CN111676205B - L-赖氨酸生产力提高的微生物和利用其生产l-赖氨酸的方法 - Google Patents
L-赖氨酸生产力提高的微生物和利用其生产l-赖氨酸的方法 Download PDFInfo
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- CN111676205B CN111676205B CN202010624521.XA CN202010624521A CN111676205B CN 111676205 B CN111676205 B CN 111676205B CN 202010624521 A CN202010624521 A CN 202010624521A CN 111676205 B CN111676205 B CN 111676205B
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Abstract
本公开涉及L‑赖氨酸生产力提高的微生物和利用其生产L‑赖氨酸的方法。本公开涉及新型修饰RNA聚合酶σ因子A(SigA)多肽；编码其的多核苷酸；包含该多肽的微生物；和利用该微生物生产L‑赖氨酸的方法。
Description
本申请是分案申请，原申请的申请日为2015年9月4日，申请号为201580047334.7，发明名称为“L-赖氨酸生产力提高的微生物和利用其生产L-赖氨酸的方法”。
技术领域
本公开涉及新型修饰RNA聚合酶σ因子A(SigA)多肽；编码其的多核苷酸；包含该多肽的微生物；和利用该微生物生产L-赖氨酸的方法。
背景技术
通过发现和适当利用糖酵解早期阶段直接/间接涉及的遗传因素，可研发生产力增强的微生物菌株，以制备大规模生产有用产物如氨基酸的菌株。基于此目的采用的技术的代表性实例可包括全局转录调控工程(global transcription machineryengineering，gTME)，一种通过对RNA聚合酶的招募蛋白诱导随机诱变来调控细胞全部基因的表达的方法。例如，麻省理工学院(Massachusetts Institute of Technology)的研究组已证明利用gTME技术(美国专利号8735132)成功显著增强了大肠杆菌(E.coli)的酪氨酸生产力。
微生物的转录步骤所利用的RNA聚合酶是由5个小亚单位——即，两个α因子、一个β因子、一个β’因子和一个ω因子——组成的大分子，并且其全酶由α2ββ’ω表示。σ(Sigma)因子连同全酶一起是转录起始步骤的必需因子，其提供RNA聚合酶的启动子结合特异性。棒杆菌(Corynebacterium)菌株具有7种σ因子(SigA、SigB、SigC、SigD、SigE、SigH和SigM)，其根据外部环境的变化调控具体基因群的转录(Journal of Biotechnology 154.2011.101-113)。具体地，SigA是大多数管家基因和核心基因的调控所涉及的σ因子中的主要调控因子。根据此前研究报道，试图通过随机诱变SigA来提高目标物质生产力(MetabolicEngineering 9.2007.258-267)，并且还有关于利用棒杆菌菌株增加L-赖氨酸生产力的研究的报道(国际公开号WO 2003-054179)。
发明内容
[技术问题]
在此情况下，发明人试图研发浓度增加而不抑制宿主细胞生长的具有L-赖氨酸生产力的微生物。结果是，其确认，通过在微生物研发后在该微生物中导入RNA聚合酶的新型修饰型SigA多肽可研发L-赖氨酸生产力增强的棒杆菌属微生物，从而完成本公开。
[技术方案]
本公开的一个目的是提供由SEQ ID NO：2的氨基酸序列组成的具有RNA聚合酶σ因子A活性的修饰多肽，其中多肽的部分氨基酸被取代。
本公开的另一目的是提供编码该修饰多肽的多核苷酸。
本公开的再一目的是提供编码该多肽的转化微生物。
本公开的还再一目的是提供生产L-赖氨酸的方法，包括通过培养微生物获得培养的培养基，和从培养的培养基或培养的微生物回收L-赖氨酸。
[本公开的有益效果]
本公开确认了是否任何具有修饰RNA聚合酶σ因子A的修饰多肽都可上调L-赖氨酸生产力。另外，基于以上，能够表达相应修饰多肽的微生物具有极其优异的L-赖氨酸生产力。因此，认为该微生物在工业方面提供生产成本降低、生产便利等效果。
具体实施方式
为实现上述目的，一方面，本公开提供具有新型RNA聚合酶σ因子A活性的修饰多肽。
如本文所用，术语“RNA聚合酶σ因子A(SigA)”指代与RNA聚合酶一起起作用的转录起始因子，并且其是相应于其中一种σ因子的蛋白质(SigA)。σ因子通过与存在于具体启动子上游的上游DNA(UP元件)和各种转录因子相互作用而涉入转录调控。具体地，SigA已知是主要调控因子，控制大多数核心基因。关于SigA蛋白的信息可由已知的数据库如NCBIGenBank获得，例如，登录号NP_601117的蛋白质。具体地，SigA蛋白可包括SEQ ID NO：2的氨基酸序列，但序列不限于此，只要蛋白质与本公开的SigA具有相同活性。
如本文所用，术语“修饰多肽”指代野生型多肽的氨基酸序列的部分或全部被取代的多肽。在本公开中，修饰多肽指代这样的具有RNA聚合酶σ因子A(SigA)活性的多肽：其中具有RNA聚合酶σ因子A(SigA)的多肽的部分氨基酸序列被取代，因而具有与野生型氨基酸序列不同的氨基酸序列。即，本公开提出有利于L-赖氨酸生产力的SigA修饰多肽，而非野生型SigA多肽。
具体地，修饰多肽可以是这样的具有RNA聚合酶σ因子A活性的多肽：其中选自由SEQ ID NO：2的氨基酸序列组成的多肽的下列位置处的氨基酸的至少一个氨基酸被另一(另外的，another)氨基酸取代：
相对于指定为第一位氨基酸的起始甲硫氨酸，第136位氨基酸；第254位氨基酸；第268位氨基酸；第281位氨基酸；第381位氨基酸；第429位氨基酸；和第445位至第495位氨基酸。
即，修饰多肽可以是其中57个氨基酸修饰位置(第136位、第254位、第268位、第281位、第381位、第429位，和第445位至第495位)中的至少一个位置被另一氨基酸取代的多肽。
更具体地，在第445位至第495位氨基酸中，选自第447位氨基酸、第451位氨基酸、第455位氨基酸、第479位氨基酸、第483位氨基酸、第488位氨基酸、和第491位氨基酸的至少一个氨基酸可被另一氨基酸取代，但不限于此。
具体地，氨基酸取代可以是下列氨基酸取代中的至少一种的组合：
相对于起始甲硫氨酸，第136位氨基酸被甘氨酸取代(D136G)；第254位氨基酸被天冬酰胺取代(I254N)；第268位氨基酸被丝氨酸取代(A268S)；第281位氨基酸被丝氨酸取代(T281S)；第381位氨基酸被精氨酸取代(L381R)；第429位氨基酸被精氨酸取代(Q429R)；第447位氨基酸被组氨酸取代(L447H)；第451位氨基酸被异亮氨酸取代(L451I)；第455位氨基酸被缬氨酸取代(M455V)；第479位氨基酸被精氨酸取代(K479R)；第483位氨基酸被精氨酸取代(K483R)；第488位氨基酸被苏氨酸取代(S488T)；和第491位氨基酸被精氨酸取代(Q491R)。
更具体地，多肽可以是这样的修饰多肽：其中相对于由SEQ ID NO：2的氨基酸序列组成的多肽的起始甲硫氨酸，第136位氨基酸和第281位氨基酸分别被甘氨酸和丝氨酸取代(D136G，T281S)；第254位氨基酸被天冬酰胺取代(I254N)；第268位氨基酸被丝氨酸取代(A268S)；第381位氨基酸被精氨酸取代(L381R)；第429位氨基酸被精氨酸取代(Q429R)；第447位氨基酸被组氨酸取代(L447H)；第451位和第491位氨基酸分别被异亮氨酸和精氨酸取代(L451I，Q491R)；第455位氨基酸被缬氨酸取代(M455V)；第479位氨基酸被精氨酸取代(K479R)；第483位氨基酸被精氨酸取代(K483R)；和第488位氨基酸被苏氨酸(S488T)取代，或组合上述11种氨基酸取代中的至少一种。
根据本公开的示例性实施方式，修饰多肽可以是具有SEQ ID NO：12至22的氨基酸序列中的至少一种氨基酸序列的多肽。
本公开的修饰多肽非限制地不仅包括SEQ ID NO：12至22所示的氨基酸序列，而且包括与上述氨基酸序列具有70％或更高，具体地80％或更高，更具体地90％或更高，还更具体地99％同源性的氨基酸序列，其中与野生型SigA蛋白相比该蛋白质有助于提高L-赖氨酸生产力。显然，除上述序列同源性外还具有与具有修饰SigA蛋白的氨基酸序列的蛋白质基本上相同或相应的生物活性的任何氨基酸序列也应属于本公开的范围，尽管氨基酸可具有部分序列的缺失、修饰、取代、或添加。
如本文所用，术语“同源性”指代编码蛋白质的基因的核苷酸或氨基酸序列的具体比较区域中两种不同核苷酸或氨基酸残基序列(在以最大匹配度对齐两序列后)之间的同一性程度。当同源性足够高时，相应基因的表达产物可具有相同或相似的活性。同源性可利用本领域已知的序列比较程序例如，BLAST(NCBI)、CLC Main Workbench(CLC bio)、MegAlignTM(DNASTAR Inc.)等来确定。
本公开的另一方面包括编码修饰多肽的多核苷酸或包括该多核苷酸的载体。
如本文所用，术语“多核苷酸”指代通过核苷酸单元共价键纵向链延伸的核苷酸多聚体，即，具有至少某长度的DNA或RNA链，并且更具体地，其指代编码修饰多肽的多核苷酸片段。
在本公开中，编码RNA聚合酶σ因子A(SigA)的氨基酸序列的基因是rpoD基因，和具体地，源自谷氨酸棒杆菌(Corynebacterium glutamicum)的基因。基于密码子简并性，编码相同氨基酸的核苷酸序列及其变体属于本公开的范围，并且具体地，其可由SEQ ID NO：1表示，但不限于此。
另外，关于修饰多肽，基于密码子简并性，编码相同氨基酸的核苷酸序列及其变体属于本公开的范围。具体地，编码氨基酸序列12至22中任一个的任何核苷酸序列及其变体可被包括在内，但不限于此。
本公开的再一方面提供包括编码修饰多肽的多核苷酸的宿主细胞、和转化有包括编码修饰多肽的多核苷酸的载体的微生物。具体地，导入可通过转化实现，但不限于此。
具体地，与包括野生型SigA多肽的微生物相比，包括SigA修饰多肽的微生物可提高L-赖氨酸生产能力，而不抑制宿主细胞生长，并且因此可高产率地获得L-赖氨酸。
如本文所用，术语“载体”指代任何将核苷酸克隆和/或转移到宿主细胞中的载体。载体可以是复制子，以允许片段组合另一DNA片段复制。术语“复制子”指代充当自我复制单位用于体内DNA复制的任何遗传单位，即，通过自我调控可复制的随机遗传单位(例如，质粒、噬菌体、粘粒、染色体、和病毒)。如本文所用，术语“载体”可包括用于体外、离体或体内将核苷酸导入宿主细胞的病毒和非病毒载体，并且其还可包括微球DNA。例如，载体可以是不带有细菌DNA序列的质粒。另外，载体可包括转座子(Annu Rev Genet.2003；37：3-29)或人工染色体。具体地，载体可包括pACYC177、pACYC184、pCL1920、pECCG117、pUC19、pBR322、pDZ和pMW118载体，但不限于此。
如本文所用，术语“转化”指代将基因导入宿主细胞从而实现基因在宿主细胞中表达的过程。转化的基因可非限制地包括插入宿主细胞染色体的基因和位于染色体之外的基因，只要其可在宿主细胞中表达。
另外，基因可以表达盒形式被导入宿主细胞，表达盒是多核苷酸构建体，包括自我表达所需的全部基本元件。表达盒可常规地包括可操作地连接至基因的启动子、转录终止信号、核糖体结合域、和翻译终止信号。表达盒可以是能够自我复制的表达载体形式。另外，多核苷酸可原样或以多核苷酸构建体形式被导入宿主细胞，并且可被可操作地连接至其在宿主细胞中表达所需的序列，但不限于此。
宿主细胞或微生物可以是包括编码修饰多肽的多核苷酸、或转化有包括编码修饰多肽的多核苷酸的载体因而能够表达修饰多肽的任何宿主细胞或微生物，并且基于本公开的目的，宿主细胞或微生物可以是通过包括SigA修饰多肽可生产L-赖氨酸的任何宿主细胞或微生物。微生物的具体实例可包括属于埃希氏菌属(Escherichia)、沙雷菌属(Serratia)、欧文氏菌属(Erwinia)、肠杆菌属(Enterobacteria)、沙门氏菌属(Salmonella)、链霉菌属(Streptomyces)、假单胞菌属(Pseudomonas)、短杆菌属(Brevibacterium)、棒杆菌属(Corynebacterium)等的微生物菌株，和具体地，属于棒杆菌属的微生物，并且更具体地，谷氨酸棒杆菌，但不限于此。
在本公开的示例性实施方式中，在具有L-赖氨酸生产力的各种谷氨酸棒杆菌菌株(KCCM11016P、KFCC10750、KCCM10770P、和CJ3P)中导入修饰多肽，该修饰多肽包括选自SEQID NO：12至22的氨基酸序列的任一氨基酸序列，并比较其L-赖氨酸生产力。结果是，确认与包括野生型SigA多肽的菌株相比所有导入有修饰多肽的菌株均显示更高L-赖氨酸生产量(实施例7至10和表8至11)。这些结果表明，包括本公开的修饰多肽的微生物是L-赖氨酸生产力增强的菌株，并且该微生物提供通过培养微生物高产率地获得L-赖氨酸的经济优势。
因此，发明人将该L-赖氨酸生产力增强的菌株“KCCM11016P：：SigA(L447H)”命名为“谷氨酸棒杆菌CA01-2277”，并于2013年11月22日将其以登录号KCCM11479P保藏于根据布达佩斯条约认定为国际保藏机构的韩国微生物保藏中心(Korean Culture Center ofMicroorganisms)。
本公开的再一方面提供生产L-赖氨酸的方法，包括培养上述棒杆菌属微生物，和从培养的微生物或培养的培养基回收L-赖氨酸。
如本文所用，术语“培养”指代使微生物在适当且人工控制的环境条件下生长。本公开的培养过程可基于本领域已知的适当培养基和培养条件进行。具体条件，如培养温度、培养时间、培养基pH等，可由本领域技术人员通过公知常识或通过本领域已知的常规方法确定，并且因此适当调节。具体地，这些已知的培养方法在参考文献中被详细描述[Chmiel；Bioprozesstechnik 1.Einfuhrung indie Bioverfahrenstechnik(Gustav FischerVerlag，Stuttgart，1991)，和Storhas；Bioreaktoren und periphere Einrichtungen(Vieweg Verlag，Braunschweig/Wiesbaden，1994)]。另外，培养方法可包括分批培养、连续培养、和补料分批培养，并且具体地，培养可在补料分批或反复补料分批过程中连续进行，但不限于此。
培养中使用的培养基必须适当地满足具体菌株的要求。培养基中所用碳源的实例可包括糖和碳水化合物，如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉、和纤维素；油和脂肪，如大豆油、葵花籽油、蓖麻油、和椰子油；脂肪酸，如棕榈酸、硬脂酸、和亚油酸；醇，如甘油和乙醇；和有机酸，如乙酸，但不限于此。这些碳源可单独或组合使用，但不限于此。培养基中所用氮源的实例可包括蛋白胨、酵母提取物、肉汁、麦芽提取物、玉米浆、大豆粉、和尿素；或无机化合物，如硫酸铵、氯化铵、磷酸铵、碳酸铵、和硝酸铵。这些氮源也可单独或组合使用，但不限于此。培养基中所用磷源的实例可包括磷酸二氢钾、磷酸氢二钾、相应的含钠盐等，但不限于此。另外，生长所需的金属盐，如硫酸镁或硫酸铁，可被包含在培养基中。最后，除上述材料外，生长必需材料，如氨基酸和维生素，也可被包含在内。另外，可使用适于培养基的前体。这些来源可通过分批培养或连续培养在培养期间以适当方式被添加至培养物中，但方法不限于此。
另外，可通过以适当方式将化合物如氢氧化铵、氢氧化钾、氨、磷酸和硫酸添加至培养物在培养期间调节培养物的pH。在培养期间，可添加消泡剂，如脂肪酸聚二醇酯，以防止泡沫生成。另外，氧气或含氧气体可被注入培养物以维持培养物的有氧状态；或氮气，氢气、或二氧化碳气体可被注入，而不注入气体，以维持培养物的无氧或微氧状态。培养温度可总体上在27℃至37℃范围内并且具体地在30℃至35℃范围内。培养可持续到获得期望量的有用物质，并且具体地持续10小时至100小时。L-赖氨酸可被释放到培养中的培养基中或可被包含在微生物内。
另外，关于本公开的生产L-赖氨酸的方法，从培养的微生物或培养物回收L-赖氨酸的方法在本领域公知。回收L-赖氨酸的方法可包括过滤、阴离子交换色谱法、结晶、HPLC等，但不限于此。
[公开详述]
下文将参考下列实施例对本公开进行更详细的描述。然而，这些实施例仅以示例为目的，而不意图本公开受限于这些实施例。
实施例1：利用人工诱变获得SigA修饰多肽
在本实施例中，通过下列方法准备获得修饰SigA的染色体内初级交叉-插入(primary crossover-insertion)的载体库。针对编码棒杆菌SigA(SEQ ID NO：2)的rpoD基因(SEQ ID NO：1)进行易错PCR，从而获得随机导入有核苷酸取代修饰的rpoD基因变体的片段(1497bp)。利用谷氨酸棒杆菌ATCC13032的基因组DNA作为模板以及引物1(SEQ ID NO：3)和引物2(SEQ ID NO：4)，利用GenemorphII随机诱变试剂盒(Stratagene)，进行易错PCR。进行易错PCR以使得修饰可以0至4.5突变/1kb扩增片段的比例被导入扩增基因片段。PCR在下列条件共进行30轮：96℃变性30sec，53℃退火30sec，和72℃聚合2min。
[表1]
| 引物编号 | 核苷酸序列 | SEQ ID NO |
| 1 | GTGGAGAGCAGCATGGTAG | 3 |
| 2 | CGCAGAGGAAAACAGTGGC | 4 |
将扩增基因片段利用pCR2.1-TOPO TA克隆试剂盒(Invitrogen)连接至pCR2.1-TOPO载体(下称，‘pCR2.1’)，转化到大肠杆菌DH5α中，并铺平在固体LB培养基上。选择由此转化的菌落中的20个，并在获得其质粒后分析其核苷酸序列。结果是，确认突变以1.5突变/kb的频率在不同位置被导入。从约20,000个转化的大肠杆菌菌落提取质粒，并将其命名为“pCR2.1-rpoD(mt)文库”。然后，制备用作对照的包括野生型rpoD基因的质粒。在上述相同条件下，利用谷氨酸棒杆菌ATCC13032的基因组DNA作为模板以及引物1(SEQ ID NO：3)和引物2(SEQ ID NO：4)，进行PCR。关于聚合酶，使用PfuUltraTM高保真度DNA聚合酶(Stratagene)，并将如此制备的质粒命名为“pCR2.1-rpoD(WT)”。
实施例2：制备L-赖氨酸生产力增强的SigA修饰菌株的文库
将利用KCCM11016P菌株(韩国专利号10-0159812)作为亲本菌株制备的pCR2.1-rpoD(mt)文库通过同源染色体重组进行转化，铺平在包含卡那霉素(25mg/L)和下述组分的复合培养基上，并从中获得约25,000个菌落。将该菌落命名为“KCCM11016P/pCR2.1-rpoD(mt)-1”至“KCCM11016P/pCR2.1-rpoD(mt)-25000”。另外，将由此制备的pCR2.1-rpoD(WT)载体转化到KCCM11016P菌株中以制备对照菌株，并将其命名为“KCCM11016P/pCR2.1-rpoD(WT)”。
由此获得的转化体的染色体上将具有2拷贝的rpoD基因。然而，pCR2.1-rpoD(mt)文库和pCR2.1-rpoD(WT)载体中插入有rpoD基因片段，而无任何启动子，因此当其通过同源重组被插入菌株染色体中时，2拷贝rpoD基因中仅1个拷贝表达，因此能够表达修饰或野生型SigA蛋白。
<复合培养基(pH 7.0)>
葡萄糖(10g)、蛋白胨(10g)、牛肉提取物(5g)、酵母提取物(5g)、脑心浸液(18.5g)、NaCl(2.5g)、尿素(2g)、山梨醇(91g)、琼脂(20g)(基于1L蒸馏水)
实施例3：筛选L-赖氨酸生产力提高的SigA修饰菌株的文库
将实施例2获得的约25,000个菌落分别接种于包含下述组分的选择培养基(300μL)中，并在32℃、1000rpm速率下在96深孔板中培养24小时。通过茚三酮法(J.Biol.Chem.1948.176：367-388)分析培养期间的L-赖氨酸生产量。在培养完成后，使10μL培养物上清液和190μL茚三酮反应溶液((63％甘油，27％茚三酮反应(在0.5M柠檬酸盐缓冲液中7.1g/L，pH 5.5))在65℃下反应30分钟。通过分光光度计测量波长570下的吸光度并与对照(即，KCCM11016P/pCR2.1-rpoD(WT))进行比较，并且选择显示吸光度至少10％增加的约935种修饰菌株。其它菌落与对照相比显示相似或减少的吸光度。
<选择培养基(pH 8.0)>
葡萄糖(10g)、硫酸铵(5.5g)、MgSO4·7H2O(1.2g)、KH2PO4(0.8g)、K2HPO4(16.4g)、生物素(100μg)、硫胺HCl(1000μg)、钙-泛酸(2000μg)、和烟酰胺(2000μg)(基于1L蒸馏水)
针对选择的935种菌株反复进行上述方法，并且前231种菌株与KCCM11016P/pCR2.1-rpoD(WT)菌株相比L-赖氨酸生产力提高15％或更高。
实施例4：分析KCCM11016P/pCR2.1-rpoD(mt)的L-赖氨酸生产力
分析实施例3选择的231种菌株——关于在将其通过下列方法培养后其L-赖氨酸生产力。
将各菌株分别接种于包含25mL种子培养基的250mL角挡板烧瓶(corner-baffleflask)中，并在摇动培养箱(200rpm)中在30℃下培养20小时。然后，将包含24mL培养物的各250mL角挡板烧瓶(包含下述组分)接种1mL种子培养液，并在摇动培养箱(200rpm)中在30℃下培养72小时。通过高效液相色谱(HPLC)分析各培养物中L-赖氨酸的浓度。
<种子培养基(pH 7.0)>
葡萄糖(20g)、蛋白胨(10g)、酵母提取物(5g)、尿素(1.5g)、KH2PO4(4g)、K2HPO4(8g)、MgSO4·7H2O(0.5g)、生物素(100μg)、硫胺HCl(1000μg)、钙-泛酸(2000μg)、和烟酰胺(2000μg)(基于1L蒸馏水)
<生产培养基(pH 7.0)>
葡萄糖(100g),(NH4)2SO4(40g)、大豆蛋白(2.5g)、玉米浆固体(5g)、尿素(3g)、KH2PO4(1g)、MgSO4·7H2O(0.5g)、生物素(100μg)、硫胺HCl(1000μg)、钙-泛酸(2000μg)、烟酰胺(3000μg)、和CaCO3(30g)(基于1L蒸馏水)
在选择的231种菌株中，选择显示与对照相比L-赖氨酸浓度可再现地增加的17种菌株，并反复进行培养和分析。L-赖氨酸浓度的分析结果显示在下表2中。
[表2]
17种选定菌株的L-赖氨酸浓度分析结果是，确认了15种菌株的L-赖氨酸生产力——不包括两种菌株，即，KCCM11016P/pCR2.1-rpoD(mt)-6589和KCCM11016P/pCR2.1-rpoD(mt)-18904——与对照(KCCM11016P/pCR2.1-rpoD(WT)菌株)相比最多增加20％。
实施例5：确认选自SigA人工诱变文库的菌株中的rpoD基因突变
为确认在15种菌株(在实施例4中，在17种选定菌株之中，显示L-赖氨酸生产力提高)的SigA中导入的突变，分析rpoD突变体的核苷酸序列。为确定核苷酸序列，利用引物1(SEQ ID NO：3)和引物3(SEQ ID NO：5)进行PCR。
[表3]
| 引物编号 | 核苷酸序列 | SEQ ID NO |
| 1 | GTGGAGAGCAGCATGGTAG | 3 |
| 3 | AACAGCTATGACCATG | 5 |
通过分析由此获得的15种菌株中的每一个的修饰rpoD基因的片段的核苷酸序列，基于NIH Genbank，确认修饰rpoD基因的核苷酸序列，并且确认修饰SigA的核苷酸序列。由此获得的15种菌株的修饰SigA氨基酸序列的分析结果显示在下表4中。
[表4]
| 菌株 | SigA的氨基酸修饰 |
| KCCM11016P/pCR2.1-rpoD(mt)-158 | D136G、T281S |
| KCCM11016P/pCR2.1-rpoD(mt)-1494 | L381R |
| KCCM11016P/pCR2.1-rpoD(mt)-1846 | M230T |
| KCCM11016P/pCR2.1-rpoD(mt)-2198 | M455V |
| KCCM11016P/pCR2.1-rpoD(mt)-2513 | S488T |
| KCCM11016P/pCR2.1-rpoD(mt)-3777 | R279L |
| KCCM11016P/pCR2.1-rpoD(mt)-4065 | L447H |
| KCCM11016P/pCR2.1-rpoD(mt)-5329 | A268S |
| KCCM11016P/pCR2.1-rpoD(mt)-9823 | L451I、Q491R |
| KCCM11016P/pCR2.1-rpoD(mt)-11267 | Q429R |
| KCCM11016P/pCR2.1-rpoD(mt)-13306 | K90E、K105Y、D250G、I254L |
| KCCM11016P/pCR2.1-rpoD(mt)-14535 | I254N |
| KCCM11016P/pCR2.1-rpoD(mt)-16323 | K483R |
| KCCM11016P/pCR2.1-rpoD(mt)-17935 | K479R |
| KCCM11016P/pCR2.1-rpoD(mt)-19803 | D238V、N263S、E358D |
结果是，确认最小1至最大4范围内的氨基酸被取代。在上表4中，数字表示SigA的氨基酸编号；数字前的字母表示取代前的氨基酸；并且数字后的字母表示取代后的氨基酸。
实施例6：制备用于导入rpoD修饰染色体以生产具有高浓度L-赖氨酸生产力的菌
株的载体
为确认应用实施例4确认的SigA修饰的效果，制备能够将SigA修饰导入染色体上的载体。
基于已报道的核苷酸序列，合成引物4(SEQ ID NO：6)(其中在5’端插入EcoRI限制位点)和引物5(SEQ ID NO：7)(其中在3’端插入SalI限制位点)。然后，利用引物对，利用各选定15种的染色体作为模板，进行PCR，从而扩增15种修饰rpoD(mt)基因片段。PCR在下列条件下进行：94℃变性5min；94℃变性30sec，56℃退火30sec，和72℃聚合2min，30个循环；和72℃聚合7min。
[表5]
| 引物No | 核苷酸序列 | SEQ ID NO |
| 4 | AAGAATTCGTGGAGAGCAGCATGGTAG | 6 |
| 5 | AAGTCGACCGCAGAGGAAAACAGTGGC | 7 |
用EcoRI和SalI处理通过PCR扩增的15种基因片段以获得各自的DNA片段，将该片段连接至用于导入染色体的pDZ载体(韩国专利号10-0924065)——在其中包括EcoRI和SalI限制位点——并转化到大肠杆菌DH5α中，并在包含卡那霉素(25mg/L)的LB固体培养基上铺涂。选择经插入有通过PCR得到的目标基因的载体转化的菌落，并通过公知的质粒提取方法获得质粒。根据各质粒的SigA上的插入修饰，质粒分别被命名为pDZ-SigA(D136G，T281S)、pDZ-SigA(L381R)、pDZ-SigA(M230T)、pDZ-SigA(M455V)、pDZ-SigA(S488T)、pDZ-SigA(R279L)、pDZ-SigA(I254N)、pDZ-SigA(A268S)、pDZ-SigA(L451I、Q491R)、pDZ-SigA(Q429R)、pDZ-SigA(K90E、K105Y、D250G、I254L)、pDZ-SigA(L447H)、pDZ-SigA(K483R)、pDZ-SigA(K479R)、和pDZ-SigA(D238V、N263S、E358D)。另外，作为上述变体的另一对照，制备引物6至9(SEQ ID NO：8至11)以构建将SigA修饰A414V插入染色体的载体——其据报道具有增加L-赖氨酸生产力的效果(国际公开WO2003-054179)。然后，分别利用谷氨酸棒杆菌ATCC13032的基因组DNA以及引物6和引物9的引物对和引物7和引物8的引物对进行PCR。PCR在下列条件下进行：94℃变性5min；94℃变性30sec，56℃退火30sec，和72℃聚合1min，30个循环；和72℃聚合7min。
[表6]
| 引物编号 | 核苷酸序列 | SEQ ID NO |
| 6 | GCAGGTCGACTCTAGACTCACCCCAGCCGTCAAGCGT | 8 |
| 7 | CCGGGGATCCTCTAGATCAAGGCGATGTCGGAAAATG | 9 |
| 8 | AAGACTCCGAAGTCGTCGTCGCAGT | 10 |
| 9 | ACTGCGACGACGACTTCGGAGTCTT | 11 |
结果是，获得约500bp大小的基因片段，并利用Infusion克隆试剂盒(Invitrogen)将扩增产物连接至pDZ载体。由此制备的载体被命名为pDZ-SigA(A414V)。
实施例7：为具有高浓度L-赖氨酸生产力的菌株制备其中导入SigA修饰多肽的
KCCM11016P菌株和比较其L-赖氨酸生产力
通过两步式同源染色体重组，将实施例6制备的导入有新型突变的15种载体和导入有之前已报道的突变的1种载体转化到谷氨酸棒杆菌KCCM11016P(L-赖氨酸生产菌株)中。然后，通过核苷酸序列分析，选择其中导入了SigA修饰的菌株。其中导入了SigA修饰的菌株分别被命名为KCCM11016P：：SigA(D136G、T281S)、KCCM11016P：：SigA(L381R)、KCCM11016P：：SigA(M230T)、KCCM11016P：：SigA(M455V)、KCCM11016P：：SigA(S488T)、KCCM11016P：：SigA(R279L)、KCCM11016P：：SigA(I254N)、KCCM11016P：：SigA(A268S)、KCCM11016P：：SigA(L451I、Q491R)、KCCM11016P：：SigA(Q429R)、KCCM11016P：：SigA(K90E、K105Y、D250G、I254L)、KCCM11016P：：SigA(L447H)、KCCM11016P：：SigA(K483R)、KCCM11016P：：SigA(K479R)、KCCM11016P：：SigA(D238V、N263S、E358D)、和KCCM11016P：：SigA(A414V)。
将该菌株以与实施例4相同的方式培养，并分析其L-赖氨酸浓度。结果显示在下表7中。
[表7]
结果是，其中导入了修饰的两种新型菌株(KCCM11016P：：SigA(M230T)和KCCM11016P：：SigA(R279L))显示与亲本菌株同等的L-赖氨酸生产力，而其中导入了突变的另外两种新型菌株(KCCM11016P：：SigA(K90E，K105Y，D250G，I254L)和KCCM11016P：：SigA(D238V，N263S，E358D))显示显著低的生长速率和显著减少的L-赖氨酸生产量。然而，其余11种新型突变菌株显示L-赖氨酸生产与亲本菌株相比最大增加19％，并且还显示与KCCM11016P：：SigA(A414V)(其中导入了之前已报道的SigA(A414V)的突变菌株)相比L-赖氨酸生产增加16％。由此，发明人将这种L-赖氨酸生产力提高的菌株“KCCM11016P：：SigA(L447H)”命名为“谷氨酸棒杆菌CA01-2277”，并将其于2013年11月22日以登录号KCCM11479P保藏于根据布达佩斯条约认定为国际保藏机构的韩国微生物保藏中心。
这些结果表明11种新型SigA修饰多肽具有优异的L-赖氨酸生产力。
实施例8：为具有高浓度L-赖氨酸生产力的菌株制备其中导入了SigA修饰多肽的
KFCC10750菌株和比较其L-赖氨酸生产力
为确认导入实施例7选择的11种SigA变体在谷氨酸棒杆菌其它菌株中的效果，以与实施例7相同的方式制备具有L-赖氨酸生产力的谷氨酸棒杆菌KFCC10750(KCCM11347P，韩国专利号10-0073610)的其中分别导入了11种SigA突变中的每一种的菌株。这些菌株分别被命名为KFCC10750：：SigA(D136G、T281S)、KFCC10750：：SigA(L381R)、KFCC10750：：SigA(M455V)、KFCC10750：：SigA(S488T)、KFCC10750：：SigA(I254N)、KFCC10750：：SigA(A268S)、KFCC10750：：SigA(L451I、Q491R)、KFCC10750：：SigA(Q429R)、KFCC10750：：SigA(L447H)、KFCC10750：：SigA(K483R)、和KFCC10750：：SigA(K479R)。另外，制备其中导入了之前已报道的SigA(A414V)突变的菌株，并命名为“KFCC10750：：SigA(A414V)”。
将该菌株以与实施例4相同的方式培养，并分析其L-赖氨酸浓度。结果显示在下表8中。
[表8]
结果是，其中导入了新型突变的11种菌株显示与亲本菌株相比L-赖氨酸生产最大增加17％，并且还显示与KFCC10750：：SigA(A414V)(其中导入了之前已报道的SigAA414V突变菌株)相比L-赖氨酸生产增加约14％。
实施例9：为具有高浓度L-赖氨酸生产力的菌株制备其中导入了SigA修饰多肽的
KCCM10770P菌株和比较其L-赖氨酸生产力
为确认导入实施例7选择的11种SigA变体在谷氨酸棒杆菌其它菌株中的效果，以与实施例7相同的方式制备具有L-赖氨酸生产力的谷氨酸棒杆菌KFCC10770(KCCM11347P，韩国专利号10-0924065)的其中分别导入了SigA修饰的菌株。这些菌株分别被命名为KCCM10770P：：SigA(D136G、T281S)、KCCM10770P：：SigA(L381R)、KCCM10770P：：SigA(M455V)、KCCM10770P：：SigA(S488T)、KCCM10770P：：SigA(I254N)、KCCM10770P：：SigA(A268S)、KCCM10770P：：SigA(L451I、Q491R)、KCCM10770P：：SigA(Q429R)、KCCM10770P：：SigA(L447H)、和KCCM10770P：：SigA(K483R)、KCCM10770P：：SigA(K479R)。另外，制备其中导入了之前已报道的SigA(A414V)突变的菌株，并命名为“KFCC10770：：SigA(A414V)”。
将该菌株以与实施例4相同的方式培养，并分析其L-赖氨酸浓度。结果显示在下表9中。
[表9]
结果是，其中导入了新型突变的11种菌株显示与亲本菌株相比L-赖氨酸生产最大增加20％，并且还显示与KFCC10770：：SigA(A414V)(其中导入了之前已报道的SigAA414V突变的菌株)相比L-赖氨酸生产增加约16％。
实施例10：为具有高浓度L-赖氨酸生产力的菌株制备其中导入了SigA修饰多肽的
CJ3P菌株和比较其L-赖氨酸生产力
为确认在谷氨酸棒杆菌其它菌株中的效果，以与实施例7相同的方式制备其中SigA修饰分别被导入至具有L-赖氨酸生产力的谷氨酸棒杆菌CJ3P(Binder et al.GenomeBiology 2012，13：R40)的菌株。这些菌株分别被命名为CJ3P：：SigA(D136G、T281S)、CJ3P：：SigA(L381R)、CJ3P：：SigA(M455V)、CJ3P：：SigA(S488T)、CJ3P：：SigA(I254N)、CJ3P：：SigA(A268S)、CJ3P：：SigA(L451I、Q491R)、CJ3P：：SigA(Q429R)、CJ3P：：SigA(L447H)、CJ3P：：SigA(K483R)、和CJ3P：：SigA(K479R)。另外，制备其中导入了之前已报道的SigA(A414V)突变的菌株，并命名为“CJ3P：：SigA(A414V)”。CJ3P是谷氨酸棒杆菌菌株，其通过基于已知技术将3种突变导入野生型微生物而具有L-赖氨酸生产力。
将该菌株以与实施例4相同的方式培养，并分析其L-赖氨酸浓度。结果显示在下表10中。
[表10]
结果是，其中导入了新型突变的11种菌株显示与亲本菌株相比L-赖氨酸生产最大增加20％，并且还显示与CJ3P：：SigA(A414V)(其中导入了之前已报道的SigAA414V突变的菌株)相比L-赖氨酸生产增加约15％。
综上结果，本公开获得的具有SigA活性的11种新型修饰多肽(即、SigA(D136G、T281S)、SigA(L381R)、SigA(M455V)、SigA(S488T)、SigA(I254N)、SigA(A268S)、SigA(L451I、Q491R)、SigA(Q429R)、SigA(L447H)、SigA(K483R)、和SigA(K479R))分别显示在各种棒杆菌属微生物中对提高L-赖氨酸生产具有优异效果，并且这些结果表明该修饰多肽与之前已报道的修饰SigA(A414V)相比对提高L-赖氨酸生产具有优异效果。
基于以上，本公开所属领域技术人员将能够理解，在不改变本公开的技术思路或重要特征的情况下，本公开可以其它具体形式实施。关于这点，本文公开的示例性实施方式仅以示例为目的，不应被解释为限制本公开的范围。相反，本公开意图不仅包括示例性实施方式，而且包括可包括在所附权利要求限定的本公开的精神和范围之内的各种替代形式、改动、等同形式和其它实施方式。
<110> CJ第一制糖株式会社
<120> L-赖氨酸生产力增强的微生物和利用其生产L-赖氨酸的方法
<130> OPA15239-PCT
<150> KR 10-2014-0119137
<151> 2014-09-05
<160> 22
<170> KopatentIn 2.0
<210> 1
<211> 1497
<212> DNA
<213> 谷氨酸棒杆菌
<400> 1
gtggagagca gcatggtaga aaacaacgta gcaaaaaaga cggtcgctaa aaagaccgca 60
cgcaagaccg cacgcaaagc agccccgcgc gtggcaaccc cattgggagt cgcatctgag 120
tctcccattt cggccacccc tgcgcgcagc atcgatggaa cctcaacccc tgttgaagct 180
gctgacacca tagagaccac cgcccctgca gcgaaggctc ctgcggccaa ggctcccgct 240
aaaaaggttg ccaagaagac agctcgcaag gcacctgcga aaaagactgt cgccaagaaa 300
gccacaaccg ccaaggctgc acctgcaact gccaaggacg aaaacgcacc tgttgatgac 360
gacgaggaga acctcgctca ggatgaacag gacttcgacg gcgatgactt cgtagacggc 420
atcgaagacg aagaagatga agacggcgtc gaagccctcg gtgaagaaag cgaagacgac 480
gaagaggacg gctcatccgt ttgggatgaa gacgaatccg caaccctgcg tcaggcacgt 540
aaagatgccg agctcaccgc ttccgccgac tctgttcgcg cttacctgaa gcaaatcggt 600
aaagttgccc tgctgaacgc tgaacaggaa gtctccctgg caaagcgcat cgaagcaggc 660
ctttacgcca cccaccgcat ggaggaaatg gaagaagctt tcgcagccgg tgacaaggac 720
gcgaaactca ccccagccgt caagcgtgac ctccgcgcca tcgctcgtga cggccgcaag 780
gcgaaaaacc acctcctgga agccaacctt cgtctggttg tctccctggc aaagcgctac 840
accggccgtg gcatggcatt cctggacctc atccaggaag gcaacctcgg tctgattcgt 900
gccgtagaga agttcgacta ctccaagggc tacaagttct ccacctacgc aacctggtgg 960
atccgtcagg caatcacccg cgccatggcc gaccaagcac gaaccatccg tatcccagtc 1020
cacatggttg aagtgatcaa caaacttggt cgcatccaac gtgaactcct tcaggaactc 1080
ggccgcgaac caaccccaca ggaactgtcc aaagaaatgg acatctccga ggaaaaggta 1140
ctggaaatcc agcagtacgc ccgcgaacca atctccctgg accaaaccat cggcgacgaa 1200
ggcgacagcc agctcggcga cttcatcgaa gactccgaag ccgtcgtcgc agtcgacgcc 1260
gtctcattca ccctgctgca agaccagcta caggacgtcc tagagaccct ctccgaacgt 1320
gaagccggcg tggttaaact ccgcttcgga ctcaccgacg gaatgccacg cactttagac 1380
gaaatcggcc aagtttacgg tgtcacccgt gagcgcatcc gccagattga gtccaagacc 1440
atgtctaagc tgcgccaccc atcacgctcc caggtccttc gcgactacct ggactaa 1497
<210> 2
<211> 498
<212> PRT
<213> 谷氨酸棒杆菌
<400> 2
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 3
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物1
<400> 3
gtggagagca gcatggtag 19
<210> 4
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物2
<400> 4
cgcagaggaa aacagtggc 19
<210> 5
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 引物3
<400> 5
aacagctatg accatg 16
<210> 6
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 引物4
<400> 6
aagaattcgt ggagagcagc atggtag 27
<210> 7
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 引物5
<400> 7
aagtcgaccg cagaggaaaa cagtggc 27
<210> 8
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物6
<400> 8
gcaggtcgac tctagactca ccccagccgt caagcgt 37
<210> 9
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物7
<400> 9
ccggggatcc tctagatcaa ggcgatgtcg gaaaatg 37
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物8
<400> 10
aagactccga agtcgtcgtc gcagt 25
<210> 11
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物9
<400> 11
actgcgacga cgacttcgga gtctt 25
<210> 12
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 12
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Gly Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Ser Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 13
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 13
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Arg Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 14
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 14
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Val Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 15
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 15
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Thr Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 16
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 16
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys His Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 17
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 17
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ser Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 18
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 18
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Ile Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Arg Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 19
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 19
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Arg Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 20
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 20
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Asn Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 21
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 21
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Lys Thr
465 470 475 480
Met Ser Arg Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
<210> 22
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> SigA变体
<400> 22
Met Glu Ser Ser Met Val Glu Asn Asn Val Ala Lys Lys Thr Val Ala
1 5 10 15
Lys Lys Thr Ala Arg Lys Thr Ala Arg Lys Ala Ala Pro Arg Val Ala
20 25 30
Thr Pro Leu Gly Val Ala Ser Glu Ser Pro Ile Ser Ala Thr Pro Ala
35 40 45
Arg Ser Ile Asp Gly Thr Ser Thr Pro Val Glu Ala Ala Asp Thr Ile
50 55 60
Glu Thr Thr Ala Pro Ala Ala Lys Ala Pro Ala Ala Lys Ala Pro Ala
65 70 75 80
Lys Lys Val Ala Lys Lys Thr Ala Arg Lys Ala Pro Ala Lys Lys Thr
85 90 95
Val Ala Lys Lys Ala Thr Thr Ala Lys Ala Ala Pro Ala Thr Ala Lys
100 105 110
Asp Glu Asn Ala Pro Val Asp Asp Asp Glu Glu Asn Leu Ala Gln Asp
115 120 125
Glu Gln Asp Phe Asp Gly Asp Asp Phe Val Asp Gly Ile Glu Asp Glu
130 135 140
Glu Asp Glu Asp Gly Val Glu Ala Leu Gly Glu Glu Ser Glu Asp Asp
145 150 155 160
Glu Glu Asp Gly Ser Ser Val Trp Asp Glu Asp Glu Ser Ala Thr Leu
165 170 175
Arg Gln Ala Arg Lys Asp Ala Glu Leu Thr Ala Ser Ala Asp Ser Val
180 185 190
Arg Ala Tyr Leu Lys Gln Ile Gly Lys Val Ala Leu Leu Asn Ala Glu
195 200 205
Gln Glu Val Ser Leu Ala Lys Arg Ile Glu Ala Gly Leu Tyr Ala Thr
210 215 220
His Arg Met Glu Glu Met Glu Glu Ala Phe Ala Ala Gly Asp Lys Asp
225 230 235 240
Ala Lys Leu Thr Pro Ala Val Lys Arg Asp Leu Arg Ala Ile Ala Arg
245 250 255
Asp Gly Arg Lys Ala Lys Asn His Leu Leu Glu Ala Asn Leu Arg Leu
260 265 270
Val Val Ser Leu Ala Lys Arg Tyr Thr Gly Arg Gly Met Ala Phe Leu
275 280 285
Asp Leu Ile Gln Glu Gly Asn Leu Gly Leu Ile Arg Ala Val Glu Lys
290 295 300
Phe Asp Tyr Ser Lys Gly Tyr Lys Phe Ser Thr Tyr Ala Thr Trp Trp
305 310 315 320
Ile Arg Gln Ala Ile Thr Arg Ala Met Ala Asp Gln Ala Arg Thr Ile
325 330 335
Arg Ile Pro Val His Met Val Glu Val Ile Asn Lys Leu Gly Arg Ile
340 345 350
Gln Arg Glu Leu Leu Gln Glu Leu Gly Arg Glu Pro Thr Pro Gln Glu
355 360 365
Leu Ser Lys Glu Met Asp Ile Ser Glu Glu Lys Val Leu Glu Ile Gln
370 375 380
Gln Tyr Ala Arg Glu Pro Ile Ser Leu Asp Gln Thr Ile Gly Asp Glu
385 390 395 400
Gly Asp Ser Gln Leu Gly Asp Phe Ile Glu Asp Ser Glu Ala Val Val
405 410 415
Ala Val Asp Ala Val Ser Phe Thr Leu Leu Gln Asp Gln Leu Gln Asp
420 425 430
Val Leu Glu Thr Leu Ser Glu Arg Glu Ala Gly Val Val Lys Leu Arg
435 440 445
Phe Gly Leu Thr Asp Gly Met Pro Arg Thr Leu Asp Glu Ile Gly Gln
450 455 460
Val Tyr Gly Val Thr Arg Glu Arg Ile Arg Gln Ile Glu Ser Arg Thr
465 470 475 480
Met Ser Lys Leu Arg His Pro Ser Arg Ser Gln Val Leu Arg Asp Tyr
485 490 495
Leu Asp
Claims (7)
1.具有RNA聚合酶σ因子A活性的修饰多肽，其中由SEQ ID NO：2所示的氨基酸序列组成的多肽的氨基酸被取代，并且所述氨基酸的取代是第488位氨基酸被苏氨酸取代。
2.根据权利要求1所述的修饰多肽，其中所述修饰多肽由SEQ ID NO：15所示的氨基酸序列组成。
3.多核苷酸，其编码权利要求1-2中任一项所述的修饰多肽。
4.微生物细胞，其包括权利要求3所述的多核苷酸。
5.L-赖氨酸生产力增强的棒杆菌属微生物，其包括具有RNA聚合酶σ因子A活性的修饰多肽，其中由SEQ ID NO：2所示的氨基酸序列组成的多肽的氨基酸被取代，并且所述氨基酸的取代是第488位氨基酸被苏氨酸取代。
6.根据权利要求5所述的微生物，其中所述微生物是谷氨酸棒杆菌(Corynebacteriumglutamicum)。
7.生产L-赖氨酸的方法，包括：
(a)在培养基中培养根据权利要求5至6中任一项所述的微生物；和
(b)从培养的微生物或培养的培养基回收L-赖氨酸。
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| KR10-2014-0119137 | 2014-09-05 | ||
| KR1020140119137A KR101755767B1 (ko) | 2014-09-05 | 2014-09-05 | L-라이신 생산능이 향상된 미생물 및 이를 이용한 l-라이신 생산방법 |
| PCT/KR2015/009353 WO2016036191A1 (ko) | 2014-09-05 | 2015-09-04 | L-라이신 생산능이 향상된 미생물 및 이를 이용한 l-라이신 생산방법 |
| CN202010624521.XA CN111676205B (zh) | 2014-09-05 | 2015-09-04 | L-赖氨酸生产力提高的微生物和利用其生产l-赖氨酸的方法 |
| CN201580047334.7A CN106715688A (zh) | 2014-09-05 | 2015-09-04 | L‑赖氨酸生产力提高的微生物和利用其生产l‑赖氨酸的方法 |
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| CN1201465A (zh) * | 1995-05-30 | 1998-12-09 | 阿斯特拉公司 | 新的dna分子 |
| CN1606617A (zh) * | 2001-12-20 | 2005-04-13 | 德古萨股份公司 | 来自棒状细菌的sigA基因的等位基因 |
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| MXPA06003775A (es) * | 2004-01-30 | 2006-06-14 | Ajinomoto Kk | Microorganismo productor de l-aminoacido y metodo para la produccion de l-aminoacido. |
| CN1930289B (zh) | 2004-03-05 | 2012-09-05 | 花王株式会社 | 变异芽孢杆菌属细菌 |
| US8735132B2 (en) * | 2010-05-07 | 2014-05-27 | Massachusetts Institute Of Technology | Mutations and genetic targets for enhanced L-tyrosine production |
| RU2615454C1 (ru) | 2011-12-21 | 2017-04-04 | СиДжей ЧеилДжеданг Корпорейшн | Способ получения L-лизина с использованием микроорганизмов, обладающих способностью продуцировать L-лизин |
| CN108467848A (zh) | 2012-01-10 | 2018-08-31 | Cj第制糖株式会社 | 可利用木糖的棒状杆菌微生物和利用其产生l-赖氨酸的方法 |
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| CN1201465A (zh) * | 1995-05-30 | 1998-12-09 | 阿斯特拉公司 | 新的dna分子 |
| CN1606617A (zh) * | 2001-12-20 | 2005-04-13 | 德古萨股份公司 | 来自棒状细菌的sigA基因的等位基因 |
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| Publication number | Publication date |
|---|---|
| WO2016036191A1 (ko) | 2016-03-10 |
| EP3196300A1 (en) | 2017-07-26 |
| MY180044A (en) | 2020-11-20 |
| PL3196300T3 (pl) | 2022-08-01 |
| BR112017004368A2 (pt) | 2018-02-27 |
| DK3196300T3 (da) | 2022-07-25 |
| US20180037615A1 (en) | 2018-02-08 |
| JP2017525381A (ja) | 2017-09-07 |
| EP3196300A4 (en) | 2018-01-31 |
| RU2668176C1 (ru) | 2018-09-26 |
| JP6464260B2 (ja) | 2019-02-06 |
| EP3196300B1 (en) | 2022-05-11 |
| US10676512B2 (en) | 2020-06-09 |
| KR20160029964A (ko) | 2016-03-16 |
| CN111676205A (zh) | 2020-09-18 |
| KR101755767B1 (ko) | 2017-07-10 |
| ES2917498T3 (es) | 2022-07-08 |
| CN106715688A (zh) | 2017-05-24 |
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