CN106929459A - 一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖二酸的方法 - Google Patents

一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖二酸的方法 Download PDF

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CN106929459A
CN106929459A CN201710200371.8A CN201710200371A CN106929459A CN 106929459 A CN106929459 A CN 106929459A CN 201710200371 A CN201710200371 A CN 201710200371A CN 106929459 A CN106929459 A CN 106929459A
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娄文勇
苏慧慧
宗敏华
徐培
杨继国
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Abstract

本发明公开了一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖二酸的方法，属于代谢工程领域。本发明通过在大肠杆菌中表达来源于植物的肌醇‑1‑磷酸合成酶基因（Ino1）、肌醇氧化酶基因（MIOX），实现葡萄糖醛酸的生产途径，同时表达来源于根癌农杆菌（Agrobacterium tumefaciens）的醛酸脱氢酶基因（Udh），成功构建葡萄糖二酸的合成途径。其中，重组大肠杆菌在LB‑G（LB培养基中加入10g/L葡萄糖）发酵培养基中生成葡萄糖二酸2.53g/L。本发明提出的重组大肠杆菌发酵生产葡萄糖二酸的方法，具有广阔的发展前景，为生物合成葡萄糖二酸奠定基础。

Description

一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖 二酸的方法
技术领域
本发明属于代谢工程领域，具体涉及一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖二酸的方法。
背景技术
葡萄糖二酸(glucuric acid,saccharic acid)又名葡萄糖质酸，是含有4个手性碳原子的化合物，通常以手性化合物D-葡萄糖二酸的形式存在，在水溶液中自发氧化，形成单内酯D-葡萄糖二酸-1,4内酯和D-葡萄糖二酸-3,6-内酯以及少量的双内酯D-葡萄糖二酸-1,4；3,6-内酯。
葡萄糖二酸是一种天然、无毒的化合物，少量生产于包括人类的哺乳动物和一些植物中，如番茄、葡萄柚等。葡萄糖二酸具有重要的生物功能，被美国能源部确定为“最有价值的生物炼制产品”。它和许多营养物质一样，可以帮助和增强免疫及身体排毒，进而减少人类疾病，研究称葡萄糖二酸有药理作用，它能特异性的强烈抑制一些与癌症发生有关的酶如β-葡萄糖醛酸苷酶的活性。它还可以降低胆固醇，同时其钙盐也是一种食品添加剂。葡萄糖二酸及其衍生物能明显抑制凝血酶诱导花生四烯酸过氧化反应，可以起到明显的抗炎症作用；葡萄糖二酸及其衍生物影响血小板在氧化应激条件的活化，通过抗氧化机制有助于预防过度血小板激活，因此葡萄糖二酸及其衍生物可能是预防心血管疾病风险的顶尖保健品，而一些水果蔬菜中含有大量的葡萄糖二酸及其衍生物，可作为膳食预防心血管疾病风险。
葡萄糖二酸由于对人体无毒，其结构与单糖结构相似，可以通过人体的糖转运***进入细胞内部，且能够聚集在病灶和少量被缺血组织快速吸收等优点，因此可以作为显像剂应用于心肌梗死和肿瘤的研究中。另外，葡萄糖二酸也可应用于生活生产中普遍用到的聚合物单体的生产中，且这些聚合物在自然界中水解为小分子单体可以被植物和微生物吸收利用，所以它作为合成多种高效环保的新兴生物质能源的原料，具有巨大的潜在经济价值。
目前葡萄糖二酸的制备方法主要是化学法，以硝酸氧化法和TEMPO为催化剂的氧化法等。硝酸氧化法主要将葡萄糖氧化为葡萄糖及小分子物质。此种方法生产的副产物多且复杂，同时有大量NO和NO2等有害气体排放，对环境产生污染，所以逐渐被新方法替代。TEMPO氧化法是指利用2,2,6,6-四甲基-1-哌啶自由基(TEMPO)介导的电化学氧化葡萄糖合成葡萄糖二酸的方法。由于它摧毁的氧化反应物金属参与反应且反应条件温和，对产物具有选择性并能够限制不可回收的副产物的产物，便于操作，是目前常用的一种方法；但是由于在反应过程中要控制反应温度和pH值以达到最佳，还有昂贵的催化剂也是需要改进的方面。
长期以来，微生物发酵法来实现有机酸高效环保生产的常用方法。同化学法相比，微生物发酵法在原料损耗、产品纯度等方面有很大改善，成为一种更优的合成方法。葡萄糖二酸是存在于哺乳动物体内作为代谢终产物被发现，在体内，从葡萄糖到葡萄糖二酸，至少需要10步反应。Prather等从酿酒酵母(Saccaromyces cerevisiae)和小鼠获取肌醇-1-磷酸合成酶(Ino1)和肌醇氧化酶(MIOX)，及丁香假单胞菌(Pseudomonas syringae)获得醛酸脱氢酶(Udh)，克隆到大肠杆菌中，实现了葡萄糖二酸的生物合成。
目前尚未发现有通过重组植物来源的肌醇氧化酶的大肠杆菌来发酵生产葡萄糖二酸的报道，植物中葡萄糖二酸含量少，且提取分离纯化较难，鉴于此，本发明中构建的植物来源基因异构重组在大肠杆菌，在微生物发酵生产葡萄糖二酸方面，提供了新的方法与思路。
发明内容
本发明通过将不同植物来源的肌醇-1-磷酸合成酶基因(Ino1)、肌醇氧化酶基因(MIOX)及来源于根癌农杆菌的醛酸脱氢酶基因(Udh)克隆表达至大肠菌株中，构建重组菌，该重组菌能以葡萄糖、甘油、蔗糖或者肌醇为底物合成葡萄糖二酸。
本发明通过以下技术方案实现。
一种重组大肠杆菌，该重组大肠杆菌同时表达肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX、醛酸脱氢酶基因Udh。
优选的，所述肌醇-1-磷酸合成酶基因Ino1来源于以下任意一种：酿酒酵母(Saccaromyces cerevisiae)、毕赤酵母(Pichia pastoris)、番茄(SolanaceaeLycopersicon 1706)、玉米(Zea mays)、大豆(Glycine max)。
进一步优选的，所述肌醇-1-磷酸合成酶基因Ino1，在本发明的一种实施方式中，来源于番茄(Solanaceae Lycopersicon 1706)。
优选的，所述肌醇氧化酶基因MIOX来源于以下任意一种：毕赤酵母(Pichiapastoris GS115)、番茄(Solanaceae Lycopersicon 1706)、玉米(Zea mays)、大豆(Glycine max)。
进一步优选的，所述肌醇氧化酶基因MIOX，在本发明的一种实施方式中，来源于番茄(Solanaceae Lycopersicon 1706)。
优选的，所述醛酸脱氢酶基因Udh来源于根癌农杆菌(Agrobacteriumtumefaciens GV3103)。
优选的，所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX、醛酸脱氢酶基因Udh，在本发明的一种实施方式中，其核苷酸序列分别是SEQ ID NO.1、SEQ ID NO.2、SEQID NO.3所示的序列。
优选的，所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX、醛酸脱氢酶基因Udh表达的蛋白质，在本发明的一种实施方式中，其氨基酸序列分别是SEQ ID NO.4、SEQID NO.5、SEQ ID NO.6所示的序列。
优选的，所述重组大肠杆菌的出发宿主菌为大肠杆菌。
优选的，所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX及醛酸脱氢酶基因Udh为整合表达。
以上所述的重组大肠杆菌的构建方法，所述方法包括如下步骤：
(1)将肌醇-1-磷酸合成酶基因Ino1和肌醇氧化酶基因MIOX连接到双元表达载体上转化到大肠杆宿主菌种，得到重组大肠杆菌1；
(2)将醛酸脱氢酶基因Udh连接到表达载体后转化到步骤(1)得到的重组大肠杆菌1中，得重组大肠杆菌。
优选的，该方法具体包括如下步骤：
(1)以番茄(Solanaceae Lycopersicon 1706)基因组为模板扩增MIOX基因、Ino1基因，并分别加上合适酶切位点，将片段连接到双元表达载体pETDuet1后，用化学法转化DH5α中，筛选获得正确的重组质粒，命名为pETDuet1-Ino1-MIOX，将重组质粒转化到宿主菌E.coli BL21(DE3)中既得到E.coli BL21(DE3)/Ino1-MIOX；
(2)以根癌农杆菌(Agrobacterium tumefaciens GV3103)基因组为模板扩增Udh基因，连接到pET28a后，用化学法转化步骤(1)构建的表达宿主E.coli BL21(DE3)/Ino1-MIOX中，筛选正确的重组大肠杆菌，命名为E.coli BL21(DE3)/Ino1-MIOX-Udh。
一种利用所述以上重组大肠杆菌生产葡萄糖二酸的方法，该方法是以葡萄糖、甘油、蔗糖或者肌醇为底物催化合成葡萄糖二酸。
优选的，所述方法具体是将重组大肠杆菌种子液以2％-5％的接种量接种到发酵培养基中，于OD600约为0.6，加入终浓度为0.1-1mM IPTG和10g/L葡萄糖于25℃-30℃，160rpm-200rpm，培养60-100h。
进一步优选的，所述发酵培养基，在本发明的一种实施方式中，其碳源为葡萄糖和/或肌醇。
进一步优选的，所述方法，在本发明的一种实施方式中，具体是将种子培养液按2％接种量接种到含有50ml发酵培养基的量为250ml摇瓶中，于OD600约为0.6，加入终浓度为0.1-1mM IPTG和10g/L葡萄糖，温度为30℃，摇床转速为180rpm，培养时间为100小时。
与现有技术相比，本发明具有如下优点：
(1)本发明提供了一种通过异构植物来源关键酶基因重组大肠杆菌发酵生产葡萄糖二酸的方法，重组大肠杆菌在LB培养基中生产葡萄糖二酸2.53g/L，对照大肠杆菌BL21(DE3)中，没有检测到葡萄糖二酸。
(2)本发明所用的来源于番茄中的肌醇-1-磷酸合成酶基因和肌醇氧化酶基因与现有报道的来源于小鼠的MIOX氨基酸差异较大，并且目前没有来源于番茄中的Ino1基因和MIOX基因能够将葡萄糖转化为醛酸的相关报道。
(3)本发明的重组大肠杆菌合成葡萄糖二酸，还具有培养时营养要求低、生产快、培养廉价、遗传稳定、表达水平高等许多优点。
(4)本发明采用代谢工程的策略，改造微生物菌株来合成目的产物葡萄糖二酸，为生物法高效生产葡萄糖二酸打下了坚实的基础。
附图说明
图1为重组大肠杆菌摇瓶发酵葡萄糖二酸生成量随发酵时间增加的曲线图；
图2a为葡萄糖二酸标样的HPLC谱图；
图2b为重组大肠杆菌发酵液的HPLC谱图。
具体实施方式
以下结合实例与附图对本发明的具体实施作进一步的说明，但本发明的实施方式不限于此，对未特别说明的工艺参数，可参照常规技术进行。
葡萄糖二酸的检测：Waser1525(Binary HPLC Pump)，2414时差检测器和2487紫外检测器。葡萄糖二酸标样的配制：准确称取50mg葡萄糖二酸溶于5mM H2SO4中，将溶解后的溶液转移至10ml容量瓶中定容，其浓度为100mg/L。再用5mM H2SO4分别稀释至4mg/ml、3mg/ml、2mg/ml、1mg/ml。
样品制备：1ml发酵液在12000rpm下离心5min，取上清经0.22um滤膜过滤，滤液经液相分析。
分析条件：
流动相：5mM H2SO4，等度洗脱
色谱柱：HPX-87H
检测器：2414时差检测器和2487紫外检测器
实施例1重组大肠杆菌E.coli BL21(DE3)/Ino1-MIOX的构建
以番茄(Solanaceae Lycopersicon 1706)基因组为模板扩增Ino1基因、MIOX基因，用Ino1-F(序列如SEQ ID NO.7所示)、Ino1-R(序列如SEQ ID NO.8所示)为引物扩增Ino1基因，用MIOX-F(序列如SEQ ID NO.9所示)、MIOX-R(序列如SEQ ID NO.10所示)为引物扩增MIOX基因。将获得的Ino1基因、MIOX基因，通过引物两端的酶切位点，经双酶切，连接到具有相应切口的表达载体pETDuet1(购于Novegen公司)，转化到DH5α(购于全式金公司)中，在确保阅读框正确的前体下，鉴定出重组表达质粒pETDuet1-Ino1-MIOX，经测序比对，序列正确。重组质粒经化学法转入表达宿主E.coli BL21(DE3)(购于全式金公司)中，重组克隆子经PCR验证正确，命名为E.coli BL21(DE3)/Ino1-MIOX。
实施例2重组大肠杆菌E.coli BL21(DE3)/Ino1-MIOX-Udh的构建
以根癌农杆菌(Agrobacterium tumefaciens GV3103)基因组为模板扩增Udh基因，Udh-F(序列如SEQ ID NO.11所示)、Udh-R(序列如SEQ ID NO.12所示)为引物扩增Udh基因，通过引物上的双酶切位点，连接至具有相应切口的表达载体pET28a(购于BioVector质粒载体菌种细胞基因保藏中心)上，转化至E.coli DH5α(购于全式金公司)中，在确保阅读框正确的前提下鉴定出重组表达质粒pET28a-Udh，经DNA测序比对，重组序列正确。重组质粒经化学法转入实施例1构建的表达宿主E.coli BL21(DE3)/Ino1-MIOX中，筛选正确的重组大肠杆菌，命名为E.coli BL21(DE3)/Ino1-MIOX-Udh。
表1为实施例1、2中用到的引物。
表1
引物 序列
Udh-F CGCAAGCTTATGAAACGGCTTCTTGTTACC
Udh-R CGCTCGAGCGGTGTCGTCTCGGTTATAT
Ino1-F CGCGGATCCGATGTTTATTGAAAATTTTAAGGT
Ino1-R GCGCTGCAGGATTTGTATTCCAAAATCATG
MIOX-F CGGATATCGATGACTATTCTCATTGAGCAGCCT
MIOX-R CCGCTCGAGACCACCTCAGCTTTGTTGGAAAAT
实施例3重组大肠杆菌发酵生产葡萄糖二酸
将重组大肠杆菌E.coli BL21(DE3)/Ino1-MIOX-Udh进行发酵培养。将单克隆重组大肠杆菌E.coli BL21(DE3)/Ino1-MIOX-Udh接种于25ml的LB培养基(胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L，pH7)中，在37℃、180rpm培养16h。然后再按2％接种量接种至50ml(摇瓶容量为500ml)发酵培养基(胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L)中发酵，于OD600为0.6时加入诱导剂IPTG使其浓度为0.2mM，同时加入葡萄糖使其浓度为10g/L，在温度为30℃，摇床转速为180rpm下，培养100小时。培养结束后，取1ml发酵液在12000rpm下离心5min，取上清经0.22um滤膜过滤，通过HPLC检测产物。图2a为葡萄糖二酸标样的HPLC谱图，图2b为重组大肠杆菌发酵液的HPLC谱图。由标样可以看出葡萄糖二酸的出峰时间为10.058min，在重组菌株发酵液中可以检测到具有同样出峰时间的葡萄糖二酸。
图1为重组大肠杆菌摇瓶发酵葡萄糖二酸生成量随发酵时间增加的曲线图；由图1可知，随着摇瓶发酵时间的增加，葡萄糖的量减少，葡萄糖二酸的生成量增加，达到2.53g/L。
SEQUENCE LISTING
<110> 华南理工大学
<120> 一种重组大肠杆菌及其构建方法与通过代谢工程生产葡萄糖二酸的方法
<130>
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1533
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<213> 人工序列
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Ala Thr Gly Thr Thr Thr Ala Thr Thr Gly Ala Ala Ala Ala Thr Thr
1 5 10 15
Thr Thr Ala Ala Gly Gly Thr Gly Gly Ala Ala Ala Gly Cys Cys Cys
20 25 30
Ala Ala Ala Thr Gly Thr Gly Ala Ala Gly Thr Ala Thr Ala Thr Thr
35 40 45
Gly Ala Gly Ala Ala Thr Gly Ala Ala Ala Thr Thr Cys Ala Thr Thr
50 55 60
Cys Thr Gly Thr Gly Thr Ala Thr Gly Ala Thr Thr Ala Thr Gly Ala
65 70 75 80
Ala Ala Cys Cys Ala Cys Ala Gly Ala Gly Cys Thr Thr Gly Thr Thr
85 90 95
Cys Ala Thr Gly Ala Ala Gly Ala Gly Ala Gly Ala Ala Ala Thr Gly
100 105 110
Gly Ala Ala Cys Thr Thr Ala Thr Cys Ala Ala Thr Gly Gly Ala Thr
115 120 125
Thr Gly Thr Thr Ala Ala Gly Cys Cys Thr Ala Ala Ala Ala Cys Thr
130 135 140
Gly Thr Cys Ala Ala Ala Thr Ala Thr Gly Ala Ala Thr Thr Thr Ala
145 150 155 160
Ala Ala Ala Cys Thr Gly Ala Thr Ala Cys Cys Cys Ala Thr Gly Thr
165 170 175
Gly Cys Cys Ala Ala Ala Ala Thr Thr Gly Gly Gly Gly Gly Thr Thr
180 185 190
Ala Thr Gly Cys Thr Thr Gly Thr Thr Gly Gly Ala Thr Gly Gly Gly
195 200 205
Gly Ala Gly Gly Ala Ala Ala Cys Ala Ala Thr Gly Gly Thr Thr Cys
210 215 220
Ala Ala Cys Ala Thr Thr Gly Ala Cys Thr Gly Gly Ala Gly Gly Thr
225 230 235 240
Gly Thr Thr Ala Thr Thr Gly Cys Gly Ala Ala Thr Cys Gly Ala Gly
245 250 255
Ala Ala Gly Gly Ala Ala Thr Thr Thr Cys Ala Thr Gly Gly Gly Cys
260 265 270
Ala Ala Cys Gly Ala Ala Ala Gly Ala Ala Ala Ala Ala Gly Thr Gly
275 280 285
Cys Ala Ala Cys Ala Ala Gly Cys Cys Ala Ala Thr Thr Ala Thr Thr
290 295 300
Thr Thr Gly Gly Gly Thr Cys Thr Cys Thr Thr Ala Cys Thr Cys Ala
305 310 315 320
Gly Gly Cys Ala Thr Cys Ala Ala Cys Cys Ala Thr Thr Cys Gly Ala
325 330 335
Gly Thr Thr Gly Gly Gly Thr Cys Thr Thr Thr Cys Ala Ala Thr Gly
340 345 350
Gly Cys Gly Ala Ala Gly Ala Gly Ala Thr Cys Thr Ala Thr Gly Cys
355 360 365
Ala Cys Cys Cys Thr Thr Cys Ala Ala Ala Ala Gly Cys Cys Thr Cys
370 375 380
Cys Thr Thr Cys Cys Cys Ala Thr Gly Gly Thr Cys Ala Ala Cys Cys
385 390 395 400
Cys Ala Gly Ala Cys Gly Ala Thr Gly Thr Ala Gly Thr Ala Thr Thr
405 410 415
Thr Gly Gly Ala Gly Gly Ala Thr Gly Gly Gly Ala Cys Ala Thr Thr
420 425 430
Ala Gly Cys Ala Ala Cys Ala Thr Gly Ala Ala Thr Thr Thr Gly Gly
435 440 445
Cys Ala Gly Ala Thr Gly Cys Thr Ala Thr Gly Gly Thr Cys Ala Gly
450 455 460
Gly Gly Cys Thr Ala Ala Gly Gly Thr Thr Thr Thr Cys Gly Ala Ala
465 470 475 480
Gly Thr Thr Gly Ala Thr Cys Thr Gly Cys Ala Ala Ala Ala Gly Cys
485 490 495
Ala Gly Cys Thr Gly Ala Gly Gly Cys Cys Cys Thr Ala Cys Ala Thr
500 505 510
Gly Gly Ala Ala Thr Cys Cys Ala Thr Gly Gly Thr Thr Cys Cys Cys
515 520 525
Cys Thr Thr Cys Cys Thr Gly Gly Thr Ala Thr Cys Thr Ala Thr Gly
530 535 540
Ala Cys Cys Cys Thr Gly Ala Cys Thr Thr Cys Ala Thr Thr Gly Cys
545 550 555 560
Gly Gly Cys Thr Ala Ala Cys Cys Ala Ala Gly Ala Ala Gly Cys Ala
565 570 575
Cys Gly Thr Gly Cys Cys Ala Ala Cys Ala Ala Cys Gly Thr Gly Ala
580 585 590
Thr Cys Ala Ala Ala Gly Gly Ala Ala Cys Cys Ala Ala Gly Ala Ala
595 600 605
Ala Gly Ala Ala Cys Ala Ala Gly Thr Thr Gly Ala Ala Cys Ala Ala
610 615 620
Ala Thr Thr Gly Thr Thr Ala Ala Ala Gly Ala Thr Ala Thr Thr Ala
625 630 635 640
Gly Gly Gly Ala Gly Thr Thr Cys Ala Ala Gly Gly Ala Gly Ala Ala
645 650 655
Gly Ala Ala Cys Ala Ala Gly Gly Thr Ala Gly Ala Cys Ala Ala Gly
660 665 670
Ala Thr Ala Gly Thr Gly Gly Thr Thr Cys Thr Ala Thr Gly Gly Ala
675 680 685
Cys Thr Gly Cys Cys Ala Ala Cys Ala Cys Cys Gly Ala Ala Ala Gly
690 695 700
Gly Thr Ala Cys Ala Gly Thr Ala Ala Thr Gly Thr Gly Gly Thr Thr
705 710 715 720
Gly Thr Thr Gly Gly Cys Cys Thr Thr Ala Ala Thr Gly Ala Cys Ala
725 730 735
Cys Cys Ala Thr Gly Gly Ala Ala Ala Ala Cys Cys Thr Thr Thr Thr
740 745 750
Ala Gly Cys Thr Gly Cys Thr Gly Thr Gly Gly Ala Thr Ala Gly Ala
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Ala Ala Thr Gly Ala Gly Gly Cys Thr Gly Ala Ala Ala Thr Ala Thr
770 775 780
Cys Thr Cys Cys Thr Thr Cys Thr Ala Cys Ala Thr Thr Gly Cys Ala
785 790 795 800
Thr Gly Cys Thr Ala Thr Thr Gly Cys Thr Thr Gly Thr Ala Thr Thr
805 810 815
Ala Thr Gly Gly Ala Ala Ala Ala Thr Gly Thr Gly Cys Cys Thr Thr
820 825 830
Thr Cys Ala Thr Cys Ala Ala Cys Gly Gly Ala Ala Gly Cys Cys Cys
835 840 845
Thr Cys Ala Ala Ala Ala Cys Ala Cys Thr Thr Thr Thr Gly Thr Thr
850 855 860
Cys Cys Ala Gly Gly Thr Cys Thr Thr Ala Thr Thr Gly Ala Thr Thr
865 870 875 880
Thr Gly Gly Cys Cys Ala Thr Ala Ala Ala Gly Ala Gly Ala Ala Ala
885 890 895
Cys Ala Cys Thr Thr Thr Ala Ala Thr Thr Gly Gly Thr Gly Gly Thr
900 905 910
Gly Ala Thr Gly Ala Cys Thr Thr Thr Ala Ala Gly Ala Gly Thr Gly
915 920 925
Gly Thr Cys Ala Ala Ala Cys Cys Ala Ala Gly Ala Thr Gly Ala Ala
930 935 940
Gly Thr Cys Cys Gly Thr Gly Thr Thr Gly Gly Thr Thr Gly Ala Thr
945 950 955 960
Thr Thr Cys Cys Thr Thr Gly Thr Thr Gly Gly Ala Gly Cys Cys Gly
965 970 975
Gly Thr Ala Thr Thr Ala Ala Gly Cys Cys Ala Ala Cys Gly Thr Cys
980 985 990
Ala Ala Thr Ala Gly Thr Gly Ala Gly Cys Thr Ala Cys Ala Ala Thr
995 1000 1005
Cys Ala Cys Thr Thr Gly Gly Gly Thr Ala Ala Cys Ala Ala Thr
1010 1015 1020
Gly Ala Thr Gly Gly Ala Ala Thr Gly Ala Ala Thr Cys Thr Thr
1025 1030 1035
Thr Cys Thr Gly Cys Thr Cys Cys Thr Cys Ala Ala Ala Cys Cys
1040 1045 1050
Thr Thr Cys Cys Gly Gly Thr Cys Thr Ala Ala Gly Gly Ala Gly
1055 1060 1065
Ala Thr Cys Thr Cys Ala Ala Ala Ala Ala Gly Thr Ala Ala Thr
1070 1075 1080
Gly Thr Thr Gly Thr Thr Gly Ala Thr Gly Ala Cys Ala Thr Gly
1085 1090 1095
Gly Thr Thr Gly Cys Thr Ala Gly Cys Ala Ala Cys Gly Cys Cys
1100 1105 1110
Ala Thr Thr Cys Thr Cys Thr Ala Thr Gly Ala Ala Thr Cys Thr
1115 1120 1125
Gly Gly Cys Gly Ala Gly Cys Ala Cys Cys Cys Thr Gly Ala Cys
1130 1135 1140
Cys Ala Thr Gly Thr Thr Gly Thr Cys Gly Thr Gly Ala Thr Cys
1145 1150 1155
Ala Ala Gly Thr Ala Thr Gly Thr Thr Cys Cys Ala Thr Ala Thr
1160 1165 1170
Gly Thr Thr Gly Gly Ala Gly Ala Cys Ala Gly Cys Ala Ala Gly
1175 1180 1185
Ala Gly Gly Gly Cys Ala Ala Thr Gly Gly Ala Thr Gly Ala Gly
1190 1195 1200
Thr Ala Cys Ala Thr Gly Thr Cys Ala Gly Ala Gly Ala Thr Thr
1205 1210 1215
Thr Thr Cys Ala Thr Gly Gly Gly Cys Gly Gly Ala Ala Ala Gly
1220 1225 1230
Ala Gly Cys Ala Cys Thr Ala Thr Ala Gly Thr Thr Ala Thr Gly
1235 1240 1245
Cys Ala Cys Ala Ala Cys Ala Cys Thr Thr Gly Thr Gly Ala Gly
1250 1255 1260
Gly Ala Cys Thr Cys Thr Cys Thr Thr Thr Thr Gly Gly Cys Ala
1265 1270 1275
Gly Cys Thr Cys Cys Ala Ala Thr Thr Ala Thr Cys Thr Thr Gly
1280 1285 1290
Gly Ala Thr Thr Thr Gly Gly Thr Cys Cys Thr Thr Cys Thr Cys
1295 1300 1305
Gly Cys Thr Gly Ala Ala Cys Thr Cys Ala Gly Cys Ala Cys Thr
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Cys Gly Cys Ala Thr Thr Cys Ala Gly Cys Thr Cys Ala Ala Ala
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Gly Cys Thr Gly Ala Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly
1340 1345 1350
Ala Ala Gly Thr Thr Cys Cys Ala Cys Thr Cys Cys Thr Thr Cys
1355 1360 1365
Cys Ala Cys Cys Cys Thr Gly Thr Gly Gly Cys Thr Ala Cys Thr
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Ala Thr Thr Cys Thr Cys Ala Gly Cys Thr Ala Cys Cys Thr Cys
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Ala Cys Cys Ala Ala Gly Gly Cys Thr Cys Cys Cys Cys Thr Gly
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Gly Thr Ala Cys Cys Ala Cys Cys Ala Gly Gly Thr Ala Cys Thr
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Cys Cys Ala Gly Thr Gly Gly Thr Gly Ala Ala Thr Gly Cys Cys
1430 1435 1440
Cys Thr Thr Thr Cys Ala Ala Ala Gly Cys Ala Gly Ala Gly Gly
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Gly Cys Ala Ala Thr Gly Cys Thr Thr Gly Ala Gly Ala Ala Thr
1460 1465 1470
Ala Thr Ala Ala Thr Gly Ala Gly Ala Gly Cys Thr Thr Gly Thr
1475 1480 1485
Gly Thr Thr Gly Gly Thr Thr Thr Gly Gly Cys Ala Cys Cys Ala
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Gly Ala Gly Ala Ala Cys Ala Ala Cys Ala Thr Gly Ala Thr Thr
1505 1510 1515
Thr Thr Gly Gly Ala Ala Thr Ala Cys Ala Ala Ala Thr Gly Ala
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<213> 人工序列
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Ala Thr Gly Ala Cys Thr Ala Thr Thr Cys Thr Cys Ala Thr Thr Gly
1 5 10 15
Ala Gly Cys Ala Gly Cys Cys Thr Gly Ala Ala Thr Thr Thr Gly Gly
20 25 30
Ala Thr Cys Ala Cys Ala Ala Gly Thr Gly Gly Ala Gly Gly Ala Gly
35 40 45
Ala Ala Ala Ala Ala Ala Gly Thr Cys Thr Cys Ala Thr Thr Cys Ala
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Ala Thr Gly Cys Cys Ala Ala Thr Gly Ala Ala Cys Thr Thr Ala Thr
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Thr Thr Thr Gly Gly Ala Thr Gly Gly Thr Gly Gly Ala Thr Thr Thr
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Ala Thr Gly Gly Thr Ala Cys Cys Ala Ala Ala Gly Ala Cys Ala Thr
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Thr Gly Thr Cys Thr Thr Cys Thr Cys Ala Ala Gly Ala Thr Gly Ala
115 120 125
Ala Ala Thr Ala Thr Thr Thr Gly Ala Ala Gly Thr Gly Cys Cys Ala
130 135 140
Gly Ala Cys Ala Thr Ala Ala Ala Thr Gly Cys Ala Thr Thr Thr Gly
145 150 155 160
Gly Thr Cys Ala Ala Thr Cys Ala Thr Thr Thr Ala Gly Gly Gly Ala
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Thr Thr Ala Thr Ala Ala Thr Gly Thr Ala Gly Ala Ala Ala Gly Thr
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Gly Ala Gly Ala Gly Ala Cys Ala Ala Ala Ala Ala Thr Cys Ala Gly
195 200 205
Thr Gly Gly Ala Ala Gly Ala Ala Thr Thr Thr Thr Ala Thr Ala Gly
210 215 220
Gly Gly Thr Thr Cys Ala Ala Cys Ala Cys Ala Thr Thr Ala Ala Thr
225 230 235 240
Cys Ala Ala Ala Cys Ala Thr Ala Thr Gly Ala Cys Thr Ala Thr Gly
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Thr Gly Ala Ala Ala Ala Ala Ala Ala Thr Gly Ala Gly Ala Ala Ala
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Ala Gly Ala Ala Thr Ala Thr Gly Gly Ala Ala Ala Ala Thr Thr Gly
275 280 285
Ala Ala Cys Ala Ala Ala Ala Thr Thr Gly Ala Ala Ala Thr Gly Ala
290 295 300
Gly Thr Ala Thr Thr Thr Gly Gly Gly Ala Thr Thr Gly Thr Thr Gly
305 310 315 320
Thr Gly Ala Ala Cys Thr Thr Thr Thr Gly Ala Ala Thr Gly Ala Thr
325 330 335
Gly Thr Ala Gly Thr Thr Gly Ala Thr Gly Ala Thr Ala Gly Thr Gly
340 345 350
Ala Thr Cys Cys Thr Gly Ala Thr Thr Thr Gly Gly Ala Thr Gly Ala
355 360 365
Ala Cys Cys Ala Cys Ala Ala Ala Thr Thr Gly Ala Gly Cys Ala Thr
370 375 380
Thr Thr Gly Thr Thr Ala Cys Ala Ala Ala Cys Thr Gly Cys Thr Gly
385 390 395 400
Ala Ala Gly Cys Thr Ala Thr Thr Ala Gly Ala Ala Ala Ala Gly Ala
405 410 415
Thr Thr Ala Thr Cys Cys Ala Ala Ala Thr Gly Ala Ala Gly Ala Thr
420 425 430
Thr Gly Gly Cys Thr Thr Cys Ala Thr Thr Thr Gly Ala Cys Cys Gly
435 440 445
Gly Cys Cys Thr Cys Ala Thr Thr Cys Ala Cys Gly Ala Cys Cys Thr
450 455 460
Ala Gly Gly Thr Ala Ala Ala Gly Thr Ala Cys Thr Thr Cys Thr Thr
465 470 475 480
Cys Ala Thr Cys Cys Ala Ala Gly Thr Thr Thr Thr Gly Gly Ala Gly
485 490 495
Gly Gly Cys Thr Thr Cys Cys Thr Cys Ala Ala Thr Gly Gly Gly Cys
500 505 510
Thr Gly Thr Thr Gly Thr Thr Gly Gly Ala Gly Ala Cys Ala Cys Ala
515 520 525
Thr Thr Thr Cys Cys Thr Cys Thr Thr Gly Gly Thr Thr Gly Thr Gly
530 535 540
Cys Thr Thr Thr Thr Gly Ala Thr Gly Ala Ala Thr Cys Ala Ala Thr
545 550 555 560
Thr Gly Thr Thr Cys Ala Cys Cys Ala Cys Ala Ala Gly Thr Ala Thr
565 570 575
Thr Thr Thr Ala Ala Gly Gly Ala Ala Ala Ala Thr Cys Cys Ala Gly
580 585 590
Ala Cys Ala Thr Cys Ala Ala Cys Ala Ala Cys Ala Ala Thr Ala Thr
595 600 605
Thr Thr Ala Thr Ala Ala Thr Ala Cys Ala Ala Ala Ala Ala Ala Thr
610 615 620
Gly Gly Thr Gly Thr Ala Thr Ala Thr Gly Ala Ala Gly Ala Ala Gly
625 630 635 640
Gly Thr Thr Gly Thr Gly Gly Ala Cys Thr Thr Gly Ala Cys Ala Ala
645 650 655
Ala Gly Thr Thr Gly Thr Thr Ala Thr Gly Thr Cys Ala Thr Gly Gly
660 665 670
Gly Gly Ala Cys Ala Thr Gly Ala Thr Gly Ala Thr Thr Ala Thr Ala
675 680 685
Thr Gly Thr Ala Thr Thr Thr Ala Ala Thr Thr Gly Cys Ala Ala Ala
690 695 700
Gly Gly Ala Ala Ala Ala Thr Ala Ala Ala Ala Cys Thr Ala Cys Thr
705 710 715 720
Cys Thr Thr Cys Cys Thr Thr Cys Thr Gly Cys Thr Gly Cys Thr Thr
725 730 735
Thr Ala Thr Thr Thr Gly Thr Cys Ala Thr Ala Cys Gly Thr Thr Ala
740 745 750
Cys Cys Ala Cys Thr Cys Thr Thr Thr Cys Thr Ala Thr Gly Cys Ala
755 760 765
Thr Thr Ala Cys Ala Thr Ala Gly Ala Thr Cys Ala Gly Gly Ala Gly
770 775 780
Cys Ala Thr Ala Thr Ala Cys Ala Cys Ala Cys Thr Thr Gly Ala Thr
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Gly Ala Ala Thr Gly Ala Gly Gly Ala Gly Gly Ala Cys Ala Ala Ala
805 810 815
Gly Ala Gly Ala Ala Cys Ala Thr Gly Ala Ala Gly Thr Gly Gly Cys
820 825 830
Thr Cys Ala Ala Cys Ala Thr Thr Thr Thr Thr Ala Ala Thr Ala Ala
835 840 845
Ala Thr Ala Thr Gly Ala Thr Thr Thr Ala Thr Ala Thr Ala Gly Cys
850 855 860
Ala Ala Gly Ala Gly Thr Ala Ala Ala Gly Thr Thr Cys Gly Ala Ala
865 870 875 880
Thr Thr Gly Ala Thr Gly Thr Gly Gly Ala Ala Ala Ala Ala Gly Thr
885 890 895
Cys Ala Ala Gly Cys Cys Ala Thr Ala Cys Thr Ala Thr Cys Thr Cys
900 905 910
Thr Cys Thr Cys Thr Thr Ala Thr Cys Gly Ala Ala Ala Ala Gly Thr
915 920 925
Ala Thr Thr Thr Thr Cys Cys Ala Ala Cys Ala Ala Ala Gly Cys Thr
930 935 940
Gly Ala Gly Gly Thr Gly Gly Thr Ala Ala
945 950
<210> 3
<211> 798
<212> PRT
<213> 人工序列
<400> 3
Ala Thr Gly Ala Ala Ala Cys Gly Gly Cys Thr Thr Cys Thr Thr Gly
1 5 10 15
Thr Thr Ala Cys Cys Gly Gly Thr Gly Cys Gly Gly Cys Gly Gly Gly
20 25 30
Cys Cys Ala Gly Cys Thr Thr Gly Gly Cys Cys Gly Cys Gly Thr Cys
35 40 45
Ala Thr Gly Cys Gly Cys Gly Ala Gly Cys Gly Thr Cys Thr Cys Gly
50 55 60
Cys Ala Cys Cys Gly Ala Thr Gly Gly Cys Gly Gly Ala Gly Ala Thr
65 70 75 80
Ala Cys Thr Gly Cys Gly Cys Cys Thr Thr Gly Cys Cys Gly Ala Thr
85 90 95
Cys Thr Cys Thr Cys Cys Cys Cys Gly Cys Thr Cys Gly Ala Cys Cys
100 105 110
Cys Gly Gly Cys Ala Gly Gly Gly Cys Cys Gly Ala Ala Cys Gly Ala
115 120 125
Ala Gly Ala Ala Thr Gly Cys Gly Thr Gly Cys Ala Ala Thr Gly Cys
130 135 140
Gly Ala Cys Cys Thr Thr Gly Cys Cys Gly Ala Thr Gly Cys Cys Ala
145 150 155 160
Ala Thr Gly Cys Cys Gly Thr Gly Ala Ala Thr Gly Cys Cys Ala Thr
165 170 175
Gly Gly Thr Cys Gly Cys Cys Gly Gly Thr Thr Gly Cys Gly Ala Cys
180 185 190
Gly Gly Thr Ala Thr Thr Gly Thr Thr Cys Ala Thr Cys Thr Cys Gly
195 200 205
Gly Cys Gly Gly Cys Ala Thr Cys Thr Cys Gly Gly Thr Gly Gly Ala
210 215 220
Gly Ala Ala Gly Cys Cys Cys Thr Thr Cys Gly Ala Ala Cys Ala Ala
225 230 235 240
Ala Thr Cys Cys Thr Thr Cys Ala Gly Gly Gly Cys Ala Ala Thr Ala
245 250 255
Thr Cys Ala Thr Cys Gly Gly Gly Cys Thr Thr Cys Ala Thr Ala Ala
260 265 270
Thr Cys Thr Cys Thr Ala Cys Gly Ala Gly Gly Cys Cys Gly Cys Cys
275 280 285
Cys Gly Cys Gly Cys Cys Cys Ala Thr Gly Gly Ala Cys Ala Gly Cys
290 295 300
Cys Ala Cys Gly Cys Ala Thr Cys Gly Thr Cys Thr Thr Thr Gly Cys
305 310 315 320
Cys Ala Gly Cys Thr Cys Cys Ala Ala Cys Cys Ala Cys Ala Cys Gly
325 330 335
Ala Thr Cys Gly Gly Cys Thr Ala Thr Thr Ala Thr Cys Cys Gly Cys
340 345 350
Ala Gly Ala Cys Cys Gly Ala Ala Cys Gly Gly Cys Thr Cys Gly Gly
355 360 365
Thr Cys Cys Gly Gly Ala Thr Gly Thr Thr Cys Cys Gly Gly Cys Gly
370 375 380
Cys Gly Gly Cys Cys Gly Gly Ala Cys Gly Gly Thr Cys Thr Thr Thr
385 390 395 400
Ala Cys Gly Gly Cys Gly Thr Cys Thr Cys Cys Ala Ala Ala Thr Gly
405 410 415
Thr Thr Thr Cys Gly Gly Cys Gly Ala Ala Ala Ala Cys Cys Thr Cys
420 425 430
Gly Cys Cys Cys Gly Cys Ala Thr Gly Thr Ala Thr Thr Thr Cys Gly
435 440 445
Ala Thr Ala Ala Ala Thr Thr Cys Gly Gly Gly Cys Ala Gly Gly Ala
450 455 460
Gly Ala Cys Gly Gly Cys Gly Cys Thr Gly Gly Thr Gly Cys Gly Cys
465 470 475 480
Ala Thr Cys Gly Gly Cys Thr Cys Cys Thr Gly Thr Ala Cys Gly Cys
485 490 495
Cys Gly Gly Ala Ala Cys Cys Cys Ala Ala Cys Ala Ala Thr Thr Ala
500 505 510
Cys Cys Gly Cys Ala Thr Gly Cys Thr Gly Thr Cys Cys Ala Cys Cys
515 520 525
Thr Gly Gly Thr Thr Thr Thr Cys Gly Cys Ala Cys Gly Ala Thr Gly
530 535 540
Ala Thr Thr Thr Cys Gly Thr Gly Thr Cys Gly Cys Thr Gly Ala Thr
545 550 555 560
Cys Gly Ala Gly Gly Cys Gly Gly Thr Gly Thr Thr Thr Cys Gly Cys
565 570 575
Gly Cys Gly Cys Cys Gly Gly Thr Gly Cys Thr Cys Gly Gly Cys Thr
580 585 590
Gly Cys Cys Cys Gly Gly Thr Cys Gly Thr Cys Thr Gly Gly Gly Gly
595 600 605
Gly Gly Cys Ala Thr Cys Gly Gly Cys Cys Ala Ala Thr Gly Ala Thr
610 615 620
Gly Cys Gly Gly Gly Cys Thr Gly Gly Thr Gly Gly Gly Ala Cys Ala
625 630 635 640
Ala Thr Thr Cys Gly Cys Ala Thr Cys Thr Thr Gly Gly Cys Thr Thr
645 650 655
Thr Cys Thr Gly Gly Gly Cys Thr Gly Gly Ala Ala Ala Cys Cys Gly
660 665 670
Gly Ala Gly Gly Ala Thr Ala Ala Thr Gly Cys Cys Gly Ala Gly Gly
675 680 685
Cys Cys Thr Thr Cys Cys Gly Gly Cys Gly Gly Cys Ala Thr Ala Thr
690 695 700
Ala Ala Cys Cys Gly Ala Gly Ala Cys Gly Ala Cys Ala Cys Cys Gly
705 710 715 720
Cys Cys Ala Cys Cys Gly Gly Ala Cys Cys Cys Gly Ala Ala Thr Gly
725 730 735
Ala Cys Gly Cys Gly Thr Thr Gly Gly Thr Gly Cys Gly Gly Thr Thr
740 745 750
Cys Cys Ala Gly Gly Gly Cys Gly Gly Thr Ala Cys Gly Thr Thr Thr
755 760 765
Gly Thr Cys Gly Ala Cys Ala Ala Cys Cys Cys Gly Ala Thr Cys Thr
770 775 780
Thr Cys Ala Ala Ala Cys Ala Gly Ala Gly Cys Thr Gly Ala
785 790 795
<210> 4
<211> 510
<212> PRT
<213> 人工序列
<400> 4
Met Phe Ile Glu Asn Phe Lys Val Glu Ser Pro Asn Val Lys Tyr Ile
1 5 10 15
Glu Asn Glu Ile His Ser Val Tyr Asp Tyr Glu Thr Thr Glu Leu Val
20 25 30
His Glu Glu Arg Asn Gly Thr Tyr Gln Trp Ile Val Lys Pro Lys Thr
35 40 45
Val Lys Tyr Glu Phe Lys Thr Asp Thr His Val Pro Lys Leu Gly Val
50 55 60
Met Leu Val Gly Trp Gly Gly Asn Asn Gly Ser Thr Leu Thr Gly Gly
65 70 75 80
Val Ile Ala Asn Arg Glu Gly Ile Ser Trp Ala Thr Lys Glu Lys Val
85 90 95
Gln Gln Ala Asn Tyr Phe Gly Ser Leu Thr Gln Ala Ser Thr Ile Arg
100 105 110
Val Gly Ser Phe Asn Gly Glu Glu Ile Tyr Ala Pro Phe Lys Ser Leu
115 120 125
Leu Pro Met Val Asn Pro Asp Asp Val Val Phe Gly Gly Trp Asp Ile
130 135 140
Ser Asn Met Asn Leu Ala Asp Ala Met Val Arg Ala Lys Val Phe Glu
145 150 155 160
Val Asp Leu Gln Lys Gln Leu Arg Pro Tyr Met Glu Ser Met Val Pro
165 170 175
Leu Pro Gly Ile Tyr Asp Pro Asp Phe Ile Ala Ala Asn Gln Glu Ala
180 185 190
Arg Ala Asn Asn Val Ile Lys Gly Thr Lys Lys Glu Gln Val Glu Gln
195 200 205
Ile Val Lys Asp Ile Arg Glu Phe Lys Glu Lys Asn Lys Val Asp Lys
210 215 220
Ile Val Val Leu Trp Thr Ala Asn Thr Glu Arg Tyr Ser Asn Val Val
225 230 235 240
Val Gly Leu Asn Asp Thr Met Glu Asn Leu Leu Ala Ala Val Asp Arg
245 250 255
Asn Glu Ala Glu Ile Ser Pro Ser Thr Leu His Ala Ile Ala Cys Ile
260 265 270
Met Glu Asn Val Pro Phe Ile Asn Gly Ser Pro Gln Asn Thr Phe Val
275 280 285
Pro Gly Leu Ile Asp Leu Ala Ile Lys Arg Asn Thr Leu Ile Gly Gly
290 295 300
Asp Asp Phe Lys Ser Gly Gln Thr Lys Met Lys Ser Val Leu Val Asp
305 310 315 320
Phe Leu Val Gly Ala Gly Ile Lys Pro Thr Ser Ile Val Ser Tyr Asn
325 330 335
His Leu Gly Asn Asn Asp Gly Met Asn Leu Ser Ala Pro Gln Thr Phe
340 345 350
Arg Ser Lys Glu Ile Ser Lys Ser Asn Val Val Asp Asp Met Val Ala
355 360 365
Ser Asn Ala Ile Leu Tyr Glu Ser Gly Glu His Pro Asp His Val Val
370 375 380
Val Ile Lys Tyr Val Pro Tyr Val Gly Asp Ser Lys Arg Ala Met Asp
385 390 395 400
Glu Tyr Met Ser Glu Ile Phe Met Gly Gly Lys Ser Thr Ile Val Met
405 410 415
His Asn Thr Cys Glu Asp Ser Leu Leu Ala Ala Pro Ile Ile Leu Asp
420 425 430
Leu Val Leu Leu Ala Glu Leu Ser Thr Arg Ile Gln Leu Lys Ala Glu
435 440 445
Gly Glu Gly Lys Phe His Ser Phe His Pro Val Ala Thr Ile Leu Ser
450 455 460
Tyr Leu Thr Lys Ala Pro Leu Val Pro Pro Gly Thr Pro Val Val Asn
465 470 475 480
Ala Leu Ser Lys Gln Arg Ala Met Leu Glu Asn Ile Met Arg Ala Cys
485 490 495
Val Gly Leu Ala Pro Glu Asn Asn Met Ile Leu Glu Tyr Lys
500 505 510
<210> 5
<211> 317
<212> PRT
<213> 人工序列
<400> 5
Met Thr Ile Leu Ile Glu Gln Pro Glu Phe Gly Ser Gln Val Glu Glu
1 5 10 15
Lys Lys Val Ser Phe Asn Ala Asn Glu Leu Ile Leu Asp Gly Gly Phe
20 25 30
Met Val Pro Lys Thr Leu Ser Ser Gln Asp Glu Ile Phe Glu Val Pro
35 40 45
Asp Ile Asn Ala Phe Gly Gln Ser Phe Arg Asp Tyr Asn Val Glu Ser
50 55 60
Glu Arg Gln Lys Ser Val Glu Glu Phe Tyr Arg Val Gln His Ile Asn
65 70 75 80
Gln Thr Tyr Asp Tyr Val Lys Lys Met Arg Lys Glu Tyr Gly Lys Leu
85 90 95
Asn Lys Ile Glu Met Ser Ile Trp Asp Cys Cys Glu Leu Leu Asn Asp
100 105 110
Val Val Asp Asp Ser Asp Pro Asp Leu Asp Glu Pro Gln Ile Glu His
115 120 125
Leu Leu Gln Thr Ala Glu Ala Ile Arg Lys Asp Tyr Pro Asn Glu Asp
130 135 140
Trp Leu His Leu Thr Gly Leu Ile His Asp Leu Gly Lys Val Leu Leu
145 150 155 160
His Pro Ser Phe Gly Gly Leu Pro Gln Trp Ala Val Val Gly Asp Thr
165 170 175
Phe Pro Leu Gly Cys Ala Phe Asp Glu Ser Ile Val His His Lys Tyr
180 185 190
Phe Lys Glu Asn Pro Asp Ile Asn Asn Asn Ile Tyr Asn Thr Lys Asn
195 200 205
Gly Val Tyr Glu Glu Gly Cys Gly Leu Asp Lys Val Val Met Ser Trp
210 215 220
Gly His Asp Asp Tyr Met Tyr Leu Ile Ala Lys Glu Asn Lys Thr Thr
225 230 235 240
Leu Pro Ser Ala Ala Leu Phe Val Ile Arg Tyr His Ser Phe Tyr Ala
245 250 255
Leu His Arg Ser Gly Ala Tyr Thr His Leu Met Asn Glu Glu Asp Lys
260 265 270
Glu Asn Met Lys Trp Leu Asn Ile Phe Asn Lys Tyr Asp Leu Tyr Ser
275 280 285
Lys Ser Lys Val Arg Ile Asp Val Glu Lys Val Lys Pro Tyr Tyr Leu
290 295 300
Ser Leu Ile Glu Lys Tyr Phe Pro Thr Lys Leu Arg Trp
305 310 315
<210> 6
<211> 265
<212> PRT
<213> 人工序列
<400> 6
Met Lys Arg Leu Leu Val Thr Gly Ala Ala Gly Gln Leu Gly Arg Val
1 5 10 15
Met Arg Glu Arg Leu Ala Pro Met Ala Glu Ile Leu Arg Leu Ala Asp
20 25 30
Leu Ser Pro Leu Asp Pro Ala Gly Pro Asn Glu Glu Cys Val Gln Cys
35 40 45
Asp Leu Ala Asp Ala Asn Ala Val Asn Ala Met Val Ala Gly Cys Asp
50 55 60
Gly Ile Val His Leu Gly Gly Ile Ser Val Glu Lys Pro Phe Glu Gln
65 70 75 80
Ile Leu Gln Gly Asn Ile Ile Gly Leu His Asn Leu Tyr Glu Ala Ala
85 90 95
Arg Ala His Gly Gln Pro Arg Ile Val Phe Ala Ser Ser Asn His Thr
100 105 110
Ile Gly Tyr Tyr Pro Gln Thr Glu Arg Leu Gly Pro Asp Val Pro Ala
115 120 125
Arg Pro Asp Gly Leu Tyr Gly Val Ser Lys Cys Phe Gly Glu Asn Leu
130 135 140
Ala Arg Met Tyr Phe Asp Lys Phe Gly Gln Glu Thr Ala Leu Val Arg
145 150 155 160
Ile Gly Ser Cys Thr Pro Glu Pro Asn Asn Tyr Arg Met Leu Ser Thr
165 170 175
Trp Phe Ser His Asp Asp Phe Val Ser Leu Ile Glu Ala Val Phe Arg
180 185 190
Ala Pro Val Leu Gly Cys Pro Val Val Trp Gly Ala Ser Ala Asn Asp
195 200 205
Ala Gly Trp Trp Asp Asn Ser His Leu Gly Phe Leu Gly Trp Lys Pro
210 215 220
Glu Asp Asn Ala Glu Ala Phe Arg Arg His Ile Thr Glu Thr Thr Pro
225 230 235 240
Pro Pro Asp Pro Asn Asp Ala Leu Val Arg Phe Gln Gly Gly Thr Phe
245 250 255
Val Asp Asn Pro Ile Phe Lys Gln Ser
260 265
<210> 7
<211> 33
<212> PRT
<213> 人工序列
<400> 7
Cys Gly Cys Gly Gly Ala Thr Cys Cys Gly Ala Thr Gly Thr Thr Thr
1 5 10 15
Ala Thr Thr Gly Ala Ala Ala Ala Thr Thr Thr Thr Ala Ala Gly Gly
20 25 30
Thr
<210> 8
<211> 30
<212> PRT
<213> 人工序列
<400> 8
Gly Cys Gly Cys Thr Gly Cys Ala Gly Gly Ala Thr Thr Thr Gly Thr
1 5 10 15
Ala Thr Thr Cys Cys Ala Ala Ala Ala Thr Cys Ala Thr Gly
20 25 30
<210> 9
<211> 33
<212> PRT
<213> 人工序列
<400> 9
Cys Gly Gly Ala Thr Ala Thr Cys Gly Ala Thr Gly Ala Cys Thr Ala
1 5 10 15
Thr Thr Cys Thr Cys Ala Thr Thr Gly Ala Gly Cys Ala Gly Cys Cys
20 25 30
Thr
<210> 10
<211> 33
<212> PRT
<213> 人工序列
<400> 10
Cys Cys Gly Cys Thr Cys Gly Ala Gly Ala Cys Cys Ala Cys Cys Thr
1 5 10 15
Cys Ala Gly Cys Thr Thr Thr Gly Thr Thr Gly Gly Ala Ala Ala Ala
20 25 30
Thr
<210> 11
<211> 30
<212> PRT
<213> 人工序列
<400> 11
Cys Gly Cys Ala Ala Gly Cys Thr Thr Ala Thr Gly Ala Ala Ala Cys
1 5 10 15
Gly Gly Cys Thr Thr Cys Thr Thr Gly Thr Thr Ala Cys Cys
20 25 30
<210> 12
<211> 28
<212> PRT
<213> 人工序列
<400> 12
Cys Gly Cys Thr Cys Gly Ala Gly Cys Gly Gly Thr Gly Thr Cys Gly
1 5 10 15
Thr Cys Thr Cys Gly Gly Thr Thr Ala Thr Ala Thr
20 25

Claims (10)

1.一种重组大肠杆菌，其特征在于，所述重组大肠杆菌同时表达肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX和醛酸脱氢酶基因Udh。
2.根据权利要求1所述的重组大肠杆菌，其特征在于，所述肌醇-1-磷酸合成酶基因Ino1来源于酿酒酵母、毕赤酵母、番茄、玉米和大豆中的任意一种；所述肌醇氧化酶基因MIOX来源于酿酒酵母、毕赤酵母、番茄、玉米和大豆中的任意一种；所述醛酸脱氢酶基因Udh来源于根癌农杆菌。
3.根据权利要求1所述的重组大肠杆菌，其特征在于，所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX、醛酸脱氢酶基因Udh的核苷酸序列分别是SEQ ID NO. 1、SEQID NO. 2、SEQ ID NO. 3所示的序列。
4.根据权利要求1或3所述的重组大肠杆菌，其特征在于，所述重组大肠杆菌的出发宿主菌为大肠杆菌。
5.根据权利要求1或3所述的重组大肠杆菌，其特征在于，所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX及醛酸脱氢酶基因Udh为整合表达。
6.一种权利要求1或3所述重组大肠杆菌的构建方法，其特征在于，所述方法包括如下步骤：
（1）将肌醇-1-磷酸合成酶基因Ino1和肌醇氧化酶基因MIOX连接到双元表达载体上转化到大肠杆宿主菌种，得到重组大肠杆菌1；
（2）将醛酸脱氢酶基因Udh连接到表达载体后转化到步骤（1）得到的重组大肠杆菌1中，得重组大肠杆菌。
7.根据权利要求6所述的构建方法，其特征在于，所述方法具体包括如下步骤：
（1）以番茄Solanaceae Lycopersicon 1706基因组为模板扩增肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX，并分别加上合适酶切位点，将片段连接到双元表达载体pETDuet1后，用化学法转化到DH5α中，筛选获得正确的重组质粒，命名为pETDuet1-Ino1-MIOX，将重组质粒转化到宿主菌E.coli BL21(DE3)中既得到E.coli BL21(DE3)/Ino1-MIOX；
（2）以根癌农杆菌Agrobacterium tumefaciens GV3103基因组为模板扩增醛酸脱氢酶基因Udh，连接到pET28a后，用化学法转化到步骤（1）构建的表达宿主E.coli BL21(DE3)/Ino1-MIOX中，筛选正确的重组大肠杆菌，命名为E.coli BL21(DE3)/Ino1-MIOX-Udh。
8.一种利用权利要求1所述的重组大肠杆菌生产葡萄糖二酸的方法，其特征在于，该方法是以葡萄糖、甘油、蔗糖或者肌醇为底物催化合成葡萄糖二酸。
9.根据权利要求8所述的方法，其特征在于，所述方法具体是将重组大肠杆菌种子液以2% - 5%的接种量接种到发酵培养基中，于25℃ - 30℃，160 rpm - 200 rpm下，培养60-100 h。
10.根据权利要求9所述的方法，其特征在于，所述发酵培养基的碳源为葡萄糖和粗甘油中的一种或两种。
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CN116814519A (zh) * 2023-08-29 2023-09-29 中国农业科学院北京畜牧兽医研究所 一种利用蔗糖生产肌醇的大肠杆菌工程菌株、及其构建方法和应用
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