CN114480458A - 一种用于表达角鲨烯合成酶的载体及其构建方法和应用 - Google Patents
一种用于表达角鲨烯合成酶的载体及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及一种用于表达角鲨烯合成酶的载体及其构建方法和应用。所述载体包含强启动子pTAC控制下的角鲨烯合酶基因hSQS,hSQS基因通过依据参照大肠杆菌密码子偏好性所优化以提高蛋白表达量。本发明为大肠杆菌发酵生产角鲨烯提供了有效的载体工具。
Description
技术领域
本发明属于基因工程领域,具体涉及一种用于表达角鲨烯合成酶的载体及其构建方法和应用。
背景技术
角鲨烯(Squalene),又称三十碳六烯,是一种不饱和脂肪烯烃。角鲨烯是所有细胞中甾醇的前体,是一种非常重要的功能油脂,具有强携氧能力、抗氧化、抗辐射、解毒作用、抑制微生物的生长、调控动物体中胆固醇的代谢以及防癌和抗癌作用等重要生理活性,在食品、医药、化妆品等领域具有包括以下方面的广泛应用:①角鲨烯抗氧化、抵御紫外线伤害,可用作化妆品;②角鲨烯可作为药物缓释剂,增强药物的治疗效果;③角鲨烯可摄取大量的氧,加强细胞新陈代谢消除疲劳,已成为功能明确的活性成分,在功能性食品中应用广泛;④角鲨烯是一种良好的自由基清除剂,因其具有抗氧化、降血糖、降血脂等生物活性,可用于保健品的生产加工,如角鲨烯软胶囊(角鲨烯含量为100-500mg/粒)等;⑤角鲨烯常被添加于大豆油、花生油等食用植物油中,抑制或延缓油脂的氧化,从而提高食用植物油的稳定性,延长产品的货架期。
通常情况下,角鲨烯大量存在于深海鲨鱼肝脏中,本着保护动物、保护环境的理念,研究人员都在积极探索其他角鲨烯来源。此外,海洋动物肝脏中存在的类似化合物,如胆固醇,使角鲨烯纯化困难,难以大规模生产应用。角鲨烯也可以从植物来源获得,但由于含量低,纯化条件复杂,难以满足制药和工业应用的需求。
由于微生物发酵存在着生长周期短,产量高、培养方法简单,原料来源丰富,价格低廉,经济效益高,受环境因素限制小等优点,因而成为目前国际上的一个新研究领域。
角鲨烯的前体物质为法尼基二磷酸酯(FPP),主要有两种合成途径:甲基赤藓糖-4-磷酸(MEP)途径和甲羟戊酸(MVA)途径,其中MEP途径主要存在于植物、细菌和藻类等,而MVA途径主要存在于真核生物、古细菌和高等植物的细胞液中。
大肠杆菌不含有角鲨烯合酶基因但具有角鲨烯合成前体物质法尼基焦磷酸合成途径,更有利于活性验证及代谢途径改造,同时大肠杆菌基遗传背景清楚,易于培养,繁殖速度快,是良好的模式生物。
发明内容
本发明的技术目的是通过构建载体以便在大肠杆菌中引入角鲨烯合成酶基因从而高效生产角鲨烯。
本发明的另一目的是提供通过微生物发酵法生产角鲨烯的方法,其中,所述方法利用上述构建的载体。
一方面,本发明提供一种用于表达角鲨烯合成酶的载体,其含有角鲨烯合成酶基因,特别是优化的角鲨烯合成酶基因(SEQ ID NO:9)。
在具体实施方式中,所述载体含有强启动子pTAC,以控制角鲨烯合酶基因。
在具体实施方式中,所述载体的序列为SEQ ID NO:8。
另一方面,本发明提供一种上述载体的构建方法,所述方法包括以下步骤:
1).构建重组质粒pTYT,
其中,以质粒载体pET-28b(+)为模板并进行反向PCR获得线性化载体片段H1,以质粒载体pRG为模板通过PCR获得片段H2,将H1、H2通过Gibson Assembly方法连接,得到重组质粒pTYT;
2).构建pTYT-hSQS载体,
通过Golden Gate组装将hSQS片段H3(优选SEQ ID NO:9)与重组质粒pTYT连接,得到重组载体pTYT-hSQS。
在具体实施方式中,在步骤1)中,使用的引物的序列如下:
H1-primer 1:SEQ ID NO:1
H1-primer 2:SEQ ID NO:2
H2-primer 1:SEQ ID NO:3
H2-primer 2:SEQ ID NO:4。
在具体实施方式中,在步骤1)中,构建的重组质粒pTYT的序列为SEQ ID NO:7。
另一方面,本发明提供一种重组菌,其含有权利要求1所述的用于表达角鲨烯合成酶的载体,优选地,所述重组菌中还转入了pMevT和pMBIS质粒。
另一方面,本发明提供一种通过微生物发酵法生产角鲨烯的方法,其中,所述方法利用上述载体。
在具体实施方式中,所述微生物为大肠杆菌DH10B。
在具体实施方式中,上述方法中,当发酵中添加抗生素时,异丙基-β-D-硫代半乳糖苷(IPTG)的浓度为20μM,在不添加抗生素时,异丙基-β-D-硫代半乳糖苷(IPTG)的浓度为100μM。
在具体实施方式中,所述抗生物可为卡那霉素、氯霉素或四环素。
在具体实施方式中,上述大肠杆菌中转入了pMevT和pMBIS质粒。
再一方面,本发明提供上述用于表达角鲨烯合成酶的载体在微生物发酵法生产角鲨烯中的用途。
有益效果
本发明构建了可表达角鲨烯合酶使大肠杆菌合成角鲨烯的质粒,该质粒包含强启动子pTAC控制下的角鲨烯合酶基因hSQS,hSQS基因通过依据参照大肠杆菌密码子偏好性所优化以提高蛋白表达量。本发明为大肠杆菌发酵生产角鲨烯提供了有效的载体工具。
附图说明
图1示意性地示出本申请的重组质粒pTYT的结构(A)以及载体pTYT-hSQS的构建过程(B)。
图2示出了发酵实验中测定的角鲨烯的产量(纵轴左,图中以柱状图表示)和相对应的生物量(纵轴右,图中以空心圆表示)。
图3示出在添加(A)和不添加抗生素(B)时,不同的IPTG浓度对于角鲨烯产量的影响。
具体实施方式
以下通过具体实施方式,详细说明本发明的技术内容。
在下文中,将通过实施例详细描述本发明。然而,在此提供的实施例仅用于说明目的,并不用于限制本发明。
下述实施例所使用的实验方法如无特殊说明,均为常规方法。
下述实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
所用酶试剂采购自ThermoFisher公司和New England Biolabs(NEB)公司,提取质粒所用的试剂盒购自天根生化科技(北京)有限公司,回收DNA片段的试剂盒购自美国omega公司,相应的操作步骤严格按照产品说明书进行,所有培养基如无特殊说明均用去离子水配制。
培养基配方:
LB培养基:5g/L酵母提取物(购自英国OXID公司,产品目录号LP0021),10g/L蛋白胨(购自英国OXID公司,产品目录号LP0042),10g/L NaCl,其余为水。调pH值至7.0-7.2,高压蒸汽灭菌。
TB培养基:24g/L酵母提取物,12g/L胰蛋白胨,0.4%(V/V)甘油,其余为水,高压蒸汽灭菌。另配制17mM磷酸二氢钾和72mM磷酸氢二钾混合溶液,高压蒸汽灭菌。将前者与后者在冷却后以10:1在无菌条件下混合。
在实际培养过程中,可向上述LB和TB培养基中加入一定浓度的抗生素以维持质粒的稳定性,所使用的抗生素终浓度为:50μg/mL卡那霉素、25μg/mL氯霉素或10μg/mL四环素。
在以下实施例中,主要载体pTYT质粒通过Gibson组装构建。增强FPP合成途径的质粒pMevT和pMBIS来自Jay D Keasling s group(Martin,Pitera et al.2003)。人角鲨烯合酶基因(hSQS)由商业公司优化合成。宿主细胞为大肠杆菌DH10b。
实施例1:构建pTYT载体
pTYT质粒由两个公开质粒提供组件:
包含抗性基因、复制起始位点和lacI表达单元的部分源自pET-28b(+)质粒;包含pTAC启动子、RiboJ隔离子、BsaI酶切位点、lacZα标记和终止子的部分源自pRG质粒。可通过Golden gate组装法,用一个或多个基因片段置换lacZα片段。插入的基因片段由pTAC强启动子控制,可由IPTG诱导表达。
以质粒载体pET-28b(+)为模板并进行反向PCR获得线性化载体片段H1,以质粒载体pRG(Zong,Zhang et al.2017)为模板通过PCR获得片段H2,将H1、H2通过GibsonAssembly方法连接,得到重组质粒pTYT(图1)。使用的引物如下表1:
表1
将重组质粒pTYT转入E.coli DH10中,用含有50μg/mL卡那霉素的LB平板筛选出阳性克隆。选其中数个单克隆提取质粒并测序验证,获得序列与设计一致的质粒。重组质粒pTYT的序列为SEQ ID NO:7。
实施例2:构建pTYT-hSQS载体
从GeneBank获取人类角鲨烯合成酶(FDFT1,索引号AC069185.7)的cDNA序列,由商业公司对基因编码区进行大肠杆菌密码子优化和全合成,优化后的序列为SEQ ID NO:9。以全合成获得的片段为模板进行PCR扩增获得hSQS片段H3,通过Golden Gate组装将H3与上文得到的重组质粒pTYT连接,得到重组载体pTYT-hSQS。使用的引物如下表2:
表2
将重组质粒pTYT-hSQS转化至E.coli DH10中,用含有50μg/mL卡那霉素的LB平板,结合蓝白斑筛选法筛选出阳性克隆。选其中数个单克隆提取质粒并对插入片段测序,获得序列与设计一致的质粒。重组载体pTYT-hSQS的序列为SEQ ID NO:8。由此,得到重组菌pTYT-hSQS/DH10B。
实施例3:pTYT-hSQS的转化、表达和检测
重组菌pTYT-hSQS/DH10B在LB培养基中220rpm,37℃培养12小时后提取质粒。将质粒转化至带有质粒pMevT和pMBIS的E.coli DH10B中,用含有50mg/mL卡那霉素、34mg/mL氯霉素和5mg/mL四环素的LB平板筛选出阳性克隆,选其中数个单克隆进行菌落PCR并将产物进行测序验证,确定hSQS已转入宿主细胞,获得重组菌株。
从固体LB平板上取3个单菌落,在3mL TB培养基中培养3小时,37℃,1000rpm。培养至OD600=0.4添加加入终浓度为100μM的IPTG诱导角鲨烯合酶蛋白表达。48h后离心收集上清,萃取产物角鲨烯进行UPLC检测,方法如下:
样品制备:收集2ml菌液放入离心管,10000rpm离心2min收集菌体。用1mL 1%(w/v)生理盐水重悬细胞洗涤一次,离心弃上清获得细胞沉淀,重复洗涤一次。加入100μL盐水短暂涡旋使菌体悬浮,通过添加1mL丙酮使细胞内容物流出,轻轻摇匀后静置2min,10000r离心1min弃菌体收集上清。用无菌注射器小心吸取上清,经有机滤膜过滤后后置于干净的液相小瓶中用于液相色谱法分析。
标品制备:取1微升角鲨烯加入1mL丙酮完全溶解。
UPLC检测:仪器:Shimazu LC-20A。固定相:GIST-HP(3μm C18,2.1×50mm)。流动相:100%甲醇。流速:0.3mL/min。
绘制不同浓度角鲨烯标准溶液的峰面积曲线。是否有产物生成是根据峰时间生成的,根据峰面积计算产物的产率。
图2示出发酵实验中测定的角鲨烯的产量(柱状图,左纵轴)和相对应的生物量(空心圆,右纵轴)。结果表明,当只将pTYT-hSQS直接转化到大肠杆菌的宿主细胞时(hSQSonly),检测到4.1±0.1mg/L的角鲨烯。向菌株中转入了来自Keasling实验室的pMevT和pMBIS质粒,以引入异源MVA途径,增强角鲨烯前体FPP的供应(hSQS+MVA),不管诱导剂IPTG是否添加,角鲨烯的产量均有了一定的提高。
在接下来的实验中,对同时引入了pMevT、pMBIS和pTYT-hSQS质粒的菌株进行发酵实验,通过控制抗生素的添加与否以及诱导剂的含量等变量,测定生物量和角鲨烯的产量,从图3的结果中发现在添加抗生素情况下,当IPTG浓度仅为20μM时,角鲨烯的单位产量最高,达到了11.1±0.1mg/L。在不添加抗生素,IPTG浓度为100μM时角鲨烯总产量最高,达到了16.0±2.0mg/L。证明了质粒pTYT-hSQS催化角鲨烯合成的有效性。
序列表
<110> 深圳蓝晶生物科技有限公司
<120> 一种用于表达角鲨烯合成酶的载体及其构建方法和应用
<130> DI20-1794-XC03
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> H1-引物 1
<400> 1
acgcgccctg tagcggcgca 20
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<212> DNA
<213> 人工序列
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atttcgcggg atcgagatct 20
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<211> 40
<212> DNA
<213> 人工序列
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<223> H2-引物 3
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agatctcgat cccgcgaaat ttcgtcaggc cacatagctt 40
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<212> DNA
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tgcgccgcta cagggcgcgt gagagcgttc accgacaaac aa 42
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cgggtctcat taagaaagag gagaaatact agatggaatt tgtaaaatgt ctaggacac 59
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cgggtctcaa gtattagtgc tcaccagtct gaacat 36
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<213> 人工序列
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acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 240
catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 300
gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 360
aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 420
acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg 480
tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 540
attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga 600
ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg 660
cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca 720
atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga 780
gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca 840
acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt 900
cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca 960
ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa 1020
tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac 1080
catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc 1140
agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt 1200
ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat 1260
tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt 1320
aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta 1380
ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc 1440
gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 1500
tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 1560
gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 1620
accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 1680
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 1740
gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 1800
ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 1860
atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 1920
gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 1980
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 2040
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 2100
gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 2160
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 2220
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 2280
tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct 2340
gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 2400
cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 2460
ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 2520
catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt 2580
cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg 2640
tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg 2700
cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga 2760
tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta 2820
aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc 2880
gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga 2940
tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga 3000
aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc 3060
acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta 3120
gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga 3180
taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga 3240
gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa 3300
agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga 3360
taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc 3420
tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct 3480
aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 3540
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg 3600
gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc 3660
tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg 3720
atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc 3780
gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc 3840
atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg 3900
gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt 3960
tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt 4020
aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc 4080
agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca 4140
tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca 4200
tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc 4260
gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt 4320
tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg 4380
gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg 4440
ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg 4500
tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg 4560
acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc 4620
tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc 4680
tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac 4740
cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg 4800
gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc 4860
ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat 4920
gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa atttcgtcag 4980
gccacatagc tttcttgttc tgatcggaac gatcgttggc tgtgttgaca attaatcatc 5040
ggctcgtata atgtgtggaa ttgtgagcgc tcacaattag ctgtcaccgg atgtgctttc 5100
cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt taatgagacc 5160
gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 5220
gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 5280
ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 5340
ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 5400
gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggt 5460
aaggtctcct actagtccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 5520
tttcgtttta tctgttgttt gtcggtgaac gctctc 5556
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<211> 6513
<212> DNA
<213> 人工序列
<220>
<223> pTYT-hSQS
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acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 240
catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 300
gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 360
aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 420
acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg 480
tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 540
attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga 600
ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg 660
cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca 720
atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga 780
gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca 840
acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt 900
cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca 960
ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa 1020
tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac 1080
catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc 1140
agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt 1200
ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat 1260
tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt 1320
aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta 1380
ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc 1440
gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 1500
tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 1560
gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 1620
accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 1680
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 1740
gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 1800
ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 1860
atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 1920
gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 1980
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 2040
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 2100
gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 2160
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 2220
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 2280
tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct 2340
gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 2400
cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 2460
ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 2520
catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt 2580
cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg 2640
tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg 2700
cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga 2760
tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta 2820
aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc 2880
gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga 2940
tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga 3000
aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc 3060
acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta 3120
gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga 3180
taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga 3240
gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa 3300
agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga 3360
taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc 3420
tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct 3480
aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 3540
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg 3600
gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc 3660
tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg 3720
atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc 3780
gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc 3840
atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg 3900
gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt 3960
tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt 4020
aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc 4080
agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca 4140
tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca 4200
tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc 4260
gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt 4320
tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg 4380
gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg 4440
ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg 4500
tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg 4560
acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc 4620
tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc 4680
tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac 4740
cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg 4800
gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc 4860
ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat 4920
gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa atttcgtcag 4980
gccacatagc tttcttgttc tgatcggaac gatcgttggc tgtgttgaca attaatcatc 5040
ggctcgtata atgtgtggaa ttgtgagcgc tcacaattag ctgtcaccgg atgtgctttc 5100
cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt taagaaagag 5160
gagaaatact agatggaatt tgtaaaatgt ctaggacacc ccgaggagtt ctacaacctg 5220
gttcgttttc gtattggcgg taaacgtaaa gttatgccga aaatggatca ggatagcttg 5280
agcagcagct tgaagacgtg ctacaagtac ctgaaccaaa ccagccgctc cttcgcagct 5340
gtgatccagg ctctggacgg cgagatgcgt aacgcggttt gtatttttta ccttgtcctg 5400
cgcgccctcg acaccctgga ggacgatatg accatctccg tggaaaagaa ggtgccgctg 5460
ctgcacaact tccacagctt cctgtatcag ccggactggc gtttcatgga gtccaaagaa 5520
aaagatcgtc aagtcttgga ggactttccg accatttctt tagagttccg caacttggct 5580
gaaaagtacc aaaccgttat cgcggacatc tgtagacgta tgggcattgg catggcggag 5640
ttcctggaca agcatgttac gtctgaacag gagtgggata aatactgcca ttatgtggcg 5700
ggcctggttg gcatcggtct gtcccgcctg tttagcgcaa gcgaatttga agacccgctg 5760
gtcggtgagg acaccgaacg ggcaaatagc atgggtctgt ttttgcaaaa gacgaacatc 5820
atccgtgatt acttggagga ccagcaaggt ggccgtgagt tctggcctca agaggtgtgg 5880
tcacgttacg tgaagaaact gggtgatttt gcgaaaccgg aaaatatcga tctggcggtg 5940
caatgtctga atgaactgat taccaacgcc ctgcaccaca ttccggacgt tattacctat 6000
ttgtcgcgcc tgcgtaatca aagcgttttc aacttttgcg cgatcccaca ggtaatggcg 6060
attgcgaccc ttgcggcctg ctataacaac cagcaagttt ttaaaggtgc agtcaaaatc 6120
cgcaagggtc aggccgtgac cctgatgatg gatgctacga atatgccggc agtgaaagcg 6180
atcatctatc aatacatgga ggagatctac catcgtattc cggactcgga tccgagtagc 6240
tctaagacgc gtcagattat ctccaccatt cgcacccaaa acctgccgaa ttgccagctg 6300
atcagccgta gccattactc cccgatttac ttgtcattcg tgatgctctt ggcggcttta 6360
tcttggcagt atctgaccac cctgagccag gtgactgaag actatgttca gactggtgag 6420
cactaatact agtccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt 6480
cgttttatct gttgtttgtc ggtgaacgct ctc 6513
<210> 9
<211> 1254
<212> DNA
<213> 人工序列
<220>
<223> hSQS
<400> 9
atggaatttg taaaatgtct aggacacccc gaggagttct acaacctggt tcgttttcgt 60
attggcggta aacgtaaagt tatgccgaaa atggatcagg atagcttgag cagcagcttg 120
aagacgtgct acaagtacct gaaccaaacc agccgctcct tcgcagctgt gatccaggct 180
ctggacggcg agatgcgtaa cgcggtttgt attttttacc ttgtcctgcg cgccctcgac 240
accctggagg acgatatgac catctccgtg gaaaagaagg tgccgctgct gcacaacttc 300
cacagcttcc tgtatcagcc ggactggcgt ttcatggagt ccaaagaaaa agatcgtcaa 360
gtcttggagg actttccgac catttcttta gagttccgca acttggctga aaagtaccaa 420
accgttatcg cggacatctg tagacgtatg ggcattggca tggcggagtt cctggacaag 480
catgttacgt ctgaacagga gtgggataaa tactgccatt atgtggcggg cctggttggc 540
atcggtctgt cccgcctgtt tagcgcaagc gaatttgaag acccgctggt cggtgaggac 600
accgaacggg caaatagcat gggtctgttt ttgcaaaaga cgaacatcat ccgtgattac 660
ttggaggacc agcaaggtgg ccgtgagttc tggcctcaag aggtgtggtc acgttacgtg 720
aagaaactgg gtgattttgc gaaaccggaa aatatcgatc tggcggtgca atgtctgaat 780
gaactgatta ccaacgccct gcaccacatt ccggacgtta ttacctattt gtcgcgcctg 840
cgtaatcaaa gcgttttcaa cttttgcgcg atcccacagg taatggcgat tgcgaccctt 900
gcggcctgct ataacaacca gcaagttttt aaaggtgcag tcaaaatccg caagggtcag 960
gccgtgaccc tgatgatgga tgctacgaat atgccggcag tgaaagcgat catctatcaa 1020
tacatggagg agatctacca tcgtattccg gactcggatc cgagtagctc taagacgcgt 1080
cagattatct ccaccattcg cacccaaaac ctgccgaatt gccagctgat cagccgtagc 1140
cattactccc cgatttactt gtcattcgtg atgctcttgg cggctttatc ttggcagtat 1200
ctgaccaccc tgagccaggt gactgaagac tatgttcaga ctggtgagca ctaa 1254
Claims (10)
1.一种用于表达角鲨烯合成酶的载体,其含有角鲨烯合成酶基因,特别是优化的角鲨烯合成酶基因(SEQ ID NO:9)。
2.根据权利要求1所述的载体,其中,所述载体的序列为SEQ ID NO:8。
3.一种如权利要求1所述的载体的构建方法,所述方法包括以下步骤:
1).构建重组质粒pTYT,
其中,以质粒载体pET-28b(+)为模板并进行反向PCR获得线性化载体片段H1,以质粒载体pRG为模板通过PCR获得片段H2,将H1、H2通过Gibson Assembly方法连接,得到重组质粒pTYT;
2).构建pTYT-hSQS载体,
通过Golden Gate组装将hSQS片段H3(优选SEQ ID NO:9)与重组质粒pTYT连接,得到重组载体pTYT-hSQS。
4.根据权利要求3所述的方法,其中,在步骤1)中,使用的引物的序列如下:
H1-primer 1:SEQ ID NO:1
H1-primer 2:SEQ ID NO:2
H2-primer 1:SEQ ID NO:3
H2-primer 2:SEQ ID NO:4。
5.根据权利要求3所述的方法,其中,在步骤1)中,构建的重组质粒pTYT的序列为SEQID NO:7。
6.一种重组菌,其含有权利要求1所述的用于表达角鲨烯合成酶的载体,优选地,所述重组菌中还转入了pMevT和pMBIS质粒。
7.一种通过微生物发酵法生产角鲨烯的方法,其中,所述方法利用如权利要求1所述的载体。
8.根据权利要求7所述的方法,其中,所述微生物为大肠杆菌DH10B。
9.根据权利要求7所述的方法,其中,在所述方法中,当发酵中添加抗生素时,异丙基-β-D-硫代半乳糖苷(IPTG)的浓度为20μM,在不添加抗生素时,异丙基-β-D-硫代半乳糖苷(IPTG)的浓度为100μM。
10.根据权利要求8所述的方法,其中,所述大肠杆菌中还转入了pMevT和pMBIS质粒。
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