籽甾醇中的应用。
本发明提供了一种产生菜籽甾醇的酿酒酵母工程菌及其构建方法和应用,所述酿酒酵母工程菌含有甾醇Δ7-还原酶基因和葡萄糖脱氢酶基因。本发明提供的酿酒酵母工程菌能够将麦角甾醇还原成菜籽甾醇。在本发明中,所述酿酒酵母工程菌中共表达甾醇Δ7-还原酶和葡萄糖脱氢酶,能够以葡萄糖脱氢酶形成的NADPH作为辅因子,利用甾醇Δ7-还原酶将酿酒酵母固有的麦角甾醇还原成菜籽甾醇,从而达到生物制备菜籽甾醇的目的。
附图说明
图1为甾醇Δ7-还原酶Δ7-red基因PCR扩增结果,其中1:Δ7-red基因 PCR扩增产物;M:DNA分子量标准;
图2为质粒pESC-Trp双酶切结果,其中1:pESC-Trp酶切结果;M: DNA分子质量标准;
图3为GDH基因PCR扩增结果,其中M:DNA质量标准DL2000,1: GDH基因PCR扩增结果。
图4为质粒酶切结果,其中1:pESCTrp-Δ7Red2酶切结果;M:DNA 分子量标准;
图5为工程菌中Δ7-red基因PCR扩增结果,其中1-8:1-8号克隆;M: DNA分子量标准;箭头表示目的条带;
图6为工程菌中GDH基因PCR扩增结果,其中1-8:1-8号克隆;M: DNA分子量标准;箭头表示目的条带;
图7为酵母工程发酵产物HPLC检测结果,其中A,1: W303-1B[TrpΔ7-red+GDH]发酵产物检测结果;2:W303-1B发酵产物检测结果;3:标准品菜籽甾醇HPLC检测结果,箭头所指为新的产物峰;B:产物和标准品的紫外图谱;
图8为发酵产物的高分辨质谱检测结果。
具体实施方式
本发明提供了一种产生菜籽甾醇的酿酒酵母工程菌,所述酿酒酵母工程菌含有甾醇Δ7-还原酶基因和葡萄糖脱氢酶基因。
在本发明中,所述甾醇Δ7-还原酶基因的核苷酸序列如SEQ ID No.1所示,具体如下:
atggcggagactgtacattctccgatcgttacttacgcatcgatgttatcgcttctcgccttctgtccacctttcgtcattct cctatggtacacaatggttcatcaggatggttctgttactcagacctttggcttcttttgggagaatggagttcaaggact tatcaacatatggccaagacccactttgattgcttggaaaattatattttgctatggagcatttgaagctattcttcagctg cttctgcctggtaaaagagttgagggtccaatatctccagccggaaaccgaccagtttacaaggccaatggtctggct gcttactttgtgacactagcaacccatcttggtctttggtggtttggaatcttcaaccctgcaattgtctatgatcacttgg gtgaaatattttcggcactaatattcggaagcttcatattttgtgttttgttgtacataaaaggccatgttgcaccttcatca agtgactctggttcatgtggtaacctaataattgacttctattggggcatggagttgtaccctcgaattggtaagagcttt gacatcaaggtgtttactaattgcagattcggaatgatgtcttgggcagttcttgcagtcacgtactgcataaaacagta tgaaataaatggcaaagtatctgattcaatgctggtgaacaccatcctgatgctggtgtatgtcacaaaattcttctggt gggaagctggttattggaacaccatggacattgcacatgaccgagctggattctatatatgctggggttgtctagtgtg ggtgccttctgtctacacttctccaggcatgtaccttgtgaaccaccccgtcgaactcggaactcagttggcaatatac attctcgttgcaggaattctgtgcatttacataaagtatgactgtgatagacaaaggcaagagttcaggaggacaaac gggaaatgtttggtttggggaagagccccgtcaaagattgtggcgtcgtatactacaacatctggtgaaactaaaactagtcttctcttaacgtctggatggtggggattggctcgtcatttccattatgttcctgagatcttaagtgctttcttctggac cgtaccggctctcttcgataacttcttggcatacttctacgtcctcacccttcttctctttgatcgagccaagagagacga tgaccgatgccgatcaaagtatgggaaatattggaagctgtattgtgagaaagtcaaatacaggatcattccgggaat ttattga。
在本发明中,所述甾醇Δ7-还原酶基因编码的甾醇Δ7-还原酶的氨基酸序列如SEQ ID No.2所示,具体如下所示:
MAETVHSPIVTYASMLSLLAFCPPFVILLWYTMVHQDGSVTQTFGFF WENGVQGLINIWPRPTLIAWKIIFCYGAFEAILQLLLPGKRVEGPISPAGNR PVYKANGLAAY FVTLATHLGLWWFGIFNPAI VYDHLGEIFSALIFGSFIFC VLLYIKGHVA PSSSDSGSCG NLIIDFYWGMELYPRIGKSF DIKVFTNCRF GMMSWAVLAVTYCIKQYEINGKVSDSMLVNTILMLVYVTKFFWWEAGY WNTMDIAHDRAGFYICWGCLVWVPSVYTSPGMYLVNHPVELGTQLAIYI LVAGILCIYIKYDCDRQRQEFRRTNGKCLVWGRAPSKIVASYTTTSGETKT SLLLTSGWWGLARHFHYVPEILSAFFWTVPALFDNFLAYFYVLTLLLFDR AK RDDDRCRSKY GKYWKLYCEKVKYRIIPGIY。
在本发明中,所述葡萄糖脱氢酶基因的核苷酸序列如SEQ ID No.3所示,具体如下:
atgtatccggatttaaaaggaaaagtcgtcgctattacaggagctgcttcagggctcggaaaggcgatggcc attcgcttcggcaaggagcaggcaaaagtggttatcaactattatagtaataaacaagatccgaacgaggtaaaaga agaggtcatcaaggcgggcggtgaagctgttgtcgtccaaggagatgtcacgaaagaggaagatgtaaaaaatat cgtgcaaacggcaattaaggagttcggcacactcgatattatgattaataatgccggtcttgaaaatcctgtgccatct cacgaaatgccgctcaaggattgggataaagtcatcggcacgaacttaacgggtgcctttttaggaagccgtgaagc gattaaatatttcgtagaaaacgatatcaagggaaatgtcattaacatgtccagtgtgcacgcgtttccttggccgttatt tgtccactatgcggcaagtaaaggcgggataaagctgatgacagaaacattagcgttggaatacgcgccgaaggg cattcgcgtcaataatattgggccaggtgcgatcaacacgccaatcaatgctgaaaaattcgctgaccctaaacaga aagctgatgtagaaagcatgattccaatgggatatatcggcgaaccggaggagatcgccgcagtagcagcctggct tgcttcgaaggaagccagctacgtcacaggcatcacgttattcgcggacggcggtatgacacaatatccttcattcca ggcaggccgcggttaa。
在本发明中,所述葡萄糖脱氢酶基因编码的葡萄糖脱氢酶的氨基酸序列如SEQ IDNo.4所示,具体如下:
MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDP NEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGL ENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSS VHAFPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINA EKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFAD GGMT QYPSFQAGRG。
本发明还提供了上述技术方案所述的酿酒酵母工程菌的构建方法,包括以下步骤:
1)将甾醇Δ7-还原酶基因插入到pESC-Trp质粒中,得到pESCTrp-Δ7Red2 质粒;
2)将葡萄糖脱氢酶基因插入到步骤1)得到的pESCTrp-Δ7Red2质粒中,得到pESCTrp-Δ7Red2-GDH质粒;
3)将所述步骤2)得到的pESCTrp-Δ7Red2-GDH质粒导入酿酒酵母中,得到酿酒酵母工程菌。
本发明将甾醇Δ7-还原酶基因插入到pESC-Trp质粒中,得到pESCTrp-Δ 7Red2质粒。
本发明优选根据已发表的甾醇Δ7-还原酶基因(Δ7-red,ATU49398)序列,人工合成,含有甾醇Δ7-还原酶基因的质粒优选命名为pGSI-Δ7-Red2。以pGSI-Δ7-Red2为模板,用Fhis49398引物和Rhis49398引物采用常规方法进行扩增,得到甾醇Δ7-还原酶基因。
在本发明中,所述Fhis49398引物的核苷酸序列如SEQ ID No.5所示,具体如下所示:
5'-ttgaaaattc gaattcatgg cggagactgt acat-3'
所述Rhis49398引物的核苷酸序列如SEQ ID No.6所示,具体如下所示:
5'-atccatcgat actagtcaat aaattcccgg aat-3'。
在本发明中,扩增甾醇Δ7-还原酶基因使用的程序优选为:94℃2min; 94℃20s,55℃30s,72℃1min,30个循环;72℃7min。
本发明优选将pESC-Trp采用EcoR I和Spe I进行双酶切后,得到酶切质粒,将所述甾醇Δ7-还原酶基因插入到酶切质粒。本发明对采用EcoR I和Spe I双酶切pESC-Trp的体系和条件没有特殊限定,采用上述两种酶常规酶切质粒的体系和条件即可。在本发明中,所述甾醇Δ7-还原酶基因插入到 pESC-Trp质粒的体系每10μl优选包括3.5μl甾醇Δ7-还原酶基因、1.5μl酶切质粒和5μl 2×Seamless MasterMix。本发明对所述2×SeamlessMasterMix 的来源没有特殊限定,采用常规市售产品即可。在本发明中,所述插入的条件优选包括:所述插入的温度优选为50min,所述插入的时间优选为15min。
本发明将葡萄糖脱氢酶基因插入到pESCTrp-Δ7Red2质粒中,得到 pESCTrp-Δ7Red2-GDH质粒。
本发明优选根据已发表的葡萄糖脱氢酶序列(M12276.1)人工合成葡萄糖脱氢酶基因,含有葡萄糖脱氢酶基因的质粒命名为pHSI-GDH。本发明优选以pHSI-GDH为模板,用FBamH12276引物和RSal12276引物采用常规方法扩增得到葡萄糖脱氢酶基因。在本发明中,所述FBamH12276引物的核苷酸序列如SEQ ID No.7所示,具体如下所示:
5'-ggagaaaaaa ccccggatcc atgtatccgg atttaaaag-3';
所述RSal12276引物的核苷酸序列如SEQ ID No.8所示,具体如下所示:
5'-caacttctgt tccatgtcga cttaaccgcg gcctgcctgg-3'。
在本发明中,扩增葡萄糖脱氢酶基因使用的程序优选为:94℃2min; 94℃20s,60℃30s,72℃30s,30个循环;72℃7min。
本发明优选将pESCTrp-Δ7Red2采用BamHⅠ和SalⅠ进行双酶切后,得到酶切质粒,将所述葡萄糖脱氢酶基因插入到酶切质粒。本发明对采用 BamHⅠ和SalⅠ双酶切pESCTrp-Δ7Red2的体系和条件没有特殊限定,采用上述两种酶常规酶切质粒的体系和条件即可。在本发明中,所述葡萄糖脱氢酶基因插入到pESCTrp-Δ7Red2质粒的体系每10μl优选包括3.5μl葡萄糖脱氢酶基因、1.5μl酶切质粒和5μl 2×Seamless Master Mix。本发明对所述 2×Seamless MasterMix的来源没有特殊限定,采用常规市售产品即可。在本发明中,所述插入的条件优选包括:所述插入的温度优选为50min,所述插入的时间优选为15min。
本发明将得到的pESCTrp-Δ7Red2-GDH质粒导入酿酒酵母中,得到酿酒酵母工程菌。
本发明对pESCTrp-Δ7Red2-GDH质粒导入酿酒酵母中的方法没有特殊限定,采用常规即可,在本发明具体实施方式中优选采用LiAc介导的方法将pESCTrp-Δ7Red2-GDH质粒导入酿酒酵母中。
本发明还提供了上述技术方案所述的酿酒酵母工程菌在制备菜籽甾醇中的应用。本发明对所述酿酒酵母工程菌在制备菜籽甾醇的方法没有特殊限定,采用常规培养酿酒酵母的方法后获得产物即可。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
Δ7-Red酵母表达载体的构建
根据已经发表的甾醇Δ7-还原酶基因(Δ7-red,ATU49398)序列,人工合成Δ7-red基因(SEQ ID No.1),含有Δ7-red基因的质粒分别命名为 pGSI-Δ7-Red2。设计引物Fhis49398(SEQ ID No.5)/Rhis49398(SEQ ID No.6),以pGSI-Δ7-Red2为模板,KOD PlusTaq为聚合酶,按照如下程序进行PCR: 94℃2min;94℃20sec,55℃30sec,72℃1min,30个循环;72℃7min; 4℃保存。反应结束后,取10μl进行电泳,结果如图1所示。从结果可以看出,大小为1293bp的条带出现在了泳道上,与预计的大小一致,表明Δ7-red 基因已经被成功扩增。
用EcoRⅠ和SpeⅠ(宝日医生物技术(北京)有限公司)按如下双酶切体系酶切pESC-Trp(50ng/μl)质粒。
酶切结束后,取5μl进行电泳,结果如图2所示,酶切以后,出现了大于5.0kb的条带,与pESC-Trp(图2)的大小吻合,表明质粒pESC-Trp的酶切成功。
选取PCR产物以及酶切的产物按如下程序和体系进行无缝连接 (Seamlessassembly cloning kit,Clone Smart Technologies Co.Ltd,美国):
50℃反应15min后,反应产物转化大肠杆菌,转化产物在37℃培养过夜,长出克隆。挑取10个单克隆,用引物Fhis49398/Rhis49398进行菌落 PCR。结果表明,选择的重组克隆中均扩增出了和理论大小一致的条带,证明重组质粒已经构建成功。从菌落PCR鉴定为阳性的克隆中挑取三个进行测序,结果表明,质粒构建成功,将构建正确的重组质粒命名为pESCTrp-Δ7Red2。
实施例2
含GDH基因的酵母表达载体的构建
根据已经发表的葡萄糖脱氢酶序列(glucose dehydrogenase,GDH, M12276.1),人工合成GDH基因(SEQ ID No.3),获得含GDH基因的质粒pGSI-GDH。设计引物FBamH12276(SEQ ID No.7)和RSal12276(SEQ ID No.8),以pGSI-GDH为模板,KOD Plus Taq为聚合酶,按照如下程序进行PCR:94℃2min;94℃20sec,60℃30sec,72℃30sec,30个循环;72℃7min;4℃保存。反应结束后,取10μl进行电泳,结果如图3所示。一条大小约为750bp的特异性条带出现在泳道上,与GDH的理论大小吻合,表明GDH基因被成功的克隆。
用BamHⅠ和SalⅠ(宝日医生物技术(北京)有限公司)按如下双酶切体系酶切pESCTrp-Δ7Red2(40ng/μl)重组质粒。
37℃反应2h后,取5μl进行电泳,结果表明,质粒pESCTrp-Δ7Red2被切出了理论大小的条带,表明质粒pESCTrp-Δ7Red2酶切成功(图4)。选取PCR产物以及酶切的产物按如下程序和体系进行无缝连接(Seamless assembly cloning kit,Clone Smart TechnologiesCo.Ltd,美国):
50℃反应15min后,反应产物转化大肠杆菌,转化产物在37℃培养过夜,重组克隆均长出了菌落。从重组菌株中挑取12个单克隆进行菌落PCR。然后从重组菌株中挑取PCR鉴定为阳性的3个克隆进行测序,结果表明,重组质粒构建成功,命名为pESCTrp-Δ7Red2-GDH。
实施例3
含Δ7-red基因和GDH基因的酵母工程菌的构建
提取pESCTrp-Δ7Red2-GDH质粒。利用LiAc介导的方法将上述质粒导入酿酒酵母W303-1B中,28℃倒置培养2-3天,工程菌长出了克隆。挑取8 个克隆进行质粒提取。以提取的质粒为模板,Fhis49398/Rhis49398(为引物,按照如下程序和体系进行PCR:94℃2min;94℃20sec,55℃30sec,72℃ 1min,30个循环;72℃7min;4℃保存。反应结束后,取10μl进行电泳。从结果可以看出,克隆1,2,4,5,6出现了大小为1.3kb的条带,与预计的大小一致,表明Δ7-red基因已经导入酿酒酵母(图5)。再以FBamH12276 和RSal12276为引物,提取的酵母质粒为模板,按照如下程序和体系进行菌落PCR:94℃2min;94℃20sec,60℃30sec,72℃30sec,30个循环;72℃ 7min;4℃保存。反应结束后,取10μl进行电泳。一条大小约为750bp的特异性条带出现在泳道上,与GDH的理论大小吻合,表明GDH基因也已经被导入同一个酿酒酵母中(图6)。上述结果表明,在重组克隆1,2,4, 5,6中同时导入了Δ7-red和GDH基因,工程菌构建成功,命名为 W303-1B[TrpΔ7-red+GDH]。
实施例4
工程菌产物的检测
挑取构建成功的酵母工程菌W303-1B[TrpΔ7-red+GDH]接种到10ml SC-Trp液体培养基中,28℃震荡培养1-2天。吸取1ml的菌液,12000g离心5min,弃上清,沉淀用无菌的ddH2O重悬,12000g离心5min,弃上清。沉淀转入50ml液体YPG(酵母提取物,10克/升;细菌蛋白胨,20克/升;半乳糖20克/升)培养基中,诱导培养2-3天。12000g离心5min,获得酵母沉淀。酵母沉淀用10ml的无菌ddH2O涮洗两遍,然后烘干。菌体用3M氢氧化钾甲醇皂化液在80℃皂化5h。加入正己烷,萃取皂化后的样品三次。合并萃取液,旋转蒸干。样品用甲醇溶解,过滤,按照如下条件进行HPLC 检测。
色谱柱:Silgreen ODS C18柱(5μm,4.6mm×250mm)。流动相:甲醇: 水=99:1;柱温:常温;流速:1.0ml/min;进样量:30μl;检测波长:210nm。
HPLC检测结果表明,和对照W303-1B相比,样品中出现了一个新的产物峰,保留时间以及紫外图谱都和标准品一致(图7)。断定在工程菌中已经产生了菜籽甾醇。对产物进行高分辨质谱检测表明,其[M+H]+m/z=399.28946 (图8),和菜籽甾醇的分子量(MW=398)一致,表明该产物为菜籽甾醇。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 河北兰升生物科技有限公司
河北谷之润科技有限公司
<120> 一种产生菜籽甾醇的酿酒酵母工程菌及其构建方法和应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1293
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcggaga ctgtacattc tccgatcgtt acttacgcat cgatgttatc gcttctcgcc 60
ttctgtccac ctttcgtcat tctcctatgg tacacaatgg ttcatcagga tggttctgtt 120
actcagacct ttggcttctt ttgggagaat ggagttcaag gacttatcaa catatggcca 180
agacccactt tgattgcttg gaaaattata ttttgctatg gagcatttga agctattctt 240
cagctgcttc tgcctggtaa aagagttgag ggtccaatat ctccagccgg aaaccgacca 300
gtttacaagg ccaatggtct ggctgcttac tttgtgacac tagcaaccca tcttggtctt 360
tggtggtttg gaatcttcaa ccctgcaatt gtctatgatc acttgggtga aatattttcg 420
gcactaatat tcggaagctt catattttgt gttttgttgt acataaaagg ccatgttgca 480
ccttcatcaa gtgactctgg ttcatgtggt aacctaataa ttgacttcta ttggggcatg 540
gagttgtacc ctcgaattgg taagagcttt gacatcaagg tgtttactaa ttgcagattc 600
ggaatgatgt cttgggcagt tcttgcagtc acgtactgca taaaacagta tgaaataaat 660
ggcaaagtat ctgattcaat gctggtgaac accatcctga tgctggtgta tgtcacaaaa 720
ttcttctggt gggaagctgg ttattggaac accatggaca ttgcacatga ccgagctgga 780
ttctatatat gctggggttg tctagtgtgg gtgccttctg tctacacttc tccaggcatg 840
taccttgtga accaccccgt cgaactcgga actcagttgg caatatacat tctcgttgca 900
ggaattctgt gcatttacat aaagtatgac tgtgatagac aaaggcaaga gttcaggagg 960
acaaacggga aatgtttggt ttggggaaga gccccgtcaa agattgtggc gtcgtatact 1020
acaacatctg gtgaaactaa aactagtctt ctcttaacgt ctggatggtg gggattggct 1080
cgtcatttcc attatgttcc tgagatctta agtgctttct tctggaccgt accggctctc 1140
ttcgataact tcttggcata cttctacgtc ctcacccttc ttctctttga tcgagccaag 1200
agagacgatg accgatgccg atcaaagtat gggaaatatt ggaagctgta ttgtgagaaa 1260
gtcaaataca ggatcattcc gggaatttat tga 1293
<210> 2
<211> 430
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Glu Thr Val His Ser Pro Ile Val Thr Tyr Ala Ser Met Leu
1 5 10 15
Ser Leu Leu Ala Phe Cys Pro Pro Phe Val Ile Leu Leu Trp Tyr Thr
20 25 30
Met Val His Gln Asp Gly Ser Val Thr Gln Thr Phe Gly Phe Phe Trp
35 40 45
Glu Asn Gly Val Gln Gly Leu Ile Asn Ile Trp Pro Arg Pro Thr Leu
50 55 60
Ile Ala Trp Lys Ile Ile Phe Cys Tyr Gly Ala Phe Glu Ala Ile Leu
65 70 75 80
Gln Leu Leu Leu Pro Gly Lys Arg Val Glu Gly Pro Ile Ser Pro Ala
85 90 95
Gly Asn Arg Pro Val Tyr Lys Ala Asn Gly Leu Ala Ala Tyr Phe Val
100 105 110
Thr Leu Ala Thr His Leu Gly Leu Trp Trp Phe Gly Ile Phe Asn Pro
115 120 125
Ala Ile Val Tyr Asp His Leu Gly Glu Ile Phe Ser Ala Leu Ile Phe
130 135 140
Gly Ser Phe Ile Phe Cys Val Leu Leu Tyr Ile Lys Gly His Val Ala
145 150 155 160
Pro Ser Ser Ser Asp Ser Gly Ser Cys Gly Asn Leu Ile Ile Asp Phe
165 170 175
Tyr Trp Gly Met Glu Leu Tyr Pro Arg Ile Gly Lys Ser Phe Asp Ile
180 185 190
Lys Val Phe Thr Asn Cys Arg Phe Gly Met Met Ser Trp Ala Val Leu
195 200 205
Ala Val Thr Tyr Cys Ile Lys Gln Tyr Glu Ile Asn Gly Lys Val Ser
210 215 220
Asp Ser Met Leu Val Asn Thr Ile Leu Met Leu Val Tyr Val Thr Lys
225 230 235 240
Phe Phe Trp Trp Glu Ala Gly Tyr Trp Asn Thr Met Asp Ile Ala His
245 250 255
Asp Arg Ala Gly Phe Tyr Ile Cys Trp Gly Cys Leu Val Trp Val Pro
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Ser Val Tyr Thr Ser Pro Gly Met Tyr Leu Val Asn His Pro Val Glu
275 280 285
Leu Gly Thr Gln Leu Ala Ile Tyr Ile Leu Val Ala Gly Ile Leu Cys
290 295 300
Ile Tyr Ile Lys Tyr Asp Cys Asp Arg Gln Arg Gln Glu Phe Arg Arg
305 310 315 320
Thr Asn Gly Lys Cys Leu Val Trp Gly Arg Ala Pro Ser Lys Ile Val
325 330 335
Ala Ser Tyr Thr Thr Thr Ser Gly Glu Thr Lys Thr Ser Leu Leu Leu
340 345 350
Thr Ser Gly Trp Trp Gly Leu Ala Arg His Phe His Tyr Val Pro Glu
355 360 365
Ile Leu Ser Ala Phe Phe Trp Thr Val Pro Ala Leu Phe Asp Asn Phe
370 375 380
Leu Ala Tyr Phe Tyr Val Leu Thr Leu Leu Leu Phe Asp Arg Ala Lys
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Arg Asp Asp Asp Arg Cys Arg Ser Lys Tyr Gly Lys Tyr Trp Lys Leu
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Tyr Cys Glu Lys Val Lys Tyr Arg Ile Ile Pro Gly Ile Tyr
420 425 430
<210> 3
<211> 783
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtatccgg atttaaaagg aaaagtcgtc gctattacag gagctgcttc agggctcgga 60
aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120
aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180
gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240
aaggagttcg gcacactcga tattatgatt aataatgccg gtcttgaaaa tcctgtgcca 300
tctcacgaaa tgccgctcaa ggattgggat aaagtcatcg gcacgaactt aacgggtgcc 360
tttttaggaa gccgtgaagc gattaaatat ttcgtagaaa acgatatcaa gggaaatgtc 420
attaacatgt ccagtgtgca cgcgtttcct tggccgttat ttgtccacta tgcggcaagt 480
aaaggcggga taaagctgat gacagaaaca ttagcgttgg aatacgcgcc gaagggcatt 540
cgcgtcaata atattgggcc aggtgcgatc aacacgccaa tcaatgctga aaaattcgct 600
gaccctaaac agaaagctga tgtagaaagc atgattccaa tgggatatat cggcgaaccg 660
gaggagatcg ccgcagtagc agcctggctt gcttcgaagg aagccagcta cgtcacaggc 720
atcacgttat tcgcggacgg cggtatgaca caatatcctt cattccaggc aggccgcggt 780
taa 783
<210> 4
<211> 260
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala
1 5 10 15
Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala
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Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val
35 40 45
Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly
50 55 60
Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile
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Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu
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Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val
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Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
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Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser
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165 170 175
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180 185 190
Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp Val
195 200 205
Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile Ala
210 215 220
Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr Gly
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<210> 5
<211> 34
<212> DNA
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ttgaaaattc gaattcatgg cggagactgt acat 34
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atccatcgat actagtcaat aaattcccgg aat 33
<210> 7
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggagaaaaaa ccccggatcc atgtatccgg atttaaaag 39
<210> 8
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
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caacttctgt tccatgtcga cttaaccgcg gcctgcctgg 40
Claims (10)
1.一种产生菜籽甾醇的酿酒酵母工程菌,其特征在于,所述酿酒酵母工程菌含有甾醇Δ7-还原酶基因和葡萄糖脱氢酶基因。
2.根据权利要求1所述的酿酒酵母工程菌,其特征在于,所述甾醇Δ7-还原酶基因的核苷酸序列如SEQ ID No.1所示。
3.根据权利要求1或2所述的酿酒酵母工程菌,其特征在于,所述甾醇Δ7-还原酶基因编码的甾醇Δ7-还原酶的氨基酸序列如SEQ ID No.2所示。
4.根据权利要求1所述的酿酒酵母工程菌,其特征在于,所述葡萄糖脱氢酶基因的核苷酸序列如SEQ ID No.3所示。
5.根据权利要求1或4所述的酿酒酵母工程菌,其特征在于,所述葡萄糖脱氢酶基因编码的葡萄糖脱氢酶的氨基酸序列如SEQ ID No.4所示。
6.权利要求1~5任一项所述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)将甾醇Δ7-还原酶基因插入到pESC-Trp质粒中,得到pESCTrp-Δ7Red2质粒;
2)将葡萄糖脱氢酶基因插入到步骤1)得到的pESCTrp-Δ7Red2质粒中,得到pESCTrp-Δ7Red2-GDH质粒;
3)将所述步骤2)得到的pESCTrp-Δ7Red2-GDH质粒导入酿酒酵母中,得到酿酒酵母工程菌。
7.根据权利要求6所述的构建方法,其特征在于,扩增甾醇Δ7-还原酶基因使用的引物包括Fhis49398引物和Rhis49398引物;
所述Fhis49398引物的核苷酸序列如SEQ ID No.5所示,所述Rhis49398引物的核苷酸序列如SEQ ID No.6所示。
8.根据权利要求7所述的构建方法,其特征在于,扩增甾醇Δ7-还原酶基因使用的程序为:94℃2min;94℃20s,55℃30s,72℃1min,30个循环;72℃7min。
9.根据权利要求6所述的构建方法,其特征在于,扩增葡萄糖脱氢酶基因使用的引物包括FBamH12276引物和RSal12276引物;
所述FBamH12276引物的核苷酸序列如SEQ ID No.7所示,所述RSal12276引物的核苷酸序列如SEQ ID No.8所示。
10.权利要求1~5任一项所述的酿酒酵母工程菌在制备菜籽甾醇中的应用。
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CN113913317A (zh) * | 2021-10-21 | 2022-01-11 | 厦门大学 | 一种重组酿酒酵母及提高重组酿酒酵母表面展示葡萄糖脱氢酶的酶活性的方法 |
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