CN112812981B - 合成番茄红素的酿酒酵母基因工程菌及其构建方法与应用 - Google Patents
合成番茄红素的酿酒酵母基因工程菌及其构建方法与应用 Download PDFInfo
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- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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- C12Y103/99031—Phytoene desaturase (lycopene-forming) (1.3.99.31)
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Abstract
本发明公开了合成番茄红素的酿酒酵母基因工程菌,属于微生物基因工程技术领域。本发明在酿酒酵母中共同表达来源于Deinococcus wulumuqiensis R12的crtE、crtB、crtI基因;所述的crtE的核苷酸序列如SEQ ID NO.1所示;所述的crtB的核苷酸序列如SEQ ID NO.2所示;所述的crtI的核苷酸序列如SEQ ID NO.3所示。本发明中使用的表达策略在酿酒酵母中实现了番茄红素的高效合成,筛选出来适合萜类化合物生产的工程菌宿主,为进一步优化人工合成细胞生产番茄红素奠定了基础。
Description
技术领域
本发明属于微生物基因工程技术领域,具体涉及合成番茄红素的酿酒酵母基因工程菌及其构建方法与应用。
背景技术
番茄红素(lycopene)是一种典型的不含氧类胡萝卜素,呈深红色,具有较强抗氧化能力,能够高效清除活性氧自由基,有效延缓衰老和降低多种疾病的患病率,逐渐成为研究的热点。目前生产番茄红素常用的方法有直接提取法、化学合成法和微生物发酵法。直接提取法的原料番茄往往受到气候与季节的影响,导致合成成本较高,而化学合成法合成过程步骤繁琐,且化学试剂的残留问题还会进一步限制产品的使用;微生物发酵生产类胡萝卜素具有可持续、环境友好且产品安全性高等多种优势,成为研究最多且最具有市场前景的方法。随着类胡萝卜素合成途径机制的解析、基因工程的快速发展以及合成生物学的异军突起,构建微生物细胞工厂成为天然产物的高效生产方式。
目前,以番茄红素为主的类胡萝卜素已经成功在大肠杆菌、酿酒酵母、毕赤酵母、解酯耶氏酵母、乳酸乳球菌等多种微生物中高效生产。在几种构建的工程菌中,酿酒酵母是GRAS认定的安全模式菌株,广泛应用于生产安全性要求严格的天然产物,如乙醇、紫衫醇、青蒿素等。此外,酿酒酵母遗传信息清晰、基因操作简便、培养方式简单、生长周期短、成本低且可工业化应用,逐渐成为类胡萝卜素及其他天然次级代谢产物细胞工厂构建的优选宿主细胞。通过优化番茄红素生物合成途径的构建策略和代谢调控方法获取高量生产番茄红素菌株,为酿酒酵母高效合成其他类胡萝卜素提供技术支持。
发明内容
发明目的:本发明要解决的技术问题是提供一种合成番茄红素的酿酒酵母基因工程菌,以解决现有技术中番茄红素在植物中积累量低和化学合成困难、副产物多的问题。
本发明还要解决的技术问题是提供上述合成番茄红素的酿酒酵母基因工程菌的构建方法。
本发明最后要解决的技术问题是提供上述合成番茄红素的酿酒酵母基因工程在发酵制备番茄红素中的应用。
技术方案:为解决上述技术问题,本发明提供如下技术方案:
合成番茄红素的酿酒酵母基因工程菌,在酿酒酵母中表达来源于Deinococcus wulumuqiensis R12 的crtI基因,或者在酿酒酵母中共同表达来源于Deinococcus wulumuqiensis R12 的crtE基因和crtB基因,或者在酿酒酵母中共同表达来源于Deinococcus wulumuqiensis R12 的crtE基因、crtB和crtI基因;
所述crtE、crtB、crtI基因分别为牻牛儿基牻牛儿基焦磷酸合成酶的编码基因、八氢番茄红素合成酶的编码基因以及八氢番茄红素脱氢酶的编码基因。
所述的crtE的核苷酸序列如SEQ ID NO.1所示;
所述的crtB的核苷酸序列如SEQ ID NO.2所示;
所述的crtI的核苷酸序列如SEQ ID NO.3所示。
合成番茄红素的酿酒酵母基因工程菌的构建方法,包括如下步骤:
(1)将crtI基因连接到酵母表达质粒中,得到pESC-I重组质粒;或者将连接了组成型强启动子PTDH3的crtI基因连接到酵母表达质粒中,得到pESC-CI重组质粒;
(2)去除crtE基因的终止密码子,并在crtE基因后连接link序列(5’-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCA-3’)和crtB基因,得到重组片段Ⅰ,将重组片段Ⅰ连接到pESC-I重组质粒中,得到pEBI重组质粒;
或者去除crtE基因的终止密码子,将组成型强启动子PTDH3、crtE基因、link序列(5’-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCA-3’)以及crtB基因依次连接,得到重组片段Ⅱ,然后将重组片段Ⅱ连接到pESC-CI重组质粒中,得到pCEBI重组质粒;
或者去除crtE基因的终止密码子,将组成型强启动子PTDH3、crtE基因、终止子TCYC1、组成型强启动子PTDH3、以及crtB基因依次连接,得到重组片段Ⅲ,然后将重组片段Ⅲ连接到pESC-CI重组质粒中,得到pCIETB重组质粒;
(3)将步骤(2)得到的重组质粒pEBI、pCEBI或pCIETB转化酿酒酵母菌,得到合成番茄红素的酿酒酵母基因工程菌。
步骤(1)中,所述的表达质粒为pESC-LEU、pESC-HIS、pESC-TRP或pESC-URA。
步骤(2)中,所述终止子TCYC1的核苷酸序列如SEQ ID NO.4所示;
所述强启动子PTDH3的核苷酸序列如SEQ ID NO.5所示。
步骤(3)中,所述的酿酒酵母菌包括:酿酒酵母YPH499和酿酒酵母BY4741。
合成番茄红素的酿酒酵母基因工程菌在发酵制备番茄红素中的应用在本发明的保护范围之内。
进一步地,以SP-Leu培养基的发酵培养基,以合成番茄红素的酿酒酵母基因工程菌为发酵菌株,发酵制备番茄红素。
进一步地,在培养基中加入脂肪酸,所述的脂肪酸为棕榈油酸、油酸、亚油酸、亚麻酸中的一种或几种的混合物,优选棕榈油酸和亚麻酸。
进一步地,所述SP-Leu培养基的配方为:20g/L蛋白胨,20 g/L葡萄糖,0.69g/LCSM-Leu,所述发酵的发酵温度为28℃、发酵时间为36h。
上述合成番茄红素的酿酒酵母基因工程菌在发酵制备番茄红素中的应用在本发明的保护范围之内。
有益效果
1、本发明构建了公开了一种合成番茄红素的酿酒酵母基因工程菌,利用本发明构建的基因工程菌在外源添加100 mg/L 亚麻酸的SP-Leu培养基中,28℃进行发酵36小时,番茄红素产量达到301 mg/L(86.04 mg/g),产率达到8.36 mg/L/h,实现了番茄红素的高产,为之后的进一步优化奠定了实验基础;
2、本发明构建的酿酒酵母工程菌bpCIETB、ypCIETB合成番茄红素途径受组成型启动子控制,发酵过程无需额外添加诱导剂,发酵时间短,节约了发酵成本,且发酵操作更加简单。
附图说明
图1 显示为重组质粒的构建过程
图2显示为目的片段的琼脂糖凝胶电泳检测结果。
依次为a.crtI基因片段;b.crtE基因片段;c.crtB、TCYC1、PTDH3基因片段
图3显示为质粒电泳图谱及酶切验证图。
依次为a.1:质粒pESC-IBamHI/HindIII双酶切;2:质粒pESC-CIBamHI/HindIII双酶切;3:质粒pESC-I ;4和7:质粒pEBIBamHI/HindIII和SacI/SpeI双酶切;8:质粒pESC-Leu;9:质粒pESC-CI;10:质粒pEBI;11:质粒pCEBI;12:质粒pCIETB;b.质粒pCIETBBamHI/HindIII和SacI/SpeI双酶切。
图4显示为6株重组菌在SG-Leu培养基和在YPG培养基上的番茄红素合成产量结果。
图5显示为6株重组菌在SD-Leu培养基和在YPD培养基上的番茄红素合成产量结果。
图6显示为bpEBI、ypEBI、bpCEBI、ypCEBI的基因表达情况。
图7显示为bpCIETB、ypCIETB的基因表达情况。
图8显示为bpCIETB菌株在不同碳源、氮源、pH、温度(pH为6)下的番茄红素产量。
图9显示为bpCIETB菌株在不同发酵时间下番茄红素合成的产量变化。
图10显示为bpCIETB菌株外源添加多不饱和脂肪酸的番茄红素产量变化结果。依次为NO:无添加不饱和脂肪酸;PA:添加棕榈油酸;OC:添加油酸;LA:添加亚油酸;ALA:添加亚麻酸。
实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明,但有必要指出以下实施例只用于对发明内容的描述,并不构成对本发明保护范围的限制。
实施例1:番茄红素生物合成途径的构建:
1.诱导型融合蛋白模块质粒的构建
将crtE、crtB、crtI基因进行密码子优化并利用软件Vector NTI 9.0设计引物,以基因优化后的基因为模板,根据图1所示,依次扩增出crtI和重组片段Ⅰ(图2),将crtI基因和重组片段Ⅰ分别通过BamHI/HindIII和SpeI/SacI双酶切-连接的方法将片段依次连接到质粒pESC-Leu和pESC-I上,得到重组质粒pESC-I和融合蛋白模块质粒pEBI。
重组片段Ⅰ是诱导型融合蛋白模块,是通过同源重组方法将crtE和crtB进行融合连接(去除掉crtE基因上的TAA,加入5’-GGTGGTGGTGGTTCTGGTGGTGGTGGTTCA-3’的Linker序列)。
引物序列为:
IF:TATGGATCC ATGACTTCTCCATTGCCAT
IR:TATAAGCTT TTATCTTCTGATATCTGCAT
EF:GGACTAGT ATGAGACCAGAATTGTTATC
ELR:ACCACCACCACCAGAACCACCACCACCTTTTTCTCTTGTAGC
BLF:GGTGGTGGTGGTTCTGGTGGTGGTGGTTCAATGGTTACTGAATTTTCTCC
BR:CGAGAGCTC TTAACCATGTGCAGCATCCA
2.组成型融合蛋白模块质粒的构建
组成型融合蛋白模块质粒pCEBI是通过同源重组方法将组成型强启动子PTDH3和crtI进行融合连接,和重组片段Ⅱ分别通过BamHI/HindIII和SpeI/SacI双酶切-连接的方法将片段依次连接到质粒pESC-Leu和pESC-CI上,得到重组质粒pESC-CI和组成型融合蛋白模块质粒pCEBI。
重组片段Ⅱ是组成型融合蛋白模块,是通过同源重组方法将组成型强启动子PTDH3和重组片段Ⅰ进行融合连接。
引物序列为:
TF:TATGGATCC ATACTAGCGTTGAATGTTAG
TR:CAATGGAGAAGTCATTAACCATTTTGTTTGTTTATGTGTG
TIF:ATGACTTCTCCATTGCCAT
IR:TATAAGCTT TTATCTTCTGATATCTGCAT
TEF:TATACTAGT ATACTAGCGTTGAATGTTAG
TER:TAACAATTCTGGTCTCATTAACCATTTTGTTTGTTTATGTGTG
EBF:ATGAGACCAGAATTGTTATC
BR:CGAGAGCTC TTAACCATGTGCAGCATCCA
3.组成型启动子模块重组质粒的构建
组成型启动子模块质粒pCIETB是将重组片段Ⅲ通过SpeI/SacI双酶切-连接的方法将片段连接到质粒pESC-CI上,得到重组质粒组成型启动子模块质粒pCIETB。
重组片段Ⅲ是组成型启动子模块,是通过同源重组方法将组成型强启动子PTDH3、crtE基因、终止子TCYC1、组成型强启动子PTDH3、以及crtB基因进行融合连接。
引物序列为:
TEF:TATACTAGT ATACTAGCGTTGAATGTTAG
ECR:TTTTTCTCTTGTAGC
CF:TAGCTACAAGAGAAAAAATCCGCTCTAACCGAAAAGG
CR:CTAACATTCAACGCTAGTATCTTCGAGCGTCCCAAAACCT
TBF:ATACTAGCGTTGAATGTTAG
BR:CGAGAGCTC TTAACCATGTGCAGCATCCA
4.酿酒酵母工程菌的验证
利用酵母转化试剂盒分别将质粒pEBI、pCEBI和pCIETB转化入酿酒酵母YPH499和BY4741中,根据载体抗性筛选,获得重组菌ypEBI、bpEBI、ypCEBI、bpCEBI、ypCIETB及bpCIETB。从固体培养基SD-Leu中挑取单个阳性克隆子,经双酶切验证,如图3所示,以及测序验证(插入的基因序列见序列表),表明插入的基因序列及方向均正确。
实施例2:重组菌株表达类胡萝卜素关键酶生产番茄红素的验证。
YPD培养基:10 g/L酵母浸粉,20 g/L蛋白胨,20 g/L葡萄糖。
YPG培养基:10 g/L酵母浸粉,20 g/L蛋白胨,20 g/L半乳糖。
SD-Leu培养基:6.7 g/L无氨基酵母氮源(YNB),20 g/L葡萄糖,0.69g/L CSM-Leu。
SG-Leu培养基:6.7 g/L 无氨基酵母氮源(YNB),20 g/L半乳糖,0.69g/L CSM-Leu。
将阳性克隆接种至50 mL的SD-Leu液体培养基中,置于30℃摇床,200 rpm 培养至OD600值约为4-6。4000 r/min离心10 min,收集所有菌体,将菌体全部转入50 mL SG-Leu和YPG培养基或者新鲜的SD-Leu和YPD培养基中继续培养48-96 h。菌体培养结束后,4°C,5000rpm离心10 min收集菌体,蒸馏水洗涤两次,冷冻干燥获得干菌体,-20°C保存。称取0.05 g干菌体,加入3 mL盐酸(3 mol/L)溶液,置于25°C水浴摇床中振荡1 h,沸水浴煮沸4 min,冰浴5 min,离心弃上清,蒸馏水洗涤两次,加入丙酮溶液(含1% BHT),25°C振荡至菌体无色,离心取上清液,经0.22 μM有机膜过滤,获得番茄红素提取液。采用高效液相色谱对番茄红素进行分析。利用Venusil XBP C18柱(4.6×150 mm,5 μm)于波长472 nm和30°C柱温条件下,使用体积比80:15:5的乙腈:甲醇:异丙醇作为流动相,流速1 mL/min,进样量20μL进行检测。如图4所示,重组菌在YPG和SG-Leu培养基中均能产生番茄红素,在SG-Leu培养基中的番茄红素产量高于YPG培养基中产量,并且以BY4741为底盘菌株的重组菌产量均高于以YPH499为底盘菌株的重组菌,含有诱导型融合蛋白模块的重组菌bpEBI在含有半乳糖诱导下,在SG-Leu培养基中产量最高。如图5所示,含有诱导型融合蛋白模块的重组菌bpEBI和ypEBI在YPD和SD-Leu培养基中由于没有半乳糖进行诱导,无法进行基因表达而产生番茄红素;含有组成型融合蛋白模块和组成型启动子模块由于是采用的组成型启动子,无需诱导即可产生番茄红素,并且在SD-Leu培养基中的番茄红素产量高于YPD培养基中产量,以BY4741为底盘菌株的重组菌产量均高于以YPH499为底盘菌株的重组菌,其中重组菌bpCIETB无需诱导在SD-Leu培养基中产量最高。
实施例3:番茄红素合成基因mRNA含量分析。
采用TRIzol法提取重组菌发酵48-96 h中的总RNA,将RNA反转录为cDNA,以cDNA为模板,设计相应的引物,进行荧光定量PCR的测定。每个样品重复3次。内参基因选择18SrRNA基因,采用2-△△Ct法对数据进行分析。不同菌株中基因表达量比较参照图6、图7。
引物序列为:
crtB-F:TGGTTTTATGGTTGCTCC
crtB-R:CCAAGTCACCCAAATGTCT
crtE-F:TGGTGTTCCAGTTGCTAT
crtE-R:GTTGACCTTCTGCTGTTCT
crtI-F:CAGCTCCATCACCATACAC
crtI-R:CTAATACCCAAAGCCAAAC
crtEB-F:GCTGCTCCAGATCCACAA
crtEB-R:AGCGTAAACTGCCCAAAC
18S-F:GTTGGTGGAGTGATTTGTCTGC
18S-R:GCACGACGGAGTTTCACAAGAT
crtEB、crtI的表达含量在bpEBI菌株中最高,高于不诱导的基因表达量。此外相较于YPH499底盘菌株,以BY4741作为底盘菌株的重组菌基因表达量更高。这些均与番茄红素的产量具有相同的趋势,也侧面反应了BY4741更适合作为发酵番茄红素的底盘菌株。
实施例4:优化重组菌发酵条件以高效生产番茄红素。
鉴于之前在SD-Leu培养基中bpCIETB重组菌获得了最高的番茄红素产量(59.2mg/L),后续将对该重组菌进行发酵条件的优化。
不同的碳源、氮源对bpCIETB合成番茄红素的影响
碳源优化则在SD-Leu培养基中分别使用葡萄糖、果糖、蔗糖、木糖、淀粉和甘油作为培养基碳源。随后在确定最优碳源之后,分别以YNB、2%尿素、蛋白胨、硫酸铵、硝酸铵、牛肉膏、玉米浆作为培养基的氮源。从固体培养基SD-Leu中挑取重组菌株bpCIETB的单个阳性克隆子,接种至相应的液体培养基中,30℃、200 rpm 培养至OD600值约为4-6时,4000 r/min 离心10 min收集所有菌体。将菌体全部转入新鲜培养基中继续培养48-96 h后,4℃、5000 rpm离心10 min收集菌体,用蒸馏水洗涤两次,冷冻干燥获得干菌体,-20℃保存。对番茄红素产量进行分析,结果如图8所示。在葡萄糖为碳源,蛋白胨为氮源的培养基中番茄红素产量最高,优化之后的培养基命名为SP-Leu(20g/L蛋白胨,20 g/L葡萄糖,0.69g/L CSM-Leu)。
诱导pH、温度对bpCIETB合成番茄红素的影响
在确定培养基氮源为蛋白胨、碳源为葡萄糖后,对菌株发酵时的初始pH及温度进行了优化。从固体培养基SD-Leu中挑取重组菌株bpCIETB的单个阳性克隆子,接种至SP-Leu液体培养基中,30℃,200 rpm至OD600约为4-6时,将离心获取的菌体全部接种至新鲜的培养基中,继续培养48-96 h后,分别考察在不同pH、温度条件下的番茄红素产量。结果如图8所示,在培养基初始pH为6时获得了番茄红素合成的最大产量,以此为基础探究了发酵温度对番茄红素合成的影响,在28℃时番茄红素合成产量较优。
不同的发酵时间对bpCIETB合成番茄红素的影响
利用SP-Leu作为发酵培养基,在初始pH为6、发酵温度为28℃时,分别发酵24、36、48、72、96 h,探究发酵时间对番茄红素合成的影响,结果如图9所示。在发酵36 h后获得了139mg/L的番茄红素产量,较之前提高了2.3倍。
(4)添加不饱和脂肪酸对bpCIETB合成番茄红素的影响
从固体培养基SD-Leu中挑取bpCIETB重组菌株的单个阳性克隆子,接种至50 mL液体培养基中,置于30℃摇床,200 rpm 培养至OD600值为4-6时,4000 r/min 离心10 min,收集所有菌体,将菌体全部转入50 mL 含有100 mg/L不同脂肪酸(棕榈油酸、油酸、亚油酸和亚麻酸)的SP-Leu培养基中28℃发酵培养36小时。如图10结果显示添加不饱和脂肪酸可以显著增加番茄红素的产量,而对菌体生长影响较小。在分别添加棕榈油酸、油酸、亚油酸和亚麻酸后,番茄红素产量增加至289 mg/L(75.75 mg/g)、254 mg/L(79.74 mg/g)、258 mg/L(77.59 mg/g)和301 mg/L(86.04 mg/g),最高产率达到8.36 mg/L/h,实现了番茄红素的高产量合成。
最后应说明的是:以上所述仅为本发明的优选实施方式,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,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。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 南京师范大学
<120> 合成番茄红素的酿酒酵母基因工程菌及其构建方法与应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 990
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgagaccag aattgttatc tagagctttg tcattgttac cagaaagatc tgcaactcca 60
gaattagcta gattctacgg tttgttgaga gattacccac aaagaggtgg taaaggtgtt 120
agatctgaat tgttattggc ttcagcaaga gcacatggtt tgagagatac tgatccaggt 180
tgggaaagag ctttatggtt ggcaacagct ttggaattgt tccaaaactg ggttttgatc 240
catgatgata tcgaagatga ttcagaagaa agaagaggtc aaccagcatt acatagattg 300
tgtggtgttc cagttgctat taatgttggt gacgcattgc atgcttatat gtgggctgca 360
gttggtagag cagatgttcc aggtgctttc gaagaattct tgactatgat ccatagaaca 420
gcagaaggtc aacatttgga tttggcttgg gttgaaggta gagaatggaa tttggcacca 480
gctgattatt tgcaaatggt tgaattaaag actgctcatt acacagttat tgttccattg 540
agattgggtg cattggctgc aggtgttatt ccatctgaag cattgactcc agcaggttta 600
gctttgggta cagcattcca aatcagagat gatgttttga atttggctgg tgacccagtt 660
aaatatggta aagaaattgg tggtgacttg ttggaaggta aaagaacttt gatcgttttg 720
cattggttag gtgctgcacc agaatcacaa aaagctgcat ttttggatca aatgagaaga 780
gatagagcag ataaagatcc agctgcaatc gatagaattc atagatggtt gttggattct 840
ggttcagttc aacatgctca agattacgca caagctcaag caacagaagg tttgaaattg 900
ttggaaagag ctttagcagg tgctccagat ccacaagctg cagctgcatt attgacttct 960
gttagagaat tagctacaag agaaaaataa 990
<210> 2
<211> 927
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gttactgaat tttctccagc tttgccatca acagaattat gtagaccacc attggctcaa 60
gcagttcaat tctgtagaga tttgactaga catcattcta agacattcta tttgggttct 120
agattgtttt caccaccaga aagagctgca gtttgggcag tttacgctgc atgtagagct 180
ggtgacgata ttgttgatga agctggttct ggtgactcag gtcatgaatt aagagaatgg 240
agaggtagaa ttgatgctgc atttgctggt catccagcag atgatccaat ttctagagct 300
ttggcatggg ctgttgcaca ttatccaatt ccacattcag ctttcgcaga attacatgaa 360
ggtttgaaca tggatttgag aggtcatgaa taccatgaat tagatgattt gttgttgtac 420
tgtagaagag ttgcaggtgt tgttggtttt atggttgctc caatttcagg ttatagaggt 480
ggtgctgaaa ctttgcatta cgcattacaa ttgggtcaag ctatgcaatt gacaaacgtt 540
ttgagagatg ttggtgaaga tttgggtaga ggtagagttt atttgccaca atctttattg 600
gctgaatacg gtttatcaag agctgcattg gaaagatgta gacaaggtga accattaggt 660
ccaggttata gagcattgat gagacatttg ggtgacttgg ctagagaagg ttacgctgaa 720
ggtagagcag gtattccaag attagaaggt agaggtccat tagcagtttt gactgctgca 780
agagcttacg aaggtatttt agatgatttg gaaagagcag gttatgataa ttttggtaga 840
agagcttacg tttctggtag aagaaaattg ttgatgttgc cacaagcatg gtgggaatta 900
agaacattgg atgctgcaca tggttaa 927
<210> 3
<211> 1647
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgacttctc cattgccatg gccagctcca tcaccataca ctagaagaaa aacagcatta 60
gttgttggtt ctggttttgg tggtttggct ttgggtatta gattgcaatc attaggtttc 120
gatactacaa tcttggaaag attggatggt ccaggtggta gagcatatca aaagagaaca 180
ccagatggtt acgtttttga tatgggtcca actgttatta cagttccaca tttcatcgaa 240
gaattgttcg ctttggaaag agatagagca catttggatg ctccagatta cccaccagaa 300
gttttatctg gtgaaagagt tagaggtggt gtttctggtg gtccaagaac ttcagaatac 360
gttacattgg ttccaatctt gccattctac agaatcgttt tccatgatgg tactttcttt 420
gattatgatg gtgacccaga ttcaacaaga agacaaattg cagaattagc tccacaagat 480
ttggctggtt atgaaagatt tcatgctgat gcagaagcta tttttagaag aggtttcttg 540
gaattaggtt acactcattt tggtgacgtt ccaacaatgt tgagagttgt tccagatttg 600
ttgaaattgg atgctgttag aactttattt tcttttacat caaagtactt tcaatctgat 660
aagatgagac aagttttctc tttcgaaact ttgttagttg gtggtaatcc attaaatgtt 720
ccagcaatct atgctatgat ccatttcgtt gaaaagactt ggggtattca ttacgctttg 780
ggtggtacag gtgcattggt tagaggttta gttagaaagt tcggtgaatt aggtggtact 840
attagatatg gtgctggtgt taaggaaatc gttgttgatg gtagattgcc aggtcaaaga 900
acagcaagag gtgttagatt agaatctggt gaagaattgg aagcagattt ggttgcttca 960
aatggtgact gggctaatac atacttgaaa agagttccag caagagctag attagttaac 1020
tctgatttga gagttagagc tgcaagacaa tcaatgggtt tgttagttgt ttacttcggt 1080
tttagaggtg gtgacgaatt gccattgaag catcataaca tcttgttagg tccaagatac 1140
gaagcattgt tgactgaaat ctttggtaaa aaggttttgg gtgaagattt ctctcaatac 1200
ttacatgttc caactttgac agatccagat ttggcaccag ctggtcatca tgctgcatat 1260
acattagttc cagttccaca taatggttca ggtattgatt ggaaagttga aggtccaaga 1320
ttggctgatg ctgcattagc agatattgaa gctagaggtt tgattccagg tttgagaggt 1380
agattgactc atttcgaatt cgttacacca gattacttcg aaggtacttt ggattcttac 1440
ttgggtaatg cttttggtcc agaaccacaa ttggttcaat ctgctttctt tagaccacat 1500
aacagatcag aagatgttag aaatttgtat ttggttggtg caggtgctca accaggtgct 1560
ggtactccat ctgttatgat gtcagcaaaa atgacagcta gattgatcgc agaagatttc 1620
ggtattcatg cagatatcag aagataa 1647
<210> 4
<211> 190
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60
ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120
gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180
acgctcgaag 190
<210> 5
<211> 807
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atactagcgt tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca 60
aaaagattcc ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt 120
cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 180
agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 240
acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 300
acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 360
aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 420
ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 480
ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 540
atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 600
agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 660
atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 720
tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 780
aacacacata aacaaacaaa atggtta 807
Claims (6)
1.合成番茄红素的酿酒酵母基因工程菌的构建方法,其特征在于,包括如下步骤:
(1)将连接了组成型强启动子PTDH3的crtI基因连接到酵母表达质粒中,得到pESC-CI重组质粒;
(2)将组成型强启动子PTDH3、crtE基因、终止子TCYC1、组成型强启动子PTDH3、以及crtB基因依次连接,得到重组片段Ⅲ,然后将重组片段Ⅲ连接到pESC-CI重组质粒中,得到pCIETB重组质粒;
(3)将步骤(2)得到的重组质粒pCIETB转化酿酒酵母菌,得到合成番茄红素的酿酒酵母基因工程菌;
所述的crtE的核苷酸序列如SEQ ID NO.1所示;
所述的crtB的核苷酸序列如SEQ ID NO.2所示;
所述的crtI的核苷酸序列如SEQ ID NO.3所示;
步骤(1)中,所述的表达质粒为pESC-LEU;
步骤(2)中,所述终止子TCYC1的核苷酸序列如SEQ ID NO.4所示;
步骤(2)中,所述强启动子PTDH3的核苷酸序列如SEQ ID NO.5所示;
步骤(3)中,所述的酿酒酵母菌为酿酒酵母BY4741。
2.权利要求1所述合成番茄红素的酿酒酵母基因工程菌的构建方法构建得到的合成番茄红素的酿酒酵母基因工程菌。
3.权利要求2所述合成番茄红素的酿酒酵母基因工程菌在发酵制备番茄红素中的应用。
4.根据权利要求3所述的应用,其特征在于,以SD-Leu培养基的发酵培养基,以权利要求1所述的合成番茄红素的酿酒酵母基因工程菌为发酵菌株,发酵制备番茄红素。
5.根据权利要求4所述的应用,其特征在于,在培养基中加入脂肪酸,所述的脂肪酸为棕榈油酸、油酸、亚油酸、亚麻酸中的一种或几种的混合物。
6. 根据权利要求4所述的应用,其特征在于,所述SD-Leu培养基的配方为:20g/L蛋白胨,20 g/L葡萄糖,0.69g/L CSM-Leu;
所述发酵的发酵温度为28℃、发酵时间为36h。
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