CN114480153A - 一种生产维生素a的酿酒酵母菌及其构建方法 - Google Patents
一种生产维生素a的酿酒酵母菌及其构建方法 Download PDFInfo
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Abstract
本发明涉及一种生产维生素A的酿酒酵母菌及其构建方法,属于发酵工程技术领域。本发明以酿酒酵母为出发菌株,在酵母细胞中构建维生素A合成途径,过表达酿酒酵母内源维生素A合成前体异戊二烯焦磷酸的甲羟戊酸途径,同时强化酿酒酵母外源类胡萝卜素合成途径关键基因表达,得到一种酿酒酵母基因工程菌。利用该酿酒酵母基因工程菌发酵生产维生素A,摇瓶产量可达到193.9mg/L,维生素A产量显著提升。
Description
技术领域
本发明涉及发酵工程技术领域,尤其是指一种生产维生素A的酿酒酵母菌及其构建方法。
背景技术
维生素A(VitaminA,VA),被称为“美容维生素”和“抗干眼病维生素”,是人体必需微量营养素,不能在体内合成,需要通过外源性的方式(主要是饮食)获得。维生素A的主要生物活性形式是视黄醇、视黄醛和视黄酸,其中视黄醇主要存在于动物肝脏中,是维生素A主要的天然存在形式。
维生素A可从动物组织中提取,但资源分散,步骤繁杂,成本高。目前,商品维生素A都是化学合成产品,关键中间体柠檬醛生产技术缺乏控制力,大多数维生素产业的上游中间体供应受制于巴斯夫、日本第一制药等国际维生素中间体供应商。不仅如此,化学合成维生素A步骤繁琐且技术门槛较高,容易生成有毒害的异构体,对能源的消耗较大。
通过合成生物学技术构建微生物细胞工厂,反应条件温和、选择专一性强,能够将糖类等可再生的生物资源转化为化学产品,替代化学合成,实现绿色清洁生产,可以摆脱对石油资源的依赖,解决石化制造过程中的高耗能和高污染问题。
酿酒酵母是异源合成天然产物的重要宿主,通过合成生物学和代谢工程对酿酒酵母中所具有的甲羟戊酸(MVA)途径进行改造,引入外源类胡萝卜素合成的相关基因,实现萜类物质维生素A的合成,但维生素A合成途径复杂内源类异戊二烯前体供应不足、外源类胡萝卜素途径相关限速酶表达量低仍然是维生素A合成过程中需要解决的问题。
鉴于上述原因,本发明人积极加以研究创新,以期通过生物合成方法构建高效生产维生素A的菌株,使其更具有产业上的利用价值,为生物合成维生素产业提供理论与实践参考。
发明内容
为此,本发明所要解决的技术问题在于克服现有技术中酿酒酵母合成维生素A时存在的内源类异戊二烯前体供应不足、外源类胡萝卜素合成途径相关限速酶表达量低以及代谢途径复杂而导致产量实现不了突破等缺陷。
为解决上述技术问题,本发明提供了一种生产维生素A的酿酒酵母菌及其构建方法,利用该酿酒酵母基因工程菌发酵生产维生素A,摇瓶产量可达到193.9mg/L,维生素A产量显著提升。
本发明采用的技术方案如下。
本发明提供了一种生产维生素A的酿酒酵母基因工程菌,所述基因工程菌以酿酒酵母为出发菌株,在酵母细胞中构建维生素A合成途径,和/或强化酿酒酵母维生素A合成的内源甲羟戊酸途径,和/或同时强化酿酒酵母外源类胡萝卜素合成途径关键基因表达。
在本发明的一个实施例中,所述基因工程菌以酿酒酵母BY4741为出发菌株。
在本发明的一个实施例中,所述在酵母细胞中构建维生素A合成途径是将外源的香叶基香叶基二磷酸合酶(GGPP合酶)crtE、八氢番茄红素合酶crtB、八氢番茄红素去饱和酶crtI、番茄红素环化酶crtYB和β-胡萝卜素15,15′-单加氧酶BCMO编码基因整合到酿酒酵母基因组中。
优选地,所述crtE来源于曼地亚红豆杉(Taxus x media)。
优选地,所述crtB来源于成团泛菌(Pantoea agglomerans)。
优选地,所述crtI来源于三孢布拉氏霉菌(Blakeslea trispora)。
优选地,所述crtYB来源于红法夫酵母(Xanthophyllomyces Dendrorhous)。
优选地,所述BCMO来源于海洋细菌(uncultured marine bacterium)66A03。
在本发明的一个实施例中,所述强化酿酒酵母维生素A合成的内源甲羟戊酸途径包括:强化表达3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20、内质网大小调节因子的相关基因INO2、敲除ROX1基因和并且下调角鲨烯合酶ERG9的表达。
在本发明的一个实施例中,所述强化酿酒酵母外源类胡萝卜素合成途径关键基因为crtE、crtI时,采用双向诱导型启动子启动。
本发明还提供了一种生产维生素A的方法,包括以下步骤:
(1)将上述酿酒酵母基因工程菌接种到种子培养基中进行种子培养;
(2)将制备得到的种子液接种于发酵培养基,发酵培养60~120h,制备得到发酵液。
在本发明的一个实施例中,所述方法具体包括以下步骤:
(1)将上述酿酒酵母基因工程菌在25~35℃,200~250rpm条件下培养16~24h,制备得到种子液;
(2)将制备得到的种子液按1~5%(v/v)的接种量接种于发酵培养基;在25~35℃,200~250rpm条件下培养80~120h,制备得到发酵液。
优选地,种子培养基的成分包括:10g/L酵母膏、20g/L蛋白胨和20g/L葡萄糖。
优选地,发酵培养基的成分包括:25g/L葡萄糖、25g/L甘油、50g/L大豆蛋白胨、0.6g/L磷酸氢二钾、25g/L蔗糖。
优选地,在发酵培养基中还加入了十二烷,其中发酵培养基与十二烷的体积比是1:10-1:1。由于产物维生素A是亲脂性产物,过量积累对细胞有毒害作用,而且胞内对亲脂性物质储存能力有限,所以加入十二烷的作用是将胞内的维生素A萃取到十二烷中,利于胞内后续产物的合成与积累。
本发明还提供了上述酿酒酵母基因工程菌在食品、化工或制药领域的应用。
本发明的上述技术方案相比现有技术具有以下优点:
本发明提供了一种提高维生素A产量的基因工程菌及其构建方法与应用,该构建方法是以酿酒酵母BY4741为出发菌株,将外源的香叶基香叶基二磷酸合酶(GGPP合酶,crtE)、八氢番茄红素合酶(crtB)、八氢番茄红素去饱和酶(crtI)、番茄红素环化酶(crtYB)和β-胡萝卜素15,15′-单加氧酶(BCMO)编码基因整合到酿酒酵母基因组中,在酵母细胞中构建维生素A合成途径,摇瓶发酵产量达到8.5mg/L。过表达酿酒酵母内源维生素A合成前体异戊二烯焦磷酸的甲羟戊酸(MVA)途径,其具体措施为强化表达3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20、内质网大小调节因子的相关基因INO2、敲除了ROX1基因和并且下调角鲨烯合酶ERG9的表达。使摇瓶发酵产量达到37.17mg/L。强化酿酒酵母外源类胡萝卜素合成途径关键基因crtE、crtI表达,并采用双向诱导型启动子启动,在保留更多酿酒酵母整合位点的同时简化基因整合操作,摇瓶发酵产量达到55.46mg/L融合crtYB和BCMO基因,摇瓶产量达到193.9mg/L。
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,其中
图1是本发明强化酿酒酵母中维生素合成的原理图。
具体实施方式
(1)培养基
种子培养基的成分包括:10g/L酵母膏、20g/L蛋白胨和20g/L葡萄糖。
发酵培养基的成分包括:25g/L葡萄糖、25g/L甘油、50g/L大豆蛋白胨、0.6g/L磷酸氢二钾、25g/L蔗糖,另外在发酵培养基中还需要加入十二烷,其中发酵培养基与十二烷的比例是1:10。
SD Trp平板:YNB培养基6.7g/L,葡萄糖20g/L,L-亮氨酸50mg/L,L-组氨酸50mg/L,尿嘧啶50mg/L,琼脂粉20g/L。
SD Leu平板:YNB培养基6.7g/L,葡萄糖20g/L,L-色氨酸50mg/L,L-组氨酸50mg/L,尿嘧啶50mg/L,琼脂粉20g/L。
SD Trp Leu平板:YNB培养基6.7g/L,葡萄糖20g/L,L-组氨酸50mg/L,尿嘧啶50mg/L,琼脂粉20g/L。
YPD固体平板:1%酵母粉,2%蛋白胨,2%葡萄糖,1.5%琼脂粉。
(2)维生素A萃取剂:使用乙酸乙酯进行胞内产物的萃取,使用十二烷进行胞外产物的萃取。
(3)发酵液处理:将发酵96h的酿酒酵母菌液在12000r/min条件下离心10min,吸取上清十二烷过膜后装于液相瓶进行胞外产物的测定;离心后收集菌体,用与发酵液同体积的灭菌超纯水水洗两次,吸取600μL水洗后的重悬菌液和600μL的乙酸乙酯加入到含有玻璃珠放入破碎管中,使用破壁机进行酵母细胞的破碎,涡旋振荡提取15min后,此时胞内产物基本溶于乙酸乙酯中,12000r/min离心10min,吸取上清过膜后装于液相瓶中,进行胞内产物的测定。
(4)HPLC检测维生素A产量:采用AgilentZORBAX EclipseXDB-C18分离柱(5μm,250×4.6mm),检测的温度在40℃,流动相使用甲醇:乙腈=95:5,流速为1mL/min,检测波长352nm,进样量10μL。
(5)菌株生长情况检测:使用紫外-可见分光光度计定时测定发酵液的吸光光度值OD600。
(6)基因及质粒的构建方法
基因片段T-X-P-Y-T表示终止子-目的基因-启动子-目的基因-终止子,如TADH1-crtE-PGAL10-crtI-TCYC1表示ADH1终止子-crtE基因-GAL10启动子-crtI基因-CYC1终止子;-表示连接,Δ表示敲除。
pMHyLp-Trp质粒的序列见SEQ ID No.1,pMHyLp-Leu质粒SEQ ID No.2,pMHyLp-His/PY26-Cre质粒的构建方法记载于专利CN113502235A中。
SEQ ID No.1:
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataaacgacattactatatatataatataggaagcatttaatagacagcatcgtaatatatgtgtactttgcagttatgacgccagatggcagtagtggaagatattctttattgaaaaatagcttgtcaccttacgtacaatcttgatccggagcttttctttttttgccgattaagaattaattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatgtctgttattaatttcacaggtagttctggtccattggtgaaagtttgcggcttgcagagcacagaggccgcagaatgtgctctagattccgatgctgacttgctgggtattatatgtgtgcccaatagaaagagaacaattgacccggttattgcaaggaaaatttcaagtcttgtaaaagcatataaaaatagttcaggcactccgaaatacttggttggcgtgtttcgtaatcaacctaaggaggatgttttggctctggtcaatgattacggcattgatatcgtccaactgcatggagatgagtcgtggcaagaataccaagagttcctcggtttgccagttattaaaagactcgtatttccaaaagactgcaacatactactcagtgcagcttcacagaaacctcattcgtttattcccttgtttgattcagaagcaggtgggacaggtgaacttttggattggaactcgatttctgactgggttggaaggcaagagagccccgaaagcttacattttatgttagctggtggactgacgccagaaaatgttggtgatgcgcttagattaaatggcgttattggtgttgatgtaagcggaggtgtggagacaaatggtgtaaaagactctaacaaaatagcaaatttcgtcaaaaatgctaagaaataggttattactgagtagtatttatttaagtattgtttgtgcacttgcctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtacccaggaaacagctatgaccatgattacgcctaggataacttcgtatagcatacattatacgaagttatgacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccaaacgacattactatatatataatataggaagcatttaatagacagcatcgtaatatatgtgtactttgcagttatgacgccagatggcagtagtggaagatattctttattgaaaaatagcttgtcaccttacgtacaatcttgatccggagcttttctttttttgccgattaagaattaattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatgtctgttattaatttcacaggtagttctggtccattggtgaaagtttgcggcttgcagagcacagaggccgcagaatgtgctctagattccgatgctgacttgctgggtattatatgtgtgcccaatagaaagagaacaattgacccggttattgcaaggaaaatttcaagtcttgtaaaagcatataaaaatagttcaggcactccgaaatacttggttggcgtgtttcgtaatcaacctaaggaggatgttttggctctggtcaatgattacggcattgatatcgtccaactgcatggagatgagtcgtggcaagaataccaagagttcctcggtttgccagttattaaaagactcgtatttccaaaagactgcaacatactactcagtgcagcttcacagaaacctcattcgtttattcccttgtttgattcagaagcaggtgggacaggtgaacttttggattggaactcgatttctgactgggttggaaggcaagagagccccgaaagcttacattttatgttagctggtggactgacgccagaaaatgttggtgatgcgcttagattaaatggcgttattggtgttgatgtaagcggaggtgtggagacaaatggtgtaaaagactctaacaaaatagcaaatttcgtcaaaaatgctaagaaatagggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttaataacttcgtatagcatacattatacgaagttatgctagcgtccggagcggccgcGCATGCaagtcgaccttggcactggccgtcgttttacaaggccgcaaattaaagccttcgagcgtcccaaaaccttctcaagcaaggttttcagtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataaataacgttcttaatactaacataactataaaaaaataaatagggacctagacttcaggttgtctaactccttccttttcggttagagcggatgtggggggagggcgtgaatgtaagcgtgacataactaattacatgactcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccgggggatccactagttctagaaaacttagattagattgctatgctttctttctaatgagcaagaagtaaaaaaagttgtaatagaacaagaaaaatgaaactgaaacttgagaaattgaagaccgtttattaacttaaatatcaatgggaggtcatcgaaagagaaaaaaatcaaaaaaaaaattttcaagaaaaagaaacgtgataaaaatttttattgcctttttcgacgaagaaaaagaaacgaggcggtctcttttttcttttccaaacctttagtacgggtaattaacgacaccctagaggaagaaagaggggaaatttagtatgctgtgcttgggtgttttgaagtggtacggcgatgcgcggagtccgagaaaatctggaagagtaaaaaaggagtagaaacattttgaagctatgagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacataggagccggaagcataaagtgtaaagcctggggtgcctaatgagtgaggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgggtccttttcatcacgtgctataaaaataattataatttaaattttttaatataaatatataaattaaaaatagaaagtaaaaaaagaaattaaagaaaaaatagtttttgttttccgaagatgtaaaagactctagggggatcgccaacaaatactaccttttatcttgctcttcctgctctcaggtattaatgccgaattgtttcatcttgtctgtgtagaagaccacacacgaaaatcctgtgattttacattttacttatcgttaatcgaatgtatatctatttaatctgcttttcttgtctaataaatatatatgtaaagtacgctttttgttgaaattttttaaacctttgtttatttttttttcttcattccgtaactcttctaccttctttatttactttctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacgaggcgcgtgtaagttacaggcaagcgatccgtcctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc.
SEQ ID No.2:
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatcgactacgtcgtaaggccgtttctgacagagtaaaattcttgagggaactttcaccattatgggaaatggttcaagaaggtattgacttaaactccatcaaatggtcaggtcattgagtgttttttatttgttgtatttttttttttttagagaaaatcctccaatatcaaattaggaatcgtagtttcatgattttctgttacacctaactttttgtgtggtgccctcctccttgtcaatattaatgttaaagtgcaattctttttccttatcacgttgagccattagtatcaatttgcttacctgtattcctttactatcctcctttttctccttcttgataaatgtatgtagattgcgtatatagtttcgtctaccctatgaacatattccattttgtaatttcgtgtcgtttctattatgaatttcatttataaagtttatgtacaaatatcataaaaaaagagaatctttttaagcaaggattttcttaacttcttcggcgacagcatcaccgacttcggtggtactgttggaaccacctaaatcaccagttctgatacctgcatccaaaacctttttaactgcatcttcaatggccttaccttcttcaggcaagttcaatgacaatttcaacatcattgcagcagacaagatagtggcgatagggtcaaccttattctttggcaaatctggagcagaaccgtggcatggttcgtacaaaccaaatgcggtgttcttgtctggcaaagaggccaaggacgcagatggcaacaaacccaaggaacctgggataacggaggcttcatcggagatgatatcaccaaacatgttgctggtgattataataccatttaggtgggttgggttcttaactaggatcatggcggcagaatcaatcaattgatgttgaaccttcaatgtagggaattcgttcttgatggtttcctccacagtttttctccataatcttgaagaggccaaaacattagctttatccaaggaccaaataggcaatggtggctcatgttgtagggccatgaaagcggccattcttgtgattctttgcacttctggaacggtgtattgttcactatcccaagcgacaccatcaccatcgtcttcctttctcttaccaaagtaaatacctcccactaattctctgacaacaacgaagtcagtacctttagcaaattgtggcttgattggagataagtctaaaagagagtcggatgcaaagttacatggtcttaagttggcgtacaattgaagttctttacggatttttagtaaaccttgttcaggtctaacactaccggtaccccatttaggaccacccacagcacctaacaaaacggcatcaaccttcttggaggcttccagcgcctcatctggaagtgggacacctgtagcatcgatagcagcaccaccaattaaatgattttcgaaatcgaacttgacattggaacgaacatcagaaatagctttaagaaccttaatggcttcggctgtgatttcttgaccaacgtggtcacctggcaaaacgacgatcttcttaggggcagacataggggcagacattagaatggtatatccttgaaatatatatatatattgctgaaatgtaaaaggtaagaaaagttagaaagtaagacgattgctaaccacctattggaaaaaacaataggtccttaaataatattgtcaacttcaagtattgtgatgcaagcatttagtcatgaacgcttctctattctatatgaaaagccggttccggcctctcacctttcctttttctcccaatttttcagttgaaaaaggtatatgcgtcaggcgacctctgaaattaacaaaaaatttccagtcatcgaatttgattctgtgcgatagcgcccctgtgtgttctcgttatgttgaggaaaaaaataatggttgctaagagattcgaactcttgcatcttacgatacctgagtattcccacagttaactgcggtcaagatatttcttgaatcaggcgccttagaccgctcggccaaacaaccaattacttgttgagaaatagagtataattatcctataaatataacgtttttgaacacacatgaacaaggaagtacaggacaattgattttgaagagaatgtggattttgatgtaattgttgggattccatttttaataaggcaataatattaggtatgtggatatactagaagttctcctcgaccgtcgatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtacccaggaaacagctatgaccatgattacgcctaggataacttcgtatagcatacattatacgaagttatgacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccattaagcaaggattttcttaacttcttcggcgacagcatcaccgacttcggtggtactgttggaaccacctaaatcaccagttctgatacctgcatccaaaacctttttaactgcatcttcaatggccttaccttcttcaggcaagttcaatgacaatttcaacatcattgcagcagacaagatagtggcgatagggtcaaccttattctttggcaaatctggagcagaaccgtggcatggttcgtacaaaccaaatgcggtgttcttgtctggcaaagaggccaaggacgcagatggcaacaaacccaaggaacctgggataacggaggcttcatcggagatgatatcaccaaacatgttgctggtgattataataccatttaggtgggttgggttcttaactaggatcatggcggcagaatcaatcaattgatgttgaaccttcaatgtagggaattcgttcttgatggtttcctccacagtttttctccataatcttgaagaggccaaaacattagctttatccaaggaccaaataggcaatggtggctcatgttgtagggccatgaaagcggccattcttgtgattctttgcacttctggaacggtgtattgttcactatcccaagcgacaccatcaccatcgtcttcctttctcttaccaaagtaaatacctcccactaattctctgacaacaacgaagtcagtacctttagcaaattgtggcttgattggagataagtctaaaagagagtcggatgcaaagttacatggtcttaagttggcgtacaattgaagttctttacggatttttagtaaaccttgttcaggtctaacactaccggtaccccatttaggaccacccacagcacctaacaaaacggcatcaaccttcttggaggcttccagcgcctcatctggaagtgggacacctgtagcatcgatagcagcaccaccaattaaatgattttcgaaatcgaacttgacattggaacgaacatcagaaatagctttaagaaccttaatggcttcggctgtgatttcttgaccaacgtggtcacctggcaaaacgacgatcttcttaggggcagacataggggcagacattagaatggtatatccttgaaatatatatatatattgctgaaatgtaaaaggtaagaaaagttagaaagtaagacgattgctaaccacctattggaaaaaacaataggtccttaaataatattgtcaacttcaagtattgtgatgcaagcatttagtcatgaacgcttctctattctatatgaaaagccggttccggcctctcacctttcctttttctcccaatttttcagttgaaaaaggtatatgcgtcaggcgacctctgaaattaacaaaaaatttccagtcatcgaatttgattctgtgcgatagcgcccctgtgtgttctcgttatgttgaggaaaaaaataatggttgctaagagattcgaactcttgcatcttacgatacctgagtattcccacagttaactgcggtcaagatatttcttgaatcaggcgccttagaccgctcggccaaacaaccaattacttgttgagaaatagagtataattatcctataaatataacgttttagggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttaataacttcgtatagcatacattatacgaagttatgctagcgtccggagcggccgcGCATGCaagtcgaccttggcactggccgtcgttttaggccgcaaattaaagccttcgagcgtcccaaaaccttctcaagcaaggttttcagtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataaataacgttcttaatactaacataactataaaaaaataaatagggacctagacttcaggttgtctaactccttccttttcggttagagcggatgtggggggagggcgtgaatgtaagcgtgacataactaattacatgactcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccgggggatccactagttctagaaaacttagattagattgctatgctttctttctaatgagcaagaagtaaaaaaagttgtaatagaacaagaaaaatgaaactgaaacttgagaaattgaagaccgtttattaacttaaatatcaatgggaggtcatcgaaagagaaaaaaatcaaaaaaaaaattttcaagaaaaagaaacgtgataaaaatttttattgcctttttcgacgaagaaaaagaaacgaggcggtctcttttttcttttccaaacctttagtacgggtaattaacgacaccctagaggaagaaagaggggaaatttagtatgctgtgcttgggtgttttgaagtggtacggcgatgcgcggagtccgagaaaatctggaagagtaaaaaaggagtagaaacattttgaagctatgagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacataggagccggaagcataaagtgtaaagcctggggtgcctaatgagtgaggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgggtccttttcatcacgtgctataaaaataattataatttaaattttttaatataaatatataaattaaaaatagaaagtaaaaaaagaaattaaagaaaaaatagtttttgttttccgaagatgtaaaagactctagggggatcgccaacaaatactaccttttatcttgctcttcctgctctcaggtattaatgccgaattgtttcatcttgtctgtgtagaagaccacacacgaaaatcctgtgattttacattttacttatcgttaatcgaatgtatatctatttaatctgcttttcttgtctaataaatatatatgtaaagtacgctttttgttgaaattttttaaacctttgtttatttttttttcttcattccgtaactcttctaccttctttatttactttctaaaatccaaatacaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacgaggcgcgtgtaagttacaggcaagcgatccgtcctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc.
下面将结合本发明中的实施例,对本发明作进一步详细描述,但不应将此理解为本发明上述主题的范围仅限于下述实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1.重组菌A1的构建
基于酿酒酵母表达的偏好性,分别人工合成编码香叶基香叶基二磷酸合酶(GGPP合酶,crtE)、八氢番茄红素合酶(crtB)、八氢番茄红素去饱和酶(crtI)、番茄红素环化酶(crtYB)和β-胡萝卜素15,15′-单加氧酶(BCMO)的基因片段,其中来源于曼地亚红豆杉(Taxus x media)的Crt E,来源于成团泛菌(Pantoea agglomerans)的Crt B,来源于三孢布拉氏霉菌(Blakeslea trispora)的Crt I,来源于红法夫酵母(XanthophyllomycesDendrorhous)的crtYB以及来源于海洋细菌66A03(uncultured marine bacterium 66A03)的BCMO在酿酒酵母中表达量较高。
具体构建过程如下:
(1)人工合成基因片段TADH1-crtE-PGAL10-crtI-TCYC1,以酿酒酵母BY4741基因组为模板,采用引物208a-U-F、208a-U-R扩增得到基因208a-U,采用引物208a-D-F、208a-D-R扩增得到基因208a-D,以pMHyLp-Trp质粒为模板,使用引物loxT-1F、loxT-1R扩增得到loxT-1基因片段。
(2)将步骤(1)中获得的三个基因片段TADH1-crtE-PGAL10-crtI-Tcyc1、308a-U和308a-D进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段208a-U-TADH1-crtE-PGAL10-crtI-Tcyc1-208a-D基因片段。
(3)制备酿酒酵母工程菌感受态,并将构建好的融合基因片段208a-U-TADH1-crtE-PGAL10-crtI-Tcyc1-208a-D转化进入酿酒酵母CEN.PK102菌株的感受态中,涂布在SD Trp平板上,
30℃培养2-3天,使用引物V-EI-1F、V-EI-1R进行菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD Trp、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ208a-TADH1-crtE-PGAL10-crtI-Tcyc1,命名为酿酒酵母重组菌A1。
引物序列
208a-U-F:ctttttattagtggcattttttatatattgtaacattagggttctg
208a-U-R:aggttttgggacgctcgaagctattggcaccgactctgctagtat
208a-D-F:cactggccgtcgttttacaacttgtagtttatgtgctttatagttaatgatcacgac
208a-D-R:ttgggaaatgggattttaagttttgttagcattc
loxT-1F:aggtatagcatgaggtcgcctccaggaaacagctatgaccatgattacgc
loxT-1R:tcattaactataaagcacataaactacaagttgtaaaacgacggccagtgc
V-EI-1F:gaattgctactgaagcttctaaa
V-EI-1R:caagaagtacggtgctgaat
实施例2.重组菌A2的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-crtB-TAOX1。以酿酒酵母BY4741基因组为模板,采用引物1021b-U-F、1021b-U-R扩增得到基因1021b-U,采用引物1021b-D-F、1021b-D-R扩增得到基因1021b-D,以pMHyLp-His质粒为模板,使用引物loxH-1F、loxH-1R扩增得到loxH-1基因片段。
(2)将步骤(1)中获得的三个基因片段PGAL7-crtB-TAOX1、1021b-U和1021b-D进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段1021b-U-PGAL7-crtB-TAOX1-1021b-D基因片段。
(3)制备酿酒酵母工程菌感受态,并将构建好的融合基因片段1021b-U-PGAL7-crtB-TAOX1-1021b-D转化进入到实施例1中酿酒酵母A1菌株的感受态中,涂布在SD His平板上,30℃培养2-3天,使用引物V-B-1F、V-B-1R进行菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD His、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ1021b-PGAL7-crtB-TAOX1,命名为酿酒酵母重组菌A2。
引物序列
1021b-U-F:attttggtaacagaagatggcagtatttcca
1021b-U-R:ccgaagtatagctttccaaaaggacaggaatggtttaactgctaagagacactg
1021b-D-F:cactggccgtcgttttatggcattatgagttaagagataatacgcacg
1021b-D-R:tgtcccttgtcagtgctgagttatt
loxH-1F:tcagagtacagaagaagattaagtgagagctccaggaaacagctatgaccatgattacgc
loxH-1R:cactggccgtcgttttatggcattatgagttaagagataatacgc
V-B-1F:tattatctggttccttcttg
V-B-1R:atcttcaacaatatctctag
实施例3.重组菌A3的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-crtYB-TAOX1,以酿酒酵母BY4741基因组为模板,采用引物1622b-U-F、1622b-U-R扩增得到基因1622b-U,采用引物1622b-D-F、1622b-D-R扩增得到基因1622b-D,以pMHyLp-Leu质粒为模板,使用引物loxL-1F、loxL-1R扩增得到loxL-1基因片段。
(2)将步骤(1)中获得的三个基因片段PGAL7-crtYB-TAOX1、1622b-U和1622b-D进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段1622b-U-PGAL7-crtYB-TAOX1-1622b-D基因片段。
(3)将步骤(2)中得到的融合基因片段1622b-U-PGAL7-crtYB-TAOX1-1622b-D转化入实施例2制备得到的A2菌株的感受态中,涂布在SD Leu平板上于30℃培养2-3天,使用引物V-YB-1F、V-YB-1R进行单菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD Leu、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ1622a-PGAL7-crtYB-TAOX1,命名为酿酒酵母重组菌A3。
引物序列
1622b-U-F:atagatacattgacaaggtaaataaagacattgcg
1622b-U-R:ggttttgggacgctcgaaggtgaactatttgtttttgctgttaccttctg
1622b-D-F:cactggccgtcgttttatggaactttatgtcgcctggc
1622b-D-R:attcttatttccatgaaatttaatttaaaacacagatatggtatttctat
loxL-1F:caggtatagcatgaggtcgctccaggaaacagctatgaccatgattacgc
loxL-1R:taaaacgacggccagtgcctggaactttatgtcgcctgg
V-YB-1F:ttgttcgggacgttgttccct
V-YB-1R:tttctctaacttcagatgga
实施例4.重组菌A4的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-BCMO-TAOX1,以酿酒酵母BY4741基因组为模板,采用引物1414a-U-F、1014a-U-R扩增得到基因1414a-U,采用引物1414a-D-F、1414a-D-R扩增得到基因1414a-D,以pMHyLp-His质粒为模板,使用引物loxH-2F、loxH-2R扩增得到loxH-2基因片段。
(2)将步骤(1)中获得的三个基因片段PGAL7-BCMO-TAOX1、1414a-U和1414a-D进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段1414a-U-PGAL7-BCMO-TAOX1-1414a-D基因片段。1622b-U-PGAL7-crtYB-TAOX1-1622b-D
(3)将步骤(2)中得到的融合基因片段1414a-U-PGAL7-BCMO-TAOX1-1414a-D转化入实施例3制备得到的A3菌株的感受态中,涂布在SD His平板上于30℃培养2-3天,使用引物V-BCMO-1F、V-BCMO-1R进行单菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD His、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ1414a-PGAL7-BCMO-TAOX1,命名为酿酒酵母重组菌A4。
引物序列
1414a-U-F:gaaggcggaaattcccttaatgacc
1414a-U-R:tccgaagtatagctttccaaaaggacggcggataatttgagcaatgatagta
1414a-D-F:ctggccgtcgttttacaatttttaagacgcatctccaaaaaaagaaaaagaac
1414a-D-R:tatgtaaacggatggcaattggtctca
loxH-2F:aggctttaatttgcggcccaggaaacagctatgaccatgattacgc
loxH-2R:taaaacgacggccagtgcctttttaagacgcatctccaaaaaaagaaa
V-BCMO-1F:aatatttgaaagttttaaat
V-BCMO-1R:agcttgctcctgatcagccta
实施例5.重组菌A5的构建
具体构建过程如下:
(1)人工合成基因片段PGPD-tHMG1-TADH1-PTEF1-IDI-TAOX1,以酿酒酵母BY4741基因组为模板,采用引物ROX1-U-F、ROX1-U-R扩增得到基因片段ROX1-U,采用引物ROX1-D-F、ROX1-D-R扩增得到基因片段ROX1-D,以质粒pMHyLp-Trp为模板,使用引物loxT-2F、loxT-2R扩增得到loxT-2片段。
(2)将步骤(1)中的四个片段PGPD-tHMG1-TADH1-PTEF1-IDI-TAOX1、ROX1-U、ROX1-D、loxH-2进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段ROX1-U-PGPD-tHMG1-TADH1-PTEF1-IDI-TAOX1-ROX1-D基因片段。
(3)将步骤(2)中得到的融合基因片段ROX1-U-PGPD-tHMG1-TADH1-PTEF1-IDI-TAOX1-ROX1-D转化入实施例4制备得到的A4菌株的感受态中,涂布在SD Trp平板上于30℃培养2-3天,使用引物V-tI-1F、V-tI-1R进行单菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD Trp、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株ΔROX1-PGPD-tHMG1-TADH1-PTEF1-IDI-TAOX1,命名为酿酒酵母重组菌A5。
引物序列
ROX1-U-F:actttgcgtaatgtaacggtct
ROX1-U-R:cgtaatcatggtcatagctgtttcctggcgtcaaaggtagtccacttaa
ROX1-D-F:caggttgctttctcaggtatagcatgaggtcgctctcctcttctccgaactcctctgt
ROX1-D-R:gttggttagctcgattgtgtct
loxT-2F:ttaagtggactacctttgacgc caggaaacagctatgaccatgattacgc
loxT-2F:cactggccgtcgttttacaaaacgacggccagtgcca
V-tI-1F:gaggttgcctattccttctgtt
V-tI-1F:catctgctttagctgctggta
实施例6.重组菌A6的构建
具体构建过程如下:
(1)人工合成基因片段PPGK1-INO2-TINO2、PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1,以酿酒酵母BY4741基因组为模板,使用引物911b-U-F、911b-U-R扩增得到基因片段911b-U,用引物911b-D-F、911b-D-R扩增得到基因片段911b-D,以质粒pMHyLp-Leu为模板,采用引物loxL-2F、loxL-2R扩增得到loxL-2片段。
(2)将步骤(1)中的五个片段PPGK1-INO2-TINO2、PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1、911b-U、1622b-D、loxL-2进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段911b-U-PPGK1-INO2-TINO2-PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1-911b-D。
(3)将步骤(2)中得到的融合基因片段911b-U-PPGK1-INO2-TINO2-PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1-911b-D。转化入实施例5制备得到的A5菌株的感受态中,涂布在SD Leu平板上于30℃培养2-3天,使用引物V-ITE-1F、V-ITE-1R进行单菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD Leu、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ911b-PPGK1-INO2-TINO2-PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1,命名为酿酒酵母重组菌A6。
引物序列
911b-U-F:tctctgctggtcggtacttaa
911b-U-R:gctcgaaggctttaatttgcggcctctgtcaccaagaaatgtcctt
911b-D-F:cgtaatcatggtcatagctgtttcctggatgagggtgaagggaaacag
911b-D-R:ccaacaatatgggtacgagaga
loxL-2F:acccattaagcaaggattttcttaaccaggaaacagctatgaccatgattacgc
loxL-2F:taaaacgacggccagtgcccgtaatcatggtcatagctgtttc
V-ITE-1F:gtttaacaagttcttgatatcata
V-ITE-1F:actgctgaaccagtcaagatc
实施例7.重组菌A7的构建
具体构建过程如下:
(1)人工合成基因片段Phxt1-ERG9-TAOX1,以酿酒酵母BY4741基因组为模板,采用引物ERG9-U-F、ERG9-U-R扩增得到基因片段ERG9-U,采用引物ERG9-D-F、ERG9-D-R扩增得到基因片段ERG9-D,以质粒pMHyLp-Leu为模板,使用引物loxL-3F、loxL-3R扩增得到loxL-3片段。
(2)将步骤(1)中的四个片段Phxt1-ERG9-TAOX1、ERG9-U、ERG9-R、loxL-3进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段ERG9-U-Phxt1-ERG9-TAOX1-ERG9-D基因片段。
(3)将步骤(2)中得到的融合基因片段ERG9-U-Phxt1-ERG9-TAOX1-ERG9-D转化入实施例6制备得到的A6菌株的感受态中,涂布在SD Leu平板上于30℃培养2-3天,使用引物V-E9-1F、V-E9-1R进行单菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD Leu、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株ΔERG9-U-Phxt1-ERG9-TAOX1,命名为酿酒酵母重组菌A7。
引物序列
ERG9-U-F:aaaagtgcagctcagagccc
ERG9-U-R:acgtcacatatcacacacacacagtaaatgtccacttaa
ERG9-D-F:tgctttctcaggtaagtctgcgccaaataacataaacaaacaa
ERG9-D-R:ttgggctgaatgatagtgataattcttttttctatca
loxL-3F:gcagtggtagtagcattagtgccaggaaacagctatgaccatgattacgc
loxL-3R:taaaacgacggccagtgccacggccaactca
V-E9-1F:tggcactagcgttggtatttt
V-E9-1F:ccaagccatgttgtctcttac
实施例8.重组菌A8的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-crtE-PGAL10-crtI-Tcyc1。以酿酒酵母BY4741基因组为模板,采用引物416d-U-F、416d-U-R扩增得到基因416d-U,采用引物416d-D-F、416d-D-R扩增得到基因416d-D,以pMHyLp-Trp质粒为模板,使用引物loxT-3F、loxT-3R扩增得到loxT-3基因片段。
(2)将步骤(1)中获得的三个基因片段TADH1-crtE-PGAL10-crtI-Tcyc1、416d-U和416d-D进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段416d-U-TADH1-crtE-PGAL10-crtI-Tcyc1-416d-D基因片段。
(3)制备酿酒酵母工程菌感受态,并将构建好的融合基因片段416d-U-TADH1-crtE-PGAL10-crtI-Tcyc1-416d-D转化进入到实施例7中酿酒酵母A7菌株的感受态中,涂布在SD Trp平板上,30℃培养2-3天,使用引物V-EI-2F、V-EI-2R进行菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD Trp、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ416d-TADH1-crtE-PGAL10-crtI-Tcyc1,命名为酿酒酵母重组菌A8。
引物序列
416d-U-F:taacaagatgtaaagataatgctaaatcatttggctttt
416d-U-R:gagatccgtttaaccggaccccttcgagcgtcccaaaccttc
416d-D-F:gcactggccgtcgttttacaacggtccactgtgtgccg
416d-D-R:ccattagccattgatgtggatatgctttg
loxT-3F:aggtagcatgaggtcgctccaggaaacagctatgaccatgattacgc
loxT-3R:tcattaactataaagcacataaactacaagttgtaaaacgacggccagtgc
V-EI-2F:caaatggtgtaaaagactct
V-EI-2R:aattcagcaccgtacttcttg
实施例9.重组菌A9的构建
具体构建过程如下:(酶融合)
(1)以人工合成基因片段PGAL7-crtYB和BCMO-TAOX1为模板,使用引物GSGGSG-1F和GSGGSG-1R扩增得到GSGGSG序列,在两个目的基因序列之间插入GSGGSG序列实现融合表达。以酿酒酵母BY4741基因组为模板,采用引物805a-U-F、805a-U-R扩增得到基因805a-U,采用引物805a-D-F、805a-D-R扩增得到基因805a-D,以pMHyLp-His质粒为模板,使用引物loxH-3F、loxH-3R扩增得到loxH-3基因片段。
(2)将步骤(1)中获得的六个基因片段PGAL7-crtYB、BCMO-TAOX1、805a-U、805a-D、GSGGSG和loxH-3进行重叠延伸PCR,PCR条件为98℃,5min预变性,然后98℃,变性10s,55℃,退火5s,7℃,延伸2min,共计30个循环,1%的琼脂糖凝胶电泳验证正确后,切胶回收片段,得到融合基因片段805a-U-PGAL7-crtYB-GSGGSG-BCMO-TAOX1-805a-D基因片段。
(3)制备酿酒酵母工程菌感受态,并将构建好的融合基因片段805a-U-PGAL7-crtYB-GSGGSG-BCMO-TAOX1-805a-D转化进入到实施例8中酿酒酵母A8菌株的感受态中,涂布在SD His平板上,30℃培养2-3天,使用引物V-YBB-1F、V-YBB-1R进行菌落PCR验证得到菌株。
(4)将步骤(3)得到的菌株制备成感受态,转化入PY26-Cre质粒,在SD Ura平板上于30℃培养2-3天,取单菌落接入YPD培养基内培养15-24h,划线于含有5-FOA的YPD平板上,于30℃培养2-3天。将长出的单菌落分别在SD Ura、SD His、YPD固体平板上进行点板验证,仅在YPD培养基上生长的单菌落为正确的酿酒酵母菌株Δ805a-PGAL7-crtYB-GSGGSG-BCMO-TAOX1,命名为酿酒酵母重组菌A9。
引物序列
GSGGSG-1F:aacttctggtcacggttcaggg
GSGGSG-1R:atcgctgcactggcctaggg
805a-U-F:ataacgtccagccactggacta
805a-U-R:aatcttaagggtgaagacaaatatgtgaggtccttttggaaagctatacttcg
805a-D-F:gaaacagctatgaccatgattacgctggatataatcactaaataaagtagattttccttgggtagtaga
805a-D-R:ggggtggtaattaaattcaagtccagattattatt
loxH-3F:caggtatagcatgaggtcgctccaggaaacagctatgaccatgattacgc
loxH-3R:taaaacgacggccagtgcctaaaacgacggccagtgcc
V-YBB-1F:aaaatggaattagacagggct
V-YBB-1R:ctttaatctttgttgattactc
实施例10.摇瓶发酵条件下重组菌株的维生素A产量
具体步骤如下:
(1)培养基的配制
种子培养基的成分包括:10g/L酵母膏、20g/L蛋白胨和20g/L葡萄糖。
发酵培养基的成分包括:25g/L葡萄糖、25g/L甘油、50g/L大豆蛋白胨、0.6g/L磷酸氢二钾、25g/L蔗糖,另外在发酵培养基中还需要加入十二烷,其中发酵培养基与十二烷的比例是1:10。
(2)菌株发酵参数
分别将上述重组酿酒酵母菌株在30℃,220rpm条件下培养16~24h,制备得到种子液,将制备得到的种子液按2%(v/v)的接种量接种于装有25mL发酵培养基和2.5mL十二烷的250mL锥形瓶,在30℃,220rpm条件下培养96h,制备得到发酵液。
(3)计算重组菌维生素A的产量:
重组菌株的产量包括两部分,一部分是细胞内含有产物的产量,另一部分是分泌到细胞外的产物含量,对于脂溶性的维生素A来说,绝大部分产物在发酵过程中已经被萃取到上层有机相十二烷中。
将发酵96h的酿酒酵母菌液在12000r/min条件下离心10min,吸取上清十二烷过膜后装于液相瓶进行胞外产物的测定;离心后收集菌体,用与发酵液同体积的灭菌超纯水水洗两次,吸取600μL水洗后的重悬菌液和600μL的乙酸乙酯加入到含有玻璃珠放入破碎管中,使用破壁机进行酵母细胞的破碎,涡旋振荡提取15min后,此时胞内产物基本溶于乙酸乙酯中,12000r/min离心10min,吸取上清过膜后装于液相瓶中,进行胞内产物的测定。通过与维生素A标品峰面积进行换算得出工程菌株的发酵产量。将吸净十二烷的发酵液重悬,吸取发酵液稀释100倍后使用紫外分光光度计测量OD600。
结果如表1所示,强化酿酒酵母内源MVA途径,过表达外源类胡萝卜素关键基因、运用多酶融合表达体系,重组菌A9合成维生素A的产量达到193.9mg/L,OD600达到97.42。
表1
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。
SEQUENCE LISTING
<110> 江南大学、好想你健康食品股份有限公司
<120> 一种生产维生素A的酿酒酵母菌及其构建方法
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 7060
<212> DNA
<213> (人工合成)
<400> 1
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat 240
atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa 300
aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa 360
gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat 420
tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta 480
atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg 540
cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa 600
agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa 660
atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg 720
ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt 780
ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag 840
actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg 900
attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg 960
gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa 1020
atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg 1080
agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat 1140
aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga 1200
aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt 1260
ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 1320
atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 1380
gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 1440
gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 1500
aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 1560
ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 1620
gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 1680
ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 1740
tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 1800
ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 1860
cgcgcgtaat acgactcact atagggcgaa ttgggtaccc aggaaacagc tatgaccatg 1920
attacgccta ggataacttc gtatagcata cattatacga agttatgacg acagagaccg 1980
ggttggcggc gcatttgtgt cccaaaaaac agccccaatt gccccaattg accccaaatt 2040
gacccaaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat 2100
atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa 2160
aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa 2220
gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat 2280
tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta 2340
atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg 2400
cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa 2460
agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa 2520
atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg 2580
ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt 2640
ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag 2700
actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg 2760
attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg 2820
gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa 2880
atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg 2940
agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat 3000
agggtaccga ctagttccat ggcctgtccc cacgttgccg gtcttgcctc ctactacctg 3060
tccatcaatg acgaggttct cacccctgcc caggtcgagg ctcttattac tgagtccaac 3120
accggtgttc ttcccaccac caacctcaag ggctctccca acgctgttgc ctacaacggt 3180
gttggcattt aggcaattaa cagatagttt gccggtgata attctcttaa cctcccacac 3240
tcctttgaca taacgattta tgtaacgaaa ctgaaatttg accagatatt gttgtaaata 3300
gaaaatctgg cttgtaggtg gcaaaatgcg gcgtctttgt tcatcaattc cctctgtgac 3360
tactcgtcat ccctttatgt tcgactgtcg tatttcttat tttccataca tatgcaagtg 3420
agatgcccgt gtccgttatc aaatctagtt aataacttcg tatagcatac attatacgaa 3480
gttatgctag cgtccggagc ggccgcgcat gcaagtcgac cttggcactg gccgtcgttt 3540
tacaaggccg caaattaaag ccttcgagcg tcccaaaacc ttctcaagca aggttttcag 3600
tataatgtta catgcgtaca cgcgtctgta cagaaaaaaa agaaaaattt gaaatataaa 3660
taacgttctt aatactaaca taactataaa aaaataaata gggacctaga cttcaggttg 3720
tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat gtaagcgtga 3780
cataactaat tacatgactc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc 3840
agcccggggg atccactagt tctagaaaac ttagattaga ttgctatgct ttctttctaa 3900
tgagcaagaa gtaaaaaaag ttgtaataga acaagaaaaa tgaaactgaa acttgagaaa 3960
ttgaagaccg tttattaact taaatatcaa tgggaggtca tcgaaagaga aaaaaatcaa 4020
aaaaaaaatt ttcaagaaaa agaaacgtga taaaaatttt tattgccttt ttcgacgaag 4080
aaaaagaaac gaggcggtct cttttttctt ttccaaacct ttagtacggg taattaacga 4140
caccctagag gaagaaagag gggaaattta gtatgctgtg cttgggtgtt ttgaagtggt 4200
acggcgatgc gcggagtccg agaaaatctg gaagagtaaa aaaggagtag aaacattttg 4260
aagctatgag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat 4320
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatag 4380
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa 4440
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 4500
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 4560
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4620
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4680
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4740
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4800
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4860
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4920
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4980
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5040
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5100
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5160
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5220
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5280
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5340
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5400
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5460
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5520
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5580
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5640
cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5700
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 5760
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 5820
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 5880
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 5940
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6000
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6060
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6120
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6180
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6240
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6300
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6360
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6420
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt 6480
ccttttcatc acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa 6540
attaaaaata gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat 6600
gtaaaagact ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct 6660
caggtattaa tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct 6720
gtgattttac attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg 6780
tctaataaat atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt 6840
tttttttctt cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac 6900
aaaacataaa aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata 6960
cgaggcgcgt gtaagttaca ggcaagcgat ccgtcctaag aaaccattat tatcatgaca 7020
ttaacctata aaaataggcg tatcacgagg ccctttcgtc 7060
<210> 2
<211> 8955
<212> DNA
<213> (人工合成)
<400> 2
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc 240
accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca 300
ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat 360
taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc 420
ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc 480
aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt 540
agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg 600
tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct 660
ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg 720
ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct 780
tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac 840
aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat 900
ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc 960
aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg 1020
ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca 1080
gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc 1140
acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata 1200
ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact 1260
tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc 1320
ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca 1380
aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt 1440
aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca 1500
ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg 1560
gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca 1620
attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga 1680
accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc 1740
ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata 1800
tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat 1860
tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat 1920
ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct 1980
ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca 2040
aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat 2100
gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga 2160
gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg 2220
gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt 2280
ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg 2340
atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta 2400
gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa 2460
ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt 2520
taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa 2580
gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag 2640
aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt 2700
gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 2760
cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag 2820
gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg 2880
cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg 2940
ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 3000
cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 3060
cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc 3120
gaattgggta cccaggaaac agctatgacc atgattacgc ctaggataac ttcgtatagc 3180
atacattata cgaagttatg acgacagaga ccgggttggc ggcgcatttg tgtcccaaaa 3240
aacagcccca attgccccaa ttgaccccaa attgacccat taagcaagga ttttcttaac 3300
ttcttcggcg acagcatcac cgacttcggt ggtactgttg gaaccaccta aatcaccagt 3360
tctgatacct gcatccaaaa cctttttaac tgcatcttca atggccttac cttcttcagg 3420
caagttcaat gacaatttca acatcattgc agcagacaag atagtggcga tagggtcaac 3480
cttattcttt ggcaaatctg gagcagaacc gtggcatggt tcgtacaaac caaatgcggt 3540
gttcttgtct ggcaaagagg ccaaggacgc agatggcaac aaacccaagg aacctgggat 3600
aacggaggct tcatcggaga tgatatcacc aaacatgttg ctggtgatta taataccatt 3660
taggtgggtt gggttcttaa ctaggatcat ggcggcagaa tcaatcaatt gatgttgaac 3720
cttcaatgta gggaattcgt tcttgatggt ttcctccaca gtttttctcc ataatcttga 3780
agaggccaaa acattagctt tatccaagga ccaaataggc aatggtggct catgttgtag 3840
ggccatgaaa gcggccattc ttgtgattct ttgcacttct ggaacggtgt attgttcact 3900
atcccaagcg acaccatcac catcgtcttc ctttctctta ccaaagtaaa tacctcccac 3960
taattctctg acaacaacga agtcagtacc tttagcaaat tgtggcttga ttggagataa 4020
gtctaaaaga gagtcggatg caaagttaca tggtcttaag ttggcgtaca attgaagttc 4080
tttacggatt tttagtaaac cttgttcagg tctaacacta ccggtacccc atttaggacc 4140
acccacagca cctaacaaaa cggcatcaac cttcttggag gcttccagcg cctcatctgg 4200
aagtgggaca cctgtagcat cgatagcagc accaccaatt aaatgatttt cgaaatcgaa 4260
cttgacattg gaacgaacat cagaaatagc tttaagaacc ttaatggctt cggctgtgat 4320
ttcttgacca acgtggtcac ctggcaaaac gacgatcttc ttaggggcag acataggggc 4380
agacattaga atggtatatc cttgaaatat atatatatat tgctgaaatg taaaaggtaa 4440
gaaaagttag aaagtaagac gattgctaac cacctattgg aaaaaacaat aggtccttaa 4500
ataatattgt caacttcaag tattgtgatg caagcattta gtcatgaacg cttctctatt 4560
ctatatgaaa agccggttcc ggcctctcac ctttcctttt tctcccaatt tttcagttga 4620
aaaaggtata tgcgtcaggc gacctctgaa attaacaaaa aatttccagt catcgaattt 4680
gattctgtgc gatagcgccc ctgtgtgttc tcgttatgtt gaggaaaaaa ataatggttg 4740
ctaagagatt cgaactcttg catcttacga tacctgagta ttcccacagt taactgcggt 4800
caagatattt cttgaatcag gcgccttaga ccgctcggcc aaacaaccaa ttacttgttg 4860
agaaatagag tataattatc ctataaatat aacgttttag ggtaccgact agttccatgg 4920
cctgtcccca cgttgccggt cttgcctcct actacctgtc catcaatgac gaggttctca 4980
cccctgccca ggtcgaggct cttattactg agtccaacac cggtgttctt cccaccacca 5040
acctcaaggg ctctcccaac gctgttgcct acaacggtgt tggcatttag gcaattaaca 5100
gatagtttgc cggtgataat tctcttaacc tcccacactc ctttgacata acgatttatg 5160
taacgaaact gaaatttgac cagatattgt tgtaaataga aaatctggct tgtaggtggc 5220
aaaatgcggc gtctttgttc atcaattccc tctgtgacta ctcgtcatcc ctttatgttc 5280
gactgtcgta tttcttattt tccatacata tgcaagtgag atgcccgtgt ccgttatcaa 5340
atctagttaa taacttcgta tagcatacat tatacgaagt tatgctagcg tccggagcgg 5400
ccgcgcatgc aagtcgacct tggcactggc cgtcgtttta ggccgcaaat taaagccttc 5460
gagcgtccca aaaccttctc aagcaaggtt ttcagtataa tgttacatgc gtacacgcgt 5520
ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg ttcttaatac taacataact 5580
ataaaaaaat aaatagggac ctagacttca ggttgtctaa ctccttcctt ttcggttaga 5640
gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa ctaattacat gactcgaggt 5700
cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag 5760
aaaacttaga ttagattgct atgctttctt tctaatgagc aagaagtaaa aaaagttgta 5820
atagaacaag aaaaatgaaa ctgaaacttg agaaattgaa gaccgtttat taacttaaat 5880
atcaatggga ggtcatcgaa agagaaaaaa atcaaaaaaa aaattttcaa gaaaaagaaa 5940
cgtgataaaa atttttattg cctttttcga cgaagaaaaa gaaacgaggc ggtctctttt 6000
ttcttttcca aacctttagt acgggtaatt aacgacaccc tagaggaaga aagaggggaa 6060
atttagtatg ctgtgcttgg gtgttttgaa gtggtacggc gatgcgcgga gtccgagaaa 6120
atctggaaga gtaaaaaagg agtagaaaca ttttgaagct atgagctcca gcttttgttc 6180
cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg 6240
aaattgttat ccgctcacaa ttccacacaa cataggagcc ggaagcataa agtgtaaagc 6300
ctggggtgcc taatgagtga ggtaactcac attaattgcg ttgcgctcac tgcccgcttt 6360
ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 6420
cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 6480
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 6540
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 6600
aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 6660
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 6720
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 6780
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 6840
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 6900
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 6960
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 7020
agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 7080
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 7140
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 7200
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 7260
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 7320
aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 7380
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 7440
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 7500
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 7560
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 7620
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 7680
cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 7740
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 7800
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 7860
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 7920
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 7980
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 8040
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 8100
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 8160
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 8220
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 8280
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 8340
tccgcgcaca tttccccgaa aagtgccacc tgggtccttt tcatcacgtg ctataaaaat 8400
aattataatt taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga 8460
aattaaagaa aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca 8520
acaaatacta ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca 8580
tcttgtctgt gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt 8640
aatcgaatgt atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac 8700
gctttttgtt gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt 8760
ctaccttctt tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag 8820
agtaaattcc caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa 8880
gcgatccgtc ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 8940
cgaggccctt tcgtc 8955
Claims (12)
1.一种生产维生素A的酿酒酵母基因工程菌,其特征在于,所述基因工程菌以酿酒酵母为出发菌株,在酵母细胞中构建维生素A合成途径,和/或强化酿酒酵母维生素A合成的内源甲羟戊酸途径,和/或同时强化酿酒酵母外源类胡萝卜素合成途径关键基因表达。
2.根据权利要求1所述的酿酒酵母基因工程菌,其特征在于,所述在酵母细胞中构建维生素A合成途径是将外源的香叶基香叶基二磷酸合酶crtE、八氢番茄红素合酶crtB、八氢番茄红素去饱和酶crtI、番茄红素环化酶crtYB和β-胡萝卜素15,15′-单加氧酶BCMO编码基因整合到酿酒酵母基因组中。
3.根据权利要求1所述的酿酒酵母基因工程菌,其特征在于,所述强化酿酒酵母维生素A合成的内源甲羟戊酸途径包括:强化表达3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、异戊烯基焦磷酸异构酶IDI、法尼基焦磷酸合酶ERG20、内质网大小调节因子的相关基因INO2、敲除ROX1基因和并且下调角鲨烯合酶ERG9的表达。
4.根据权利要求1所述的酿酒酵母基因工程菌,其特征在于,所述强化酿酒酵母外源类胡萝卜素合成途径关键基因为crtE、crtI时,采用双向诱导型启动子启动。
5.根据权利要求1~4任一项所述的酿酒酵母基因工程菌,其特征在于,所述基因工程菌以酿酒酵母BY4741为出发菌株。
6.根据权利要求2所述的酿酒酵母基因工程菌,其特征在于,所述crtE来源于曼地亚红豆杉;所述crtB来源于成团泛菌;所述crtI来源于三孢布拉氏霉菌;所述crtYB来源于红法夫酵母;所述BCMO来源于海洋细菌66A03。
7.一种生产维生素A的方法,其特征在于,包括以下步骤:
(1)将上述酿酒酵母基因工程菌接种到种子培养基中进行种子培养;
(2)将制备得到的种子液接种于发酵培养基,发酵培养,制备得到发酵液。
8.根据权利要求7所述的方法,其特征在于,步骤(1)中,所述种子培养的条件:温度25~35℃,200~250rpm条件下培养16~24h。
9.根据权利要求7所述的方法,其特征在于,步骤(2)中,所述种子液按1~5%的接种量接种于发酵培养基。
10.根据权利要求9所述的方法,其特征在于,在发酵培养基中还加入了十二烷,其中发酵培养基与十二烷的体积比是1:10-1:1。
11.根据权利要求7所述的方法,其特征在于,步骤(2)中,发酵培养条件:培养温度为25~35℃,200~250rpm条件下培养60~120h。
12.权利要求1~6任一项所述的酿酒酵母基因工程菌在食品、化工或制药领域的应用。
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