CN113832087B - 一种利用大肠杆菌全生物合成丙二酸的方法 - Google Patents

一种利用大肠杆菌全生物合成丙二酸的方法 Download PDF

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CN113832087B
CN113832087B CN202010577670.5A CN202010577670A CN113832087B CN 113832087 B CN113832087 B CN 113832087B CN 202010577670 A CN202010577670 A CN 202010577670A CN 113832087 B CN113832087 B CN 113832087B
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邓禹
赵运英
张祺玥
付雯宣
许铭清
刘源
赵润芝
朱桐江
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Abstract

本发明公开了一种利用大肠杆菌全生物合成丙二酸的方法，属于生物工程领域。本发明是以大肠杆菌BL21(DE3)为宿主，将大肠杆菌自身的磷酸烯醇丙酮酸羧化酶基因和天冬氨酸转氨酶共表达，将来自谷氨酸棒杆菌的天冬氨酸‑α‑脱氢酶基因单独进行表达，将大肠杆菌自身的琥珀酸半醛脱氢酶基因和来自铜绿假单胞菌的异源基因β‑丙氨酸丙酮酸转氨酶基因共表达，构建了经由草酰乙酸‑天冬氨酸的丙二酸全生物合成途径，使工程菌积累丙二酸的能力进一步提高，发酵36小时丙二酸产量可达0.74g/L，较前期构建菌株丙二酸最高产量0.23g/L提高了3倍，发酵时间提前了12小时。

Description

一种利用大肠杆菌全生物合成丙二酸的方法
技术领域
本发明涉及一种利用大肠杆菌全生物合成丙二酸的方法，属于生物工程领域。
背景技术
丙二酸主要用于香料、胶粘剂和树脂添加剂等的生产，也可用于皮革制品或铝制品表面处理剂、泡沫塑料发泡剂和核反应器的化学清洗剂，如丙二酸是生产农药杀菌剂富士一号、除草剂枯杀达、植物生长调节剂吲熟酯的原料；医药行业使用丙二酸制备利尿药苯磺唑酮、抗炎药羟苯保泰松、镇静药溴甲辛托品等；丙二酸加热后生成二氧化碳和水，没有污染问题，所以可直接用作铝表面处理剂。此外，丙二酸及其酯类也可广泛应用作医药中间体，丙二酸的两个羧基中的羟基全被乙氧基取代而生成的化合物丙二酸二乙酯或丙二酸二乙酯是重要的有机合成原料，用于染料和药物合成，如制造巴比妥类药物，维生素B1和B6等。随着目前国内和国际化工行业的快速发展，加上丙二酸的用途的大力开发，丙二酸的产量和品质在日剧上升。
丙二酸现在应用于实际生产中的主要合成方法为化学合成法。主要有两种生产路线，一是丙二酸酯用硫酸在80-90℃水解，该法特点是工艺路线及生产周期短、三废少，但该水解过程属于可逆反应，且丙二酸受高温容易发生脱羧反应分解生成乙酸、水和二氧化碳，故产品收率低，但该方法易控制杂质的产生，所以产品的纯度较高；二是二是酯交换工艺，只要控制原料的纯度，就可以制备含量高、杂质少、水份低的高纯度丙二酸且得到的副产可以制备纯度较高的乙酸乙酯，此法的缺点是：需要用到乙酸、有刺鼻酸味、另有副产乙酸乙醋和乙醇的混合溶液产生，不好处理。
丙二酸的生物合成途径还没有得到很好的发展，已知的方法是通过丙二酰-CoA水解酶活性从丙二酰-CoA产生丙二酸的方法(WO 2013134424)。但是，丙二酰-CoA途径的代谢物生产效率比天冬氨酸途径低，因此，利用天冬氨酸途径生产丙二酸是一种效率更高的方法。
发明内容
针对上述问题，本发明的第一个目的是提供一种由草酰乙酸-天冬氨酸途径生物合成丙二酸的重组大肠杆菌，所述大肠杆菌分模块过量表达来自大肠杆菌的磷酸烯醇丙酮酸羧化酶，天冬氨酸转氨酶，琥珀酸半醛脱氢酶，来自谷氨酸棒杆菌的异源基因天冬氨酸-α-脱氢酶和来自铜绿假单胞菌的异源基因β-丙氨酸丙酮酸转氨酶。
在一种实施方式中，所述重组大肠杆菌以大肠杆菌BL21(DE3)为宿主。
在一种实施方式中，所述分模块过量表达是将基因ppc和aspC共表达，将基因panD单独表达，将基因yne1和pa0123共表达。
在一种实施方式中，所述大肠杆菌以大肠杆菌BL21(DE3)为宿主，分模块过量表达来自大肠杆菌的磷酸烯醇丙酮酸羧化酶基因(ppc)，天冬氨酸转氨酶基因(aspC)，琥珀酸半醛脱氢酶基因(yne1)，来自谷氨酸棒杆菌的异源基因天冬氨酸-α-脱氢酶基因(panD)和来自铜绿假单胞菌的异源基因β-丙氨酸丙酮酸转氨酶基因(pa0123)。
在一种实施方式中，所述磷酸烯醇丙酮酸羧化酶基因(ppc)的核苷酸序列如SEQID NO.1所示；氨基酸序列如SEQ ID NO.7所示。
在一种实施方式中，所述天冬氨酸转氨酶基因(aspC)的核苷酸序列如SEQ IDNO.2所示；氨基酸序列如SEQ ID NO.8所示。
在一种实施方式中，所述天冬氨酸-α-脱氢酶基因(panD)的核苷酸序列如SEQ IDNO.3所示；氨基酸序列如SEQ ID NO.9所示。
在一种实施方式中，所述β-丙氨酸丙酮酸转氨酶基因(pa0123)的核苷酸序列如SEQ ID NO.4所示，氨基酸序列如SEQ ID NO.10所示。
在一种实施方式中，所述琥珀酸半醛脱氢酶基因(yne1)的核苷酸序列如SEQ IDNO.5所示；氨基酸序列如SEQ ID NO.11所示。
在一种实施方式中，琥珀酸脱氢酶基因(sdhC)的核苷酸序列如SEQ ID NO.6所示。
在一种实施方式中，所述分模块表达是将基因ppc和aspC以共同的质粒表达，并将基因yne1和pa0123以共同的质粒表达。
在一种实施方式中，所述分模块表达是将基因ppc和aspC以pCDFDuet-1为表达载体表达，将基因panD以pTrc99a为表达载体表达，并将基因yne1和pa0123以pRSFDuet-1为表达载体表达。
本发明还提供一种构建所述重组大肠杆菌的方法，包括以下步骤：
(1)以质粒pCDFDuet-1为骨架载体，连接基因片段ppc和aspC，得到重组质粒pCDF-ppc-aspC；
(2)以质粒pTrc99a为骨架载体，连接基因片段panD，得到重组质粒pTrc99a-panD；
(3)以质粒pRSFDuet-1为骨架载体，连接基因片段yne1、pa0123，得到重组质粒pRSF-yne1-pa0123；
(4)将pCDF-ppc-aspC、pTrc99a-panD和pRSF-yne1-pa0123共同转入大肠杆菌BL21(DE3)，得到重组大肠杆菌。
在一种实施方式中，步骤(1)使用引物(aspC-F，aspC-R)从大肠杆菌BL21的基因组中扩增基因aspC，以EcoRI，SalI对pCDF-sdhC-ppc质粒及aspC片段的PCR纯化产物进行双酶切及胶回收。用T4DNA连接酶将载体和片段进行连接，并转化到大肠杆菌JM109感受态细胞中，通过菌落PCR和Sanger测序(pCDF-R，pCDF-F)进行验证筛选。得到重组质粒pCDF-ppc-aspC。
在一种实施方式中，步骤(2)使用引物(panD-F，panD-R)从实验室保存的pTrc99A-aspA-panD质粒扩增基因panD，以EcoRI，HindⅢ对pTrc99a质粒及panD片段的PCR纯化产物进行双酶切及胶回收。用T4DNA连接酶将载体和片段进行连接，并转化到大肠杆菌JM109感受态细胞中，通过菌落PCR和Sanger测序(pTrc99a-R，pTrc99a-F)进行验证筛选。得到重组质粒pTrc99a–panD。
在一种实施方式中，步骤(3)将密码子优化后的基因片段pa0123及质粒pRSFDuet-1都用Nco I和Hind III双酶酶切处理后，以T4 DNA连接酶连接得到重组质粒pRSF-pa0123；将yne1及重组质粒pRSF-pa0123都用Xho I和Nde I双酶切处理，再用T4 DNA连接酶连接，得到重组质粒pRSF-yne1-pa0123。
本发明还提供了一种应用所述重组大肠杆菌发酵生产丙二酸的方法，是以葡萄糖为碳源，在有氧的环境下培养所述重组大肠杆菌。
在一种实施方式中，所述发酵是以SOB培养基为发酵培养基，将重组大肠杆菌于35～37℃、200～280rpm培养至OD600为0.8-1.0时加入IPTG，于28～30℃诱导培养至少24h。
在一种实施方式中，所述SOB培养基的成分为2g/100ml胰蛋白胨、0.5g/100ml酵母粉、0.05g/100ml NaCl、2.5mM KCl、10mM MgCl2、0.8g/100ml葡萄糖、50μg/ml硫酸卡那霉素、50μg/ml氨苄青霉素、50μg/ml链霉素。
在一种实施方式中，所述发酵接种重组大肠杆菌种子液；所述种子液的制备方法是将甘油保藏的菌种于平板上划线，挑取单菌落接种于盛有50ml的LB液体培养基的250ml锥形瓶中，37℃、250rpm摇瓶过夜。次日取1ml菌液转接于50ml LB液体培养基中，37℃、250rpm培养至OD600达到0.8-1.0，获得种子液。
本发明还要求保护所述大肠杆菌丙二酸生物合成基因工程菌或所述方法在制备丙二酸及其衍生产品方面的应用。
在一种实施方式中，所述衍生产品包括但不限于丙二酸二乙酯或丙二酸二乙酯。
在一种实施方式中，所述衍生产品包括但不限于巴比妥类药物，维生素B1或维生素B6。
有益效果：本发明提供了一种可利用葡萄糖，经由草酰乙酸-天冬氨酸途径发酵生产丙二酸的重组大肠杆菌。通过向大肠杆菌中引入两个外源基因，即来自谷氨酸棒杆菌的天冬氨酸-α-脱氢酶基因(panD)和来自铜绿假单胞菌的β-丙氨酸丙酮酸转氨酶基因(pa0123)，然后再过量表达大肠杆菌自身的磷酸烯醇丙酮酸羧化酶基因(ppc)、天冬氨酸转氨酶(aspC)和琥珀酸半醛脱氢酶基因(yne1)三个基因，实现丙二酸的高效生物合成。该方法操作简单方便，成本低，发酵36小时的丙二酸产量可达0.74g/L，较前期构建的重组大肠杆菌的丙二酸最高产量0.23g/L提高了3倍。
附图说明
图1为丙二酸合成途径示意图。
图2为pCDF–ppc-aspC、pTrc99a-panD和pRSF-yne1-pa0123的质粒图谱。
图3为pCDF–ppc-aspC质粒菌落PCR验证电泳图；其中泳道1为5000bp标准分子量的DNA标记，2-4为正确转化子的菌落PCR电泳检测aspC结果。
图4为pTrc99a-panD质粒菌落PCR验证电泳图；其中泳道1为5000bp标准分子量的DNA标记，2-6为正确转化子的菌落PCR电泳检测panD结果。
图5为pRSF-yne1-pa0123质粒菌落PCR验证电泳图；其中泳道1为5000bp标准分子量的DNA标记，2-6为正确转化子的菌落PCR电泳检测yne1和pa0123结果。
图6为ppc和aspC基因的表达蛋白检测结果。
图7为panD基因的表达蛋白检测结果。
图8为yne1和pa0123基因的表达蛋白检测结果。
图9为工程菌在不同条件下丙二酸发酵结果。
具体实施方式
SOB培养基：20g/L胰蛋白胨，5g/L酵母粉，0.5g/LNaCl，2.5mM KCl，10mM MgCl2，4g/L葡萄糖，50μg/ml硫酸卡那霉素，50μg/ml氨苄青霉素，50μg/ml链霉素。
丙二酸液相质谱检测及结果分析：
预处理：发酵样品12,000rpm离心2min将发酵液与菌体分离，0.22μm滤膜处理发酵液，供液相质谱检测。
液相质谱条件：检测波长：200---400nm，分析柱：BEH C18(2.1x150 mm 1.7μm)，柱温45℃，流速：0.3ml/min，进样量：5μL，检测器：Waters Acquity PDA(200-400nm)；流动相A相为0.1％(v/v)甲酸，B相为乙腈。
流动相条件如下：
表1实施例涉及的引物序列表
实施例1：重组质粒pCDF-ppc-sdhC的构建
用Nco I和EcoR I双酶切质粒pCDFDuet-1，切胶回收目的载体DNA片段pCDF-1(3744bp)。以大肠杆菌BL21基因组为模板，用引物ppc-F/R，PCR扩增得到的ppc片段(如SEQID NO.1所示)，然后将pCDF-1和ppc这两个目的片段用T4 DNA连接酶连接，化学转化法转化至JM109感受态细胞，菌落PCR挑取阳性转化子，并提取质粒酶切和PCR验证，验证正确后的质粒命名为pCDF-ppc。
用EcoR I和Sal I双酶切质粒pCDF-ppc，切胶回收目的DNA片段pCDF-2(6379bp)，以大肠杆菌BL21基因组为模板，用引物sdhC-F/R，PCR扩增得到的sdhC片段(如SEQ ID NO.6所示)，然后将pCDF-2和sdhC片段用T4 DNA连接酶连接，化学转化法转化至JM109感受态细胞中，菌落PCR挑取阳性转化子，并提取质粒酶切和PCR验证，验证引物是veri-pCDF-F/R,验证正确后的质粒命名为pCDF-ppc-sdhC。
实施例2重组质粒pCDF–ppc-aspC的构建
以大肠杆菌BL21的基因组为模板，以引物aspC-F和aspC-R扩增基因aspC(如SEQID NO.2所示)，用EcoRI和SalI双酶切实施例1构建的质粒pCDF-ppc-sdhC，切胶回收目的载体DNA片段pCDF-ppc(约6400bp)，然后将基因aspC与pCDF-ppc用T4 DNA连接酶连接，化学转化法转化至JM109感受态细胞中，菌落PCR挑取阳性转化子，并提取质粒酶切和PCR验证，验证引物为veri-pCDF-F/R，验证正确的质粒命名为pCDF–ppc-aspC(质粒图如图2所示，菌落PCR检测结果如图3所示)。
实施例3重组质粒pTrc99a-panD的构建
使用引物(panD-F，panD-R)从实验室保存的pTrc99A-aspA-panD质粒扩增基因panD(序列如SEQ ID NO.3所示)，以EcoRI，HindⅢ对pTrc99A质粒及panD片段的PCR纯化产物进行双酶切及胶回收。用T4DNA连接酶将载体和片段进行连接，并转化到大肠杆菌JM109感受态细胞中，通过菌落PCR和Sanger测序(pTrc99A-R，pTrc99A-F)进行验证筛选。验证正确后的质粒命名为pTrc99a-panD(质粒图如图2所示,菌落PCR检测结果如图4所示)。
实施例4重组质粒pRSF-yne1-pa0123的构建
用Nco I和Hind III双酶切质粒pRSFDuet-1，切胶回收目的基因片段pRSF-1(3761bp)，以金唯智公司合成的质粒pUC57-pa0123(将SEQ ID NO.4所示的pa0123基因克隆在pUC57质粒的Sma I位点)为模板，用引物pa0123-F/R，PCR扩增得到目的基因片段pa0123(序列如SEQ ID NO.4所示)，然后将pRSF-1和pa0123片段用T4 DNA连接酶连接，化转至大肠杆菌JM109感受态细胞中，菌落PCR挑取阳性转化子，并提取质粒进行酶切和PCR验证，验证引物为veri-pRSF-F/R，验证正确后的质粒命名为pRSF-pa0123。
用Xho I和Nde I酶切质粒pRSF-pa0123，切胶回收目的基因片段pRSF-2(5101bp)。,以大肠杆菌BL21基因组为模板，用引物yne1-F/R，PCR扩增得到的yne1片段(如SEQ ID NO.6所示)，然后将pRSF-2和yne1这两个目的片段用T4 DNA连接酶连接，化转至大肠杆菌JM109感受态细胞，菌落PCR挑取阳性转化子，并提取质粒酶切和PCR验证，验证引物为veri-pRSF-F/R。验证正确后的质粒命名为pRSF-yne1-pa0123(质粒图如图2所示,菌落PCR检测结果如图5所示)。
实施例5：重组大肠杆菌的构建
将pCDF-ppc-aspC，pTrc99a–panD和pRSF-yne1-pa0123三个表达质粒共同转入大肠杆菌BL21(DE3)感受态细胞中，制备重组大肠杆菌MAEC-2。
实施例6：表达不同基因的重组大肠杆菌的蛋白表达
(1)ppc和aspC基因的表达检测
首先制备大肠杆菌BL21的感受态细胞，用化学转化法转入pCDF-ppc-aspC质粒。挑取单菌落接种于50ml的LB液体培养基中，37℃、250rpm摇瓶过夜培养。按2％接种量接种于50ml的SOB培养基中，使其初始OD600为0.1。再于37℃、250rpm培养至OD600为0.8～1.0时加入1.0mM IPTG，于30℃、250rpm诱导蛋白的表达。以培养12h的大肠杆菌BL21发酵液做对照，选取发酵4h，8h，12h，24h，36h，48h的发酵液进行SDS聚丙烯酰胺凝胶电泳，电泳结果如图6所示，结果显示ppc和aspC基因成功表达。
(2)panD基因的表达检测
首先用化学转化法将pTrc99a-panD质粒转入大肠杆菌BL21感受态细胞，挑取单菌落接种于50ml的LB液体培养基中，37℃、250rpm摇瓶过夜。按2％接种量接种于50ml的SOB培养基中，使其初始OD600为0.1。37℃、250rpm培养至OD600分别为0.8～1.0时，加入1.0mM IPTG诱导，于30℃、250rpm诱导蛋白的表达。同时以12h的大肠杆菌BL21发酵液做对照，选取发酵4h，8h，12h，24h，36h，48h的发酵液进行SDS聚丙烯酰胺凝胶电泳，电泳结果如图7所示，结果显示panD基因成功表达。
(3)yne1和pa0123基因的表达检测
首先用化学转化法将pRSF-yne1-pa0132质粒转入大肠杆菌BL21感受态细胞，挑取单菌落接种于50ml的LB液体培养基中，37℃、250rpm摇瓶过夜。按2％接种量接种于50ml的SOB养基中，使其初始OD600为0.1。于37℃、250rpm培养至OD600分别为0.8～1.0时加入1.0mMIPTG诱导重组菌，于30℃、250rpm诱导蛋白的表达。同时以12h的大肠杆菌BL21发酵液做对照，选取发酵4h，8h，12h，24h，36h，48h的发酵液进行SDS聚丙烯酰胺凝胶电泳，电泳结果如图8所示，结果显示yne1和pa0123基因成功表达。
实施例7：重组大肠杆菌MAEC-2的摇瓶发酵
发酵培养基：SOB培养基。
种子液制备：将用甘油保藏的实施例6构建的重组大肠杆菌MAEC-2于平板上划线，挑取单菌落接种于盛有50ml的LB液体培养基的250ml锥形瓶中，37℃、250rpm摇瓶过夜培养。
发酵条件：按体积计，以2％接种量(1ml)将种子液接种于摇瓶发酵培养基中，使其初始OD600为0.1。于37℃、250rpm培养至OD600为0.8～1.0时，加入1.0mM IPTG，于30℃、250rpm诱导蛋白的表达。分别在有氧(培养箱通入正常空气)、厌氧(培养箱通入含5％CO2的空气)及前24h供氧后厌氧培养。
结果分析：发酵过程中在4h，8h，12h，24h，36h，48h，60h和72h各取一次样，12,000rpm离心5min将发酵液与菌体分离，0.22μm滤膜处理发酵液，将上清液用于进行HPLC(高效液相色谱法，美国伯Bio-Rad伯乐Aminex HPX-87H有机酸柱)检测，HPLC检测中流动相为5mM H2SO4，柱温为30℃，示差检测器。检测结果如图9所示。
根据液相结果，重组大肠杆菌MAEC-2在有氧的条件下发酵时在36h丙二酸产量最高，为0.74g/L；在厌氧发酵条件下，发酵48h时丙二酸产量最高，为0.08g/L；在前24h供氧，24h后厌氧的条件下，在发酵72h时丙二酸产量最高，为0.04g/L。由上述实验结果可知，工程菌有氧发酵条件下丙二酸生产能力最高，为0.74g/L，较前期申请号为201911420851.0的专利申请中构建的工程菌丙二酸产量0.23g/L提高了3倍。由于摇瓶发酵的供氧能力有限，可以预期的是，将重组大肠杆菌MAEC-2用于更大体量的发酵罐中进行培养，可获得比摇瓶水平提高更多的丙二酸产量。
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> 一种利用大肠杆菌全生物合成丙二酸的方法
<160> 11
<170> PatentIn version 3.3
<210> 1
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atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60
gaaaccatca aggatgcgtt gggagaacac attcttgaac gcgtagaaac tatccgtaag 120
ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180
caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240
ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300
ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360
accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420
gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480
ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540
ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600
gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660
ctgcgcgaac tgaacgaaca actggaagag aacctcggct acaaactgcc cgtcgaattt 720
gttccggtcc gttttacttc gtggatgggc ggcgaccgcg acggcaaccc gaacgtcact 780
gccgatatca cccgccacgt cctgctactc agccgctgga aagccaccga tttgttcctg 840
aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900
gcgctggttg gcgaagaagg tgccgcagaa ccgtatcgct atctgatgaa aaacctgcgt 960
tctcgcctga tggcgacaca ggcatggctg gaagcgcgcc tgaaaggcga agaactgcca 1020
aaaccagaag gcctgctgac acaaaacgaa gaactgtggg aaccgctcta cgcttgctac 1080
cagtcacttc aggcgtgtgg catgggtatt atcgccaacg gcgatctgct cgacaccctg 1140
cgccgcgtga aatgtttcgg cgtaccgctg gtccgtattg atatccgtca ggagagcacg 1200
cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260
tggtcagagg ccgacaaaca ggcgttcctg atccgcgaac tgaactccaa acgtccgctt 1320
ctgccgcgca actggcaacc aagcgccgaa acgcgcgaag tgctcgatac ctgccaggtg 1380
attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440
tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500
gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560
ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620
tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680
caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740
cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800
ccaggaagcc tgaaaggcgg cctgcgcgta accgaacagg gcgagatgat ccgctttaaa 1860
tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920
gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980
tcagtcatct cctgcgatgt ctaccgcggc tacgtacgtg aaaacaaaga ttttgtgcct 2040
tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100
gcgaaacgtc gcccaaccgg cggcgtcgag tcactacgcg ccattccgtg gatcttcgcc 2160
tggacgcaaa accgtctgat gctccccgcc tggctgggtg caggtacggc gctgcaaaaa 2220
gtggtcgaag acggcaaaca gagcgagctg gaggctatgt gccgcgattg gccattcttc 2280
tcgacgcgtc tcggcatgct ggagatggtc ttcgccaaag cagacctgtg gctggcggaa 2340
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gatctgccgt ggattgcaga gtctattcag ctacggaata tttacaccga cccgctgaac 2520
gtattgcagg ccgagttgct gcaccgctcc cgccaggcag aaaaagaagg ccaggaaccg 2580
gatcctcgcg tcgaacaagc gttaatggtc actattgccg ggattgcggc aggtatgcgt 2640
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atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60
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ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180
accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240
ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300
ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360
aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420
ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480
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tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660
ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720
gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780
gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840
gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900
gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960
atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020
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ggcgagaaag tggcgattgt agacattacc aacggggctc gcttggaaac ttatgtcatt 180
gtgggtgatg cgggaactgg gaacatctgc attaacgggg ccgcagctca tctgatcaat 240
ccgggcgatt tggtgatcat catgtcatat ttgcaagcga cggatgcaga agctaaagca 300
tatgagccga agatcgtcca tgtcgacgct gataaccgca ttgtggcgct gggaaacgac 360
ctggctgagg ccttgccagg ttcaggcctt ttaaccagtc gctcgatcta g 411
<210> 4
<211> 1347
<212> DNA
<213> Pseudomona aeruginosa
<400> 4
atgaatcagc ccctgaatgt cgctccgccc gtgagctcgg aattaaacct gcgcgcccac 60
tggatgccat tttcggctaa ccgcaatttc caaaaagacc cgcgtattat cgtcgcggcg 120
gagggctcct ggctgaccga cgacaagggc cgtaaagtat acgatagcct gtcaggatta 180
tggacctgcg gtgcggggca tagccgcaag gaaattcagg aagcggttgc tcgtcaactg 240
gggactttgg actattcgcc aggattccaa tatggacatc cattgtcttt ccagttggcc 300
gagaagattg ctgggttatt acctggggaa ttaaaccatg tcttttttac gggatcaggg 360
tcggagtgcg cagacacttc gattaagatg gcccgcgcct actggcgctt aaagggacaa 420
ccccagaaga ctaagctgat tggacgcgca cgcggttacc acggcgtgaa tgtcgcgggc 480
acaagccttg gagggatcgg ggggaaccgc aagatgttcg gacagctgat ggatgtggac 540
catcttcccc atacccttca gccaggtatg gcattcactc gcgggatggc gcagacagga 600
ggcgttgaac tggcgaatga gttattaaag ttaattgaat tgcacgatgc gtctaacatc 660
gcagcggtta ttgtcgagcc catgtccggt tccgcaggag ttttagtgcc acccgtgggc 720
tatctgcagc gtttgcgcga aatctgtgac caacacaata ttctgcttat ctttgatgaa 780
gtgattacgg ctttcgggcg tctgggtact tactcgggag ccgaatactt cggggtcacg 840
ccggacttga tgaatgttgc aaaacaggtc acgaatggtg cagtacctat gggtgctgta 900
atcgcctcta gcgagattta cgatactttc atgaaccagg cgctgcctga acatgcggtt 960
gaattttccc acggttatac atattcagcg cacccagtgg catgtgctgc gggattagca 1020
gcactggaca tcttggcgcg cgataactta gtacagcagt cagcagagtt agctccacac 1080
ttcgagaagg gattgcatgg ccttcaaggt gccaagaatg ttatcgacat tcgcaactgc 1140
ggcttagcag gcgcgatcca gatcgctccc cgtgatgggg atccgacagt tcgccccttt 1200
gaagccggga tgaaactgtg gcaacaaggg ttttacgtcc gtttcggcgg cgacactctg 1260
cagtttgggc caacatttaa tgcacgccca gaggaattgg accgtctttt tgacgctgta 1320
ggtgaggctc tgaacggaat tgcctga 1347
<210> 5
<211> 1389
<212> DNA
<213> Escherichia coli
<400> 5
atgaccatta ctccggcaac tcatgcaatt tcgataaatc ctgccacggg tgaacaactt 60
tctgtgctgc cgtgggctgg cgctgacgat atcgaaaacg cacttcagct ggcggcagca 120
ggctttcgcg actggcgcga gacaaatata gattatcgtg ctgaaaaact gcgtgatatc 180
ggtaaggctc tgcgcgctcg tagcgaagaa atggcgcaaa tgatcacccg cgaaatgggc 240
aaaccaatca accaggcgcg cgctgaagtg gcgaaatcgg cgaatttgtg tgactggtat 300
gcagaacatg gtccggcaat gctgaaggcg gaacctacgc tggtggaaaa tcagcaggcg 360
gttattgagt atcgaccgtt ggggacgatt ctggcgatta tgccgtggaa ttttccgtta 420
tggcaggtga tgcgtggcgc tgttcccatc attcttgcag gtaacggcta cttacttaaa 480
catgcgccga atgtgatggg ctgtgcacag ctcattgccc aggtgtttaa agatgcgggt 540
atcccacaag gcgtatatgg ctggctgaat gccgacaacg acggtgtcag tcagatgatt 600
aaagactcgc gcattgctgc tgtcacggtg accggaagtg ttcgtgcggg agcggctatt 660
ggcgcacagg ctggagcggc actgaaaaaa tgcgtactgg aactgggcgg ttcggatccg 720
tttattgtgc ttaacgatgc cgatctggaa ctggcggtga aagcggcggt agccggacgt 780
tatcagaata ccggacaggt atgtgcagcg gcaaaacgct ttattatcga agagggaatt 840
gcttcggcat ttaccgaacg ttttgtggca gctgcggcag ccttgaaaat gggcgatccc 900
cgtgacgaag agaacgctct cggaccaatg gctcgttttg atttacgtga tgagctgcat 960
catcaggtgg agaaaaccct ggcgcagggt gcgcgtttgt tactgggcgg ggaaaagatg 1020
gctggggcag gtaactacta tccgccaacg gttctggcga atgttacccc agaaatgacc 1080
gcgtttcggg aagaaatgtt tggccccgtt gcggcaatca ccattgcgaa agatgcagaa 1140
catgcactgg aactggctaa tgatagtgag ttcggccttt cagcgaccat ttttaccact 1200
gacgaaacac aggccagaca gatggcggca cgtctggaat gcggtggggt gtttatcaat 1260
ggttattgtg ccagcgacgc gcgagtggcc tttggtggcg tgaaaaagag tggctttggt 1320
cgtgagcttt cccatttcgg cttacacgaa ttctgtaata tccagacggt gtggaaagac 1380
cggatctga 1389
<210> 6
<211> 390
<212> DNA
<213> 人工序列
<400> 6
atgataagaa atgtgaaaaa acaaagacct gttaatctgg acctacagac catccggttc 60
cccatcacgg cgatagcgtc cattctccat cgcgtttccg gtgtgatcac ctttgttgca 120
gtgggcatcc tgctgtggct tctgggtacc agcctctctt cccctgaagg tttcgagcaa 180
gcttccgcga ttatgggcag cttcttcgtc aaatttatca tgtggggcat ccttaccgct 240
ctggcgtatc acgtcgtcgt aggtattcgc cacatgatga tggattttgg ctatctggaa 300
gaaacattcg aagcgggtaa acgctccgcc aaaatctcct ttgttattac tgtcgtgctt 360
tcacttctcg caggagtcct cgtatggtaa 390
<210> 7
<211> 883
<212> PRT
<213> Escherichia coli
<400> 7
Met Asn Glu Gln Tyr Ser Ala Leu Arg Ser Asn Val Ser Met Leu Gly
1 5 10 15
Lys Val Leu Gly Glu Thr Ile Lys Asp Ala Leu Gly Glu His Ile Leu
20 25 30
Glu Arg Val Glu Thr Ile Arg Lys Leu Ser Lys Ser Ser Arg Ala Gly
35 40 45
Asn Asp Ala Asn Arg Gln Glu Leu Leu Thr Thr Leu Gln Asn Leu Ser
50 55 60
Asn Asp Glu Leu Leu Pro Val Ala Arg Ala Phe Ser Gln Phe Leu Asn
65 70 75 80
Leu Ala Asn Thr Ala Glu Gln Tyr His Ser Ile Ser Pro Lys Gly Glu
85 90 95
Ala Ala Ser Asn Pro Glu Val Ile Ala Arg Thr Leu Arg Lys Leu Lys
100 105 110
Asn Gln Pro Glu Leu Ser Glu Asp Thr Ile Lys Lys Ala Val Glu Ser
115 120 125
Leu Ser Leu Glu Leu Val Leu Thr Ala His Pro Thr Glu Ile Thr Arg
130 135 140
Arg Thr Leu Ile His Lys Met Val Glu Val Asn Ala Cys Leu Lys Gln
145 150 155 160
Leu Asp Asn Lys Asp Ile Ala Asp Tyr Glu His Asn Gln Leu Met Arg
165 170 175
Arg Leu Arg Gln Leu Ile Ala Gln Ser Trp His Thr Asp Glu Ile Arg
180 185 190
Lys Leu Arg Pro Ser Pro Val Asp Glu Ala Lys Trp Gly Phe Ala Val
195 200 205
Val Glu Asn Ser Leu Trp Gln Gly Val Pro Asn Tyr Leu Arg Glu Leu
210 215 220
Asn Glu Gln Leu Glu Glu Asn Leu Gly Tyr Lys Leu Pro Val Glu Phe
225 230 235 240
Val Pro Val Arg Phe Thr Ser Trp Met Gly Gly Asp Arg Asp Gly Asn
245 250 255
Pro Asn Val Thr Ala Asp Ile Thr Arg His Val Leu Leu Leu Ser Arg
260 265 270
Trp Lys Ala Thr Asp Leu Phe Leu Lys Asp Ile Gln Val Leu Val Ser
275 280 285
Glu Leu Ser Met Val Glu Ala Thr Pro Glu Leu Leu Ala Leu Val Gly
290 295 300
Glu Glu Gly Ala Ala Glu Pro Tyr Arg Tyr Leu Met Lys Asn Leu Arg
305 310 315 320
Ser Arg Leu Met Ala Thr Gln Ala Trp Leu Glu Ala Arg Leu Lys Gly
325 330 335
Glu Glu Leu Pro Lys Pro Glu Gly Leu Leu Thr Gln Asn Glu Glu Leu
340 345 350
Trp Glu Pro Leu Tyr Ala Cys Tyr Gln Ser Leu Gln Ala Cys Gly Met
355 360 365
Gly Ile Ile Ala Asn Gly Asp Leu Leu Asp Thr Leu Arg Arg Val Lys
370 375 380
Cys Phe Gly Val Pro Leu Val Arg Ile Asp Ile Arg Gln Glu Ser Thr
385 390 395 400
Arg His Thr Glu Ala Leu Gly Glu Leu Thr Arg Tyr Leu Gly Ile Gly
405 410 415
Asp Tyr Glu Ser Trp Ser Glu Ala Asp Lys Gln Ala Phe Leu Ile Arg
420 425 430
Glu Leu Asn Ser Lys Arg Pro Leu Leu Pro Arg Asn Trp Gln Pro Ser
435 440 445
Ala Glu Thr Arg Glu Val Leu Asp Thr Cys Gln Val Ile Ala Glu Ala
450 455 460
Pro Gln Gly Ser Ile Ala Ala Tyr Val Ile Ser Met Ala Lys Thr Pro
465 470 475 480
Ser Asp Val Leu Ala Val His Leu Leu Leu Lys Glu Ala Gly Ile Gly
485 490 495
Phe Ala Met Pro Val Ala Pro Leu Phe Glu Thr Leu Asp Asp Leu Asn
500 505 510
Asn Ala Asn Asp Val Met Thr Gln Leu Leu Asn Ile Asp Trp Tyr Arg
515 520 525
Gly Leu Ile Gln Gly Lys Gln Met Val Met Ile Gly Tyr Ser Asp Ser
530 535 540
Ala Lys Asp Ala Gly Val Met Ala Ala Ser Trp Ala Gln Tyr Gln Ala
545 550 555 560
Gln Asp Ala Leu Ile Lys Thr Cys Glu Lys Ala Gly Ile Glu Leu Thr
565 570 575
Leu Phe His Gly Arg Gly Gly Ser Ile Gly Arg Gly Gly Ala Pro Ala
580 585 590
His Ala Ala Leu Leu Ser Gln Pro Pro Gly Ser Leu Lys Gly Gly Leu
595 600 605
Arg Val Thr Glu Gln Gly Glu Met Ile Arg Phe Lys Tyr Gly Leu Pro
610 615 620
Glu Ile Thr Val Ser Ser Leu Ser Leu Tyr Thr Gly Ala Ile Leu Glu
625 630 635 640
Ala Asn Leu Leu Pro Pro Pro Glu Pro Lys Glu Ser Trp Arg Arg Ile
645 650 655
Met Asp Glu Leu Ser Val Ile Ser Cys Asp Val Tyr Arg Gly Tyr Val
660 665 670
Arg Glu Asn Lys Asp Phe Val Pro Tyr Phe Arg Ser Ala Thr Pro Glu
675 680 685
Gln Glu Leu Gly Lys Leu Pro Leu Gly Ser Arg Pro Ala Lys Arg Arg
690 695 700
Pro Thr Gly Gly Val Glu Ser Leu Arg Ala Ile Pro Trp Ile Phe Ala
705 710 715 720
Trp Thr Gln Asn Arg Leu Met Leu Pro Ala Trp Leu Gly Ala Gly Thr
725 730 735
Ala Leu Gln Lys Val Val Glu Asp Gly Lys Gln Ser Glu Leu Glu Ala
740 745 750
Met Cys Arg Asp Trp Pro Phe Phe Ser Thr Arg Leu Gly Met Leu Glu
755 760 765
Met Val Phe Ala Lys Ala Asp Leu Trp Leu Ala Glu Tyr Tyr Asp Gln
770 775 780
Arg Leu Val Asp Lys Ala Leu Trp Pro Leu Gly Lys Glu Leu Arg Asn
785 790 795 800
Leu Gln Glu Glu Asp Ile Lys Val Val Leu Ala Ile Ala Asn Asp Ser
805 810 815
His Leu Met Ala Asp Leu Pro Trp Ile Ala Glu Ser Ile Gln Leu Arg
820 825 830
Asn Ile Tyr Thr Asp Pro Leu Asn Val Leu Gln Ala Glu Leu Leu His
835 840 845
Arg Ser Arg Gln Ala Glu Lys Glu Gly Gln Glu Pro Asp Pro Arg Val
850 855 860
Glu Gln Ala Leu Met Val Thr Ile Ala Gly Ile Ala Ala Gly Met Arg
865 870 875 880
Asn Thr Gly
<210> 8
<211> 396
<212> PRT
<213> Escherichia coli
<400> 8
Met Phe Glu Asn Ile Thr Ala Ala Pro Ala Asp Pro Ile Leu Gly Leu
1 5 10 15
Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Leu Gly
20 25 30
Ile Gly Val Tyr Lys Asp Glu Thr Gly Lys Thr Pro Val Leu Thr Ser
35 40 45
Val Lys Lys Ala Glu Gln Tyr Leu Leu Glu Asn Glu Thr Thr Lys Asn
50 55 60
Tyr Leu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Gln Glu
65 70 75 80
Leu Leu Phe Gly Lys Gly Ser Ala Leu Ile Asn Asp Lys Arg Ala Arg
85 90 95
Thr Ala Gln Thr Pro Gly Gly Thr Gly Ala Leu Arg Val Ala Ala Asp
100 105 110
Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro
115 120 125
Ser Trp Pro Asn His Lys Ser Val Phe Asn Ser Ala Gly Leu Glu Val
130 135 140
Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu Asn His Thr Leu Asp Phe Asp
145 150 155 160
Ala Leu Ile Asn Ser Leu Asn Glu Ala Gln Ala Gly Asp Val Val Leu
165 170 175
Phe His Gly Cys Cys His Asn Pro Thr Gly Ile Asp Pro Thr Leu Glu
180 185 190
Gln Trp Gln Thr Leu Ala Gln Leu Ser Val Glu Lys Gly Trp Leu Pro
195 200 205
Leu Phe Asp Phe Ala Tyr Gln Gly Phe Ala Arg Gly Leu Glu Glu Asp
210 215 220
Ala Glu Gly Leu Arg Ala Phe Ala Ala Met His Lys Glu Leu Ile Val
225 230 235 240
Ala Ser Ser Tyr Ser Lys Asn Phe Gly Leu Tyr Asn Glu Arg Val Gly
245 250 255
Ala Cys Thr Leu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe
260 265 270
Ser Gln Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Asn Pro Pro Ala
275 280 285
His Gly Ala Ser Val Val Ala Thr Ile Leu Ser Asn Asp Ala Leu Arg
290 295 300
Ala Ile Trp Glu Gln Glu Leu Thr Asp Met Arg Gln Arg Ile Gln Arg
305 310 315 320
Met Arg Gln Leu Phe Val Asn Thr Leu Gln Glu Lys Gly Ala Asn Arg
325 330 335
Asp Phe Ser Phe Ile Ile Lys Gln Asn Gly Met Phe Ser Phe Ser Gly
340 345 350
Leu Thr Lys Glu Gln Val Leu Arg Leu Arg Glu Glu Phe Gly Val Tyr
355 360 365
Ala Val Ala Ser Gly Arg Val Asn Val Ala Gly Met Thr Pro Asp Asn
370 375 380
Met Ala Pro Leu Cys Glu Ala Ile Val Ala Val Leu
385 390 395
<210> 9
<211> 136
<212> PRT
<213> Corynebacterium glutamicum
<400> 9
Met Leu Arg Thr Ile Leu Gly Ser Lys Ile His Arg Ala Thr Val Thr
1 5 10 15
Gln Ala Asp Leu Asp Tyr Val Gly Ser Val Thr Ile Asp Ala Asp Leu
20 25 30
Val His Ala Ala Gly Leu Ile Glu Gly Glu Lys Val Ala Ile Val Asp
35 40 45
Ile Thr Asn Gly Ala Arg Leu Glu Thr Tyr Val Ile Val Gly Asp Ala
50 55 60
Gly Thr Gly Asn Ile Cys Ile Asn Gly Ala Ala Ala His Leu Ile Asn
65 70 75 80
Pro Gly Asp Leu Val Ile Ile Met Ser Tyr Leu Gln Ala Thr Asp Ala
85 90 95
Glu Ala Lys Ala Tyr Glu Pro Lys Ile Val His Val Asp Ala Asp Asn
100 105 110
Arg Ile Val Ala Leu Gly Asn Asp Leu Ala Glu Ala Leu Pro Gly Ser
115 120 125
Gly Leu Leu Thr Ser Arg Ser Ile
130 135
<210> 10
<211> 448
<212> PRT
<213> Pseudomona aeruginosa
<400> 10
Met Asn Gln Pro Leu Asn Val Ala Pro Pro Val Ser Ser Glu Leu Asn
1 5 10 15
Leu Arg Ala His Trp Met Pro Phe Ser Ala Asn Arg Asn Phe Gln Lys
20 25 30
Asp Pro Arg Ile Ile Val Ala Ala Glu Gly Ser Trp Leu Thr Asp Asp
35 40 45
Lys Gly Arg Lys Val Tyr Asp Ser Leu Ser Gly Leu Trp Thr Cys Gly
50 55 60
Ala Gly His Ser Arg Lys Glu Ile Gln Glu Ala Val Ala Arg Gln Leu
65 70 75 80
Gly Thr Leu Asp Tyr Ser Pro Gly Phe Gln Tyr Gly His Pro Leu Ser
85 90 95
Phe Gln Leu Ala Glu Lys Ile Ala Gly Leu Leu Pro Gly Glu Leu Asn
100 105 110
His Val Phe Phe Thr Gly Ser Gly Ser Glu Cys Ala Asp Thr Ser Ile
115 120 125
Lys Met Ala Arg Ala Tyr Trp Arg Leu Lys Gly Gln Pro Gln Lys Thr
130 135 140
Lys Leu Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Val Ala Gly
145 150 155 160
Thr Ser Leu Gly Gly Ile Gly Gly Asn Arg Lys Met Phe Gly Gln Leu
165 170 175
Met Asp Val Asp His Leu Pro His Thr Leu Gln Pro Gly Met Ala Phe
180 185 190
Thr Arg Gly Met Ala Gln Thr Gly Gly Val Glu Leu Ala Asn Glu Leu
195 200 205
Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile
210 215 220
Val Glu Pro Met Ser Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly
225 230 235 240
Tyr Leu Gln Arg Leu Arg Glu Ile Cys Asp Gln His Asn Ile Leu Leu
245 250 255
Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Tyr Ser
260 265 270
Gly Ala Glu Tyr Phe Gly Val Thr Pro Asp Leu Met Asn Val Ala Lys
275 280 285
Gln Val Thr Asn Gly Ala Val Pro Met Gly Ala Val Ile Ala Ser Ser
290 295 300
Glu Ile Tyr Asp Thr Phe Met Asn Gln Ala Leu Pro Glu His Ala Val
305 310 315 320
Glu Phe Ser His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala
325 330 335
Ala Gly Leu Ala Ala Leu Asp Ile Leu Ala Arg Asp Asn Leu Val Gln
340 345 350
Gln Ser Ala Glu Leu Ala Pro His Phe Glu Lys Gly Leu His Gly Leu
355 360 365
Gln Gly Ala Lys Asn Val Ile Asp Ile Arg Asn Cys Gly Leu Ala Gly
370 375 380
Ala Ile Gln Ile Ala Pro Arg Asp Gly Asp Pro Thr Val Arg Pro Phe
385 390 395 400
Glu Ala Gly Met Lys Leu Trp Gln Gln Gly Phe Tyr Val Arg Phe Gly
405 410 415
Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Ala Arg Pro Glu Glu
420 425 430
Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Ala Leu Asn Gly Ile Ala
435 440 445
<210> 11
<211> 462
<212> PRT
<213> Escherichia coli
<400> 11
Met Thr Ile Thr Pro Ala Thr His Ala Ile Ser Ile Asn Pro Ala Thr
1 5 10 15
Gly Glu Gln Leu Ser Val Leu Pro Trp Ala Gly Ala Asp Asp Ile Glu
20 25 30
Asn Ala Leu Gln Leu Ala Ala Ala Gly Phe Arg Asp Trp Arg Glu Thr
35 40 45
Asn Ile Asp Tyr Arg Ala Glu Lys Leu Arg Asp Ile Gly Lys Ala Leu
50 55 60
Arg Ala Arg Ser Glu Glu Met Ala Gln Met Ile Thr Arg Glu Met Gly
65 70 75 80
Lys Pro Ile Asn Gln Ala Arg Ala Glu Val Ala Lys Ser Ala Asn Leu
85 90 95
Cys Asp Trp Tyr Ala Glu His Gly Pro Ala Met Leu Lys Ala Glu Pro
100 105 110
Thr Leu Val Glu Asn Gln Gln Ala Val Ile Glu Tyr Arg Pro Leu Gly
115 120 125
Thr Ile Leu Ala Ile Met Pro Trp Asn Phe Pro Leu Trp Gln Val Met
130 135 140
Arg Gly Ala Val Pro Ile Ile Leu Ala Gly Asn Gly Tyr Leu Leu Lys
145 150 155 160
His Ala Pro Asn Val Met Gly Cys Ala Gln Leu Ile Ala Gln Val Phe
165 170 175
Lys Asp Ala Gly Ile Pro Gln Gly Val Tyr Gly Trp Leu Asn Ala Asp
180 185 190
Asn Asp Gly Val Ser Gln Met Ile Lys Asp Ser Arg Ile Ala Ala Val
195 200 205
Thr Val Thr Gly Ser Val Arg Ala Gly Ala Ala Ile Gly Ala Gln Ala
210 215 220
Gly Ala Ala Leu Lys Lys Cys Val Leu Glu Leu Gly Gly Ser Asp Pro
225 230 235 240
Phe Ile Val Leu Asn Asp Ala Asp Leu Glu Leu Ala Val Lys Ala Ala
245 250 255
Val Ala Gly Arg Tyr Gln Asn Thr Gly Gln Val Cys Ala Ala Ala Lys
260 265 270
Arg Phe Ile Ile Glu Glu Gly Ile Ala Ser Ala Phe Thr Glu Arg Phe
275 280 285
Val Ala Ala Ala Ala Ala Leu Lys Met Gly Asp Pro Arg Asp Glu Glu
290 295 300
Asn Ala Leu Gly Pro Met Ala Arg Phe Asp Leu Arg Asp Glu Leu His
305 310 315 320
His Gln Val Glu Lys Thr Leu Ala Gln Gly Ala Arg Leu Leu Leu Gly
325 330 335
Gly Glu Lys Met Ala Gly Ala Gly Asn Tyr Tyr Pro Pro Thr Val Leu
340 345 350
Ala Asn Val Thr Pro Glu Met Thr Ala Phe Arg Glu Glu Met Phe Gly
355 360 365
Pro Val Ala Ala Ile Thr Ile Ala Lys Asp Ala Glu His Ala Leu Glu
370 375 380
Leu Ala Asn Asp Ser Glu Phe Gly Leu Ser Ala Thr Ile Phe Thr Thr
385 390 395 400
Asp Glu Thr Gln Ala Arg Gln Met Ala Ala Arg Leu Glu Cys Gly Gly
405 410 415
Val Phe Ile Asn Gly Tyr Cys Ala Ser Asp Ala Arg Val Ala Phe Gly
420 425 430
Gly Val Lys Lys Ser Gly Phe Gly Arg Glu Leu Ser His Phe Gly Leu
435 440 445
His Glu Phe Cys Asn Ile Gln Thr Val Trp Lys Asp Arg Ile
450 455 460

Claims (7)

1.一种重组大肠杆菌，其特征在于，以葡萄糖为原料，由草酰乙酸-天冬氨酸途径合成丙二酸；所述重组大肠杆菌分模块表达了磷酸烯醇丙酮酸羧化酶、天冬氨酸转氨酶、琥珀酸半醛脱氢酶、天冬氨酸-α-脱氢酶和β-丙氨酸丙酮酸转氨酶；所述分模块表达是将磷酸烯醇丙酮酸羧化酶基因ppc和天冬氨酸转氨酶基因aspC共表达，将天冬氨酸-α-脱氢酶基因panD单独表达，将琥珀酸半醛脱氢酶基因yne1和β-丙氨酸丙酮酸转氨酶基因pa0123共表达；
所述磷酸烯醇丙酮酸羧化酶基因的核苷酸序列如SEQ ID NO.1所示；
所述天冬氨酸转氨酶基因的核苷酸序列如SEQ ID NO.2所示；
所述天冬氨酸-α-脱氢酶基因的核苷酸序列如SEQ ID NO.3所示；
所述β-丙氨酸丙酮酸转氨酶基因的核苷酸序列如SEQ ID NO.4所示；
所述琥珀酸半醛脱氢酶基因的核苷酸序列如SEQ ID NO.5所示。
2.根据权利要求1所述的重组大肠杆菌，其特征在于，以大肠杆菌BL21(DE3)为宿主。
3.一种构建权利要求1或2所述重组大肠杆菌的方法，其特征在于，包括以下步骤：
（1）以质粒pCDFDuet-1为骨架载体，连接基因片段ppc和aspC，得到重组质粒pCDF-ppc-aspC；
（2）以质粒pTrc99a为骨架载体，连接基因片段panD，得到重组质粒pTrc99a-panD；
（3）以质粒pRSFDuet-1为骨架载体，连接基因片段yne1、pa0123，得到重组质粒pRSF-yne1-pa0123；
（4）将pCDF-ppc-aspC、pTrc99a-panD和pRSF-yne1-pa0123共同转入大肠杆菌BL21(DE3)，得到重组大肠杆菌。
4.一种发酵生产丙二酸的方法，其特征在于，将权利要求1或2所述的重组大肠杆菌接种于含葡萄糖的培养基中，于在有氧的环境下，35~37℃发酵至少24 h。
5.根据权利要求4所述的方法，其特征在于，将所述重组大肠杆菌于35~37℃、200~280rpm培养至OD600为0.8-1.0时加入IPTG，于28~30℃诱导培养至少24 h。
6.根据权利要求5所述的方法，其特征在于，所述重组大肠杆菌还经过种子液培养；所述种子液培养是将所述重组大肠杆菌在LB培养集中培养至OD600达到0.8-1.0，获得种子液，再接种至发酵培养基中发酵。
7.权利要求1或2所述的重组大肠杆菌或权利要求4~6任一所述方法在制备丙二酸及含丙二酸的产品方面的应用。
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