CN107922954A - 具有l‑赖氨酸生产力的微生物和使用其生产l‑赖氨酸的方法 - Google Patents
具有l‑赖氨酸生产力的微生物和使用其生产l‑赖氨酸的方法 Download PDFInfo
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- CN107922954A CN107922954A CN201680040171.4A CN201680040171A CN107922954A CN 107922954 A CN107922954 A CN 107922954A CN 201680040171 A CN201680040171 A CN 201680040171A CN 107922954 A CN107922954 A CN 107922954A
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Abstract
本发明涉及具有L‑赖氨酸生产力的微生物和使用其生产L‑赖氨酸的方法。更具体地,本发明涉及:棒状杆菌属某种微生物,其被突变使得与细胞壁水解相关的蛋白质的活性与其内源活性相比失活;和通过使用其生产L‑赖氨酸的方法。
Description
技术领域
本公开内容涉及生产L-赖氨酸的微生物和使用其生产L-赖氨酸的方法。
背景技术
L-氨基酸——具体而言L-赖氨酸——在动物饲料、人用治疗剂或化妆品行业中使用,主要使用棒状杆菌属的菌株或埃希氏杆菌属的菌株通过发酵生产。因此,各种研究正在研发以高产率生产L-赖氨酸的菌株和其发酵技术。但是,对于控制细胞裂解——其可能在发酵的后期造成降低的生产力——的研究仍然是不足的。
同时,细胞壁水解酶已知为降解细菌细胞壁并且在具有肽聚糖的所有微生物中存在的酶(Rice KC&Bayles KW.Microbiol Mol Biol Rev.2008.72:85-109)。虽然对于这类细胞壁水解酶的研究已经在各种细菌中进行,但是它们的精确控制机制是仍然未知的。
在微生物的培育期间发生的细胞裂解机制的模型最近已经在肺炎球菌属中提出(Mellroth P et al.J Biol Chem.2012.287:11018-29)。具体而言,当细胞暴露于各种类型的应激时,存在于细胞的外壁上的细胞壁水解酶的活性增加,从而开始细胞壁分解。当细胞通过这类细胞壁水解酶的连续作用而溶解时,存在于细胞质中的细胞壁水解酶暴露于细胞外。已经报道,当一系列过程连续发生使得细胞壁水解酶的量超过细胞外的阈值时,周围细胞被溶解。但是,在发酵培养过程期间生成的细胞裂解与氨基酸生产之间的关联是仍然未知的。
公开内容
技术问题
本发明人已经进行了努力以持续地寻找能够增加棒状杆菌属的微生物——其是代表性的产L-赖氨酸的菌株——中的L-赖氨酸生产力的有效特性。结果,本发明人已经确认当编码参与细胞壁水解的蛋白质的基因有缺陷时L-赖氨酸生产力增加,并且当编码具有类似功能的蛋白质的另外基因有缺陷时赖氨酸生产力的增加受到影响,从而完成本公开内容。
技术方案
本公开内容的目标是提供生产L-赖氨酸的棒状杆菌属的微生物。
本公开内容的另一个目标是提供使用生产L-赖氨酸的棒状杆菌属的微生物制备L-赖氨酸的方法。
有益效果
根据本公开内容的微生物是棒状杆菌属的微生物,其被修饰使得参与细胞壁水解的蛋白质的活性与其内源活性相比降低或失活。即,根据本公开内容的微生物是导致发酵后期中生产力增加的新菌株,并且因而被用作生产L-赖氨酸的棒状杆菌属的微生物的新型范例,从而提供了能够以高产率生产L-赖氨酸的微生物。因此,制备的L-赖氨酸不仅可以应用于动物饲料或者动物饲料添加剂,而且也可以应用于各种产品比如人食物、食物添加剂、药物等。
最佳模式
为了实现上述目标,本公开内容的方面提供了生产L-赖氨酸的棒状杆菌属的微生物,其被修饰使得参与细胞壁水解的蛋白质的活性与其内源活性相比失活。
如本文所使用,术语“参与细胞壁水解的蛋白质”指的是能够水解棒状杆菌属的微生物中的细胞壁的相关蛋白质。参与细胞壁水解的蛋白质可以是细胞壁相关的水解酶或者N-乙酰胞壁酰(acetylmuramoyl)-L-丙氨酸酰胺酶,但是不限于此。
如上面所描述的,只要蛋白质具有能够水解微生物中的细胞壁的相关蛋白质的活性,蛋白质和基因序列可以从已知数据库获得。此外,NCBI的基因库(Genbank)等可以被用作已知数据库的实例,但是这些不限于此。
细胞壁相关的水解酶可以是衍生自棒状杆菌属的微生物——具体地衍生自谷氨酸棒状杆菌——的NCgl1480基因编码的蛋白质、NCgl2107基因编码的蛋白质或NCgl2108基因编码的蛋白质,但是不限于此。作为具体实例,细胞壁相关的水解酶可以具有SEQ ID NO:1、2或3的氨基酸,但是可以非限制性地包括具有上述活性的蛋白质序列。另外,任何核苷酸序列可以非限制性地包括在其中,只要其是编码具有细胞壁相关的水解酶活性的蛋白质的核苷酸序列。作为具体实例,其可以是由SEQ ID NO:5、6或7的核苷酸序列编码的蛋白质,但是不限于此。
N-乙酰胞壁酰-L-丙氨酸酰胺酶可以是衍生自棒状杆菌属的微生物——具体地衍生自谷氨酸棒状杆菌——的NCgl2986基因编码的蛋白质。具体地,N-乙酰胞壁酰-L-丙氨酸酰胺酶可以具有SEQ ID NO:4的氨基酸序列,但是可以非限制性地包括具有上述活性的蛋白质的任何氨基酸序列。此外,可以非限制性地包括任何核苷酸序列,只要该核苷酸序列编码具有N-乙酰胞壁酰-L-丙氨酸酰胺酶的活性的蛋白质。例如,其可以是由SEQ ID NO:8的核苷酸序列编码的蛋白质,但是不限于此。
除了由SEQ ID NO表示的氨基酸序列之外,上面描述的蛋白质中的每一种还可以非限制性地包括与上面列举的氨基酸序列具有80%或更高、优选地90%或更高、更优选地95%或更高、并且甚至更优选地97%或更高的同源性的任何氨基酸序列,只要该氨基酸序列编码具有与蛋白质的每种基本上相同或相应的作用的蛋白质。另外地,显而易见的是具有上面描述的同源性的任何修饰的蛋白质可以属于本公开内容的范围,但是蛋白质可以具有在其中含有部分缺失、修饰、置换或添加的氨基酸序列。
另外地,除了由SEQ ID NO描述的核苷酸序列之外,编码本公开内容的蛋白质中的每种的基因还可以非限制性地包括编码蛋白质的与上面列举的核苷酸序列中的每种具有80%或更高、优选地90%或更高、更优选地95%或更高、甚至更优选地98%或更高、并且最优选地99%或更高的同源性的任何基因序列,只要该基因序列编码具有与蛋白质的每种基本上相同或相应的作用的蛋白质。另外地,显而易见的是具有上述同源性的任何核苷酸序列可以属于本公开内容的范围,但是序列可以在其中具有部分缺失、修饰、置换或添加。
如本文所使用,“同源性”指的是编码蛋白质的基因的核苷酸序列或氨基酸序列的相似性。当同源性足够高时,相应基因的产物可以是相同的或者具有相似活性。即,其指的是两个多核苷酸或核苷酸部分之间的同一性的百分比。一个部分与另一个部分的序列对应性可以由本领域中已知的技术测定。例如,可以通过使用容易获得的且能够比对序列信息的计算机程序直接地比对两个多核苷酸分子或两个多肽分子的序列信息来测定同源性。计算机程序可以是BLAST(NCBI)、CLC Main Workbench(CLC bio)、MegAlignTM(DNASTAR Inc)等。此外,可以通过在同源区中形成稳定双链的条件下杂交多核苷酸,并且然后通过单链特异性核酸酶消化杂交链以测定消化片段的大小来测定同源性。
如本文所使用的,术语“内源活性”指的是微生物修饰前的状态下或其天然状态下的蛋白质活性。
如本文所使用的,术语“酶的活性被修饰为与其内源活性相比失活”指的是如此活性:与野生型菌株或者修饰前的菌株相比编码酶的基因根本不表达,或者指的是即使当基因表达时活性降低或消除。
“与其内源活性相比活性失活”指的是当与在其自然状态或修饰前的状态下具备的活性相比时,活性降低或消除。降低是指如下情况的概念:由于编码酶的基因的修饰,与微生物原始具备的活性相比酶的活性降低;总体酶表达水平低于微生物的自然类型菌株或者修饰前的菌株的总体酶表达水平;或者其组合。
“活性消除”指的是如下情况:与自然类型菌株或者修饰前的菌株相比,编码酶的基因根本不表达,和/或基因表达但是不展现活性。
修饰以使酶活性失活的方法可以通过应用本领域中熟知的各种方法实现。方法的实例可以包括将染色体上编码酶的基因替换为突变的基因,使得酶活性可以降低的方法,其包括酶活性被去除时的情况;在染色体上编码酶的基因的表达调控序列上引入修饰的方法;将编码酶的基因的表达调控序列替换为具有弱活性或无活性的序列的方法;使染色体上编码酶的部分或全部基因缺失的方法;引入反义寡核苷酸(例如反义RNA)的方法,该反义寡核苷酸经由互补结合至染色体上的基因的转录物抑制从mRNA翻译为酶;通过在编码酶的基因的SD序列的前端上人工地添加Shine-Dalgarno(SD)序列和其互补序列形成二级结构使得核糖体的附着不可能的方法;逆转录工程学(RTE)——其添加启动子从而在相应序列的开放阅读框(ORF)的3′末端上相反地转录——的方法等,并且还包括其组合,但是不限于此。
具体地,使编码酶的部分或全部基因缺失的方法可以通过将多核苷酸——其经由将染色体插入微生物的载体编码染色体内的内源靶蛋白——替换为核酸序列的部分缺失的多核苷酸或标记物来执行。例如,可以使用经由同源重组基因缺失的方法。
如本文所使用的,术语“部分”虽然可以根据多核苷酸的种类变化,但是其可以具体地指1个核苷酸至300个核苷酸、更具体地1个核苷酸至100个核苷酸、并且甚至更具体地1个核苷酸至50个核苷酸,但是不限于此。
如本文所使用,术语“同源重组”指的是在具有相互同源性的基因链的基因座处经由交换发生的遗传重组。
根据本公开内容的示例性实施方式,蛋白质通过同源重组失活。
具体地,修饰表达调控序列的方法可以通过经由表达调控序列的核苷酸序列的缺失、插入、非保守性或保守性置换,或其组合在表达调控序列上引入修饰来实施;或者可以通过将序列替换为较弱启动子来实施。表达调控序列包括启动子、操纵基因序列、编码核糖体结合位点的序列、和用于调控转录和翻译的终止的序列。
另外,修饰染色体上的基因序列的方法可以通过经由基因序列的缺失、插入、非保守性或保守性置换,或其组合在序列中引入修饰从而进一步降低酶活性来实施;或者通过将序列替换为改进以具有另外较弱活性的基因序列或者替换为改进以不具有活性的基因序列来实施。
如本文所使用,术语“生产L-赖氨酸的微生物”指的是能够通过发酵生产L-赖氨酸的微生物菌株。例如,其包括通过经由本公开内容的操作来修饰序列使得参与细胞壁水解的蛋白质的活性与其内源活性相比失活而能够增加L-赖氨酸生产力的菌株;和通过调控在发酵期间发生的用于生产赖氨酸的细胞裂解而能够增加L-赖氨酸生产力的菌株,但是不限于此。
在本公开内容中,生产L-赖氨酸的微生物可以包括棒状杆菌属的所有微生物——其能够被修饰使得参与细胞壁水解的蛋白质的活性与其内源活性相比失活。例如,可以使用谷氨酸棒状杆菌、产氨棒状杆菌(Corynebacterium ammoniagenes)、嗜热产氨棒状杆菌(Corynebacterium thermoaminogenes)、黄色短杆菌(Brevibacterium flavum)、或发酵短杆菌(Brevibacterium fermentum),但是微生物不限于此。例如,谷氨酸棒状杆菌可以被用于棒状杆菌属的微生物。修饰的棒状杆菌属的微生物的特征在于L-赖氨酸生产力与不被修饰的微生物相比增强,使得参与细胞壁水解的蛋白质的活性与其内源活性相比失活。
本公开内容的另一个方面提供了用于制备L-赖氨酸的方法,其包括:(i)培养棒状杆菌属的微生物,其被修饰使得参与细胞壁水解的蛋白质的活性与其内源活性相比失活;和(ii)从培养基或微生物回收L-赖氨酸。
L-赖氨酸生产力增加的棒状杆菌属的微生物如上面所描述。
如本文所使用,术语“培养”指的是在人工控制的环境条件下培养微生物。在本公开内容中,使用棒状杆菌属的微生物培养L-赖氨酸的方法可以使用本领域中广泛公知的方法进行。具体地,培养的实例包括分批工艺和以连续方式的补料分批或重复补料分批工艺,但是不限于此。
用于培养的培养基应当以适合方式满足具体菌株的需要(例如,Manual ofMethods for General Bacteriology.American Society for Bacteriology.WashingtonD.C.,USA,1981)。可以在本公开内容中使用的碳源可以包括糖类和碳水化合物比如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉和纤维素;油脂比如豆油、葵花油、蓖麻油和椰子油;脂肪酸比如棕榈酸、硬脂酸和亚油酸;醇类比如甘油和乙醇;和有机酸比如葡糖酸、乙酸和丙酮酸,但是这些不限于此。这些物质可以单独地或在混合物中使用。可以在本公开内容中使用的氮源可以包括蛋白胨、酵母提取物、肉膏、麦芽汁、玉米浆、脱脂豆饼和尿素,或无机化合物比如硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵,但是这些不限于此。这些氮源也可以单独或在混合物中使用。可以在本公开内容中使用的磷源可以包括磷酸二氢钾或磷酸氢二钾,或相应的含钠盐,但是这些不限于此。此外,培养基可以包含金属盐,比如硫酸镁或硫酸铁,其对于生长是需要的。最后,除了上面描述的物质外,可以使用必需生长因子比如氨基酸和维生素。另外地,可以在培养基中使用适合的前体。这些物质可以在培养期间以分批或连续方式添加至培养基。例如在文献(“Biochemical Engineering”by James M.Lee,Prentice-Hall International Editions,pp 138-176)中公开了这样的多种培养方法。
碱性化合物比如氢氧化钠、氢氧化钾或氨,或酸性化合物比如磷酸或硫酸可以以适合方式添加至培养基以调节培养基的pH。此外,消泡剂比如脂肪酸聚乙二醇酯可以被用于抑制泡沫的形成。为了维持培养基处于需氧状态,氧或含氧气体(例如空气)可以被注入培养基。培养基的温度可以通常为20℃至45℃,优选地25℃至40℃,但是可以根据条件而改变。培养可以持续直到产生最大量的期望的L-氨基酸,并且其通常可以在10小时至160小时内实现。L-赖氨酸可以被释放入培养基或者包含在细胞中。
用于生产L-赖氨酸的本公开内容的方法可以包括从微生物或培养基回收赖氨酸的步骤。在本领域中已知的方法,比如离心、过滤、阴离子交换色谱、结晶、HPLC等可以被用于从微生物或培养基回收L-赖氨酸的方法,但是方法不限于此。
回收步骤可以包括纯化过程。
发明的模式
在下文,将伴随示例性实施方式详细地描述本公开内容。但是,本文公开的示例性实施方式仅仅出于说明性目的并且不应当被解释为限制本公开内容的范围。
实施例1:使用转座子制备随机突变体文库
为了获得增加赖氨酸生产力的基因,通过下述方法制备载体文库。使用菌株KCCM11016P(该微生物已经被命名为KFCC10881并且利用根据布达佩斯条约的国际保藏机构重新保藏,并且然后被指定KCCM11016P的保藏登录号;韩国专利号10-0159812)作为亲本菌株转化使用EZ-Tn5TM<R6Kγori/KAN-2>Tnp TransposomeTM试剂盒(Epicentre)获得的质粒,并且然后涂布在含有卡那霉素(25mg/L)的复合培养板上以获得20,000个菌落。
<复合培养板(pH 7.0)>
10g葡萄糖、10g蛋白胨、5g牛肉膏、5g酵母提取物、18.5g脑心浸液、2.5g NaCl、2g尿素、91g山梨醇、20g琼脂(基于1L蒸馏水)
实施例2:使用转座子筛选随机突变体文库
将在实施例1中获得的大约20,000个菌落中的每个接种入选择培养基(300μL),并且在32℃、1000rpm下在96深孔板中培养大约24小时。茚三酮方法被用于分析在培养基中产生的L-赖氨酸的量(Moore,S.,Stein,W.H.,Photometric ninhydrin method for use inthe chromatography of amino acids.J.Biol.Chem.1948,176,367-388)。在培养完成时,将培养物上清液(10μL)和茚三酮反应溶液(190μL)在65℃下反应30分钟。随后,使用分光光度计在570nm的波长下测量吸光度,并且大约60种菌落被选择为修饰菌株,其与对照KCCM11016P相比显示了高吸光度。其它菌落与对照KCCM11016P菌株相比显示了类似的或降低的吸光度。
60种选择的菌株以与上面相同的方式培养,并且重复进行茚三酮反应。结果,选择与菌株KCCM11016P相比具有提高的L-赖氨酸生产力的前10种菌株。
<选择培养基(pH 8.0)>
10g葡萄糖、5.5g硫酸铵、1.2g MgSO4 7H2O、0.8g KH2PO4、16.4g K2HPO4、100μg生物素、1000μg HCl硫胺素、2000μg泛酸钙、2000μg烟酰胺(基于1L蒸馏水)
实施例3:分析选择的随机突变体菌株的L-赖氨酸生产力
为了最终选择具有增加的L-赖氨酸生产力的菌株,对于在实施例2中选择的10种菌株使用下面的培养基在烧瓶中实施再现性试验。将10种菌株和对照接种在含有下面的种子培养基(25mL)的三角挡板烧瓶(250mL)中,并且在振荡的同时在30℃和200rpm下培养20小时。种子培养基和生产培养基具有下列组成。在完成培养之后,使用HPLC分析培养液中的L-赖氨酸浓度,并且突变体菌株中每种的L-赖氨酸浓度在下面的表1中示出。
<种子培养基(pH 7.0)>
20g葡萄糖、10g蛋白胨、5g酵母提取物、1.5g尿素、4g KH2PO4、8g K2HPO4、0.5gMgSO4·7H2O、100μg生物素、1000μg HCl硫胺素、2000μg泛酸钙、2000μg烟酰胺(基于1L蒸馏水)
<生产培养基(pH 7.0)>
100g葡萄糖、40g(NH4)2SO4、2.5g大豆蛋白、5g玉米浆固体、3g尿素、1g KH2PO4、0.5gMgSO4·7H2O、100μg生物素、1000μg HCl硫胺素、2000μg泛酸钙、3000μg烟酰胺、30g CaCO3(基于1L蒸馏水)
[表1]10种选择的突变体菌株的L-赖氨酸浓度
在上面的10种选择的突变体中,KCCM11016P/mt-1和KCCM11016P/mt-8最终被选择为具有显著提高的L-赖氨酸生产力的菌株。
实施例4:确认在最终选择的菌株中涉及L-赖氨酸生产力的基因和选择另外的候选基因
在该实施例中,由在实施例3中最终选择的菌株尝试基因的鉴定,由于转座子的随机插入这些基因是有缺陷的。KCCM11016P/mt-1和KCCM11016P/mt-8的基因组DNA被提取并且然后被消化。随后,生成物被连接,转化入大肠杆菌DH5a,并且然后平板接种在含有卡那霉素(25mg/L)的LB固体培养基上。在选择20种转化的菌落之后,获得含有未知基因的部分的质粒,并且使用SEQ ID NO:9和SEQ ID NO:10的序列在EZ-Tn5TM<R6Kγori/KAN-2>TnpTransposomeTM试剂盒中分析核苷酸序列(表2)。结果,确认了NCgl2108和NCgl2986基因中的每种在突变体菌株中失活。
[表2]
序列 | SEQ ID NO | |
试剂盒引物 | ACCTACAACAAAGCTCTCATCAACC | 9 |
试剂盒引物 | CTACCCTGTGGAACACCTACATCT | 10 |
在实施例3中选择的突变体菌株中被鉴定为缺陷的NCgl2108和NCgl2986基因内源性地存在于棒状杆菌属中,并且因而被鉴定为参与细胞壁水解的蛋白质。
基于使用转座子在随机突变体菌株中选择参与细胞壁水解的2种蛋白质的结果,认为参与细胞壁水解的基因的缺陷在增加L-赖氨酸生产力中将是有效的。因此,在国家生物技术信息中心(NCBI)中进行参与细胞壁水解的基因——非NCgl2108和NCgl2986基因——的搜索。
作为搜索的结果,内源地存在于棒状杆菌属中的NCgl1480和NCgl2107基因被另外地选择为参与细胞壁水解的蛋白质。因此,为了确认NCgl1480和NCgl2107基因的缺失是否影响L-赖氨酸生产力,这些基因被选择为另外的缺失候选基因。
实施例5:生产用于NCgl1480、NCgl2107、NCgl2108和NCgl2986基因失活的重组质
粒
在该实施例中,为了确认NCgl1480、NCgl2107、NCgl2108和NCgl2986基因的失活是否将影响L-赖氨酸生产,生产在棒状杆菌属中产L-赖氨酸菌株的染色体上用于缺失在实施例4中选择的NCgl1480、NCgl2107、NCgl2108和NCgl2986基因的重组质粒。
基于在美国国立卫生研究院基因库(NIH基因库)中报道的核苷酸序列,获得NCgl1480、NCgl2107、NCgl2108和NCgl2986的SEQ ID NO:1、2、3和4的氨基酸序列,以及编码其的SEQ ID NO:5、6、7和8的核苷酸序列。为了生产基因片段,其中NCgl1480、NCgl2107、NCgl2108和NCgl2986的每种的开放阅读框内部缺失,基于上述SEQ ID NO:5、6、7和8生产NCgl1480的SEQ ID NO:11至14、NCgl2107的15至18、NCgl2108的19至22和NCgl2986的23至26的引物。其序列在下面的表3中示出。
[表3]
使用谷氨酸棒状杆菌ATCC13032的基因组DNA作为模板,连同SEQ ID NO:11和12、13和14、15和16、17和18、19和20、21和22、23和24、以及25和26的核苷酸序列对作为引物进行PCR(Sambrook et al.,Molecular Cloning,a Laboratory Manual(1989),Cold SpringHarbor Laboratories)。在下述条件下进行PCR:30个循环,每个由在95℃下变性30秒、在50℃下退火30秒和在72℃下延伸1分钟组成。
结果,获得两对含有NCg11480基因的上游和下游区域的319bp和410bp(分别地NCg11480-A和NCg11480-B)的DNA片段;两对含有NCgl2107基因的上游和下游区域的324bp和300bp(分别地NCgl2107-A和NCgl2107-B)的DNA片段;两对含有NCgl2108基因的上游和下游区域的381bp和377bp(分别地NCgl2108-A和NCgl2108-B)的DNA片段;和两对含有NCgl2986基因的上游和下游区域的356bp和374bp(分别地NCgl2986-A和NCgl2986-B)的DNA片段。将通过PCR扩增的DNA片段使用Infusion Cloning试剂盒(Invitrogen)缀合至pDZ质粒(韩国专利号10-0924065),转化入大肠杆菌DH5α,并且然后平板接种在含有卡那霉素(25mg/L)的LB固体培养基上。在选择转化有质粒——期望基因通过PCR插入其中——的菌落之后,使用本领域中常规知晓的质粒提取方法获得质粒。如此获得的质粒分别被命名为pDZ-ΔNCgl1480、pDZ-ΔNCgl2107、pDZ-ΔNCgl2108和pDZ-ΔNCgl2986。在pDZ-ΔNCgl1480中,NCgl1480基因的1672bp基因片段缺失;在pDZ-ΔNCgl2107中,NCgl2107基因的1026bp基因片段缺失;在pDZ-ΔNCgl2108中,NCgl2108基因的576bp基因片段缺失;并且在pDZ-ΔNCgl2986中,基因NCgl2986的1092bp基因片段缺失。
实施例6:生产和评估衍生自产赖氨酸菌株KCCM11016P的细胞壁水解相关的蛋白
基因失活的菌株
基于KCCM11016P,制备棒状杆菌属的代表性的产L-赖氨酸菌株——选自上面的细胞壁水解相关的蛋白基因失活的菌株,并且尝试其赖氨酸生产力的评估。
将在实施例5中产生的4种重组质粒(pDZ-ΔNCgl1480、pDZ-ΔNCgl2107、pDZ-ΔNCgl2108和pDZ-ΔNCgl2986)中的每种通过电脉冲方法转化入谷氨酸棒状杆菌KCCM11016P,并且通过PCR方法制备其中靶基因通过同源重组失活的菌株。制备的失活菌株分别被命名为KCCM11016P::ΔNCgl1480、KCCM11016P::ΔNCgl2107、KCCM11016P::ΔNCgl2108和KCCM11016P::ΔNCgl2986。
将4种菌株中的每种和对照菌株接种入含有25mL下述种子培养基的三角挡板烧瓶(25mL),并且在振荡的同时在30℃和200rpm下培养20小时。随后将种子培养物(1mL)接种在含有24mL下述生产培养基的三角挡板烧瓶(1mL)中,并且在振荡的同时在37℃和200rpm下培养96小时。种子培养基和生产培养基中每种的组成如下。
<种子培养基(pH 7.0)>
20g葡萄糖、10g(NH4)2SO4、10g蛋白胨、5g酵母提取物、1.5g尿素、4g KH2PO4、8gK2HPO4、0.5g MgSO4·H2O、100μg生物素、1000μg HCl硫胺素、2000μg泛酸钙、2000μg烟酰胺(基于1L蒸馏水)
<生产培养基(pH 7.0)>
100g葡萄糖、40g(NH4)2SO4、2.5g大豆蛋白、5g玉米浆固体、3g尿素、1g KH2PO4、0.5gMgSO4·H2O、100μg生物素、1000μg HCl硫胺素、2000μg泛酸钙、3000μg烟酰胺、30g CaCO3(基于1L蒸馏水)
在培养完成之后,使用HPLC分析L-赖氨酸浓度,并且浓度在下面的表4中示出。表4中的结果是三组重复实验的结果,并且生产力基于平均值评估。
[表4]
结果,如在上面的表4中所示出的,与亲本菌株KCCM11016P的赖氨酸生产力相比,菌株——其中NCgl1480、NCgl2107、NCgl2108和NCgl2986基因中的每种失活——的赖氨酸生产力分别增加至3.2%、5.4%、13%和15%。
这些结果表明可以通过使参与细胞壁水解——其可以造成棒状杆菌属的微生物的细胞融合——的蛋白质失活来提高L-赖氨酸生产力。
因此,如下进行实验以确定类似作用是否可以在如下情况中展现:其中参与细胞壁水解的蛋白质在多种棒状杆菌属的微生物中失活。
实施例7:生产和评估衍生自产L-赖氨酸菌株KCCM10770P的细胞壁水解相关的蛋
白质失活的菌株
为了检查在具有增强的赖氨酸生物合成途径的产L-赖氨酸菌株谷氨酸棒状杆菌KCCM10770P(韩国专利号10-0924065)中细胞壁水解相关的蛋白质失活的作用是否类似于实施例6的实验结果,以与实施例6中描述的相同方式制备菌株——其中参与细胞壁水解的4种蛋白质中的每种失活。制备的菌株被命名为KCCM10770P::ΔNCgl1480、KCM10770P::ΔNCgl2107、KCCM10770P::ΔNCgl2108和KCM10770P::ΔNCgl2986。比较它们之间的L-赖氨酸生产力。
为了比较上述菌株的赖氨酸生产力,以与在实施例6中相同的方式培养这些菌株和对照菌株。在培养完成之后,使用HPLC分析的L-赖氨酸浓度在下面的表5中示出。表5中的结果是三组重复实验的结果,并且基于平均值评估生产力。
[表5]
结果,如在上面的表5中示出的,与亲本菌株KCCM10770P的赖氨酸生产力相比,菌株——其中NCgl1480、NCgl2107、NCgl2108和NCgl2986基因中的每种失活——的赖氨酸生产力分别增加至2.2%、4.3%、12.1%和14.2%。
因此,发现了在谷氨酸棒状杆菌KCCM10770P(韩国专利号10-0924065)中,通过与在实施例6中相同的方式使参与细胞壁水解的蛋白质失活也提高了L-赖氨酸生产力。
实施例8:生产和评估衍生自产L-赖氨酸菌株KCCM11347P的细胞壁水解相关的蛋
白质失活的菌株
为了检查通过人工修饰制备的产L-赖氨酸菌株谷氨酸棒状杆菌KCCM11347P(该微生物已经被命名为KFCC10750并且利用根据布达佩斯条约的国际保藏机构重新保藏,并且然后被指定KCCM11347P的保藏登录号;韩国专利号10-0073610)中细胞壁水解相关的蛋白质失活的作用,以与在实施例6中描述的相同方式制备菌株——其中参与细胞壁水解的4种蛋白质中的每种失活。制备的菌株被命名为KCCM11347P::ΔNCgl1480、KCCM11347P:ΔNCgl2107、KCCM11347P::ΔNCgl2108和KCCM11347P:ΔNCgl2986。比较它们之间的L-赖氨酸生产力。
为了比较上述菌株的赖氨酸生产力,以与在实施例6中相同的方式培养这些菌株和对照菌株。在培养完成之后,使用HPLC分析L-赖氨酸浓度,并且在下面的表6中示出。表6中的结果是三组重复实验的结果,并且基于平均值评估生产力。
[表6]
结果,如在上面的表6中示出的,与亲本菌株KCCM11347P的赖氨酸生产力相比,菌株——其中NCgl1480、NCgl2107、NCgl2108和NCgl2986基因中的每种失活——的赖氨酸生产力分别增加至2%、2.4%、11.7%和14.4%。
因此,发现了在谷氨酸棒状杆菌KCCM11347P(韩国专利号10-0073610)中,通过与在实施例6和7中相同的方式使参与细胞壁水解的蛋白质失活也提高了L-赖氨酸生产力。
实施例9:生产和评估衍生自产L-赖氨酸菌株CJ3P的细胞壁水解相关的蛋白质失
活的菌株
为了检查谷氨酸棒状杆菌CJ3P(Binder et al.Genome Biology 2012,13:R40)——其通过在野生型谷氨酸棒状杆菌中引入3种修饰[pyc(P458S)、hom(V59A)和lysC(T311I)]而生产L-赖氨酸——中细胞壁水解相关的蛋白质失活的作用是否类似于实施例6、7和8的实验结果,以与在实施例6中描述的相同方式制备菌株——其中参与细胞壁水解的4种蛋白质中的每种失活。制备的菌株被命名为CJ3P::ΔNCgl1480、CJ3P::ΔNCgl2107、CJ3P::ΔNCgl2108和CJ3P::ΔNCgl2986。比较它们之间的L-赖氨酸生产力。
为了比较上述菌株的赖氨酸生产力,以与在实施例6中相同的方式培养这些菌株和对照菌株。在培养完成之后,使用HPLC分析的L-赖氨酸浓度在下面的表7中示出。表7中的结果是三组重复实验的结果,并且基于平均值评估生产力。
[表7]
结果,如在上面的表7中示出的,与亲本菌株CJ3P的赖氨酸生产力相比,菌株——其中NCgl1480、NCgl2107、NCgl2108和NCgl2986基因中的每种失活——的赖氨酸生产力分别增加至3%、1.3%、12.7%和16%。
因此,发现了在谷氨酸棒状杆菌CJ3P中,通过与在实施例6、7和8中相同的方式使参与细胞壁水解的蛋白质失活也提高了L-赖氨酸生产力。
实施例10:生产和评估衍生自产L-赖氨酸菌株KCCM11016P的细胞壁水解相关的蛋
白质同时失活的菌株
在从上述实施例确认了当在产L-赖氨酸菌株棒状杆菌属中使参与细胞壁水解的蛋白质中的每种失活时,L-赖氨酸生产力增加,当2种相关蛋白质同时失活时,尝试鉴定L-赖氨酸生产力是否也将增加。
因此,实施下列实验以确认参与产L-赖氨酸菌株棒状杆菌属中细胞壁水解的蛋白质同时失活的作用。以与在实施例6中相同的方式制备菌株,其中参与细胞壁水解的两种类型的蛋白基因(NCgl2108和NCgl2986)同时失活,当蛋白质中的每种是有缺陷的时,它们在增强L-赖氨酸生产力中是高效的。制备的菌株被命名为KCCM11016P::ΔNCgl2108/ΔNCgl2986。比较L-赖氨酸生产力。
为了比较上述菌株的赖氨酸生产力,以与在实施例6中相同的方式培养该菌株和对照菌株。在培养完成之后,使用HPLC分析的L-赖氨酸浓度在下面的表8中示出。表8中的结果是三组重复实验的结果,并且基于平均值评估生产力。
[表8]
结果,如在表8中示出的,与亲本菌株KCCM11016P的赖氨酸生产力相比,菌株——其中NCgl2108和NCgl2986基因同时失活——的赖氨酸生产力增加至21.6%。
该结果表明甚至当参与棒状杆菌属微生物中细胞壁水解的不仅一种蛋白质而且两种或更多种蛋白质同时失活时,L-赖氨酸生产力可以提高。
就此而言,上述菌株KCCM11016P-ΔNCgl2986被命名为CA01-2292。CA01-2292利用根据布达佩斯条约的国际保藏机构韩国微生物培养中心(KCCM)保藏,并且然后指定KCCM11627P的保藏登录号。
基于这些结果,确认了产L-赖氨酸菌株通过调节发酵期间的细胞裂解具有增强L-赖氨酸生产力的作用,其中参与细胞壁水解的蛋白质与它们的内源活性相比失活。另外,也确认了当参与细胞壁水解的不仅一种而且两种或更多种蛋白质同时失活时,L-赖氨酸生产力可以提高,从而提供了生产L-赖氨酸的新型菌株。
虽然已经参照具体的说明性实施方式描述了本公开内容,但是本公开内容所属领域的技术人员将理解本公开内容可以以其它具体形式体现而不背离本公开内容的技术精神或基本特征。因此,上面描述的实施方式被在所有方面被视为是说明性的并且不是限制性的。而且,本公开内容的范围由所附权利要求书而不是详细描述限定,并且应当理解,源自这些含义和本公开内容的范围的所有修改或变化和其等价物包含在所附权利要求书的范围内。
国际承认用于专利程序的微生物保藏布达佩斯条约
国际表格
原始保藏接收证明
由该页下部确定的国际保藏机构根据第7.1条发出
至:CJ第一制糖株式会社
CJ第一制糖中心
330,DONGHO-RO,JUNG-GU,首尔,
100-400,大韩国民
证明上述翻译与原文内容没有相互违背
2015年7月3日
专利代理人Son Min印
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Ala Gln Ala Ala Glu Val Val Val Pro Gly Thr Gly Ile Ser Val Asp
35 40 45
Ile Ala Gly Ile Glu Thr Thr Pro Gly Leu Asn Asn Val Pro Gly Ile
50 55 60
Asp Gln Trp Ile Pro Ser Leu Ser Ser Gln Ala Ala Pro Thr Ala Tyr
65 70 75 80
Ala Ala Val Ile Asp Ala Pro Ala Ala Gln Ala Ala Pro Ala Ala Ser
85 90 95
Thr Gly Gln Ala Ile Val Asp Ala Ala Arg Thr Lys Ile Gly Ser Pro
100 105 110
Tyr Gly Trp Gly Ala Thr Gly Pro Asn Ala Phe Asp Cys Ser Gly Leu
115 120 125
Thr Ser Trp Ala Tyr Ser Gln Val Gly Lys Ser Ile Pro Arg Thr Ser
130 135 140
Gln Ala Gln Ala Ala Gln Gly Thr Pro Val Ala Tyr Ser Asp Leu Gln
145 150 155 160
Ala Gly Asp Ile Val Ala Phe Tyr Ser Gly Ala Thr His Val Gly Ile
165 170 175
Tyr Ser Gly His Gly Thr Val Ile His Ala Leu Asn Ser Ser Thr Pro
180 185 190
Leu Ser Glu His Ser Leu Asp Tyr Met Pro Phe His Ser Ala Val Arg
195 200 205
Phe
<210> 4
<211> 414
<212> PRT
<213> 棒状杆菌属
<220>
<221> 肽
<222> (1)..(414)
<223> NCgl2986
<400> 4
Met Asp Glu Leu Tyr Pro Leu Ile Ile Leu Asn Met Asn Asp Gly Arg
1 5 10 15
Ser Arg Val Ser Lys Val Leu Arg Val Gly Asp Arg Ser Pro Arg Val
20 25 30
Ala Glu Val Arg Thr Thr Leu Ala Arg Leu Gly Val Ile Glu Gly Tyr
35 40 45
Ser Arg Glu Met Ser Ala Lys Thr Glu Ser Gln Lys Phe His Glu Glu
50 55 60
Glu Thr Leu Phe Asp Glu Glu Leu Ser Leu Ser Ile Lys Ser Phe Gln
65 70 75 80
Gln Ala Arg Gly Val Val Pro Ser Gly Leu Ile Asp Asp Pro Thr Leu
85 90 95
Arg Ala Ile Arg Glu Ala Ser Tyr Thr Leu Gly Thr Arg Val Leu Ala
100 105 110
Tyr Gln Pro Gly Asn Gln Leu Val Gly Asp Asp Val Val Glu Ile Gln
115 120 125
Ser His Leu Gln Glu Leu Gly Phe Tyr Ala Asp Arg Val Asp Gly His
130 135 140
Phe Gly Glu Leu Thr His Lys Ala Val Met Asn Tyr Gln Leu Asn Tyr
145 150 155 160
Gly Met Gln Val Asp Gly Ile Cys Gly Pro Asp Thr Ile Arg Ala Leu
165 170 175
Ser Arg Leu Gly Leu Arg Ile Lys Gly Gly Ser Ala Gln Ala Ile Arg
180 185 190
Glu Arg Glu Arg Met Arg Asn Ala Gly Pro Arg Leu Ala Gly Lys Arg
195 200 205
Val Val Ile Asp Pro Ala Leu Gly Gly Ser Asn Lys Gly Gln Ile Val
210 215 220
Lys Gly Pro Tyr Gly Glu Ile Ser Glu Glu Glu Ile Leu Trp Asp Leu
225 230 235 240
Ala Thr Arg Leu Glu Gly Arg Met Ile Ala Thr Gly Met Glu Thr Ile
245 250 255
Leu Ser Arg Pro His Met Asp Asp Pro Ser Ser Arg Asp Arg Ala Ser
260 265 270
Ile Ala Asn Ala Phe Gly Ala Asp Leu Met Leu Ser Leu His Cys Asp
275 280 285
Ser Tyr Pro Asn Glu Lys Ala Asn Gly Val Ala Ser Phe Tyr Phe Gly
290 295 300
Ser Glu Asn Gly Thr Asn Ser Leu Thr Gly Glu Thr Leu Ser Ala Tyr
305 310 315 320
Ile Gln Lys Glu Ile Val Ala Arg Thr Pro Leu Asn Asn Cys Gly Ser
325 330 335
His Ala Arg Thr Trp Asp Leu Leu Arg Leu Thr Arg Met Pro Met Val
340 345 350
Glu Val Val Thr Gly Tyr Leu Thr Asn Pro Asp Asp Leu Ala Val Leu
355 360 365
Thr Asp Pro Gln Met Arg Asp His Ile Ala Glu Ala Ile Val Val Ala
370 375 380
Val Lys Arg Leu Tyr Leu Leu Asp Glu Glu Ala Gln Pro Lys Thr Gly
385 390 395 400
Thr Phe Lys Phe Ser Glu Leu Leu Gln Ser Glu Gln Ala Gly
405 410
<210> 5
<211> 1815
<212> DNA
<213> 棒状杆菌属
<220>
<221> 基因
<222> (1)..(1815)
<223> NCgl1480
<400> 5
ttgaccaggg cgttgattgc gcttgcagta agcggagctt tgcttagttc catgactccg 60
gcggtggcgc agccacagaa tccggatgac gcagccattg cacaggcaga ggaaaatgtt 120
tcggcgggcg atggggaagt cgcccgcctg gcaggatctt tgtccagcac tgacgcggaa 180
attaaccgcg tcgagctgga aatgggtgct ctgcgtgaag aagtgaacaa gtccctcgtg 240
gatttgcatg atgcgcaggc aatcgccgag caggcccgcc aagatgcact tgcagccaag 300
aaggatctcg atgattctca agcgcagatc gaagcagccc aagagcgcct tgatgagatt 360
tcacgtgcag cgtatcgcca aaacggaacc tccaaggggc tttcaggcat ctcgggcaat 420
ggaaattctg aagatgcgct agatcgtcag acttacctgc gaaccagtgc ggaaaagcag 480
caggcagctg ttgaagagct tgatcgcctc cgtacggaaa acgccaacaa ggaatcggtg 540
ttgcgccagg cccgcatcgt tgctgagcag cgtgaggcgg aagccgtcga aaagcaagtc 600
cagaccgagg ctgcaattgc cgcaaacagc gagcagctca atgtcttgac taacaatcgc 660
agtaccttgg ttgcccagcg tgatggggct gagcgcaact tggccatcgc tcgtgcgcag 720
gcggataatc tgcaaggtca gcgtgctgag tacgaggaat tccagcaggc agagcaggct 780
cgcatccagg cggaagcgga agctcaggct gctgcggagg agaagcgtcg tgccgatgag 840
gctgctgcac aggcagccgc tgaagctcaa gaagctgccc agcaagctca ggcggcggag 900
gaagcccaag ccgcgcaagc agctgagaca gcacaagccc aagccgcgca agctgcggaa 960
acccaagctg cacaagccgc gcaagctcag gcagaagcga atgatcgtgc cgccgcgcaa 1020
cagcgtgctg cagaggctca agcagcagcg gaacaggcgc aacgtgaggc tgacgctcag 1080
gcggccaacg atgcccaagc tcaggcactg cgtgaacagg cgctcaccgc agcctccatc 1140
gctgcggctg ctctaattgc ggcgagccag tccagccatg ccactactca aaatccttac 1200
ccaactgatg aagacgcgga tccgaccgat attgcggaca tccaaggccc aacgcagcca 1260
ggtacgggtg agtctggaga ttcccagagc aactccagcg acaacgattc cacaggcaac 1320
gattccacag gctctgactc ttcagattca gattcctccg gcaacgattc ttcagaggtt 1380
atttccggcg atcgttccgc tcagattgag actgtgattg cgcgcgccat gagccagttg 1440
ggtgtgcagt acgcatgggg tggcggtaac gctaatggcc caactctggg tatccgtgac 1500
ggtggcgtgg cggactctta cggcgattac aacaaggttg gcttcgactg ctctggactg 1560
accttgtatg cgtttgcggg tgtgggaatt tcacttcctc actacacggg ctaccagtac 1620
cagcacggca ccaaggtgtc gccttctgag atgcaacgtg gcgatctgat cttctatggt 1680
ccgggagcgt ctcagcacgt ggcaatttac ctcggtgatg gtcagatgat tgaggctccg 1740
aattcgggtt ctgtcgtgaa gatttctcct gttcgctgga gcggaatgac cgagagcgtg 1800
gtacgcctca tttag 1815
<210> 6
<211> 1050
<212> DNA
<213> 棒状杆菌属
<220>
<221> 基因
<222> (1)..(1050)
<223> NCgl2107
<400> 6
ttgaatgagg ttgatagggg atttttgaag atgtttggtc gccgttgggt gagcgttgtg 60
gcgtcatgtg ttatcgcaag cacgctgatt ctggtgcctt cgcattccgg tgcggaggaa 120
gtcgatcaac tgattgctga tatcgagcat gtctctcagg aaacgtctgc ccagaatgag 180
gaagtcaaac agcttgagat tgatattgag gctcgtgagg tcacgatcaa ggaagttcag 240
gagcagtcgg taagctaccg tgaggcggct gatcaagcat cggagaatgt cgaagcttat 300
cgttcggaga tcaatcggat cgctcaggcg aagtatcgtg gcacagtcac ggatcctttg 360
agcattgcgg tgtctgcaga agatccacaa aacgtgattg atcggatgag ctacctttca 420
acgttgacta agtccactag tgatgtggtt gaatccctca acgcggagac tgagaagtcc 480
gcagaagctg tgtatcaagc aaaccgtact aaggcggaag cggagttcca gttggggcag 540
ctgaaggtac gccaggcgga gcttgaatct gaaaaggaag cattggatgg tcgaaaatcg 600
gagatccgag accgggtgga tgccctgacg ccacaggagc gggaaatgtg ggttgctaag 660
aatggtccat tggacattga tctgactgat ttgcttggtc tttccgctgc gacttcgggt 720
gcggtggatg ctgccttgtc taagttggga agcccttatg gttggggtgg cattggccca 780
aatgagtttg attgctcagg tttgatctat tgggcgtatc agcagatggg taagactttg 840
ccacgtacgt ctcaagctca gatggctggc ggaacgccgg tgagcagaga tgagctgcag 900
cctggcgatg tcattggata ttacccaggt gctactcacg tgggactgta tattggggac 960
ggaaagattg tgcacgcctc agactacgga atccctgtgc aggtggtatc tgttgattca 1020
gcaccgtttt atggtgcgcg tcgctactaa 1050
<210> 7
<211> 630
<212> DNA
<213> 棒状杆菌属
<220>
<221> 基因
<222> (1)..(630)
<223> NCgl2108
<400> 7
gtgggtaagc accgtcgcaa caattcaaac gcaactcgca aggctgtagc agcatctgca 60
gttgcgcttg gagcaaccgc agctatcgcc tccccagcac aggcagctga ggttgttgtt 120
cctggcaccg gaatcagcgt tgacatcgct ggcatcgaga ccactccagg tcttaacaac 180
gttccaggaa tcgatcagtg gatcccttcc cttagcagcc aggcagctcc tactgcttac 240
gcagccgtca ttgatgcacc tgcagcacag gctgcacctg cagcaagcac cggtcaggca 300
atcgttgatg cagcgcgcac caagattggt tccccatacg gttggggtgc taccggtcct 360
aacgctttcg actgctccgg ccttacctca tgggcataca gccaggttgg caagtccatc 420
ccacgtacct cccaggctca ggctgcacag ggcacccctg ttgcttactc tgaccttcag 480
gctggcgaca tcgttgcgtt ctactccggc gctacccacg ttggtatcta ctccggccac 540
ggcaccgtta tccacgcact gaacagcagc acccctctgt ctgagcactc cttggattac 600
atgccattcc actctgcagt tcgtttctaa 630
<210> 8
<211> 1245
<212> DNA
<213> 棒状杆菌属
<220>
<221> 基因
<222> (1)..(1245)
<223> NCgl2986
<400> 8
gtggatgaac tatatccatt gataattttg aacatgaatg atggaaggag cagggtgtct 60
aaagtcctga gagttggcga tcgcagcccg cgcgtggcag aagtgcgcac tacgctcgct 120
cgcctcggtg tgattgaagg ctattccagg gagatgtctg caaagacaga atcccagaag 180
ttccacgaag aagagacgct tttcgacgaa gaactcagcc tcagcatcaa gtcattccag 240
caagctcgag gagtcgttcc ctccgggctt attgacgacc ccaccctgcg cgcaatccgc 300
gaagcctcct acaccctggg aacccgcgtg ctggcctacc agcccggcaa ccagcttgtt 360
ggtgacgacg ttgtagaaat ccaatcccat ctccaagagc tcggcttcta cgccgaccgt 420
gtggatggac attttggcga gctcacacac aaagctgtga tgaactacca actcaactac 480
ggcatgcagg tagacggcat ctgtggccct gacaccatcc gtgcgctgtc ccgacttggt 540
ctgcgcatca agggtggctc tgctcaagct atccgtgaac gcgaacgcat gcgcaatgca 600
ggcccacgtc ttgctggcaa gcgtgtggtc attgatcctg cgcttggggg ctccaacaag 660
ggtcagatcg tgaaaggccc ctacggtgag atctctgagg aagaaatcct ctgggatttg 720
gccacccgcc tggaaggtcg catgatcgca acaggcatgg aaaccattct gtcgcgcccg 780
cacatggatg atcccagcag ccgtgatcgc gcgtcgatcg cgaatgcttt cggcgctgac 840
ctcatgctga gcctgcactg cgattcctac ccgaatgaaa aagctaacgg cgtggccagc 900
ttctacttcg gttcggaaaa cggcaccaac tccttgaccg gtgaaacgct ctccgcgtac 960
atccaaaaag agatcgttgc ccgcacccca ctgaacaact gtggcagcca tgcccgtacc 1020
tgggatctgc tgcgcctcac gcgcatgccc atggtggaag ttgtcaccgg ttacctcacc 1080
aaccccgatg acctggcagt tctgactgat ccacaaatgc gtgatcacat tgccgaagcc 1140
atcgttgtcg ccgtcaagcg cctgtacctc cttgatgagg aagcacagcc caagaccgga 1200
accttcaagt tctctgagct gttgcaatca gagcaggctg gctaa 1245
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 试剂盒引物
<400> 9
acctacaaca aagctctcat caacc 25
<210> 10
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 试剂盒引物
<400> 10
ctaccctgtg gaacacctac atct 24
<210> 11
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl1480引物
<400> 11
ccggggatcc tctagaacct tgaaacttcc actc 34
<210> 12
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl1480引物
<400> 12
ctcctgacga actatttcaa atcccctatc aacctc 36
<210> 13
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl1480引物
<400> 13
caccgaggta aattgccatg caagcgcaat caacgc 36
<210> 14
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl1480引物
<400> 14
gcaggtcgac tctagaaacc acacattatc gatc 34
<210> 15
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> NCgl2107引物
<400> 15
ccggggatcc tctagagcac agggcacccc tgttg 35
<210> 16
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl2107引物
<400> 16
ctcctgacga actatttcaa atcccctatc aacctc 36
<210> 17
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl2107引物
<400> 17
gaggttgata ggggatttga aatagttcgt caggag 36
<210> 18
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl2107引物
<400> 18
gcaggtcgac tctagaaacc acacattatc gatc 34
<210> 19
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl2108引物
<400> 19
ccggggatcc tctagagaac ccttagtagt tggg 34
<210> 20
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl2108引物
<400> 20
gtaatccaag gagtgctcac ccactgatga aactcc 36
<210> 21
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl2108引物
<400> 21
ggagtttcat cagtgggtga gcactccttg gattac 36
<210> 22
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl2108引物
<400> 22
gcaggtcgac tctagacgag cctcaatatc aatc 34
<210> 23
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl2986引物
<400> 23
ccggggatcc tctagattag gagaaaccat gagc 34
<210> 24
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl2986引物
<400> 24
atcagtcaga actgccagga ctgcagtaag aatacc 36
<210> 25
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> NCgl2986引物
<400> 25
ggtattctta ctgcagtcct ggcagttctg actgat 36
<210> 26
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> NCgl2986引物
<400> 26
gcaggtcgac tctagagttg aggcgtttgg atac 34
Claims (4)
1.一种生产L-赖氨酸的棒状杆菌属的微生物,其被修饰使得参与细胞壁水解的蛋白质的活性与其内源活性相比失活。
2.根据权利要求1所述的微生物,其中参与细胞壁水解的所述蛋白质是选自包括SEQID NO:1至4的氨基酸序列的蛋白质的至少一种蛋白质。
3.根据权利要求1所述的微生物,其中所述棒状杆菌属的微生物是谷氨酸棒状杆菌。
4.用于制备L-赖氨酸的方法,其包括:
(i)在培养基中培养权利要求1-3中任一项所述的棒状杆菌属的微生物;和
(ii)从所述培养基或所述微生物回收L-赖氨酸。
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KR10-2015-0095528 | 2015-07-03 | ||
PCT/KR2016/006833 WO2017007159A1 (ko) | 2015-07-03 | 2016-06-27 | L-라이신 생산능을 갖는 미생물 및 이를 이용한 l-라이신 생산 방법 |
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CN114107158A (zh) * | 2021-12-22 | 2022-03-01 | 广东省科学院生物与医学工程研究所 | 一种高产高纯度异麦芽酮糖的重组谷氨酸棒杆菌及其应用 |
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KR101793328B1 (ko) * | 2015-07-03 | 2017-11-03 | 씨제이제일제당 (주) | L-라이신 생산능을 갖는 미생물 및 이를 이용한 l-라이신 생산 방법 |
KR101947945B1 (ko) * | 2018-01-25 | 2019-02-13 | 씨제이제일제당 (주) | L-아미노산을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아미노산의 생산방법 |
EP3599282B1 (en) | 2018-07-24 | 2021-03-17 | Evonik Operations GmbH | Method for the fermentative production of l-lysine |
CN110106206B (zh) * | 2019-05-14 | 2023-05-02 | 诸城东晓生物科技有限公司 | 一种提高l-赖氨酸产量及稳定性的谷氨酸棒状杆菌构建方法 |
CN112877269B (zh) * | 2020-01-15 | 2021-12-24 | 中国科学院天津工业生物技术研究所 | 生产赖氨酸的微生物以及赖氨酸的生产方法 |
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RU2683551C1 (ru) | 2019-03-28 |
ES2790382T3 (es) | 2020-10-27 |
BR112018000074B1 (pt) | 2024-03-05 |
EP3318637A1 (en) | 2018-05-09 |
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PL3318637T3 (pl) | 2020-08-24 |
MY171648A (en) | 2019-10-22 |
HUE048882T2 (hu) | 2020-08-28 |
US11104925B2 (en) | 2021-08-31 |
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CN107922954B (zh) | 2022-01-11 |
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