CN117165545A - α-异丙基苹果酸合酶突变体、重组微生物及其应用 - Google Patents
α-异丙基苹果酸合酶突变体、重组微生物及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,具体涉及α‑异丙基苹果酸合酶突变体、重组微生物及其应用。本发明提供α‑异丙基苹果酸合酶突变体,以谷氨酸棒杆菌野生型α‑异丙基苹果酸合酶为参考序列,其含有第87位脯氨酸被除脯氨酸以外的氨基酸取代的突变。该突变体能够使微生物的亮氨酸合成能力显著下降,减少发酵过程中亮氨酸的积累,同时使微生物合成缬氨酸、异亮氨酸的能力显著增强,缬氨酸、异亮氨酸的产量和转化率显著提高。本发明提供的重组微生物能够高效地合成缬氨酸,缬氨酸产量和转化率均显著提高,且具有较好的生长性能,同时副产物亮氨酸的积累量显著降低,更有利于目标产物的分离纯化。
Description
技术领域
本发明涉及微生物技术领域,具体涉及α-异丙基苹果酸合酶突变体、重组微生物及其应用。
背景技术
支链氨基酸(branch chain amino acid,BCAA)包括缬氨酸、亮氨酸和异亮氨酸。其中,L-缬氨酸(L-valine),化学名称为L-α-氨基异戊酸,分子式为C5H11NO2,相对分子质量为117.15。L-缬氨酸呈白色结晶或结晶性粉末,无臭,味苦,在水中25℃的溶解度为88.5g/L,50℃的溶解度为96.2g/L,不溶于冷乙醇、乙醚、丙酮,等电点为5.96,熔点为315℃。
L-缬氨酸是人体八种必需氨基酸之一,因其特殊的结构和功能,在人类生命代谢中具有特别重要的地位。L-缬氨酸可以广泛应用于医药工业、食品工业和饲料工业等。其中,在医药工业中,L-缬氨酸可作为氨基酸输液、综合氨基酸制剂的主要成分,可治疗肝功能衰竭、中枢神经系统功能紊乱。在食品工业中,L-缬氨酸可用作食品添加剂、营养增补液及风味剂等。L-缬氨酸也可用作氨基酸功能饮料与运动员饮料,具有形成肌肉、强化肝功能、减轻肌肉疲劳等作用。在饲料工业中,L-缬氨酸对动物的乳腺组织分泌乳汁具有重要的促进作用。
目前,L-缬氨酸的生产方法主要包括三种:提取法、化学合成法、微生物发酵法。提取法和化学合成法由于原料来源受到限制、生产成本高、污染环境等问题,难以实现工业化生产。微生物发酵法生产L-缬氨酸具有原料成本低、反应条件温和、容易实现大规模生产等优点,是目前生产L-缬氨酸最主要的方法。但目前L-缬氨酸菌种的发酵性能仍较差,且副产物亮氨酸的含量较高,导致转化率仍较低,较难满足大规模工业化生产的需求。
发明内容
本发明的目的之一是提供α-异丙基苹果酸合酶突变体及其应用。本发明的另一目的是提供表达α-异丙基苹果酸合酶突变体的重组微生物及其应用。
具体地,本发明提供以下技术方案:
本发明提供α-异丙基苹果酸合酶突变体,以谷氨酸棒杆菌野生型α-异丙基苹果酸合酶为参考序列,α-异丙基苹果酸合酶突变体含有第87位脯氨酸被除脯氨酸以外的氨基酸取代的突变。
本发明中,α-异丙基苹果酸合酶由leuA基因编码,催化α-酮基异戊酸与乙酰-CoA反应生成a-异丙基苹果酸,是亮氨酸合成末端途径中的第一个酶,也是亮氨酸合成途径的关键酶,该酶受到亮氨酸的反馈抑制和转录弱化调控。谷氨酸棒杆菌野生型α-异丙基苹果酸合酶的氨基酸序列如SEQ ID NO.1所示,其编码基因leuA的核苷酸序列如SEQ ID NO.2所示。
本发明发现,α-异丙基苹果酸合酶第87位脯氨酸突变为其他氨基酸能够显著降低该酶的催化活性,减少微生物中由α-酮基异戊酸、乙酰-CoA反应生成a-异丙基苹果酸的代谢通量,进而减少亮氨酸的合成,增加缬氨酸和异亮氨酸合成途径的代谢通量,进而增强缬氨酸和异亮氨酸以及以缬氨酸、异亮氨酸作为前体物质的下游代谢产物的合成。此外,本发明还发现,α-异丙基苹果酸合酶第87位脯氨酸突变为其他氨基酸,不会导致α-异丙基苹果酸合酶的完全失活,其能够少量合成亮氨酸供菌株生长。
优选地,以谷氨酸棒杆菌野生型α-异丙基苹果酸合酶为参考序列,α-异丙基苹果酸合酶突变体含有第87位脯氨酸被亮氨酸、缬氨酸或异亮氨酸取代的突变。
在第87位脯氨酸突变的各种突变体中,上述三种突变体不仅能够显著降低α-异丙基苹果酸合酶的活性,减少菌株的亮氨酸合成,促进缬氨酸、异亮氨酸及其衍生物的合成,而且还能够很好地保证菌株的生长性能,表达这三个突变体的工程菌株具有优异的发酵性能。
具体地,所述α-异丙基苹果酸合酶突变体具有如SEQ ID NO.3-8任一所示的氨基酸序列。
上述突变体序列中,SEQ ID NO.3-5所示序列为将SEQ ID NO.1所示序列的第87位分别替换为亮氨酸、缬氨酸、异亮氨酸(编码基因序列相应地由CCT突变为CTT、GTT、ATT),同时将其第1位氨基酸替换为缬氨酸得到。SEQ ID NO.6-8所示序列为将SEQ ID NO.1所示序列的第87位分别替换为亮氨酸、缬氨酸、异亮氨酸得到。
上述α-异丙基苹果酸合酶突变体中,第87位分别突变为亮氨酸、缬氨酸、异亮氨酸的突变体能够显著降低α-异丙基苹果酸合酶的活性,减少菌株的亮氨酸合成,促进缬氨酸、异亮氨酸及其衍生物的合成;在此基础上,联合第1位氨基酸突变为缬氨酸,两者能够产生叠加效应,进一步降低亮氨酸的合成能力并促进缬氨酸、异亮氨酸及其衍生物的高效合成。
此外,本领域技术人员应该理解,在上述α-异丙基苹果酸合酶突变体序列的N端或C端添加标签蛋白或将其与其他蛋白进行融合形成融合蛋白,在不改变上述α-异丙基苹果酸合酶突变体本身结构的情况下,其活性不会发生明显改变,因此,上述添加标签的蛋白或融合蛋白也在本发明的保护范围内。
本发明还提供编码所述α-异丙基苹果酸合酶突变体的基因。
根据上述提供的α-异丙基苹果酸合酶突变体的氨基酸序列,本领域技术人员能够获得其编码基因的序列。基于密码子的简并性,编码上述氨基酸序列的基因序列不止一种,所有能够编码上述突变体的基因均在本发明的保护范围内。
作为本发明的一种实施方式,编码α-异丙基苹果酸合酶突变体的基因的核苷酸序列如SEQ ID NO.9-11任一所示。
本发明还提供含有所述基因的生物材料,所述生物材料为重组DNA、载体或宿主细胞。
其中,所述重组DNA可为在所述基因的上游或下游连接用于驱动其转录、翻译的元件得到的重组DNA。所述载体可为表达载体或克隆载体,包括但不限于质粒载体、噬菌体载体、转座子等。所述宿主细胞可为微生物细胞。
进一步地,本发明提供一种重组微生物,所述重组微生物表达所述α-异丙基苹果酸合酶突变体,且不表达其出发菌株所具有的α-异丙基苹果酸合酶。
优选地,所述重组微生物中,编码α-异丙基苹果酸合酶的基因被编码所述α-异丙基苹果酸合酶突变体的基因取代。
优选地,所述重组微生物为棒杆菌属细菌或短杆菌属细菌。
其中,所述棒杆菌属细菌优选为选自谷氨酸棒杆菌(Corynebacteriumglutamicum)、北京棒杆菌(Corynebacterium pekinense)、有效棒杆菌(Corynebacteriumefficiens)、钝齿棒杆菌(Corynebacterium crenatum)、嗜热产氨棒杆菌(Corynebacterium thermoaminogenes)、产氨棒杆菌(Corynebacterium aminogenes)的细菌。所述短杆菌属细菌优选为选自黄色短杆菌(Breviabacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)的细菌。
本发明中,出发菌株是指用于将编码α-异丙基苹果酸合酶的基因替换为编码所述α-异丙基苹果酸合酶突变体的基因的起始菌株,即:将出发菌株的编码α-异丙基苹果酸合酶的基因替换为编码所述α-异丙基苹果酸合酶突变体的基因可得到所述重组微生物。
优选地,所述出发菌株为能够积累缬氨酸或异亮氨酸的细菌。
作为本发明的一种实施方式,所述出发菌株为MHZ-1012-2或MHZ-1012-3。MHZ-1012-2菌株已于2016年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏中心地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.13406,分类命名为:谷氨酸棒杆菌Corynebacterium glutamicum,该菌株公开于专利申请CN201611250330.1中。MHZ-1012-3菌株为一株缬氨酸产生菌,为将MHZ-1012-2菌株的α-异丙基苹果酸合酶基因leuA的编码区第一个碱基由A变为G得到,可采用本领域常规的基因突变方法构建得到,或者可参考专利申请CN201911370732.9的实施例1中的构建方法。
本发明提供的上述重组微生物的亮氨酸积累明显减少,缬氨酸、异亮氨酸的合成能力显著增强,能够高效积累缬氨酸、异亮氨酸,且具有较好的生长性能。
本发明还提供所述重组微生物的构建方法,其包括:将所述重组微生物的出发菌株中编码α-异丙基苹果酸合酶的基因替换为编码所述α-异丙基苹果酸合酶突变体的基因。
上述基因的替换可采用本领域的常规技术手段实现,例如:采用同源重组的方法将染色体上编码α-异丙基苹果酸合酶的基因替换为编码所述α-异丙基苹果酸合酶突变体的基因。
基于本发明提供的α-异丙基苹果酸合酶突变体、重组微生物的功能,本发明提供所述α-异丙基苹果酸合酶突变体或编码所述α-异丙基苹果酸合酶突变体的基因或含有所述基因的生物材料或所述重组微生物的如下任一种应用:
(1)在工程菌构建中的应用;
(2)在发酵生产缬氨酸或其衍生物、异亮氨酸或其衍生物中的应用;
(3)在提高菌株的缬氨酸或其衍生物、异亮氨酸或其衍生物的产量和/或转化率中的应用;
(4)在减少菌株的亮氨酸积累中的应用。
优选地,以上(1)中所述的应用中,所述工程菌为缬氨酸或其衍生物、异亮氨酸或其衍生物的工程菌。优选为谷氨酸棒杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)、黄色短杆菌(Breviabacterium flavum)。
本发明的缬氨酸衍生物、异亮氨酸衍生物为以缬氨酸、异亮氨酸为前体合成的物质。在重组微生物合成缬氨酸、异亮氨酸的能力显著提高的情况下,以缬氨酸、异亮氨酸为前体的下游产物的合成能力也会增强。其中,缬氨酸的衍生物包括但不限于泛解酸、2-氨基异戊酸等,异亮氨酸的衍生物包括但不限于4-羟基异亮氨酸、2-氨基丁酸、2-羟基酮丁酸等。
本发明提供一种发酵生产氨基酸或其衍生物的方法,包括:培养所述重组微生物得到培养物,将培养物经分离提取得到氨基酸或其衍生物。
优选地,所述氨基酸或其衍生物为缬氨酸或其衍生物,或者为异亮氨酸或其衍生物。
具体地,所述发酵生产氨基酸或其衍生物的方法包括:将所述重组微生物接种于种子培养基中进行种子培养,得到种子液,将种子液接种于发酵培养基中培养,得到发酵液,将发酵液经分离提取得到所述氨基酸或其衍生物。
优选地,所述发酵培养基包括如下组分:豆粕抽提物10-20g/L,葡萄糖10-60g/L,硫酸铵5-20g/L,硫酸镁0.2-0.8g/L,磷酸二氢钾0.5-2g/L,磷酸氢二钾0.5-2g/L,尿素1-3g/L,VB3 10-20μg/L,VH 40-60μg/L,VB1·HCl80-120μg/L,pH 6.8-7.2。
本发明的有益效果在于:本发明提供的α-异丙基苹果酸合酶突变体能够使得微生物的亮氨酸合成能力显著下降,减少发酵过程中亮氨酸的积累,同时使得微生物合成缬氨酸、异亮氨酸的能力显著增强,缬氨酸、异亮氨酸的产量和转化率显著提高。
本发明提供的表达α-异丙基苹果酸合酶突变体的重组微生物能够高效地合成缬氨酸,缬氨酸产量和转化率均显著提高,且具有较好的生长性能,同时副产物亮氨酸的积累量显著降低,更有利于目标产物的分离纯化。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
以下实施例中使用的引物名称及序列如表1所示。
表1引物序列
引物名称 | 引物序列 |
LeuAP87L-UP-1F | acaacgtcgtgactgggaaaacccaagtcgtggactgggaatgg |
LeuAP87L-UP-1R | aacatgcggcgcttacgctcaagagacatcggatcaatcagagcctggttg |
LeuAP87L-DN-2F | caaccaggctctgattgatccgatgtctcttgagcgtaagcgccgcatgt |
LeuAP87L-DN-2R | cgtaatcatgtcatagctgtttccagattgcggtgcatccattc |
LeuAP87L-pk18-3F | ccattcccagtccacgacttgggttttcccagtcacgacgttgt |
LeuAP87L-pk18-3R | gaatggatgcaccgcaatctggaaacagctatgacatgattacg |
LeuAP87V-UP-1R | aacatgcggcgcttacgctcaacagacatcggatcaatcagagcctggttg |
LeuAP87V-DN-2F | caaccaggctctgattgatccgatgtctgttgagcgtaagcgccgcatgt |
LeuAP87I-UP-1R | aacatgcggcgcttacgctcaatagacatcggatcaatcagagcctggttg |
LeuAP87I-DN-2F | caaccaggctctgattgatccgatgtctattgagcgtaagcgccgcatgt |
以下实施例中使用的出发菌株MHZ-1012-3为一株缬氨酸产生菌,通过将MHZ-1012-2菌株的α-异丙基苹果酸合酶基因leuA的编码区第一个碱基由A变为G得到,其构建方法可采用本领域常规的基因突变方法或者可参见专利申请CN201911370732.9的实施例1。MHZ-1012-2菌株已于2016年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏中心地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏编号为CGMCC No.13406,分类命名为:谷氨酸棒杆菌Corynebacteriumglutamicum。该菌株公开于专利申请CN201611250330.1中。
实施例1构建α-异丙基苹果酸合酶突变菌株2-leuAP87L、3-leuAP87L
1、质粒pK18mobsacB-leuAP87L的构建
利用Phusion超保真聚合酶(New England BioLabs),以出发菌株MHZ-1012-3的基因组为模板,以LeuAP87L-UP-1F/LeuAP87L-UP-1R为引物,制备重组片段UP-1,以LeuAP87L-DN-2F/LeuAP87L-DN-2R为引物,制备重组片段DOWN-1;以质粒pk18-mob-sacB为模板,以LeuAP87L-pk18-3F/LeuAP87L-pk18-3R为引物获得片段pk18-1,经琼脂糖凝胶回收试剂盒(天根)纯化,随后按照吉普森组装试剂盒配置体系进行反应,反应体系表2所示。
表2吉普森组装反应体系
组分 | UP-1 | DOWN-1 | pk18-1 | CE Buffer | CE Exnase | 无菌水 |
体积/μL | 1 | 1 | 2 | 4 | 2 | 9 |
将配制的反应体系于37℃反应30min,吸取10μL转化Trans1T1感受态细胞(TransGen Biotech),挑取单克隆,通过菌落PCR鉴定插入的片段正确,进一步酶切鉴定得到片段插入pK18mobsacB的阳性克隆,最后将质粒送至金唯智测序公司进行测序,将所得到的测序正确的质粒命名为pK18mobsacB-leuAP87L。
2、重组菌株leuAP87L的构建
将pK18mobsacB-leuAP87L分别转入出发菌株MHZ-1012-2、MHZ-1012-3中,在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃,倒置培养。将筛得的转化子过夜培养于普通液体脑心浸液培养基中,培养温度为30℃,回转摇床220rpm振荡培养。此培养过程中,转化子发生第二次重组,通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4),稀释液涂布在含有10%蔗糖的普通固体脑心浸液培养基上,33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列,核苷酸测序分析,获得目的突变菌株,分别命名为2-leuAP87L、3-leuAP87L,两菌株的α-异丙基苹果酸合酶的第87位均由脯氨酸突变为亮氨酸,差别在于菌株2-leuAP87L的α-异丙基苹果酸合酶的第1位为甲硫氨酸,起始密码子为ATG;而菌株3-leuAP87L的α-异丙基苹果酸合酶的第1位为缬氨酸,起始密码子为GTG。
实施例2构建α-异丙基苹果酸合酶突变菌株2-leuAP87V、3-leuAP87V
1、质粒pK18mobsacB-leuAP87V的构建
构建过程同实施例1,利用Phusion超保真聚合酶(New England BioLabs),以出发菌株MHZ-1012-3的基因组为模板,以LeuAP87L-UP-1F/LeuAP87V-UP-1R为引物,制备重组片段UP-2,以LeuAP87V-DN-2F/LeuAP87L-DN-2R为引物,制备重组片段DOWN-2;以质粒pk18-mob-sacB为模板,以LeuAP87L-pk18-3F/LeuAP87L-pk18-3R为引物获得片段pk18-2,经琼脂糖凝胶回收试剂盒(天根)纯化,随后按照吉普森组装试剂盒配置体系进行反应,反应体系如表3所示。
表3吉普森组装反应体系
组分 | UP-2 | DOWN-2 | pk18-2 | CE Buffer | CE Exnase | 无菌水 |
体积/μL | 1 | 1 | 2 | 4 | 2 | 9 |
将配制的反应体系于37℃反应30min,吸取10μL转化Trans1T1感受态细胞(TransGen Biotech),挑取单克隆,通过菌落PCR鉴定插入的片段正确,进一步酶切鉴定得到片段插入pK18mobsacB的阳性克隆,最后将质粒送至金唯智测序公司进行测序,将所得到的测序正确的质粒命名为pK18mobsacB-leuAP87V。
2、重组菌株leuAP87V的构建
将pK18mobsacB-leuAP87V分别转入出发菌株MHZ-1012-2、MHZ-1012-3中,在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃,倒置培养。将筛得的转化子过夜培养于普通液体脑心浸液培养基中,培养温度为30℃,回转摇床220rpm振荡培养。此培养过程中,转化子发生第二次重组,通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4),稀释液涂布在含有10%蔗糖的普通固体脑心浸液培养基上,33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列,核苷酸测序分析,获得目的突变菌株,分别命名为2-leuAP87V、3-leuAP87V,两菌株的α-异丙基苹果酸合酶的第87位均由脯氨酸突变为缬氨酸,差别在于菌株2-leuAP87V的α-异丙基苹果酸合酶的第1位为甲硫氨酸,起始密码子为ATG;而菌株3-leuAP87V的α-异丙基苹果酸合酶的第1位为缬氨酸,起始密码子为GTG。
实施例3构建α-异丙基苹果酸合酶突变菌株2-leuAP87I、3-leuAP87I
1、质粒pK18mobsacB-leuAP87I的构建
构建过程同实施例1,利用Phusion超保真聚合酶(New England BioLabs),以出发菌株MHZ-1012-3的基因组为模板,以LeuAP87L-UP-1F/LeuAP87I-UP-1R为引物,制备重组片段UP-3,以LeuAP87I-DN-2F/LeuAP87L-DN-2R为引物,制备重组片段DOWN-3;以质粒pk18-mob-sacB为模板,以LeuAP87L-pk18-3F/LeuAP87L-pk18-3R为引物获得片段pk18-3,经琼脂糖凝胶回收试剂盒(天根)纯化,随后按照吉普森组装试剂盒配置体系进行反应,反应体系如表4所示。
表4吉普森组装反应体系
组分 | UP-3 | DOWN-3 | pk18-3 | CE Buffer | CE Exnase | 无菌水 |
体积/μL | 1 | 1 | 2 | 4 | 2 | 9 |
将配制的反应体系于37℃反应30min,吸取10μL转化Trans1T1感受态细胞(TransGen Biotech),挑取单克隆,通过菌落PCR鉴定插入的片段正确,进一步酶切鉴定得到片段插入pK18mobsacB的阳性克隆,最后将质粒送至金唯智测序公司进行测序,将所得到的测序正确的质粒命名为pK18mobsacB-leuAP87I。
2、重组菌株leuAP87I的构建
将pK18mobsacB-leuAP87I分别转入出发菌株MHZ-1012-2、MHZ-1012-3中,在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃,倒置培养。将筛得的转化子过夜培养于普通液体脑心浸液培养基中,培养温度为30℃,回转摇床220rpm振荡培养。此培养过程中,转化子发生第二次重组,通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4),稀释液涂布在含有10%蔗糖的普通固体脑心浸液培养基上,33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列,核苷酸测序分析,获得目的突变菌株,分别命名为2-leuAP87I、3-leuAP87I,两菌株的α-异丙基苹果酸合酶的第87位均由脯氨酸突变为异亮氨酸,差别在于菌株2-leuAP87I的α-异丙基苹果酸合酶的第1位为甲硫氨酸,起始密码子为ATG;而菌株3-leuAP87I的α-异丙基苹果酸合酶的第1位为缬氨酸,起始密码子为GTG。
实施例4菌株发酵生产L-缬氨酸
对实施例1-3构建的α-异丙基苹果酸合酶突变菌株进行发酵验证,具体方法如下:
1、培养基
种子培养基:豆粕抽提物15g/L,葡萄糖20g/L,硫酸铵7g/L,硫酸镁0.5g/L,磷酸二氢钾1g/L,磷酸氢二钾1g/L,尿素2g/L,余量为水,pH 7.2。
发酵培养基:豆粕抽提物15g/L,葡萄糖60g/L,硫酸铵20g/L,硫酸镁0.5g/L,磷酸二氢钾1g/L,磷酸氢二钾1g/L,尿素2g/L,碳酸钙40g/L,VB3 15mg/L,VH 50μg/L,VB1·HCl100μg/L,余量为水,pH 7.2。
2、摇瓶发酵生产L-缬氨酸
(1)种子培养:挑取斜面种子1环接至装有50mL种子培养基的500mL三角瓶中,30℃、220r/min振荡培养10-12h;
(2)发酵培养:将5mL种子液接种至装有50mL发酵培养基的500mL三角瓶中,30℃、220r/min振荡培养48h;
(3)取1mL发酵液离心(12000rpm,2min),收集上清液,用HPLC检测酵液中的L-缬氨酸、L-亮氨酸。同时采用分光光度法检测发酵液在562nm下的OD值,结果如表5所示。
表5菌株生长和缬氨酸产量检测结果
注:*表示与出发菌株相比具有显著差异。
结果显示,出发菌株MHZ-1012-2的L-缬氨酸产量仅为5.9g/L,菌株2-leuAP87L、2-leuAP87V、2-leuAP87I的L-缬氨酸产量和转化率较出发菌株显著提高,且L-亮氨酸产量大幅下降;出发菌株MHZ-1012-3的L-缬氨酸积累量仅为7.5g/L,而与出发菌株相比,α-异丙基苹果酸合酶突变菌株3-leuAP87L、3-leuAP87V、3-leuAP87I的L-缬氨酸产量和转化率均显著提高,且L-亮氨酸产量大幅下降,部分菌株的生长略有下降,但仍能够保持较好的生长性能。其中,以α-异丙基苹果酸合酶突变菌株3-leuAP87L表现最为突出,缬氨酸产量为9.2g/L,比出发菌株产量提高1.7g/L,提高了22.6%;副产物亮氨酸产量为0.1g/L,较出发菌株亮氨酸产量下降89%。由此可见,本发明提供的α-异丙基苹果酸合酶突变体及α-异丙基苹果酸合酶突变菌株对目标产物缬氨酸的产量提升具有显著的促进作用,对副产物亮氨酸产量有显著的降低作用。该α-异丙基苹果酸合酶突变体及其重组微生物为产缬氨酸异亮氨酸及以其为前体的衍生物的生产菌株的构建提供借鉴。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 胡丹
<120> α-异丙基苹果酸合酶突变体、重组微生物及其应用
<130> KHP211120021.0
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 616
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<400> 1
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
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Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
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Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
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Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
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Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
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Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
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Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
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Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
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<210> 2
<211> 1851
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atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
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gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
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gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 3
<211> 616
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Val Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
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Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
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Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
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Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
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Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
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Leu Ile Asp Pro Met Ser Leu Glu Arg Lys Arg Arg Met Phe Glu Leu
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Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 4
<211> 616
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Val Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Val Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 5
<211> 616
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Val Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Ile Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 6
<211> 616
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Leu Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 7
<211> 616
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Val Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 8
<211> 616
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Ile Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Asp Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Val Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
His Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser
545 550 555 560
Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 9
<211> 1851
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gtgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctct tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 10
<211> 1851
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctgt tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 11
<211> 1851
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gtgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaatgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctat tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggattcaagg aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaagga catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactcaac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactga tcaagaccgt cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggcg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccacggc aacggcccac tggctgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680
ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
<210> 12
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
acaacgtcgt gactgggaaa acccaagtcg tggactggga atgg 44
<210> 13
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aacatgcggc gcttacgctc aagagacatc ggatcaatca gagcctggtt g 51
<210> 14
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caaccaggct ctgattgatc cgatgtctct tgagcgtaag cgccgcatgt 50
<210> 15
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgtaatcatg tcatagctgt ttccagattg cggtgcatcc attc 44
<210> 16
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccattcccag tccacgactt gggttttccc agtcacgacg ttgt 44
<210> 17
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gaatggatgc accgcaatct ggaaacagct atgacatgat tacg 44
<210> 18
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aacatgcggc gcttacgctc aacagacatc ggatcaatca gagcctggtt g 51
<210> 19
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
caaccaggct ctgattgatc cgatgtctgt tgagcgtaag cgccgcatgt 50
<210> 20
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aacatgcggc gcttacgctc aatagacatc ggatcaatca gagcctggtt g 51
<210> 21
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
caaccaggct ctgattgatc cgatgtctat tgagcgtaag cgccgcatgt 50
Claims (10)
1.α-异丙基苹果酸合酶突变体,其特征在于,以谷氨酸棒杆菌野生型α-异丙基苹果酸合酶为参考序列,所述α-异丙基苹果酸合酶突变体含有第87位脯氨酸被除脯氨酸以外的氨基酸取代的突变。
2.根据权利要求1所述的α-异丙基苹果酸合酶突变体,其特征在于,以谷氨酸棒杆菌野生型α-异丙基苹果酸合酶为参考序列,所述α-异丙基苹果酸合酶突变体含有第87位脯氨酸被亮氨酸、缬氨酸或异亮氨酸取代的突变。
3.根据权利要求1或2所述的α-异丙基苹果酸合酶突变体,其特征在于,所述α-异丙基苹果酸合酶突变体具有如SEQ ID NO.3-8任一所示的氨基酸序列。
4.编码权利要求1~3任一项所述的α-异丙基苹果酸合酶突变体的基因。
5.含有权利要求4所述的基因的生物材料,其特征在于,所述生物材料为重组DNA、载体或宿主细胞。
6.一种重组微生物,其特征在于,所述重组微生物表达权利要求1~3任一项所述的α-异丙基苹果酸合酶突变体,且不表达其出发菌株所具有的α-异丙基苹果酸合酶;
优选地,所述重组微生物中,编码α-异丙基苹果酸合酶的基因被权利要求4所述的基因取代。
7.根据权利要求6所述的重组微生物,其特征在于,所述重组微生物为棒杆菌属细菌或短杆菌属细菌;
优选地,所述棒杆菌属细菌为选自谷氨酸棒杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)、有效棒杆菌(Corynebacterium efficiens)、钝齿棒杆菌(Corynebacterium crenatum)、嗜热产氨棒杆菌(Corynebacteriumthermoaminogenes)、产氨棒杆菌(Corynebacterium aminogenes)中的细菌;
所述短杆菌属细菌为选自黄色短杆菌(Breviabacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)中的细菌。
8.权利要求6或7所述的重组微生物的构建方法,其特征在于,包括:将所述重组微生物的出发菌株中编码α-异丙基苹果酸合酶的基因替换为权利要求4所述的基因。
9.权利要求1~3任一项所述的α-异丙基苹果酸合酶突变体或权利要求4所述的基因或权利要求5所述的生物材料或权利要求6或7所述的重组微生物的如下任一种应用:
(1)在工程菌构建中的应用;
(2)在发酵生产缬氨酸或其衍生物、异亮氨酸或其衍生物中的应用;
(3)在提高菌株的缬氨酸或其衍生物、异亮氨酸或其衍生物的产量和/或转化率中的应用;
(4)在减少菌株的亮氨酸积累中的应用。
10.一种发酵生产氨基酸或其衍生物的方法,其特征在于,包括:培养权利要求6或7所述的重组微生物得到培养物,将培养物经分离提取得到氨基酸或其衍生物;
优选地,所述氨基酸或其衍生物为缬氨酸或其衍生物,或者为异亮氨酸或其衍生物。
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