CN111041040A - 产左旋海松二烯和左旋海松酸的重组酿酒酵母及构建方法 - Google Patents
产左旋海松二烯和左旋海松酸的重组酿酒酵母及构建方法 Download PDFInfo
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- CN111041040A CN111041040A CN201911305260.9A CN201911305260A CN111041040A CN 111041040 A CN111041040 A CN 111041040A CN 201911305260 A CN201911305260 A CN 201911305260A CN 111041040 A CN111041040 A CN 111041040A
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Abstract
本发明公开了产左旋海松二烯和左旋海松酸的重组酿酒酵母及构建方法，步骤为：向酿酒酵母中导入改造的左旋海松二烯合酶编码基因TΔLPS、优化的细胞色素P450酶编码基因CYP720B1和优化的细胞色素P450还原酶编码基因TcCPR，得到重组菌1；向重组菌1中导入3‑羟基‑3‑甲基戊二酰辅酶A还原酶编码基因tHMG1，得到重组菌2；向重组菌2中导入法呢基焦磷酸合酶编码基因Erg20，得到重组菌3；将重组菌3中野生型鲨烯合酶启动子替换成Met3启动子，得到重组菌4；实验证明，本发明的重组菌发酵可以提高左旋海松二烯和左旋海松酸的产量；为人工细胞合成左旋海松二烯和左旋海松酸奠定了基础。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种产左旋海松二烯和左旋海松酸的重组酿酒酵母及构建方法。
背景技术
左旋海松二烯(levopimaradiene)是合成银杏内酯的前提物质，银杏内酯具有抗血小板凝集、抗炎症、抗血栓等药理作用。左旋海松酸(levopimaric acid)是一种重要的松香树脂酸，左旋海松酸具有同环共轭双键结构，在常温下能与马来酸酐发生Diels-Alder加成反应，生成马来海松酸，广泛应用于涂料，印染、油墨等行业，可以作为增塑剂。马来海松酸的酰胺和亚酰胺具有抗兽类肝炎的作用，同时它的衍生物具有抗炎症、抗溃疡作用。左旋海松酸与2-(2,4-二氧-5-噻唑烷二酮)乙酸合成一种左旋海松酸衍生物，其具有抗癌症、抗菌、抗痨、抗HIV作用。左旋海松酸的制备现在主要是以松脂为原材料，从中分离出左旋海松酸，分离工艺比较复杂。
左旋海松酸属于二萜类化合物，在烯丙基焦磷酸(DMAPP)的基础上，逐步叠加3个异戊烯基焦磷酸(IPP)，生成前体化合物GGPP(C20)。2001年Schepmann等人在银杏幼苗根部所构建的cDNA文库中克隆并鉴定了左旋海松二烯合酶LPS，LPS催化GGPP环化形成85％的左旋海松二烯，15％的副产物abietadiene，及微量的neo-abietadiene和palustradiene。催化原理为，GGPP在LPS的催化下首先生成C8-sandaracopimarenyl阳离子中间体，通过氢重排，进一步形成C15-sandaracopimarenyl阳离子中间体。然后13位上的甲基转移到15位上生成abietenyl阳离子中间体，最后通过不同的氢重排和去质子化作用，分别形成abietadiene，levopimaradiene，palustradiene和neo-abietadiene。2010年Leonard等在大肠杆菌中表达LPS基因，最终在生物反应器中发酵得到了约700mg/L的左旋海松二烯(levopimaradiene)。原始的左旋海松二烯合酶LPS的酶活很低，需要进行改造才能提高左旋海松二烯的产量。
细胞色素P450酶的CYP720B亚家族在二萜松脂酸的生物合成中扮演着重要的作用，CYP720B亚家族中的两种P450酶，来源Loblollypine的CYP720B1以及来源Sitka spruce的CYP720B4已经经过鉴定，可以三步氧化左旋海松二烯，依次形成醇、醛中间体，最后生成左旋海松酸。
目前未见在酿酒酵母中异源合成左旋海松二烯和左旋海松酸的报道。
发明内容
本发明的一个目的是克服现有技术的不足，提供一种产左旋海松二烯和左旋海松酸的重组酿酒酵母的构建方法。
本发明的第二个目的是提供一种产左旋海松二烯和左旋海松酸的重组酿酒酵母。
本发明的技术方案概述如下：
一种产左旋海松二烯和左旋海松酸的重组酿酒酵母的构建方法，包括如下步骤：
(1)向酿酒酵母(Saccharomyces cerevisiaeW303-1a)中导入改造的左旋海松二烯合酶编码基因TΔLPS、优化的细胞色素P450酶编码基因CYP720B1和优化的细胞色素P450还原酶编码基因TcCPR，得到重组菌1；
所述改造的左旋海松二烯合酶编码基因TΔLPS是左旋海松二烯合酶编码基因LPS编码的氨基酸序列的N端截短79个氨基酸并且左旋海松二烯合酶编码基因LPS编码的氨基酸序列的第593的甲硫氨酸密码子突变为异亮氨酸密码子以及第700的酪氨酸的密码子突变为苯丙氨酸密码子；所述改造的左旋海松二烯合酶编码基因TΔLPS的核苷酸序列如SEQID NO.1所示；所述优化的细胞色素P450酶编码基因CYP720B1的核苷酸序列如SEQ ID NO.2所示；所述优化的细胞色素P450还原酶编码基因TcCPR的核苷酸序列如SEQ ID NO.3所示；
(2)向重组菌1中导入3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1，得到重组菌2；
所述3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1的核苷酸序列如SEQ IDNO.4所示。
(3)向重组菌2中导入法呢基焦磷酸合酶编码基因Erg20，得到重组菌3；
所述法呢基焦磷酸合酶编码基因Erg20的核苷酸序列如SEQ ID NO.5所示；
(4)将重组菌3中野生型鲨烯合酶启动子替换成Met3启动子，得到重组菌4；
所述Met3启动子的核苷酸序列如SEQ ID NO.6所示。
上述方法制备的产左旋海松二烯和左旋海松酸的重组酿酒酵母菌。
实验证明，本发明的重组菌发酵可以提高左旋海松二烯和左旋海松酸的产量；本发明成功构建的产左旋海松二烯和左旋海松酸的重组酿酒酵母菌株，为人工细胞合成左旋海松二烯和左旋海松酸奠定了基础。
具体实施方式
酿酒酵母(Saccharomyces cerevisiae)W303-1a，美国ATCC208352
购买时间，2016.6网址：https://www.atcc.org/
下面通过具体实施例对本发明作进一步的说明。
下面实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、各片段来源及质粒制备方法
(1)本发明所用的基因原件LPS序列(levopimaradiene synthase LPS，GenPept:Q947C4.1)来源于植物银杏(Ginkgo biloba)；CYP720B1序列(cytochrome P450)来源于火炬松(Loblolly pine)、TcCPR序列(plant P450 s depend on cytochrome P450reductase CPR)来源于植物东北红豆杉(Taxus cuspidate)，由武汉金开瑞生物工程有限公司通过化学合成的方法合成，并针对酿酒酵母进行密码子优化，并连接在大肠杆菌质粒上，保存在大肠杆菌中。得到优化的细胞色素P450酶编码基因CYP720B1的核苷酸序列如SEQID NO.2所示和优化的细胞色素P450还原酶编码基因TcCPR的核苷酸序列如SEQ ID NO.3所示。
(2)将编码左旋海松二烯合酶的基因LPS，改造成截短突变的TΔLPS的方法如下：
TΔLPS(左旋海松二烯合酶编码基因LPS编码的氨基酸序列的N端截短79个氨基酸，左旋海松二烯合酶编码基因LPS编码的氨基酸序列的第593的甲硫氨酸密码子突变为异亮氨酸密码子、第700的酪氨酸的密码子突变为苯丙氨酸密码子)，将5’端237个碱基切除，并在5’端加上起始密码子ATG，然后将LPS的第1777-1779bp碱基由ATG突变成ATC，将LPS的第2098-2100bp碱基由TAT突变成TTC，得到改造后的碱基序列TΔLPS。所述LPS序列在NCBI中可查(GenPept:Q947C4.1)，改造的左旋海松二烯合酶编码基因TΔLPS序列如SEQ IDNO.1所示。
用表1描述的引物进行PCR。以pUC57-LPS为模板，Tef1p-T79LPS-F(SEQ ID NO.7)为前引，LPS-R-M593I(SEQ ID NO.8)为后引，PCR扩增得到片段T79LPS；
以pUC57-LPS为模板，LPS-M593I-F(SEQ ID NO.9)为前引，LPS-R-Y700F(SEQ IDNO.10)为后引，扩增片段LPSM593I；
以pUC57-LPS为模板，以LPS-Y700F-F(SEQ ID NO.11)为前引，LPS-R-Adh2t(SEQID NO.12)为后引，扩增出基因片段LPSY700F.
本发明所用PCR酶为南京诺唯赞生物科技有限公司的
Max Super-Fidelity聚合酶。50μL的PCR扩增体系如下：DNA模板，1μL；前引(10μM)和后引(10μM)各2μL；dNTP(10mM)，1μL；2×Phanta Max Buffer，25μL；
Max Super-Fidelity聚合酶，1μL；最后用双蒸水补齐50μL。在PCR仪上设置扩增程序。扩增条件为95℃预变性4min(1个循环)；95℃变性15sec、退火60℃15sec、72℃延伸1min(34个循环)；72℃延伸5min(1个循环)。
本发明所用融合PCR体系如下：DNA片段总量800ng，摩尔比1:1；dNTP(10mM)，1μL；2×Phanta Max Buffer，25μL；
Max Super-Fidelity聚合酶，1μL；最后用双蒸水补齐50μL。在PCR仪上设置扩增程序。扩增条件为95℃预变性4min(1个循环)；95℃变性15sec、60℃退火30sec、72℃延伸1min(11个循环)，72℃延伸5min(1个循环)。
将以上扩增得到的T79LPS、LPSM593I、LPSY700F片段进行纯化回收，然后进行融合PCR，将三个片段融合成一条，即得到突变且截短79aa的TΔLPS基因，完成第593个氨基酸密码子从Met到Ile的突变，以及第700个氨基酸密码子从Tyr到Phe的突变。
表1引物序列
(3)本发明所用酿酒酵母内源片段，通过提取酿酒酵母基因组，以其为模板，通过PCR扩增得到。
酿酒酵母基因组的提取方法为：将酿酒酵母接种于液体YPD培养基中，30℃摇床过夜培养；吸取2mL过夜培养的酿酒酵母到离心管中，10000rpm转速下离心1min，弃上清液，收集底部菌体沉淀；向离心管中加入与菌体体积相等的石英砂，然后加入400μL的STES溶液，400μL酚/氯仿/异戊醇(体积比为25：24：1)；将上述混合物置于振荡器，振荡10min；向振荡结束的离心管中加入400μL的TE溶液，混合均匀，离心取上清液，将上清液转移到新的2mL离心管中；然后向收集的上清液中加入1/10体积的3M NaAc，2倍体积的无水乙醇，混合均匀后置于-20℃放置1h，10000rpm离心10min，可看见离心管底部有少量白色沉淀，即基因组DNA，除去上清液，用体积浓度为75％的乙醇水溶液漂洗一次；在通风处，使离心管中残余的酒精充分挥发，然后加入100μL水，即完成酵母基因组提取，用微量分光光度计测DNA浓度，保存于-20℃冰箱中备用。
所用到的溶液配方如下：
1.Tris·HCl(50mmol/L)溶液：50mL的0.1mol/L Tris溶液与29.2mL的0.1mol/L盐酸混合均匀，然后用水定容至100mL。
2.EDTA(0.5mol/L)溶液：18.16g Na2EDTA·2H2O溶解于水中，并定容至100mL，用固体NaOH调节PH至8.0。
3.TE溶液的配方：200ml的Tris·HCl(50mmol/L)溶液，2mL的EDTA(0.5mol/L)溶液，混合后加水，定容至1L。
4.STES裂解液的配方：取20mL的Tris·HCl(50mmol/L)溶液，2mL的Triton X-100，0.584gNaCl，200μL的EDTA(0.5mol/L)溶液，将其混匀后，用水定容至100mL。
5.酚/氯仿/异戊醇混合液中苯酚：氯仿：异戊醇＝25:24:1。
(4)保存在大肠杆菌中的质粒选用天根质粒小提试剂盒进行提取，步骤如下：
1.将大肠杆菌接种到含有抗生素(氨苄)的LB液体培养基中培养，收集3mL菌液于离心管中，12000rpm离心1min，弃上清液，收集离心管底部的菌体(上清液要尽量除尽)；
2.将吸附柱用500μL的BL平衡液进行平衡，12000rpm离心，倒掉收集管中的废液；
3.向有菌体沉淀的离心管中加入250μL的P1溶液，并使菌体沉淀完全悬浮；
4.再向离心管中加入250μL的P2裂解溶液，温和地翻转离心管，使菌体充分裂解，此时菌液会变得清亮，裂解时间不能太长避免质粒破环；
5.再向离心管中加入350μL的P3溶液，上下翻转离心管，使蛋白沉淀；
6.将上述离心管12000rpm离心10min，将沉淀彻底离心到底部，取上清液于用BL溶液平衡过的吸附柱中，于-20℃冰箱中吸附5min,12000rpm离心1min，倒去收集管中废液；
7.将已经吸附质粒的吸附柱用含酒精的PW溶液漂洗两次，以除去杂质，然后12000rpm转速下离心2min，尽量除去PW，然后吸附柱置于通风处使酒精挥发完毕。
8.向吸附柱中加入50μL去离子水，置于37℃下10min，使质粒溶解于水中，将吸附柱放在离心管中，12000rpm离心2min，收集质粒。
实施例2、酿酒酵母重组菌株1的构建
向酿酒酵母(Saccharomyces cerevisiaeW303-1a，美国ATCC208352)中导入改造的左旋海松二烯合酶编码基因TΔLPS、优化的细胞色素P450酶编码基因CYP720B1和优化的细胞色素P450还原酶编码基因TcCPR，得到重组菌1。所述改造的左旋海松二烯合酶编码基因TΔLPS的核苷酸序列如SEQ ID NO.1所示，所述优化的细胞色素P450酶编码基因CYP720B1的核苷酸序列如SEQ ID NO.2所示，所述优化的细胞色素P450还原酶编码基因TcCPR的核苷酸序列如SEQ ID NO.3所示。
一、模块构建
rDNA-up(SEQ ID NO.13)，启动子TEF1(SEQ ID NO.14)，终止子ADH2(SEQ IDNO.15)，启动子PGK1(SEQ ID NO.16)，终止子ADH1(SEQ ID NO.17)，启动子TDH3(SEQ IDNO.18)，终止子TDH2(SEQ ID NO.19)，rDNA-down(SEQ ID NO.20)均来自酿酒酵母ATCC208352基因组，筛选标记基因URA3(SEQ ID NO.21)序列来自酿酒酵母ATCC 208352，TΔLPS(SEQ ID NO.1)来自含LPS基因的质粒，CYP720B1(SEQ ID NO.2)来自含CYP720B1基因的质粒，TcCPR(SEQ ID NO.3)来自含TcCPR基因的质粒，基因由武汉金开瑞生物工程有限公司通过化学合成的方法合成，
以酿酒酵母ATCC208352基因组为模板，以R1-F(SEQ ID NO.22)，R1-R-Tef1p(SEQID NO.23)为引物，扩增rDNA-up；以R1-Tef1p-F(SEQ ID NO.24)，Tef1p-R-TΔLPS(SEQ IDNO.25)为引物扩增启动子TEF1；以Tef1p-TΔLPS-F(SEQ ID NO.26)，TΔLPS-R-ADH2(SEQID NO.27)为引物扩增TΔLPS；以TΔLPS-Adh2t-F(SEQ ID NO.28)，Adh2t-R-Pgk1p(SEQ IDNO.29)为引物扩增终止子ADH2；
以Adh2t-Pgk1p-F(SEQ ID NO.30)，Pgk1p-R-CYP720B1(SEQ ID NO.31)为引物扩增启动子PGK1；以Pgk1p-CYP720B1-F(SEQ ID NO.32)，CYP720B1-R-Adh1t(SEQ ID NO.33)为引物扩增CYP720B1；以CYP720B1-Adh1t-F(SEQ ID NO.34)，Adh1t-R-Tdh3p(SEQ IDNO.35)为引物扩增终止子ADH1；
以Adh1t-Tdh3p-F(SEQ ID NO.36)，Tdh3p-R-TcCPR(SEQ ID NO.37)为引物扩增启动子TDH3；以Tdh3p-TcCPR-F(SEQ ID NO.38)，TcCPR-R-Tdh2t(SEQ ID NO.39)为引物扩增TcCPR；以TcCPR-Tdh2t-F(SEQ ID NO.40)，Tdh2t-R-Ura3(SEQ ID NO.41)为引物扩增终止子TDH2；
以Tdh2t-Ura3-F(SEQ ID NO.42)，Ura3-R-R2(SEQ ID NO.43)为引物扩增URA3；
以Ura3-R2-F(SEQ ID NO.44)，R2-R(SEQ ID NO.45)为引物，扩增rDNA-down。
本发明所用PCR酶为南京诺唯赞生物科技有限公司的
Max Super-Fidelity聚合酶。50μL的PCR扩增体系如下：DNA模板，1μL；前引(10μM)和后引(10μM)各2μL；dNTP(10mM)，1μL；2×Phanta Max Buffer，25μL；
Max Super-Fidelity聚合酶，1μL；最后用双蒸水补齐50μL。在PCR仪上设置扩增程序。扩增条件为95℃预变性4min(1个循环)；95℃变性15sec、退火60℃15sec、72℃延伸1min(34个循环)；72℃延伸5min(1个循环)。
本发明所用融合PCR体系如下：DNA片段总量800ng，摩尔比1:1；dNTP(10mM)，1μL；2×Phanta Max Buffer，25μL；
Max Super-Fidelity聚合酶，1μL；最后用双蒸水补齐50μL。在PCR仪上设置扩增程序。扩增条件为95℃预变性4min(1个循环)；95℃变性15sec、60℃退火30sec、72℃延伸1min(11个循环)，72℃延伸5min(1个循环)。
最后得到转化酿酒酵母所用的DNA片段，上游同源臂rDNA-up、TΔLPS表达盒PTef1-TΔLPS-TAdh2、基因CYP720B1表达盒PPgk1-CYP720B1-TAdh1、基因TcCPR表达盒PTdh3-TcCPR-TTdh2和含有筛选标记基因URA3的下游同源臂URA3-rDNAdown。
二、酿酒酵母转化
1.出发菌酿酒酵母Saccharomyces cerevisiae W303-1a接种于试管YPD液体培养基中，30℃摇床过夜培养；
2.将过夜培养的酵母菌以1/10的体积比，转接到新的试管YPD液体培养基中，30℃摇床培养4h，使其达到对数生长期；
3.取1mL菌液于无菌的离心管中，5000rpm离心3min，除去上清液，收集底部菌液，并用1mL无菌水将菌体洗一次；
4.将洗涤过的酵母菌用1mL 100mM LiAc重悬，并静置5min；
5. 5000rpm离心3min，除去LiAc溶液，保留底部酵母菌；
6.在含有酵母菌的离心管中配制转化体系，具体加入试剂及顺序如下表：
表2转化体系
转化的DNA片段包括rDNA-up，PTef1-TΔLPS-TAdh2，PPgk1-CYP720B1-TAdh1，PTdh3-TcCPR-TTdh2，Ura3-rDNA-down，每种片段大于300ng；
7.其中鲑鱼精DNA需要在沸水中煮沸5min，使其解链，然后迅速转移到冰上冰浴，用以酵母转化；
8.将配好的转化体系，用移液器吹吸，或者放在漩涡振荡器上震荡1min，使体系充分混匀，放入42℃水浴锅中，热激30min；
9.将热激后的酵母菌离心，用移液器移去上清液，加入1mLYPD培养基，在30℃摇床中复苏2h；
10. 5000rpm离心3min，移去YPD液体培养基，并用无菌水洗涤2次；
11.加入100μL无菌水，将酵母细胞重悬，并涂布在缺失尿嘧啶Ura的SC选择培养基上，30℃培养箱培养2d。
12.待长出单菌落后，进行菌落PCR，需要对基因组进行粗提。本发明采用冻融法粗提酿酒酵母基因组，首先将单菌落挑取到10μL浓度为10mM的NaOH溶液中，在沸水中煮沸10min，再放入-20℃冰箱中冷冻10min，反复冻融三次，即将基因组粗提，可直接当做模板，进行菌落验证。验证正确的菌株即为重组菌1。
SC选择培养基配方：6.7g/L Yeast Nitrogen Base(YNB)，20g/L葡萄糖或半乳糖，2g/L相应缺省氨基酸混合物。混合物成分包括：
本发明出发菌酿酒酵母Saccharomyces cerevisiae W303-1a缺失基因Leu2、Trp1、Ura3、Ade2、His3，且被用于筛选标记，所以在配制SC选择培养基时，根据需要补齐相应成分，终浓度为：
表3引物序列
实施例3、酿酒酵母重组菌株2的构建
向重组菌1中导入3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1，得到重组菌2，所述3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1的核苷酸序列如SEQ ID NO.4所示。
一、质粒pRS304-tHMG1的制备方法
根据表6，以酿酒酵母基因组为模板，ApaI-Tdh3p-F(SEQ ID NO.46)为前引，Tdh3p-R-tHMG1(SEQ ID NO.47)为后引，PCR扩增出片段PTdh3；以酿酒酵母基因组为模板，Tdh3p-tHMG1-F(SEQ ID NO.48)为前引，tHMG1-R-Cyc1t(SEQ ID NO.49)为后引，PCR扩增出片段tHMG1，(将编码HMGR1N端氨基酸的序列截短，只保留编码HMGR1蛋白C末端的503个氨基酸的序列)；以酿酒酵母基因组为模板，tHMG1-Cyc1t-F(SEQ ID NO.50)为前引，Cyc1t-R-Pts1(SEQ ID NO.51)为后引，PCR扩增出片段TCyc1。将片段PTdh3、tHMG1、TCyc1根据实施例2中的融合PCR方法，融合成表达框PTdh3-tHMG1-TCyc1。
将表达框PTdh3-tHMG1-TCyc1和载体pRS304(购自BioVector质粒载体菌种细胞基因保藏中心NTCC)通过酶切连接的方法，进行连接，构建表达载体pRS304-tHMG1。
本文选用Thermo Scientific公司的限制性内切酶及T4连接酶构建质粒。酶切体系如下：
表4酶切体系
这里选用的限制性内切酶为ApaI和PstI；
将配好的酶切体系置于37℃水浴锅中进行酶切1h，酶切好的质粒和片段进行纯化回收，进行连接反应。
表5连接体系如下：
将配好的连接体系置于22℃下连接1h。构建好的质粒转化到大肠杆菌中，转化方法：
大肠杆菌感受态细胞的化学转化法(热激法)
1.从-80℃冰箱中取出Trans5α化学感受态细胞成品，在冰盒中放置10min解冻，无菌环境下吸取50μL菌液至灭菌的1.5mLPE离心管中；
2.向离心管中加入5μL重组体系溶液，温和混匀，在冰盒中放置30min；
3.从冰盒中取出离心管，在42℃恒温水浴中热激30sec，取出并立刻在冰盒中放置2min；
4.向离心管中加入500μL灭菌的LB液体培养基，放入设定条件为37℃，200rpm的摇床中复苏1h；
5.在4000rmp条件下离心3min，吸去上清液350μL，收集菌体并重悬；
6.在无菌条件下，将菌液涂布在含有抗生素Amp的LB平板培养基上，在37℃培养箱中倒置过夜培养。
7.挑取转化子菌落PCR验证，得到正确目的片段的为正确转化子，提取质粒ApaI和PstI酶切验证正确的质粒为正确质粒，即pRS304-tHMG1构建成功。
二、酿酒酵母转化
将含有步骤一获得的基因tHMG1的表达盒导入重组菌1中，得到重组菌2。
采取与实施例2中步骤二相同的方法，进行重组菌感受态细胞的制备及转化，转入pRS304-tHMG1，在缺失色氨酸的培养基中筛选培养，得到转化子。筛选培养条件为30℃，培养48h以上，挑取转化子过夜培养，提基因组，并用check引物验证，得到正确目的片段的为正确克隆，命名为重组菌2。TRP1的核苷酸序列如SEQ ID NO.52所示。
表6引物序列
实施例4、酿酒酵母重组菌株3的构建
向重组菌2中导入法呢基焦磷酸合酶编码基因Erg20，得到重组菌3，所述法呢基焦磷酸合酶编码基因Erg20的核苷酸序列如SEQ ID NO.5所示。
一、质粒pRS405-Erg20的制备方法
以酿酒酵母基因组为模板，Pst1-Pgk1p-F(SEQ ID NO.53)为前引，Pgk1p-R-Erg20(SEQ ID NO.54)为后引，PCR扩增出片段PPgk1；以酿酒酵母基因组为模板，Pgk1p-Erg20-F(SEQ ID NO.55)为前引，Erg20-R-Cyc1t(SEQ ID NO.56)为后引，PCR扩增出片段Erg20；以酿酒酵母基因组为模板，Erg20-Cyc1t-F(SEQ ID NO.57)为前引，Cyc1t-R-BamHI(SEQ IDNO.58)为后引，PCR扩增出片段TCyc1。将片段PPgk1、Erg20、TCyc1根据实施例2中的融合PCR方法，融合成表达框PPgk1-Erg20-TCyc1。
将表达框PPgk1-Erg20-TCyc1和空载体pRS405(购自生物风)通过酶切连接的方法，进行连接，构建表达载体pRS405-Erg20。
本文选用Thermo Scientific公司的限制性内切酶及T4连接酶构建质粒。酶切体系如下：
表7酶切体系
这里选用的限制性内切酶为PstI和BamHI，BamHI具有星号活性。
将配好的酶切体系置于37℃水浴锅中进行酶切1h，酶切好的质粒和片段进行纯化回收，进行连接反应。
表8连接体系如下：
将配好的连接体系置于22℃下连接1h。构建好的质粒转化到大肠杆菌中，转化方法如下：
大肠杆菌感受态细胞的化学转化法(热激法)
7.从-80℃冰箱中取出Trans5α化学感受态细胞成品，在冰盒中放置10min解冻，无菌环境下吸取50μL菌液至灭菌的1.5mLPE离心管中；
8.向离心管中加入5μL重组体系溶液，温和混匀，在冰盒中放置30min；
9.从冰盒中取出离心管，在42℃恒温水浴中热激30sec，取出并立刻在冰盒中放置2min；
10.向离心管中加入500μL灭菌的LB液体培养基，放入设定条件为37℃，200rpm的摇床中复苏1h；
11.在4000rmp条件下离心3min，吸去上清液350μL，收集菌体并重悬；
12.在无菌条件下，将菌液涂布在含有抗生素Amp的LB平板培养基上，在37℃培养箱中倒置过夜培养。
7.挑取转化子菌落PCR验证，得到正确目的片段的为正确转化子，提取质粒BamHI和PstI酶切验证正确的质粒为正确质粒，即pRS405-Erg20构建成功。
二、酿酒酵母转化
将含有步骤一获得的基因Erg20的表达盒导入重组菌2中，得到重组菌3。
采取与实施例2中步骤二相同的方法，进行重组菌感受态细胞的制备及转化，转入pRS405-Erg20，在缺失亮氨酸的培养基中筛选培养，得到转化子。筛选培养条件为30℃，培养48h以上，挑取转化子过夜培养，提基因组，并用check引物验证，得到正确目的片段的为正确克隆，命名为重组菌3。LEU2的核苷酸序列如SEQ ID NO.59所示。
表9引物序列
实施例5、酿酒酵母重组菌株4的构建
将重组菌3中野生型鲨烯合酶启动子替换成Met3启动子，得到重组菌4，所述Met3启动子的核苷酸序列如SEQ ID NO.6所示。
一、模块构建
Erg9-up(SEQ ID NO.60)，启动子Met3(SEQ ID NO.6)，Erg9-down(SEQ ID NO.61)均来自酿酒酵母ATCC208352基因组，筛选标记基因Ade2(SEQ ID NO.62)序列来自酿酒酵母ATCC 208352。基因由武汉金开瑞生物工程有限公司通过化学合成的方法合成。
以酿酒酵母ATCC208352基因组为模板，以9up-F(SEQ ID NO.63)，9up-R-Ade2(SEQID NO.64)为引物，扩增Erg9-up；
以9up-Ade2-F(SEQ ID NO.65)，Ade2-R-Met3p(SEQ ID NO.66)为引物扩增筛选标记基因Ade2；
以酿酒酵母ATCC208352基因组为模板，以Ade2-Met3p-F(SEQ ID NO.67)，Met3p-R-9down(SEQ ID NO.68)为引物扩增启动子Met3；
以酿酒酵母ATCC208352基因组为模板，以Met3p-9down-F(SEQ ID NO.69)，9down-R(SEQ ID NO.70)为引物扩增Erg9-down
上述引物如表10所示。
本发明所用PCR酶为南京诺唯赞生物科技有限公司的
Max Super-Fidelity聚合酶。50μL的PCR扩增体系如下：DNA模板，1μL；前引(10μM)和后引(10μM)各2μL；dNTP(10mM)，1μL；2×PhantaMax Buffer，25μL；
Max Super-Fidelity聚合酶，1μL；最后用双蒸水补齐50μL。在PCR仪上设置扩增程序。扩增条件为95℃预变性4min(1个循环)；95℃变性15sec、退火60℃15sec、72℃延伸1min(34个循环)；72℃延伸5min(1个循环)。
本发明所用融合PCR体系如下：DNA片段总量800ng，摩尔比1:1；dNTP(10mM)，1μL；2×Phanta Max Buffer，25μL；
Max Super-Fidelity聚合酶，1μL；最后用双蒸水补齐50μL。在PCR仪上设置扩增程序。扩增条件为95℃预变性4min(1个循环)；95℃变性15sec、60℃退火30sec、72℃延伸1min(11个循环)，72℃延伸5min(1个循环)。
最后得到转化酿酒酵母所用的DNA片段，分别连有上游同源臂Erg9-up的标记基因Ade2表达盒Erg9-up-Ade2、和含有启动子Met3的下游同源臂PMet3-Erg9-down。
二、酿酒酵母转化
将含有步骤一获得的PMet3基因的表达盒导入重组菌3中，得到重组菌4。转化的DNA片段包括Erg9-up-Ade2、PMet3-Erg9-down，每种片段大于300ng。
采取与实施例2中步骤二相同的方法，进行重组菌感受态细胞的制备及转化，转入Erg9-up-Ade2、PMet3-Erg9-down，在缺失腺嘌呤的培养基中筛选培养，得到转化子。筛选培养条件为30℃，培养48h以上，挑取转化子过夜培养，提基因组，并用check引物验证，得到正确目的片段的为正确克隆，命名为重组菌4。ADE2的核苷酸序列如SEQ ID NO.62所示。
表10引物序列
实施例6、重组菌生产左旋海松二烯和左旋海松酸
1、重组菌培养及产物提取
首先将实施例2、3、4、5中得到的重组菌1，重组菌2，重组菌3、重组菌4，连同ATCC208352，在固体筛选培养基上活化；然后将各个重组菌接种到试管YPD液体培养基中，过夜培养，待OD600长到约为4.0，接种到含30mLYPD培养基的摇瓶中，使初始OD600为0.05，由于左旋海松二烯是左旋海松酸的前体物质，为了防止双相发酵过程中，前体左旋海松二烯被吸收到正十二烷相，影响左旋海松酸的生成，所以发酵过程中不添加正十二烷。Erg9弱化的重组菌需加入30g/L无菌Met溶液300μL，30℃，220rpm发酵5d后，测左旋海松二烯和左旋海松酸产量。发酵后的酿酒酵母菌，通过12000rpm离心，将发酵液与菌体分离开。发酵液中的左旋海松二烯通过加入正己烷进行萃取，菌体中的左旋海松二烯通过加入石英砂和正己烷，在振荡器中振荡30min，进行酵母破壁提取，通过离心，收集上清正己烷进行检测；发酵液中的左旋海松酸通过加入乙酸乙酯进行萃取，菌体中的左旋海松酸通过加入石英砂和乙酸乙酯，在振荡器中振荡30min，进行酵母破壁提取，通过离心，收集上清乙酸乙酯进行检测。样品需要将上清过0.22mm有机膜后备用。
2、GC-MS鉴定左旋海松二烯、LC-MS鉴定左旋海松酸
(1)GC-MS鉴定左旋海松二烯
左旋海松二烯的气相色谱检测条件：色谱柱Rxi-1HT，氮气流速1mL/min，进样温度250℃，进样分流比1:5，进样量1μL；炉温：150℃保持5min，5℃/min升高至250℃，250℃保持5min；FID检测器：260℃。左旋海松二烯的GC-MS检测条件：色谱柱HP-5ms，氦气流速1mL/min，进样温度250℃，分流比1:5，进样量1μL；炉温：150℃保持5min，5℃/min升高至250℃，250℃保持5min；离子源温度230℃，离子扫描范围50-600m/z。左旋海松二烯的保留时间为16.37min。
(2)LC-MS鉴定左旋海松酸
左旋海松酸液相检测方法：流动相，甲醇：水：甲酸＝87：13：0.02；流速1mL/min；色谱柱：依利特C18(4.6mm×250mm)；检测波长275nm，进样量20μL；左旋海松酸LC-MS检测方法：流动相，甲醇：水：甲酸＝87：13：0.02；流速0.2mL/min；色谱柱：C18；离子源：ESI，正离子模式；扫描范围：50-1800m/z。左旋海松酸保留时间为25.8min。
3、检测结果
A.酿酒酵母W303-1aATCC208352没有检测到左旋海松二烯和左旋海松酸；
B.重组菌1：提取重组菌1的发酵产物，可以检测到微量的左旋海松二烯和左旋海松酸，结果为左旋海松二烯含量为2.2mg/L，左旋海松酸含量为4.0mg/L；
C.重组菌2：提取重组菌2发酵产物，可以检测到左旋海松二烯的含量为5.7mg/L,左旋海松酸含量为9.4mg/L；
D.重组菌3：提取重组菌3发酵产物，可以检测到左旋海松二烯的含量为10.3mg/L,左旋海松酸含量为20.7mg/L；
E.重组菌4：提取重组菌4发酵产物，可以检测到左旋海松二烯的含量为16.2mg/L,左旋海松酸含量为46.5mg/L；
将上述重组菌的结果统计见表11。
表11重组菌株生产左旋海松二烯及左旋海松酸能力
实施例7、重组菌4的5L罐发酵
首先将重组菌4的单菌落接种到200mLYPD培养基中，30℃的摇床，220rpm转速下培养24h，制备种子液。发酵罐初始装入体积为2L的YPD培养基，补充碳源、氮源、无机盐、生物素、微量元素等生长要素，溶氧量保持在40％，控制发酵pH在5.5，发酵6d，收集发酵后的菌液，提取发酵产物，进行气相色谱以及高效液相色谱检测，得到左旋海松二烯的产量为142mg/L，左旋海松酸的产量为408mg/L。
补充的碳源为500mL的600g/L葡萄糖溶液；
补充的氮源为250mL的300g/L的谷氨酸钠溶液；
微量元素母液的配方为：ZnSO4·7H2O 10.2g/L，EDTANa2·2H2O 15g/L，FeSO4·7H2O 5.12g/L，无水CuSO40.5g/L，MnCl2·4H2O 0.5g/L，CoCl2·6H2O 0.86g/L，CaCl2·2H2O3.84g/L，Na2MoO4·2H2O 0.56g/L。
维生素母液的配方为：肌醇25g/L，生物素0.05g/L，烟碱酸1g/L，泛酸钙1g/L，thiamine HCl 1g/L，pyridoxol HCl 1g/L，对氨基苯甲酸0.2g/L。
无机盐配方：KH2PO49g/L，K2SO43.5g/L，Na2SO40.28g/L，MgSO4·7H2O 0.5g/L。
在灭菌的无机盐溶液中，每1L加入10mL的微量元素母液，及12mL的维生素溶液母液，配制成无机盐/微量元素/维生素混合溶液。流加补料过程中补充500mL的无机盐/微量元素/维生素混合溶液。
上述发酵采用的培养基为YPD培养基：20g/L蛋白胨，10g/L酵母浸粉，20g/L葡萄糖，固体培养基需另外添加2％琼脂粉。
本发明证明发酵过程中溶氧量对于P450对底物的彻底氧化有重要作用，只有保证足够溶氧量，P450才能更好地发挥催化氧化作用。
序列表
<110> 天津大学
<120> 产左旋海松二烯和左旋海松酸的重组酿酒酵母及构建方法
<160> 70
<170> SIPOSequenceListing 1.0
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atgttgaatg cagattatca tccagctgtt tggaaggatg atttcatcga ttctttgact 60
tcaccaaatt ctcatgctac atctaaatct tcagttgatg aaactattaa taagagaata 120
caaacattgg ttaaagaaat tcaatgtatg tttcaatcaa tgggtgacgg tgaaactaat 180
ccatctgcat acgatacagc ttgggttgca agaataccat caattgatgg ttctggtgct 240
ccacaattcc cacaaacttt gcaatggatt ttgaacaacc aattgccaga tggttcttgg 300
ggtgaagaat gtattttctt ggcatacgat agagttttga acactttggc ttgtttgttg 360
acattgaaga tctggaataa gggtgacatc caagttcaaa agggtgttga atttgttaga 420
aagcatatgg aagaaatgaa ggatgaagct gataaccata gaccatcagg ttttgaagtt 480
gtttttccag caatgttgga tgaagctaaa tcattgggtt tagatttgcc ataccatttg 540
ccttttattt ctcaaatcca tcaaaagaga caaaagaaat tgcaaaagat tccattgaat 600
gttttgcata accatcaaac tgctttgttg tactctttag aaggtttgca agatgttgtt 660
gattggcaag aaatcacaaa tttgcaatca agagatggtt catttttgtc ttctccagct 720
tcaactgcat gtgtttttat gcatacacaa aataagagat gtttgcattt cttgaacttc 780
gttttgtcta aatttggtga ctacgttcca tgtcattacc cattagattt gttcgaaaga 840
ttgtgggctg ttgatactgt tgaaagattg ggtatcgata gatacttcaa gaaagaaatt 900
aaagaatctt tggattatgt ttacagatat tgggatgcag aaagaggtgt tggttgggct 960
agatgtaatc caattccaga tgttgatgat acagctatgg gtttgagaat tttaagattg 1020
catggttaca acgtttcttc agatgttttg gaaaacttca gagatgaaaa gggtgacttt 1080
ttctgttttg ctggtcaaac tcaaatcggt gttacagata atttgaattt gtacagatgt 1140
tctcaagttt gttttccagg tgaaaagatt atggaagaag caaagacttt tactacaaac 1200
catttgcaaa atgctttggc taaaaataac gcattcgata agtgggctgt taagaaagat 1260
ttgccaggtg aagttgaata cgcaattaaa tatccatggc atagatcaat gccaagattg 1320
gaagctagat catacatcga acaattcggt tctaacgatg tttggttggg taaaactgtt 1380
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ggttttaatg atttgacttt tacaagacaa agaccagttg aaatctactt ttcagttgca 1560
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ttggctgtta ttttagatga tttgtatgat acacatggtt cattggatga tttgaagttg 1680
ttttctgaag cagttagaag atgggatatc tcagttttgg attctgttag agataaccaa 1740
ttgaaggttt gtttcttggg tttgtacaac acagttaacg gtttcggtaa agatggtttg 1800
aaagaacaag gtagagatgt tttaggttac ttgagaaaag tttgggaagg tttgttagca 1860
tcattcacta aagaagctga atggtctgct gcaaaatacg ttccaacttt taatgaatac 1920
gttgaaaacg ctaaagtttc aattgctttg gcaactgttg ttttaaattc tattttcttt 1980
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catttggttt ctttgactgg tagattgatt aatgatacta agacatacca agctgaaaga 2100
aaccgtggtg aattagtttc ttcagttcaa tgttacatga gagaaaaccc agaatgtaca 2160
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atggctgatc aaatttcttt gttgttggtt gttttcaccg ctgctgttgc tttgttgcat 60
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caagagttgc acttgccacc aggttctact ggttggccat tgattggtga aacttactct 180
tactacaggt ccatgacttc taatagacca agacaattca tcgacgacag agagaaaaga 240
tacgattccg atgttttcgt gtctcacttg tttggttctc aagccgttat ttcttctgac 300
ccacaattca acaaatacgt cttgcaaaac gagggtagat tcttccaagc tcattatcca 360
aaagctttga aggctttgat cggtgattat ggtttgttgt ctgttcatgg tgacttgcaa 420
agaaagttgc atggtattgc tgtcaacttg ttgagattcg aaagattgaa gttcgacttc 480
atggaagaga tccaaaactt ggtccattct actttggata gatgggtcga caaaaaagaa 540
attgccttgc agaacgaatg ccaccaaatg gttttgaatt tgatggctaa gcagttgttg 600
gacttgtccc catctaaaga aactaacgaa atttgcgagt tgttcgtcga ttacaccaat 660
gctgttattg ccattccaat caagattcca ggttccactt atgctaaagg tttgaaagcc 720
agagagttgt tgatcagaaa gatctccaac atgatcaaag agagaaggga tcatccacat 780
atcgttcaca aagatttgtt gaccaagttg ctagaagagg actctatttc cgacgaaatt 840
atctgcgatt tcatcttgtt cttgttgttc gctggtcacg aaacatcttc tagagctatg 900
actttcgcca ttaagttctt gactacttgt ccaaaggcat tgacccaaat gaaggaagaa 960
cacgacgcta tcttgaaagc aaaaggtggt cacaaaaagt tggaatggga tgactacaag 1020
tctatgaagt tcactcaatg cgttatcaac gaaaccttga gattgggtaa ttttggtcca 1080
ggtgttttca gggaaacaaa agaagatacc aaggttaagg attgcttgat tccaaaaggt 1140
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ttaactttta atccttggag atgggaattg gaccaagatg tttctaacaa ccatttgttc 1260
tctccatttg gtggtggtgc tagattgtgt cctggttcac atttggctag attagaattg 1320
gccttattct tgcacatttt catcaccaga ttcaggtggg aagctttggc tgatgaacat 1380
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atgcaagcca attccaatac agtagaaggt gcctctcaag gtaaaagttt gttagacata 60
agtagattag accacatatt cgcattattg ttaaatggta aaggtggtga cttgggtgcc 120
atgactggta gtgctttgat cttgacagaa aactctcaaa acttgatgat cttaactaca 180
gccttggctg tcttagtagc atgtgttttc tttttcgtct ggagaagagg tggtagtgac 240
acacaaaagc cagctgtcag acctacccca ttggtaaagg aagaagatga agaagaagaa 300
gatgactctg ctaaaaagaa agttactatt ttctttggta cacaaaccgg tactgctgaa 360
ggttttgcaa aggccttagc tgaagaagca aaagccagat atgaaaaggc tgttttcaaa 420
gttgttgatt tggataacta tgctgcagat gacgaacaat acgaagaaaa gttgaagaaa 480
gaaaagttgg cattctttat gttggccacc tatggtgacg gtgaacctac tgataacgcc 540
gctagatttt acaagtggtt cttagagggt aaagaaagag aaccatggtt gtctgatttg 600
acttatggtg tatttggttt gggtaataga caatacgaac atttcaacaa ggttgctaaa 660
gcagtcgatg aagtattaat cgaacaaggt gcaaaaagat tagttcctgt cggtttgggt 720
gacgacgatc aatgtattga agacgatttt acagcttgga gagaacaagt ttggccagaa 780
ttggaccaat tgttaagaga tgaagacgat gaacctacat cagcaacccc atacactgca 840
gccatcccag aatacagagt agaaatctat gattcagtag tttccgttta cgaagaaact 900
catgccttga agcaaaatgg tcaagctgtt tacgatattc atcacccttg tagatccaac 960
gtagctgtta gaagagaatt gcatacacca ttaagtgata gatcttgcat ccacttagaa 1020
tttgacatta gtgataccgg tttgatctat gaaactggtg accatgtcgg tgtacacaca 1080
gaaaattcta ttgaaaccgt cgaagaagct gcaaagttgt taggttacca attggatacc 1140
atattttctg ttcatggtga caaagaagat ggtactcctt taggtggttc ttcattgcca 1200
cctccattcc ctggtccatg tacattgaga accgccttag ctagatatgc tgatttgtta 1260
aaccctccaa gaaaggccgc atttttggct ttagcagccc acgcatctga ccctgcagaa 1320
gccgaaagat tgaagttctt atccagtcca gccggtaaag atgaatactc acaatgggtt 1380
actgcttcac aaagatcctt gttagaaatt atggccgaat ttccatccgc taaacctcca 1440
ttaggtgttt tctttgctgc aattgctcct agattgcaac caagatatta cagtatatct 1500
tcatccccta gattcgctcc atctagaatc catgtaactt gcgcattagt ttatggtcct 1560
tcaccaacag gtagaattca caagggtgtt tgttccaatt ggatgaaaaa cagtttgcct 1620
tctgaagaaa ctcatgactg ctcatgggca ccagttttcg tcagacaatc aaacttcaag 1680
ttacctgccg attccaccac tccaatagta atggttggtc ctggtacagg tttcgcacct 1740
tttagaggtt tcttacaaga aagagcaaag ttgcaagaag ccggtgaaaa attaggtcca 1800
gctgttttgt tctttggttg tagaaacaga caaatggact acatctatga agatgaattg 1860
aagggttacg tcgaaaaggg tattttgaca aacttaatcg tagctttttc aagagaaggt 1920
gcaaccaagg aatacgttca acataagatg ttggaaaagg catcagatac atggtcctta 1980
atagcccaag gtggttattt gtacgtttgc ggtgacgcta agggtatggc aagagatgtc 2040
catagaactt tgcacacaat cgttcaagaa caagaatctg tcgatagttc taaggctgaa 2100
ttcttggtta agaaattgca aatggacggt agatatttga gagatatttg gtaa 2154
<210> 4
<211> 1512
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 4
atgccagttt taaccaataa aacagtcatt tctggatcga aagtcaaaag tttatcatct 60
gcgcaatcga gctcatcagg accttcatca tctagtgagg aagatgattc ccgcgatatt 120
gaaagcttgg ataagaaaat acgtccttta gaagaattag aagcattatt aagtagtgga 180
aatacaaaac aattgaagaa caaagaggtc gctgccttgg ttattcacgg taagttacct 240
ttgtacgctt tggagaaaaa attaggtgat actacgagag cggttgcggt acgtaggaag 300
gctctttcaa ttttggcaga agctcctgta ttagcatctg atcgtttacc atataaaaat 360
tatgactacg accgcgtatt tggcgcttgt tgtgaaaatg ttataggtta catgcctttg 420
cccgttggtg ttataggccc cttggttatc gatggtacat cttatcatat accaatggca 480
actacagagg gttgtttggt agcttctgcc atgcgtggct gtaaggcaat caatgctggc 540
ggtggtgcaa caactgtttt aactaaggat ggtatgacaa gaggcccagt agtccgtttc 600
ccaactttga aaagatctgg tgcctgtaag atatggttag actcagaaga gggacaaaac 660
gcaattaaaa aagcttttaa ctctacatca agatttgcac gtctgcaaca tattcaaact 720
tgtctagcag gagatttact cttcatgaga tttagaacaa ctactggtga cgcaatgggt 780
atgaatatga tttctaaagg tgtcgaatac tcattaaagc aaatggtaga agagtatggc 840
tgggaagata tggaggttgt ctccgtttct ggtaactact gtaccgacaa aaaaccagct 900
gccatcaact ggatcgaagg tcgtggtaag agtgtcgtcg cagaagctac tattcctggt 960
gatgttgtca gaaaagtgtt aaaaagtgat gtttccgcat tggttgagtt gaacattgct 1020
aagaatttgg ttggatctgc aatggctggg tctgttggtg gatttaacgc acatgcagct 1080
aatttagtga cagctgtttt cttggcatta ggacaagatc ctgcacaaaa tgttgaaagt 1140
tccaactgta taacattgat gaaagaagtg gacggtgatt tgagaatttc cgtatccatg 1200
ccatccatcg aagtaggtac catcggtggt ggtactgttc tagaaccaca aggtgccatg 1260
ttggacttat taggtgtaag aggcccgcat gctaccgctc ctggtaccaa cgcacgtcaa 1320
ttagcaagaa tagttgcctg tgccgtcttg gcaggtgaat tatccttatg tgctgcccta 1380
gcagccggcc atttggttca aagtcatatg acccacaaca ggaaacctgc tgaaccaaca 1440
aaacctaaca atttggacgc cactgatata aatcgtttga aagatgggtc cgtcacctgc 1500
attaaatcct aa 1512
<210> 5
<211> 1059
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 5
atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60
gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120
gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180
gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240
aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttacttctt ggtcgccgat 300
gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360
gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420
aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480
accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540
gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600
tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660
gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720
gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780
gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840
aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900
attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960
gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020
actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059
<210> 6
<211> 534
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 6
tggtataagg tgagggggtc cacagatata acatcgttta atttagtact aacagagact 60
tttgtcacaa ctacatataa gtgtacaaat atagtacaga tatgacacac ttgtagcgcc 120
aacgcgcatc ctacggattg ctgacagaaa aaaaggtcac gtgaccagaa aagtcacgtg 180
taattttgta actcaccgca ttctagcggt ccctgtcgtg cacactgcac tcaacaccat 240
aaaccttagc aacctccaaa ggaaatcacc gtataacaaa gccacagttt tacaacttag 300
tctcttatga agttacttac caatgagaaa tagaggctct ttctcgagaa atatgaatat 360
ggatatatat atatatatat atatatatat atatatatat gtaaacttgg ttctttttta 420
gcttgtgatc tctagcttgg gtctctctct gtcgtaacag ttgtgatatc gtttcttaac 480
aattgaaaag gaactaagaa agtataataa taacaagaat aaagtataat taac 534
<210> 7
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
taatctaagt tttaattaca aaatgttgaa tgcagattat catccag 47
<210> 8
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
actgcaactg aaaagtagat ttcaactggt ctttgtc 37
<210> 9
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gacaaagacc agttgaaatc tacttttcag ttgcagt 37
<210> 10
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccattcagct tctttagtga atgatgctaa caaaccttc 39
<210> 11
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaaggtttgt tagcatcatt cactaaagaa gctgaatgg 39
<210> 12
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gtaaagacat aagagatccg cttatgcaac tggatcgaac aa 42
<210> 13
<211> 548
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 13
atgagagtag caaacgtaag tctaaaggtt gttttatagt agttaggatg tagaaaatgt 60
attccgatag gccattttac atttggaggg acggttgaaa gtggacagag gaaaaggtgc 120
ggaaatggct gattttgatt gtttatgttt tgtgtgatga ttttacattt ttgcatagta 180
ttaggtagtc agatgaaaga tgaatagaca taggagtaag aaaacataga atagttaccg 240
ttattggtag gagtgtggtg gggtggtata gtccgcattg ggatgttact ttcctgttat 300
ggcatggatt tccctttagg gtctctgaag cgtatttccg tcaccgaaaa aggcagaaaa 360
agggaaactg aagggaggat agtagtaaag tttgaatggt ggtagtgtaa tgtatgatat 420
ccgttggttt tggtttcggt tgtgaaaagt tttttggtat gatattttgc aagtagcata 480
tatttcttgt gtgagaaagg tatattttgt atgttttgta tgttcccgcg cgtttccgta 540
ttttccgc 548
<210> 14
<211> 430
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 14
agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60
tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120
ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180
aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240
tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300
tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360
ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420
taattacaaa 430
<210> 15
<211> 450
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 15
gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60
tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120
ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180
attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240
acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300
tttccttcgc atgccagcat tgaaatgatc gaagttcaat gatgaaacgg taattcttct 360
gtcatttact catctcatct catcaagtta tataattcta tacggatgta atttttcact 420
tttcgtcttg acgtccaccc tataatttca 450
<210> 16
<211> 778
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 16
tattttagat tcctgacttc aactcaagac gcacagatat tataacatct gcataatagg 60
catttgcaag aattactcgt gagtaaggaa agagtgagga actatcgcat acctgcattt 120
aaagatgccg atttgggcgc gaatccttta ttttggcttc accctcatac tattatcagg 180
gccagaaaaa ggaagtgttt ccctccttct tgaattgatg ttaccctcat aaagcacgtg 240
gcctcttatc gagaaagaaa ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact 300
gaaaaaaccc agacacgctc gacttcctgt cttcctattg attgcagctt ccaatttcgt 360
cacacaacaa ggtcctagcg acggctcaca ggttttgtaa caagcaatcg aaggttctgg 420
aatggcggga aagggtttag taccacatgc tatgatgccc actgtgatct ccagagcaaa 480
gttcgttcga tcgtactgtt actctctctc tttcaaacag aattgtccga atcgtgtgac 540
aacaacagcc tgttctcaca cactcttttc ttctaaccaa gggggtggtt tagtttagta 600
gaacctcgtg aaacttacat ttacatatat ataaacttgc ataaattggt caatgcaaga 660
aatacatatt tggtcttttc taattcgtag tttttcaagt tcttagatgc tttctttttc 720
tcttttttac agatcatcaa ggaagtaatt atctactttt tacaacaaat ataaaaca 778
<210> 17
<211> 437
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 17
gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60
tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120
ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180
tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata 240
tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttatgtcct cagaggacaa 300
cacctgttgt aatcgttctt ccacacggat ccacagccta gccttcagtt gggctctatc 360
ttcatcgtca ttcattgcat ctactagccc cttacctgag cttcaagacg ttatatcgct 420
tttatgtatc atgatct 437
<210> 18
<211> 800
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 18
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 800
<210> 19
<211> 450
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 19
gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60
tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120
ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180
attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240
acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300
tttccttcgc atgccagcat tgaaatgatc gaagttcaat gatgaaacgg taattcttct 360
gtcatttact catctcatct catcaagtta tataattcta tacggatgta atttttcact 420
tttcgtcttg acgtccaccc tataatttca 450
<210> 20
<211> 521
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 20
gggggtttag tcatggagta caagtgtgag gaaaagtagt tgggaggtac ttcatgcgaa 60
agcagttgaa gacaagttcg aaaagagttt ggaaacgaat tcgagtaggc ttgtcgttcg 120
ttatgttttt gtaaatggcc tcgtcaaacg gtggagagag tcgctaggtg atcgtcagat 180
ctgcctagtc tctatacagc gtgtttaatt gacatgggtt gatgcgtatt gagagataca 240
atttgggaag aaattcccag agtgtgtttc ttttgcgttt aacctgaaca gtctcatcgt 300
gggcatcttg cgattccatt ggtgagcagc gaaggatttg gtggattact agctaatagc 360
aatctatttc aaagaattca aacttggggg aatgccttgt tgaatagccg gtcgcaagac 420
tgtgattctt caagtgtaac ctcctctcaa atcagcgata tcaaacgtac cattccgtga 480
aacaccgggg tatctgtttg gtggaacctg attagaggaa a 521
<210> 21
<211> 1117
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tcagggtcca taaagctttt caattcatct tttttttttt tgttcttttt tttgattccg 60
gtttctttga aatttttttg attcggtaat ctccgagcag aaggaagaac gaaggaagga 120
gcacagactt agattggtat atatacgcat atgtggtgtt gaagaaacat gaaattgccc 180
agtattctta acccaactgc acagaacaaa aacctgcagg aaacgaagat aaatcatgtc 240
gaaagctaca tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt 300
taatatcatg cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa 360
ggaattactg gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt 420
ggatatcttg actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc 480
caagtacaat tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa 540
attgcagtac tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca 600
cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag gcggcggaag aagtaacaaa 660
ggaacctaga ggccttttga tgttagcaga attgtcatgc aagggctccc tagctactgg 720
agaatatact aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt 780
tattgctcaa agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc 840
cggtgtgggt ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga 900
tgtggtctct acaggatctg acattattat tgttggaaga ggactatttg caaagggaag 960
ggatgctaag gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag 1020
atgcggccag caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca 1080
aattagagct tcaatttaat tatatcagtt attaccc 1117
<210> 22
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccggggcacc tgtcactttg gaaaaaaaat atacgctaag a 41
<210> 23
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ctatggtgtg tgggggatca cttttcctct aatcaggttc cacc 44
<210> 24
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggtggaacct gattagagga aaagtgatcc cccacacacc atag 44
<210> 25
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gcgaacaaaa cacctgccat tttgtaatta aaacttagat 40
<210> 26
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atctaagttt taattacaaa atggcaggtg ttttgttcgc 40
<210> 27
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gtaaagacat aagagatccg cttatgcaac tggatcgaac aa 42
<210> 28
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ttgttcgatc cagttgcata agcggatctc ttatgtcttt ac 42
<210> 29
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
agttgaagtc aggaatctaa aatatgaaat tatagggtgg acgtc 45
<210> 30
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gacgtccacc ctataatttc atattttaga ttcctgactt caact 45
<210> 31
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
acaaagaaat ttgatcagcc attgttttat atttgttgta aaaag 45
<210> 32
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ctttttacaa caaatataaa acaatggctg atcaaatttc tttgt 45
<210> 33
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atcataaatc ataagaaatt cgcttactct ctattgtaca acctc 45
<210> 34
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gaggttgtac aatagagagt aagcgaattt cttatgattt atgat 45
<210> 35
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gctaacattc aacgctagta tagatcatga tacataaaag cg 42
<210> 36
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
tcgcttttat gtatcatgat ctatactagc gttgaatgtt agc 43
<210> 37
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ctgtattgga attggcttgc attttgtttg tttatgtgtg ttta 44
<210> 38
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
taaacacaca taaacaaaca aaatgcaagc caattccaat acag 44
<210> 39
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
aagtaactta aggagttaaa tttaccaaat atctctcaaa ta 42
<210> 40
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tatttgagag atatttggta aatttaactc cttaagttac tt 42
<210> 41
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gaattgaaaa gctgtggtat ggtggcgaaa agccaattag tgtgatac 48
<210> 42
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
gtatcacact aattggcttt tcgccaccat accacagctt ttcaattc 48
<210> 43
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gacttacgtt tgctactctc atgggtaata actgatataa ttaa 44
<210> 44
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ttaattatat cagttattac ccatgagagt agcaaacgta agtc 44
<210> 45
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gcggaaaata cggaaacgcg cgggaacata caa 33
<210> 46
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
aagggcccat actagcgttg aatgttagcg 30
<210> 47
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ctgttttatt ggttaaaact ggcattttgt ttgtttatgt gtgtttattc 50
<210> 48
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
gaataaacac acataaacaa acaaaatgcc agttttaacc aataaaacag 50
<210> 49
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
cgacaaagga aaaggggcct gtttaggatt taatgcaggt gacgg 45
<210> 50
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
ccgtcacctg cattaaatcc taaacaggcc ccttttcctt tgtcg 45
<210> 51
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
tgcctgcaga agcagacgct actaaggaaa ac 32
<210> 52
<211> 1003
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc gtaatatatg 60
tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta ttgaaaaata 120
gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg attaagaatt 180
aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg actattgagc 240
acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt tattaatttc 300
acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga ggccgcagaa 360
tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa tagaaagaga 420
acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata taaaaatagt 480
tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga ggatgttttg 540
gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga gtcgtggcaa 600
gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc aaaagactgc 660
aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt gtttgattca 720
gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg ggttggaagg 780
caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc agaaaatgtt 840
ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg tgtggagaca 900
aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa gaaataggtt 960
attactgagt agtatttatt taagtattgt ttgtgcactt gcc 1003
<210> 53
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
taactgcagt attttagatt cctgacttca actc 34
<210> 54
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
ctaatttctt tttctgaagc cattgtttta tatttgttgt aaaaagtag 49
<210> 55
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ctacttttta caacaaatat aaaacaatgg cttcagaaaa agaaattag 49
<210> 56
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
cgacaaagga aaaggggcct gtctatttgc ttctcttgta aactttg 47
<210> 57
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
caaagtttac aagagaagca aatagacagg ccccttttcc tttgtcg 47
<210> 58
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
cgggatccaa gcagacgcta ctaaggaaaa c 31
<210> 59
<211> 3379
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
cggccaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 60
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 120
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 180
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 240
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 300
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 360
actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 420
catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 480
ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 540
aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agcttgcatg 600
cctgcaggtc gactctagag gatccccggg ataacttcgt atagcataca ttatacgaag 660
ttatcgtttt aagagcttgg tgagcgctag gaacctaata ttattgcctt attaaaaatg 720
gaatcccaac aattacatca aaatccacat tctcttcaaa atcaattgtc ctgtacttcc 780
ttgttcatgt gtgttcaaaa acgttatatt tataggataa ttatactcta tttctcaaca 840
agtaattggt tgtttggccg agcggtctaa ggcgcctgat tcaagaaata tcttgaccgc 900
agttaactgt gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc 960
attatttttt tcctcaacat aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt 1020
cgatgactgg aaattttttg ttaatttcag aggtcgcctg acgcatatac ctttttcaac 1080
tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc atatagaata 1140
gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa tattatttaa 1200
ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa cttttcttac 1260
cttttacatt tcagcaatat atatatatat atttcaagga tataccattc taatgtctgc 1320
ccctaagaag atcgtcgttt tgccaggtga ccacgttggt caagaaatca cagccgaagc 1380
cattaaggtt cttaaagcta tttctgatgt tcgttccaat gtcaagttcg atttcgaaaa 1440
tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtt ccacttccag atgaggcgct 1500
ggaagcctcc aagaaggctg atgccgtttt gttaggtgct gtgggtggtc ctaaatgggg 1560
taccggtagt gttagacctg aacaaggttt actaaaaatc cgtaaagaac ttcaattgta 1620
cgccaactta agaccatgta actttgcatc cgactctctt ttagacttat ctccaatcaa 1680
gccacaattt gctaaaggta ctgacttcgt tgttgtcaga gaattagtgg gaggtattta 1740
ctttggtaag agaaaggaag acgatggtga tggtgtcgct tgggatagtg aacaatacac 1800
cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc atggccctac aacatgagcc 1860
accattgcct atttggtcct tggataaagc taatgttttg gcctcttcaa gattatggag 1920
aaaaactgtg gaggaaacca tcaagaacga attccctaca ttgaaggttc aacatcaatt 1980
gattgattct gccgccatga tcctagttaa gaacccaacc cacctaaatg gtattataat 2040
caccagcaac atgtttggtg atatcatctc cgatgaagcc tccgttatcc caggttcctt 2100
gggtttgttg ccatctgcgt ccttggcctc tttgccagac aagaacaccg catttggttt 2160
gtacgaacca tgccacggtt ctgctccaga tttgccaaag aataaggtca accctatcgc 2220
cactatcttg tctgctgcaa tgatgttgaa attgtcattg aacttgcctg aagaaggtaa 2280
ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt atcagaactg gtgatttagg 2340
tggttccaac agtaccaccg aagtcggtga tgctgtcgcc gaagaagtta agaaaatcct 2400
tgcttaaaaa gattctcttt ttttatgata tttgtacata aactttataa atgaaattca 2460
taatagaaac gacacgaaat tacaaaatgg aatatgttca tagggtagac gaaactatat 2520
acgcaatcta catacattta tcaagaagga gaaaaaggag gatgtaaagg aatacaggta 2580
agcaaattga tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat 2640
attgacaagg aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactat 2700
gattcctaat ttatatattg gaggattttc tctaaaaaaa aaaaaataca acaaataaaa 2760
aacactcaat gacctgacca tttgatggag tttaagtcaa taccttcttg aactcttgaa 2820
ctcgaataac ttcgtatagc atacattata cgaagttatc ccgggtaccg agctcgaatt 2880
catcacgtgc tataaaaata attataattt aaatttttta atataaatat ataaattaaa 2940
aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa 3000
gactctaggg ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta 3060
ttaatgccga attgtttcat cttgtctgtg tagaagacca cacacgaaaa tcctgtgatt 3120
ttacatttta cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat 3180
aaatatatat gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt 3240
tcttcattcc gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca 3300
taaaaataaa taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc 3360
gcgtgtaagt tacgaattc 3379
<210> 60
<211> 141
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 60
tcagtacatt tcatagccca tcttcaacaa caataccgac ttaccatcct atttgctttg 60
ccctttttct tttccactgc actttgcatc ggaaggcgtt atcggttttg ggtttagtgc 120
ctaaacgagc agcgagaaca c 141
<210> 61
<211> 119
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 61
gaccacgggc tatataaatg gaaagttagg acaggggcaa agaataagag cacagaagaa 60
gagaaaagac gaagagcaga agcggaaaac gtatacacgt cacatatcac acacacaca 119
<210> 62
<211> 3926
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
cggccaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 60
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 120
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 180
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 240
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 300
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 360
actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 420
catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 480
ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 540
aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agcttgcatg 600
cctgcaggtc gactctagag gatccccggg ataacttcgt atagcataca ttatacgaag 660
ttatcgtttt aagagcttgg tgagcgctag gaattaacgt attacataag ttacaggatt 720
catgcttatg ggttagctat ttcgcccaat gtgtccatct gacattacta ttttgcattt 780
taatttaatt agaacttgac tagcgcacta ccagtatatc atctcatttc cgtaaatacc 840
aaatgtatta tatattgaaa gcttttgacc aggttattat aaaagaaact tcatgctcga 900
aaaagatcat ttcgaaaagt tgcctagttt catgaaattt taaagcagtt tatataaatt 960
ttaccttttg atgcggaatt gactttttct tgaataatac ataacttttc ttaaaagaat 1020
caaagacaga taaaatttaa gagatattaa atattagtga gaagccgaga attttgtaac 1080
accaacataa cactgacatc tttaacaact tttaattatg atacatttct tacgtcatga 1140
ttgattatta cagctatgct gacaaatgac tcttgttgca tggctacgaa ccgggtaata 1200
ctaagtgatt gactcttgct gaccttttat taagaactaa atggacaata ttatggagca 1260
tttcatgtat aaattggtgc gtaaaatcgt tggatctctc ttctaagtac atcctactat 1320
aacaatcaag aaaaacaaga aaatcggaca aaacaatcaa gtatggattc tagaacagtt 1380
ggtatattag gagggggaca attgggacgt atgattgttg aggcagcaaa caggctcaac 1440
attaagacgg taatactaga tgctgaaaat tctcctgcca aacaaataag caactccaat 1500
gaccacgtta atggctcctt ttccaatcct cttgatatcg aaaaactagc tgaaaaatgt 1560
gatgtgctaa cgattgagat tgagcatgtt gatgttccta cactaaagaa tcttcaagta 1620
aaacatccca aattaaaaat ttacccttct ccagaaacaa tcagattgat acaagacaaa 1680
tatattcaaa aagagcattt aatcaaaaat ggtatagcag ttacccaaag tgttcctgtg 1740
gaacaagcca gtgagacgtc cctattgaat gttggaagag atttgggttt tccattcgtc 1800
ttgaagtcga ggactttggc atacgatgga agaggtaact tcgttgtaaa gaataaggaa 1860
atgattccgg aagctttgga agtactgaag gatcgtcctt tgtacgccga aaaatgggca 1920
ccatttacta aagaattagc agtcatgatt gtgagatctg ttaacggttt agtgttttct 1980
tacccaattg tagagactat ccacaaggac aatatttgtg acttatgtta tgcgcctgct 2040
agagttccgg actccgttca acttaaggcg aagttgttgg cagaaaatgc aatcaaatct 2100
tttcccggtt gtggtatatt tggtgtggaa atgttctatt tagaaacagg ggaattgctt 2160
attaacgaaa ttgccccaag gcctcacaac tctggacatt ataccattga tgcttgcgtc 2220
acttctcaat ttgaagctca tttgagatca atattggatt tgccaatgcc aaagaatttc 2280
acatctttct ccaccattac aacgaacgcc attatgctaa atgttcttgg agacaaacat 2340
acaaaagata aagagctaga aacttgcgaa agagcattgg cgactccagg ttcctcagtg 2400
tacttatatg gaaaagagtc tagacctaac agaaaagtag gtcacataaa tattattgcc 2460
tccagtatgg cggaatgtga acaaaggctg aactacatta caggtagaac tgatattcca 2520
atcaaaatct ctgtcgctca aaagttggac ttggaagcaa tggtcaaacc attggttgga 2580
atcatcatgg gatcagactc tgacttgccg gtaatgtctg ccgcatgtgc ggttttaaaa 2640
gattttggcg ttccatttga agtgacaata gtctctgctc atagaactcc acataggatg 2700
tcagcatatg ctatttccgc aagcaagcgt ggaattaaaa caattatcgc tggagctggt 2760
ggggctgctc acttgccagg tatggtggct gcaatgacac cacttcctgt catcggtgtg 2820
cccgtaaaag gttcttgtct agatggagta gattctttac attcaattgt gcaaatgcct 2880
agaggtgttc cagtagctac cgtcgctatt aataatagta cgaacgctgc gctgttggct 2940
gtcagactgc ttggcgctta tgattcaagt tatacaacga aaatggaaca gtttttatta 3000
aagcaagaag aagaagttct tgtcaaagca caaaagttag aaactgtcgg ttacgaagct 3060
tatctagaaa acaagtaata tataagttta ttgatatact tgtacagcaa ataattataa 3120
aatgatatac ctatttttta ggctttgtta tgattacatc aaatgtggac ttcatacata 3180
gaaatcaacg cttacaggtg tcctttttta agaatttcat acataagatc acttattata 3240
catacataca tatccagtaa caagaagcaa ggaataatta cctgcttaag tctgcgatta 3300
aaaaaataac gtttcgatac agttcatata aggcggctca atgcagaacc gaggatagcc 3360
tcttgaactc gaataacttc gtatagcata cattatacga agttatcccg ggtaccgagc 3420
tcgaattcat cacgtgctat aaaaataatt ataatttaaa ttttttaata taaatatata 3480
aattaaaaat agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga 3540
tgtaaaagac tctaggggga tcgccaacaa atactacctt ttatcttgct cttcctgctc 3600
tcaggtatta atgccgaatt gtttcatctt gtctgtgtag aagaccacac acgaaaatcc 3660
tgtgatttta cattttactt atcgttaatc gaatgtatat ctatttaatc tgcttttctt 3720
gtctaataaa tatatatgta aagtacgctt tttgttgaaa ttttttaaac ctttgtttat 3780
ttttttttct tcattccgta actcttctac cttctttatt tactttctaa aatccaaata 3840
caaaacataa aaataaataa acacagagta aattcccaaa ttattccatc attaaaagat 3900
acgaggcgcg tgtaagttac gaattc 3926
<210> 63
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
actgtcacta cgtagctggt ttggtcg 27
<210> 64
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
catcaaaagg taaaatttat accacgcaaa gtccttgatt tc 42
<210> 65
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
gaaatcaagg actttgcgtg gtataaattt taccttttga tg 42
<210> 66
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
ggaccccctc accttatacc accaggtttg attcgtgatt tc 42
<210> 67
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
gaaatcacga atcaaacctg gtggtataag gtgagggggt cc 42
<210> 68
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
gccaattgta atagctttcc catgttaatt atactttatt cttg 44
<210> 69
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
caagaataaa gtataattaa catgggaaag ctattacaat tggc 44
<210> 70
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
acacgtcgta gtcgtggacg gtttgcaac 29
Claims (2)
1.一种产左旋海松二烯和左旋海松酸的重组酿酒酵母的构建方法，其特征是包括如下步骤：
(1)向酿酒酵母(Saccharomyces cerevisiaeW303-1a)中导入改造的左旋海松二烯合酶编码基因TΔLPS、优化的细胞色素P450酶编码基因CYP720B1和优化的细胞色素P450还原酶编码基因TcCPR，得到重组菌1；
所述改造的左旋海松二烯合酶编码基因TΔLPS是左旋海松二烯合酶编码基因LPS编码的氨基酸序列的N端截短79个氨基酸并且左旋海松二烯合酶编码基因LPS编码的氨基酸序列的第593的甲硫氨酸密码子突变为异亮氨酸密码子以及第700的酪氨酸的密码子突变为苯丙氨酸密码子；所述改造的左旋海松二烯合酶编码基因TΔLPS的核苷酸序列如SEQ IDNO.1所示；所述优化的细胞色素P450酶编码基因CYP720B1的核苷酸序列如SEQ ID NO.2所示；所述优化的细胞色素P450还原酶编码基因TcCPR的核苷酸序列如SEQ ID NO.3所示；
(2)向重组菌1中导入3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1，得到重组菌2；
所述3-羟基-3-甲基戊二酰辅酶A还原酶编码基因tHMG1的核苷酸序列如SEQ ID NO.4所示。
(3)向重组菌2中导入法呢基焦磷酸合酶编码基因Erg20，得到重组菌3；
所述法呢基焦磷酸合酶编码基因Erg20的核苷酸序列如SEQ ID NO.5所示；
(4)将重组菌3中野生型鲨烯合酶启动子替换成Met3启动子，得到重组菌4；
所述Met3启动子的核苷酸序列如SEQ ID NO.6所示。
2.权利要求1的方法制备的产左旋海松二烯和左旋海松酸的重组酿酒酵母菌。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440733A (zh) * | 2020-02-07 | 2020-07-24 | 天津大学 | 产松油醇的重组酿酒酵母及构建方法及应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020164736A1 (en) * | 2001-01-05 | 2002-11-07 | Matsuda Seiichi P.T. | Ginkgo biloba levopimaradiene synthase |
| CN107746815A (zh) * | 2017-09-11 | 2018-03-02 | 天津大学 | 生产13r‑泪柏醚的重组酿酒酵母菌及其构建方法 |
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2019
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| CN111440733A (zh) * | 2020-02-07 | 2020-07-24 | 天津大学 | 产松油醇的重组酿酒酵母及构建方法及应用 |
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Application publication date: 20200421 |