CN116144518A - 一种生产视黄醇的酿酒酵母菌株及其应用 - Google Patents
一种生产视黄醇的酿酒酵母菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种生产视黄醇的酿酒酵母菌株及其应用,构建方法为:以酿酒酵母为出发菌株,引入外源基因crtE、crtB、crtI、crtYB、blh,在酵母细胞中构建视黄醇合成途径,通过多元模块化工程优化视黄醇的代谢网络,将视黄醇合成途径分为5个模块进行代谢调控,再通过转运体工程敲除转运蛋白Pdr5p,最后利用酶的半理性改造提高关键酶的催化效率,将突变后的脱氢酶基因ENV9与大肠杆菌来源的脱氢酶基因ybbo组合整合在酿酒酵母基因组中,视黄醇产量达到387.4mg/L。最后优化培养基发酵条件,添加橄榄油作为萃取剂,摇瓶水平上视黄醇的产量达到1.2g/L。
Description
技术领域
本发明涉及一种生产视黄醇的酿酒酵母菌株及其应用,属于生物技术领域。
背景技术
视黄醇(Retinol)是维生素A的主要活性形式,又名维生素A1,是一种单环二萜类化合物,参与视觉循环,是构成视觉细胞感光物质的成分,提高眼睛对黑暗弱光的敏感度。除此之外,视黄醇在增强机体免疫、修复皮肤屏障、促进生长发育、抑制肿瘤生长、改善贫血等方面也发挥非常重要的作用,广泛应用于医药、食品、化妆品以及动物饲料等领域。
目前,视黄醇的合成主要包括自然提取法和化学合成法。然而从海洋鱼类肝脏中通过分子蒸馏和层析提取视黄醇资源分散,成本高,提取效率低;化学合成法合成视黄醇技术门槛高,对能源的消耗较大。微生物合成由于具备周期短、成本低、发酵条件温和、安全性高、可控性强且不受气候影响等诸多优势,因此构建微生物细胞工厂成为合成多种高价值化学品的有效手段之一。
酿酒酵母是公认的安全(GRAS)模式菌株,具备生长周期短、遗传操作简易等优势,除了自身具有很好的鲁棒性外,还具有以中心代谢物乙酰辅酶A为前体的甲羟戊酸途径,可提供合成二萜类物质视黄醇的关键前体法尼烯基焦磷酸。然而,生物合成生产视黄醇仍然存在以下问题:代谢途径复杂且不易系统优化;前体视黄醛的外排,不利于视黄醇的积累;视黄醛到视黄醇的转化效率低;发酵过程中产物不稳定。
鉴于上述原因,本发明人积极加以研究创新,以期构建一株高产视黄醇的酿酒酵母菌株,实现视黄醇在酿酒酵母中的高效合成,为工业化生产视黄醇奠定基础。
发明内容
为解决上述问题,本发明以酿酒酵母为出发菌株,引入外源基因crtE、crtB、crtI、crtYB、blh,在酵母细胞中构建视黄醇合成途径,通过多元模块化工程优化视黄醇的代谢网络,将视黄醇合成途径分为5个模块进行代谢调控,再通过转运体工程敲除转运蛋白Pdr5p,最后利用酶的半理性改造提高关键酶的催化效率,将突变后的脱氢酶基因ENV9与大肠杆菌来源的脱氢酶基因ybbo组合整合在酿酒酵母基因组中,实现视黄醇的高效生产。最后优化培养基发酵条件,添加橄榄油作为萃取剂,摇瓶水平上视黄醇的产量达到1.2g/L。
本发明的第一个目的是提供一种重组酿酒酵母,所述重组酿酒酵母异源表达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-胡萝卜素加氧酶Blh,过表达异戊烯基焦磷酸异构酶IDI、3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、法尼基焦磷酸合酶ERG20、乙醇脱氢酶ADH2、乙醛脱氢酶ALD6、乙酰辅酶A连接酶ACS1、GGPP合酶CrtE、八氢番茄红素去饱和酶CrtI、番茄红素环化酶CrtYB、β-胡萝卜素加氧酶Blh和NADH激酶POS5,弱化表达角鲨烯合成酶ERG9,敲除了柠檬酸合酶CIT2、苹果酸合酶MLS1、调节因子YPL062W和ABC转运体蛋白Pdr5p,将内源视黄醇脱氢酶ENV9第99位的苏氨酸突变为丙氨酸并引入异源视黄醇脱氢酶YBBO。
进一步地,所述重组酿酒酵母强化表达了磷脂酸磷酸酶PAH1和磷脂二酰基甘油酰基转移酶LRO1。
进一步地,强化表达为至少增加一个拷贝的磷脂酸磷酸酶PAH1和磷脂二酰基甘油酰基转移酶LRO1。
进一步地,所述弱化表达角鲨烯合成酶ERG9为替换原启动子为PHXT1启动子并添加降解标签CLN2。
进一步地,PHXT1启动子的核苷酸序列如SEQ ID NO.1所示;降解标签CLN2的核苷酸序列如SEQ ID NO.2所示。
进一步地,过表达GGPP合酶CrtE、八氢番茄红素去饱和酶CrtI、番茄红素环化酶CrtYB为增加三个拷贝的GGPP合酶CrtE和八氢番茄红素去饱和酶CrtI、增加一个拷贝的番茄红素环化酶CrtYB。
进一步地,所述内源视黄醇脱氢酶ENV9的氨基酸序列如SEQ ID NO.3所示。
进一步地,编码GGPP合酶CrtE的基因如SEQ ID NO.5所示。
进一步地,编码八氢番茄红素合酶CrtB的基因如SEQ ID NO.6所示。
进一步地,编码八氢番茄红素去饱和酶CrtI的基因如SEQ ID NO.7所示。
进一步地,编码番茄红素环化酶CrtYB的基因如SEQ ID NO.8所示。
进一步地,编码视黄醇脱氢酶YBBO的基因如SEQ ID NO.9所示。
进一步地,编码异戊烯基焦磷酸异构酶IDI的基因如SEQ ID NO.10所示。
进一步地,编码3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1的基因如SEQ ID NO.11所示。
进一步地,编码法尼基焦磷酸合酶ERG20的基因如SEQ ID NO.12所示。
进一步地,编码乙醇脱氢酶ADH2的基因如SEQ ID NO.13所示。
进一步地,编码乙醛脱氢酶ALD6的基因如SEQ ID NO.14所示。
进一步地,编码乙酰辅酶A连接酶ACS1的基因如SEQ ID NO.15所示。
进一步地,编码β-胡萝卜素加氧酶Blh的基因如SEQ ID NO.16所示。
进一步地,编码NADH激酶POS5的基因如SEQ ID NO.17所示。
进一步地,编码柠檬酸合酶CIT2的基因如SEQ ID NO.18所示。
进一步地,编码苹果酸合酶MLS1的基因如SEQ ID NO.19所示。
进一步地,编码调节因子YPL062W的基因如SEQ ID NO.20所示。
进一步地,编码ABC转运体蛋白Pdr5p的基因如SEQ ID NO.21所示。
进一步地,编码磷脂酸磷酸酶PAH1的基因如SEQ ID NO.22所示。
进一步地,编码磷脂二酰基甘油酰基转移酶LRO1的基因如SEQ ID NO.23所示。
本发明的第二个目的是提供一种视黄醇脱氢酶突变体,所述突变体由氨基酸序列如SEQ ID NO.1所示的视黄醇脱氢酶的第99位苏氨酸突变为丙氨酸得到。
本发明的第三个目的是提供编码上述视黄醇脱氢酶突变体的基因。
进一步地,基因序列如SEQ ID NO.4所示。
本发明的第四个目的是提供携带上述基因的重组质粒。
本发明的第五个目的是提供表达所述视黄醇脱氢酶突变体的细胞。
进一步地,所述细胞为细菌、真菌、植物细胞或动物细胞。
本发明的第六个目的是提供一种生产视黄醇的方法,应用上述重组酿酒酵母进行发酵。
进一步地,将所述重组酿酒酵母在种子培养基中活化得到种子液,再将种子液接种至发酵培养基中进行发酵培养。
进一步地,种子培养基包括5-15g/L酵母膏、10-30g/L蛋白胨和10-30g/L葡萄糖。
进一步地,发酵培养基包括15-35g/L葡萄糖、15-35g/L甘油、40-60g/L大豆蛋白胨、0.1-2g/L磷酸氢二钾和15-35g/L蔗糖。
进一步地,向所述发酵培养基中加入萃取剂。
进一步地,所述萃取剂选自十二烷或橄榄油。
进一步地,所述发酵培养基中含有二丁基羟基甲苯、没食子酸酯和乙氧基喹啉中的一种或几种。
本发明的有益效果:
本发明通过多元模块工程,强化甲羟戊酸合成模块,视黄醇产量提高3.18倍,达到3.6mg/L;强化中心碳代谢模块,视黄醇产量提高11.7倍达到45.8mg/L;弱化角鲨烯竞争支路,角鲨烯产量下降58.7%,视黄醇产量提高41%,达到64.6mg/L;优化β-胡萝卜素合成模块,产量提高29.4%;强化视黄醇合成模块,视黄醇产量提高83.7%,达到153.6mg/L。通过转运体工程抑制视黄醛的分泌,促进视黄醇的积累,胞内视黄醛产量提高170%,视黄醇产量提高7.2%。通过酶工程对关键脱氢酶进行半理性改造,视黄醇产量提高18.2%。最后选用橄榄油进行双相发酵,相较于十二烷萃取视黄醇产量提高了181.8%,相较于添加抗氧化剂BHT萃取视黄醇,产量提高了93.1%,最终视黄醇达到1.2g/L。
附图说明
图1为本发明重组菌的构建路径;
图2为重组菌RT1-RT7的结果;
图3为重组菌RP1-RP5的结果;
图4为重组菌RMA1-RMA23的结果。
具体实施方式
下面结合附图和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。
本发明的方案如下:
以酿酒酵母CEN.PK2-1C为宿主,视黄醇为目标产物,以提高视黄醇发酵产量以及稳定性为目的,成功在酿酒酵母中构建视黄醇从头合成途径,通过模块化工程、转运体工程以及酶工程等手段结合发酵条件优化,最终得到一株高产视黄醇的酿酒酵母重组菌株。主要研究结论如下:
(1)通过模块化工程,将酿酒酵母中复杂的视黄醇代谢途径分为以下五个代谢模块,逐一细致调控。
内源甲羟戊酸合成模块:过表达MVA途径关键基因IDI、tHMG1、ERG20,视黄醇产量达3.6mg/L。
中心碳代谢模块:过表达ADH2、ALD6、ACS1提高碳代谢中乙醇利用率,敲除CIT2、MLS1、YPL062W增强乙酰辅酶A代谢通量,视黄醇产量达到45.8mg/L。
角鲨烯合成模块:弱启动子PHXT1动态调控结合添加降解标签CLN2,下调角鲨烯竞争支路,视黄醇产量达到64.6mg/L。
外源β-胡萝卜素合成模块:采用不同基因拷贝数过表达crtE、crtI、crtYB并进行组合优化,增加3个crtE、crtI拷贝数,增加1个crtYB拷贝数时,视黄醇产量最佳,达到83.6mg/L。
视黄醇合成模块:过表达β-胡萝卜素加氧酶Blh、NADH激酶POS5,提高视黄醇的代谢通量,视黄醇产量达到153.6mg/L。
(3)利用转运体工程,敲除ABC转运体蛋白Pdr5p,减少前体视黄醛的外排,促进视黄醇的积累,视黄醇产量达164.6mg/L。
(4)通过酶工程改造,对酿酒酵母内源关键视黄醇脱氢酶ENV9进行半理性改造,利用分子对接预测关键位点,将第99位的苏氨酸突变为丙氨酸,与基因ybbo组合优化整合到酿酒酵母基因组中,视黄醇产量提高至387.4mg/L。
(5)在摇瓶水平进行发酵条件的优化,在培养基中加入萃取剂橄榄油,提高产物稳定性,遮光发酵,最终视黄醇产量达到1.2g/L。
下述实施例中涉及的材料及方法:
(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。
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)视黄醇萃取剂:使用乙酸乙酯进行胞内产物的萃取,使用橄榄油进行胞外产物的萃取。
(3)发酵液处理:将发酵120h的酿酒酵母重悬液在12000r/min条件下离心10min,吸取上清橄榄油经乙酸乙酯稀释10倍后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600uL水洗后的重悬菌液和600uL的乙酸乙酯加入到含有玻璃珠放入破碎管中,使用高压匀浆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检测视黄醇产量:采用Agilent ZORBAX EclipseXDB-C18分离柱(5μm,250×4.6mm),检测的温度在40℃,流动相使用乙腈:甲醇=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。
实施例1.重组菌R1的构建
基于酿酒酵母表达的偏好性,人工合成基因片段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-胡萝卜素加氧酶(Blh),其中CrtE来源于曼地亚红豆杉(Taxus x media),CrtB来源于成团泛菌(Pantoeaagglomerans),CrtI来源于三孢布拉氏霉菌(Blakeslea trispora),CrtYB来源于红法夫酵母(Xanthophyllomyces Dendrorhous),Blh来源于海洋细菌66A03(Uncultured marinebacterium66A03),并进行密码子优化。
具体构建过程如下:
(1)人工合成基因片段TADH1-crtE-PGAL10-crtI-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物308a-U-F、308a-U-R扩增得到基因308a-U,采用引物308a-D-F、308a-D-R扩增得到基因308a-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-TAOXI、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35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308a-U-TADH1-crtE-PGAL10-crtI-TAOXI-308a-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308a-U-TADH1-crtE-PGAL10-crtI-TAOXI-308a-D转化进入酿酒酵母CEN.PK2-1C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Δ308a-TADH1-crtE-PGAL10-crtI-TAOXI,命名为酿酒酵母重组菌R1。
引物序列:
308a-U-F:aagccagttttaaggaaccgagata
308a-U-R:tcgaatttttttctttttatctaagaattcatggcttacactgctatggc
308a-D-F:cttggcactggccgtcgttttacaaaggattacttgtcttctttgctaca
308a-D-R:aaaaggtaatgattgaaaaagtttt
loxT-1F:aggtatagcatgaggtcgctccaggaaacagctatgaccatgattacgc
loxT-1R:gcactggccgtcgttttacaacttcagaactaaaaaaataataaggaagaaaaa
V-EI-1F:cagagcatgtagtatgggac
V-EI-1R:caagaagtacggtgctgaat
实施例2.重组菌R2的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-crtB-TAOX1-PGAL1-crtYB-TCYC1。以酿酒酵母CEN.PK2-1C基因组为模板,采用引物607c-U-F、607c-U-R扩增得到基因607c-U,采用引物607c-D-F、607c-D-R扩增得到基因607c-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-PGAL1-crtYB-TCYC1、607c-U和607c-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35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607c-U-PGAL7-crtB-TAOX1-PGAL1-crtYB-TCYC1-607c-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607c-U-PGAL7-crtB-TAOX1-PGAL1-
crtYB-TCYC1-607c-D转化进入到实施例1中酿酒酵母R1重组菌株的感受态中,涂布在SD-His平板上,30℃培养2-3天,使用引物V-BYB-1F、V-BYB-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Δ607c-U-PGAL7-crtB-TAOX1-PGAL1-crtYB-TCYC1,命名为酿酒酵母重组菌R2。
引物序列
607c-U-F:ggcagtataagttcaaaaaaatcca
607c-U-R:ttgtgtcgtaccgaagaaaaagcct
607c-D-F:gagtacagaagattaagtgagagctctacacttctagccttgattaccaa
607c-D-R:gttactgaagaaactttaaaagggat
loxH-1F:atcgccttcagacaaaactgagagctc caggaaacagctatgaccatgattacgc
loxH-1R:cttggcactggccgtcgttttatacacttctagccttgattaccaa
V-BYB-1F:ggtgaagacagcttctttttccactat
V-BYB-1R:aagacaatctacttctactccta
实施例3.重组菌R3的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-blh-TADH1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物1309a-U-F、1309a-U-R扩增得到基因片段1309a-U,采用引物1309a-D-F、1309a-D-R扩增得到基因片段1309a-D,以质粒pMHyLp-Leu为模板,使用引物loxL-1F、loxL-1R扩增得到loxL-1片段。
(2)将步骤(1)中的四个片段PGAL7-blh-TADH1、1309a-U、1309a-D、loxL-1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35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1309a-U-PGAL7-blh-TADH1-1309a-D基因片段。
(3)将步骤(2)中得到的融合基因片段1309a-U-PGAL7-blh-TADH1-1309a-D转化入实施例2制备得到的R2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-BL-1F、V-BL-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Δ1309a-PGAL7-blh-TADH1,命名为酿酒酵母重组菌R3。
引物序列:
1309a-U-F:cgttgttgatcttgtagtctggg
1309a-U-R:tctccctatttataaacgtcactaa tccttttggaaagctatacttcggagc
1309a-D-F:ggcactggccgtcgttttaaggtctactactccatcgtaaa
1309a-D-R:ttgccgctacccgaaaattttgg
loxL-1F:caggaaacagctatgaccatgattacgc
loxL-1R:ggcactggccgtcgttttaaggtctactactccatcgtaaa
V-BL-1F:aaacagaagtaagtagcaaaagta
V-BL-1R:tcaatcaacatcaaacccattttt
实施例4.重组菌RM1的构建
具体构建过程如下:
(1)人工合成基因片段PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物911b-U-F、911b-U-R扩增得到基因片段911b-U,采用引物911b-D-F、911b-D-R扩增得到基因片段911b-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-ERG20-TCYC1、911b-U、911b-D、loxT-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35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-PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1-911b-D基因片段。
(3)将步骤(2)中得到的融合基因片段911b-U-PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1-911b-D转化入实施例3制备得到的R3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-HE-1F、V-HE-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Δ911b-PGPD-tHMG1-TADH1-PTEF1-ERG20-TCYC1,命名为酿酒酵母重组菌RM1。
引物序列:
911b-U-F:gatgagatatggaggtgttttt
911b-U-R:agaaaaaaatcggatgttgaatgggcataaatataaatgtatatataa
911b-D-F:gcactggccgtcgttttacaatggagaagtaaatgaaaaatgaaatagcatacaaaacag
911b-D-R:aagggaaaattgagccgggctaatgaa
loxT-2F:caggaaacagctatgaccatgattacgcct
loxT-2R:gcactggccgtcgttttacaaaatgaaaaatgaaatagcatacaaaacag
V-HE-1F:taaataaagtattggacataaa
V-HE-1R:catctgatcgtactggtaatgtg
实施例5.重组菌RM2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-TCYC1,以酿酒酵母CEN.PK2-1C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-His为模板,使用引物loxH-2F、loxH-2R扩增得到loxH-2片段。
(2)将步骤(1)中的四个PGPD-tHMG1-TADH1-PTEF1-IDI-TCYC1、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35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-TCYC1-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-TCYC1-ROX1-D转化入实施例4制备得到的RM1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-HI-1F、V-HI-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ΔROX1-PGPD-tHMG1-TADH1-PTEF1-IDI-TCYC1,命名为酿酒酵母重组菌RM2。
引物序列:
ROX1-U-F:tcccctggatatcattggcctgtcg
ROX1-U-R:acaaaagaacgcagttagacaatcaacagagctctcacttaatcttctgtactctgaaga
ROX1-D-F:ggcactggccgtcgttttattttttttttccatttcttctttccgttatattata
ROX1-D-R:tttgacaatgttaagctttgttaaacaggttcatatt
loxH-2F:caggaaacagctatgaccatgattacgcc
loxH-2R:ggcactggccgtcgttttagctctcacttaatcttctgtactctgaaga
V-HI-1F:tctttgaaagaattgctactg
VHI-1R:cgttgggtagatacgttgacac
实施例6.重组菌RC1的构建
具体构建过程如下:
(1)人工合成基因片段PTEF1-ADH2-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物CIT2-U-F、CIT2-U-R扩增得到基因片段CIT2-U,采用引物CIT2-D-F、CIT2-D-R扩增得到基因片段CIT2-D,以质粒pMHyLp-Leu为模板,使用引物loxL-2F、loxL-2R扩增得到loxL-2片段。
(2)将步骤(1)中的四个片段PTEF1-ADH2-TAOX1、CIT2-U、CIT2-R、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35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CIT2-U-PTEF1-ADH2-TAOX1-CIT2-D基因片段。
(3)将步骤(2)中得到的融合基因片段CIT2-U-PTEF1-ADH2-TAOX1-CIT2-D转化入实施例5制备得到的RM2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-AH-1F、V-AH-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ΔCIT2-PTEF1-ADH2-TAOX1,命名为酿酒酵母重组菌RC1。
引物序列:
CIT2-U-F:ataaaaatgaacgtaaaacaagtaaaaatgtaggatgt
CIT2-U-R:gaggggtgtaaaagtaggatgtaatccaa
CIT2-D-F:ttttcttgttactagtattattaaaacaaaaagttttg
CIT2-D-R:gcttatgttgtggcaacaactctg
loxL-2F:caggaaacagctatgaccatgattacg
loxL-2R:ggcactggccgtcgttttattttcttgttactagtattattaaa
V-AH-1F:tccagaaactcaaaaagccattatcttctac
V-AH-1R:agaagccttagatttctttgccagag
实施例7.重组菌RC2的构建
具体构建过程如下:
(1)人工合成基因片段PGPD-ALD6-TADH1-PTEF1-ACS1-TCYC1。以酿酒酵母CEN.PK2-1C基因组为模板,采用引物MLS1-U-F、MLS1-U-R扩增得到基因MLS1-U,采用引物MLS1-D-F、MLS1-D-R扩增得到基因MLS1-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PGPD-ALD6-TADH1-PTEF1-ACS1-TCYC1、MLS1-U和MLS1-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35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MLS1-U-PGPD-ALD6-TADH1-PTEF1-ACS1-TCYC1-MLS1-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MLS1-U-PGPD-ALD6-TADH1-PTEF1
-ACS1-TCYC1-MLS1-D转化进入到实施例6中酿酒酵母RC1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-AA-1F、V-AA-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ΔMLS1-PGPD-ALD6-TADH1-PTEF1-ACS1-TCYC1,命名为酿酒酵母重组菌RC2。
引物序列
MLS1-U-F:ttgctatcggctcaatggaaatccccat
MLS1-U-R:ttgaactaaacaaagtagtaaaagcacataaaagaattaagaaagagctctcacttaatcttctgtactctgaaga
MLS1-D-F:gcactggccgtcgttttacaaatctcccttgccccagtgtacacatatataaaaatata
MLS1-D-R:gcgtcgggtatcagttcgttt
loxT-3F:caggaaacagctatgaccatgattacgcct
loxT-3R:gcactggccgtcgttttacaaccagtgtacacatatataaaaatata
V-AA-1F:actttgacactgctgaaccagtc
V-AA-1R:gaaatcaaagttggtaatccattt
实施例8.重组菌RC3的构建
具体构建过程如下:
(1)以酿酒酵母CEN.PK2-1C基因组为模板,采用引物YPL062W-U-F、YPL062W-U-R扩增得到基因YPL062W-U,采用引物YPL062W-D-F、YPL062W-D-R扩增得到基因YPL062W-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)中获得的两个基因片段YPL062W-U和YPL062W-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35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YPL062W-U-YPL062W-D基因片段。
(3)将步骤(2)中得到的融合基因片段YPL062W-U-YPL062W-D转化入实施例8制备得到的RC2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-YP-1F、V-YP-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ΔYPL062W,命名为酿酒酵母重组RC3。
引物序列:
YPL062W-U-F:taaaccctcttctcatgtatctacgtat
YPL062W-U-R:tgcccctcacgtaagggccaggaaacagctatgaccatg
YPL062W-D-F:ggcactggccgtcgttttaaccgaccatgtgggcaaatt
YPL062W-D-R:gaagatgttgaatatgctatcgaatgtgc
loxH-3F:caggaaacagctatgaccatgattacgcc
loxH-3R:ggcactggccgtcgttttacgaccatgtgggcaaatt
V-YP-1F:ccctcttctcatgtatctacgtat
V-YP-1R:cgacaaaagaaaaacgaccgaa
实施例9.重组菌RS1的构建
具体构建过程如下:
(1)人工合成基因片段PHXT1-ERG9-TADH1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物PERG9-U-F、PERG9-U-R扩增得到基因PERG9-U,采用引物PERG9-D-F、PERG9-D-R扩增得到基因PERG9-D,以pMHyLp-Leu质粒为模板,使用引物loxL-3F、loxL-3R扩增得到loxL-3基因片段。
(2)将步骤(1)中获得的三个基因片段PHXT1-ERG9-TADH1、PERG9-U和PERG9-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35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PERG9-U-PHXT1-ERG9-TADH1-PERG9-D基因片段。
(3)将步骤(2)中得到的融合基因片段PERG9-U-PHXT1-ERG9-TADH1-PERG9-D转化入实施例8制备得到的RC3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-PH-1F、V-PH-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PERG9::PHXT1-ERG9-TADH1,命名为酿酒酵母重组RS1。
引物序列:
PERG9-U-F:agggctatatatgctggagctgcta
PERG9-U-R:gcgtgctctgactcagtacatttcatag
PERG9-D-F:atgggaaagctattacaattggcattgc
PERG9-D-R:gatgaaaattacaacttgaatgggttgca
loxL-3F:caggaaacagctatgaccatgattacgcct
loxL-3R:ggcactggccgtcgttttaatgggaaagctattacaatt
V-PH-1F:tttgcagaacaccgctattctccatctat
V-PH-1R:tggctattggaatcggcaga
实施例10.重组菌RS2的构建
具体构建过程如下:
(1)人工合成基因片段PERG1-ERG9-TADH1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物PERG9-U-F、PERG9-U-R扩增得到基因PERG9-U,采用引物PERG9-D-F、PERG9-D-R扩增得到基因PERG9-D,以pMHyLp-Trp质粒为模板,使用引物loxT-4F、loxT-4R扩增得到loxT-4基因片段。
(2)将步骤(1)中获得的三个基因片段PERG1-ERG9-TADH1、PERG9-U和PERG9-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35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PERG9-U-PERG1-ERG9-TADH1-PERG9-D基因片段。
(3)将步骤(2)中得到的融合基因片段PERG9-U-PERG1-ERG9-TADH1-PERG9-D转化入实施例9制备得到的RS1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-PE-1F、V-PE-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PERG9::PERG1-ERG9-TADH1,命名为酿酒酵母重组RS2。
引物序列:
PERG9-U-F:agggctatatatgctggagctgcta
PERG9-U-R:gcgtgctctgactcagtacatttcatag
PERG9-D-F:atgggaaagctattacaattggcattgc
PERG9-D-R:gatgaaaattacaacttgaatgggttgca
loxT-4F:caggaaacagctatgaccatgattacgcct
loxT-4R:gcactggccgtcgttttacaa
V-PE-1F:gagcgtgtcccggtgggttctggga
V-PE-1R:tatgtccatcgaacacgattt
实施例11.重组菌RS3的构建
具体构建过程如下:
(1)人工合成基因片段PERG1-ERG9-TADH1-CLN2,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物PERG9-U-F、PERG9-U-R扩增得到基因PERG9-U,采用引物PERG9-D-F、PERG9-D-R扩增得到基因PERG9-D,以pMHyLp-His质粒为模板,使用引物loxH-4F、loxH-4R扩增得到loxH-4基因片段。
(2)将步骤(1)中获得的三个基因片段PERG1-ERG9-TADH1-CLN2、PERG9-U和PERG9-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35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PERG9-U-PERG1-ERG9-TADH1-CLN2-PERG9-D基因片段。
(3)将步骤(2)中得到的融合基因片段PERG9-U-PERG1-ERG9-TADH1-CLN2-PERG9-D转化入实施例10制备得到的RS2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-PL-1F、V-PL-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PERG9::PERG1-ERG9-TADH1-CLN2,命名为酿酒酵母重组RS3。
引物序列:
PERG9-U-F:agggctatatatgctggagctgcta
PERG9-U-R:gcgtgctctgactcagtacatttcatag
PERG9-D-F:atgggaaagctattacaattggcattgc
PERG9-D-R:gatgaaaattacaacttgaatgggttgca
loxH-4F:caggaaacagctatgaccatgattacgcc
loxH-4R:ggcactggccgtcgttttaatgggaaagctattacaa
V-PL-1F:gagcgtgtcccggtgggttctggga
V-PL-1R:tatgtccatcgaacacgattt
实施例12.重组菌RR1的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-crtE-PGAL10-crtI-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物1414a-U-F、1414a-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-Leu质粒为模板,使用引物loxL-4F、loxL-4R扩增得到loxL-4基因片段。
(2)将步骤(1)中获得的三个基因片段TADH1-crtE-PGAL10-crtI-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35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-TADH1-crtE-PGAL10-crtI-TAOX1-1414a-D基因片段。
(3)将步骤(2)中得到的融合基因片段1414a-U-TADH1-crtE-PGAL10-crtI-TAOX1-1414a-D转化入实施例11制备得到的RS3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-EI-2F、V-EI-2R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-TADH1-crtE-PGAL10-crtI-TAOX1,命名为酿酒酵母重组RR1。
引物序列:
1414a-U-F:atccatatcaactgatgacactttagaaa
1414a-U-R:actatcattgctcaaattatccgccg
1414a-D-F:tttttaagacgcatctccaaaaaaagaaaaagaac
1414a-D-R:gccatccgtttacataccacagc
loxL-4F:caggaaacagctatgaccatgattacg
loxL-4R:ggcactggccgtcgtttta
V-EI-2F:aattgagaaccgttgcttcttacca
V-EI-2R:ctgacaaagctacttacccaaagttgatgg
实施例13.重组菌RR2的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-crtE-PGAL10-crtI-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物720a-U-F、720a-U-R扩增得到基因720a-U,采用引物720a-D-F、720a-D-R扩增得到基因720a-D,以pMHyLp-Trp质粒为模板,使用引物loxT-5F、loxT-5R扩增得到loxT-5基因片段。
(2)将步骤(1)中获得的三个基因片段TADH1-crtE-PGAL10-crtI-TAOX1、720a-U和720a-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35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720a-U-TADH1-crtE-PGAL10-crtI-TAOX1-720a-D基因片段。
(3)将步骤(2)中得到的融合基因片段720a-U-TADH1-crtE-PGAL10-crtI-TAOX1-720a-D转化入实施例12制备得到的RR1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-EI-3F、V-EI-3R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Δ720a-TADH1-crtE-PGAL10-crtI-TAOX1,命名为酿酒酵母重组RR2。
引物序列:
720a-U-F:tccatataaacaaatgttgaaaacttcttcgaac
720a-U-R:ttactgttgattgttcgtttatttgtataattgagtttaca
720a-D-F:tggccgtcgttttacaatgggtaattagggccaagagagtgac
720a-D-R:ttttctttacttatttgtctttgctcataaatttctacataatg
loxT-5F:caggaaacagctatgaccatgattacgcct
loxT-5R:gcactggccgtcgttttacaatagggccaagagagtgac
V-EI-3F:aattgagaaccgttgcttcttacca
V-EI-3R:ctgacaaagctacttacccaaagttgatgg
实施例14.重组菌RR3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CEN.PK2-1C基因组为模板,采用引物1114a-U-F、1114a-U-R扩增得到基因1114a-U,采用引物1114a-D-F、1114a-D-R扩增得到基因720a-D,以pMHyLp-His质粒为模板,使用引物loxH-5F、loxH-5R扩增得到loxH-5基因片段。
(2)将步骤(1)中获得的三个基因片段PGAL7-crtYB-TAOX1、1114a-U和1114a-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35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1114a-U-PGAL7-crtYB-TAOX1-1114a-D基因片段。
(3)将步骤(2)中得到的融合基因片段1114a-U-PGAL7-crtYB-TAOX1-1114a-D转化入实施例13制备得到的RR2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-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-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Δ1114a-PGAL7-crtYB-TAOX1,命名为酿酒酵母重组RR3。
引物序列:
1114a-U-F:agataattagttctatggaatgggctttttcgt
1114a-U-R:atttactatcaacgctatgtaagtttgtactattatctccttttggaaagctatacttcggagc
1114a-D-F:actggccgtcgttttaaggacagtcaatagcatcatctaacat
1114a-D-R:gtacctccttcatctggtgaagc
loxH-5F:caggaaacagctatgaccatgattacgcc
loxH-5R:ggcactggccgtcgttttataaggacagtcaatagcat
V-YB-1F:cttattatcaaattcatttgat
V-YB-1R:tttgactttgttgtttggtccaccatt
实施例15.重组菌RR4的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-crtE-PGAL10-crtI-TAOX1,以酿酒酵母CEN.PK2-1C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-Leu质粒为模板,使用引物loxL-5F、loxL-5R扩增得到loxL-5基因片段。
(2)将步骤(1)中获得的三个基因片段TADH1-crtE-PGAL10-crtI-TAOX1、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35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-TAOX1-416d-D基因片段。
(3)将步骤(2)中得到的融合基因片段416d-U-TADH1-crtE-PGAL10-crtI-TAOX1-416d-D转化入实施例14制备得到的RR3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-EI-4F、V-EI-4R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Δ416d-TADH1-crtE-PGAL10-crtI-TAOX1,命名为酿酒酵母重组RR4。
引物序列:
416d-U-F:ataacaagatgtaaagataatgctaaatcatttggctt
416d-U-R:cgagatccgtttaaccggaccc gagctctcacttaatcttctgtactctgaaga
416d-D-F:ctggccgtcgttttacggtccactgtgtgccgaa
416d-D-R:gccattagccattgatgtggatatgct
loxL-5F:caggaaacagctatgaccatgattacg
loxL-5R:ggcactggccgtcgttttacggtccactgtgt
V-EI-4F:aattgagaaccgttgcttcttacca
V-EI-4R:ctgacaaagctacttacccaaagttgatgg
实施例16.重组菌RR5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CEN.PK2-1C基因组为模板,采用引物106a-U-F、106a-U-R扩增得到基因106a-U,采用引物106a-D-F、106a-D-R扩增得到基因106a-D,以pMHyLp-Trp质粒为模板,使用引物loxT-6F、loxT-6R扩增得到loxT-6基因片段。
(2)将步骤(1)中获得的三个基因片段PGAL7-crtYB-TAOX1、106a-U和106a-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35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106a-U-PGAL7-crtYB-TAOX1-106a-D基因片段。
(3)将步骤(2)中得到的融合基因片段106a-U-PGAL7-crtYB-TAOX1-106a-D转化入实施例15制备得到的RR4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-YB-2F、V-YB-2R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Δ106a-PGAL7-crtYB-TAOX1,命名为酿酒酵母重组RR5。
引物序列:
106a-U-F:gagggtctgtccagcgaataag
106a-U-R:ccccggatcgtcggttgtg tccttttggaaagctatacttcggagc
106a-D-F:ccgtcgttttacaatggtcaaacttcagaactaaaaaaataataaggaaga
106a-D-R:cttgtgatgtcttccaagtgatttcc
loxT-6F:caggaaacagctatgaccatgattacgcct
loxT-6R:gcactggccgtcgttttacaaggtcaaacttcagaactaa
V-YB-2F:cttattatcaaattcatttgat
V-YB-2R:tttgactttgttgtttggtccaccatt
实施例17.重组菌RA1的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-blh-PGAL10-blh-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物1206a-U-F、1206a-U-R扩增得到基因1206a-U,采用引物1206a-D-F、1206a-D-R扩增得到基因1206a-D,以pMHyLp-His质粒为模板,使用引物loxH-6F、loxH-6R扩增得到loxH-6基因片段。
(2)将步骤(1)中获得的三个基因片段TADH1-blh-PGAL10-blh-TAOX1、1206a-U和1206a-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35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1206a-U-TADH1-blh-PGAL10-blh-TAOX1-1206a-D基因片段。
(3)将步骤(2)中得到的融合基因片段1206a-U-TADH1-blh-PGAL10-blh-TAOX1-1206a-D转化入实施例16制备得到的RR5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-bl-1F、V-bl-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Δ1206a-TADH1-blh-PGAL10-blh-TAOX1,命名为酿酒酵母重组RA1。
引物序列:
1206a-U-F:tttttaaaaactttcgaacaagaatcagtaatataa
1206a-U-R:gtaatattaataatcacttgtgccaatataatgttacag
1206a-D-F:ttggaaaattgaatgatagaaaattcgaacagcga
1206a-D-R:ttccaacagatataagaattacaaaaagagaactgttatttcta
loxH-6F:caggaaacagctatgaccatgattacgcc
loxH-6R:ggcactggccgtcgtttta
V-bl-1F:aaataaaatcaatcaaaatcatatgtgga
V-bl-1R:acaccaatcaatcaacatcaaac
实施例18.重组菌RA2的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-POS5-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物CAN1y-U-F、CAN1y-U-R扩增得到基因CAN1y-U,采用引物CAN1y-D-F、CAN1y-D-R扩增得到基因CAN1y-D,以pMHyLp-Leu质粒为模板,使用引物loxL-6F、loxL-6R扩增得到loxL-6基因片段。
(2)将步骤(1)中获得的三个基因片段PGAL7-POS5-TAOX1、CAN1y-U和CAN1y-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35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CAN1y-U-PGAL7-POS5-TAOX1-CAN1y-D基因片段。
(3)将步骤(2)中得到的融合基因片段CAN1y-U-PGAL7-POS5-TAOX1-CAN1y-D转化入实施例17制备得到的RA1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-PS-1F、V-PS-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ΔCAN1y-PGAL7-POS5-TAOX1,命名为酿酒酵母重组RA2。
引物序列:
CAN1y-U-F:cagtatattagaaacccgataatg
CAN1y-U-R:ttctccttcatcttcatcacctatgccat
CAN1y-D-F:gccgtcgttttactcgccatttactctcgtcgggaa
CAN1y-D-R:ttcattgatagagacaactgcaaaaaaagaaattc
loxL-6F:caggaaacagctatgaccatgattacg
loxL-6R:ggcactggccgtcgttttactcgccatttactctc
V-PS-1F:ttaaattgaataaaccagtaaaat
V-PS-1R:tattcttacataggggtaattcccctca
实施例19.重组菌RT1-RT7的构建
具体构建过程如下:
(1)以酿酒酵母CEN.PK2-1C基因组为模板,采用引物Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-U-F、Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-U-R扩增得到基因Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-U,采用引物Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-D-F、Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-D-R扩增得到基因Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-D,以pMHyLp-Trp质粒为模板,使用引物loxT-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-7F、loxT-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-7R扩增得到loxT-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-7基因片段。
(2)将步骤(1)中获得的两个基因片段Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-U和Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-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35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Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-U-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-D基因片段。
(3)将步骤(2)中得到的融合基因片段Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-U-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-D转化入实施例18制备得到的RA2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-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-1F、V-Pdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1-1R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ΔPdr5/Snq2/Adp1/Pdr10/Mdl1/Ste6/Yor1,命名为酿酒酵母重组RT1-RT7。
引物序列:
Pdr5-U-F:cccacggaacgagtggactg
Pdr5-U-R:cgctcgttcgaaagactttagacaaaacaggaaacagct
Pdr5-D-F:tcgttttacaatagaattttgaatttggttaagaaaagaaacttaccaag
Pdr5-D-R:cgtgcagaaagaacattcctcaccac
LoxT-Pdr5-7F:aagactttagacaacaggaaacagctatgaccatgattacgcct
loxT-Pdr5-7R:gcactggccgtcgttttacaa
V-Pdr5-1F:gatatctgttgaacgtaatctgag
V-Pdr5-1R:gaaacagctatgaccatgattacg
Snq2-U-F:tccgttttctattttctttaggtctttccattg
Snq2-U-R:aataacacagctaccaaaatacgtaaagagaattcacaggaaacagctat
Snq2-D-F:ccgtcgttttacaatgtgggcttcagataacttaacattttgtg
Snq2-D-R:aaaagaaaataaaaaaattggacacaggaaaacaac
LoxT-Snq2-7F:cgtaaagagaattcacaggaaacagctatgaccatgattacgcct
loxT-Snq2-7R:gcactggccgtcgttttacaatgtgggcttcag
V-Snq2-1F:gaataagacaaaaaaactgaggctcc
V-Snq2-1R:ggaaacagctatgaccatgatt
Adp1-U-F:ccttcgattgcttgttacaaaacctgt
Adp1-U-R:catggtcatagctgtttcctgactgtaattgcttttagttgtgtatttttagtgtgc
Adp1-D-F:cactggccgtcgttttaataggcgtcgtatatagtctcttcttttacataaga
Adp1-D-R:tagtagtttctgttgtattgttattgctattggacg
LoxT-Adp1-7F:caggaaacagctatgaccatgattacgcct
loxT-Adp1-7R:gcactggccgtcgttttacaa
V-Adp1-1F:aaatgtaagtttcacgaggtt
V-Adp1-1R:tgggtcaatttggggtcaattg
Pdr10-U-F:atccgaagaattttgagccacttttgc
Pdr10-U-R:aaatcaaacaataatttctggtcttacatagttgttagg
Pdr10-D-F:ttaaatattactggctacattcattgtatatgcaaaattag
Pdr10-D-R:gccaccatatattcgttgttgagtttgt
LoxT-Pdr10-7F:caggaaacagctatgaccatgattacgcct
loxT-Pdr10-7R:gcactggccgtcgttttacaa
V-Pdr10-1F:acatctgcagtgaatggaaattgctg
V-Pdr10-1R:tgggtcaatttggggtcaattg
Mdl1-U-F:agatctccaataccgcaaacacagc
Mdl1-U-R:tactaagttaaagaagaggaagggctcca
Mdl1-D-F:gaagttgccgcagtatggaatcacttt
Mdl1-D-R:cgcggagttttcactcttgataaagtaac
LoxT-Mdl1-7F:caggaaacagctatgaccatgattacgcct
loxT-Mdl1-7R:gcactggccgtcgttttacaa
V-Mdl1-1F:tatcgttttcatatctttcctgag
V-Mdl1-1R:tgggtcaatttggggtcaattg
Ste6-U-F:agggagaatataacaacaagaaatatgataaaatttgggat
Ste6-U-R:tttgagtatgttttagttttttgttttatattttctttgggt
Ste6-D-F:gacgtagcttgttctttgtttcctagtg
Ste6-D-R:acttggccaaaagggctgagtaa
LoxT-Ste6-7F:caggaaacagctatgaccatgattacgcct
loxT-Ste6-7R:gcactggccgtcgttttacaa
V-Ste6-1F:aacggcaataatgcaacagttt
V-Ste6-1R:tgggtcaatttggggtcaattg
Yor1-U-F:ttctattttcctccccaccgcgag
Yor1-U-R:ctgtttttatattcaaaaagagtaaagccgttgctatatacgaat
Yor1-D-F:tttatattatttgttgcatgatttttctcttttatttatttatatgttgcc
Yor1-D-R:agccattcttgtttaaagagtatttttaaagcgttcat
LoxT-Yor1-7F:caggaaacagctatgaccatgattacgcct
loxT-Yor1-7R:gcactggccgtcgttttacaa
V-Yor1-1F:aataaagtatacacaaaatacgaat
V-Yor1-1R:tgggtcaatttggggtcaattg
实施例20.重组菌RP1-RP5的构建
具体构建过程如下:
(1)人工合成基因片段PGAL7-CYC2/FOX2/ENV9/IFA38/ybbo-TAOX1以酿酒酵母CEN.PK2-1C基因组为模板,采用引物1622b-CYC2/FOX2/ENV9/IFA38/ybbo-U-F、1622b-CYC2/FOX2/ENV9/IFA38/ybbo-U-R扩增得到基因1622b-CYC2/FOX2/ENV9/IFA38/ybbo-U,采用引物1622b-CYC2/FOX2/ENV9/IFA38/ybbo-D-F、1622b-CYC2/FOX2/ENV9/IFA38/ybbo-D-R扩增得到基因1622b-CYC2/FOX2/ENV9/IFA38/ybbo-D,以pMHyLp-His质粒为模板,使用引物loxH-CYC2/FOX2/ENV9/IFA38/ybbo-7F、loxH-CYC2/FOX2/ENV9/IFA38/ybbo-7R扩增得到loxH-CYC2/FOX2/ENV9/IFA38/ybbo-7基因片段。
(2)将步骤(1)中获得的两个基因片段1622b-CYC2/FOX2/ENV9/IFA38/ybbo-U和1622b-CYC2/FOX2/ENV9/IFA38/ybbo-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35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-CYC2/FOX2/ENV9/IFA38/ybbo-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-CYC2/FOX2/ENV9/IFA38/ybbo-TAOX1-1622b-D转化入实施例19制备得到的RT1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-CYC2/FOX2/ENV9/IFA38/ybbo-1F、V-CYC2/FOX2/ENV9/IFA38/ybbo-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Δ1622b-PGAL7-CYC2/FOX2/ENV9/IFA38/ybbo-TAOX1-1622b-D,命名为酿酒酵母重组RP1-RP5。
引物序列:
1622b-CYC2-U-F:gaataatcgccttcttactttgtcatttat
1622b-CYC2-U-R:cagaaggtaacagcaaaaacaaatagttcac
1622b-CYC2-D-F:ctggccgtcgttttatggaactttatgtcgcctggcac
1622b-CYC2-D-R:catatctgtgttttaaattaaatttcatggaaataagaatagtaaca
loxH-CYC2-7F:caggaaacagctatgaccatgattacgcc
loxH-CYC2-7R:ggcactggccgtcgttttatggaactttatgtcgcctg
1622b-FOX2-U-F:gaataatcgccttcttactttgtcatttat
1622b-FOX2-U-R:cagaaggtaacagcaaaaacaaatagttcac
1622b-FOX2-D-F:ctggccgtcgttttatggaactttatgtcgcctggcac
1622b-FOX2-D-R:catatctgtgttttaaattaaatttcatggaaataagaatagtaaca
loxH-FOX2-7F:caggaaacagctatgaccatgattacgcc
loxH-FOX2-7R:ggcactggccgtcgttttatggaactttatgtcgcctg
1622b-ENV9-U-F:gaataatcgccttcttactttgtcatttat
1622b-ENV9-U-R:cagaaggtaacagcaaaaacaaatagttcac
1622b-ENV9-D-F:ctggccgtcgttttatggaactttatgtcgcctggcac
1622b-ENV9-D-R:catatctgtgttttaaattaaatttcatggaaataagaatagtaaca
loxH-ENV9-7F:caggaaacagctatgaccatgattacgcc
loxH-ENV9-7R:ggcactggccgtcgttttatggaactttatgtcgcctg
1622b-IFA38-U-F:gaataatcgccttcttactttgtcatttat
1622b-IFA38-U-R:cagaaggtaacagcaaaaacaaatagttcac
1622b-IFA38-D-F:ctggccgtcgttttatggaactttatgtcgcctggcac
1622b-IFA38-D-R:catatctgtgttttaaattaaatttcatggaaataagaatagtaaca
loxH-IFA38-7F:caggaaacagctatgaccatgattacgcc
loxH-IFA38-7R:ggcactggccgtcgttttatggaactttatgtcgcctg
1622b-ybbo-U-F:gaataatcgccttcttactttgtcatttat
1622b-ybbo-U-R:cagaaggtaacagcaaaaacaaatagttcac
1622b-ybbo-D-F:ctggccgtcgttttatggaactttatgtcgcctggcac
1622b-ybbo-D-R:catatctgtgttttaaattaaatttcatggaaataagaatagtaaca
loxH-ybbo-7F:caggaaacagctatgaccatgattacgcc
loxH-ybbo-7R:ggcactggccgtcgttttatggaactttatgtcgcctg
实施例21.重组菌RMA1-RMA23的构建
具体构建过程如下:
(1)将底物视黄醛与ENV9进行分子对接,预测出23个关键位点,THR22,GLY23,ASN25,THR26,ILE28,CYS46,GLY47,ARG48,ASN49,LEU96,ASP97,LEU98,THR99,ASN124,GLY126,ILE127,THR145,LEU171,PRO230,VAL233,THR236,ASN237,LEU238。并将上述位点突变为丙氨酸
以JM-ENV9质粒为模板构建单位点突变体,定点突变引物中分别引入丙氨酸密码子。
(2)将步骤(1)中质粒模板与引物进行重叠延伸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35个循环,1%的琼脂糖凝胶电泳验证正确后,得到融合基因片段。
(3)将步骤(2)中得到的融合基因片段加入DpnI消化1h,去除质粒模板。
(4)将步骤(3)的消化产物转化大肠杆菌感受态中,冰浴30min,42℃热激90s后冰浴2min,加入500uL LB液体培养基,在37℃,220rpm/min条件下,培养1h。涂布于含有Amp抗性的LB固体培养基中,菌落PCR验证后,挑取正确的菌落。即可得到突变后的重组质粒。
(5)将突变后的重组质粒转化至实施例20的AT1菌株中,得到RMA1-RMA23。(酿酒酵母转化方法参考实施例20)
实施例22.重组菌AX1的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-ybbo-PGAL10-ENV9(T99A)-TAOX1,以酿酒酵母CEN.PK2-1C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-His质粒为模板,使用引物loxH-7F、loxH-7R扩增得到loxH-7基因片段。
(2)将步骤(1)中获得的两个基因片段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35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-TADH1-ybbo-PGAL10-ENV9(T99A)-TAOX1-1622b-D基因片段。
(3)将步骤(2)中得到的融合基因片段1622b-U-TADH1-ybbo-PGAL10-ENV9(T99A)-TAOX1-1622b-D转化入实施19制备得到的RT1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-yE-1F、V-yE-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Δ1622b-TADH1-ybbo-PGAL10-ENV9(T99A)-TAOX1-1622b-D,命名为酿酒酵母重组AX1。
引物序列:
1622b-U-F:gaataatcgccttcttactttgtcatttat
1622b-U-R:cagaaggtaacagcaaaaacaaatagttcac
1622b-D-F:ctggccgtcgttttatggaactttatgtcgcctggcac
1622b-D-R:catatctgtgttttaaattaaatttcatggaaataagaatagtaaca
loxH-7F:caggaaacagctatgaccatgattacgcc
loxH-7R:ggcactggccgtcgttttatggaactttatgtcgcctg
V-yE-1F:cgaatattgccatactacgacccggct
V-yE-1R:gacgaagcggcatcgacttggat
实施例23.重组菌AX2的构建
具体构建过程如下:
(1)人工合成基因片段TADH1-PAH1-PGAL10-LRO1-TAOX1,以酿酒酵母CEN.PK2-1C基因组为模板,采用引物HIS3b-U-F、HIS3b-U-R扩增得到基因HIS3b-U,采用引物HIS3b-D-F、HIS3b-D-R扩增得到基因HIS3b-D,以pMHyLp-Leu质粒为模板,使用引物loxL-7F、loxL-7R扩增得到loxL-7基因片段。
(2)将步骤(1)中获得的两个基因片段HIS3b-U和HIS3b-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35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HIS3b-U-TADH1-PAH1-PGAL10-LRO1-TAOX1-HIS3b-D基因片段。
(3)将步骤(2)中得到的融合基因片段HIS3b-U-TADH1-PAH1-PGAL10-LRO1-TAOX1-HIS3b-D转化入实施例19制备得到的RT1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-D-1F、V-D-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ΔHIS3b-TADH1-PAH1-PGAL10-LRO1-TAOX1,命名为酿酒酵母重组AX2。
引物序列:
HIS3b-U-F:tgaagaaatttactaggaatagtgccatgg
HIS3b-U-R:cgcaattttctttttctattactcttggcctgagctctcacttaatcttc
HIS3b-D-F:ctggccgtcgttttatttttatgcctcggtaatgattttcatttttttt
HIS3b-D-R:taggggccgtgcgtg
loxL-7F:caggaaacagctatgaccatgattacg
loxL-7R:ggcactggccgtcgttttatttttatgcctcggtaatgatt
V-D-1F:ctaaatcctctacacctaagattccaa
V-D-1R:acattcctctacatcaaattat
实施例24.摇瓶发酵条件下重组菌株的视黄醇产量
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,(由于视黄醇易氧化,因此我们在发酵培养基中加入十二烷/十二烷+不同抗氧化剂/橄榄油去验证不同发酵培养基条件对视黄醇产量的影响)
(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30mL发酵培养基和3mL十二烷的250mL锥形瓶中,在30℃,220rpm条件下培养120h,制备得到发酵液。
(3)计算重组菌视黄醇的产量:
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,对于脂溶性的视黄醇来说,绝大部分产物分泌到胞外,小部分积累于细胞内部。
将发酵120h的酿酒酵母菌液在12000r/min条件下离心10min,吸取萃取剂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600uL水洗后的重悬菌液和600uL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。通过与视黄醇标品峰面积进行换算得出工程菌株的发酵产量。使用紫外可见分光光度计测定发酵液的吸光度值(OD 600)并检测酿酒酵母的生长。
结果如表1所示,模块化工程强化内源甲羟戊酸合成模块、中心碳代谢模块、角鲨烯合成模块、外源β-胡萝卜素合成模块和视黄醇合成模块;敲除转运体抑制剂前体分泌促进产物积累;半理性改造关键脱氢酶ENV9,十二烷萃取双相摇瓶发酵,菌株视黄醇的产量最高达到425.7mg/L,OD600达到42.7。
表1不同重组菌的产量及OD600(以10%(v/v)添加十二烷作为萃取剂)
视黄醇是一种侧链含有四个双键的不饱和的一元醇,化学性质不稳定。视黄醇分子结构的羟甲基末端易氧化成醛基,暴露在光线下会迅速分解。添加十二烷作为萃取剂在促进细胞外视黄醇积累的同时也会产生视黄醛。十二烷的存在可能会抑制视黄醛向视黄醇的转化,我们选择了两种策略来提高视黄醇的转化率。第一种是在萃取剂中添加1%抗氧化剂(包括二丁基羟基甲苯(BHT),没食子酸酯(PG),乙氧基喹啉(EQ)和叔丁基对苯二酚(TBHQ)),第二种是选用橄榄油代替十二烷进行发酵萃取。以菌株AX2作为出发菌株,在不同萃取剂双相发酵下,实验结果如表2所示。
表2重组菌AX2在不同萃取条件下的产量及OD600
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。
Claims (12)
1.一种重组酿酒酵母,其特征在于:所述重组酿酒酵母异源表达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-胡萝卜素加氧酶Blh,过表达异戊烯基焦磷酸异构酶IDI、3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1、法尼基焦磷酸合酶ERG20、乙醇脱氢酶ADH2、乙醛脱氢酶ALD6、乙酰辅酶A连接酶ACS1、GGPP合酶CrtE、八氢番茄红素去饱和酶CrtI、番茄红素环化酶CrtYB、β-胡萝卜素加氧酶Blh和NADH激酶POS5,弱化表达角鲨烯合成酶ERG9,敲除了柠檬酸合酶CIT2、苹果酸合酶MLS1、调节因子YPL062W和ABC转运体蛋白Pdr5p,将内源视黄醇脱氢酶ENV9第99位的苏氨酸突变为丙氨酸并引入异源视黄醇脱氢酶YBBO。
2.根据权利要求1所述的重组酿酒酵母,其特征在于:所述重组酿酒酵母强化表达了磷脂酸磷酸酶PAH1和磷脂二酰基甘油酰基转移酶LRO1。
3.根据权利要求1所述的重组酿酒酵母,其特征在于:所述弱化表达角鲨烯合成酶ERG9为替换原启动子为PHXT1启动子并添加降解标签CLN2。
4.根据权利要求1所述的重组酿酒酵母,其特征在于:增加三个拷贝的GGPP合酶CrtE和八氢番茄红素去饱和酶CrtI、增加一个拷贝的番茄红素环化酶CrtYB。
5.一种视黄醇脱氢酶突变体,其特征在于:所述突变体由氨基酸序列如SEQ ID NO.1所示的视黄醇脱氢酶的第99位苏氨酸突变为丙氨酸得到。
6.编码权利要求5所述视黄醇脱氢酶突变体的基因。
7.携带权利要求6所述基因的重组质粒。
8.表达权利要求5所述视黄醇脱氢酶突变体的细胞。
9.一种生产视黄醇的方法,其特征在于:应用权利要求1-4任一项所述的重组酿酒酵母进行发酵。
10.根据权利要求9所述的方法,其特征在于:向发酵培养基中加入萃取剂。
11.根据权利要求10所述的方法,其特征在于:所述萃取剂选自十二烷或橄榄油。
12.根据权利要求9所述的方法,其特征在于:向发酵培养基中加入二丁基羟基甲苯、没食子酸酯和乙氧基喹啉中的一种或几种。
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