CN114606149A - 一种生产麦角甾醇的酿酒酵母工程菌株及其应用 - Google Patents
一种生产麦角甾醇的酿酒酵母工程菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种生产麦角甾醇的酿酒酵母工程菌株及其应用，属于基因工程和生物工程技术领域。本发明通过敲除酿酒酵母基因组中的非路径基因ROX1、DOS2、YJL064W、YPL062W和NEM1，使麦角甾醇积累量大幅度提升，并在此基础上对基因ERG1、ERG2、ERG3、ERG4、ERG11、CTT1加强表达，进一步提高胞内麦角甾醇的积累量，并将基因tHMG1、IDI多拷贝整合，加强前体乙酰CoA的向下转化以加强前体角鲨烯的供应，并且将POS5、ERG4、ERG2、ERG3多拷贝整合，使构建的酿酒酵母发酵72h的麦角甾醇产量可达292.60mg/L，为甾体类化合物的生物合成奠定了基础。
Description
技术领域
本发明涉及一种生产麦角甾醇的酿酒酵母工程菌株及其应用，属于基因工程及生物工程技术领域。
背景技术
麦角甾醇(ergosterol)，又称麦角固醇，系统命名为24β-甲基胆固醇-5，7烯-3β-羟基，分子式为C28H44O，分子量396.66，熔点156-158℃。麦角甾醇常温下为白色针状或片状结晶，不溶于水，易溶于有机溶剂，且在空气中可被氧化。麦角甾醇对酵母的应激适应起着至关重要的作用，其衍生物具有显著的抗肿瘤和抗HIV活性；麦角甾醇也常作为药物中间体被广泛使用，如生产可的松和孕酮等药物。此外，麦角甾醇是麦角钙化醇的维生素原，具有抗氧化、抗炎等特性。在紫外照射下，麦角甾醇可以转化为维生素D2，可以应用于软骨病、骨质疏松、手脚抽搐等病的治疗。
目前，麦角甾醇的生产方法主要有2种，包括化学合成法、微生物发酵法。化学合成法制备麦角甾醇的纯度高但生产过程能耗高，对温度要求严格，污染环境，安全性差，成本高，不符合绿色生产的理念，因此这些生产工艺不被业内所推崇。微生物发酵法是利用微生物本身的内源代谢路径来天然合成麦角甾醇，该方法具有低能耗、低成本、产量高等优点，比化学合成法更适用于大规模产业化生产。但目前微生物法生产麦角甾醇仍然存在产量低的问题。
发明内容
本发明提供了一株高产麦角甾醇的酿酒酵母工程菌，以酿酒酵母SQ11为出发菌株，进行了如下至少一种改进：
(1)敲除了酿酒酵母内源基因YPL062W、ROX1；
(2)敲除了酿酒酵母内源基因NEM1；
(3)将基因ERG1、ERG2、ERG3、ERG4、ERG11、CTT1单拷贝整合到酿酒酵母的YJL064W、DOS2位点；
(4)将tHMG1、IDI基因在Ty12位点多拷贝整合；
(5)将POS5、ERG4、ERG2、ERG3在Ty3位点多拷贝整合。
在一种实施方式中，所述酿酒酵母SQ11已公开于论文《Enhancing SqualeneProduction inSaccharomyces cerevisiae by Metabolic Engineering and RandomMutagenesis》中。
在一种实施方式中，所述基因YPL062W的核苷酸序列如SEQ ID NO.1所示；所述基因ROX1的核苷酸序列如SEQ ID NO.2所示。
在一种实施方式中，所述基因NEM1的核苷酸序列如SEQ ID NO.3所示。
在一种实施方式中，所述基因ERG1、ERG2、ERG3、ERG4、ERG11、CTT1、YJL064W、DOS2的核苷酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示。
在一种实施方式中，所述基因YJL064W、DOS2被敲除后，在DOS2位点整合基因ERG2、ERG3、ERG4和CTT1，在YJL064W位点整合基因ERG1、ERG11和ERG4。
在一种实施方式中，所述基因tHMG1的核苷酸序列如SEQ ID NO.12所示、所述基因IDI的核苷酸序列如SEQ ID NO.13所示。
在一种实施方式中，所述基因POS5的核苷酸序列如SEQ ID NO.14所示。
在一种实施方式中，所述基因tHMG1、IDI整合在酿酒酵母Ty12位点上。
在一种实施方式中，所述基因POS5、ERG4、ERG2、ERG3还整合在酿酒酵母多拷贝位点Ty3。
在一种实施方式中，用启动子PTDH1起始ERG1基因的表达。
在一种实施方式中，用启动子PGAL7起始ERG2基因的表达。
在一种实施方式中，用启动子PTDH3起始ERG3基因的表达。
在一种实施方式中，用启动子PGAL1起始ERG3基因的表达。
在一种实施方式中，用启动子PTDH1和启动子PTDH3起始不同位点上ERG4基因的表达。
在一种实施方式中，用启动子PGAL7起始ERG11基因的表达。
在一种实施方式中，启动子PGAL1/10为双向启动子，用PGAL1起始tHMG1基因的表达，用PGAL10起始IDI基因的表达。
在一种实施方式中，用PGAL10起始CTT1基因的表达。
在一种实施方式中，用启动子PPGK1起始POS5基因的表达。
在一种实施方式中，所述启动子PTDH1的核苷酸序列如SEQ ID NO.15所示；所述启动子PPGK1的核苷酸序列如SEQ ID NO.16所示；所述启动子PGAL7的核苷酸序列如SEQ IDNO.17所示；所述启动子PTDH3的核苷酸序列如SEQ ID NO.18所示；所述启动子PGAL1/10的核苷酸序列如SEQ ID NO.19所示。
本发明还提供了所述酿酒酵母工程菌在生产麦角甾醇中的应用。
在一种实施方式中，将所述酿酒酵母工程菌接种于YPD培养基中，于30℃发酵72～100h。
在一种实施方式中，所述应用是用于制备含有麦角甾醇的产品。
有益效果：
本发明利用多基因编辑技术将酿酒酵母菌株SQ11的内源基因YPL062W、ROX1进行组合敲除，并敲除了内源基因NEM1，并将基因ERG1、ERG2、ERG3、ERG4、ERG11、CTT1单拷贝整合到酿酒酵母的YJL064W、DOS2位点，并在Ty12位点将tHMG1、IDI基因进行了多拷贝整合，并将POS5、ERG4、ERG2、ERG3多拷贝整合于Ty3位点，构建获得了麦角淄醇产量提高的酿酒酵母工程菌。将构建的酿酒酵母工程菌在YPD培养基中摇瓶发酵72h的麦角甾醇产量可达292.60mg/L，相比于原始菌株提高了250mg/L。
附图说明
图1为酿酒酵母中合成麦角甾醇的代谢示意图。
图2为工程菌株在YPD培养下的麦角甾醇液相图。
图3为麦角甾醇标准品的液相图。
图4为工程菌株在YPD培养下的麦角甾醇产量图。
具体实施方式
(一)培养基
LB培养基：蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L。加入20g/L琼脂粉，以配制LB固体培养基。
YNB培养基：Yeast Nutrition Base 67.4g/L，葡萄糖20g/L，氨基酸(5g/L尿嘧啶，10g/L色氨酸，10g/L亮氨酸，10g/L组氨酸，根据需要适当缺失相应氨基酸)。
YPD培养基：蛋白胨20g/L，酵母粉10g/L，葡萄糖20g/L。
(二)酿酒酵母的感受态制备：酿酒酵母感受态制备使用Frozen-EZ YeastTransformation II化转试剂盒，30℃，与10mL YPD培养基培养酿酒酵母至中数量级(OD600＝0.8-1.0)。以下步骤室温进行。
1、3500rpm离心细胞5min，吸掉上清液；
2、加入10mL EZ1溶液清洗沉淀，重新离心沉淀细胞，吸掉上清液；
3、加入1mL EZ2溶液重悬沉淀细胞。
(三)酿酒酵母的转化：
1、取50μL感受态细胞与0.2-1μg DNA(体积少于5μl)混合；加入500μL EZ3溶液，完全混合；
2、30℃孵育45min，孵育过程中用手指轻弹或低俗涡旋2-3次让其混匀；
3、从转化混合液中取50-150μL到适当营养缺陷型平板上。
4、30℃平板孵育3-5天长出转化子。
(四)麦角甾醇测定：使用高效液相色谱进行测定。条件：色谱柱：InertSustainC18 250mm×4.6mm column(particle size 5μm)；流动相B，含有1‰三氟乙酸的甲醇；流速：1mL/min；柱温：30℃；进样量：10μL；检测器波长：281nm。
(五)菌株信息如表1所示。
表1本发明中涉及的菌株
表2引物序列
实施例1抑制因子YPL062W、ROX1的敲除
菌株SQ11是在酿酒酵母CENPK2-1D的基础上在Ty2位点进行了PTEF-tHMGR-PGAL7-IDI1-LEU2-Degradation tag的多拷贝整合，在GAL2位点完成了PTEF-NADH-HMG1的单拷贝整合，并公开于论文《Enhancing Squalene Production inSaccharomyces cerevisiae byMetabolic Engineering and Random Mutagenesis》中。利用多基因编辑技术将酿酒酵母菌株SQ11的内源基因YPL062W(核苷酸序列如SEQ ID NO.1所示)、ROX1(核苷酸序列如SEQID NO.2所示)进行组合敲除。用引物YPL062W-armup-F、ROX1-armup-F、YPL062W-armup-R、ROX1-armup-R和YPL062W-armdown-F、ROX1-armdown-F、YPL062W-armdown-R、ROX1-armdown-R分别扩增出YPL062W、ROX1位点的上下游同源臂，利用融合PCR的方法将上下游同源臂分别进行融合，融合后的片段成为ROX1-1和YPL062W-1。利用引物3-1-ROX1-PAM-1-F、3-1-ROX1-PAM-1-R、3-2-ROX1-PAM-2-F、3-2-ROX1-PAM-2-R、3-3-YP-PAM-1-F、3-3-YP-PAM-1-R、3-4-YP-PAM-2-F、1-4-R，参考《CRISPR-Cas9系统:应用于酿酒酵母的一步多靶点基因编辑技术》的方法构建基因敲除质粒3-1-1，用于敲除Rox1的20nt为CACGACCCTTCAACGAGACA，CGGTGTCAAGCTCGAACAGC；用于敲除YPL062W的20nt为AAGCAACCAGCACGTCGCCG，CACGGGAATAAGGCAGCCGA。将敲除质粒3-1-1和融合片段ROX1-1和YPL062W-1转化至SQ11中，获得工程菌株WQ2。将菌株WQ2在YPD培养基中，于30℃，220rpm条件下发酵72h，结果显示，工程菌WQ2可以生产麦角甾醇，发酵72h的麦角甾醇产量为144.85mg/L。
实施例2敲除影响脂质代谢基因NEM1
利用多基因编辑技术将酿酒酵母菌株WQ2的内源基因NEM1(核苷酸序列如SEQ IDNO.3所示)进行敲除。用引物NEM1-armup-F、NEM1-armup-R和NEM1-armdown-F、NEM1-armdown-R分别扩增出NEM1位点的上下游同源臂，利用融合PCR的方法将上下游同源臂进行融合，融合后的片段成为NEM1-1。利用引物7-3-NEM1-PAM-1-F、7-3-NEM1-PAM-1-R、7-4-NEM1-PAM-2-F、1-4-R，通过golden gate的方法构建基因敲除质粒7-1-1，用于敲除NEM1的20nt为cgacgttatggtgaactctg，cgacgttatggtgaactctg。将敲除质粒7-1-1和片段NEM1-1共同转化进酿酒酵母WQ2进行基因敲除，获得的菌株命名为WQ3。将获得的菌株WQ3在YPD培养基中，30℃，220rpm条件下发酵72h时麦角甾醇的产量为150.78mg/L。
实施例3：YJL064W、DOS2位点关键基因的单拷贝整合
利用多基因编辑技术将酿酒酵母菌株WQ3的内源基因YJL064W(核苷酸序列如SEQID NO.10)、DOS2(核苷酸序列如SEQ ID NO.11)进行敲除。并在YJL064W位点完成ERG1、ERG11、ERG4的单拷贝过表达，在DOS2位点完成基因ERG2、ERG3、ERG4、CTT1的单拷贝过表达。用引物ERG1-F/ERG1-R从酿酒酵母基因组上扩增出基因ERG1(核苷酸序列如SEQ ID NO.4所示)，用引物ERG11-F/ERG11-R从酿酒酵母基因组上扩增出基因ERG11(核苷酸序列如SEQ IDNO.8所示)，用引物ERG2-F/ERG2-R从酿酒酵母基因组上扩增出基因ERG2(核苷酸序列如SEQID NO.5所示)，ERG3-F/ERG3-R从酿酒酵母基因组上扩增出基因ERG3(核苷酸序列如SEQ IDNO.6所示)，ERG4-F/ERG4-R从酿酒酵母基因组上扩增出基因ERG4(核苷酸序列如SEQ IDNO.7所示)，CTT1-F/CTT1-R从酿酒酵母基因组上扩增出基因CTT1(核苷酸序列如SEQ IDNO.9所示)。用引物TEF1t-F/TEF1t-R扩增终止子TTEF1，用引物GAL1/10p-F、GAL1/10p-R扩增双向启动子PGAL1/10(核苷酸序列如SEQ ID NO.19所示)，用引物TER22t-F/TER22t-R扩增终止子TTER22，引物TEF1t-F/TEF1t-R扩增终止子TTEF1，用引物TDH1p-F/TDH1p-R扩增启动子PTDH1(核苷酸序列如SEQ ID NO.15所示)，GAL7p-F/GAL7p-R扩增启动子PGAL7(核苷酸序列如SEQ ID NO.17所示)，用引物TDH3p-F/TDH3p-R扩增启动子PTDH3(核苷酸序列如SEQ IDNO.18所示)。用引物YJL064W-armup-F/YJL064W-armup-R和YJL064W-armdown-F/YJL064W-armdown-R分别扩增出YJL064W位点的上下游同源臂，引物DOS2-armup-F/DOS2-armup-R和DOS2-armdown-F/DOS2-armdown-R分别扩增出DOS2位点的上下游同源臂。将YJL064W的上下游同源臂与片段PTDH3，ERG4,TTEF1,ERG11,PGAL7,TTER22,ERG11和TDH1利用吉布森组装构建在质粒上，构成的质粒称之为YJ-1用引物YJL064W-armup-F和YJL064W-armdown-R从YJ-1上P下来整合片段YJL064W-PD。将DOS2的上下游同源臂与片段ERG2,ERG3,ERG4,CTT1,TDH1,PGAL1/10,PGAL7,TTER22,TTEF1利用吉布森组装构建在质粒上，构成的质粒称之为DOS-1,利用引物DOS2-armup-F和DOS2-armdown-R从DOS-1上P下来整合片段DOS-PD。利用引物6-1-DOS2-PAM-1-F/6-1-DOS2-PAM-1-R/6-2-DOS2-PAM-2-F/6-2-DOS2-PAM-2-R/6-3-YJL064W-PAM-1-F/6-3-YJL064W-PAM-1-R/6-4-YJL064W-PAM-2-F/1-4-R，利用golden gate的方法构建基因敲除质粒6-3-3，用于敲除DOS2的20nt为TGACGTGGATGAAAAAACTG，AGACGAGAATATTCACAGCG；用于敲除YJL064W的20nt为GAATTCTGTAGCAAACGCTG，GTGAGTTCATCTGGGAGCGG。将敲除质粒6-3-3与片段YJL064W-PD，DOS-PD共同转化进菌株WQ3,获得工程菌株WQ4。将菌株WQ4在YPD培养基中，30℃，220rpm条件下发酵72h时麦角甾醇的产量为214.07mg/L。
表3引物序列
实施例4：加强前体的供应
为促进前体角鲨烯的供给，将酿酒酵母内源基因tHMG1(核苷酸序列如SEQ IDNO.12所示)、IDI(核苷酸序列如SEQ ID NO.13所示)整合在酿酒酵母的多克隆位点Ty12上，以实现tHMG1、IDI的过表达。用引物tHMG1-F/tHMG1-R从酿酒酵母基因组上扩增出基因tHMG1，用引物IDI1-F/IDI1-R扩增基因IDI1。用引物GAL1/10p-F、GAL1/10p-R扩增双向启动子PGAL1/10。利用Ty12-armup-F/Ty12-armup-R和Ty12-armdown-F/Ty12-armdown-R分别扩增出Ty12位点的上下游同源臂。PCR产物通过乙醇沉淀法回收，利用吉布森组装上述片段tHMG1、IDI，PGAL1/10与载体Pct125(公开于公开号为CN113403334A的专利申请文件中)，获得的质粒命名为Ty12-1。引物Ty12-armup-F/Ty12-armdown-R从构好的质粒Ty12-1上获取片段Ty12-PD。约10μg的整合片段Ty12-PD与约500ng的sgRNA利用酿酒酵母高效转化法转化至实施例3构建的酿酒酵母工程菌株WQ4，涂布于筛选YNB固体培养基上，30℃培养3-5天，直至出现菌落，验证正确的克隆命名为WQ5。挑取菌株WQ5单菌落转接于5mL YPD培养基中，于30℃，220rpm发酵17-24h后按1％接种量转接于25mL YPD培养基中，在30℃，220rpm发酵72h时麦角甾醇的产量为246.40mg/L。
表4引物序列
实施例5：加强后路径基因的表达
为促进前体向目标产物麦角甾醇的转化，将酿酒酵母内源基因ERG2(核苷酸序列如SEQ ID NO.5所示)、ERG3(核苷酸序列如SEQ ID NO.6所示)、POS5(核苷酸序列如SEQ IDNO.14所示)、ERG4(核苷酸序列如SEQ ID NO.7所示)整合在酿酒酵母多克隆位点Ty3上，以实现ERG2、ERG3、POS5、ERG4的过表达。用引物GAL7p-F/GAL7p-R扩增启动子PGAL7。用引物ERG2-F/ERG2-R从酿酒酵母基因组上扩增出基因ERG2，用引物TEF1t-F/TEF1t-R扩增终止子TTEF1，用引物ERG4-F/ERG4-R从酿酒酵母基因组上扩增出基因ERG4，用引物ERG3-F/ERG3-R从酿酒酵母基因组上扩增出基因ERG3，用引物GAL7p-F、GAL7p-R扩增双向启动子PGAL7。用引物POS5-F/POS5-R从酿酒酵母基因组上扩增出基因POS5(核苷酸序列如SEQ ID NO.14所示)，用引物TER22t-F/TER22t-R扩增终止子TTER22，用引物TDH1p-F/TDH1p-R扩增启动子PTDH1，用引物TDH3p-F/TDH3p-R扩增启动子PTDH3(核苷酸序列如SEQ ID NO.18所示)，用引物PGK1p-F/PGK1p-R扩增启动子PPGK1(核苷酸序列如SEQ ID NO.16所示)。PCR产物通过乙醇沉淀法回收，利用吉布森组装上述片段与载体Pct31(载体Pct31公开于公开号为CN113403334A的专利申请文件中)，构成质粒Ty3-1，用引物Ty3-armup-F/Ty3-armdown-R从构好的质粒Ty3-1上获取片段Ty3up-ERG2-ERG3-POS5。约10μg的整合片段Ty3up-ERG2-ERG3-POS5与约500ng的sgRNA利用酿酒酵母高效转化法转化至酿酒酵母工程菌株WQ5，涂布于筛选YNB固体培养基上，30℃培养3-5天，直至出现菌落，验证正确的克隆命名为WQ6。挑取菌株WQ6单菌落转接于5mL YPD培养基中，于30℃，220rpm培养17-24h后按1％接种量转接于25mL YPD培养基中，于30℃，220rpm发酵72h的麦角甾醇产量为292.60mg/L。
进一步优化发酵条件，将菌株WQ6单菌落转接于5mL YPD培养基中，于30℃，220rpm培养17-24h后按1％接种量转接于25mL YPD培养基中，于30℃，220rpm发酵96h，并分别于发酵第12、24、36、48h各补加125μL乙醇(共0.5mL)。结果如图4所示，经发酵优化96h时麦角甾醇产量为447.91mg/L。
表5引物序列
虽然本发明已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，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种生产麦角甾醇的酿酒酵母工程菌株及其应用
<130> BAA220221A
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 405
<212> DNA
<213> 人工序列
<400> 1
atgatagaat tggattatgt aaaaggtgaa gataccattg tagaagcaac cagcacgtcg 60
ccgtggctga tgaggtctcc tcttgcccgg gccgcagaaa agaggggcag tggcctgttt 120
ttcgacataa atgaggggca tggccagcac cgagacgtca ttgttgcata tggcgtatcc 180
aagccgaaac ggcgctcgcc tcatccccac gggaataagg cagccgacaa aagaaaaacg 240
accgaaaagg aaccagaaag aaaaaagagg gtgggcgcgc cgcggacgtg taaaaagata 300
tgcatccagc ttctatatcg ctttaacttt accgttttgg gcatcgggaa cgtatgtaac 360
attgatctcc tcttgggaac ggtgagtgca acgaatgcga tatag 405
<210> 2
<211> 1107
<212> DNA
<213> 人工序列
<400> 2
atgaatccta aatcctctac acctaagatt ccaagaccca agaacgcatt tattctgttc 60
agacagcact accacaggat cttaatagac gaatggaccg ctcaaggtgt ggaaataccc 120
cataattcaa acatttctaa aattattggt acgaagtgga agggcttaca accggaagat 180
aaggcacact gggaaaatct agcggagaag gagaaactag aacataaaag gaagtatcct 240
gaatacaaat acaagccggt aagaaagtct aagaagaagc aactactttt gaaggaaatc 300
gagcaacagc agcagcagca acagaaagaa cagcagcagc agaaacagtc acaaccgcaa 360
ttacaacagc cctttaacaa caatatagtt cttatgaaaa gagcacattc tctttcacca 420
tcttcctcgg tgtcaagctc gaacagctat cagttccaat tgaacaatga tcttaagagg 480
ttgcctattc cttctgttaa tacttctaac tatatggtct ccagatcttt aagtggacta 540
cctttgacgc atgataagac ggcaagagac ctaccacagc tgtcatctca actaaattct 600
attccatatt actcagctcc acacgaccct tcaacgagac atcattacct caacgtcgct 660
caagctcaac caagggctaa ctcgacccct caattgccct ttatttcatc cattatcaac 720
aacagcagtc aaacaccggt aactacaact accacatcca caacaactgc gacatcttct 780
cctgggaaat tctcctcttc tccgaactcc tctgtactgg agaacaacag attaaacagt 840
atcaacaatt caaatcaata tttacctccc cctctattac cttctctgca agattttcaa 900
ctggatcagt accagcagct aaagcagatg ggaccaactt atattgtcaa accactgtct 960
cacaccagga acaatctatt gtccacaact acccctacgc atcatcacat tcctcatata 1020
ccaaaccaaa acattcctct acatcaaatt ataaactcaa acaacactga ggtcaccgct 1080
aaaactagcc tagtttctcc gaaatga 1107
<210> 3
<211> 1341
<212> DNA
<213> 人工序列
<400> 3
atgaatgccc taaaatattt ctcaaatcat ttaataacta caaagaaaca aaaaaaaatc 60
aatgttgagg tgacaaagaa tcaagatcta ctaggccctt caaaggaagt ttcgaacaaa 120
tatactagtc acagcgaaaa tgattgcgtt ggtgaggtgg accaacagta cgaccactcc 180
tcaagccact taaaagaatc tgatcaaaat caagagcgca aaaattctgt tcctaaaaaa 240
ccaaaagctt tacgttccat cctaaaagag aaaatagcgt caattttgtg ggcactacta 300
ctttttcttc cgtattatct gataatcaag cccttgatgt ctttatggtt tgtttttacc 360
ttcccactaa gtgtcatcga gcgtcgtgta aaacacaccg ataagagaaa caggggttca 420
aatgctagtg agaatgagct gcctgtcagt tcaagcaata ttaacgattc tagcgagaaa 480
acaaatccta aaaattgcaa tctaaatacg attcccgagg cagttgagga tgacttaaat 540
gccagtgatg agataattct acaaagggat aatgttaaag gctcattact tagagctcaa 600
tcagtcaaat caaggccaag aagttattcc aaatcagaac tatcactttc taatcattca 660
agttctaata ccgtatttgg tacaaaacga atgggaaggt tcttatttcc aaagaagctg 720
atacctaaat ccgtgcttaa cacacaaaag aaaaagaaac tggttataga tctcgatgag 780
accttaattc actcggcttc tcgaagtaca acgcatagta attcttctca gggtcacttg 840
gtggaagtga agtttggatt gagcggaatt cgtacactat actttattca caagaggcct 900
tactgtgatt tatttttgac aaaagttagc aaatggtacg accttatcat ttttacagca 960
tccatgaaag aatacgctga tcctgtgata gattggttag aaagctcctt cccatccagt 1020
ttttcaaaaa gatattaccg ttctgattgc gttctaaggg atggtgttgg atacattaaa 1080
gatttgagca ttgtcaagga ctccgaagaa aacgggaaag gcaactcctc ttctttggat 1140
gatgtcatta ttatagataa cagcccggta agttacgcaa tgaatgtaga taacgctatt 1200
caagtagaag gatggataag tgatccaact gatacggact tactgaacct gttacctttc 1260
ttggaggcta tgaggtattc aacagatgtc aggaatatac tggcattgaa acatggagag 1320
aaggcattca acataaactg a 1341
<210> 4
<211> 1491
<212> DNA
<213> 人工序列
<400> 4
atgtctgctg ttaacgttgc acctgaattg attaatgccg acaacacaat tacctacgat 60
gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg ctactggtct agcaagaaag 120
ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc ctgatagaat tgttggtgaa 180
ttgatgcaac caggtggtgt tagagcattg agaagtctgg gtatgattca atctatcaac 240
aacatcgaag catatcctgt taccggttat accgtctttt tcaacggcga acaagttgat 300
attccatacc cttacaaggc cgatatccct aaagttgaaa aattgaagga cttggtcaaa 360
gatggtaatg acaaggtctt ggaagacagc actattcaca tcaaggatta cgaagatgat 420
gaaagagaaa ggggtgttgc ttttgttcat ggtagattct tgaacaactt gagaaacatt 480
actgctcaag agccaaatgt tactagagtg caaggtaact gtattgagat attgaaggat 540
gaaaagaatg aggttgttgg tgccaaggtt gacattgatg gccgtggcaa ggtggaattc 600
aaagcccact tgacatttat ctgtgacggt atcttttcac gtttcagaaa ggaattgcac 660
ccagaccatg ttccaactgt cggttcttcg tttgtcggta tgtctttgtt caatgctaag 720
aatcctgctc ctatgcacgg tcacgttatt cttggtagtg atcatatgcc aatcttggtt 780
taccaaatca gtccagaaga aacaagaatc ctttgtgctt acaactctcc aaaggtccca 840
gctgatatca agagttggat gattaaggat gtccaacctt tcattccaaa gagtctacgt 900
ccttcatttg atgaagccgt cagccaaggt aaatttagag ctatgccaaa ctcctacttg 960
ccagctagac aaaacgacgt cactggtatg tgtgttatcg gtgacgctct aaatatgaga 1020
catccattga ctggtggtgg tatgactgtc ggtttgcatg atgttgtctt gttgattaag 1080
aaaataggtg acctagactt cagcgaccgt gaaaaggttt tggatgaatt actagactac 1140
catttcgaaa gaaagagtta cgattccgtt attaacgttt tgtcagtggc tttgtattct 1200
ttgttcgctg ctgacagcga taacttgaag gcattacaaa aaggttgttt caaatatttc 1260
caaagaggtg gcgattgtgt caacaaaccc gttgaatttc tgtctggtgt cttgccaaag 1320
cctttgcaat tgaccagggt tttcttcgct gtcgcttttt acaccattta cttgaacatg 1380
gaagaacgtg gtttcttggg attaccaatg gctttattgg aaggtattat gattttgatc 1440
acagctatta gagtattcac cccatttttg tttggtgagt tgattggtta a 1491
<210> 5
<211> 669
<212> DNA
<213> 人工序列
<400> 5
atgaagtttt tcccactcct tttgttgatt ggtgttgtag gctacattat gaacgtattg 60
ttcactacct ggttgccaac caattacatg ttcgatccaa aaactttgaa cgaaatatgt 120
aactcggtga ttagcaaaca caacgcagca gaaggtttat ccactgaaga cctgttacag 180
gatgtcagag acgcacttgc ctctcattac ggggacgaat acatcaacaa gtacgtcaaa 240
gaagaatggg tcttcaacaa tgctggtggt gcgatgggcc aaatgatcat cctacacgct 300
tccgtatccg agtacttaat tctattcgga accgctgttg gtactgaagg gcacacaggt 360
gttcactttg ctgacgacta ttttaccatc ttacatggta cgcaaatcgc agcattgcca 420
tatgccactg aagccgaagt ttacactcct ggtatgactc atcacttgaa gaagggatac 480
gccaagcaat acagcatgcc aggtggttcc tttgcccttg aattggctca aggctggatt 540
ccatgtatgt tgccattcgg gtttttggac actttctcca gtactcttga tttatacact 600
ctatatagaa ctgtctacct gactgccagg gacatgggta agaacttgtt gcaaaacaaa 660
aagttctaa 669
<210> 6
<211> 1098
<212> DNA
<213> 人工序列
<400> 6
atggatttgg tcttagaagt cgctgaccat tatgtcttag acgacttgta cgctaaagtt 60
ctgcccgctt cgttggcagc caatattcct gtcaagtggc agaaattgct agggttgaac 120
agtgggttca gcaattctac gattttgcag gagactttga actccaagaa tgccgtcaaa 180
gaatgtagaa ggttctacgg gcaggtgcca ttcctgtttg atatgtcgac gacgtctttt 240
gcatcgctat tgcctcgttc cagcatcttg agagaattcc tctcactatg ggttattgtt 300
acgatctttg gtttactact ttacttattc acggctagtc tcagctacgt gtttgtgttt 360
gacaagtcga ttttcaacca tcctcgttac ttgaaaaacc aaatggcaat ggaaatcaag 420
ttggcagtca gtgctatccc atggatgtcg atgttgaccg ctccatggtt tgttatggaa 480
ttgaacggcc attctaaact atacatgaag attgattatg aaaaccacgg tgtaaggaag 540
ctcattatcg agtacttcac tttcatcttt ttcactgatt gcggtgtgta tttagcgcac 600
agatggttgc attggccaag ggtctaccgt gctctgcaca agcctcatca caagtggctg 660
gtctgcacac ctttcgcatc tcattctttc catcctgtag acgggttttt gcaatccatc 720
tcgtaccaca tctacccatt gattctgccg ttacacaagg tttcttattt gattctgttc 780
acttttgtta acttttggac tgttatgatt catgacggtc aatacctatc aaacaatcct 840
gccgtcaacg gtactgcctg ccacacggtt caccatctat atttcaacta caactacggt 900
caattcacca ctttgtggga cagactaggg ggttcttacc gtagaccaga tgactcattg 960
tttgatccta agttaagaga tgctaaggag acctgggacg ctcaagttaa ggaagttgaa 1020
catttcatca aggaggtcga aggtgatgat aatgatagaa tctatgaaaa cgacccaaat 1080
accaagaaga acaactga 1098
<210> 7
<211> 1422
<212> DNA
<213> 人工序列
<400> 7
atggcaaagg ataatagtga gaagctgcag gtgcagggag aggagaaaaa gtccaagcaa 60
ccggttaatt tcctgcctca gggtaaatgg ctgaagccaa atgaaatcga atatgagttt 120
ggtgggacta ctggtgttat tggtatgctg atcgggtttc cactgctaat gtactatatg 180
tggatttgtg cggaatttta tcacggtaag gttgccctac ccaaggctgg tgaatcgtgg 240
atgcacttta tcaagcacct ataccagtta gtcttggaga acggtatccc agaaaagtat 300
gactggacta ttttcttaac attttgggtg tttcagatca ttttctacta tacgttgccc 360
gggatttgga caaaaggtca accattgtct catttgaagg gaaaacaatt gccttacttt 420
tgtaatgcca tgtggacctt gtatgtaact accactttgg tcttggtttt gcactttacc 480
aatcttttta gattgtatgt cattattgac cgttttggga ggatcatgac atgtgccatt 540
atttcagggt ttgccttctc catcatattg tacttatgga ctttatttat ctcacatgac 600
tatcatagaa tgacaggaaa ccatctatat gatttcttca tgggagctcc actaaaccct 660
aggtggggga ttttggactt gaagatgttt ttcgaggtta gattaccttg gttcaccctt 720
tactttatca ctttgggtgc ctgtttgaag cagtgggaga cttacggcta tgtgacacca 780
caattggggg ttgtcatgtt agctcattgg ttgtacgcga acgcatgtgc taaaggtgaa 840
gaattgattg ttccaacctg ggacatggct tacgaaaagt ttggatttat gctgatcttc 900
tggaatattg ccggtgtccc atacacttac tgtcattgta cgttgtattt gtactaccat 960
gacccatctg aatatcactg gtctacactg tacaatgttt cgctgtacgt tgttctatta 1020
tgcgcctact acttctttga cacggcaaat gctcagaaaa atgccttcag aaagcaaatg 1080
tctggtgaca agacaggtag gaagactttc ccatttttgc cataccaaat tttgaagaat 1140
ccaaagtata tggttacctc caatggatcg tacctattga ttgatggttg gtacactttg 1200
gctagaaaaa ttcactacac tgccgattgg actcaatctc tcgtttgggc cttgtcttgc 1260
gggttcaact cggtgttccc atggtttttc ccagtattct tccttgttgt cctgattcac 1320
agagccttca gagaccaagc aaaatgtaag agaaagtacg gaaaagattg ggatgagtat 1380
tgtaaacatt gcccttacgt ctttattcct tatgttttct ag 1422
<210> 8
<211> 1593
<212> DNA
<213> 人工序列
<400> 8
atgtctgcta ccaagtcaat cgttggagag gcattggaat acgtaaacat tggtttaagt 60
catttcttgg ctttaccatt ggcccaaaga atctctttga tcataataat tcctttcatt 120
tacaatattg tatggcaatt actatattct ttgagaaagg accgtccacc tctagtgttt 180
tactggattc catgggtcgg tagtgctgtt gtgtacggta tgaagccata cgagtttttc 240
gaagaatgtc aaaagaaata cggtgatatt ttttcattcg ttttgttagg aagagtcatg 300
actgtgtatt taggaccaaa gggtcacgaa tttgtcttca acgctaagtt ggcagatgtt 360
tcagcagaag ctgcttacgc tcatttgact actccagttt tcggtaaagg tgttatttac 420
gattgtccaa attctagatt gatggagcaa aagaagtttg ttaagggtgc tctaaccaaa 480
gaagccttca agagctacgt tccattgatt gctgaagaag tgtacaagta cttcagagac 540
tccaaaaact tccgtttgaa tgaaagaact actggtacta ttgacgtgat ggttactcaa 600
cctgaaatga ctattttcac cgcttcaaga tcattattgg gtaaggaaat gagagcaaaa 660
ttggataccg attttgctta cttgtacagt gatttggata agggtttcac tccaatcaac 720
ttcgtcttcc ctaacttacc attggaacac tatagaaaga gagatcacgc tcaaaaggct 780
atctccggta cttacatgtc tttgattaag gaaagaagaa agaacaacga cattcaagac 840
agagatttga tcgattcctt gatgaagaac tctacctaca aggatggtgt gaagatgact 900
gatcaagaaa tcgctaactt gttaattggt gtcttaatgg gtggtcaaca tacttctgct 960
gccacttctg cttggatttt gttgcacttg gctgaaagac cagatgtcca acaagaattg 1020
tacgaagaac aaatgcgtgt tttggatggt ggtaagaagg aattgaccta cgatttatta 1080
caagaaatgc cattgttgaa ccaaactatt aaggaaactc taagaatgca ccatccattg 1140
cactctttgt tccgtaaggt tatgaaagat atgcacgttc caaacacttc ttatgtcatc 1200
ccagcaggtt atcacgtttt ggtttctcca ggttacactc atttaagaga cgaatacttc 1260
cctaatgctc accaattcaa cattcaccgt tggaacaaag attctgcctc ctcttattcc 1320
gtcggtgaag aagtcgatta cggtttcggt gccatttcta agggtgtcag ctctccatac 1380
ttacctttcg gtggtggtag acacagatgt atcggtgaac actttgctta ctgtcagcta 1440
ggtgttctaa tgtccatttt tatcagaaca ttaaaatggc attacccaga gggtaagacc 1500
gttccacctc ctgactttac atctatggtt actcttccaa ccggtccagc caagatcatc 1560
tgggaaaaga gaaatccaga acaaaagatc taa 1593
<210> 9
<211> 615
<212> DNA
<213> 人工序列
<400> 9
atgacagctg ccactacatc acagccagct ttctcgcctg accaagtttc cgtgatcttc 60
gttctaggag gacccggtgc aggcaagggt actcagtgtg aaaaactagt taaggactat 120
tcatttgtcc atttgtcagc cggagacctt ctacgtgctg agcagggcag agcaggttcc 180
caatatgggg aattgatcaa gaactgcatc aaagagggcc agattgtccc tcaagagatt 240
actttggcgc ttttacgcaa cgctatttcc gataacgtca aggcgaacaa gcataagttc 300
ttaattgacg gatttcctag gaagatggat caagccattt cctttgaaag agacatcgtt 360
gaaagcaaat tcatcctgtt ctttgactgc cctgaagata tcatgttaga gagactattg 420
gagcgtggca agaccagtgg tagaagcgat gacaacattg agtccattaa gaagagattt 480
aacactttca aggagactag tatgcccgtc atcgagtact ttgaaaccaa atcgaaagtc 540
gtccgtgttc gttgcgacag atccgtcgaa gatgtgtaca aagacgtcca agacgctatc 600
cgtgatagct tatag 615
<210> 10
<211> 504
<212> DNA
<213> 人工序列
<400> 10
atgaacaacg agactagtgg taaagaaacg gcgtctgcac ctctgtgttc gcccaagtta 60
cctgtagaaa aagtgcagag aatagccaag aatgatccag aatatatgga cacttcggat 120
gacgcattcg tagccacagc gtttgctaca gaattcttcg tccaggtgct gacacatgag 180
tccctacata ggcaacagca gcagcaacaa caacaggtac cgccgctccc agatgaactc 240
acgctgtcgt acgatgacat ctctgccgca attgtgcact cttctgacgg ccatctgcag 300
tttttgaatg atgtgatacc aacaacaaag aatttgaggc ttctagtgga agaaaaccga 360
gttagatata ctacaagtgt catgccccct aatgaagttt actccgccta tgtggtgaac 420
gatacggctc cgaagcccaa cattgtcgag attgatcttg ataatgacga agacgacgac 480
gaagacgtta ctgatcaaga ataa 504
<210> 11
<211> 957
<212> DNA
<213> 人工序列
<400> 11
tcattcccag tcatcatcgt cgtcgtcgtc gtcgtcgtcg tcgtcgtcgt cgtccttgct 60
ctcatccttg ttggcgaggc caacatggtc actaacatct ttcaatccct cttgagacac 120
agcaactttc gtctctcctt tagatttgtt gtcaactact gcttctactt tgtcatcatc 180
atcatcttct tcttcgtcat cccactcaac ttctttttcc tccgtttcct tttccttttt 240
ggacaatatt tctttccttt tactttcttt atctagaatt ttgtttcttt gtaagaaata 300
gatgtgccag aaatctttat agctgatttt atggggtacg atgtcgttca ttaatttgga 360
gatatctttg tcgccctgta aaatagaaca tatctcctca gttttttcat ccacgtcaaa 420
cgggtccagt tgcaaatcca ttttattgtc taaataaacc gatttatctt tagataatgt 480
ccttagttcg gcttctgttc tatttccacc cacagcaatt tcattttctt tcgaattctc 540
atccttgtca ttagagtcat tttctgcagc attatcgaag ctactgaagc cagaccaaaa 600
attcttagtt ttcattttgc tccaatatga ctgggctaga ctttccacgc tgtgaatgtt 660
ctcgtctagt ttcttcaaat acttttgtgc agtctctgta gtttcattgc taattggcaa 720
gttaatctca attccgtcat ctttttcgat tactaacttt ttgaatgcac ttgtagtctt 780
ttcatatcgt ttattgactt cctcttctaa cttttggaag gccccatttg ttttgtcatc 840
tcggctctga agttcgttat tttcagtgtt ttctgttgat ccagtttcct tggtatgaga 900
gtcgtttatt ttatcatctt cgatacaagc tacttgctct tcataaaaga attccat 957
<210> 12
<211> 1584
<212> DNA
<213> 人工序列
<400> 12
ttaggattta atgcaggtga cggacccatc tttcaaacga tttatatcag tggcgtccaa 60
attgttaggt tttgttggtt cagcaggttt cctgttgtgg gtcatatgac tttgaaccaa 120
atggccggct gctagggcag cacataagga taattcacct gccaagacgg cacaggcaac 180
tattcttgct aattgacgtg cgttggtacc aggagcggta gcatgcgggc ctcttacacc 240
taataagtcc aacatggcac cttgtggttc tagaacagta ccaccaccga tggtacctac 300
ttcgatggat ggcatggata cggaaattct caaatcaccg tccacttctt tcatcaatgt 360
tatacagttg gaactttcaa cattttgtgc aggatcttgt cctaatgcca agaaaacagc 420
tgtcactaaa ttagctgcat gtgcgttaaa tccaccaaca gacccagcca ttgcagatcc 480
aaccaaattc ttagcaatgt tcaactcaac caatgcggaa acatcacttt ttaacacttt 540
tctgacaaca tcaccaggaa tagtagcttc tgcgacgaca ctcttaccac gaccttcgat 600
ccagttgatg gcagctggtt ttttgtcggt acagtagtta ccagaaacgg agacaacctc 660
catatcttcc cagccatact cttctaccat ttgctttaat gagtattcga cacctttaga 720
aatcatattc atacccattg cgtcaccagt agttgttcta aatctcatga agagtaaatc 780
tcctgctaga caagtttgaa tatgttgcag acgtgcaaat cttgatgtag agttaaaagc 840
ttttttaatt gcgttttgtc cctcttctga gtctaaccat atcttacagg caccagatct 900
tttcaaagtt gggaaacgga ctactgggcc tcttgtcata ccatccttag ttaaaacagt 960
tgttgcacca ccgccagcat tgattgcctt acagccacgc atggcagaag ctaccaaaca 1020
accctctgta gttgccattg gtatatgata agatgtacca tcgataacca aggggcctat 1080
aacaccaacg ggcaaaggca tgtaacctat aacattttca caacaagcgc caaatacgcg 1140
gtcgtagtca taatttttat atggtaaacg atcagatgct aatacaggag cttctgccaa 1200
aattgaaaga gccttcctac gtaccgcaac cgctctcgta gtatcaccta attttttctc 1260
caaagcgtac aaaggtaact taccgtgaat aaccaaggca gcgacctctt tgttcttcaa 1320
ttgttttgta tttccactac ttaataatgc ttctaattct tctaaaggac gtattttctt 1380
atccaagctt tcaatatcgc gggaatcatc ttcctcacta gatgatgaag gtcctgatga 1440
gctcgattgc gcagatgata aacttttgac tttcgatcca gaaatgactg ttttattggt 1500
taaaactggt gtagaagcct tttgtacagg agcagtaaaa gacttcttgg tgacttcagt 1560
tttcaccaat tggtctgcag ccat 1584
<210> 13
<211> 867
<212> DNA
<213> 人工序列
<400> 13
atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60
caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120
agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180
ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240
gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300
ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360
caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420
catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480
ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540
actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600
gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660
aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720
gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780
tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840
gacaggcaaa ttcatagaat gctataa 867
<210> 14
<211> 1245
<212> DNA
<213> 人工序列
<400> 14
atgtttgtca gggttaaatt gaataaacca gtaaaatggt ataggttcta tagtacgttg 60
gattcacatt ccctaaagtt acagagcggc tcgaagtttg taaaaataaa gccagtaaat 120
aacttgagga gtagttcatc agcagatttc gtgtccccac caaattccaa attacaatct 180
ttaatctggc agaacccttt acaaaatgtt tatataacta aaaaaccatg gactccatcc 240
acaagagaag cgatggttga attcataact catttacatg agtcataccc cgaggtgaac 300
gtcattgttc aacccgatgt ggcagaagaa atttcccagg atttcaaatc tcctttggag 360
aatgatccca accgacctca tatactttat actggtcctg aacaagatat cgtaaacaga 420
acagacttat tggtgacatt gggaggtgat gggactattt tacacggcgt atcaatgttc 480
ggaaatacgc aagttcctcc ggttttagca tttgctctgg gcactctggg ctttctatta 540
ccgtttgatt ttaaggagca taaaaaggtc tttcaggaag taatcagctc tagagccaaa 600
tgtttgcata gaacacggct agaatgtcat ttgaaaaaaa aggatagcaa ctcatctatt 660
gtgacccatg ctatgaatga catattctta cataggggta attcccctca tctcactaac 720
ctggacattt tcattgatgg ggaatttttg acaagaacga cagcagatgg tgttgcattg 780
gccactccaa cgggttccac agcatattca ttatcagcag gtggatctat tgtttcccca 840
ttagtccctg ctattttaat gacaccaatt tgtcctcgct ctttgtcatt ccgaccactg 900
attttgcctc attcatccca cattaggata aagataggtt ccaaattgaa ccaaaaacca 960
gtcaacagtg tggtaaaact ttctgttgat ggtattcctc aacaggattt agatgttggt 1020
gatgaaattt atgttataaa tgaggtcggc actatataca tagatggtac tcagcttccg 1080
acgacaagaa aaactgaaaa tgactttaat aattcaaaaa agcctaaaag gtcagggatt 1140
tattgtgtcg ccaagaccga gaatgactgg attagaggaa tcaatgaact tttaggattc 1200
aattctagct ttaggctgac caagagacag actgataatg attaa 1245
<210> 15
<211> 530
<212> DNA
<213> 人工序列
<400> 15
tttgttttgt gtgtaaattt agtgaagtac tgttttttgt gtgtgttggt gaaatatcaa 60
accaagttct tgatgaattt cttatttatg caagagagag aatagaactg tactacaaat 120
ctcattgtgt gaaaatatat tgtctattta tatgatttcg agactccagt tttggtcatt 180
atcaccaagc tcttactgct acagagaatg aacatgctcc tccccccctt cttcagacta 240
tgttgttctg cacgtggata ccgtcgcatg cacctaagaa gcagatggtg gcttgcctta 300
ctgtattgta aagatccagt ctccagatct gcgaccactc cgaaggttga aacccgagct 360
tcctgtttgc tgtctcgcgc cttttaaaaa aaaagcgcga ttatgggccg ctcgtgacag 420
taaaggaagc aagcagatcg accccctgaa aatgtggtgt ggttactaag cagaagcgtc 480
ttcgtcgcat atcctattcc tagcgcaaca aggccccacg gtgtggtttc 530
<210> 16
<211> 872
<212> DNA
<213> 人工序列
<400> 16
actgtaattg cttttagttg tgtattttta gtgtgcaagt ttctgtaaat cgattaattt 60
ttttttcttt cctcttttta ttaaccttaa tttttatttt agattcctga cttcaactca 120
agacgcacag atattataac atctgcataa taggcatttg caagaattac tcgtgagtaa 180
ggaaagagtg aggaactatc gcatacctgc atttaaagat gccgatttgg gcgcgaatcc 240
tttattttgg cttcaccctc atactattat cagggccaga aaaaggaagt gtttccctcc 300
ttcttgaatt gatgttaccc tcataaagca cgtggcctct tatcgagaaa gaaattaccg 360
tcgctcgtga tttgtttgca aaaagaacaa aactgaaaaa acccagacac gctcgacttc 420
ctgtcttcct attgattgca gcttccaatt tcgtcacaca acaaggtcct agcgacggct 480
cacaggtttt gtaacaagca atcgaaggtt ctggaatggc gggaaagggt ttagtaccac 540
atgctatgat gcccactgtg atctccagag caaagttcgt tcgatcgtac tgttactctc 600
tctctttcaa acagaattgt ccgaatcgtg tgacaacaac agcctgttct cacacactct 660
tttcttctaa ccaagggggt ggtttagttt agtagaacct cgtgaaactt acatttacat 720
atatataaac ttgcataaat tggtcaatgc aagaaataca tatttggtct tttctaattc 780
gtagtttttc aagttcttag atgctttctt tttctctttt ttacagatca tcaaggaagt 840
aattatctac tttttacaac aaatataaaa ca 872
<210> 17
<211> 725
<212> DNA
<213> 人工序列
<400> 17
tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt tcttaattgc 60
aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg agttcagtga 120
taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca agttctctgt 180
accaccatgg agacatcaaa aattgaaaat ctatggaaag atatggacgg tagcaacaag 240
aatatagcac gagccgcgga gttcatttcg ttacttttga tatcactcac aactattgcg 300
aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta gtattcgttt 360
ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt aaattgcttt 420
gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc attagatata 480
ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc gctcggacaa 540
ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc aagctgaaaa 600
taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag gtcggaaata 660
tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg aatattccct 720
caaaa 725
<210> 18
<211> 698
<212> DNA
<213> 人工序列
<400> 18
ataaaaaaca cgctttttca gttcgagttt atcattatca atactgccat ttcaaagaat 60
acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa ttagcctttt 120
aattctgctg taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca 180
tcgtaggtgt ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct 240
ttttaagctg gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc 300
aaccatcagt tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag 360
gcaaaaaacg ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac 420
aaggcaattg acccacgcat gtatctatct cattttctta caccttctat taccttctgc 480
tctctctgat ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc 540
cctacttgac taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat 600
ttcttaaact tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca 660
agaacttagt ttcgaataaa cacacataaa caaacaaa 698
<210> 19
<211> 668
<212> DNA
<213> 人工序列
<400> 19
tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60
ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120
tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180
attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccta tggttgttaa 240
tttgattcgt taatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300
accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360
ctgcgtttca ggaacgcgac cggtgaagac gaggacgcac ggaggagagt cttccgtcgg 420
agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480
gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540
catttccaca acatataagt aagattagat atggatatgt atatggtggt aatgccatgt 600
aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 660
caatataa 668
Claims (10)
1.一株产麦角甾醇的酿酒酵母工程菌，其特征在于，对出发菌株进行了如下至少一种改进：
(1)敲除酿酒酵母内源基因YPL062W、ROX1；
(2)敲除酿酒酵母内源基因NEM1；
(3)将基因ERG1、ERG2、ERG3、ERG4、ERG11、CTT1单拷贝整合到酿酒酵母的YJL064W、DOS2位点；
(4)将tHMG1、IDI基因在Ty12位点多拷贝整合；
(5)将POS5、ERG4、ERG2、ERG3在Ty3位点多拷贝整合。
2.根据权利要求1所述的酿酒酵母工程菌，其特征在于，所述基因YJL064W、DOS2被敲除后，在DOS2位点整合基因ERG2、ERG3、ERG4和CTT1，在YJL064W位点整合基因ERG1、ERG11和ERG4。
3.根据权利要求1或2所述的酿酒酵母工程菌，其特征在于，所述基因YPL062W的核苷酸序列如SEQ ID NO.1所示、所述ROX1基因的核苷酸序列如SEQ ID NO.2所示；所述基因NEM1的核苷酸序列如SEQ ID NO.3所示。
4.根据权利要求1～3任一所述的酿酒酵母工程菌，其特征在于，所述基因ERG1、ERG2、ERG3、ERG4、ERG11、CTT1、YJL064W、DOS2的核苷酸序列分别如SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示。
5.根据权利要求1～4任一所述的酿酒酵母工程菌，其特征在于，所述基因tHMG1的核苷酸序列如SEQ ID NO.12所示，所述基因IDI的核苷酸序列如SEQ ID NO.13所示；所述基因POS5的核苷酸序列如SEQ ID NO.14所示。
6.根据权利要求1～5任一所述的酿酒酵母工程菌，其特征在于，用启动子PTDH1、PGAL7、PTDH3、P GAL1、PGAL10或P PGK1起始基因的表达。
7.根据权利要求1～6任一所述的酿酒酵母工程菌，其特征在于，以酿酒酵母CENPK2-1D为出发菌株。
8.权利要求1～7任一所述的酿酒酵母工程菌在生产麦角甾醇中的应用。
9.一种产麦角甾醇的方法，其特征在于，将权利要求1～7任一所的述酿酒酵母工程菌接种于发酵培养基中，于28～30℃发酵72～100h。
10.权利要求1～7任一所述的酿酒酵母工程菌或权利要求9所述的方法在食品、医药、化工领域生产含麦角甾醇产品方面的应用。
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