CN116171327A - 具有增强的l-瓜氨酸生产能力的谷氨酸棒状杆菌变体,以及用于使用其产生l-瓜氨酸的方法 - Google Patents
具有增强的l-瓜氨酸生产能力的谷氨酸棒状杆菌变体,以及用于使用其产生l-瓜氨酸的方法 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/10—Citrulline; Arginine; Ornithine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
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- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及具有增强的L‑瓜氨酸生产能力的谷氨酸棒状杆菌(corynebacterium glutamicum)变体、以及用于使用所述变体产生L‑瓜氨酸的方法,所述谷氨酸棒状杆菌变体允许通过使由NCgl2816基因表达的转运蛋白活性减弱或失活而高产率地产生高浓度L‑瓜氨酸。
Description
技术领域
本发明涉及具有提高的L-瓜氨酸生产力的谷氨酸棒状杆菌突变体菌株,以及使用所述突变体菌株产生L-瓜氨酸的方法。
背景技术
L-瓜氨酸是一种非必需氨基酸,并且已知瓜氨酸具有有用的作用,例如促进氨代谢、通过血管舒张改善血流、降低血压、神经传递、增强免疫力以及清除活性氧物质。这样的瓜氨酸是在精氨酸生物合成期间作为中间产物合成的。微生物中精氨酸的生物合成由L-谷氨酸通过两种不同的途径(线性途径和环状途径)经过八个酶促步骤而进行。在线性途径中,L-精氨酸由L-谷氨酸经过N-乙酰谷氨酸、N-乙酰鸟氨酸、鸟氨酸、瓜氨酸和精氨琥珀酸而合成。
L-瓜氨酸通常是使用微生物(例如细菌或酵母)通过发酵而产生的,并且L-瓜氨酸的产生可使用天然存在的野生型菌株或从野生型菌株修饰以具有提高的瓜氨酸生产力的突变体菌株来进行。最近,为了改善L-瓜氨酸的生产效率,已将遗传重组技术应用于已广泛用于L-氨基酸和其他有用物质的生产的微生物,例如大肠杆菌和棒状杆菌,并且已开发了具有优异L-瓜氨酸生产力的多种重组菌株或突变体菌株、以及使用其产生L-瓜氨酸的方法。根据韩国专利No.10-1102263和10-1053429,已确定了L-瓜氨酸的生产力通过增强或减弱参与L-瓜氨酸生物合成的蛋白质(例如酶和转录因子)的活性或表达而提高。因此,可预期,L-瓜氨酸的产生也可通过调节参与L-瓜氨酸生物合成途径的多种蛋白质的活性或表达来调节。
[现有技术文件]
[专利文件]
韩国专利No.10-1102263
韩国专利No.10-1053429
发明内容
技术问题
本发明的一个目的是提供具有提高的L-瓜氨酸生产力的谷氨酸棒状杆菌(Corynebacterium glutamicum)突变体菌株。
本发明的另一个目的是提供使用所述突变体菌株产生L-瓜氨酸的方法。
技术方案
本发明人已进行了使用谷氨酸棒状杆菌菌株开发具有提高的L-瓜氨酸生产力的新的突变体菌株的研究,并且作为结果,发现当编码参与L-瓜氨酸生物合成途径的转运蛋白(transporter)或转运蛋白(transport protein)的NCgl2816基因被去除时,L-瓜氨酸的产生提高,从而完成了本发明。
本发明的一个方面提供了谷氨酸棒状杆菌突变体菌株,其中由NCgl2816基因表达的蛋白质的活性已减弱或失活,并且其具有提高的L-瓜氨酸生产力。
本文中使用的术语“NCgl2816基因”是指编码参与主动转运的多种次级转运蛋白中的推定整合膜转运蛋白的基因,所述转运蛋白克服了在物质移动通过细胞膜期间细胞外与细胞内之间的浓度梯度,并选择性地吸收或释放营养物。
根据本发明的一个实施方案,NCgl2816基因可来源于棒状杆菌(Corynebacterium)属菌株。特别地,棒状杆菌属菌株可以是谷氨酸棒状杆菌、Corynebacterium crudilactis、沙漠棒状杆菌(Corynebacterium deserti)、帚石南棒状杆菌(Corynebacterium callunae)、Corynebacterium suranareeae、Corynebacteriumlubricantis、Corynebacterium doosanense、有效棒状杆菌(Corynebacteriumefficiens)、Corynebacterium uterequi、停滞棒状杆菌(Corynebacterium stationis)、Corynebacterium pacaense、单一棒状杆菌(Corynebacterium singulare)、Corynebacterium humireducens、海洋棒状杆菌(Corynebacterium marinum)、耐盐棒状杆菌(Corynebacterium halotolerans)、企鹅棒状杆菌(Corynebacterium spheniscorum)、Corynebacterium freiburgense、纹状棒状杆菌(Corynebacterium striatum)、犬棒状杆菌(Corynebacterium canis)、产氨棒状杆菌(Corynebacterium ammoniagenes)、牛肾盂炎棒状杆菌(Corynebacterium renale)、Corynebacterium pollutisoli、汉氏棒状杆菌(Corynebacterium imitans)、黑海棒状杆菌(Corynebacterium caspium)、龟口腔棒状杆菌(Corynebacterium testudinoris)、Corynebacaterium pseudopelargi或微黄棒状杆菌(Corynebacterium flavescens),但不限于此。
根据本发明的一个实施方案,NCgl2816基因可由SEQ ID NO:1的核苷酸序列组成。根据本发明的另一个实施方案,NCgl2816基因可编码SEQ ID NO:2的氨基酸序列。
根据本发明的一个实施方案,由NCgl2816基因表达的蛋白质是参与L-瓜氨酸生物合成途径的转运蛋白,并且该转运蛋白对L-瓜氨酸的产生的作用尚未知或未研究。然而,本发明人已发现,当编码转运蛋白的NCgl2816基因缺失时,膜稳定性可改善、L-瓜氨酸的释放可提高、在L-瓜氨酸的过度产生中另外检测到的中间体6-乙酰鸟氨酸的释放可被抑制,并且因此L-瓜氨酸可以以高产率和高浓度产生。
本发明中使用的“减弱的活性”意指目的基因的表达水平与原始表达水平相比已降低。术语“减弱的活性”还包括:其中由于编码基因的核苷酸序列中的一个或更多个核苷酸的替换、插入、缺失或其组合,与亲本微生物中蛋白质的活性相比,蛋白质本身的活性降低的情况;其中由于编码酶的基因的表达或翻译降低,细胞中酶的总体活性低于天然菌株、野生型菌株或修饰之前的菌株中那些的情况;及其组合。
本发明中使用的“失活”意指编码蛋白质例如酶、转录因子或转运蛋白的基因与天然菌株、野生型菌株或修饰之前的菌株中的那些相比完全未表达,或者该基因即使表达也没有活性。
根据本发明的一个实施方案,通过从谷氨酸棒状杆菌菌株中缺失NCgl2816基因,获得了与野生型谷氨酸棒状杆菌菌株或先前开发以过度产生瓜氨酸的谷氨酸棒状杆菌突变体菌株相比具有提高的L-瓜氨酸生产力的谷氨酸棒状杆菌突变体菌株。
本文中使用的术语“提高的生产力”意指L-瓜氨酸生产力与亲本菌株中的L-瓜氨酸生产力相比有所提高。本文中使用的术语“亲本菌株”是指野生型菌株或待突变的突变体菌株,并且包括待直接突变或待用重组载体等转化的菌株。在本发明中,亲本菌株可以是野生型谷氨酸棒状杆菌菌株或从野生型菌株突变而来的菌株。例如,亲本菌株可以是谷氨酸棒状杆菌突变体菌株,其具有增强的鸟氨酸氨甲酰基转移酶和氨甲酰基磷酸合酶活性以过度产生瓜氨酸(参见韩国专利申请No.10-2019-0151321)。
根据本发明的一个实施方案,与亲本菌株相比,具有提高的L-瓜氨酸生产力的谷氨酸棒状杆菌突变体菌株表现出提高的L-瓜氨酸生产力。特别地,由谷氨酸棒状杆菌突变体菌株产生的L-瓜氨酸的量可比由亲本菌株产生的L-瓜氨酸的量高至少0.5%,特别地高0.5%至5%,而作为副产物产生的6-乙酰鸟氨酸的量可比由亲本菌株产生的6-乙酰鸟氨酸的量低至少20%,特别地低20%至50%。
根据本发明一个实施方案的谷氨酸棒状杆菌突变体菌株可通过缺失亲本菌株中NCgl2816基因的重组载体获得。
本文中使用的术语“载体”是指用于将核酸克隆和/或转移到宿主细胞中的任何载剂。载体可以是可连接另外的DNA片段以引起所连接片段复制的复制子。术语“复制子”是指作为体内DNA复制的自主单元发挥作用,即能够在其自身控制下复制的任何遗传元件(例如,质粒、噬菌体、黏粒、染色体、病毒)。在本发明中,载体没有特别限制,只要其可在宿主中复制即可,并且可使用本领域已知的任何载体。用于构建重组载体的载体可以是天然状态或重组状态的质粒、黏粒、病毒或噬菌体。可使用的噬菌体载体或黏粒载体的一些实例包括但不限于pWE15、M13、λEMBL3、λEMBL4、λFIXII、λDASHII、λZAPII、λgt10、λgt11、Charon4A和Charon21A,并且可使用的质粒载体的一些实例包括但不限于pDZ载体,以及基于pBR、基于pUC、基于pBluescriptII、基于pGEM、基于pTZ、基于pCL和基于pET的载体。
本文中使用的术语“转化”意指将基因引入到宿主细胞中,使得该基因可在宿主细胞中表达。转化的基因可包括任何基因,不管该基因是插入在宿主细胞的染色体中还是位于染色体之外,只要该基因可在宿主细胞中表达即可。
根据本发明的一个实施方案,转化方法包括将核酸引入到细胞中的任何方法,并且可使用本领域已知的根据宿主细胞选择的合适的标准技术来进行。转化方法的一些实例包括但不限于电穿孔、磷酸钙(CaPO4)沉淀、氯化钙(CaCl2)沉淀、显微注射、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法和乙酸锂-DMSO法。
根据本发明的一个实施方案,作为转化方法,可使用电穿孔法(van der Rest etal.,Appl.Microbiol.Biotechnol.,52,541-545,1999)。
宿主细胞包括在体内或体外用本发明的重组载体或多核苷酸转染、转化或感染的细胞。包含本发明的重组载体的宿主细胞是重组宿主细胞、重组细胞或重组微生物。
另外,根据本发明的重组载体可包含选择标志物。选择标志物用于选择用载体转化的转化体(宿主细胞)。由于只有表达选择标志物的细胞才可在用选择标志物处理的培养基中存活,所以可选择经转化的细胞。选择标志物的代表性实例包括但不限于卡那霉素、链霉素、氯霉素等。
插入到根据本发明的用于转化的重组载体中的基因可通过同源重组交叉整合至宿主细胞例如微生物(例如棒状杆菌属菌株或大肠杆菌)中。
本发明的另一方面提供了用于产生L-瓜氨酸的方法,其包括以下步骤:a)在培养基中培养谷氨酸棒状杆菌突变体菌株;以及b)从所培养的突变体菌株中或从培养所述突变体菌株的培养基中回收L-瓜氨酸。
可使用本领域已知的合适培养基和培养条件进行培养,并且本领域任何技术人员可容易地调整和使用培养基和培养条件。特别地,培养基可以是液体培养基,但不限于此。培养方法的一些实例包括但不限于分批培养、连续培养、补料分批培养、或其组合。
根据本发明的一个实施方案,培养基应当以适当的方式满足特定菌株的要求,并且可由本领域技术人员进行适当的修改。针对用于棒状杆菌属菌株的培养基,可参考已知文件(Manual of Methods for General Bacteriology,American Society forBacteriology,Washington D.C.,USA,1981),但不限于此。
根据本发明的一个实施方案,培养基可包含多种碳源、氮源和微量元素组分。可使用的碳源的一些实例包括:糖类和碳水化合物,例如葡萄糖、蔗糖、乳糖、果糖、麦芽糖、淀粉和纤维素;油和脂肪,例如大豆油、葵花籽油、蓖麻油和椰子油;脂肪酸,例如棕榈酸、硬脂酸和亚油酸;醇,例如甘油和乙醇;以及有机酸,例如乙酸。这些物质可单独使用或作为混合物使用,但不限于此。可使用的氮源的一些实例包括蛋白胨、酵母提取物、肉提取物、麦芽提取物、玉米浆、大豆粉、尿素,或者无机化合物例如硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵。氮源也可单独使用或作为混合物使用,但不限于此。可使用的磷源的一些实例包括但不限于磷酸二氢钾或磷酸氢二钾、或者相应的含钠盐。另外,培养基可包含但不限于生长所需的金属盐,例如硫酸镁或硫酸铁。另外,培养基可包含必需的生长物质,例如氨基酸和维生素。此外,可在培养基中使用合适的前体。可在培养期间将培养基或单独的组分通过合适的方法分批或以连续方式添加至培养基中,但不限于此。
根据本发明的一个实施方案,培养基的pH可通过在培养期间以适当方式向微生物培养基中添加化合物例如氢氧化铵、氢氧化钾、氨、磷酸和硫酸来调节。另外,在培养期间,可使用消泡剂例如脂肪酸聚乙二醇酯来抑制发泡。另外,为了将培养基保持在有氧条件下,可将氧气或含氧气体(例如,空气)注入培养基中。培养基的温度通常可以是20℃至45℃,例如25℃至40℃。可继续培养直至产生所期望量的可用物质。例如,培养时间可以是10小时至160小时。
根据本发明的一个实施方案,在从所培养的突变体菌株中或从培养所述突变体菌株的培养基中回收L-瓜氨酸的步骤中,所产生的L-瓜氨酸可使用本领域中已知的合适方法根据培养方法从培养基中收集或回收。可使用的用于回收L-瓜氨酸的方法的一些实例包括但不限于离心、过滤、萃取、喷洒、干燥、蒸发、沉淀、结晶、电泳、分级溶解(例如,硫酸铵沉淀)、色谱(例如,离子交换、亲和力、疏水性和尺寸排阻)等。
根据本发明的一个实施方案,回收L-瓜氨酸的步骤可通过将培养基在低速下离心以去除生物质并通过离子交换色谱分离所获得的上清液来进行。
根据本发明的一个实施方案,回收L-瓜氨酸的步骤可包括纯化L-瓜氨酸的过程。
有益效果
根据本发明的谷氨酸棒状杆菌突变体菌株能够以高产率和高浓度产生L-瓜氨酸,因为其中由NCgl2816基因表达的转运蛋白的活性已减弱或失活。
具体实施方式
在下文中,将更详细地描述本发明。然而,提供这些描述仅用于举例说明目的以帮助理解本发明,并且本发明的范围不受这些举例说明性描述的限制。
实施例1.对瓜氨酸产生菌株中转录物组水平变化的分析
1-1.细胞收集
为了分析瓜氨酸产生菌株中转录物组水平变化,在各菌株的生长阶段中发生变化时进行细胞收集。
首先,将谷氨酸棒状杆菌突变体菌株(参见韩国专利申请No.10-2019-0151321,在下文中称为“CT4”)(其具有增强的鸟氨酸氨甲酰基转移酶和氨甲酰基磷酸合酶活性以过度产生瓜氨酸)和野生型谷氨酸棒状杆菌菌株(ATCC13032)中各自接种在瓜氨酸种子培养基中并在30℃下培养10小时,并且随后将250mL的各培养物接种在5-L培养基中。当初始培养基中包含的葡萄糖完全耗竭时,添加另外的培养基。在接种之后培养13、28、35和68小时时,收集细胞并将其用于制备转录物组分析样品。本实验中使用的培养基的组成在下表1中示出。
[表1]
1-2.转录物组分析样品的制备
在上述实施例1-1中收集细胞之后,以相同浓度制备所培养的细胞,并使用RneasyMini试剂盒(QIAGEN,Germany)根据制造商说明从其中提取RNA。将提取的RNA储存在液氮中,并由Macrogen Co.,Ltd.进行全基因组转录物组分析。
1-3.有效候选基因的选择
基于在上述实施例1-2中进行的谷氨酸棒状杆菌的全基因组转录物组分析的结果,首先从通透酶和转运蛋白中选择40种候选基因,其RPKM值大于1000,并且其表达水平在发酵的后半部分快速提高或是野生型菌株中表达水平的至少三倍高。对于参考,已知野生型谷氨酸棒状杆菌菌株(ATCC13032)几乎没有产生瓜氨酸的能力。
将40种基因中的每种中有缺陷的谷氨酸棒状杆菌菌株接种在烧瓶滴度培养基中(参见下表2)并在30℃下在200rpm下培养30小时。在培养完成之后,将各培养物用蒸馏水稀释100倍并通过0.45μm过滤器过滤,并且随后通过使用配备有柱(DionexIonPacTM CS12A)和紫外检测器(195mm)的高效液相色谱(HPLC)测量各菌株培养物中L-瓜氨酸和副产物6-乙酰鸟氨酸的浓度。从40种候选基因中的每种中有缺陷的菌株中进一步选择5种具有低浓度6-乙酰鸟氨酸的候选菌株。野生型菌株和5种所选择候选菌株的培养物中L-瓜氨酸和6-乙酰鸟氨酸的浓度在下表3中示出。
[表2]
[表3]
作为结果,其显示出在NCgl2816基因缺陷型菌株中,L-瓜氨酸的产生最高,并且同时,6-乙酰鸟氨酸的产生最低。因此,选择NCgl2816基因作为待缺失的基因以用于提高菌株中L-瓜氨酸的产生。
实施例2.NCgl2816基因缺失突变体菌株的构建
2-1.载体的构建
野生型谷氨酸棒状杆菌菌株(ATCC13032)用于构建谷氨酸棒状杆菌突变体菌株。
首先,使用Wizard基因组DNA纯化试剂盒(Promega,USA)从野生型菌株中提取染色体DNA。使用所提取的DNA作为模板,使用引物1和2的组以及引物3和4的组中的每一种进行PCR。使用引物1和4的组通过交叉PCR再次扩增所获得的PCR产物,并且随后将其插入到重组载体pK19mobSacB的HindIII和XbaI限制酶位点中。将所得载体命名为载体pK19ms/△NCgl2816。对于载体的构建,使用表4中示出的引物。
[表4]
引物 | 序列(5′→3′) | SEQ ID NO. |
引物1 | tgatttacgccaagctccttatccg | 3 |
引物2 | ggaggggttttacttagattggtgacctcttctctgaaac | 4 |
引物3 | gtttcagagaagaggtcaccaatctaagtaaaacccctcc | 5 |
引物4 | ccggggatcctctagcgac | 6 |
使用上述引物,在以下条件下进行PCR。使用热循环仪(TP600,TAKARA BIO Inc.,Japan)在包含以下的反应溶液中进行PCR:100μM的每种脱氧核苷酸三磷酸(dATP、dCTP、dGTP、dTTP)、1pM寡核苷酸、10ng的谷氨酸棒状杆菌(C.glutamicum)ATCC13032的染色体DNA作为模板、以及1单位的PrimeSTAR Max DNA聚合酶(Takara,Japan)。PCR进行25至30个循环,每个循环由以下组成:(i)在94℃下变性30秒,(ii)在58℃下退火30秒,以及(iii)在72℃下延伸1至2分钟(聚合时间为2分钟/kb)。
将如上所述制备的基因片段通过自组装克隆克隆到pK19mobSacB载体中。将载体转化到大肠杆菌(E.coli)DH5a中,然后将其平板接种在包含50μg/ml的卡那霉素的LB-琼脂板上,并在37℃下培养24小时。分离最终形成的菌落并检查插入物是否准确地存在于载体中。接下来,分离载体并将其用于谷氨酸棒状杆菌菌株的重组。
作为上述方法中普遍进行的过程,通过PCR自谷氨酸棒状杆菌ATCC13032的基因组DNA来扩增相应基因,并根据策略通过自组装克隆方法将其插入到pK19mobSacB载体中,并在大肠杆菌DH5a中选择所得质粒。在这种情况下,为了将经扩增的基因插入到pK19mobSacB载体中,根据制造商提供的缓冲液和方案使用了DNA连接试剂盒(Takara,Japan)和限制酶HindIII和XbaI(NEB,England)。
2-2.突变体菌株的构建
通过将上述实施例2-1中构建的pK19ms/ΔNCgl2816载体引入到作为亲本菌株的突变体菌株CT4中来构建谷氨酸棒状杆菌突变体菌株。
特别地,首先将亲本菌株培养在100ml RG培养基(包含10g/L牛肉提取物、40g/LBHI和30g/L山梨糖醇)中,并将2.5g/L甘氨酸、400mg/L异烟肼和0.1ml/L Tween80添加至同一培养基中。接下来,接种种子培养物使得OD610值达到0.3,并随后在30℃下在180rpm下将其培养3至5小时使得OD610值达到1.6至1.8。将培养物在冰上保持30分钟,并随后在4℃下在3500rpm下离心15分钟。接下来,弃去上清液,并且将沉淀的亲本菌株用10%甘油溶液洗涤4次,并最终重悬于0.5ml 10%甘油溶液中。使用Bio-Rad电穿孔仪进行电穿孔。将通过上述方法制备的感受态细胞置于电穿孔比色皿(0.2mm)中,并向其中添加pK19ms/ΔNCgl2816载体,随后在2.5kV、200Ω和12.5μF的条件下进行电穿孔。在电穿孔完成之后,立即向细胞添加1ml再生培养基(包含18.5g/l脑心浸液和0.5M山梨糖醇),然后将细胞在46℃下热处理6分钟。接下来,将细胞在室温下冷却,转移到15ml盖管中,在30℃下孵育2小时,并平板接种在包含30μg/ml卡那霉素的固体培养基(包含40g/L脑心浸液、30g/L D-山梨糖醇、10g/L牛肉提取物和20g/L琼脂)上。将细胞在30℃下培养2天以获得菌落。在其中诱导了一级同源重组的菌落中,选择其中在与上述实施例2-1相同的条件下使用上表4的引物1和4通过PCR确定了扩增的菌落作为一级重组菌株,并在2YT液体培养基(包含16g/L胰蛋白胨、10g/L酵母提取物和5g/L NaCl)中培养12小时。接下来,将培养的菌落平板接种在2YT-10%蔗糖固体培养基(包含16g/L胰蛋白胨、10g/L酵母提取物、5g/L NaCl和100g/L蔗糖)上,以诱导二级同源重组,因此去除抗生素标志物。使在2YT-10%蔗糖固体培养基上培养的菌落贴附在2YT-Km培养基(包含16g/L胰蛋白胨、10g/L酵母提取物、5g/L NaCl和30μg/ml卡那霉素)上,并且最终选择对卡那霉素无抗性并可在10%蔗糖培养基中生长的菌株。在与实施例2-1相同的条件下通过使用上表4中示出的引物1和4进行PCR,最终检查NCgl2816基因是否被去除,并将NCgl2816基因缺失菌株命名为CT5。
实施例3.对NCgl2816基因缺失突变体菌株的L-瓜氨酸生产力的评价
比较了用作亲本菌株的过度产生瓜氨酸的谷氨酸棒状杆菌突变体菌株(CT4)和实施例2中构建的NCgl2816基因缺失菌株(CT5)的L-瓜氨酸生产力。
将各菌株接种到瓜氨酸种子培养基中,并在30℃下培养10小时。然后,将250mL的各培养物接种到5-L培养基中。当初始培养基中包含的葡萄糖完全耗竭时,添加另外的培养基。本实验中使用的培养基的组成在上表1中示出。在培养完成之后,将各培养物用蒸馏水稀释100倍并通过0.45μm过滤器过滤,并且随后使用配备有柱(DionexIonPacTM CS12A)和紫外检测器(195mm)的高效液相色谱(HPLC)测量各菌株培养物中L-瓜氨酸和副产物6-乙酰鸟氨酸的浓度。结果在下表5中示出。
[表5]
菌株 | L-瓜氨酸(%) | 6-乙酰鸟氨酸(%) |
CT4 | 8.56 | 1.6 |
CT5 | 8.73 | 0.7 |
如上表5中所示,确定了与先前开发的突变体菌株CT4的L-瓜氨酸生产力相比,NCgl2816基因缺失的谷氨酸棒状杆菌突变体菌株CT5的L-瓜氨酸生产力提高了约2%,而副产物6-乙酰鸟氨酸的产生降低了约44%。这些结果表明,当由NCgl2816基因表达的蛋白质的活性减弱或失活时,菌株的L-瓜氨酸生产力可提高并且副产物的产生也可降低,并且因此该菌株能够产生具有高纯度的L-瓜氨酸。
迄今为止,已参照一些优选实施方案描述了本发明。本发明所属领域的普通技术人员将理解,在不脱离本发明的实质特征的情况下,本发明可以以经修改形式实施。因此,应从解释说明的角度而不是从限制性的角度考虑所公开的实施方案。本发明的范围由权利要求书而不是以上描述来限定,并且与之等同的范围内的所有差异均应被解释为包括在本发明中。
<110> DAESANG CORPORATION
<120> 具有增强的L-瓜氨酸生产力的谷氨酸棒状杆菌突变体,以及用于使用其制备L-瓜氨酸的方法
<130> PX210021PCT
<150> KR 10-2020-0127021
<151> 2020-09-29
<160> 6
<170> KoPatentIn 3.0
<210> 1
<211> 1302
<212> DNA
<213> 人工序列
<220>
<223> NCgl2816
<400> 1
atgacaaccg cagtagatca aaactcaccg cccaagcagc aactcaacaa gcgcgtcctg 60
ctgggcagct tgagtggcag cgttatcgaa tggttcgact tcctggttta cggaaccgtc 120
gccgcgctgg tcttcaacaa gatgtacttc cccagcggca acgagttcct ctccacaatc 180
ctggcgtacg catccttctc cctgaccttc ttcttccgcc ccattggtgg cgtcatcttc 240
gcccacatcg gcgaccgcat tgggcgtaag aagaccctgt tcatcacctt gatgctcatg 300
ggtggcggca ccgtcgccat tggtttgctg cccgactaca acgccatcgg catttgggca 360
ccaatccttc tgatgttcct ccgcattttg cagggcatcg gaattggcgg cgaatggggt 420
ggcgcactgc tcctggcata cgaatacgct ccaaagaagc agcgtgggct ctacggcgca 480
gttcctcaaa tgggcatttc cctgggcatg ctgcttgcag ctggcgtgat ctctctgctc 540
accctcatgc cggaagatca gttcctcacc tggggctggc gcatcccatt cgtcggatcc 600
atcctcctag tgttcatcgg cctgttcatc cgaaacggcc ttgatgaaac ccccgagttc 660
aagcgtatcc gcgattccgg ccagcaggta aagatgcctc tgaaggaagt tctgaccaag 720
tactggccag ccgttctggt ctccatcggc gcaaaagctg ccgagaccgg ccccttctac 780
atcttcggca cctacatcgt tgcttacgca accaacttcc tgaacatccg cgacaacatt 840
gtccttctgg cagttgcttg cgccgccctc gttgccacca tctggatgcc actgttcgga 900
tccttctccg accgcgtcaa ccgtgcagtg ctctacagga tctgtgcatc cgcaaccatc 960
gtgctgattg tcccttacta cttggtcctc aacaccggcg aaatttgggc actgtttatc 1020
actaccgtga ttggcttcgg catcctctgg ggtagcgtca acgcaatcct cggaaccgtc 1080
atcgcagaaa acttcgcacc tgaggtccgc tacaccggcg ctaccctggg ttaccaagtc 1140
ggagcagcac tcttcggcgg taccgcaccc attatcgcag catggctgtt cgaaatctcc 1200
ggcggacaat ggtggccaat cgccgtctac gtcgctgcat gttgccttct ctctgtgatc 1260
gcctcgttct tcatccaacg cgtcgcgcac caagagaact aa 1302
<210> 2
<211> 433
<212> PRT
<213> 人工序列
<220>
<223> NCgl2816
<400> 2
Met Thr Thr Ala Val Asp Gln Asn Ser Pro Pro Lys Gln Gln Leu Asn
1 5 10 15
Lys Arg Val Leu Leu Gly Ser Leu Ser Gly Ser Val Ile Glu Trp Phe
20 25 30
Asp Phe Leu Val Tyr Gly Thr Val Ala Ala Leu Val Phe Asn Lys Met
35 40 45
Tyr Phe Pro Ser Gly Asn Glu Phe Leu Ser Thr Ile Leu Ala Tyr Ala
50 55 60
Ser Phe Ser Leu Thr Phe Phe Phe Arg Pro Ile Gly Gly Val Ile Phe
65 70 75 80
Ala His Ile Gly Asp Arg Ile Gly Arg Lys Lys Thr Leu Phe Ile Thr
85 90 95
Leu Met Leu Met Gly Gly Gly Thr Val Ala Ile Gly Leu Leu Pro Asp
100 105 110
Tyr Asn Ala Ile Gly Ile Trp Ala Pro Ile Leu Leu Met Phe Leu Arg
115 120 125
Ile Leu Gln Gly Ile Gly Ile Gly Gly Glu Trp Gly Gly Ala Leu Leu
130 135 140
Leu Ala Tyr Glu Tyr Ala Pro Lys Lys Gln Arg Gly Leu Tyr Gly Ala
145 150 155 160
Val Pro Gln Met Gly Ile Ser Leu Gly Met Leu Leu Ala Ala Gly Val
165 170 175
Ile Ser Leu Leu Thr Leu Met Pro Glu Asp Gln Phe Leu Thr Trp Gly
180 185 190
Trp Arg Ile Pro Phe Val Gly Ser Ile Leu Leu Val Phe Ile Gly Leu
195 200 205
Phe Ile Arg Asn Gly Leu Asp Glu Thr Pro Glu Phe Lys Arg Ile Arg
210 215 220
Asp Ser Gly Gln Gln Val Lys Met Pro Leu Lys Glu Val Leu Thr Lys
225 230 235 240
Tyr Trp Pro Ala Val Leu Val Ser Ile Gly Ala Lys Ala Ala Glu Thr
245 250 255
Gly Pro Phe Tyr Ile Phe Gly Thr Tyr Ile Val Ala Tyr Ala Thr Asn
260 265 270
Phe Leu Asn Ile Arg Asp Asn Ile Val Leu Leu Ala Val Ala Cys Ala
275 280 285
Ala Leu Val Ala Thr Ile Trp Met Pro Leu Phe Gly Ser Phe Ser Asp
290 295 300
Arg Val Asn Arg Ala Val Leu Tyr Arg Ile Cys Ala Ser Ala Thr Ile
305 310 315 320
Val Leu Ile Val Pro Tyr Tyr Leu Val Leu Asn Thr Gly Glu Ile Trp
325 330 335
Ala Leu Phe Ile Thr Thr Val Ile Gly Phe Gly Ile Leu Trp Gly Ser
340 345 350
Val Asn Ala Ile Leu Gly Thr Val Ile Ala Glu Asn Phe Ala Pro Glu
355 360 365
Val Arg Tyr Thr Gly Ala Thr Leu Gly Tyr Gln Val Gly Ala Ala Leu
370 375 380
Phe Gly Gly Thr Ala Pro Ile Ile Ala Ala Trp Leu Phe Glu Ile Ser
385 390 395 400
Gly Gly Gln Trp Trp Pro Ile Ala Val Tyr Val Ala Ala Cys Cys Leu
405 410 415
Leu Ser Val Ile Ala Ser Phe Phe Ile Gln Arg Val Ala His Gln Glu
420 425 430
Asn
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物1
<400> 3
tgatttacgc caagctcctt atccg 25
<210> 4
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物2
<400> 4
ggaggggttt tacttagatt ggtgacctct tctctgaaac 40
<210> 5
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物3
<400> 5
gtttcagaga agaggtcacc aatctaagta aaacccctcc 40
<210> 6
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物4
<400> 6
ccggggatcc tctagcgac 19
Claims (4)
1.谷氨酸棒状杆菌(Corynebacterium glutamicum)突变体菌株,其中由NCgl2816基因表达的蛋白质的活性已减弱或失活,并且其具有提高的L-瓜氨酸生产力。
2.根据权利要求1所述的谷氨酸棒状杆菌突变体菌株,其中所述NCgl2816基因由SEQID NO:1的核苷酸序列组成。
3.根据权利要求1所述的谷氨酸棒状杆菌突变体菌株,其中所述NCgl2816基因的全部或一部分被插入、替换或缺失。
4.用于产生L-瓜氨酸的方法,其包括以下步骤:
a)在培养基中培养权利要求1所述的谷氨酸棒状杆菌突变体菌株;以及
b)从所培养的突变体菌株中或从培养所述突变体菌株的培养基中回收L-瓜氨酸。
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PCT/KR2021/003602 WO2022071638A1 (ko) | 2020-09-29 | 2021-03-23 | L-시트룰린 생산능이 향상된 코리네박테리움 글루타미쿰 변이주 및 이를 이용한 l-시트룰린의 생산 방법 |
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