CN106520821B - 适用于链霉菌的T7RNA聚合酶表达盒PhT7及其表达载体和应用 - Google Patents

适用于链霉菌的T7RNA聚合酶表达盒PhT7及其表达载体和应用 Download PDF

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CN106520821B
CN106520821B CN201611117332.3A CN201611117332A CN106520821B CN 106520821 B CN106520821 B CN 106520821B CN 201611117332 A CN201611117332 A CN 201611117332A CN 106520821 B CN106520821 B CN 106520821B
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韦俊宏
周泽扬
田金金
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Abstract

本发明涉及一种适用于链霉菌的T7 RNA聚合酶表达盒PhT7及其表达载体和应用，适用于链霉菌的T7 RNA聚合酶表达盒PhT7核苷酸序列如SEQ ID NO.2所示，该序列根据链霉菌的密码子偏好优化设计，因此能够在链霉菌中高效表达，可用于提高链霉菌次级代谢产物的产量，缩短发酵时间。

Description

适用于链霉菌的T7RNA聚合酶表达盒PhT7及其表达载体和 应用
技术领域
本发明属于基因工程领域，具体涉及适用于链霉菌的T7RNA聚合酶表达盒PhT7，还涉及含有该表达盒表达载体及该载体的应用。
背景技术
链霉菌是一类重要的次级代谢产物生产菌，有重要价值的抗生素等次级代谢产物约80％由链霉菌产生。很多链霉菌产生的次级代谢产物都具有很高的应用价值，构建相应的高产菌株可以有效降低成本，提高生产效率。目前常用的构建次级代谢产物高产菌株的方法包括提升限速步骤酶活、增强基因簇内正调控基因的表达、敲除负调控基因、改造与倍增次级代谢产物生物合成基因簇等。在这些方法中，提升限速步骤酶活与增强基因簇内正调控基因的表达这两种方法最为简单易行，仅需要通过整合型载体导入对应的基因表达盒即可，也得到了广泛的应用。已有部分高效表达系统用于链霉菌次级代谢产物的产量提升等研究，但是这些系统不同程度的存在转录量不够高或者在不同种的链霉菌间通用性不好等问题。
T7RNA聚合酶来源于大肠杆菌T7噬菌体，T7RNA聚合酶-T7启动子系统由于其特异性与高效性，是目前在大肠杆菌中应用最广泛的蛋白表达系统之一。理论上，只要在链霉菌中稳定表达T7RNA聚合酶，在该菌株中即可利用T7启动子大量表达目的基因。但是与大肠杆菌相比，链霉菌的基因组GC含量更高，T7RNA聚合酶中有大量的密码子在链霉菌中的使用频率很低，这会使得T7RNA聚合酶的链霉菌中的表达受到影响。想要使T7RNA聚合酶-T7启动子系统在链霉菌中高效运行，对T7RNA聚合酶所有序列根据链霉菌的密码子使用偏好性进行优化是必需的。因此，有必要提供一套适用于链霉菌的T7RNA聚合酶-T7启动子高效表达系统用于次级代谢产物的超量表达。
发明内容
有鉴于此，本发明的目的之一在于提供一种适用于链霉菌的T7RNA聚合酶表达盒PhT7；本发明的目的之二在于提供含有所述适用于链霉菌的T7RNA聚合酶表达盒PhT7的高效表达载体；本发明的目的之三在于提供适用于链霉菌的T7RNA聚合酶表达盒PhT7的应用；本发明的目的之四在于提供高效表达载体的应用。
为达到上述目的，本发明提供如下技术方案：
适用于链霉菌的T7RNA聚合酶表达盒PhT7，核苷酸序列如SEQ ID NO.2所示。
2、含有所述适用于链霉菌的T7RNA聚合酶表达盒PhT7的高效表达载体。
优选的，所述高效表达载体由以下方法构建：将SEQ ID NO.1所示序列经SpeI和EcoRI酶切后连入经XbaI和EcoRI酶切的pSET152质粒，获得pPAT载体，然后再将SEQ IDNO.2所示序列经EcoRI酶切后连入经同样酶切的pPAT载体，得到高效表达载体pPhT7-PAT。
3、所述适用于链霉菌的T7RNA聚合酶表达盒PhT7在提高链酶菌次级代谢产物产量中的应用。
4、所述高效表达载体在提高链酶菌次级代谢产物产量或表达重组蛋白中的应用。
本发明的有益效果在于：本发明公开了适用于链霉菌的T7RNA聚合酶表达盒PhT7，该表达盒根据链霉菌的密码子偏好优化设计，能够在链霉菌中高效表达，可用于提高链霉菌次级代谢产物的产量和高效表达重组蛋白，并缩短发酵时间，具有很好的应用前景。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为高效表达激活子的重组菌株与野生型菌株的放线紫红素产量对比，WT：野生型菌株Streptomyces coelicolor M145，S1：高效表达放线紫红素的重组菌株Streptomyces coelicolor M145/pPhT7-PA2O4T。
图2为高效表达激活子的重组菌株与野生型菌株的生长量(干重)，WT：野生型菌株Streptomyces coelicolor M145，S1：高效表达放线紫红素的重组菌株Streptomycescoelicolor M145/pPhT7-PA2O4T。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。
本发明中放线紫红素(actinorhodin，ACT)产量的测定采用比色法，具体方法如下：取1ml发酵液，加入1ml 2M氢氧化钾混合均匀，5000g离心5min后，取1ml上清测定其OD640值。
实施例1、构建适用于链霉菌的高效表达载体pPhT7-PAT
扩增目的基因表达盒PAT：以pNgrR为模板，引物对PT7F与T7TER进行PCR扩增，其中PT7F与T7TER序列如下：
PT7F：aattactagtctcgatcccgcgaaattaata(下划线表示SpeI酶切位点)(SEQ IDNO.1)；
T7TER：aattgaattcatccggatatagttcctcctttc(下划线表示EcoRI酶切位点)(SEQID NO.2)；
PCR扩增程序如下：95℃5min变性；95℃30s，60℃30s，72℃30s扩增30个循环；72℃10min；
扩增获得目的基因表达盒PAT，具体如SEQ ID NO.3所示。将扩增产物用SpeI和EcoRI酶切处理后与经XbaI和EcoRI酶切的pSET152质粒相连，得到载体pPAT。
再根据模式菌株天蓝色链霉菌的密码子使用频率对大肠杆菌T7噬菌体T7RNA聚合酶进行优化，然后合成T7RNA聚合酶表达盒pPhT7，具体序列如SEQ ID NO.4所示，该序列上游包含了链霉菌组成型表达启动子PhrdB，下游含有密码子优化后的T7RNA聚合酶序列，然后将SEQ ID NO.4所示序列用EcoRI酶切后连入经同样酶切的pPAT质粒，得到链霉菌高效表达载体pPhT7-PAT。
实施例2、表达载体pPhT7-PA2O4T的构建
根据actII-ORF4序列(NCBI登录号AF335989.1)设计扩增放线紫红素生物合成的激活子actII-ORF4的引物，具体序列如下：
A2O4F:5’-aattcatatgagattcaacttattgggac-3’(SEQ ID NO.5)(下划线表示NdeI酶切位点)；
A2O4R:5’-aattagatctctacacgagcaccttctcaccg-3’(SEQ ID NO.6)(下划线表示BglII酶切位点)；
然后以天蓝色链霉菌Streptomyces coelicolor M145基因组DNA为模板，SEQ IDNO.5和SEQ ID NO.6为引物，进行PCR扩增，PCR扩增条件为：95℃5min变性；95℃30s，65℃30s，72℃30s扩增30个循环；72℃10min。将扩增产物进行琼脂糖凝胶电泳，并回收目的条带，然后将回收产物用NdeI与BglII进行双酶切，然后将酶切产物连接经NdeI与BamHI酶切的pPhT7-PAT载体，得到表达载体pPhT7-PA2O4T。
实施例3、放线紫红素的高效表达
将表达载体pPhT7-PA2O4T利用大肠杆菌-链霉菌种间接合转移的方法导入Streptomyces coelicolor M145菌株(WT)中，获得重组菌株，命名为Streptomycescoelicolor M145/pPhT7-PA2O4T(S1)。将重组菌株在种子培养液YEME中以200rpm，28℃培养48h，以1％接种量接种至R5培养基中以200rpm，28℃培养192h，每隔24h取一次样检测放线紫红素和菌株浓度。同时将野生型Streptomyces coelicolor M145菌株按照相同方式培养与取样，作为对照。
放线紫红素检测结果如图1所示。结果显示与野生型菌株相比，重组菌株在48h时即开始大量表达放线紫红素，且在144h时就能达到最高产量(约为对照菌株最高产量的15.7倍)，而对照菌株在72h后才开始大量表达放线紫红素，且在192h时才达到最高产量。菌株浓度检测结果如图2所示，结果显示重组菌株的生长状况与野生型菌株相比则没有明显差别。该结果说明本方法可以应用于链霉菌，大幅提升次级代谢产物的产量，并有效缩短发酵时间。
虽然本发明已经以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可以作各种改动与修饰，包括但不限定于替换T7RNA聚合酶启动子、替换表达的目的基因、替换作用的宿主链霉菌等。因此，本发明的保护范围应当以权利要求书界定的为准。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
<110> 西南大学；
<120> 适用于链霉菌的T7 RNA聚合酶表达盒PhT7及其表达载体和应用
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<210> 1
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 引物PT7F
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aattactagt ctcgatcccg cgaaattaat a 31
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<212> DNA
<213> 人工序列
<220>
<223> 引物PT7F
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aattgaattc atccggatat agttcctcct ttc 33
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<212> DNA
<213> 人工序列
<220>
<223> 目的基因表达盒PAT
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ctcgatcccg cgaaattaat acgactcact atagggagaa ggagacggag aatctcgacg 60
ggggcgcagc atatgaacct tggatccggt accagtactc accaccacca ccaccactga 120
gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 180
taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga 240
ggaactatat ccggatgaat tc 262
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<212> DNA
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<223> T7 RNA聚合酶表达盒pPhT7
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ccgccttccg ccggaacggc ggggtccggg cacgccaaac ccctcctgtg gctgtggccg 60
gccaccgccg tcaccttcgg accccgtgga gccgctcccg gttccacggg gtccgaaggt 120
gtgatgagca ggctgcgcct tcctcgcgcg gccgcaaggt acgagttgat gaccttgttt 180
atccgcatct gaccaatttt gatcgcttac ggggtgtgac tcgggccacg cggattgggc 240
gtaacgctct tgggaacaac acgatgacct aagaggtgac agccgcggag ggaatacgga 300
cgccgttcac ggcgctgtgc atctccccgg cccgcccgca ccgtcggccc attcccaagc 360
cggtggtcgg cccctgtccg ccgtggacgg ggccggaagc cgtttttcaa cgttccgaga 420
ggttgttcat gaataccatc aacattgcga aaaacgactt ctccgatatc gagctggccg 480
cgatcccctt caataccctc gcggaccact atggcgaacg gctggcccgg gaacaactgg 540
cgctggagca cgagtcctac gagatgggcg aggcccggtt ccggaaaatg ttcgaacggc 600
agctgaaggc cggcgaagtc gccgacaacg cggccgcgaa gcccctgatc acgaccctgc 660
tgcccaagat gattgcccgc atcaacgact ggttcgaaga ggtcaaggcg aagcgcggca 720
agcgccccac ggccttccag ttcctccagg agatcaaacc cgaagccgtc gcgtacatca 780
ccatcaagac gaccctggcc tgcctcacct ccgccgacaa tacgacggtc caggcggtgg 840
cgtccgccat cggccgcgcg atcgaggacg aagcccggtt tggccgcatc cgcgatctcg 900
aggccaagca cttcaaaaag aacgtcgaag agcaactgaa caagcgcgtc ggccacgtgt 960
acaagaaggc cttcatgcag gtggtcgagg cggacatgct gtccaagggc ctgctgggcg 1020
gcgaggcgtg gtcctcctgg cacaaggagg actcgatcca cgtgggggtg cgctgcattg 1080
agatgctgat cgagtccacg gggatggtct ccctgcaccg gcagaacgcg ggggtggtcg 1140
ggcaggattc cgagaccatc gaactggccc ccgagtatgc cgaagccatc gcgacccgcg 1200
cgggcgcgct cgccggcatc tcgccgatgt tccagccctg cgtggtgccg cccaaaccgt 1260
ggaccggcat caccgggggc ggctattggg ccaacgggcg ccggcccctg gccctcgtcc 1320
ggacccactc gaagaaggcc ctgatgcgct atgaggacgt ctatatgccg gaggtctaca 1380
aggcgatcaa catcgcccaa aataccgcgt ggaagatcaa caagaaggtc ctggccgtgg 1440
ccaatgtcat cacgaaatgg aagcactgcc ccgtggagga catcccggcc atcgagcgcg 1500
aggaactccc catgaagccc gaggacatcg acatgaaccc cgaagccctc accgcgtgga 1560
aacgcgccgc ggcggcggtc taccgcaagg acaaagcgcg gaaatcccgc cgcatctccc 1620
tggagttcat gctggagcag gcgaacaagt ttgccaacca caaggcgatc tggttcccgt 1680
acaacatgga ctggcgcggg cgcgtgtatg cggtgtccat gttcaacccg cagggcaacg 1740
atatgaccaa gggcctcctc accctcgcga agggcaagcc gatcgggaag gagggctact 1800
actggctgaa gatccatggg gcgaactgcg ccggcgtcga caaagtcccg ttcccggagc 1860
ggatcaaatt catcgaggag aaccacgaga acatcatggc gtgcgccaag tcgcccctgg 1920
aaaacacctg gtgggccgag caggattcgc ccttctgctt tctggccttc tgcttcgaat 1980
acgccggggt gcaacaccat ggcctctcct acaattgctc gctgcccctg gcctttgacg 2040
gctcctgctc cggcatccag cacttctccg ccatgctgcg ggacgaagtg ggcgggcggg 2100
ccgtcaatct gctcccgtcc gagacggtcc aggacatcta cgggatcgtg gccaagaagg 2160
tcaacgaaat tctgcaggcc gacgccatca atggcacgga caacgaggtg gtgaccgtca 2220
cggacgagaa caccggcgag atttccgaga aggtcaaact gggcaccaag gccctcgccg 2280
ggcaatggct ggcgtatggc gtcacgcggt ccgtgaccaa gcggtccgtc atgacgctgg 2340
cgtacggctc gaaggagttc ggcttccggc aacaggtcct cgaagacacg atccaaccgg 2400
ccatcgactc cgggaagggc ctgatgttca cccagccgaa ccaggccgcc ggctacatgg 2460
ccaagctgat ctgggaatcg gtctcggtga ccgtcgtcgc ggccgtcgag gccatgaact 2520
ggctgaagtc cgcggccaaa ctgctggcgg cggaagtcaa agacaagaag accggggaga 2580
tcctgcgcaa gcgctgtgcc gtccactggg tcacccccga tggcttcccg gtgtggcagg 2640
agtacaaaaa gcccatccaa acgcgcctga acctgatgtt tctgggccag ttccggctcc 2700
agccgacgat caacacgaac aaggactccg agatcgacgc gcacaagcag gagtccggca 2760
tcgcccccaa ttttgtccac tcgcaagacg gctcccacct gcgcaaaacc gtggtctggg 2820
cccacgagaa gtacggcatc gagtcgtttg ccctgatcca cgactccttc ggcacgattc 2880
cggccgacgc cgccaacctc ttcaaagccg tgcgcgaaac catggtcgac acgtatgagt 2940
cctgcgacgt cctggcggac ttctatgatc aattcgcgga ccagctgcac gagtcgcagc 3000
tggacaaaat gccggcgctc cccgccaagg ggaatctgaa tctccgggac atcctcgagt 3060
ccgacttcgc gtttgcgtga 3080
<210> 5
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物A2O4F
<400> 5
aattcatatg agattcaact tattgggac 29
<210> 6
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物A2O4R
<400> 6
aattagatct ctacacgagc accttctcac cg 32

Claims (5)

1.适用于链霉菌的T7 RNA聚合酶表达盒PhT7，其特征在于：核苷酸序列如SEQ ID NO.4所示。
2. 含有权利要求1所述适用于链霉菌的T7 RNA聚合酶表达盒PhT7的高效表达载体。
3. 根据权利要求2所述的高效表达载体，其特征在于，所述高效表达载体由以下方法构建：将SEQ ID NO.3所示序列经SpeI和EcoRI酶切后连入经XbaI和EcoRI酶切的pSET152质粒，获得pPAT载体，然后再将SEQ ID NO.4所示序列经EcoRI酶切后连入经同样酶切的pPAT载体，得到高效表达载体pPhT7-PAT。
4. 权利要求1所述适用于链霉菌的T7 RNA聚合酶表达盒PhT7在提高链酶菌次级代谢产物产量中的应用。
5.权利要求2或3所述高效表达载体在提高链酶菌次级代谢产物产量或表达重组蛋白中的应用。
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CN103421778A (zh) * 2012-05-23 2013-12-04 中国科学院微生物研究所 一种链霉菌组成型启动子及其应用
CN104531598A (zh) * 2015-01-08 2015-04-22 齐鲁工业大学 一种重组链霉菌、其构建方法及提高抗生素产量的方法

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