CN107190004A - 一种tigr调控的甲羟戊酸途径 - Google Patents

一种tigr调控的甲羟戊酸途径 Download PDF

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CN107190004A
CN107190004A CN201610142672.5A CN201610142672A CN107190004A CN 107190004 A CN107190004 A CN 107190004A CN 201610142672 A CN201610142672 A CN 201610142672A CN 107190004 A CN107190004 A CN 107190004A
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辛珉
刘建忠
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Abstract

本发明属于生物化学领域，涉及一种利用TIGR(可调控基因区域)调控MEV(甲羟戊酸)途径，生产异戊烯类化合物，尤其是玉米黄素。具体地，将外源MEV途径引入到产玉米黄素的工程大肠杆菌(ZEAX)中，尤其是引入TIGR调控的MEV的上游途径和下游途径，以协调中间产物的产生，减小中间产物积累对细胞产生毒性，最终提高玉米黄素的产量。

Description

一种TIGR调控的甲羟戊酸途径
技术领域
本发明属于生物化学领域，具体涉及利用大肠杆菌重组工程菌，生产玉米黄素。
背景技术
玉米黄素是一种高价值的类胡萝卜素，除用作保健品，化妆品，食物等，临床上还广泛用于治疗老年性黄斑病变。随着玉米黄素的市场需求不断扩大，原玉米黄素的生产方式(化学合成和直接从植物提纯)难以稳定的供给市场。因此，急需要提高玉米黄素的产量。
目前已有多个课题组报道了可用工程大肠杆菌来生产玉米黄素(Albrecht etal.Biotechnol Lett 1999,21(9):791–795；Nishizaki et al.,Appl Environ Microbiol2007；73(4):1355–1361；Li et al.,J Ind Microbiol Biot.2015,42,627-636)。其中Liet al.报道的工程大肠杆菌的玉米黄素产量最高，达到11.95mg/g DCW(菌干重)。
利用大肠杆菌生成玉米黄素，通常是先合成中间物质IPP(异戊二烯焦磷酸)，再生成玉米黄素。
IPP(异戊二烯焦磷酸)为不仅为玉米黄素的前体,还是许多萜类化合物的前体，如番茄红素、胡萝卜素、玉米黄素、虾青素等类胡萝卜素，柠檬烯和蒎烯等单萜化合物，发呢烯、没药烯和紫穗槐-4,11-二烯等类倍半萜类化合物，紫杉烯等双萜类化合物等。
大肠杆菌由糖酵解途径中间产物3-磷酸甘油醛和丙酮酸缩合通过DXP途径(图1)来合成IPP。而一些真核细胞如酵母，则是通过甲羟戊酸(MEV)途径(图1)来合成IPP。
本领域人员研究发现，在大肠杆菌中引入酿酒酵母的MEV途径，能够增加IPP的供给，从而提高目标产物异戊烯类化合物的生产效率。
然而，他们所引入的MEV途径并没有考虑途径内每个基因间的协调表达问题，MEV途径内基因间表达不协调导致MEV途径中间产物积累，对细胞产生毒性。目前为止，也没有将MEV途径引入大肠杆菌，用于生产玉米黄素。
TIGR(tunable intergenic regions，可调基因区域)是基因与基因间一段的非编码序列，利用TIGR，可以同时协同多个基因的表达。Pfleger等报道了一种TIGR(可调基因间隔区)代谢通道技术，即在两个基因间插入TIGR能很好地协调基因的表达，他们利用该技术对酿酒酵母MEV上游途径的基因间插入了TIGR后，使其合成甲羟戊酸的量提稿7倍(NatureBiotechnology 2006,24:1027-1032)。
发明内容
本发明目的在于提供一种TIGR调控MEV途径的重组基因片段，利用该重组基因片段 可以提高IPP产量。
本发明的目的还在于提供一种含有上述重组片段的产玉米黄素大肠杆菌，利用所述的大肠杆菌可以提高玉米黄素的产量。
本发明的目的通过以下技术手段实现：
本发明提供了一种TIGR序列。
所述序列选自如SEQ ID No.3所述的TIGR3序列或如SEQ ID No.4所述的TIGR4序列。
本发明提供了一种TIGR调控MEV途径的重组基因片段。
所述的MEV途径包括MEV上游途径基因和MEV下游途径基因。
所述的MEV上游途径基因包括：
atoB：乙酰辅酶A乙酰转移酶基因；
HMGS：羟甲基戊二酰辅酶A合成酶基因；
HMGR：羟甲基戊二酰辅酶A还原酶基因；
所述的MEV上游途径基因atoB与HMGS间插有TIGR1序列，HMGS与HMGR之间插有TIGR2序列。
所述的TIGR1如SEQ ID No.1所示；
所述的TIGR2如SEQ ID No.2所示；
所述的MEV下游途径基因包括：
MK：甲羟戊酸激酶基因；
PMK：磷酸甲羟戊酸激酶基因；
MVD：焦磷酸甲羟戊酸脱羧酶基因。
所述的MEV下游途径基因MK与PMK间插有TIGR3序列，PMK与MVD之间插有TIGR4序列。
所述的TIGR3如SEQ ID No.3所示；
所述的TIGR4如SEQ ID No.4所示。
所述TIGR序列既可以经全合成，也可以采用Pfleger等报道的文库方法筛选得到(Nature Biotechnology 2006,24:1027-1032)。
本发明提供的TIGR调控MEV途径的重组基因片段还包含启动子P1。
所述的启动子P1可以是P37、PgadE、Ptrc、Ptac、PBAD和PlacUV5等大肠杆菌启动子；优选地，为PgadE。
本发明提供的TIGR调控MEV途径的重组基因片段还包含idi和ispA基因。
本发明提供的TIGR调控MEV途径如图2所示。
所述的MEV途径的第一个基因atoB来自大肠杆菌；其他基因(atoB、HMGS、 HMGR、MK、PMK、MVD、idi、ispA)可来自于酿酒酵母、也可以来其他微生物，如金黄色酿脓葡萄球菌(Staphylococcus aureus)、博德特氏菌(Bordetella petrii)、食酸戴尔福特菌(Delftiaacidovorans)和甲羟戊酸假单胞菌(Pseudomonas mevalonii)等。既可以是天然基因、也可以是其突变基因。既可以全部来自于一种上述微生物、也可以来自上述两种或多种微生物的组合。
在本发明的实施例中MK、PMK和MVD均选自酵母菌，分别为ERG12，ERG8和ERG19。
利用本发明的TIGR调控MEV途径的重组基因片段，可用于产IPP为前体的异戊烯类化合物的大肠杆菌，包括番茄红素、胡萝卜素、玉米黄素、虾青素等类胡萝卜素，柠檬烯和蒎烯等单萜化合物，发呢烯、没药烯和紫穗槐-4,11-二烯等类倍半萜类化合物，紫杉烯等双萜类化合物等。在本发明优选的实施例中，用于生产玉米黄素。
本发明提供了一种含TIGR调控MEV途径的重组基因片段的质粒制备方法，具体如下：
S1.TIGR调控的MEV上游途径的构建
1)以F1/F2为引物、大肠杆菌Escherichia coli DH5基因组为模板，PCR扩增atoB基因，连接到pBAD33载体上，得到pBAD33-atoB；
2)以F3/F4为引物、酿酒酵母(Saccharomyces cerevisiae)基因组为模板，PCR扩增HMGS基因，连接到pMD18-T载体上，得到pMD-HMGS；
3)以F5/F6为引物、酿酒酵母(Saccharomyces cerevisiae)基因组为模板，PCR扩增tHMGR基因，连接到pMD18-T载体上，得到pMD-tHMGR；
4)以F7/F8为引物、pMD-HMGS为模板，PCR扩增；以F9/F6为引物、pMD-tHMGR为模板，PCR扩增；
5)以F7/F6为引物、上一步胶回收的2个PCR片段为模版，PCR扩增，连接到pBAD33-atoB的SmaI/PstI间，得到pBAD33-MevTTIGR。
6)NheI/PstI酶切pBAD33-MevTTIGR，连接到pZSBP的相应酶切位点间，得到pZS-MevTTIGR.
SEQ ID NO.5
F1:GCTGAGCTCTTTCGGAATTAAAGGAGCATCAAATATGAAAAATTGTGTCATCGTCAG(SacI)
SEQ ID NO.6
F2:GATCCCGGGTTAATTCAACCGTTCAATCACC(SmaI)
SEQ ID NO.7
F3:TCAGGATACAGTATCTGCGGTACCGGAGGACAGCTAAATGAAACTGTCCACTAAACTGT
SEQ ID NO.8
F4:GGGTGGTCGCGCACCGGGATCAGGAGATCTTGCTAGGCTTATTTTTTAACATCGTAAGAT
SEQ ID NO.9
F5:TATCGTCGCCTCCGAGACACCATCATTGTATAGGCGGAGGATTACACTATGGACCAACTGGTGAAAACTG
SEQ ID NO.10
F6:GCTACTGCAGTTAGGATTTAATGCAGGTGACGG(PstI)
SEQ ID NO.11
F7:TCTCCCGGGGCCTAGCAAGATCTCCTGATCAGGATACAGTATCTGCGGTACCG(SmaI)
SEQ ID NO.12
F8:GGCGACGATACGCCAATCCTCAGACTGGCCCAGACTATGCAGATGTCCGGGTGGTCGCGCACCGGGATCAGG
SEQ ID NO.13
F9:ATCTGCATAGTCTGGGCCAGTCTGAGGATTGGCGTATCGTCGCCTCCGAGAC
S2.MEV下游途径pBAD24-MevBIS的构建
1)分别以F10/F11、F12/F13和F14/F15为引物、酿酒酵母(Saccharomycescerevisiae)基因组为模板，PCR扩增ERG12、ERG19和ERG8基因，连接到pBAD24上，得到pBAD24-MevB。
SEQ ID NO.14
F10:CTAGCTAGCTTTCGGAATTAAAGGAGCATCAAATATGTCTCTGCCGTTCCTG(NheI)
SEQ ID NO.15
F11:CTACCCGGGAAACTCGAGTTATGAAGTCCATGGTAAATTCG(SmaI/XhoI)
SEQ ID NO.16
F12:GATCCCGGGTTTCGGAATTAAAGGAGCATCAAATATGACCGTTTACACGGCATCC(SmaI)
SEQ ID NO.17
F13:TGCCTGCAGCCAATCGATTTATTTCTTTGGTAGACCAG(PstI/ClaI)
SEQ ID NO.18
F14:ATTCTCGAGAAAAGGGCCCTTTCGGAATTAAAGGAGCATCAAATATGTCTGAGCTGCGTGCCTTCTCTGCCCCAGGT(XhoI/ApaI)
SEQ ID NO.19
F15:ATTCCCGGGAAAAACTAGTTTATTTATCAAGATAAGTTTCCGGA(SmaI/SpeI)
2)以F16/F17为引物、大肠杆菌基因组为模板，PCR扩增idi基因，连接到pMD-18T上，并点突变去除SpeI酶切位点，得到pMD-idi。
SEQ ID NO.20
F16:ATCGATTTTCGGAATTAAGGAGGTAATAAATATGCAAACGGAACACGTCATT(ClaI)
SEQ ID NO.21
F17:GTCGACAAAAGATCTTTATTTAAGCTGGGTAAATGC(PstI/BglII)
3)以F18/F19为引物、大肠杆菌基因组为模板，PCR扩增ispA基因，克隆到pMD-idi的BglII/PstI间，并并点突变去除NheI和SmaI酶切位点，得到pMD-idi-ispA。然后用ClaI/PstI酶切pMD-idi-ispA，并将idi-ispA片段连接到pBAD24-MevB上，得到pBAD24-MevBIS。
SEQ ID NO.22
F18:TTAGTCGACTTTCGGAATTAAGGAGGTAATAAATATGGACTTTCCGCAGCAACTCG(BamHI)
SEQ ID NO.23
F19:TTAAGCATGCTTATTTATTACGCTGGATGATG(PstI)
S3.TIGR调控MEV下游途径的构建
1)在pBAD24-MevBIS中插入如下TIGR序列，得到pBAD24-MevBTIGRIS
ERG12与ERG8基因间TIGR序列为SEQ ID NO.3：
TIGR3:5'-GCCTAGCAAGATCTCCTGATCCCGGTGCGCGACCACCCGGACATCTGCATAGTCTGGGTGGATCAGGTACACTTACACTTGCCTTGAATTTACAGTATTTCAGTTACCGCTCTATCCTTATCCTTATCCGCTCAAGATAACCGGATACCGGCCCGATCGGTACCGCAGATACTGAATCC-3'
ERG8与ERG19基因间TIGR序列为SEQ ID NO.4：
TIGR4:5'-GCCTAGCAAGATCTCCTGATCCACCTTTGATGGCTAGAAAAATTAAGCTGCGGACATCTGCATAGTCTGGGCCAGTCTGAGGACTGGCGGATCAGGGCCTTGAATTTACAGTATTTAATGAACTAGCGTTCCGAGTGCATGCCTTATCCGCTCAAGACATGCACTCGGAACGCATCTAGGGTACCGCAGATACTGTATCC-3'
上述TIGR序列既可以经全合成，也可以采用Pfleger等报道的文库方法筛选得到(Nature Biotechnology 2006,24:1027-1032)。
2)用NheI/PstI酶切pBAD24-MevBTIGRIS，连接到pZSBP的相应酶切位点间，得到pZS-MevBTIGRIS。
S4.TIGR调控MEV途径的构建
1)用AvrII/SalI酶切pZS-MevBTIGRIS，连接到pZS-MevTTIGR的XbaI/SalI间，得到TIGR调控MEV途径质粒pZS-MevTTIGR-MevBTIGRIS。
2)以F20/21为引物、大肠杆菌基因组为模板，PCR扩增基因gadE的启动子，连接到pZS-MevTTIGR的BglII/NheI间，得到pZSPgadE-MevTTIGR。然后按1)的方法，与pZS-MevBTIGRIS进行组装，得到pZSPgadE-MevTTIGR-MevBTIGRIS。
SEQ ID NO.24
F20:CAACAGATCTTTAATACTCTCTCCGCTACG(BglII)
SEQ ID NO.25
F21:ATATACGCTAGCTCGTTTCGAATATGTCATCC(NheI)
本发明的重组基因片段中，大肠杆菌启动子的存在对于玉米黄素的产量提高起重要作用。发明人研究发现，大肠杆菌启动子的存在，对于产量有58％-71％的提升。所述的大肠杆菌可以是P37、PgadE、Ptrc、Ptac、PBAD和PlacUV5等大肠杆菌启动子。本发明优选的实施例中，优选的启动子为PgadE。
本发明提供了一种含有上述TIGR调控MEV途径的重组基因片段的产玉米黄素大肠菌。
所述的产玉米黄素大肠杆菌中TIGR调控MEV途径的重组基因片段即可以用质粒来表达、也可以整合到染色体上进行表达.
所述的产玉米黄素大肠杆菌为ZEAX(Li XR et al.,J Ind MicrobiolBiot.2015,42,627-636).
所述的大肠杆菌生产玉米黄素采用摇床发酵培养方式(详见Li XR et al.,J IndMicrobiol Biot.2015,42,627-636).
本发明取得的有益效果:
本发明首次将外源MEV途径引入到产玉米黄素的工程大肠杆菌中，尤其时引入TIGR调控的MEV途径。并同时对MEV整个途径(包括上游的3个基因和下游的3个基因)进行调控，协调平衡基因的表达，从而避免的甲羟戊酸途径的中间产物积累及其对大肠杆菌的毒性。此外，TIGR调控的MEV途径显著提高了中间产物IPP的产量，从而提高了以IPP为前体物的玉米黄素的产量。
附图说明
图1异戊二烯焦磷酸合成途径.
atoB:乙酰辅酶A乙酰转移酶基因；HMGS:羟甲基戊二酰辅酶A合成酶基因；HMGR:羟甲基戊二酰辅酶A还原酶基因；MK(ERG12):甲羟戊酸激酶基因；PMK(ERRG8):磷酸甲羟戊酸激酶基因；MVD(ERG19):焦磷酸甲羟戊酸脱羧酶基因。
图2 TIGR调控MEV途径
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护 范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
本方案中，所采用的分子生物学实验技术包括PCR扩增、质粒提取、质粒转化、DNA片段连接、酶切、凝胶电泳等都采用常规的方法，具体可参见《分子克隆实验指南》(第三版)(Sambrook J,Russell DW，Janssen K,Argentine J.黄培堂等译，2002，北京：科学出版社)。
实施例1TIGR调控的MEV上游途径的构建
1)以F1/F2为引物、大肠杆菌Escherichia coli DH5基因组为模板，PCR扩增atoB基因，连接到pBAD33载体上，得到pBAD33-atoB；
2)以F3/F4为引物、酿酒酵母(Saccharomyces cerevisiae)基因组为模板，PCR扩增HMGS基因，连接到pMD18-T载体上，得到pMD-HMGS；
3)以F5/F6为引物、酿酒酵母(Saccharomyces cerevisiae)基因组为模板，PCR扩增tHMGR基因，连接到pMD18-T载体上，得到pMD-tHMGR；
4)以F7/F8为引物、pMD-HMGS为模板，PCR扩增；以F9/F6为引物、pMD-tHMGR为模板，PCR扩增；
5)以F7/F6为引物、上一步胶回收的2个PCR片段为模版，PCR扩增，连接到pBAD33-atoB的SmaI/PstI间，得到pBAD33-MevTTIGR。
6)NheI/PstI酶切pBAD33-MevTTIGR，连接到pZSBP的相应酶切位点间，得到pZS-MevTTIGR
SEQ ID NO.5
F1:GCTGAGCTCTTTCGGAATTAAAGGAGCATCAAATATGAAAAATTGTGTCATCGTCAG(SacI)
SEQ ID NO.6
F2:GATCCCGGGTTAATTCAACCGTTCAATCACC(SmaI)
SEQ ID NO.7
F3:TCAGGATACAGTATCTGCGGTACCGGAGGACAGCTAAATGAAACTGTCCACTAAACTGT
SEQ ID NO.8
F4:GGGTGGTCGCGCACCGGGATCAGGAGATCTTGCTAGGCTTATTTTTTAACATCGTAAGAT
SEQ ID NO.9
F5:TATCGTCGCCTCCGAGACACCATCATTGTATAGGCGGAGGATTACACTATGGACCAACTGGTGAAAACTG
SEQ ID NO.10
F6:GCTACTGCAGTTAGGATTTAATGCAGGTGACGG(PstI)
SEQ ID NO.11
F7:TCTCCCGGGGCCTAGCAAGATCTCCTGATCAGGATACAGTATCTGCGGTACCG(SmaI)
SEQ ID NO.12
F8:GGCGACGATACGCCAATCCTCAGACTGGCCCAGACTATGCAGATGTCCGGGTGGTCGCGCACCGGGATCAGG
SEQ ID NO.13
F9:ATCTGCATAGTCTGGGCCAGTCTGAGGATTGGCGTATCGTCGCCTCCGAGAC
实施例2MEV下游途径pBAD24-MevBIS
1)分别以F10/F11、F12/F13和F14/F15为引物、酿酒酵母(Saccharomycescerevisiae)基因组为模板，PCR扩增ERG12、ERG19和ERG8基因，连接到pBAD24上，得到pBAD24-MevB。
SEQ ID NO.14
F10:CTAGCTAGCTTTCGGAATTAAAGGAGCATCAAATATGTCTCTGCCGTTCCTG(NheI)
SEQ ID NO.15
F11:CTACCCGGGAAACTCGAGTTATGAAGTCCATGGTAAATTCG(SmaI/XhoI)
SEQ ID NO.16
F12:GATCCCGGGTTTCGGAATTAAAGGAGCATCAAATATGACCGTTTACACGGCATCC(SmaI)
SEQ ID NO.17
F13:TGCCTGCAGCCAATCGATTTATTTCTTTGGTAGACCAG(PstI/ClaI)
SEQ ID NO.18
F14:ATTCTCGAGAAAAGGGCCCTTTCGGAATTAAAGGAGCATCAAATATGTCTGAGCTGCGTGCCTTCTCTGCCCCAGGT(XhoI/ApaI)
SEQ ID NO.19
F15:ATTCCCGGGAAAAACTAGTTTATTTATCAAGATAAGTTTCCGGA(SmaI/SpeI)
2)以F16/F17为引物、大肠杆菌基因组为模板，PCR扩增idi基因，连接到pMD-18T上，并点突变去除SpeI酶切位点，得到pMD-idi。
SEQ ID NO.20
F16:ATCGATTTTCGGAATTAAGGAGGTAATAAATATGCAAACGGAACACGTCATT(ClaI)
SEQ ID NO.21
F17:GTCGACAAAAGATCTTTATTTAAGCTGGGTAAATGC(PstI/BglII)
3)以F18/F19为引物、大肠杆菌基因组为模板，PCR扩增ispA基因，克隆到pMD-idi的BglII/PstI间，并并点突变去除NheI和SmaI酶切位点，得到pMD-idi-ispA。然后用ClaI/PstI酶切pMD-idi-ispA，并将idi-ispA片段连接到pBAD24-MevB上，得到pBAD24-MevBIS。
SEQ ID NO.22
F18:TTAGTCGACTTTCGGAATTAAGGAGGTAATAAATATGGACTTTCCGCAGCAACTCG(BamHI)
SEQ ID NO.23
F19:TTAAGCATGCTTATTTATTACGCTGGATGATG(PstI)
实施例3TIGR调控MEV下游途径的构建
1)在pBAD24-MevBIS中插入如下TIGR序列，得到pBAD24-MevBTIGRIS
ERG12与ERG8基因间TIGR序列为SEQ ID NO.3：
TIGR3:5'-GCCTAGCAAGATCTCCTGATCCCGGTGCGCGACCACCCGGACATCTGCATAGTCTGGGTGGATCAGGTACACTTACACTTGCCTTGAATTTACAGTATTTCAGTTACCGCTCTATCCTTATCCTTATCCGCTCAAGATAACCGGATACCGGCCCGATCGGTACCGCAGATACTGAATCC-3'
ERG8与ERG19基因间TIGR序列为SEQ ID NO.4：
TIGR4:5'-GCCTAGCAAGATCTCCTGATCCACCTTTGATGGCTAGAAAAATTAAGCTGCGGACATCTGCATAGTCTGGGCCAGTCTGAGGACTGGCGGATCAGGGCCTTGAATTTACAGTATTTAATGAACTAGCGTTCCGAGTGCATGCCTTATCCGCTCAAGACATGCACTCGGAACGCATCTAGGGTACCGCAGATACTGTATCC-3'
上述TIGR序列既可以经全合成，也可以采用Pfleger等报道的文库方法筛选得到(Nature Biotechnology 2006,24:1027-1032)。
2)用NheI/PstI酶切pBAD24-MevBTIGRIS，连接到pZSBP的相应酶切位点间，得到pZS-MevBTIGRIS。
实施例4TIGR调控MEV途径的构建
1)用AvrII/SalI酶切pZS-MevBTIGRIS，连接到pZS-MevTTIGR的XbaI/SalI间，得到TIGR调控MEV途径质粒pZS-MevTTIGR-MevBTIGRIS。
2)以F20/21为引物、大肠杆菌基因组为模板，PCR扩增基因gadE的启动子，连接到pZS-MevTTIGR的BglII/NheI间，得到pZSPgadE-MevTTIGR。然后按1)的方法，与pZS-MevBTIGRIS进行组装，得到pZSPgadE-MevTTIGR-MevBTIGRIS。
SEQ ID NO.24
F20:CAACAGATCTTTAATACTCTCTCCGCTACG(BglII)
SEQ ID NO.25
F21:ATATACGCTAGCTCGTTTCGAATATGTCATCC(NheI)
实施例5生产玉米黄素的方法
将实施例4中的TIGR调控MEV途径质粒pZS-MevTTIGR-MevBTIGRIS和pZSPgadE-MevTTIGR-MevBTIGRIS分别转化到产玉米黄素的大肠杆菌ZEAX(Li XR et al.,J IndMicrobiol Biot.2015,42,627-636)，并按该文所介绍的方法进行摇床培养发酵，分析其合成玉米黄素的情况，结果见表1。
表1不同MEV途径对大肠杆菌合成玉米黄素的影响
由表1可看出，引入外源甲羟戊酸途径能促进大肠杆菌合成玉米黄素的能力，而且PgadE驱动的TIGR调控MEV途径的效果最佳，其玉米黄素的产量为22.48mg/g DCW，是Li etal(Li XR et al.,J Ind Microbiol Biot.2015,42,627-636)报道的两倍((11.95±0.21mg/g DCW)。由此可见，本发明的方案显著提高了玉米黄素的产量。

Claims (9)

1.一种TIGR序列，选自如SEQ ID No.3所述的TIGR3序列或如SEQ ID No.4所述的TIGR4序列。
2.一种TIGR调控MEV途径的重组基因片段，其特征在于包含TIGR调控MEV上游途径基因和MEV下游途径基因；
所述的MEV上游途径基因包含：atoB，HMGS，HMGR；
所述atoB与HMGS间插有TIGR1序列，HMGS与HMGR之间插有TIGR2序列；
所述的TIGR1如SEQ ID No.1所示；
所述的TIGR2如SEQ ID No.2所示；
所述的MEV下游途径基因包括：MK，PMK，MVD；
所述的MK与PMK间插有TIGR3序列，PMK与MVD之间插有TIGR4序列。
3.如权利要求1所述的重组基因片段，其特征在还包含启动子P1，所述的启动子P1选自大肠杆菌启动子P37、PgadE、Ptrc、Ptac、PBAD、PlacUV5中的一种；优选地，为PgadE。
4.如权利要求1所述的重组基因片段，其特征在还包含基因idi和ispA。
5.如权利要求1所述的重组基因片段，其特征在于所述的atoB基因来源于大肠杆菌。
6.一种包含如权利要求1所述的重组基因片段的重组质粒的制备方法，其特征在于包含以下步骤：
S1TIGR调控的MEV上游途径的构建；
S2MEV下游途径的构建；
S3TIGR调控MEV下游途径的构建；
S4TIGR调控MEV途径的构建。
7.如权利要求1所述的重组基因片段，其特征在于其可以用来生产以IPP为前体的异戊烯类化合物，所述的异戊烯类化合物为番茄红素、胡萝卜素、玉米黄素、虾青素、柠檬烯、蒎烯发呢烯、没药烯、紫穗槐-4,11-二烯或紫杉烯；优选地，用于生产玉米黄素。
8.一种含有如权利要求1所述的重组基因片段的产玉米黄素大肠杆菌，其特征在于所述的产玉米黄素大肠杆菌为ZEAX。
9.如权利要求8所述的大肠杆菌，其特征在于其含有的重组基因片段可以用质粒来表达，或整合到染色体上进行表达而生产玉米黄素。
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CN114540363A (zh) * 2022-02-17 2022-05-27 江南大学 一种类人胶原蛋白重组毕赤酵母工程菌的构建和蛋白快速纯化方法
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