CN116867895A - aroG醛缩酶变体及利用其生产支链氨基酸的方法 - Google Patents
aroG醛缩酶变体及利用其生产支链氨基酸的方法 Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01054—3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
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- C—CHEMISTRY; METALLURGY
- 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
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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Abstract
本申请涉及aroG醛缩酶(磷酸‑2‑脱氢‑3‑脱氧庚酸醛缩酶)变体、包含该变体的微生物,以及使用该微生物生产氨基酸的方法。
Description
【技术领域】
本申请涉及aroG醛缩酶(磷酸-2-脱氢-3-脱氧庚酸醛缩酶)的变体,以及使用该酶生产氨基酸的方法。
【背景技术】
L-氨基酸是蛋白质的基本组成部分,并用作医药原料、食品添加剂、动物饲料、营养补充剂、杀虫剂、杀菌剂等的重要原料。因此,从经济角度来看,氨基酸的工业生产已成为重要的工业过程。
已经进行了各种研究以有效地生产氨基酸;例如,已经努力开发微生物或发酵工艺技术,以高效生产氨基酸。特别是,已经开发了针对目标材料的特定方法,例如增强编码参与棒杆菌属(Corynebacterium)菌株中氨基酸生物合成的酶的基因表达或删除氨基酸生物合成所必需的基因(US 9109242 B2)。除了这些方法之外,还使用了去除不参与氨基酸生产的基因的方法和去除其产生氨基酸功能不明确已知的基因的方法。
同时,在氨基酸中,支链氨基酸是指缬氨酸、亮氨酸和异亮氨酸三种氨基酸,并且已知它们主要在肌肉组织中代谢并作为运动过程中的能量来源。由于已知支链氨基酸在运动期间的肌肉维持和生长中起重要作用,因此其使用正在增加。然而,由于支链氨基酸的生物合成途径中会产生大量的副产物,因此减少副产物的量以生产高产量和高纯度的支链氨基酸非常重要。
【发明内容】
【技术问题】
本发明人已经证实,已引入新开发的aroG醛缩酶变体的微生物可以高产和高纯度生产支链氨基酸,从而完成了本申请。
【技术方案】
本申请的一个目的是提供aroG醛缩酶(磷酸-2-脱氢-3-脱氧庚酸醛缩酶)变体,其中对应于SEQ ID NO:1的氨基酸序列中自N末端起第217、310、403和462位的任一个或多个氨基酸被另一种氨基酸取代。
本申请的另一个目的是提供一种编码该变体的多核苷酸及其载体。
本申请的另一个目的是提供棒杆菌属(Corynebacterium)微生物,其包括一种以上aroG醛缩酶变体、多核苷酸和载体。
本申请的另一个目的是提供一种生产支链氨基酸的方法,包括在培养基中培养所述微生物。
【有利效果】
当使用本申请的aroG醛缩酶变体时,副产物的产生可以减少,并且与不使用aroG醛缩酶变体的情况相比,可以高产率地生产支链氨基酸。
【优选实施例详细说明】
下面对本申请进行详细说明。同时,本文公开的每个描述和实施例可以分别应用于其它描述和实施例。也就是说,本文公开的各种元件的所有组合都属于本申请的范围。此外,本申请的范围不受下面描述的具体描述的限制。
此外,本领域普通技术人员可以仅使用常规实验来识别或确认许多等同于本文描述的本发明的特定方面。此外,还打算在本申请中包括这些等同物。
aroG醛缩酶变体是指其中对应于SEQ ID NO:1的氨基酸序列中自N末端起第217、310、403和462位的任一个或多个氨基酸被另一种氨基酸取代的变体,在具有aroG醛缩酶活性的多肽中或在aroG醛缩酶中。
所述aroG醛缩酶变体是指在具有aroG醛缩酶活性的多肽中或在aroG醛缩酶中中对应于N末端的任意一种以上氨基酸在SEQ ID NO:217的氨基酸序列中被另一种氨基酸取代。
本文所用的术语“aroG醛缩酶”是能够催化以下反应的酶:
磷酸烯醇式丙酮酸盐+D-赤藓糖-4-磷酸+H2O3-脱氧-D-***-庚-2-酮糖酸-7-磷酸+磷酸
本申请的aroG醛缩酶可以是对aroG醛缩酶进行修饰以制备本文提供的aroG醛缩酶变体,或者具有aroG醛缩酶活性的多肽。具体地,可以是天然存在的多肽或野生型多肽,可以是其成熟的多肽,并且可以包括其变体或其功能片段而不受限制,只要它可以是本申请的aroG醛缩酶变体的母体。
如本文所用,aroG醛缩酶可以是SEQ ID NO:1的多肽,但不限于此。在一个实施方案中,其可以是与SEQ ID NO:1的多肽具有约60%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%以上序列同一性的多肽,并且具有与由SEQ ID NO:1的氨基酸序列组成的多肽相同或对应的活性的任何多肽可以包括在aroG醛缩酶的范围内,但不限于此。
本申请的aroG醛缩酶序列可以从NCBI的GenBank获得,这是一个已知的数据库。具体地,它可以是由aroG基因编码的多肽,但不限于此。
本文所用的“变体”是指在通过保守替换和/或修饰使得多肽的功能和性质得以保留之前具有一个以上氨基酸不同于氨基酸序列的多肽。这种变异通常可以通过修饰多肽的一个或多个上述氨基酸序列并评估修饰的多肽的性质来鉴定。也就是说,在修饰之前,变体相对于多肽的能力可以增强、改变或减弱。此外,一些变体可以包括其中一个或多个部分,例如N末端前导序列或跨膜结构域已被去除的变体。其他变体可能包括已从成熟蛋白的N端和/或C末端去除一部分的变体。术语“变体”可以与诸如修饰、修饰、修饰的多肽、修饰的蛋白质、突变和突变等术语互换使用,只要这些术语用于表示变异。
此外,所述变体还可以包括对多肽的性质和二级结构具有最小影响的氨基酸的缺失或添加。例如,参与蛋白质易位的信号(或先导)序列可以在共翻译或翻译后在变体的N末端共翻译或翻译后偶联。此外,这些变体还可以与另一个序列或接头偶联以鉴定、纯化或合成多肽。
本文提供的变体可以是aroG醛缩酶变体,其中对应于SEQ ID NO:1的氨基酸序列中自N末端起第217、310、403和462位的任一个或多个氨基酸被另一种氨基酸取代。例如,在上述位置,4个氨基酸中的2个或更多、3个或更多或全部可以被取代,但不限于此。
对应于SEQ ID NO:1的氨基酸序列中从N末端起第217位的氨基酸可以是精氨酸;对应于SEQ ID NO:1的氨基酸序列中从N末端起第310位的氨基酸可以是赖氨酸;对应于SEQID NO:1的氨基酸序列中从N末端起第403位的氨基酸可以是精氨酸;和/或对应于SEQ IDNO:1的氨基酸序列中从N末端起第462位的氨基酸可以是谷氨酸。
本文提供的变体可以包含选自对应于SEQ ID NO:1的氨基酸序列中自N末端起下列位的氨基酸的一个或多个取代:第217位的氨基酸被精氨酸之外的氨基酸取代;第310位的氨基酸被赖氨酸之外的氨基酸取代;第403位的氨基酸被精氨酸之外的氨基酸取代;以及第462位的氨基酸被谷氨酸之外的氨基酸取代,但不限于此。
术语“另一种氨基酸”不受限制,只要它是不同于取代前氨基酸的氨基酸。同时,当表示“特定氨基酸已被取代”时,即使没有明确说明氨基酸已被不同的氨基酸取代,也很明显该氨基酸已被不同于取代前的氨基酸的氨基酸取代。
在一个实施方案中,本申请的变体可以是其中对应于作为参考蛋白的SEQ ID NO:1的氨基酸序列中的第217、310、403和462位的任一个或多个氨基酸被不同于取代前的氨基酸的疏水性氨基酸或脂族氨基酸取代的变体。
具体地,变体可以是其中对应于SEQ ID NO:1的氨基酸序列中的第217、310、403和462位的任一个或多个氨基酸被疏水性(非极性)氨基酸或脂族氨基酸取代的那些变体。所述脂肪族氨基酸可以是,例如,选自甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸组成的组的氨基酸,但不限于此。所述疏水性(非极性)氨基酸可以是,例如选自甘氨酸、甲硫氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、酪氨酸和色氨酸的组的氨基酸,但不限于此。
在一个实施方案中,本申请的变体可以是其中对应于SEQ ID NO:1的氨基酸序列中的第217、310、403和462位的任一个或多个氨基酸被在小尺寸氨基酸中与取代前的氨基酸不同的氨基酸取代的变体,但不限于此。
本文所用的术语“小尺寸氨基酸”可以包括20种氨基酸中具有相对小尺寸的氨基酸,例如甘氨酸、丙氨酸、丝氨酸、苏氨酸、半胱氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸和天冬酰胺,并且可以特指甘氨酸、丙氨酸、丝氨酸、苏氨酸、半胱氨酸、缬氨酸、亮氨酸、异亮氨酸和脯氨酸,但不限于此。更具体地说,可以指甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸和苏氨酸,但不限于此。
更具体地说,相对于本申请的变体,用另一种氨基酸的取代可以是用丙氨酸取代,但不限于此。
本文所用的术语“对应”是指氨基酸残基在多肽中列出的位置,或与多肽中列出的残基相似、相同或同源的氨基酸残基。识别相应位置的氨基酸可以是确定引用特定序列的序列中的特定氨基酸。如本文所用,术语“对应区域”通常是指在相关蛋白质或参比蛋白质中的相似或相应位置。
例如,任何氨基酸序列与SEQ ID NO:1比对,并且基于该比对,可以参考氨基酸残基的每个氨基酸残基的数值位置对对应于SEQ ID NO:1的氨基酸残基进行编号。例如,诸如本文描述的序列比对算法可以识别氨基酸的位置或位置与查询序列(也称为“参考序列”)相比,发生替换、***或删除等修改的情况。
比对的例子可以通过Needleman-Wunsch算法(Needleman and Wunsch,1970,J.Mol.Biol.48:443-453)来确定,该算法使用EMBOSS包的Needlemman程序(EMBOSS:TheEuropean Molecular Biology Open Software Suite,Rice et alia,2000,TrendsGenet.16:276-277)等进行,但不限于此,以及序列比对程序,可以适当地使用本领域已知的诸如成对序列比较算法等。
在一个实施方案中,本申请的变体可以是这样的变体,其中对应于SEQ ID NO:1的氨基酸序列中的第217、310、403和462位的任一个或多个氨基酸被另一种氨基酸取代,同时与SEQ ID NO:1的多肽具有约60%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%以上的序列同一性。
在一个实施方案中,本申请的变体可以包括与由SEQ ID NO:3、SEQ ID NO:5、SEQID NO:7、SEQ ID NO:9、SEQ ID NO:23或SEQ ID NO:25表示的氨基酸序列具有至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%、99.5%、99.7%或99.9%同源性或同一性的氨基酸序列。
具体地,本申请的变体可以具有下列序列、包括下列序列、由下列序列组成或基本上由下列序列组成:由SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:23或SEQ ID NO:25表示的氨基酸序列。
在一个实施方案中,本申请的变体可以包括氨基酸序列,其中对应于SEQ ID NO:1的氨基酸序列的第217、310、403和462位的任一个或多个氨基酸是丙氨酸,并且与由SEQ IDNO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:23或SEQ ID NO:25表示的氨基酸序列具有至少70%、75%、80%、85%、90%、95%、96%、97%、98%、99%、99.5%、99.7%或99.9%的同源性或同一性。此外,显而易见的是,具有氨基酸序列的变体,其中氨基酸序列的一部分被删除、修饰、取代、保守取代或添加,可以落入本申请的范围内,只要该氨基酸序列具有这样的同源性或同一性,并且显示出与本申请的变体的功效相对应的功效。
例如,可以包括序列添加或删除、自然发生的突变、沉默突变或保守替换,其不会改变本申请的变体在N末端、C末端和/或氨基酸序列内的功能。
本文所用的术语“保守取代”是指用具有相似结构和/或化学性质的另一氨基酸取代一种氨基酸。这种氨基酸取代通常可以基于残基的极性、电荷、溶解度、疏水性、亲水性和/或两亲性性质的相似性而发生。通常,保守替代可能对蛋白质或多肽的活性几乎没有影响。
本文所用的术语“同源性”或“同一性”是指两个给定氨基酸序列或核苷酸序列之间的近似程度,并且可以表示为百分比。术语同源性和同一性通常可以互换使用。
保守多肽或多核苷酸序列的序列同源性或同一性可以通过标准比对算法来确定,并且可以与由所使用的程序建立的默认空位罚分一起使用。基本上,在中等或高度严格的条件下,通常预计同源或相同的序列将与全部或部分序列的整个长度杂交。很明显,在杂交多核苷酸中,还考虑了含有简并密码子而不是密码子的多核苷酸。
任何两个多核苷酸序列是否具有同源性、相似性或同一性,例如可以通过已知的计算机算法例如“FASTA”程序使用默认参数来确定(Pearson et alia,(1988)Proc.Natl.Acad.Sci.USA 85:2444)。或者,也可以通过Needleman-Wunsch算法(Needlemanand Wunsch,1970,J.Mol.Biol.48:443-453)来确定,该算法使用EMBOSS包的Needleman程序执行(EMBOSS:The European Molecular Biology Open Software Suite,Rice etalia,2000,Trends Genet.16:276-277)(版本5.0.0或更高版本)(GCG程序包(Devereux,J.,et alia,Nucleic Acids Research 12:387(1984)),BLASTP,BLASTN,FASTA(Atschul,S.F.,et alia,J MOLEC BIOL 215:403(1990);Guide to Huge Computers,MartinJ.Bishop,ed.,Academic Press,San Diego,1994,and CARILLO et alia(1988)SIAM JApplied Math 48:1073)。例如,可以使用美国国立生物技术信息中心(NCBI)的BLAST或ClustalW来确定同源性,相似性或同一性。
多肽或多核苷酸的同源性、相似性或同一性可以通过比较序列信息来确定,例如,使用例如GAP计算机程序,如Needleman et alia(1970),J Mol Biol.48:443,如Smith andWaterman,Adv.Appl.Math(1981)2:482中公开的。总之,GAP程序将同源性,相似性或同一性定义为通过将相似排列的符号(即核苷酸或氨基酸)的数量除以两个序列中较短的符号总数而获得的值。GAP程序的默认参数可能包括(1)一元比较矩阵(包含1表示身份的值和0表示非身份的值)和Gribskov et alia(1986),Nucl.Acids Res.14:6745,如Schwartz andDayhoff,eds.,Atlas of Protein Sequence and Structure,National BiomedicalResearch Foundation,pp.353-358(1979)披露(或EDNAFULL替换矩阵(NCBI NUC4.4的EMBOSS版本));(2)每个空位罚分3.0,每个空位中每个符号额外罚分0.10(或空位开放罚分10,缺口扩大处罚0.5);(3)对末端空位不进行处罚。
在一个实施方案中,本申请的变体可具有aroG醛缩酶活性。在一个实施方案中,与野生型或非修饰的aroG醛缩酶相比,本申请的变体可能具有能够增加支链氨基酸的生产能力的活性。在一个实施方案中,与野生型或非修饰的aroG醛缩酶相比,本申请的变体可以具有能够降低支链氨基酸生产途径中副产物的生产水平的活性。在一个实施方案中,与野生型或未修饰的aroG醛缩酶相比,本申请的变体可具有减弱的活性,但不限于此。
本申请的另一方面提供了编码本申请的多核苷酸变体。
本文所用的“多核苷酸”,其是由核苷酸单体组成的聚合物,通过共价键连接在长链中,是具有至少一定长度的DNA或RNA链。更具体地说,它可以指编码变体的多核苷酸片段。
编码本申请的变体的多核苷酸序列可以包括编码由SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:23或SEQ ID NO:25表示的氨基酸序列的核苷酸序列。作为本申请的一个示例,本申请的多核苷酸可以具有或包括SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:24或SEQ ID NO:26的序列。此外,本申请的多核苷酸可以由SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:24或SEQID NO:26的序列组成或主要由上述序列组成。
本申请的多核苷酸可以在编码区域内经历各种修饰,其范围内不会改变本申请变体的氨基酸序列,由于密码子简并性或考虑到本申请的变体要表达的生物体中优选的密码子。具体地,本申请的多核苷酸可以具有或包括与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID:24或SEQ ID NO:26的序列具有70%以上、75%以上、80%以上、85%以上、90%以上、95%以上、96%以上、97%以上、98%以上和100%以下同源性或同一性的核苷酸序列,或可由或基本上由与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ IDNO:10、SEQ ID NO:24或SEQ ID NO:26的序列具有70%以上、75%以上、80%以上、85%以上、90%以上、95%以上、96%以上、97%以上、98%以上和100%以下同源性或同一性的核苷酸序列组成,但不限于此。
特别地,在具有同源性或同一性的序列中,编码对应于SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:24或SEQ ID NO:26的第217、310、403和462位的氨基酸的密码子可以是编码丙氨酸的密码子之一。
此外,本申请的多核苷酸可以包括探针,其可以从已知的基因序列制备,例如,任何序列可以与与本申请的全部或部分多核苷酸序列互补的序列杂交,严格无限制。“严格条件”是指允许多核苷酸之间进行特定杂交的条件。这些条件在文献中有具体描述(J.Sambrook et alia,Molecular Cloning,A Laboratory Manual,2nd Edition,ColdSpring Harbor Laboratory press,Cold Spring Harbor,New York,1989;F.M.Ausubelet alia,Current Protocols in Molecular Biology,John Wiley&Sons,Inc.,New York,9.50-9.51,11.7-11.8)。例如,严格条件可以包括具有70%以上、75%以上、80%以上、85%以上、90%以上、95%以上、96%以上、97%以上、98%以上或99%以上的同源性或同一性的高同源性或同一性的基因彼此杂交,并且具有低于上述同源性或同一性的同源性或同源性的基因彼此不杂交,或Southern杂交的洗涤条件,即在对应于60℃,1×SSC,0.1%SDS,具体地60℃,0.1×SSC,0.1%SDS,更具体地说是68℃,0.1×SSC,0.1%SDS的盐浓度和温度下洗涤一次,具体地,洗涤两次或三次。
杂交要求两个核酸含有互补序列,尽管碱基之间的错配取决于杂交的严格程度。术语“互补”用于描述可以相互杂交的核苷酸碱基之间的关系。例如,关于DNA,腺苷与胸腺嘧啶互补,胞嘧啶与鸟嘌呤互补。因此,本申请的多核苷酸可以包括与整个序列互补的分离核苷酸片段以及与其基本相似的核酸序列。
具体地,与本申请的多核苷酸具有同源性或同一性的多核苷酸可以使用杂交条件(包括在上述条件下在Tm值为55℃的杂交步骤)进行检测。此外,Tm值可以是60℃、63℃或65℃,但不限于此,并且可以由本领域技术人员根据其目的适当地进行调整。
杂交多核苷酸的适当严格性取决于多核苷酸的长度和互补程度,并且这些变量是本领域公知的(例如,Sambrook等人)。
本申请的又一方面提供了含有本申请的多核苷酸的载体。所述载体可以是用于在宿主细胞中表达多核苷酸的表达载体,但不限于此。
本文所用的术语“载体”是指含有编码目标多肽的核苷酸序列的DNA构建体可操作地连接到合适的表达调控区(表达调控序列),以便能够在合适的宿主细胞中表达目标多肽。所述表达调控序列可以包括能够启动转录的启动子、用于调节转录的任何操作员序列、编码合适的mRNA核糖体结合位点的序列以及用于调节转录终止和翻译的序列。一旦转化为合适的宿主细胞,载体可以独立于宿主基因组进行复制或功能,或者可以整合到宿主基因组中。
本申请中使用的载体没有特别限制,可以使用本领域已知的任何载体。通常使用的载体的例子可以包括天然或重组质粒、cosmid、病毒和噬菌体。例如,作为噬菌体载体或cosmid载体,可以使用pWE15,M13,MBL3,MBL4,IXII,ASHII,APII,t10,t11,Charon4A和Charon21A等;作为质粒载体,可以使用基于pDC,pBR,pUC,pBluescriptII,pGEM,pTZ,pCL,pET等的质粒载体。具体地,可以使用pDC,pDCM2,pACYC177,pACYC184,pCL,pECCG117,pUC19,pBR322,pMW118和pCC1BAC载体。
在一示例中,编码目标多肽的多核苷酸可以通过载体***染色体中,用于细胞内染色体***。将多核苷酸***染色体可以通过本领域已知的任何方法进行,例如通过同源重组,但不限于此。载体还可以包括选择标记以确认***到染色体中。选择标记用于选择用载体转化的细胞,即用于确认靶核酸分子是否已***,并且可以使用提供可选表型的标记,例如耐药性、营养性、对细胞毒性药物的抵抗力或表面蛋白的表达。只有表达选择标记的细胞才能在用选择性试剂处理的环境中存活或显示不同的表型,因此可以选择转化的细胞。
本文所用的术语“转化”是指将含有编码目标多肽的多核苷酸的载体引入宿主细胞或微生物中,使得由该多核苷酸编码的多肽能够在宿主细胞中表达。只要转化的多核苷酸能在宿主细胞中表达,转化后的多核苷酸是整合到宿主细胞的染色体中并位于其中还是位于染色体外并不重要,两种情况都可以包括在内。此外,所述多核苷酸可包括编码所述目标多肽的DNA和RNA。多核苷酸可以以任何形式引入,只要它可以引入宿主细胞并在其中表达。例如,多核苷酸可以以表达盒的形式引入宿主细胞,表达盒是一种基因构建体,包括其自主表达所需的所有元件。表达盒通常可以包括可操作地与多核苷酸连接的启动子、转录终止子、核糖体结合位点或翻译终止子。所述表达盒可以是可自我复制的表达载体的形式。此外,多核苷酸可以按原样引入宿主细胞中,并且可以操作地与在宿主细胞中表达所需的序列连接,但不限于此。
此外,如本文所用,术语“可操作地连接”是指多核苷酸序列在功能上与启动子序列相连,启动子序列启动和介导编码本申请的目标变体的多核苷酸的转录。
本申请的又一方面提供了棒杆菌属(Corynebacterium)微生物,包括本申请的一个或多个变体、本申请的多核苷酸和本申请的载体。
微生物可以包括本申请的多肽变体、编码多肽的多核苷酸、或含有本申请的多核苷酸的载体。
本文所用的术语“微生物”或“菌株”包括所有野生型微生物,或天然或人工转基因微生物,并且可以是其中特定机制由于***外源基因而减弱或增强的微生物,或者增强或失活的内源性基因的活性,并且可以是包括基因修饰以产生所需多肽的微生物,蛋白质或产品。
本申请的菌株可以是包括本申请的一个或多个变体、本申请的多核苷酸和含有本申请的多核苷酸的载体的菌株;为表达本申请的变体或本申请的多核苷酸而修饰的菌株;表达本申请变体或本申请多核苷酸的菌株(例如重组菌株);或具有本申请的变体活性的菌株(例如,重组菌株),但不限于此。
本申请的菌株可以是具有支链氨基酸生产能力的菌株。
本申请的菌株是具有自然产生aroG醛缩酶或支链氨基酸能力的微生物,或将本申请的变体或编码相同的多核苷酸(或含有多核苷酸的载体)引入到aroG醛缩酶或亲本菌株中具有产生支链氨基酸的能力和/或产生支链氨基酸的能力的微生物生产力已经传授,但不限于此。
在一示例中,本申请的菌株菌株是用含有本申请的多核苷酸或编码本申请变体的多核苷酸的载体转化以表达本申请的变体的细胞或微生物,并且为了本申请的目的,菌株可以包括所有能够产生支链氨基酸的微生物,包括本申请的变体。例如,本申请的菌株可以是重组菌株,其中支链氨基酸的生产能力在表达aroG醛缩酶变体时得到提高,方法是将编码本申请变体的多核苷酸引入天然野生型微生物或用于生产支链氨基酸的微生物中。支链氨基酸生产能力增加的重组菌株可以是与天然野生型微生物或aroG醛缩酶非修饰微生物(即表达野生型aroG醛缩酶的微生物)相比支链氨基酸生产能力增强的微生物,但不限于此。
在一示例中,所述aroG醛缩酶为非修饰微生物,要比较目标菌株以确定支链氨基酸生产能力的增加,可以是谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032菌株。在另一实例中,所述aroG醛缩酶为非修饰微生物,要比较的目标菌株以确定支链氨基酸生产能力的增加,可以是CJ L-8109、KCCM12739P(CA10-3101)或KCCM11201P,但不限于此。
在一示例中,重组菌株可具有与修饰前的亲本菌株或非修饰微生物相比,支链氨基酸的生产能力增加约1%以上,具体而言,约3%或约5%,但不限于此,只要其具有与修饰前的亲本菌株或非修饰微生物的生产能力相比增加+值。
在另一实例中,重组菌株的产生支链氨基酸的产生可减少约50%以下,具体而言,与修饰前的亲本菌株或非修饰微生物相比,可减少约30%以下或约10%以下,但不限于此。
本文所用的术语“约”是指包括所有±0.5、±0.4、±0.3、±0.2、±0.1等的范围,并且它包括紧跟在术语“以上”之后的所有值或那些在类似范围内,但不限于此。
本文所用的术语“支链氨基酸”是指在侧链中具有支链烷基的氨基酸,并且包括缬氨酸、亮氨酸和异亮氨酸。具体地,在本申请中,支链氨基酸可以是L-支链氨基酸,并且L-支链氨基酸可以是选自L-缬氨酸、L-亮氨酸和L-异亮氨酸中的一种以上,但仅限于此。
在本申请中,支链氨基酸生产过程中产生的副产物是指支链氨基酸以外的物质,具体地说,芳族氨基酸,更具体地说,选自L-酪氨酸和L-苯丙氨酸中的一种以上,但不限于此。
如本文所用,术语“非修饰微生物”并不排除含有可能在微生物中自然发生的突变的菌株,并且可以指野生型菌株或天然型菌株本身,或者由于自然或人为因素引起的遗传修饰而改变性状之前的菌株。例如,非改性微生物可以指在本申请的aroG醛缩酶变体中尚未引入的菌株或引入前的菌株。“非修饰微生物”可与“修饰前菌株”、“修饰前微生物”、“非突变菌株”、“非修饰菌株”、“非突变微生物”或“参考微生物”互换使用。
在一个实施方案中,本申请的微生物可以是静止棒杆菌(Corynebacteriumstationis)、粗乳棒杆菌(Corynebacterium crudilactis)、沙漠棒杆菌(Corynebacteriumdeserti)、高效棒杆菌(Corynebacterium efficiens)、美棒杆菌(Corynebacterium callunae)、谷氨酸棒杆菌(Corynebacterium glutamicum)、奇异棒杆菌(Corynebacteriumsingulare)、耐盐棒杆菌(Corynebacterium halotolerans)、纹带棒杆菌(Corynebacterium striatum)、产氨棒杆菌(Corynebacterium ammoniagenes)、污染棒杆菌(Corynebacterium pol lutisoli)、模仿棒杆菌(Corynebacterium imitans)、睾丸棒杆菌(Corynebacterium testudinoris)或微黄棒杆菌(Corynebacterium flavescens)。
本申请的微生物还可以包括修饰以增加支链氨基酸的生产能力。
在一个实施方案中,本申请的微生物可以包括一种以上活性变更的异丙基苹果酸合酶、高丝氨酸脱氢酶、苏氨酸脱水酶、支链氨基酸氨基转移酶和柠檬酸合酶。
在一个实施方案中,本申请的微生物可以是其中一种以上异丙基苹果酸合酶、支链氨基酸氨基转移酶、高丝氨酸脱氢酶和苏氨酸脱水酶的活性进一步增强的微生物。
在一个实施方案中,本申请的微生物可以是其中柠檬酸合酶活性进一步减弱的微生物。
然而,本申请不限于上述描述,并且本领域技术人员可以根据要生产的支链氨基酸适当地选择包含在微生物中的附加修饰。
如本文所用,多肽活性的“增强”意味着多肽的活性与其内源性活性相比增加。增强可以与激活、上调、过表达、增加等术语互换使用。特别是,术语激活、增强、上调、过表达和增加可以包括两种情况,其中表现出最初不具有的活动,或者与内源性活性或修饰前的活动相比活性增强。“内源性活性”是指亲本菌株或非修饰微生物在转化前原本具有的特定多肽的活性,当微生物的性状因自然或人为因素而通过基因改造而改变时,可以与“修饰前活性”互换使用。与其内源性活性相比,多肽活性的“增强”、“上调”、“过表达”或“增加”意味着与转化前最初由亲本菌株或未修饰微生物具有的特定多肽相比,活性和/或浓度(表达水平)得到改善。
增强可以通过引入外来多肽,或者通过增强内源性多肽的活性和/或浓度(表达水平)来实现。多肽活性的增强可以通过多肽活性水平,表达水平或从多肽***的产物量的增加来确认。
多肽活性的增强可以通过本领域公知的各种方法应用,并且不受限制,只要它能够增强目标多肽的活性与修饰前的微生物相比。具体地,可以使用本领域技术人员熟知的基因工程和/或蛋白质工程,这是分子生物学的常用方法,但该方法不限于此(例如,Sitnicka et alia Functional Analysis of Genes.Advances in Cell Biology.2010,Vol.2.1-16,Sambrook et alia Molecular Cloning 2012等等)。
具体地,本申请的多肽的增强可以通过以下途径实现:
(1)增加编码多肽的多核苷酸的细胞内拷贝数;
(2)用具有强活性的序列替换染色体上编码多肽的基因的表达调控区;
(3)修饰编码多肽的基因转录本的起始密码子或编码5'-UTR的核苷酸序列;
(4)修饰多肽的氨基酸序列,使其增强多肽的活性;
(5)修改编码多肽的多核苷酸序列,使得多肽的活性得到增强(例如,修改多肽基因的多核苷酸序列以编码已以增强多肽的活性的方式修饰的多肽);
(6)引入具有所述多肽活性的外源多肽或编码所述多肽的外源多核苷酸;
(7)编码多肽的多核苷酸的密码子优化;
(8)分析多肽的三级结构并由此选择和修饰暴露位点,或对其进行化学修饰;或
(9)选自上述(1)至(8)中的两个或多个的组合,但不限于此。
更具体地,
(1)增加编码多肽的基因的细胞内拷贝数的方法可以通过引入载体来实现,该载体可操作地与编码多肽的多核苷酸连接并且能够复制和发挥作用,而不管宿主细胞如何,进入宿主细胞。或者,该方法可以通过将编码多肽的多核苷酸的一个拷贝或两个拷贝引入宿主细胞的染色体来实现。引入染色体可以通过引入载体来实现,载体能够将多核苷酸***宿主细胞的染色体中,进入宿主细胞,但不限于此。载体与上述相同。
(2)将编码多肽的基因的表达调控区(或表达调控序列)替换为具有强活性的序列的方法可以通过诱导对序列的修饰来实现,通过删除、***、非保守或保守替换,或其组合来进一步增强表达调控区的活性,或者用具有更强活性的序列替换序列。所述表达调控区可包括,但不特别限于,启动子、操纵子序列、编码核糖体结合位点的序列、以及调节转录和翻译终止的序列。在一示例中,该方法可包括用强启动子替换原始启动子,但不限于此。
强启动子的实例可以包括cj1至cj7启动子(US 7662943 B2)、lac启动子、trp启动子、trc启动子、tac启动子、λ噬菌体PR启动子、PL启动子、tet启动子、gapA启动子、SPL7启动子、SPL13(sm3)启动子(US 10584338 B2)、O2启动子(US 10273491 B2)、tkt启动子、yccA启动子等,但不限于此。
(3)修饰编码起始密码子的核苷酸序列或编码多肽的基因转录本的5'-UTR的方法可以实现,例如,通过用编码另一个起始密码子的核苷酸序列代替核苷酸序列,与内源性起始密码子相比,该多肽具有更高的表达率,但不限于此。
所述(4)和(5)修饰氨基酸序列或多核苷酸序列的方法可以通过诱导对序列的修饰来实现,通过删除、***、非保守或保守地取代所述多肽的氨基酸序列或编码所述多肽的多核苷酸序列,或其组合来进一步增强所述多肽的活性,或者通过用修饰的氨基酸序列或多核苷酸序列替换序列以具有更强的活性,或者用氨基酸序列或多核苷酸序列修饰以增强活性。替换可以通过同源重组将多核苷酸***染色体来具体地进行,但不限于此。本文中使用的载体还可以包括选择标记以确认***到染色体中。选择标记与上述相同。
(6)引入具有多肽活性的外来多核苷酸的方法可以通过向宿主细胞中引入编码多肽的外源多核苷酸来实现,该多肽表现出与多肽相同或相似的活性。无论其来源或序列如何,只要外源多核苷酸表现出与多肽相同或相似的活性,都可以不受限制地使用。所述引入可以通过选择本领域已知的转化方法由本领域普通技术人员之一进行,并且引入的多核苷酸在宿主细胞中的表达能够产生多肽,从而增加其活性。
(7)编码多肽的密码子优化方法可以通过对内源性多核苷酸进行密码子优化以增加宿主细胞内的转录或翻译来实现,或者通过优化其密码子使得外来多核苷酸的优化转录和翻译可以在宿主细胞内实现。
(8)分析多肽的三级结构并由此选择和修饰暴露位点的方法,或者对该方法进行化学修饰可以实现,例如,通过将要分析的多肽的序列信息与数据库进行比较,其中存储了已知蛋白质的序列信息,根据序列相似程度确定候选蛋白模板,从而基于信息确认结构,从而选择和改造或修改要修改或化学修饰的暴露部位。
这种多肽活性的增强可能意味着多肽的活性或浓度(表达水平)相对于在改性前在野生型或微生物菌株中表达的多肽的活性或浓度增加,或者说由多肽产生的产物的量增加,但不限于此。
本申请的微生物中部分或全部多核苷酸的修饰可以通过(a)使用用于染色体***微生物的载体或使用工程核酸酶(例如CRISPR-Cas9)进行基因组编辑和/或(b)可以由光诱导,例如紫外线和辐射,等,和/或化学处理来实现,但不限于此。修饰部分或全部基因的方法可以包括使用DNA重组技术的方法。例如,可以通过将核苷酸序列或含有与靶基因同源的核苷酸序列的载体注射到微生物中来删除部分或全部基因以诱导同源重组。注射的核苷酸序列或载体可以包括显性选择标记,但不限于此。
如本文所用,术语“减弱”多肽活性是一个综合概念,包括与其内源性活性相比活性降低或无活性。减弱可以与失活、缺乏、下调、减少、减少、衰减等术语互换使用。
弱化还可以包括由于编码多肽的多核苷酸的突变而导致多肽本身的活性与微生物最初具有的多肽的活性相比降低或去除的情况;与天然菌株相比,由于编码多肽的多核苷酸基因表达受到抑制或多肽翻译受到抑制等,细胞内多肽活性和/或浓度(表达水平)的总体水平降低;多核苷酸根本不表达的情况;和/或即使多核苷酸表达也没有观察到多肽活性的情况。如本文所用,术语“内源性活性”是指当性状由于自然或人为因素引起的遗传修饰而改变时,最初由亲本菌株、野生型或非修饰的微生物在转化前具有的特定多肽的活性,并且可以与“修饰前的活性”互换使用。表达“与其内源性活性相比,多肽活性”失活、缺乏、降低、下调、降低或减弱“意味着多肽活性与转化前最初由亲本菌株或未修饰的微生物具有的特定多肽的活性相比降低。
多肽活性的减弱可以通过本领域已知的任何方法进行,但该方法不限于此,并且可以通过应用本领域公知的各种方法(例如,Nakashima N et alia,Bacterial cellularengineering by genome editing and gene silencing.Int J Mol Sci.2014;15(2):2773-2793,Sambrook et alia Molecular Cloning 2012等)。
具体地,本申请的多肽的弱化可以通过以下方法实现:
(1)删除编码多肽的部分或全部基因;
(2)修饰表达调控区(表达调控序列),使得编码多肽的基因的表达降低;
(3)修饰构成多肽的氨基酸序列,使得多肽活性被去除或削弱(例如,在氨基酸序列上删除/取代/添加一个或多个氨基酸)。
(4)修改编码多肽的基因序列,使得多肽的活性被移除或削弱(例如,在多肽基因的核苷酸序列上删除/取代/添加一个或多个核苷酸以编码已被修饰以去除或削弱多肽的活性;
(5)修饰编码多肽的基因转录本的起始密码子或编码5'-UTR的核苷酸序列;
(6)引入反义寡核苷酸(例如反义RNA),其与编码多肽的基因的转录本互补结合;
(7)在编码多肽的SD序列的前端加入与Shine-Dalgarno(SD)序列互补的序列,形成二级结构,从而抑制核糖体附着;
(8)逆转录工程(RTE),其在编码多肽的基因序列的开放阅读框(ORF)的3'末端添加启动子,其被反向转录;或
(9)选自上述方法(1)至(8)中的两种或两种以上的组合,但不限于此。
例如,
(1)删除编码多肽的部分或全部基因的方法可以通过删除染色体内编码内源性靶多肽的所有多核苷酸来实现,或者通过用具有部分删除的核酸序列的多核苷酸或标记基因替换多核苷酸。
此外,(2)修饰表达调控区(表达调控序列)的方法可以通过诱导对表达调控区(表达调控序列)的修饰来实现,通过删除、***、非保守取代或保守取代,或其组合;或者用活性较弱的序列替换序列。所述表达调控区可以包括启动子、操纵子序列、编码核糖体结合位点的序列、以及用于调节转录和翻译的序列,但不限于此。
此外,(5)修饰编码起始密码子的核苷酸序列或编码多肽的基因转录本的5'-UTR的方法可以实现,例如,通过用编码另一个起始密码子的核苷酸序列替换核苷酸序列,其多肽表达率低于内源性起始密码子,但不限于此。
此外,修饰氨基酸序列或多核苷酸序列的(3)和(4)方法可以通过对多肽的氨基酸序列或编码多肽的多核苷酸序列进行缺失、***、非保守或保守取代来诱导对序列的修饰来实现,或其组合以进一步削弱多肽的活性,或通过用被修饰为具有较弱活性的氨基酸序列或多核苷酸序列、或被修饰为不具有活性的氨基酸顺序或多核苷酸顺序取代该序列。例如,可以通过将突变引入多核苷酸序列以形成终止密码子来抑制或削弱基因的表达,但不限于此。
(6)引入反义寡核苷酸(例如反义RNA)的方法,其与编码多肽的基因的转录本互补结合可以在文献中找到[Weintraub,H.et alia,Antisense-RNA as amolecular tool forgenetic analysis,Reviews-Trends in Genetics,Vol.1(1)1986]。
(7)在编码多肽的SD序列的前端添加与Shine-Dalgarno(SD)序列互补的序列以形成二级结构的方法,从而抑制核糖体的附着可以通过抑制mRNA翻译或降低其翻译速度来实现。
(8)逆转录工程(RTE),其添加启动子,其被反向转录,在编码多肽的基因序列的开放阅读框(ORF)的3'末端,可以通过形成与编码多肽的基因转录本互补的反义核苷酸来实现削弱活性。
在本申请的微生物中,变异体、多核苷酸、载体和支链氨基酸与其它方面描述的相同。
甚至本申请的另一方面提供了生产支链氨基酸的方法,包括在培养基中培养棒杆菌属(Corynebacterium)微生物。
本文所用的术语“培养”是指本申请的棒杆菌属(Corynebacterium)微生物在适当控制的环境条件下生长。本申请的培养过程可以在合适的培养基和本领域已知的培养条件下进行。这样的培养方法可以容易地根据所选择的菌株调整供本领域技术人员使用。具体而言,培养可以是分批培养、连续培养和/或补料分批培养,但不限于此。
本文所用的术语“培养基”是指其含有培养本申请的棒杆菌属(Corynebacterium)微生物所需的营养物质作为主要成分的物质的混合物,并且它提供营养物质和生长因子,以及对于生存和生长至关重要的水。具体地,本申请中用于培养棒杆菌属(Corynebacterium)微生物的培养基和其它培养条件可以是用于常规培养微生物的任何培养基,而没有任何特别限制。然而,本申请的棒杆菌属(Corynebacterium)微生物可以在含有适当碳源、氮源、磷源、无机化合物、氨基酸和/或维生素的常规培养基中在有氧条件下培养,同时调节温度、pH等。
具体地,用于棒杆菌属(Corynebacterium)微生物的培养基可以在文献中找到[“Manual of Methods for General Bacteriology”by the American Society forBacteriology(Washington D.C.,USA,1981)]。
在本申请中,碳源可以包括碳水化合物,例如葡萄糖、蔗糖、乳糖、果糖、蔗糖、麦芽糖等;糖醇,如甘露醇、山梨糖醇等;有机酸,如丙酮酸、乳酸、柠檬酸等;氨基酸,如谷氨酸、甲硫氨酸、赖氨酸等。此外,碳源可包括天然有机营养物质,例如淀粉水解物、糖蜜、黑糖蜜、米糠、木薯、甘蔗糖蜜、玉米浸泡液等。具体地,可以使用碳水化合物如葡萄糖和灭菌的预处理糖蜜(即糖蜜转化为还原糖),此外,可以不受限制地使用适量的各种其它碳源。这些碳源可以单独使用,也可以以两种以上的组合使用,但不限于此。
所述氮源可包括无机氮源,如氨、硫酸铵、氯化铵、乙酸铵、磷酸铵、碳酸铵、硝酸铵等;氨基酸,如谷氨酸、甲硫氨酸、谷氨酰胺等;和有机氮源,如蛋白胨、NZ-胺、肉类提取物、酵母提取物、麦芽提取物、玉米浸泡液、酪蛋白水解物、鱼或其分解产物、脱脂豆饼或其分解产物等。这些氮源可以单独使用,也可以以两种以上的组合使用,但不限于此。
磷源可以包括磷酸一钾、磷酸二钾或相应的含钠盐等。无机化合物的实例可包括氯化钠、氯化钙、氯化铁、硫酸镁、硫酸铁、硫酸锰、碳酸钙等。此外,可能包括氨基酸、维生素和/或适当的前体。这些构成成分或前体可以分批或连续的方式添加到培养基中,但这些磷源不限于此。
培养基的pH值可以通过以适当的方式向培养基中加入诸如氢氧化铵、氢氧化钾、氨、磷酸、硫酸等的棒杆菌属(Corynebacterium)微生物来调节。此外,在培养过程中,可以添加消泡剂,例如脂肪酸聚乙二醇酯以防止泡沫产生。此外,可以将氧气或含氧气体注入介质中以维持介质的有氧状态;可以注入氮气、氢气或二氧化碳气体而不注入气体以维持介质的厌氧或微好氧状态,但气体不限于此。
本申请中的培养基温度可以在20℃至45℃的范围内,具体地说,在25℃至40℃的范围内,并且培养可以进行约10至160小时,但不限于此。
本申请培养产生的支链氨基酸可以释放到培养基中或保留在细胞中。
本申请的支链氨基酸的制备方法还可以包括制备本申请的棒杆菌属(Corynebacterium)微生物的步骤、制备用于培养菌株的培养基的步骤,或其组合(无论顺序如何,以任何顺序),例如在培养步骤之前。
本申请的支链氨基酸的制备方法还可以包括从本申请的棒杆菌属(Corynebacterium)的培养基(培养基)或微生物中回收支链氨基酸的步骤。回收步骤可以进一步包括在培养步骤之后。
在回收步骤中,可以使用本申请的微生物培养方法收集所需的支链氨基酸,例如,使用本领域已知的分批培养、连续培养或补料分批培养方法的合适方法。例如,可以使用离心、过滤、用蛋白质结晶沉淀剂处理(盐析法)、萃取、超声破碎、超滤、透析等方法,可以使用各种色谱法,例如分子筛色谱(凝胶过滤)、吸附色谱、离子交换色谱、亲和色谱等,可以使用HPLC或其组合,并且所需的支链氨基酸可以从培养基或微生物采用本领域已知的合适方法。
此外,本申请的支链氨基酸的制备方法还可以包括纯化过程,其可以使用本领域已知的适当方法进行。在一示例中,当本申请的支链氨基酸的制备方法包括回收步骤和纯化步骤两者时,该回收步骤和纯化步骤可以间歇地(或连续地)进行,而不管顺序如何或同时进行,或者可以整合到一个步骤中,但该方法不限于此。
在本申请的方法中,变体、多核苷酸、载体和微生物与其它方面描述相同。
本申请的又一方面提供了用于生产支链氨基酸的组合物,包括本申请的变体、编码该变体的多核苷酸、含有多核苷酸的载体或棒杆菌属(Corynebacterium)微生物,包括本申请的多核苷酸;含有相同物质的介质;或其中两种或两种以上的组合。
本申请的组合物还可以包括组合物中常用于生产支链氨基酸的任何合适的赋形剂,并且这种赋形剂可以是例如防腐剂、润湿剂、分散剂、悬浮剂、缓冲剂、稳定剂或等渗剂,但不限于此。
在本申请的组合物中,变体、多核苷酸、载体、菌株、培养基和支链氨基酸与其它方面描述的相同。
本申请的又一方面提供了利用本申请的aroG醛缩酶变体生产支链氨基酸的用途。
本申请的又一方面提供了利用包括本申请的一个或多个aroG醛缩酶变体;编码aroG醛缩酶变体的多核苷酸;和含有多核苷酸的载体中的一种以上的微生物生产支链氨基酸的用途。
在本申请的使用中,变体、多核苷酸、载体、微生物等与其它方面描述的相同。
【实施本发明的方式】
下面通过实施例对本申请进行详细描述。然而,对于本领域技术人员来说显而易见的是,给出这些实施例仅用于说明目的,并不旨在限制本发明的范围。
【实施例1:aroG醛缩酶突变的发现】
【实施例1-1:含aroG醛缩酶的载体的制备】
为了制备具有磷酸-2-脱氢-3-脱氧庚酸醛缩酶活性的aroG醛缩酶突变体文库,首先制备了含有aroG醛缩酶的重组载体。为了扩增编码源自野生型谷氨酸棒杆菌(Corynebacterium glutamicum)的aroG醛缩酶(SEQ ID NO:1,KEGG ID:NCgl2098)的aroG基因(SEQ ID NO:2),使用SEQ ID NO:11和12的引物基于野生型谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC13032的染色体作为模板进行PCR,在以下条件下:在94℃下变性1分钟,在58℃下退火30秒,以及使用Pfu DNA聚合酶在72℃下聚合1分钟的25个循环。所用引物的具体序列如表1所示。使用TOPO克隆试剂盒(Invitrogen)将扩增产物克隆到大肠杆菌(E.coli)载体pCR2.1中以获得“pCR-aroG”。
【表1】
SEQ ID NO: | 序列(5'→3') |
SEQ ID NO:11 | TGATGCGCGT CATAATTTAG |
SEQ ID NO:12 | CTCATCTCTG ACTGGACGTG |
【实施例1-2:aroG醛缩酶突变文库的制备】
基于实施例1-1中制备的载体,使用易于出错的PCR试剂盒(clontechPCR随机突变试剂盒)制备aroG醛缩酶突变文库。使用SEQ ID NO:11和SEQ ID NO:12作为引物在每1000bp可能发生0至3个突变的条件下进行PCR反应。具体而言,在94℃预热30秒后,通过在94℃下重复变性30秒和在68℃下退火1分30秒的25个循环来进行PCR。使用由此获得的PCR产物作为大引物(50至125ng),通过在95℃下重复变性50秒、在60℃下退火50秒和在68℃下聚合12分钟的25个循环进行PCR,并用DpnI处理所得产物,通过热冲击法转化大肠杆菌(E.coli)DH5α,并将其接种到含有卡那霉素(25mg/L)的LB固体培养基上。选择20种这样转化的菌落获得质粒,并分析核苷酸序列。结果,证实了以2个突变/kb的频率在不同位置引入突变。收集了大约20,000个转化的大肠杆菌(E.coli)菌落并提取了质粒,这些质粒被命名为“pTOPO-aroG文库”。
【实施例2:制备文库的评估和变体的选择】
【实施例2-1:选择具有增加L-亮氨酸产生能力的突变菌株】
将实施例1-2中制备的pTOPO-aroG文库通过电穿孔转化到野生型谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032中,然后将其接种到含有25mg/L卡那霉素的营养培养基(表2)上,以选择10,000个***突变基因的菌株菌落。每个选定的菌落被命名为ATCC13032/pTOPO_aroG(mt)1到ATCC13032/pTOPO_aroG(mt)10,000。
以下列方式评估每个菌落的发酵效价,以便在获得的10,000个菌落中鉴定其中L-亮氨酸产量增加、且芳族氨基酸中的L-酪氨酸和L-苯丙氨酸减少的菌落。
【表2】
将每个菌落用铂环接种到含有25μg/mL卡那霉素的25mL高压灭菌生产培养基(表2)中的250mL角挡板烧瓶中,然后在30℃下以200rpm振荡培养60小时。培养完成后,通过高效液相色谱法(HPLC,SHIMAZDU LC20A)测定L-亮氨酸的产量,以及芳族氨基酸中的L-酪氨酸和L-苯丙氨酸。
在由此获得的10,000个菌落中,选择了与野生型谷氨酸棒杆菌(Corynebacteriumglutamicum)菌株(ATCC13032)相比具有最改善的L-亮氨酸生产能力的4个菌株,它们是:ATCC13032/pTOPO_aroG(mt)2256、ATCC13032/pTOPO_aroG(mt)6531、ATCC13032/pTOPO_aroG(mt)8316和ATCC13032/pTOPO_aroG(mt)9426。所选菌株中产生的L-亮氨酸(Leu)、L-酪氨酸(Tyr)和L-苯丙氨酸(Phe)的浓度如下表3所示。
【表3】
菌株名称 | Leu(g/L) | Tyr(g/L) | Phe(g/L) |
ATCC13032 | 0.87 | 1.28 | 1.87 |
ATCC13032/ptopO_aroG(mt)2256 | 1.25 | 0.43 | 0.57 |
ATCC13032/ptopO_aroG(mt)6531 | 1.23 | 0.15 | 0.19 |
ATCC13032/ptopO_aroG(mt)8316 | 1.31 | 0.54 | 0.63 |
ATCC13032/ptopO_aroG(mt)9426 | 1.27 | 0.31 | 0.34 |
如以上表3所示,证实了在aroG基因中具有突变的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032/pTOPO_aroG(mt)2256与亲本菌株谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032相比显示出增加了约1.4倍的L-亮氨酸产生能力。此外,经证实,ATCC13032/pTOPO_aroG(mt)6531、ATCC13032/pTOPO_aroG(mt)8316、ATCC13032/pTOPO_aroG(mt)9426与亲本菌株相比,L-亮氨酸生成能力提高了约1.4倍。此外,确认ATCC13032/pTOPO_aroG(mt)2256、ATCC13032/pTOPO_aroG(mt)6531、ATCC13032/pTOPO_aroG(mt)8316、ATCC13032/pTOPO_aroG(mt)9426的所有4种菌株均显示L-酪氨酸产量降低了2.4至8.5倍,L-苯丙氨酸产量减少了3至10倍。
【实施例2-2:确认L-亮氨酸产量增加,芳族氨基酸产量减少的突变菌株的突变】
为了确认所选择的4个突变菌株的aroG基因突变,使用表1中列出的SEQ ID NO:11和SEQ ID NO:12的引物,基于每个突变菌株的DNA,在94℃变性5分钟,然后在94℃变性30秒,在55℃退火30秒,在72℃下聚合1分30秒的30个循环,然后在72℃聚合5分钟的条件下进行PCR,然后进行DNA测序。
作为测序的结果,确认在ATCC13032/pTOPO_aroG(mt)2256菌株中,aroG基因的第649、650和651个核苷酸,即CGC被GCG取代。这表明可以编码一种变体(以下简称R217A),其中精氨酸(aroG醛缩酶的第217个氨基酸)被丙氨酸取代。aroG醛缩酶突变体(R217A)的氨基酸序列和编码所述氨基酸序列的aroG醛缩酶突变体的核苷酸序列如SEQ ID NO:3和4表示。
此外,在ATCC13032/pTOPO_aroG(mt)6531菌株中,证实作为aroG基因的第928和第929个核苷酸的AA被GC取代。这表明可以编码用丙氨酸取代赖氨酸(即aroG醛缩酶的第310个氨基酸)的变体(以下简称K310A)。aroG醛缩酶突变体(K310A)的氨基酸序列和编码所述氨基酸序列的aroG醛缩酶突变体的核苷酸序列如SEQ ID NO:5和6表示。
在ATCC13032/pTOPO_aroG(mt)8316菌株中,确认CGC(即aroG基因的第1207至1209个核苷酸)被GCG取代。这表明可以编码用丙氨酸取代精氨酸(即aroG醛缩酶的第403个氨基酸)的变体(以下简称R403A)。aroG醛缩酶突变体(R403A)的氨基酸序列和编码所述氨基酸序列的aroG醛缩酶突变体的核苷酸序列由SEQ ID NO:7和8表示。
在ATCC13032/pTOPO_aroG(mt)9426菌株中,证实作为DAHP基因的第1385个和第1386个核苷酸的AA被CG取代。这表明可以编码谷氨酸(即aroG醛缩酶的第462个氨基酸)被丙氨酸取代的突变体(以下简称E462A)。aroG醛缩酶突变体(E462A)的氨基酸序列和编码所述氨基酸序列的aroG醛缩酶突变体的核苷酸序列由SEQ ID NO:9和10表示。
因此,在以下实施例中,进行了实验以确定突变(R217A、K310A、R403A、E462A)是否影响棒杆菌属(Corynebacterium)微生物中L-亮氨酸和芳族氨基酸的产生。
【实施例3:确认L-亮氨酸、L-酪氨酸和L-苯丙氨酸产生选定突变菌株的能力】
【实施例3-1:制备含有aroG醛缩酶突变的***载体】
为了将实施例2中选择的突变引入菌株中,制备***载体。使用定点诱变制备引入aroG(R217A,K310A,R403A,E462A)突变的载体。以野生型谷氨酸棒杆菌(Corynebacteriumglutamicum)ATCC13032的染色体为模板,使用SEQ ID NO:13和14以及SEQ ID NO:15和16的引物对进行PCR以产生R217A突变,并使用SEQ ID NO:13和17以及SEQ ID NO:15至18的引物对产生K310A突变。此外,使用SEQ ID NO:13和19以及SEQ ID NO:15和20的引物对进行PCR以产生R403A突变,并且使用SEQ ID NO:13和21以及SEQ ID NOS 15和22的引物对来产生E462A突变。具体而言,在94℃变性5分钟,然后在94℃下变性30秒,在55℃下退火30秒,并在72℃下聚合1分30秒的30个循环,然后在72℃聚合5分钟的条件下进行PCR。所用引物的具体序列如表4所示。
【表4】
SEQ ID NO: | 序列(5'→3') |
SEQ ID NO:13 | GTGAATTCGA GCTCGGTACC CAGTTGAATG CTACCAACTTG |
SEQ ID NO:14 | CACGAGCAAG AGCCTCGTAc gcTGCACCAG CTGGG |
SEQ ID NO:15 | GGTCGACTCT AGAGGATCCC CGCTGTTATT ACTGTGCCTG |
SEQ ID NO:16 | GAACTCCCCA GCTGGTGCAg cgTACGAGGC TCTTGCTCG |
SEQ ID NO:17 | GGGTGATACC AGGACCAATc gcGATGCCGA TTGGGTTAG |
SEQ ID NO:18 | CTCTAACCCA ATCGGCATCg cgATTGGTCC TGGTATCAC |
SEQ ID NO:19 | CTGGGTGGGT GCCCAATGCc gcGTGGACCT CGAAGAAG |
SEQ ID NO:20 | GGGCTTCTTC GAGGTCCACg cgGCATTGGG CACCCAC |
SEQ ID NO:21 | AAGCTTAGTT ACGCAGCATc gcTGCAACGA GGAAAGCC |
SEQ ID NO:22 | GTTGGCTTTC CTCGTTGCAg cgATGCTGCG TAACTAAGC |
利用将所得PCR产物用SmaI限制性内切酶切断的线性pDCM2载体(韩国专利公开KR10-2020-0136813A)和In-Fusion酶融合DNA片段之间的末端15个碱基的相同序列而克隆而制备了取代aroG的氨基酸的载体,即'pDCM2-aroG(R217A)'、'pDCM2-aroG(K310A)'、'pDCM2-aroG(R403A)',和“pDCM2-aroG(E462A)”。此外,通过变体的组合制备了用于取代aroG的217、310、403和462位氨基酸的载体,'pDCM2-aroG(R217A、K310A、E462A)'和'pDCM2aroG(R217-A、K310A、R403A、E462B)'。aroG醛缩酶变体(R217A、K310A、E462A)的氨基酸序列和编码其的aroG醛缩酶变体的核苷酸序列由SEQ ID NO:23和24表示。此外,aroG醛缩酶变体(R217A、K310A、R403A、E462A)的氨基酸序列和编码其的aroG醛缩酶变体的核苷酸序列由SEQ ID NO:25和26表示。
【实施例3-2:ATCC13032菌株的变体引入及其评估】
通过电穿孔将实施例3-1中制备的pDCM2-aroG(R217A),pDCM2-aroG(K310A),pDCM2-aroG(R403A),pDCM2-aroG(E462A),pDCM2-aroG(R217A,K310A,E462A)和pDCM2-aroG(R217A,K310A,R403A,E462A)载体转化进ATCC13032,在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对选定的一代菌株进行次级交叉,并选择引入靶基因突变的菌株。通过使用表1中列出的SEQ ID NO:11和SEQ ID NO:12的引物进行PCR,然后分析核苷酸序列,确认将aroG基因突变引入最终转化的菌株中。共生产了5个菌株,分别命名为ATCC13032_aroG_R217A,ATCC13032_aroG_K310A,ATCC13032_aroG_R403A,ATCC13032_aroG_E462A,ATCC13032_aroG_(R217A,K310A,E462A)和ATCC13032_aroG_(R217A,K310A,R403A,E462A)。
对烧瓶发酵效价进行评估,以评价上述制备的6种菌株中L-亮氨酸和芳族氨基酸的产生能力。将上述制备的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032和ATCC13032_aroG_R217A、ATCC13032_aroG_K310A、ATCC13032_aroG_R403A、ATCC13032_aroG_E462A、ATCC13032_aroG_(R217A、K310A、E462A)和ATCC13032_aroG_(R217A、K310A、R403A、E462A)各一个铂环接种到含有25mL生产培养基的250mL角挡烧瓶中,然后在30℃下以200rpm振荡培养60小时以产生L-亮氨酸。培养完成后,用HPLC测定L-亮氨酸,L-酪氨酸和L-苯丙氨酸的产生。每种测试菌株的培养基中的亮氨酸浓度如下表5所示。
【表5】
如上表5所示,证实ATCC13032_aroG_R217A、ATCC13032_aroG_K310A、ATCC13032_aroG_R403A、ATCC13032_aroG_E462A、ATCC13032_aroG_(R217A、K310A、E462A)和ATCC13032_aroG_(R217A、K310A、R403A、E462A)显示L-亮氨酸的产率比亲本菌株谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032提高约1.35倍至1.55倍。此外,证实L-酪氨酸的收率降低了2.5至8.5倍,L-苯丙氨酸的收率降低了约3.5至14倍。
【实施例4:亮氨酸产生菌株中aroG选择性突变产生亮氨酸、酪氨酸和苯丙氨酸能力的确认】
即使棒杆菌属(Corynebacterium)的野生型菌株能够产生亮氨酸,也只能产生非常微量的亮氨酸。因此,进行了一项实验以制备源自ATCC13032的亮氨酸产生菌株,并引入选定的突变以确认产生亮氨酸,酪氨酸和苯丙氨酸的能力。具体实验如下进行。
【实施例4-1:L-亮氨酸产生菌株CJ L-8109的制备】
作为生产高浓度L-亮氨酸的菌株,制备来自ATCC13032的菌株包含:(1)突变(R558H),其中leuA基因的1673个核苷酸G被A取代,并且leuA蛋白的第558个氨基酸精氨酸被组氨酸取代;(2)突变(G561D),其中leuA基因的1682和1683个核苷酸GC被AT取代,第561个氨基酸甘氨酸被天冬氨酸取代;和(3)突变(P247C),其中leuA基因的第739和740个核苷酸CC被TG取代,并且第247个氨基酸脯氨酸被半胱氨酸取代。
具体地,将含有leuA基因突变的载体pDCM2-leuA(P247C,R558H,G561D)通过电穿孔转化谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032,并且在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对所选择的初级菌株进行二次交叉,并选择引入leuA基因突变的菌株。通过使用表6的SEQ ID NO:27和SEQ IDNO:28的引物,在94℃变性5分钟,然后在94℃下变性30秒,在55℃下退火30秒,以及在72℃下聚合1分30秒的30个循环,然后在72℃下聚合5分钟的条件下进行PCR,确认将P247C,R558H,G561D突变引入最终转化的菌株中,并分析核苷酸序列。将用载体pDCM2-leuA_(P247C,R558H,G561D)转化的ATCC13032_leuA_(P247C,R558H,G561D)株命名为“CJ L-8105”。
【表6】
SEQ ID NO: | 序列(5'→3') |
27 | TATGCTTCAC CACATGACTT C |
28 | AAATCATTTG AGAAAACTCG AGG |
为了提高这样制备的CJ L-8105菌株中L-亮氨酸的产生能力,制备了其中编码支链氨基酸氨基转移酶的基因ilvE突变体(V156A)的菌株(韩国专利第10-2143964号)。具体而言,通过电穿孔将含有ilvE基因突变的pDCM2-ilvE(V156A)载体转化到谷氨酸棒杆菌(Corynebacterium glutamicum)CJL-8105中,并在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对所选择的原代菌株进行二次杂交,并选择引入ilvE基因突变的菌株。通过使用表7的SEQ ID NO:29和SEQ ID NO:30的引物,在94℃变性5分钟,然后在94℃下变性30秒、在55℃下退火30秒和在72℃下聚合1分30秒的30个循环,然后在72℃下聚合5分钟的条件下进行PCR,确认将突变引入最终转化的菌株中,并分析核苷酸序列,从而确认V156A突变的引入。用pDCM2 ilvE(V156A)载体转化的菌株命名为“CJL-8108”。/p>
【表7】
SEQ ID NO: | 序列(5'→3') |
29 | GTCACCCGAT CGTCTGAAG |
30 | GTCTTAAAAC CGGTTGAT |
为了在这样制备的CJL-8108菌株中增加L-亮氨酸的产生能力,制作了引入了具有减弱的柠檬酸合酶活性的gltA突变体(M312I;SEQ ID NO:41)的菌株。具体而言,通过电穿孔将含有gltA基因突变的pDCM2-gltA(M312I)载体转化到谷氨酸棒杆菌(Corynebacteriumglutamicum)CJ L-8108中,并在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对所选择的初级菌株进行二次交叉,并选择引入gltA基因突变的菌株。通过使用表8的SEQ ID NO:31和SEQ ID NO:30的引物,在94℃变性5分钟,然后在94℃下变性30秒,在55℃下退火30秒,以及在72℃下聚合1分30秒的30个循环,然后在72℃下聚合5分钟的条件下进行PCR,确认将突变引入最终转化的菌株中,并分析核苷酸序列,从而确认M312I突变的引入。用pDCM2-gltA(M312I)载体转化的菌株命名为“CJ L-8109”。
【表8】
SEQ ID NO: | 序列(5'→3') |
31 | CAATGCTGGC TGCGTACGC |
32 | CTCCTCGCGA GGAACCAACT |
【实施例4-2:CJL-8109菌株中aroG醛缩酶变体的引入及其评价】
用在实施例3-1中制备的载体pDCM2-aroG(R217A),pDCM2-aroG(K310A),pDCM2-aroG(R403A),pDCM2-aroG(E462A),pDCM2-aroG(R217A,K310A,E462A),pDCM2-aroG(R217A,K310A,R403A,E462A)载体转化亮氨酸产生菌株CJ L-8109,在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对选定的一代菌株进行次级交叉,并选择引入靶基因突变的菌株。通过使用SEQ ID NO:11和SEQ ID NO:12的引物进行PCR并分析核苷酸序列来确认aroG基因突变引入最终转化的菌株中,从而确认aroG醛缩酶突变引入菌株中。制备的CJL-8109_aroG_R217A命名为CJ L-8110,CJ L-8109_aroG_K310A命名为CJ L-8111,CJL-8109_aroG_R403A命名为CJ L-8112,CJ L-8109_aroG_E462A命名为CJL-8113,CJL-8109_aroG_(R217A,K310A,E462A)命名为CA13-8114,CJL-8109_aroG_(R217A,K310A,R403A,E462A)命名为CJ L-8115。
评价了这样制备的CJL-8110、CJL-8111、CJL-8112、CJ L-8113、CA13-8114、CJL-8115和ATCC13032、CJ L-8109菌株的产亮氨酸能力。烧瓶培养以与实施例2相同的方法进行,培养完成后用HPLC测定亮氨酸和芳族氨基酸的产生,培养结果如下表9所示。
【表9】
如表9所示,产生L-亮氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)CJL-8110、CJ L-8111、CJ L-8112、CJL-8113、CA13-8114和CJ L-8115的菌株在aroG基因中具有额外的R217A、K310A、R403A和E462A突变,与亲本菌株谷氨酸棒杆菌(Corynebacteriumglutamicum)ATCC13032相比,亮氨酸产量增加了约4至5倍。此外,生产L-亮氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)CJL-8110、CJ L-8111、CJ L-813、CA13-8114和CJ L-8115菌株与亲本菌株谷氨酸棒杆菌(CJ L-8109)相比,L-亮氨酸生产能力提高了约1.2至1.6倍,L-酪氨酸生产能力和L-苯丙氨酸生产能力降低了约10至20倍。从以上结果可以证实,aroG醛缩酶氨基酸序列中217、310、403和462位的氨基酸是对于产生L-亮氨酸和L-酪氨酸和L-苯丙氨酸(芳族氨基酸)而言重要的位置。
由此制备的菌株CA13-8114于2021年1月18日根据布达佩斯条约保藏在国际保藏机构韩国微生物保藏中心(KCCM),保藏号为KCCM12931P。
【实施例5:确认产生异亮氨酸的菌株中aroG选择突变的亮氨酸,酪氨酸和苯丙氨酸产生能力】
为了确认所选突变是否显示出对异亮氨酸的影响,异亮氨酸是作为亮氨酸的代表支链氨基酸,进行了实验以通过将突变引入产生异亮氨酸的棒杆菌属(Corynebacterium)的产生异亮氨酸的菌株中来确认异亮氨酸的产生能力。具体实验如下。
【实施例5-1:产L-异亮氨酸菌株CA10-3101的制备】
产L-异亮氨酸菌株从野生型谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032开发而成。具体而言,为了解除苏氨酸(生物合成途径中异亮氨酸的前体)的反馈抑制,用组氨酸取代编码高丝氨酸脱氢酶的基因hom的第407个氨基酸精氨酸(US 2020-0340022 A1)。具体而言,为了制备引入hom(R407H)的菌株,使用基于谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032的染色体的SEQ ID NO:33和34或SEQ ID NOC:35和36的引物对作为模板进行PCR。本文所用的引物序列如下表10所示。
【表10】
SEQ ID NO: | 序列(5'→3') |
33 | TCGAGCTCGG TACCCCGCTT TTGCACTCAT CGAGC |
34 | CACGATCAGA TGTGCATCAT CAT |
35 | ATGATGATGC ACATCTGATC GTG |
36 | CTCTAGAGGA TCCCCGAGCA TCTTCCAAAA CCTTG |
PfuUltraTM高可靠性DNA聚合酶(Stratagene)用作PCR反应的聚合酶,通过在95℃下变性30秒、在55℃下退火30秒和在72℃下聚合1分钟的28个循环来进行PCR。结果,在hom基因的突变周围分别获得了5'上游区域中的DNA片段(1000bp)和3'下游区域中的脱氧核糖核酸片段(1000BP)。基于两个扩增的DNA片段作为模板,使用SEQ ID NO:34和SEQ ID NO:35的引物,在95℃变性5分钟,然后在95℃下变性30秒,在55℃下退火30秒,并在72℃下聚合2分钟的28个循环,然后在72℃聚合5分钟的条件下进行PCR。
结果,扩增了包括hom基因突变的DNA片段(2kb),其编码高丝氨酸脱氢酶变体,其中407位的精氨酸被组氨酸取代。使用PCR纯化试剂盒(QIAGEN)纯化扩增产物,并将其用作制备载体的***DNA片段。同时,用限制性内切酶SmaI处理纯化的扩增产物后,在65℃下热处理20分钟的pDCM2载体与***DNA片段(扩增产物)的摩尔浓度(M)的比例设定为1:2,并且根据制造商的手册使用Infusion Cloning Kit(TaKaRa)克隆载体,从而制备用于将hom(R407H)突变引入染色体的pDCM2-R407H载体。
将如此制备的载体通过电穿孔转化谷氨酸棒杆菌(Corynebacteriumglutamicum)ATCC13032并进行二次交叉,得到在染色体上包含hom(R407H)突变的菌株,并将该菌株命名为谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032 hom(R407H)。
为了释放对L-异亮氨酸的反馈并增加所制备的ATCC13032 hom(R407H)中的活性,制备了一种引入作为编码L-苏氨酸脱水酶的基因的ilvA突变体(T381A,F383A)的菌株。更具体地说,为了制备引入ilvA(T381A,F383A)突变的菌株,使用SEQ ID NO:37和38或SEQ IDNO:39和40的引物对进行PCR,基于谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032的染色体作为模板。本文中使用的引物序列示于下面的表11中。
【表11】
SEQ ID NO: | 序列(5'→3') |
37 | TCGAGCTCGG TACCCATGAG TGAAACATAC GTGTC |
38 | GCGCTTGAGG TACTCtgcCA GCGcGATGTC ATCATCCGG |
39 | CCGGATGATG ACATCgCGCT GgcaGAGTAC CTCAAGCGC |
40 | CTCTAGAGGA TCCCCCGTCA CCGACACCTC CACA |
PfuUltraTM高可靠性DNA聚合酶(Stratagene)用作PCR反应的聚合酶,通过重复在95℃下变性30秒、在55℃下退火30秒和在72℃下聚合1分钟的28个循环来进行PCR。结果,在ilvA基因的突变周围分别获得了5'上游区域的DNA片段(1126bp)和3'下游区域的DNA片段(286bp)。基于两个扩增的DNA片段作为模板,使用SEQ ID NO:37和SEQ ID NO:40的引物,在95℃变性5分钟,然后在95℃下变性30秒,在55℃下退火30秒,并在72℃下聚合2分钟的28个循环,然后在72℃聚合5分钟的条件下进行PCR。
结果,扩增了包括ilvA基因突变的DNA片段(1.4kb),该基因编码苏氨酸脱氢酶变体,其中381位的苏氨酸被丙氨酸取代,383位的苯丙氨酸被丙氨酸取代。使用PCR纯化试剂盒(QIAGEN)纯化扩增产物,并将其用作制备载体的***DNA片段。同时,用限制性内切酶SmaI处理纯化的扩增产物后,在65℃下热处理20分钟的pDCM2载体与***DNA片段(扩增产物)的摩尔浓度(M)的比例设定为1:2,并且根据制造商的说明书使用Infusion CloningKit(TaKaRa)克隆载体,从而制备用于将ilvA(T381A,F383A)突变引入染色体的pDCM2 ilvA载体(T381B,F383B)。将如此制备的载体通过电穿孔转化到谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032-hom(R407H)中,并进行二次交叉,以获得在染色体上包括ilvA(T381A,F383A)突变的菌株,该菌株被命名为谷氨酸棒杆菌(Corynebacterium glutamicum)CA10-3101。
【实施例5-2:aroG醛缩酶变体在CA10-3101菌株中的引入及其评价】
用实施例3-1中制备的载体pDCM2-aroG(R217A),pDCM2-aroG(K310A),pDCM2-aroG(R403A),pDCM2-aroG(E462A),pDCM2-aroG(R217A,K310A,E462A)和pDCM2-aroG(R217A,K310A,R403A,E462A)转化作为L-异亮氨酸生产菌株的CA10-3101,在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对选定的一代菌株进行次级交叉,并选择引入靶基因突变的菌株。通过使用SEQ ID NO:11和SEQ ID NO:12的引物进行PCR并分析核苷酸序列,证实了将aroG基因突变引入最终转化的菌株中,从而确认了aroG醛缩酶突变引入菌株。
将这样制备的菌株命名为CA10-3101_aroG_R217A,CA10-3101_aroG_K310A,CA10-3101_aroG_R403A,CA10-3101_aroG_E462A,CA10-3101_aroG_(R217A,K310A,E462A)和CA10-3101_aroG_(R217A,K310A,R403A,E462A)。
对制备的菌株CA10-3101_aroG_R217A,CA10-3101_aroG_K310A,CA10-3101_aroG_R403A,CA10-3101_aroG_E462A,CA10-3101_aroG_(R217A,K310A,E462A),CA10-3101_aroG_(R217A,K310A,R403A,E462A)和ATCC13032,CA10-3101的L-异亮氨酸生成能力进行了评价。通过将亲本菌株和aroG醛缩酶突变菌株接种到含有25mL异亮氨酸生产培养基的250mL角挡板烧瓶中,然后在32℃下以200rpm振荡培养60小时来制备L-异亮氨酸。
本实施例中使用的生产介质的组成如下。
<生产培养基>
葡萄糖10%,酵母提取物0.2%,(NH4)2SO4 1.6%,KH2PO4 0.1%,MgSO4·7H2O0.1%,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,生物素200μg/L,pH 7.2
培养完成后,使用高效液相色谱(HPLC)测量L-异亮氨酸和副产物的产生,并且测试的每种菌株的培养基中L-异亮氨酸和副产物的浓度如下表12所示。
【表12】
如表12所示,与亲本菌株谷氨酸棒杆菌(Corynebacterium glutamicum)CA10-3101相比,在aroG基因中具有额外R217A、K310A、R403A和E462A突变的L-异亮氨酸生产菌株显示出L-异亮氨酸的生产能力增加了约1.1至1.2倍,并且L-酪氨酸的生产能力和L-苯丙氨酸的生产能力降低。
根据以上结果,可以确认aroG醛缩酶的氨基酸序列中的217、310、403和462位的氨基酸是对于生产作为芳族氨基酸的L-异亮氨酸、L-酪氨酸和L-苯丙氨酸而言重要的位置。
【实施例6:确认产生缬氨酸菌株中aroG选择突变体的缬氨酸产生能力】
为了确认所选突变是否对L-缬氨酸(L-缬氨酸是亮氨酸的代表支链氨基酸)有影响,还将所选突变引入到产生缬氨酸的菌株KCCM11201P的棒杆菌属(Corynebacterium)中,以确认缬氨酸的产生能力。具体实验如下。
【实施例6-1:在KCCM11201P菌株中引入aroG醛缩酶变体及其评价】
为了确定突变是否有效增加缬氨酸的产生能力,使用了产生L-缬氨酸的菌株谷氨酸棒杆菌(Corynebacterium glutamicum)KCCM11201P(US 8465962 B2)。用实施例3-1中制备的载体pDCM2-aroG(R217A)、pDCM2-aroG(K310A)、pDCM2-aroG(R403A)、pDCM2-aroG(E462A),pDCM2-aroG(R217A,K310A,E462A)和pDCM2-aroG(R217A,K310A,R403A,E462A)转化作为缬氨酸-产生株的KCCM11201P,在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对选定的一代菌株进行次级交叉,并选择引入靶基因突变的菌株。通过使用SEQ ID NO:11和SEQ ID NO:12的引物进行PCR并分析核苷酸序列来确认aroG基因突变引入最终转化的菌株中,从而确认aroG醛缩酶突变引入菌株中。这样制备的菌株分别命名为KCCM11201P-aroG(R217A),KCCM11201P-aroG(K310A),KCCM11201P-aroG(R403A),KCCM11201P-aroG(E462A),KCCM11201P-aroG(R217A,K310A,E462A)和KCCM11201P-aroG(R217A,K310A,R403A,E462A)。
对所制备的缬氨酸菌株KCCM11201P-aroG(R217A),KCCM11201P-aroG(K310A),KCCM11201P-aroG(R403A),KCCM11201P-aroG(E462A),KCCM11201P-aroG(R217A,K310A,E462A)和KCCM11201P-aroG(R217A,K310A,R403A,E462A)的产生能力进行了评价。烧瓶培养以与实施例2相同的方法进行,培养完成后用HPLC测定缬氨酸产量,培养结果如下表13所示。
【表13】
如以上表13所示,与亲本谷氨酸棒杆菌(Corynebacterium glutamicum)KCCM11201P相比,在aroG基因中具有另外的R217A,K310A,R403A和E462A突变的谷氨酸棒杆菌(Corynebacterium glutamicum)KCCM11201P-aroG(R217A),KCCM11201P-aroG(K310A),KCCM11201P-aroG(R403A),KCCM11201P-aroG(E462A),KCCM11201P-aroG(R217A,K310A,E462A)和KCCM11201P-aroG(R217A,K310A,R403A,E462A)显示缬氨酸产生能力最多增加1.11倍,L-酪氨酸产生能力和L-苯丙氨酸产生能力减少约10~20倍。
根据以上结果,可以确认aroG醛缩酶的氨基酸序列中217、310、403和462位的氨基酸是对于生产L-缬氨酸以及作为芳族氨基酸的L-酪氨酸和L-苯丙氨酸而言重要的位置。
【参考例1:确认gltA(M312I)突变对亮氨酸产生的效果】
【参考例1-1:含gltA突变的***载体的制备】
使用定点诱变来制备用于引入gltA(M312I;SEQ ID NO:41)突变的载体。
使用基于野生型谷氨酸棒杆菌(Corynebacterium glutamicum)染色体的SEQ IDNO:43和44和SEQ ID NO:45和46的引物对进行PCR。在94℃变性5分钟,然后在94℃下变性30秒,在55℃下退火30秒,并在72℃下聚合1分30秒的30个循环,然后在72℃聚合5分钟的条件下进行PCR。利用将所得基因片段用SmaI限制性内切酶酶切的线性pDCM15载体和In-Fusion酶融合DNA片段之间的末端15个碱基的同源序列而克隆,制备了用异亮氨酸取代第312个氨基酸甲硫氨酸的pDCM2-gltA(M312I)载体。
【表14】
【参考例1-2:ATCC13032菌株的变体引入及其评估】
用参考例1-1中制作的载体pDCM2-gltA(M312I)转化野生型ATCC13032,并且在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对选定的原发菌株进行次级交叉,并选择引入靶基因突变的菌株。通过使用SEQ ID NO:31和SEQID NO:30(实施例4-1,表8)的引物进行PCR并分析核苷酸序列,确认将gltA基因突变引入最终转化的菌株中,从而确认将突变(SEQ ID NO:42)引入菌株中。由此制备的菌株命名为ATCC13032_gltA_M312I。
对烧瓶发酵效价进行评估,以评价上述制备的ATCC13032_gltA_M312I菌株的亮氨酸产生能力。将每个亲本菌株谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032和上述制备ATCC13032_gltA_M312I的一个铂环接种到含有25mL生产培养基的250mL角挡板烧瓶中,然后在30℃下以200rpm振荡培养60小时以产生亮氨酸。培养完成后,通过HPLC测量亮氨酸的产生。每种测试菌株的培养基中的亮氨酸浓度如下表15所示。
●生产培养基: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,盐酸硫胺素1,000μg,泛酸钙2000μg,烟酰胺3,000μg,CaCO3 30g(基于1L的蒸馏水),pH 7.0
【表15】
菌株名称 | 亮氨酸(g/L) |
ATCC13032 | 0.87 |
ATCC13032_gltA_M312I | 1.25 |
基于这些结果,证实了gltA的M312I取代是增加亮氨酸产量的有效突变。
【参考例2:确认ilvA(T381A,F383A)突变对异亮氨酸产生的效果】
【参考例2-1:pECCG117-ilvA(F383A)的制备】
为了扩增作为编码苏氨酸脱水酶(SEQ ID NO:47)的基因的ilvA(SEQ ID NO:48),基于先前报道的引入F383A突变的ilvA序列(World J Microbiol Biotechnol(2015)31:1369-1377),在用于从启动子区域(起始密码子上游约300bp)扩增到终止子区域(终止密码子下游约100bp)的引物(SEQ ID NO:49和SEQ ID NO:50)的两端***BamHI限制性内切酶位点。此外,引物(SEQ ID NO:51和SEQ ID NO:52)用于将F383A突变引入ilvA。本文中使用的引物序列如下表16所示。
【表16】
SEQ ID NO: | 引物 | 序列 |
49 | 引物1 | ggatccGACT GAGCCTGGGC AACTGG |
50 | 引物2 | ggatccCCGT CACCGACACC TCCACA |
51 | 引物3 | ACATCACGCT GgcaGAGTAC CTCAA |
52 | 引物4 | TTGAGGTACT CtgcCAGCGT GATGT |
使用基于野生型谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032的染色体的引物对SEQ ID NO:49和52以及SEQ ID NO:50和51进行PCR。在95℃变性5分钟,然后在95℃下变性30秒,在55℃下退火30秒,并在72℃下聚合1分30秒的30个循环,然后在72℃聚合5分钟的条件下进行PCR。
结果,在ilvA基因突变附近获得了5'上游区域的DNA片段(1460bp)和3'下游区域的DNA片段(276bp)。
基于两个扩增的DNA片段作为模板,使用SEQ ID NO:49和SEQ ID NO:50的引物进行PCR。
结果,包括ilvA基因突变的DNA片段(1531bp)被扩增,其中第383个苯丙氨酸被丙氨酸取代。将pECCG117载体(韩国专利第10-0057684号)和ilvA DNA片段用限制性内切酶BamHI处理,使用DNA连接酶连接,克隆得到质粒,质粒命名为pECCG117-ilvA(F383A)。
【参考例2-2:将随机突变的附加引入pECCG117-ilvA(F383A)】
为了获得编码L-苏氨酸脱水酶的基因的变体,使用随机诱变试剂盒制备ilvA突变基因质粒(美国安捷伦科技公司)。使用基于ilvA(F383A)的染色体的SEQ ID NO:49和SEQID NO:50的引物作为模板进行PCR。在95℃变性2分钟,然后在95℃下变性30秒,在55℃下退火30秒,并在72℃下聚合1分30秒的30个循环,然后在72℃聚合10分钟的条件下进行PCR。
结果,除了第383个苯丙氨酸被丙氨酸取代的突变之外,还具有随机突变的能够编码L-苏氨酸脱水酶的ilvA变体的DNA片段(1531bp)被扩增。pECCG117载体和ilvA突变DNA片段用限制性内切酶BamHI处理,使用DNA连接酶连接,克隆得到一组质粒。
【参考例2-3:CJILE-301菌株的制备】
将pECCG117-ilvA(F383A)引入谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032 hom(R407H)菌株中,并将所得质粒引入的菌株命名为hom(R407H)/pECCG117-ilvA(F383A)。此外,将参考文献2-2中获得的变异质粒组引入谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032 hom(R407H)菌株中,并接种到最小培养基上,测定死亡率。结果,死亡率为70%,存活的细胞接种并在种子培养基中培养。最后,选择与对照ATCC13032 hom(R407H)/pECCG117-ilvA(F383A)相比具有更好异亮氨酸生成能力的突变菌株,并命名为谷氨酸棒杆菌(Corynebacterium glutamicum)CJ LE-301。
通过从CJILE-301菌株中分离质粒对ilvA基因进行测序,结果证实编码ilvA基因的第1141个核苷酸A被G取代而ilvA蛋白的第383位的F被A取代的变异之外,第381位的T进一步被A取代的变异蛋白,其由SEQ ID NO:54表示。
【参考例2-4:引入ilvA变体(T381A,F383A)】
制备SEQ ID NO:49和SEQ ID NO:52的引物(实施例5-1,表16)以便将ilvA变体(T381A,F383A)引入野生型菌株中。
为了制备引入ilvA变体(T381A、F383A)的菌株,基于从CJILE-301菌株提取的质粒DNA作为模板,使用SEQ ID NO:49和SEQ ID NO:52的引物进行PCR。
PfuUltraTM高可靠性DNA聚合酶(Stratagene)用作PCR反应的聚合酶,通过重复在95℃下变性30秒、在55℃下退火30秒和在72℃下聚合2分钟的28个循环来进行PCR。
结果,获得了包括ilvA基因(1311bp)的约100bp终止区的基因片段(1411bp)。
使用PCR纯化试剂盒(QIAGEN)纯化扩增产物,并将其用作制备载体的***DNA片段。同时,用限制性内切酶SmaI处理纯化的扩增产物后,在65℃下热处理20分钟的pDCM2载体与***DNA片段(扩增产物)的摩尔浓度(M)的比例设定为1:2,并根据制造商手册使用Infusion Cloning Kit(TaKaRa)克隆载体,从而制备用于将T381A和F383A突变引入染色体的pDCM2-T381A_F383A载体。
将如此制备的载体通过电穿孔转化谷氨酸棒杆菌(Corynebacteriumglutamicum)ATCC13032 hom(R407H)并进行二次交叉,从而得到包括ilvA(T381A、F383A;SEQ ID NO:53)染色体突变,菌株被命名为CA10-3101。
CA10-3101菌株于2020年5月27日根据《布达佩斯条约》保藏在国际保藏机构韩国微生物保藏中心(KCCM),保藏号为KCCM12739P。
将KCCM12739P菌株接种到含有25mL异亮氨酸生产培养基的250mL角挡板烧瓶中,然后在32℃下以200rpm振荡培养60小时以制备L-异亮氨酸。所使用的生产介质的组成如下。
<生产培养基>
葡萄糖10%,酵母提取物0.2%,(NH4)2SO4 1.6%,KH2PO4 0.1%,MgSO4·7H2O0.1%,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,生物素200μg/L,pH 7.2
培养完成后,使用高效液相色谱(HPLC)测定培养基中L-异亮氨酸和L-苏氨酸的浓度,结果示于下表17中。
【表17】
如表17所示,亲本菌株谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032hom(R407H)不能产生L-异亮氨酸,但是ATCC13032 hom(R407H)ilvA(T381A,F383A)突变株能够以3.9g/L的浓度产生L-异亮氨酸,从而证实L-异亮氨酸的生产率与亲本菌株相比显著提高。
基于该结果,证实ilvA(T381A,F383A)突变是用于增加异亮氨酸产量的有效突变。
【参考例3:确认leuA(P247C,R558H,G561D)对亮氨酸生成的效果】
【参考例3-1:CJL-8100菌株的制备】
具体地,通过电穿孔将包含KR 10-2018-0077008A中公开的leuA基因突变的载体pDCM2-leuA(R558H,G561D)转化到谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032中,并且在含有25mg/L卡那霉素的培养基中选择通过同源序列的重组将载体***染色体的菌株。再次对所选择的初级菌株进行二次交叉,并选择引入leuA基因突变的菌株。通过使用SEQ ID NO:55和SEQ ID NO:66的引物,在94℃变性5分钟,然后在94℃下变性30秒,在55℃下退火30秒,以及在72℃下聚合1分30秒的30个循环,然后在72℃下聚合5分钟的条件下进行PCR,确认将突变引入最终转化的菌株中并分析核苷酸序列,从而确认R558H和G561D突变的引入。用pDCM2-leuA(R558H,G561D)载体转化的ATCC13032_leuA_(R558H,G561D)菌株命名为“CJ L-8100”。
下面参考例3中使用的引物序列示于表18中。
【表18】
SEQ ID NO: | 序列(5'→3') |
SEQ ID NO:55 | AACACGACCG GCATCCCGTC GC |
SEQ ID NO:56 | AAATCATTTG AGAAAACTCG AGG |
SEQ ID NO:57 | GTGAATTCGA GCTCGGTACC CAAATCATTT GAGAAAACTC GAGGC |
SEQ ID NO:58 | GGTGATCATC TCAACGGTGG AACACAGGTT GATGATCATT GGGTT |
SEQ ID NO:59 | AACCCAATGA TCATCAACCT GTGTTCCACC GTTGAGATGA TCACC |
SEQ ID NO:60 | GGTCGACTCT AGAGGATCCC CAAGAAGGCA ACATCGGACA GC |
SEQ ID NO:61 | ATCCATTCAA TGGAGTCTGC G |
【参考例3-2:含有leuA突变的***载体的制备】
制备了用于将P247C突变引入CJ L-8100的载体,CJL-81000是一种L-亮氨酸生产菌株,其中两个突变(R558H,G561D)被引入LeuA。
以CJ L-8100菌株的染色体作为模板,使用SEQ ID NO:57和58或SEQ ID NO:59和60的引物对进行PCR。在94℃变性5分钟,然后在94℃下变性30秒,在55℃下退火30秒,并在72℃下聚合1分30秒的30个循环,然后在72℃聚合5分钟的条件下进行PCR。利用将得到的PCR产物用SmaI限制性内切酶消化的线性pDCM2载体和In-Fusion酶融合DNA片段之间的末端15个碱基的同源序列来克隆,制备了含有编码野生型菌株的LeuA氨基酸序列中第558个氨基酸精氨酸被组氨酸取代、第561个氨基酸甘氨酸被天冬氨酸取代的leuA突变体的leuA突变、且将LeuA的第247个氨基酸脯氨酸(Pro)用半胱氨酸(Cys)取代的pDCM2-leuA(P247C,R558H,G561D)载体。
【参考例3-3:将LeuA变体(P247C)引入CJ L-8100菌株及其评价】
将生产L-亮氨酸的菌株CJL-8100转化到参考例3-2中制备的pDCM2-leuA(P247C,R558H,G561D)载体中,并在含有25mg/L卡那霉素的培养基中选择通过同源序列重组将载体***染色体的菌株。再次对所选择的初级菌株进行二次交叉,并选择引入靶基因突变的菌株。通过使用SEQ ID NO:55和SEQ ID NO:61的引物,在94℃变性5分钟,随后在94℃下变性30秒、在55℃下退火30秒和在72℃下聚合1分30秒的30个循环,然后在72℃下聚合5分钟的条件下进行PCR,确认将leuA基因的突变引入最终转化的菌株中并分析核苷酸序列。作为序列分析的结果,证实了编码菌株染色体中的leuA基因的第1673个核苷酸G被A取代、第1682和1683个核苷酸GC被AT取代、第739和740个核苷酸CC被TG取代而LeuA蛋白的第558个氨基酸精氨酸被组氨酸、第561个氨基酸甘氨酸被天冬氨酸取代、第247个氨基酸脯氨酸(Pro)被半胱氨酸(Cys)取代的leuA变体(P247C、R558H、G561D)的leuA突变被导入菌株内。
由此制备的CJ L8100_leuA_P247C被命名为“CA13-8105”,并于2020年4月29日根据《布达佩斯条约》保藏在国际保藏机构韩国微生物保藏中心(KCCM),保藏号为KCCM12709P。
包括上述三个突变的LeuA变体(P247C、R558H、G561D)的氨基酸序列和编码所述氨基酸序列的LeuA变体的核苷酸序列分别用SEQ ID NO:62和SEQ ID NO:63表示。
对ATCC13032、由此生产的CJL-8100和CA13-8105菌株的L-亮氨酸产生能力进行了评价。具体地,烧瓶培养以与实施例2-1相同的方式进行,培养完成后,使用HPLC测量亲本菌株和突变菌株的L-亮氨酸产生,结果如表19所示。
【表19】
菌株名称 | L-亮氨酸(g/L) |
ATCC13032 | 0.87 |
ATCC13032_leuA_(R558H,G561D):CJL-8100 | 2.71 |
CJL8100_leuA_P247C:CA13-8105 | 3.52 |
如表19所示,谷氨酸棒杆菌(Corynebacterium glutamicum)CJL8100是一种L-亮氨酸生产菌株,与亲本菌株ATCC13032相比,其L-亮氨酸生产能力提高了约130%。此外,在CJ L8100菌株中进一步引入leuA_P247C突变的CA13-8105菌株显示出与亲本菌株CJ L8100相比提高了约150%的L-亮氨酸产生能力。
基于该结果,证实leuA(R558H,G561D,P247C)突变是用于增加亮氨酸产生的有效突变。
本领域普通技术人员将认识到,本申请可以以其它特定形式体现而不背离其精神或本质特征。所描述的实施例仅在所有方面被视为说明性的,而不是限制性的。因此,本申请的范围由所附权利要求而不是由上述描述表示。属于权利要求等同性的含义和范围的所有变更均应纳入本申请的范围。
【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
国际保藏机构对原始保藏的收条
根据细则第7.1条发给
至:CJ第一制糖株式会社
韩国,首尔市,中区,东湖路330,CJ第一制糖中心,(邮)100-400
【国际承认用于专利程序的微生物保藏布达佩斯条约】
国际表格国际保藏机构对原始保藏的收条
根据细则第7.1条发给
至:CJ第一制糖株式会社
韩国,首尔市,中区,东湖路330,CJ第一制糖中心,(邮)100-400
【国际承认用于专利程序的微生物保藏布达佩斯条约】
国际表格国际保藏机构对原始保藏的收条
根据细则第7.1条发给
至:CJ第一制糖株式会社
韩国,首尔市,中区,东湖路330,CJ第一制糖中心,(邮)100-400
/>
序列表
<110> CJ第一制糖株式会社
<120> aroG醛缩酶变体及利用其生产支链氨基酸的方法
<130> OPA21303
<150> KR10-2021-0010911
<151> 2021-01-26
<160> 63
<170> KoPatentIn 3.0
<210> 1
<211> 466
<212> PRT
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Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
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Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
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Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
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Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
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Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
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Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
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Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
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Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
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Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
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Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
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Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
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Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
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Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
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Val Ala Asn Ser Pro Ala Gly Ala Arg Tyr Glu Ala Leu Ala Arg Glu
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Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
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Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
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Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
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Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
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Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
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Asn Pro Ile Gly Ile Lys Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
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Arg Asn
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<210> 2
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<220>
<223> aroG_WT
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atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcacg ctacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcaag attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagaaatgc tgcgtaacta a 1401
<210> 3
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> aroG_R217A
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Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
1 5 10 15
Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
20 25 30
Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
35 40 45
Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
50 55 60
Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
65 70 75 80
Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
85 90 95
Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
100 105 110
Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
115 120 125
Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
130 135 140
Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
145 150 155 160
Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
165 170 175
Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
180 185 190
Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
195 200 205
Val Ala Asn Ser Pro Ala Gly Ala Ala Tyr Glu Ala Leu Ala Arg Glu
210 215 220
Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
225 230 235 240
Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
245 250 255
Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
260 265 270
Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
275 280 285
Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
290 295 300
Asn Pro Ile Gly Ile Lys Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
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Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu
325 330 335
Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro
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Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln
355 360 365
Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys
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Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu
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Val His Arg Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe
405 410 415
Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr
420 425 430
Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu
435 440 445
Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Glu Met Leu
450 455 460
Arg Asn
465
<210> 4
<211> 1401
<212> DNA
<213> 人工序列
<220>
<223> aroG_R217A_nt
<400> 4
atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcagc gtacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcaag attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagaaatgc tgcgtaacta a 1401
<210> 5
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> aroG_K310A
<400> 5
Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
1 5 10 15
Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
20 25 30
Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
35 40 45
Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
50 55 60
Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
65 70 75 80
Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
85 90 95
Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
100 105 110
Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
115 120 125
Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
130 135 140
Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
145 150 155 160
Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
165 170 175
Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
180 185 190
Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
195 200 205
Val Ala Asn Ser Pro Ala Gly Ala Arg Tyr Glu Ala Leu Ala Arg Glu
210 215 220
Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
225 230 235 240
Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
245 250 255
Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
260 265 270
Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
275 280 285
Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
290 295 300
Asn Pro Ile Gly Ile Ala Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
305 310 315 320
Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu
325 330 335
Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro
340 345 350
Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln
355 360 365
Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys
370 375 380
Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu
385 390 395 400
Val His Arg Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe
405 410 415
Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr
420 425 430
Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu
435 440 445
Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Glu Met Leu
450 455 460
Arg Asn
465
<210> 6
<211> 1401
<212> DNA
<213> 人工序列
<220>
<223> aroG_K310A_nt
<400> 6
atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcacg ctacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcgcg attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagaaatgc tgcgtaacta a 1401
<210> 7
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> aroG_R403A
<400> 7
Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
1 5 10 15
Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
20 25 30
Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
35 40 45
Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
50 55 60
Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
65 70 75 80
Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
85 90 95
Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
100 105 110
Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
115 120 125
Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
130 135 140
Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
145 150 155 160
Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
165 170 175
Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
180 185 190
Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
195 200 205
Val Ala Asn Ser Pro Ala Gly Ala Arg Tyr Glu Ala Leu Ala Arg Glu
210 215 220
Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
225 230 235 240
Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
245 250 255
Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
260 265 270
Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
275 280 285
Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
290 295 300
Asn Pro Ile Gly Ile Lys Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
305 310 315 320
Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu
325 330 335
Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro
340 345 350
Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln
355 360 365
Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys
370 375 380
Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu
385 390 395 400
Val His Ala Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe
405 410 415
Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr
420 425 430
Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu
435 440 445
Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Glu Met Leu
450 455 460
Arg Asn
465
<210> 8
<211> 1401
<212> DNA
<213> 人工序列
<220>
<223> aroG_R403A_nt
<400> 8
atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcacg ctacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcaag attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccacgcgg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagaaatgc tgcgtaacta a 1401
<210> 9
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> aroG_E462A
<400> 9
Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
1 5 10 15
Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
20 25 30
Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
35 40 45
Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
50 55 60
Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
65 70 75 80
Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
85 90 95
Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
100 105 110
Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
115 120 125
Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
130 135 140
Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
145 150 155 160
Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
165 170 175
Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
180 185 190
Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
195 200 205
Val Ala Asn Ser Pro Ala Gly Ala Arg Tyr Glu Ala Leu Ala Arg Glu
210 215 220
Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
225 230 235 240
Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
245 250 255
Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
260 265 270
Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
275 280 285
Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
290 295 300
Asn Pro Ile Gly Ile Lys Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
305 310 315 320
Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu
325 330 335
Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro
340 345 350
Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln
355 360 365
Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys
370 375 380
Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu
385 390 395 400
Val His Arg Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe
405 410 415
Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr
420 425 430
Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu
435 440 445
Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Ala Met Leu
450 455 460
Arg Asn
465
<210> 10
<211> 1401
<212> DNA
<213> 人工序列
<220>
<223> aroG_E462A_nt
<400> 10
atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcacg ctacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcaag attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagcgatgc tgcgtaacta a 1401
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 11
tgatgcgcgt cataatttag 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 12
ctcatctctg actggacgtg 20
<210> 13
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 13
gtgaattcga gctcggtacc cagttgaatg ctaccaactt g 41
<210> 14
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 14
cacgagcaag agcctcgtac gctgcaccag ctggg 35
<210> 15
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 15
ggtcgactct agaggatccc cgctgttatt actgtgcctg 40
<210> 16
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 16
gaactcccca gctggtgcag cgtacgaggc tcttgctcg 39
<210> 17
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 17
gggtgatacc aggaccaatc gcgatgccga ttgggttag 39
<210> 18
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 18
ctctaaccca atcggcatcg cgattggtcc tggtatcac 39
<210> 19
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 19
ctgggtgggt gcccaatgcc gcgtggacct cgaagaag 38
<210> 20
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 20
gggcttcttc gaggtccacg cggcattggg cacccac 37
<210> 21
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 21
aagcttagtt acgcagcatc gctgcaacga ggaaagcc 38
<210> 22
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 22
gttggctttc ctcgttgcag cgatgctgcg taactaagc 39
<210> 23
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> aroG_R217A+K310A+E462A
<400> 23
Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
1 5 10 15
Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
20 25 30
Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
35 40 45
Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
50 55 60
Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
65 70 75 80
Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
85 90 95
Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
100 105 110
Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
115 120 125
Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
130 135 140
Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
145 150 155 160
Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
165 170 175
Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
180 185 190
Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
195 200 205
Val Ala Asn Ser Pro Ala Gly Ala Ala Tyr Glu Ala Leu Ala Arg Glu
210 215 220
Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
225 230 235 240
Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
245 250 255
Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
260 265 270
Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
275 280 285
Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
290 295 300
Asn Pro Ile Gly Ile Ala Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
305 310 315 320
Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu
325 330 335
Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro
340 345 350
Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln
355 360 365
Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys
370 375 380
Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu
385 390 395 400
Val His Arg Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe
405 410 415
Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr
420 425 430
Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu
435 440 445
Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Ala Met Leu
450 455 460
Arg Asn
465
<210> 24
<211> 1401
<212> DNA
<213> 人工序列
<220>
<223> aroG_R217A+K310A+E462A_nt
<400> 24
atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcagc gtacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcgcg attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagcgatgc tgcgtaacta a 1401
<210> 25
<211> 466
<212> PRT
<213> 人工序列
<220>
<223> aroG_R217A+K310A+R403A+E462A
<400> 25
Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu
1 5 10 15
Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp
20 25 30
Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln
35 40 45
Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val
50 55 60
Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn
65 70 75 80
Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu
85 90 95
Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln
100 105 110
Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met
115 120 125
Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp
130 135 140
Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu
145 150 155 160
Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala
165 170 175
Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser
180 185 190
Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe
195 200 205
Val Ala Asn Ser Pro Ala Gly Ala Ala Tyr Glu Ala Leu Ala Arg Glu
210 215 220
Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu
225 230 235 240
Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val
245 250 255
Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn
260 265 270
Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg
275 280 285
Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser
290 295 300
Asn Pro Ile Gly Ile Ala Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala
305 310 315 320
Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu
325 330 335
Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro
340 345 350
Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln
355 360 365
Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys
370 375 380
Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu
385 390 395 400
Val His Ala Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe
405 410 415
Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr
420 425 430
Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu
435 440 445
Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Ala Met Leu
450 455 460
Arg Asn
465
<210> 26
<211> 1401
<212> DNA
<213> 人工序列
<220>
<223> aroG_R217A+K310A+R403A+E462A_nt
<400> 26
atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60
ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120
caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180
ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240
ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300
ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360
gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420
tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480
gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540
tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600
ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcagc gtacgaggct 660
cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720
tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780
tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840
caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900
tccatgatct ctaacccaat cggcatcgcg attggtcctg gtatcacccc tgaagaggct 960
gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020
cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080
tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140
aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200
gtccacgcgg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260
gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320
gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380
gcagcgatgc tgcgtaacta a 1401
<210> 27
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 27
tatgcttcac cacatgactt c 21
<210> 28
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 28
aaatcatttg agaaaactcg agg 23
<210> 29
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 29
gtcacccgat cgtctgaag 19
<210> 30
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 30
gtcttaaaac cggttgat 18
<210> 31
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 31
caatgctggc tgcgtacgc 19
<210> 32
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 32
ctcctcgcga ggaaccaact 20
<210> 33
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 33
tcgagctcgg taccccgctt ttgcactcat cgagc 35
<210> 34
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 34
cacgatcaga tgtgcatcat cat 23
<210> 35
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 35
atgatgatgc acatctgatc gtg 23
<210> 36
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 36
ctctagagga tccccgagca tcttccaaaa ccttg 35
<210> 37
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 37
tcgagctcgg tacccatgag tgaaacatac gtgtc 35
<210> 38
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 38
gcgcttgagg tactctgcca gcgcgatgtc atcatccgg 39
<210> 39
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 39
ccggatgatg acatcgcgct ggcagagtac ctcaagcgc 39
<210> 40
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 40
ctctagagga tcccccgtca ccgacacctc caca 34
<210> 41
<211> 437
<212> PRT
<213> 人工序列
<220>
<223> gltA_M312I
<400> 41
Met Phe Glu Arg Asp Ile Val Ala Thr Asp Asn Asn Lys Ala Val Leu
1 5 10 15
His Tyr Pro Gly Gly Glu Phe Glu Met Asp Ile Ile Glu Ala Ser Glu
20 25 30
Gly Asn Asn Gly Val Val Leu Gly Lys Met Leu Ser Glu Thr Gly Leu
35 40 45
Ile Thr Phe Asp Pro Gly Tyr Val Ser Thr Gly Ser Thr Glu Ser Lys
50 55 60
Ile Thr Tyr Ile Asp Gly Asp Ala Gly Ile Leu Arg Tyr Arg Gly Tyr
65 70 75 80
Asp Ile Ala Asp Leu Ala Glu Asn Ala Thr Phe Asn Glu Val Ser Tyr
85 90 95
Leu Leu Ile Asn Gly Glu Leu Pro Thr Pro Asp Glu Leu His Lys Phe
100 105 110
Asn Asp Glu Ile Arg His His Thr Leu Leu Asp Glu Asp Phe Lys Ser
115 120 125
Gln Phe Asn Val Phe Pro Arg Asp Ala His Pro Met Ala Thr Leu Ala
130 135 140
Ser Ser Val Asn Ile Leu Ser Thr Tyr Tyr Gln Asp Gln Leu Asn Pro
145 150 155 160
Leu Asp Glu Ala Gln Leu Asp Lys Ala Thr Val Arg Leu Met Ala Lys
165 170 175
Val Pro Met Leu Ala Ala Tyr Ala His Arg Ala Arg Lys Gly Ala Pro
180 185 190
Tyr Met Tyr Pro Asp Asn Ser Leu Asn Ala Arg Glu Asn Phe Leu Arg
195 200 205
Met Met Phe Gly Tyr Pro Thr Glu Pro Tyr Glu Ile Asp Pro Ile Met
210 215 220
Val Lys Ala Leu Asp Lys Leu Leu Ile Leu His Ala Asp His Glu Gln
225 230 235 240
Asn Cys Ser Thr Ser Thr Val Arg Met Ile Gly Ser Ala Gln Ala Asn
245 250 255
Met Phe Val Ser Ile Ala Gly Gly Ile Asn Ala Leu Ser Gly Pro Leu
260 265 270
His Gly Gly Ala Asn Gln Ala Val Leu Glu Met Leu Glu Asp Ile Lys
275 280 285
Ser Asn His Gly Gly Asp Ala Thr Glu Phe Met Asn Lys Val Lys Asn
290 295 300
Lys Glu Asp Gly Val Arg Leu Ile Gly Phe Gly His Arg Val Tyr Lys
305 310 315 320
Asn Tyr Asp Pro Arg Ala Ala Ile Val Lys Glu Thr Ala His Glu Ile
325 330 335
Leu Glu His Leu Gly Gly Asp Asp Leu Leu Asp Leu Ala Ile Lys Leu
340 345 350
Glu Glu Ile Ala Leu Ala Asp Asp Tyr Phe Ile Ser Arg Lys Leu Tyr
355 360 365
Pro Asn Val Asp Phe Tyr Thr Gly Leu Ile Tyr Arg Ala Met Gly Phe
370 375 380
Pro Thr Asp Phe Phe Thr Val Leu Phe Ala Ile Gly Arg Leu Pro Gly
385 390 395 400
Trp Ile Ala His Tyr Arg Glu Gln Leu Gly Ala Ala Gly Asn Lys Ile
405 410 415
Asn Arg Pro Arg Gln Val Tyr Thr Gly Asn Glu Ser Arg Lys Leu Val
420 425 430
Pro Arg Glu Glu Arg
435
<210> 42
<211> 1314
<212> DNA
<213> 人工序列
<220>
<223> gltA_M312I_nt
<400> 42
atgtttgaaa gggatatcgt ggctactgat aacaacaagg ctgtcctgca ctaccccggt 60
ggcgagttcg aaatggacat catcgaggct tctgagggta acaacggtgt tgtcctgggc 120
aagatgctgt ctgagactgg actgatcact tttgacccag gttatgtgag cactggctcc 180
accgagtcga agatcaccta catcgatggc gatgcgggaa tcctgcgtta ccgcggctat 240
gacatcgctg atctggctga gaatgccacc ttcaacgagg tttcttacct acttatcaac 300
ggtgagctac caaccccaga tgagcttcac aagtttaacg acgagattcg ccaccacacc 360
cttctggacg aggacttcaa gtcccagttc aacgtgttcc cacgcgacgc tcacccaatg 420
gcaaccttgg cttcctcggt taacattttg tctacctact accaggacca gctgaaccca 480
ctcgatgagg cacagcttga taaggcaacc gttcgcctca tggcaaaggt tccaatgctg 540
gctgcgtacg cacaccgcgc acgcaagggt gctccttaca tgtacccaga caactccctc 600
aatgcgcgtg agaacttcct gcgcatgatg ttcggttacc caaccgagcc atacgagatc 660
gacccaatca tggtcaaggc tctggacaag ctgctcatcc tgcacgctga ccacgagcag 720
aactgctcca cctccaccgt tcgtatgatc ggttccgcac aggccaacat gtttgtctcc 780
atcgctggtg gcatcaacgc tctgtccggc ccactgcacg gtggcgcaaa ccaggctgtt 840
ctggagatgc tcgaagacat caagagcaac cacggtggcg acgcaaccga gttcatgaac 900
aaggtcaaga acaaggaaga cggcgtccgc ctcatcggct tcggacaccg cgtttacaag 960
aactacgatc cacgtgcagc aatcgtcaag gagaccgcac acgagatcct cgagcacctc 1020
ggtggcgacg atcttctgga tctggcaatc aagctggaag aaattgcact ggctgatgat 1080
tacttcatct cccgcaagct ctacccgaac gtagacttct acaccggcct gatctaccgc 1140
gcaatgggct tcccaactga cttcttcacc gtattgttcg caatcggtcg tctgccagga 1200
tggatcgctc actaccgcga gcagctcggt gcagcaggca acaagatcaa ccgcccacgc 1260
caggtctaca ccggcaacga atcccgcaag ttggttcctc gcgaggagcg ctaa 1314
<210> 43
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> gltA M312I Up F
<400> 43
gtgaattcga gctcggtacc cgcgggaatc ctgcgttacc gc 42
<210> 44
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> gltA M312I Up R
<400> 44
tgtaaacgcg gtgtccgaag ccgatgaggc ggacgccgtc tt 42
<210> 45
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> gltA M312I Down F
<400> 45
aagacggcgt ccgcctcatc ggcttcggac accgcgttta ca 42
<210> 46
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> gltA M312I Down R
<400> 46
ggtcgactct agaggatccc cttagcgctc ctcgcgagga ac 42
<210> 47
<211> 436
<212> PRT
<213> 未知
<220>
<223> ilvA_WT
<400> 47
Met Ser Glu Thr Tyr Val Ser Glu Lys Ser Pro Gly Val Met Ala Ser
1 5 10 15
Gly Ala Glu Leu Ile Arg Ala Ala Asp Ile Gln Thr Ala Gln Ala Arg
20 25 30
Ile Ser Ser Val Ile Ala Pro Thr Pro Leu Gln Tyr Cys Pro Arg Leu
35 40 45
Ser Glu Glu Thr Gly Ala Glu Ile Tyr Leu Lys Arg Glu Asp Leu Gln
50 55 60
Asp Val Arg Ser Tyr Lys Ile Arg Gly Ala Leu Asn Ser Gly Ala Gln
65 70 75 80
Leu Thr Gln Glu Gln Arg Asp Ala Gly Ile Val Ala Ala Ser Ala Gly
85 90 95
Asn His Ala Gln Gly Val Ala Tyr Val Cys Lys Ser Leu Gly Val Gln
100 105 110
Gly Arg Ile Tyr Val Pro Val Gln Thr Pro Lys Gln Lys Arg Asp Arg
115 120 125
Ile Met Val His Gly Gly Glu Phe Val Ser Leu Val Val Thr Gly Asn
130 135 140
Asn Phe Asp Glu Ala Ser Ala Ala Ala His Glu Asp Ala Glu Arg Thr
145 150 155 160
Gly Ala Thr Leu Ile Glu Pro Phe Asp Ala Arg Asn Thr Val Ile Gly
165 170 175
Gln Gly Thr Val Ala Ala Glu Ile Leu Ser Gln Leu Thr Ser Met Gly
180 185 190
Lys Ser Ala Asp His Val Met Val Pro Val Gly Gly Gly Gly Leu Leu
195 200 205
Ala Gly Val Val Ser Tyr Met Ala Asp Met Ala Pro Arg Thr Ala Ile
210 215 220
Val Gly Ile Glu Pro Ala Gly Ala Ala Ser Met Gln Ala Ala Leu His
225 230 235 240
Asn Gly Gly Pro Ile Thr Leu Glu Thr Val Asp Pro Phe Val Asp Gly
245 250 255
Ala Ala Val Lys Arg Val Gly Asp Leu Asn Tyr Thr Ile Val Glu Lys
260 265 270
Asn Gln Gly Arg Val His Met Met Ser Ala Thr Glu Gly Ala Val Cys
275 280 285
Thr Glu Met Leu Asp Leu Tyr Gln Asn Glu Gly Ile Ile Ala Glu Pro
290 295 300
Ala Gly Ala Leu Ser Ile Ala Gly Leu Lys Glu Met Ser Phe Ala Pro
305 310 315 320
Gly Ser Val Val Val Cys Ile Ile Ser Gly Gly Asn Asn Asp Val Leu
325 330 335
Arg Tyr Ala Glu Ile Ala Glu Arg Ser Leu Val His Arg Gly Leu Lys
340 345 350
His Tyr Phe Leu Val Asn Phe Pro Gln Lys Pro Gly Gln Leu Arg His
355 360 365
Phe Leu Glu Asp Ile Leu Gly Pro Asp Asp Asp Ile Thr Leu Phe Glu
370 375 380
Tyr Leu Lys Arg Asn Asn Arg Glu Thr Gly Thr Ala Leu Val Gly Ile
385 390 395 400
His Leu Ser Glu Ala Ser Gly Leu Asp Ser Leu Leu Glu Arg Met Glu
405 410 415
Glu Ser Ala Ile Asp Ser Arg Arg Leu Glu Pro Gly Thr Pro Glu Tyr
420 425 430
Glu Tyr Leu Thr
435
<210> 48
<211> 1311
<212> DNA
<213> 未知
<220>
<223> ilvA_WT_nt
<400> 48
atgagtgaaa catacgtgtc tgagaaaagt ccaggagtga tggctagcgg agcggagctg 60
attcgtgccg ccgacattca aacggcgcag gcacgaattt cctccgtcat tgcaccaact 120
ccattgcagt attgccctcg tctttctgag gaaaccggag cggaaatcta ccttaagcgt 180
gaggatctgc aggatgttcg ttcctacaag atccgcggtg cgctgaactc tggagcgcag 240
ctcacccaag agcagcgcga tgcaggtatc gttgccgcat ctgcaggtaa ccatgcccag 300
ggcgtggcct atgtgtgcaa gtccttgggc gttcagggac gcatctatgt tcctgtgcag 360
actccaaagc aaaagcgtga ccgcatcatg gttcacggcg gagagtttgt ctccttggtg 420
gtcactggca ataacttcga cgaagcatcg gctgcagcgc atgaagatgc agagcgcacc 480
ggcgcaacgc tgatcgagcc tttcgatgct cgcaacaccg tcatcggtca gggcaccgtg 540
gctgctgaga tcttgtcgca gctgacttcc atgggcaaga gtgcagatca cgtgatggtt 600
ccagtcggcg gtggcggact tcttgcaggt gtggtcagct acatggctga tatggcacct 660
cgcactgcga tcgttggtat cgaaccagcg ggagcagcat ccatgcaggc tgcattgcac 720
aatggtggac caatcacttt ggagactgtt gatccctttg tggacggcgc agcagtcaaa 780
cgtgtcggag atctcaacta caccatcgtg gagaagaacc agggtcgcgt gcacatgatg 840
agcgcgaccg agggcgctgt gtgtactgag atgctcgatc tttaccaaaa cgaaggcatc 900
atcgcggagc ctgctggcgc gctgtctatc gctgggttga aggaaatgtc ctttgcacct 960
ggttctgtcg tggtgtgcat catctctggt ggcaacaacg atgtgctgcg ttatgcggaa 1020
atcgctgagc gctccttggt gcaccgcggt ttgaagcact acttcttggt gaacttcccg 1080
caaaagcctg gtcagttgcg tcacttcctg gaagatatcc tgggaccgga tgatgacatc 1140
acgctgtttg agtacctcaa gcgcaacaac cgtgagaccg gtactgcgtt ggtgggtatt 1200
cacttgagtg aagcatcagg attggattct ttgctggaac gtatggagga atcggcaatt 1260
gattcccgtc gcctcgagcc gggcacccct gagtacgaat acttgaccta a 1311
<210> 49
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 49
ggatccgact gagcctgggc aactgg 26
<210> 50
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 50
ggatccccgt caccgacacc tccaca 26
<210> 51
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 51
acatcacgct ggcagagtac ctcaa 25
<210> 52
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 52
ttgaggtact ctgccagcgt gatgt 25
<210> 53
<211> 436
<212> PRT
<213> 人工序列
<220>
<223> ilvA_T381A+F383A
<400> 53
Met Ser Glu Thr Tyr Val Ser Glu Lys Ser Pro Gly Val Met Ala Ser
1 5 10 15
Gly Ala Glu Leu Ile Arg Ala Ala Asp Ile Gln Thr Ala Gln Ala Arg
20 25 30
Ile Ser Ser Val Ile Ala Pro Thr Pro Leu Gln Tyr Cys Pro Arg Leu
35 40 45
Ser Glu Glu Thr Gly Ala Glu Ile Tyr Leu Lys Arg Glu Asp Leu Gln
50 55 60
Asp Val Arg Ser Tyr Lys Ile Arg Gly Ala Leu Asn Ser Gly Ala Gln
65 70 75 80
Leu Thr Gln Glu Gln Arg Asp Ala Gly Ile Val Ala Ala Ser Ala Gly
85 90 95
Asn His Ala Gln Gly Val Ala Tyr Val Cys Lys Ser Leu Gly Val Gln
100 105 110
Gly Arg Ile Tyr Val Pro Val Gln Thr Pro Lys Gln Lys Arg Asp Arg
115 120 125
Ile Met Val His Gly Gly Glu Phe Val Ser Leu Val Val Thr Gly Asn
130 135 140
Asn Phe Asp Glu Ala Ser Ala Ala Ala His Glu Asp Ala Glu Arg Thr
145 150 155 160
Gly Ala Thr Leu Ile Glu Pro Phe Asp Ala Arg Asn Thr Val Ile Gly
165 170 175
Gln Gly Thr Val Ala Ala Glu Ile Leu Ser Gln Leu Thr Ser Met Gly
180 185 190
Lys Ser Ala Asp His Val Met Val Pro Val Gly Gly Gly Gly Leu Leu
195 200 205
Ala Gly Val Val Ser Tyr Met Ala Asp Met Ala Pro Arg Thr Ala Ile
210 215 220
Val Gly Ile Glu Pro Ala Gly Ala Ala Ser Met Gln Ala Ala Leu His
225 230 235 240
Asn Gly Gly Pro Ile Thr Leu Glu Thr Val Asp Pro Phe Val Asp Gly
245 250 255
Ala Ala Val Lys Arg Val Gly Asp Leu Asn Tyr Thr Ile Val Glu Lys
260 265 270
Asn Gln Gly Arg Val His Met Met Ser Ala Thr Glu Gly Ala Val Cys
275 280 285
Thr Glu Met Leu Asp Leu Tyr Gln Asn Glu Gly Ile Ile Ala Glu Pro
290 295 300
Ala Gly Ala Leu Ser Ile Ala Gly Leu Lys Glu Met Ser Phe Ala Pro
305 310 315 320
Gly Ser Val Val Val Cys Ile Ile Ser Gly Gly Asn Asn Asp Val Leu
325 330 335
Arg Tyr Ala Glu Ile Ala Glu Arg Ser Leu Val His Arg Gly Leu Lys
340 345 350
His Tyr Phe Leu Val Asn Phe Pro Gln Lys Pro Gly Gln Leu Arg His
355 360 365
Phe Leu Glu Asp Ile Leu Gly Pro Asp Asp Asp Ile Ala Leu Ala Glu
370 375 380
Tyr Leu Lys Arg Asn Asn Arg Glu Thr Gly Thr Ala Leu Val Gly Ile
385 390 395 400
His Leu Ser Glu Ala Ser Gly Leu Asp Ser Leu Leu Glu Arg Met Glu
405 410 415
Glu Ser Ala Ile Asp Ser Arg Arg Leu Glu Pro Gly Thr Pro Glu Tyr
420 425 430
Glu Tyr Leu Thr
435
<210> 54
<211> 1311
<212> DNA
<213> 人工序列
<220>
<223> ilvA_T381A+F383A_nt
<400> 54
atgagtgaaa catacgtgtc tgagaaaagt ccaggagtga tggctagcgg agcggagctg 60
attcgtgccg ccgacattca aacggcgcag gcacgaattt cctccgtcat tgcaccaact 120
ccattgcagt attgccctcg tctttctgag gaaaccggag cggaaatcta ccttaagcgt 180
gaggatctgc aggatgttcg ttcctacaag atccgcggtg cgctgaactc tggagcgcag 240
ctcacccaag agcagcgcga tgcaggtatc gttgccgcat ctgcaggtaa ccatgcccag 300
ggcgtggcct atgtgtgcaa gtccttgggc gttcagggac gcatctatgt tcctgtgcag 360
actccaaagc aaaagcgtga ccgcatcatg gttcacggcg gagagtttgt ctccttggtg 420
gtcactggca ataacttcga cgaagcatcg gctgcagcgc atgaagatgc agagcgcacc 480
ggcgcaacgc tgatcgagcc tttcgatgct cgcaacaccg tcatcggtca gggcaccgtg 540
gctgctgaga tcttgtcgca gctgacttcc atgggcaaga gtgcagatca cgtgatggtt 600
ccagtcggcg gtggcggact tcttgcaggt gtggtcagct acatggctga tatggcacct 660
cgcactgcga tcgttggtat cgaaccagcg ggagcagcat ccatgcaggc tgcattgcac 720
aatggtggac caatcacttt ggagactgtt gatccctttg tggacggcgc agcagtcaaa 780
cgtgtcggag atctcaacta caccatcgtg gagaagaacc agggtcgcgt gcacatgatg 840
agcgcgaccg agggcgctgt gtgtactgag atgctcgatc tttaccaaaa cgaaggcatc 900
atcgcggagc ctgctggcgc gctgtctatc gctgggttga aggaaatgtc ctttgcacct 960
ggttctgtcg tggtgtgcat catctctggt ggcaacaacg atgtgctgcg ttatgcggaa 1020
atcgctgagc gctccttggt gcaccgcggt ttgaagcact acttcttggt gaacttcccg 1080
caaaagcctg gtcagttgcg tcacttcctg gaagatatcc tgggaccgga tgatgacatc 1140
gcgctggcag agtacctcaa gcgcaacaac cgtgagaccg gtactgcgtt ggtgggtatt 1200
cacttgagtg aagcatcagg attggattct ttgctggaac gtatggagga atcggcaatt 1260
gattcccgtc gcctcgagcc gggcacccct gagtacgaat acttgaccta a 1311
<210> 55
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 55
aacacgaccg gcatcccgtc gc 22
<210> 56
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 56
aaatcatttg agaaaactcg agg 23
<210> 57
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 57
gtgaattcga gctcggtacc caaatcattt gagaaaactc gaggc 45
<210> 58
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 58
ggtgatcatc tcaacggtgg aacacaggtt gatgatcatt gggtt 45
<210> 59
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 59
aacccaatga tcatcaacct gtgttccacc gttgagatga tcacc 45
<210> 60
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 60
ggtcgactct agaggatccc caagaaggca acatcggaca gc 42
<210> 61
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 61
atccattcaa tggagtctgc g 21
<210> 62
<211> 616
<212> PRT
<213> 人工序列
<220>
<223> leuA_P247C+R558H+G561D
<400> 62
Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr
1 5 10 15
Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg
20 25 30
Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val
35 40 45
Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr
50 55 60
Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala
65 70 75 80
Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu
85 90 95
Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala
100 105 110
Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Gly Met
115 120 125
Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His
130 135 140
Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile
145 150 155 160
Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe
165 170 175
Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu
180 185 190
Leu Ile Lys Thr Ile Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp
195 200 205
Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys
210 215 220
Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn
225 230 235 240
Pro Met Ile Ile Asn Leu Cys Ser Thr Val Glu Met Ile Thr Pro Asn
245 250 255
Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg
260 265 270
Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly
275 280 285
Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu
290 295 300
Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val
305 310 315 320
Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp
325 330 335
Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln
340 345 350
Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr
355 360 365
Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala
370 375 380
Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp
385 390 395 400
Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp
405 410 415
Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser
420 425 430
Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly
435 440 445
Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln
450 455 460
Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp
465 470 475 480
Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln
485 490 495
Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser
500 505 510
Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly
515 520 525
Arg Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu
530 535 540
Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala His Thr Ser
545 550 555 560
Asp Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly
565 570 575
Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser
580 585 590
Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His
595 600 605
Glu Ala Val Leu Ala Gly Gly Val
610 615
<210> 63
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> leuA_P247C+R558H+G561D_nt
<400> 63
atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60
cgcaacgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120
tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180
aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240
ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300
ggcttcaaag aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360
cgtgagatca tcgaaaaggg catgatccct gacgatgtca ccattcaggt tctggttcag 420
gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480
gtgcacttct acaactccac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540
gtgcaggtga agaagctggc taccgatgcc gctgaactaa tcaagaccat cgctcaggat 600
tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660
gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720
ccaatgatca tcaacctgtg ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780
tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840
ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900
gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960
accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020
cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080
tacggcggtg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140
ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200
gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260
ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320
tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380
accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440
gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500
gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560
ggcaaggacg tcaccgtcga tggccgcggc aacggcccac tggccgctta cgccaacgcg 1620
ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccacacctcg 1680
gatgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740
ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800
aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851
Claims (13)
1.aroG醛缩酶(磷酸-2-脱氢-3-脱氧庚酸醛缩酶)变体,其中对应于SEQ ID NO:1的氨基酸序列中自N末端起第217、310、403和462位的任一个或多个氨基酸被另一种氨基酸取代。
2.根据权利要求1所述的变体,其中所述变体包含选自对应于SEQ ID NO:1的氨基酸序列中自N末端起下列位的氨基酸的一个或多个取代:
第217位的氨基酸被精氨酸之外的氨基酸取代;
第310位的氨基酸被赖氨酸之外的氨基酸取代;
第403位的氨基酸被精氨酸之外的氨基酸取代;以及
第462位的氨基酸被谷氨酸之外的氨基酸取代。
3.根据权利要求1所述的变体,其中被另一种氨基酸取代是被非极性氨基酸或小尺寸氨基酸取代。
4.根据权利要求1所述的变体,其中另一种氨基酸是丙氨酸(Ala)。
5.根据权利要求1所述的变体,其中所述aroG醛缩酶变体与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:23或SEQ ID NO:25具有99%以上的同源性或同一性。
6.根据权利要求1所述的变体,其中,与SEQ ID NO:1的aroG醛缩酶相比,所述aroG醛缩酶变体具有减弱的活性。
7.多核苷酸,其编码权利要求1~6之任一项所述的aroG醛缩酶变体。
8.载体,其含有权利要求7所述的多核苷酸。
9.棒杆菌属(Corynebacterium)微生物,其包含下列中的一种以上:
权利要求1~6之任一项所述的aroG醛缩酶变体,
编码所述aroG醛缩酶变体的多核苷酸;和
含有所述多核苷酸的载体。
10.根据权利要求9所述的微生物,其中所述棒杆菌属(Corynebacterium)微生物是谷氨酸棒杆菌(Corynebacterium glutamicum)。
11.生产支链氨基酸的方法,其包括在培养基中培养根据权利要求9所述的微生物。
12.根据权利要求11所述的方法,其中所述方法还包括从微生物或培养基中回收支链氨基酸。
13.根据权利要求12所述的方法,其中所述支链氨基酸是选自下列的一种以上:L-亮氨酸、L-异亮氨酸和L-缬氨酸。
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KR1020210010911A KR102495918B1 (ko) | 2021-01-26 | 2021-01-26 | aroG 알돌라아제 (Phospho-2-dehydro-3-deoxyheptonate aldolase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
KR10-2021-0010911 | 2021-01-26 | ||
PCT/KR2021/016966 WO2022163981A1 (ko) | 2021-01-26 | 2021-11-18 | Arog 알돌라아제 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
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US (1) | US20240093163A1 (zh) |
EP (1) | EP4249593A4 (zh) |
JP (1) | JP2024502989A (zh) |
KR (1) | KR102495918B1 (zh) |
CN (1) | CN116867895A (zh) |
AR (1) | AR124413A1 (zh) |
AU (1) | AU2021423180A1 (zh) |
CA (1) | CA3205759A1 (zh) |
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JP3185261B2 (ja) * | 1991-02-05 | 2001-07-09 | 味の素株式会社 | 発酵法による芳香族アミノ酸の製造法 |
JP3225597B2 (ja) * | 1992-06-15 | 2001-11-05 | 味の素株式会社 | 発酵法によるl−フェニルアラニンの製造法 |
SK18872001A3 (sk) * | 1999-06-25 | 2002-12-03 | Basf Aktiengesellschaft | Gény Corynebacterium glutamicum kódujúce proteíny zapojené do metabolizmu uhlíka a produkcie energie |
RU2229514C2 (ru) * | 2000-11-13 | 2004-05-27 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" | 3-дезокси-d-арабиногептулозонат-7-фосфатсинтаза и фрагмент днк, кодирующий 3-дезокси-d-арабиногептулозонат-7-фосфатсинтазу из methylophilus methylotrophus |
KR100620092B1 (ko) | 2004-12-16 | 2006-09-08 | 씨제이 주식회사 | 코리네박테리움 속 세포로부터 유래된 신규한 프로모터서열, 그를 포함하는 발현 카세트 및 벡터, 상기 벡터를포함하는 숙주 세포 및 그를 이용하여 유전자를 발현하는방법 |
CN101573438B (zh) | 2006-09-15 | 2013-01-02 | Cj第一制糖株式会社 | 具有增加的l-赖氨酸生产力的棒状杆菌和使用其生产l-赖氨酸的方法 |
JP2010110216A (ja) * | 2007-02-20 | 2010-05-20 | Ajinomoto Co Inc | L−アミノ酸または核酸の製造方法 |
DE102008040352A1 (de) * | 2008-07-11 | 2010-01-14 | Evonik Degussa Gmbh | Verfahren zur Herstellung von L-Tryptophan unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae |
KR101117022B1 (ko) | 2011-08-16 | 2012-03-16 | 씨제이제일제당 (주) | L-발린 생산능이 향상된 미생물 및 이를 이용한 l-발린 제조방법 |
KR101632642B1 (ko) | 2015-01-29 | 2016-06-22 | 씨제이제일제당 주식회사 | 신규한 프로모터 및 그의 용도 |
CN108559747B (zh) | 2016-08-31 | 2021-08-31 | Cj第一制糖株式会社 | 新型启动子及其应用 |
PL3858987T3 (pl) | 2016-12-28 | 2023-05-22 | Cj Cheiljedang Corporation | Nowy wariant syntazy izopropylojabłczanowej i sposób wytwarzania l-leucyny z jego wykorzystaniem |
KR101996769B1 (ko) | 2018-12-21 | 2019-10-01 | 씨제이제일제당 (주) | 변이형 호모세린 디하이드로게나제 및 이를 이용한 호모세린 또는 호모세린 유래 l-아미노산의 생산 방법 |
KR102143964B1 (ko) | 2019-12-06 | 2020-08-12 | 씨제이제일제당 주식회사 | 신규한 분지쇄 아미노산 아미노트랜스퍼라제 변이체 및 이를 이용한 류신 생산방법 |
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CA3205759A1 (en) | 2022-08-04 |
KR102495918B1 (ko) | 2023-02-06 |
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