CN114249803A - 一种高产精氨酸的工程菌及其构建方法与应用 - Google Patents
一种高产精氨酸的工程菌及其构建方法与应用 Download PDFInfo
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- CN114249803A CN114249803A CN202111676693.2A CN202111676693A CN114249803A CN 114249803 A CN114249803 A CN 114249803A CN 202111676693 A CN202111676693 A CN 202111676693A CN 114249803 A CN114249803 A CN 114249803A
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- arginine
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Abstract
本发明公开了一种高产精氨酸的工程菌及其构建方法与应用。本发明公开的YH66_03760突变体是将YH66_03760蛋白质第282位氨基酸残基由甘氨酸突变为精氨酸得到的蛋白质。本发明首先通过对YH66_03760基因进行单点突变得到YH66_03760G844A，然后通过对构建的YH66_03760或突变基因的过表达重组菌以及YH66_03760敲除重组菌进行发酵培养发现，YH66_03760基因或其突变基因可以调控细菌L‑精氨酸产量。本发明首次发现YH66_03760基因参与精氨酸的生物合成，对于培育符合工业化生产的高产、高质量菌种，以及精氨酸工业化生产具有重大的应用价值。
Description
技术领域
本发明属于生物技术领域，具体涉及一种高产精氨酸的工程菌及其构建方法与应用。
背景技术
L-精氨酸(L-argnine，简称L-Arg)是人体内所需半必需碱性氨基酸之一，作为一种含有胍基的碱性氨基酸，是生物体尿素循环的一种重要中间代谢物，具有多种独特的生理和药理作用，对于治疗生理功能、心血管疾病、激励免疫系统、维持婴儿的营养平衡、促进人体解毒等都具有良好的疗效，被专家称为是机体内运输和贮存氨基酸的重要载体，在肌内代谢中极为重要。它是合成胞浆蛋白和核蛋白的必需氨基酸；作为唯一的氨来源参与肌酸的合成；作为尿素循环的重要中间体，在肝脏中扮演着排除多余氨的角色，防止氨过量积累而引起中毒；它还具有调节人体免疫力的功能，可抑制肿瘤生长、促进受伤组织愈合等。并且，精氨酸是一氧化氮、尿素、鸟氨酸及肌丁胺的直接前体，是合成肌肉素的重要原素，且被用作聚胺、瓜氨酸及谷氨酰胺的合成。因此，L-精氨酸在医药、食品及化工领域方面具有重要而广泛的应用。据统计，目前全世界L-精氨酸需求量在15000吨以上，并且需求量以每年12％-15 ％的速度增长。
L-精氨酸的生产方法主要有两种：一是蛋白质水解提取法，二是微生物发酵法。水解法存在操作费时，收率和产量低，成本高等问题，并且存在严重的污染而不适合大规模的生产。发酵法生产L-精氨酸工艺相对简单，且对环境友好，因此具有很大的发展潜力，成为国内外氨基酸工业的一个重要趋势。但目前国内的微生物发酵生产L-精氨酸的产量普遍较低，远远不能满足国内需求。
基因工程技术对于精氨酸高产菌选育具有重要的推动作用，如何利用基因工程技术构建适合于工业规模生产的高产重组菌对于提高L-精氨酸产量具有重要意义。
发明内容
本发明所要解决的技术问题是如何利用YH66_03760基因构建重组载体、重组菌和/或提高L-精氨酸的产量。所要解决的技术问题不限于如所描述的技术主题，本领域技术人员通过以下描述可以清楚地理解本文未提及的其它技术主题。
为了解决上述技术问题，本发明首先提供了一种YH66_03760突变体。
本发明提供的YH66_03760突变体是将YH66_03760蛋白质第282位氨基酸残基由甘氨酸突变为其他氨基酸残基得到的蛋白质；
所述YH66_03760蛋白质为如下A1)-A3)中的任一种：
A1)SEQ ID No.2所示的氨基酸序列组成的蛋白质；
A2)将A1)所示的氨基酸序列经过除第282位氨基酸残基以外的一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与细菌产精氨酸相关的蛋白质；
A3)来源于细菌且与A1)或A2)具有95％以上同一性且与细菌产精氨酸相关的蛋白质。
上述A2)所述的蛋白质中，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述A3)所述的蛋白质中，这里使用的术语“同一性”指与天然氨基酸序列的序列相似性。“同一性”包括与本发明的SEQ ID No.2所示的氨基酸序列具有95％或更高，或96％或更高，或 97％或更高，或98％或更高，或99％或更高同一性的氨基酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述A1)或A2)或A3)所述的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
进一步的，所述YH66_03760突变体是将YH66_03760蛋白质第144位氨基酸残基由甘氨酸突变为精氨酸得到的蛋白质(对应本发明实施例中的YH66_03760G884A蛋白质)。
更进一步的，所述YH66_03760突变体(YH66_03760G884A蛋白质)是SEQ ID No.4所示的氨基酸序列组成的蛋白质。
为了解决上述技术问题，本发明又提供了与YH66_03760突变体相关的生物材料。
本发明提供的与YH66_03760突变体相关的生物材料为如下B1)至B4)中的任一种：
B1)编码上述YH66_03760突变体的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物。
为了解决上述技术问题，本发明还提供了上述YH66_03760蛋白质或与上述YH66_03760 蛋白质相关的生物材料或上述YH66_03760突变体或与上述YH66_03760突变体相关的生物材料的新用途。
本发明提供了上述YH66_03760蛋白质或与上述YH66_03760蛋白质相关的生物材料或上述YH66_03760突变体或与上述YH66_03760突变体相关的生物材料在如下X1)至X4)中任一种中的应用：
X1)调控细菌精氨酸产量；
X2)构建产精氨酸工程菌；
X3)制备精氨酸；
与YH66_03760蛋白质相关的生物材料为如下D1)至D4)中的任一种：
D1)编码所述YH66_03760蛋白质的核酸分子；
D2)含有D1)所述核酸分子的表达盒；
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体；
D4)含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物。
上述生物材料或应用中，B1)所述编码YH66_03760突变体的核酸分子为如下C1)或C2)中的任一种：
C1)核苷酸序列为SEQ ID No.3的DNA分子；
C2)将SEQ ID No.3所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与C1)所示的DNA分子具有90％以上的同一性，且具有相同功能的DNA 分子。
D1)所述编码YH66_03760蛋白质的核酸分子为如下E1)或E2)中的任一种：
E1)核苷酸序列为SEQ ID No.1的DNA分子；
E2)将SEQ ID No.1所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与E1)所示的DNA分子具有90％以上的同一性，且具有相同功能的DNA分子。
其中，SEQ ID No.1所示的DNA分子为谷氨酸棒杆菌(Corynebacteriumglutamicum) CGMCC No.20516中的YH66_03760基因，其编码的YH66_03760蛋白质的氨基酸序列如SEQ ID No.2所示。在本发明中通过引入点突变，得到SEQ ID No.3所示的YH66_03760G844A基因，其编码的YH66_03760G884A蛋白质的氨基酸序列如SEQ ID No.4所示。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码YH66_03760蛋白质或YH66_03760突变体的核苷酸序列进行突变。那些经过人工修饰的，具有编码YH66_03760蛋白质或YH66_03760突变体的核苷酸序列90％或者更高同一性的核苷酸，只要编码YH66_03760蛋白质或YH66_03760突变体且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码SEQ ID No.2或SEQ ID No.4所示的氨基酸序列组成的蛋白质的核苷酸序列具有90％或更高，或91％或更高，或92％或更高，或93％或更高，或94％或更高，或95％或更高，或96％或更高，或97％或更高，或98％或更高，或99％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比 (％)表示，其可以用来评价相关序列之间的同一性。
所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min；或，0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE (或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述生物材料或应用中，B2)所述含有编码YH66_03760突变体的核酸分子的表达盒是指能够在宿主细胞中表达YH66_03760突变体的DNA，该DNA不但可包括启动YH66_03760突变体基因转录的启动子，还可包括终止YH66_03760突变体基因转录的终止子。进一步，所述表达盒还可包括增强子序列。D2)所述含有编码YH66_03760蛋白质的核酸分子的表达盒是指能够在宿主细胞中表达YH66_03760蛋白质的DNA，该DNA不但可包括启动 YH66_03760基因转录的启动子，还可包括终止YH66_03760基因转录的终止子。进一步，所述表达盒还可包括增强子序列。
上述生物材料或应用中，B3)或D3)所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒具体可为pK18mobsacB质粒或pXMJ19质粒。
在本发明的一个具体实施例中，所述重组载体为重组载体pK18-YH66_03760G844A。
在本发明的另一个具体实施例中，所述重组载体为重组载体pK18-YH66_03760OE或重组载体pK18-YH66_03760G844AOE。
在本发明的又一个具体实施例中，所述重组载体为重组载体pXMJ19-YH66_03760或重组载体pXMJ19-YH66_03760G844A。
上述生物材料中，B4)或D4)所述微生物可为酵母、细菌、藻或真菌。
进一步的，细菌可为任一具有产精氨酸能力的细菌，如来自短杆菌属(Brevibacterium)、棒杆菌属(Corynebacterium)、埃希氏菌属(Escherichia)、气杆菌属(Aerobacter)、微球菌属(Micrococcus)、黄杆菌属(Flavobacterium)或芽胞杆菌属(Bacillus)的细菌等。
更进一步的，所述细菌包括但不限于谷氨酸棒杆菌(Corynebacteriumglutamicum)、黄色短杆菌(Brevibacterium flavum)、乳酸发酵短杆菌(Brevibacteriumlactofermentum)、产谷氨酸微球菌(Micrococcus glutamicus)、产氨短杆菌(Brevibacterum ammoniagenes)、大肠杆菌(Escherichia coli)、产气气杆菌(Aerobacteraerogenes)。
在本发明的一个具体实施例中，所述微生物为谷氨酸棒杆菌(Corynebacteriumglutamicum)CGMCC No.20516，该菌株名称为YPARG01，已于2020年8月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏登记号为CGMCC No.20516。
上述应用中，调控为正调控。具体体现为当细菌中YH66_03760蛋白质或YH66_03760 突变体含量或活性提高时，所述细菌精氨酸产量提高；当细菌中YH66_03760蛋白质含量或活性降低时，所述细菌精氨酸产量降低。
为了解决上述技术问题，本发明还提供了提高YH66_03760蛋白质或YH66_03760突变体含量和/或活性的物质或提高YH66_03760基因或YH66_03760突变体基因表达量的物质的新用途。
本发明提供了提高YH66_03760蛋白质或YH66_03760突变体含量和/或活性的物质或提高YH66_03760基因或YH66_03760突变体基因表达量的物质在如下Y1)至Y4)中任一种中的应用：
Y1)提高细菌精氨酸产量；
Y2)构建产精氨酸工程菌；
Y3)制备精氨酸。
进一步的，所述提高YH66_03760基因表达量的物质可为YH66_03760基因或含有所述YH66_03760基因的重组载体。
所述提高YH66_03760突变体基因表达量的物质可为YH66_03760突变体基因或含有所述YH66_03760突变体基因的重组载体。
更进一步的，含有所述YH66_03760基因的重组载体具体可为重组载体pK18-YH66_03760OE或重组载体pXMJ19-YH66_03760。
含有所述YH66_03760突变体基因的重组载体具体可为重组载体pK18-YH66_03760G844A OE或重组载体pXMJ19-YH66_03760G844A。
为了解决上述技术问题，本发明还提供了一种提高细菌精氨酸产量的方法。
本发明提供的提高细菌精氨酸产量的方法为如下M1)或M2)：
所述M1)包括如下步骤：将细菌基因组中的YH66_03760基因替换为YH66_03760突变体基因，实现细菌精氨酸产量的提高；
所述M2)包括如下步骤：提高细菌中YH66_03760蛋白质或YH66_03760突变体含量和/ 或活性，或提高细菌中YH66_03760基因或YH66_03760突变体基因表达量，实现细菌精氨酸产量的提高。
为了解决上述技术问题，本发明还提供了一种产精氨酸工程菌的构建方法。
本发明提供的产精氨酸工程菌的构建方法为如下N1)或N2)：
所述N1)包括如下步骤：将细菌基因组中的YH66_03760基因替换为YH66_03760突变体基因，得到所述产精氨酸工程菌；
所述N2)包括如下步骤：提高细菌中YH66_03760蛋白质或YH66_03760突变体含量和/ 或活性，或提高细菌中YH66_03760基因或YH66_03760突变体基因表达量，得到所述产精氨酸工程菌；
上述任一所述应用或方法中，所述YH66_03760突变体具体为YH66_03760G884A蛋白质，具体为SEQ ID No.4所示的氨基酸序列组成的蛋白质。
所述YH66_03760突变体基因具体为YH66_03760G844A基因，具体为SEQ ID No.3所示的 DNA分子。
按照上述产精氨酸工程菌的构建方法构建得到的产精氨酸工程菌在制备精氨酸中的应用也属于本发明的保护范围。
为了解决上述技术问题，本发明最后提供了一种制备精氨酸的方法。
本发明提供的制备精氨酸的方法包括如下步骤：发酵培养按照上述产精氨酸工程菌的构建方法构建得到的产精氨酸工程菌，得到所述精氨酸。
所述发酵培养方法可按照现有技术中的常规试验方法进行。也可采用优化和改进后的常规试验方法进行。所述发酵培养条件如实施例中的表1所示。
上述任一所述应用或方法中，所述精氨酸具体为L-精氨酸。
本发明首先通过对YH66_03760基因进行单点突变得到YH66_03760G844A基因，然后通过对构建的YH66_03760或突变基因的过表达重组菌以及YH66_03760敲除重组菌进行发酵培养发现，YH66_03760基因或其突变基因可以调控细菌L-精氨酸产量。本发明首次发现YH66_03760基因参与精氨酸的生物合成，对于培育符合工业化生产的高产、高质量菌种，以及精氨酸工业化生产具有重大的应用价值。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、构建包含点突变的YH66_03760基因编码区的重组载体
依据NCBI公布的黄色短杆菌(Brevibacterium flavum)ATCC15168基因组序列，设计并合成两对扩增YH66_03760基因编码区的引物，以等位基因置换的方式在谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC 20516(经测序确认该菌株染色体上保留有野生型的 YH66_03760基因)的YH66_03760基因编码区(SEQ ID No.1)中引入点突变，所述点突变为将YH66_03760基因的核苷酸序列(SEQ ID No.1)中的第844位鸟嘌呤(G)突变为腺嘌呤(A)，得到SEQ ID No.3所示的DNA分子(突变的YH66_03760基因，名称为 YH66_03760G844A)。
其中，SEQ ID No.1所示的DNA分子编码氨基酸序列为SEQ ID No.2的蛋白质(所述蛋白质名称为蛋白质YH66_03760)。
SEQ ID No.3所示的DNA分子编码氨基酸序列为SEQ ID No.4的突变蛋白质(所述突变蛋白质名称为YH66_03760G884A)。所述突变蛋白质YH66_03760G884A氨基酸序列(SEQ IDNo.4)中的第282位精氨酸(R)由甘氨酸(G)突变而来。
采用NEBuilder重组技术进行基因定点突变，引物设计如下(上海invitrogen公司合成)，加粗字体的碱基为突变位置：
P1:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGATTGGTACTGAAGGCT CACC-3'，
P2:5'-AAGAATTCCACGGTTCTCGCGCCCTGGTAACCAATGG-3'，
P3:5'-CCATTGGTTACCAGGGCGCGAGAACCGTGGAATTCTT-3'，
P4:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGCAGATCCTTGAT GTTGGG-3'。
构建方法：以黄色短杆菌ATCC15168为模板，分别采用引物P1/P2和P3/P4进行PCR扩增，获得两条分别带有突变碱基，大小分别为716bp和735bp的YH66_03760基因编码区的DNA片段(YH66_03760Up和YH66_03760Down)。
PCR扩增体系为：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM) 4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
PCR扩增反应程序为：94℃预变性5min，(94℃变性30s；52℃退火30s；72℃延伸40s；30个循环)，72℃过度延伸10min。
将上述两条DNA片段(YH66_03760Up和YH66_03760Down)经琼脂糖凝胶电泳分离纯化后，与经过酶切(Xbal I/BamH I)后纯化的pK18mobsacB质粒(购自Addgene公司，用 XbalI/BamH I酶切)用NEBuilder酶(购自NEB公司)50℃连接30min，连接产物转化后长出的单克隆经PCR鉴定获得阳性重组载体pK18-YH66_03760G844A，该重组载体上含有卡那霉素抗性标记。将酶切正确的重组载体pK18-YH66_03760G844A送测序公司测序鉴定，并将含有正确点突变(G-A)的重组载体pK18-YH66_03760G844A保存备用。
重组载体pK18-YH66_03760G844A中YH66_03760G844A Up-Down DNA片段(YH66_03760Up-Down，SEQ ID No.5)大小为1414bp，由于含有突变位点，导致菌株谷氨酸棒杆菌CGMCC20516中YH66_03760基因编码区的第844位鸟嘌呤(G)变为腺嘌呤(A)，最终导致编码蛋白的第282位甘氨酸(G)变为精氨酸(R)。
所述重组载体pK18-YH66_03760G844A是将pK18mobsacB载体的Xbal I和/BamH I识别位点间的片段(小片段)替换为序列表中SEQ ID No.5的第37-1376位所示的DNA片段，且保持pK18mobsacB载体的其他序列不变，得到的重组载体。
所述重组载体pK18-YH66_03760G844A含有SEQ ID No.3所示的突变基因 YH66_03760G844A的第181-1520位所示的DNA分子。
实施例2、构建包含基因YH66_03760G844A的工程菌株YPR-007
构建方法：将实施例1中的等位替换质粒(pK18-YH66_03760G844A)通过电击转化入谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC 20516中后，在培养基中进行培养，培养基配方如下：溶剂为水，溶质及其浓度分别如下：蔗糖10g/L、多聚蛋白胨10g/L、牛肉膏10g/L、酵母粉5g/L、尿素2g/L、氯化钠2.5g/L、琼脂粉20g/L，pH7.0；培养条件如下： 32℃。对培养产生的单菌落分别通过实施例1中的引物P1和通用引物M13R进行鉴定，能扩增出1421bp大小条带的菌株为阳性菌株。将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落分别在含有卡那霉素和不含卡那霉素的培养基上培养，选择在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用如下引物(上海invitrogen公司合成)进行PCR鉴定：
P5:5'-GTCACCAAAAAGTTGTCGAA-3'；
P6:5'-GGTTGCACCAGCAGCCAAGC-3'。
将得到的PCR扩增产物(260bp)通过95℃高温变性10min、冰浴5min后进行SSCP(Single-Strand Conformation Polymorphis)电泳(以质粒pK18-YH66_03760G844A扩增片段为阳性对照，黄色短杆菌ATCC15168扩增片段为阴性对照，水作为空白对照)，SSCP电泳的PAGE的制备成分及用量分别如下：40％丙烯酰胺(丙烯酰胺终浓度为8％)8mL、ddH2O 26mL、甘油4mL、10×TBE 2mL、TEMED 40μL、10％APS 600μL，电泳条件如下：将电泳槽置入冰中，使用1×TBE缓冲液，电压120V，电泳时间10h。由于片段结构不同，电泳位置不同，因此片段电泳位置与阴性对照片段位置不一致且与阳性对照片段位置一致的菌株为等位替换成功的菌株。再次通过引物P5/P6 PCR扩增阳性菌株YH66_03760基因片段，并连接到PMD19-T载体进行测序，通过序列比对，碱基序列发生突变(G-A)的菌株为等位替换成功的阳性菌株，并被命名为YPR-007。
重组菌YPR-007是将谷氨酸棒杆菌CGMCC 20516基因组中的YH66_03760基因进行单点突变(对应于SEQ ID No.1所示YH66_03760基因的第844位的碱基G突变为碱基A)，且保持谷氨酸棒杆菌CGMCC 20516的基因组中的其它序列不变得到的重组菌。
实施例3、构建基因组上过表达YH66_03760基因或YH66_03760G844A基因的工程菌株YPR-008和YPR-009
依据NCBI公布的黄色短杆菌ATCC15168基因组序列，设计并合成三对扩增上下游同源臂片段及YH66_03760或YH66_03760G844A基因编码区及启动子区的引物，以同源重组的方式在谷氨酸棒杆菌CGMCC 20516中引入YH66_03760或YH66_03760G844A基因。
引物设计如下(上海invitrogen公司合成)：
P7:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCATGACGGCTGACTGG ACTC-3'；
P8:5'-AAAATCTAAGACTCGGAAAAAATCGGACTCCTTAAATGGG-3'；
P9:5'-CCCATTTAAGGAGTCCGATTTTTTCCGAGTCTTAGATTTT-3'；
P10:5'-CTATGTGAGTAGTCGATTTATTAGGAAACGACGACGATCA-3'；
P11:5'-TGATCGTCGTCGTTTCCTAATAAATCGACTACTCACATAG-3'；
P12:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCATAAGAAACAA CCACTT-3'。
构建方法：分别以黄色短杆菌ATCC15168或YPR-007为模板，分别采用引物P7/P8、P9/P10和P11/P12进行PCR扩增，获得上游同源臂片段806bp(对应于谷氨酸棒杆菌CGMCC20516YH66_03350基因及其启动子区或者是与上一个基因的间隔区，序列如SEQ ID No.6所示)，YH66_03760基因及其启动子片段3858bp(序列如SEQ ID No.7所示)或YH66_03760G844A基因及其启动子片段3858bp(序列如SEQ ID No.8所示)及下游同源臂片段783bp(对应于谷氨酸棒杆菌CGMCC 20516YH66_03355基因及其与YH66_03350基因的部分间隔区，序列如SEQID No.9所示)。PCR反应结束后，对每个模板扩增得到的3个片段采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3个片段与经过Xbal I/BamH I酶切后纯化的 pK18mobsacB质粒(购自Addgene公司)用NEBuilder酶(购自NEB公司)50℃连接30min，连接产物转化后长出的单克隆用M13引物经PCR鉴定获得阳性整合质粒(重组载体)，分别为pK18-YH66_03760OE、pK18-YH66_03760G844AOE，该阳性整合质粒上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得质粒整合到基因组上的重组子。
PCR反应体系为：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM) 4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
PCR反应程序为：94℃预变性5min，94℃变性30s；52℃退火30s；72℃延伸60s (30个循环)，72℃过度延伸10min。
将测序正确的整合质粒(pK18-YH66_03760OE、pK18-YH66_03760G844AOE)分别电转化入谷氨酸棒杆菌CGMCC 20516，在培养基中进行培养，对培养产生的单菌落通过P13/P14引物进行PCR鉴定，PCR扩增出大小2245bp的片段的菌株为阳性菌株，扩增不到片段的菌株为原菌。将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落进一步采用P15/P16 引物进行PCR鉴定，PCR扩增出大小为3453bp的片段的菌株为YH66_03760或 YH66_03760G844A基因整合到谷氨酸棒杆菌CGMCC 20516基因组上同源臂YH66_03350和下同源臂YH66_03355的间隔区上的阳性菌株，分别命名为YPR-008(不含突变点)和YPR-009 (含突变点)。
PCR鉴定引物如下所示：
P13:5'-GTCCGCTCTGTTGGTGTTCA-3'(对应上同源臂YH66_03350的外侧)；
P14:5'-CTTATCTTGGGTCAGACCCA-3'(对应YH66_03760基因内部)；
P15:5'-CACAGCATTTGGATCCAGAA-3'(对应YH66_03760基因内部)；
P16:5'-TGGAGGAATATTCGGCCCAG-3'(对应下同源臂YH66_03355的外侧)。
重组菌YPR-008含有双拷贝的SEQ ID No.1所示的YH66_03760基因；具体地，重组菌 YPR-008是将谷氨酸棒杆菌CGMCC 20516的基因组中上同源臂YH66_03350和下同源臂YH66_03355的间隔区替换为YH66_03760基因，保持谷氨酸棒杆菌CGMCC 20516的基因组中的其它核苷酸不变得到的重组菌。含有双拷贝YH66_03760基因的重组菌可以显著和稳定地提高YH66_03760基因的表达量。重组菌YPR-009含有SEQ ID No.3所示的突变的 YH66_03760G844A基因；具体地，重组菌YPR-009是将谷氨酸棒杆菌CGMCC 20516的基因组中上同源臂YH66_03350和下同源臂YH66_03355的间隔区替换为YH66_03760G844A基因，保持谷氨酸棒杆菌CGMCC 20516的基因组中的其它核苷酸不变得到的重组菌。
实施例4、构建质粒上过表达YH66_03760基因或YH66_03760G844A基因的工程菌株YPR-010和YPR-011
依据NCBI公布的黄色短杆菌ATCC15168基因组序列，设计并合成一对扩增YH66_03760 或YH66_03760G844A基因编码区及启动子区的引物，引物设计如下(上海invitrogen公司合成)：
P17:5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTTTCCGAGTCTTAGAT TTT-3'(带下划线的核苷酸序列为pXMJ19上的序列)，
P18:5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTAGGAAACGACGACGAT CA-3'(带下划线的核苷酸序列为pXMJ19上的序列)。
构建方法：分别以黄色短杆菌ATCC15168或YPR-007为模板，以引物P17/P18进行PCR 扩增，获得YH66_03760基因及其启动子片段3888bp和YH66_03760G844A基因及其启动子片段3888bp，对扩增产物进行电泳并采用柱式DNA凝胶回收试剂盒进行纯化回收，回收的DNA 片段与经EcoR I酶切回收的穿梭质粒pXMJ19用NEBuilder酶(购自NEB公司)50℃连接30min，连接产物转化后长出的单克隆用M13引物经PCR鉴定获得阳性过表达质粒 pXMJ19-YH66_03760(含有YH66_03760基因)和pXMJ19-YH66_03760G844A(含有 YH66_03760G844A基因)，将该质粒送测序。因质粒上含有氯霉素抗性标记，可以通过氯霉素来筛选质粒是否转化到菌株中。
PCR反应体系为：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM) 4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
PCR反应程序为：94℃预变性5min，94℃变性30s；52℃退火30s；72℃延伸60s (30个循环)，72℃过度延伸10min。
将测序正确的pXMJ19-YH66_03760和pXMJ19-YH66_03760G844A质粒分别电转化入谷氨酸棒杆菌CGMCC 20516中，在培养基中进行培养，对培养产生的单菌落通过引物M13R(-48) /P18进行PCR鉴定，PCR扩增出大小为3927bp的片段的菌株为阳性菌株，其被命名为YPR-010(不含突变点)和YPR-011(含突变点)。
重组菌YPR-010含有SEQ ID No.1所示的YH66_03760基因，是通过质粒表达 YH66_03760基因的重组菌。重组菌YPR-011含有SEQ ID No.3所示的突变的 YH66_03760G844A基因，是通过质粒表达YH66_03760G844A基因的重组菌。
实施例5、构建基因组上缺失YH66_03760基因的工程菌株YPR-012
根据NCBI公布的黄色短杆菌ATCC15168的基因组序列，合成两对扩增YH66_03760基因编码区两端片段的引物，作为上下游同源臂片段。引物设计如下(上海invitrogen公司合成)：
P19:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCCTTTGGCTACTAAC CCAC-3'，
P20:5'-GGGGCTTTTTACAGAAAGGTTAGAGTAATTGTTCCTTTCA-3'，
P21:5'-TGAAAGGAACAATTACTCTAACCTTTCTGTAAAAAGCCCC-3'，
P22:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTCTATTTGCTTATCG ACGT-3'。
构建方法：以黄色短杆菌ATCC15168为模板，分别采用引物P19/P20和P21/P22进行PCR 扩增，获得YH66_03760的上游同源臂片段716bp及YH66_03760的下游同源臂片段755bp。对扩增的产物进行电泳并采用柱式DNA凝胶回收试剂盒进行纯化，回收的DNA片段与经过 Xbal I/BamH I酶切后纯化的pK18mobsacB质粒(购自Addgene公司)用NEBuilder酶(购自 NEB公司)50℃连接30min，连接产物转化后长出的单克隆用M13引物经PCR鉴定获得阳性敲除载体pK18-ΔYH66_03760，该质粒含有整个敲除YH66_03760同源臂片段1431bp(序列如SEQ ID No.10所示)和卡那霉素抗性作为筛选标记，将该质粒送测序。
PCR扩增反应体系为：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL。
PCR扩增反应程序为：94℃预变性5min，94℃变性30s；52℃退火30s；72℃延伸 90s(30个循环)，72℃过度延伸10min。
将测序正确的敲除质粒pK18-ΔYH66_03760电转化入谷氨酸棒杆菌CGMCC 20516，在培养基中进行培养，对培养产生的单菌落通过如下引物(上海invitrogen公司合成)进行PCR 鉴定：
P23:5'-TCCTTTGGCTACTAACCCAC-3'(对应于谷氨酸棒杆菌CGMCC 20516 YH66_03755基因内部)；
P24:5'-GTCTATTTGCTTATCGACGT-3'(对应于谷氨酸棒杆菌CGMCC 20516 YH66_03765 基因内部)。
上述PCR同时扩增出大小1357bp及4780bp的条带的菌株为阳性菌株，只扩增出4780bp 条带的菌株为原菌。阳性菌株在15％蔗糖培养基上筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养，选择在不含卡那霉素的培养基上生长，而在含卡那霉素的培养基上不生长的菌株进一步采用P23/P24引物进行PCR鉴定，扩增出大小为1357bp条带的菌株为YH66_03760基因编码区被敲除的阳性菌株。再次通过P23/P24引物PCR扩增阳性菌株YH66_03760片段，并连接到pMD19-T载体进行测序，将测序正确的菌株命名为YPR-012(谷氨酸棒杆菌CGMCC 20516上的基因组上的YH66_03760基因被敲除)。
实施例6、L-精氨酸发酵实验
将上述实施例构建的菌株(YPR-007、YPR-008、YPR-009、YPR-010、YPR-011和YPR-012) 和原始菌株谷氨酸棒杆菌CGMCC 20516在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表1所示的控制工艺进行发酵实验。每个菌株重复三次。完成发酵后，收集上清，采用HPLC检测上清中的L-精氨酸产量。发酵培养基配方如下：溶剂为水，溶质及其浓度分别如下：硫酸铵14g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，硫酸镁0.5g/L，酵母粉2g/L，硫酸亚铁18mg/L，硫酸锰4.2mg/L，生物素0.02mg/L，维生素B1 2mg/L， antifoam(CB-442)消泡0.5mL/L，70％葡萄糖(底糖)40g/L。
结果如表2所示，在谷氨酸棒杆菌中对YH66_03760基因编码区进行点突变 YH66_03760G844A及过表达，有助于L-精氨酸产量及转化率的提高，而对基因进行敲除或弱化，不利于L-精氨酸的积累。
表1、发酵控制工艺
表2、L-精氨酸发酵实验结果
| 菌株 | OD<sub>562nm</sub> | L-精氨酸产量(g/L) |
| 谷氨酸棒杆菌CGMCC 20516 | 74.6 | 86.5 |
| YPR-007 | 77.9 | 89.9 |
| YPR-008 | 78.0 | 89.0 |
| YPR-009 | 80.1 | 90.2 |
| YPR-010 | 79.2 | 89.7 |
| YPR-011 | 80.8 | 90.6 |
| YPR-012 | 73.8 | 87.1 |
序列表
<110> 宁夏伊品生物科技股份有限公司
<120> 一种高产精氨酸的工程菌及其构建方法与应用
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 3423
<212> DNA
<213> Artificial Sequence
<400> 1
gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180
attggtactg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240
gctaaaaaag ttaaagcaga tgctatttac ccgggatatg gcttcctgtc tgaaaatgcc 300
cagcttgccc gcgagtgcgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360
cttgatctca ccggtgataa gtctcgtgcg gtaaccgccg cgaagaaggc tggtctgcca 420
gttttggcgg aatccacccc gagcaaaaac atcgatgaca tcgttaaaag cgctgaaggc 480
cagacttacc ccatctttgt aaaggcagtt gccggtggtg gcggacgcgg tatgcgcttt 540
gtttcttcac ctgatgagct ccgcaaattg gcaacagaag catctcgtga agctgaagcg 600
gcattcggcg acggttcggt atatgtcgaa cgtgctgtga ttaaccccca gcacattgaa 660
gtgcagatcc ttggcgatcg cactggagaa gttgtacacc tttatgaacg tgactgctca 720
ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780
ctgcgtgatc gcatttgcgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840
gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgttttcat cgaaatgaac 900
ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960
gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020
atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080
ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140
cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200
aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260
gctgagttca ccgtgtctgg tgttgcaacc aacattggct tcttgcgcgc gctgctgcgg 1320
gaagaggact tcacttccaa gcgcatcgcc accggattta tcggcgatca cccacacctc 1380
cttcaggctc cacctgcgga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440
accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagcaccaat cgataagctg 1500
cccaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560
ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620
ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680
gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740
gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800
ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860
ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccacctcc 1920
ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980
atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040
gatctctctg atccaaatga aaagctctac accctggatt actacctgaa gatggcagag 2100
gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160
ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacaccgc gggtggccag ctggctacct actttgctgc agctcaagct 2280
ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340
ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400
gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460
tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagctg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgctg atcgcttcga gctcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctg gttggtgcgg gtgtagatcc agcagacttt 2700
gctgcagacc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760
cttggtaacc ctccaggtgg ctggccagaa ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880
gatgattcca aggaacgtcg caacagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000
ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060
ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120
gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180
accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240
ggtgttgtca ctgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300
atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420
taa 3423
<210> 2
<211> 1140
<212> PRT
<213> Artificial Sequence
<400> 2
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Asp Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ser Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ser Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp Arg Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Gly Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Thr Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 3
<211> 3423
<212> DNA
<213> Artificial Sequence
<400> 3
gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180
attggtactg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240
gctaaaaaag ttaaagcaga tgctatttac ccgggatatg gcttcctgtc tgaaaatgcc 300
cagcttgccc gcgagtgcgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360
cttgatctca ccggtgataa gtctcgtgcg gtaaccgccg cgaagaaggc tggtctgcca 420
gttttggcgg aatccacccc gagcaaaaac atcgatgaca tcgttaaaag cgctgaaggc 480
cagacttacc ccatctttgt aaaggcagtt gccggtggtg gcggacgcgg tatgcgcttt 540
gtttcttcac ctgatgagct ccgcaaattg gcaacagaag catctcgtga agctgaagcg 600
gcattcggcg acggttcggt atatgtcgaa cgtgctgtga ttaaccccca gcacattgaa 660
gtgcagatcc ttggcgatcg cactggagaa gttgtacacc tttatgaacg tgactgctca 720
ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780
ctgcgtgatc gcatttgcgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840
gcgagaaccg tggaattctt ggtcgatgaa aagggcaacc acgttttcat cgaaatgaac 900
ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960
gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020
atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080
ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140
cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200
aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260
gctgagttca ccgtgtctgg tgttgcaacc aacattggct tcttgcgcgc gctgctgcgg 1320
gaagaggact tcacttccaa gcgcatcgcc accggattta tcggcgatca cccacacctc 1380
cttcaggctc cacctgcgga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440
accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagcaccaat cgataagctg 1500
cccaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560
ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620
ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680
gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740
gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800
ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860
ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccacctcc 1920
ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980
atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040
gatctctctg atccaaatga aaagctctac accctggatt actacctgaa gatggcagag 2100
gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160
ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacaccgc gggtggccag ctggctacct actttgctgc agctcaagct 2280
ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340
ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400
gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460
tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagctg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgctg atcgcttcga gctcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctg gttggtgcgg gtgtagatcc agcagacttt 2700
gctgcagacc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760
cttggtaacc ctccaggtgg ctggccagaa ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880
gatgattcca aggaacgtcg caacagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000
ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060
ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120
gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180
accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240
ggtgttgtca ctgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300
atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420
taa 3423
<210> 4
<211> 1140
<212> PRT
<213> Artificial Sequence
<400> 4
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
1 5 10 15
Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
20 25 30
Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly
35 40 45
Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu
50 55 60
Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala
65 70 75 80
Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu
85 90 95
Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr
100 105 110
Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser
115 120 125
Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu
130 135 140
Ser Thr Pro Ser Lys Asn Ile Asp Asp Ile Val Lys Ser Ala Glu Gly
145 150 155 160
Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg
165 170 175
Gly Met Arg Phe Val Ser Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr
180 185 190
Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ser Val Tyr
195 200 205
Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu
210 215 220
Gly Asp Arg Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser
225 230 235 240
Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His
245 250 255
Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe
260 265 270
Cys Arg Ser Ile Gly Tyr Gln Gly Ala Arg Thr Val Glu Phe Leu Val
275 280 285
Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln
290 295 300
Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys
305 310 315 320
Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu
325 330 335
Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile
340 345 350
Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile
355 360 365
Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala
370 375 380
Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val
385 390 395 400
Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala
405 410 415
Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile
420 425 430
Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg
435 440 445
Ile Ala Thr Gly Phe Ile Gly Asp His Pro His Leu Leu Gln Ala Pro
450 455 460
Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
465 470 475 480
Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
485 490 495
Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
500 505 510
Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
515 520 525
Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala
530 535 540
His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro
545 550 555 560
Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu
565 570 575
Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu
580 585 590
Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val
595 600 605
Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro
610 615 620
Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Thr Ser
625 630 635 640
Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
645 650 655
Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala
660 665 670
Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys
675 680 685
Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys
690 695 700
Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg
705 710 715 720
Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp
725 730 735
Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala
740 745 750
Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala
755 760 765
Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile
770 775 780
Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu
785 790 795 800
Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr
805 810 815
Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg
820 825 830
His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr
835 840 845
Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala
850 855 860
Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser
865 870 875 880
Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp
885 890 895
Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser
900 905 910
Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp
915 920 925
Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys
930 935 940
Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala
945 950 955 960
Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro
965 970 975
Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr
980 985 990
Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg
995 1000 1005
Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val
1010 1015 1020
Arg Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn
1025 1030 1035
Val Val Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg
1040 1045 1050
Asp Arg Ser Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp
1055 1060 1065
Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
1070 1075 1080
Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
1085 1090 1095
Val Ala Ile Ile Glu Ala Met Lys Met Glu Ala Thr Ile Thr Ala
1100 1105 1110
Ser Val Asp Gly Lys Ile Asp Arg Val Val Val Pro Ala Ala Thr
1115 1120 1125
Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser
1130 1135 1140
<210> 5
<211> 1414
<212> DNA
<213> Artificial Sequence
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctagattg gtactgaagg ctcaccagtc 60
aaggcgtacc tggacatcga tgaaattatc ggtgcagcta aaaaagttaa agcagatgct 120
atttacccgg gatatggctt cctgtctgaa aatgcccagc ttgcccgcga gtgcgcggaa 180
aacggcatta cttttattgg cccaacccca gaggttcttg atctcaccgg tgataagtct 240
cgtgcggtaa ccgccgcgaa gaaggctggt ctgccagttt tggcggaatc caccccgagc 300
aaaaacatcg atgacatcgt taaaagcgct gaaggccaga cttaccccat ctttgtaaag 360
gcagttgccg gtggtggcgg acgcggtatg cgctttgttt cttcacctga tgagctccgc 420
aaattggcaa cagaagcatc tcgtgaagct gaagcggcat tcggcgacgg ttcggtatat 480
gtcgaacgtg ctgtgattaa cccccagcac attgaagtgc agatccttgg cgatcgcact 540
ggagaagttg tacaccttta tgaacgtgac tgctcactgc agcgtcgtca ccaaaaagtt 600
gtcgaaattg cgccagcaca gcatttggat ccagaactgc gtgatcgcat ttgcgcggat 660
gcagtaaagt tctgccgctc cattggttac cagggcgcga gaaccgtgga attcttggtc 720
gatgaaaagg gcaaccacgt tttcatcgaa atgaacccac gtatccaggt tgagcacacc 780
gtgactgaag aagtcaccga ggtggacctg gtgaaggcgc agatgcgctt ggctgctggt 840
gcaaccttga aggaattggg tctgacccaa gataagatca agacccacgg tgcagcactg 900
cagtgccgca tcaccacgga agatccaaac aacggcttcc gcccagatac cggaactatc 960
accgcgtacc gctcaccagg cggagctggc gttcgtcttg acggtgcagc tcagctcggt 1020
ggcgaaatca ccgcacactt tgactccatg ctggtgaaaa tgacctgccg tggttccgac 1080
tttgaaactg ctgttgctcg tgcacagcgc gcgttggctg agttcaccgt gtctggtgtt 1140
gcaaccaaca ttggcttctt gcgcgcgctg ctgcgggaag aggacttcac ttccaagcgc 1200
atcgccaccg gatttatcgg cgatcaccca cacctccttc aggctccacc tgcggatgat 1260
gagcagggac gcatcctgga ttacttggca gatgtcaccg tgaacaagcc tcatggtgtg 1320
cgtccaaagg atgttgcagc accaatcgat aagctgccca acatcaagga tctgccgggt 1380
accgagctcg aattcgtaat catggtcata gctg 1414
<210> 6
<211> 806
<212> DNA
<213> Artificial Sequence
<400> 6
cagtgccaag cttgcatgcc tgcaggtcga ctctagcatg acggctgact ggactcgact 60
tccatacgag gttctggaga agatctccac ccgcatcacc aacgaagttc cagatgtgaa 120
ccgcgtggtt ttggacgtaa cctccaagcc accaggaacc atcgaatggg agtaggcctt 180
aaatgagcct tcgttaagcg gcaatcacct tattggagat tgtcgctttt cccatttctc 240
cgggttttct ggaacttttt gggcgtatgc tgggaatgat tctattattg ccaaatcaga 300
aagcaggaga gacccgatga gcgaaatcct agaaacctat tgggcacccc actttggaaa 360
aaccgaagaa gccacagcac tcgtttcata cctggcacaa gcttccggcg atcccattga 420
ggttcacacc ctgttcgggg atttaggttt agacggactc tcgggaaact acaccgacac 480
tgagattgac ggctacggcg acgcattcct gctggttgca gcgctatccg tgttgatggc 540
tgaaaacaaa gcaacaggtg gcgtgaatct gggtgagctt gggggagctg ataaatcgat 600
ccggctgcat gttgaatcca aggagaacac ccaaatcaac accgcattga agtattttgc 660
gctctcccca gaagaccacg cagcagcaga tcgcttcgat gaggatgacc tgtctgagct 720
tgccaacttg agtgaagagc tgcgcggaca gctggactaa ttgtctccca tttaaggagt 780
ccgatttttt ccgagtctta gatttt 806
<210> 7
<211> 3858
<212> DNA
<213> Artificial Sequence
<400> 7
cccatttaag gagtccgatt ttttccgagt cttagatttt gagaaaaccc aggattgctt 60
tgtgcactcc tgggttttca ctttgttaag cagttttggg gaaaagtgca aagtttgcaa 120
agtttagaaa tattttaaga ggtaagatgt ctgcaggtgg aagcgtttaa atgcgttaaa 180
cttggccaaa tgtggcaacc tttgcaaggt gaaaaactgg ggcggggtta gatcctgggg 240
ggtttatttc attcactttg gcttgaagtc gtgcaggtca gaggagtgtt gcccgaaaac 300
attgagagga aaacaaaaac cgatgtttga ttgggggaat cgggggttac gatactagga 360
cgaagtgact gctatcaccc ttggcggtct cttgttgaaa ggaacaatta ctctagtgtc 420
gactcacaca tcttcaacgc ttccagcatt caaaaagatc ttggtagcaa accgcggcga 480
aatcgcggtc cgtgctttcc gtgcagcact cgaaaccggt gcagccacgg tagctattta 540
cccccgtgaa gatcggggat cattccaccg ctcttttgct tctgaagctg tccgcattgg 600
tactgaaggc tcaccagtca aggcgtacct ggacatcgat gaaattatcg gtgcagctaa 660
aaaagttaaa gcagatgcta tttacccggg atatggcttc ctgtctgaaa atgcccagct 720
tgcccgcgag tgcgcggaaa acggcattac ttttattggc ccaaccccag aggttcttga 780
tctcaccggt gataagtctc gtgcggtaac cgccgcgaag aaggctggtc tgccagtttt 840
ggcggaatcc accccgagca aaaacatcga tgacatcgtt aaaagcgctg aaggccagac 900
ttaccccatc tttgtaaagg cagttgccgg tggtggcgga cgcggtatgc gctttgtttc 960
ttcacctgat gagctccgca aattggcaac agaagcatct cgtgaagctg aagcggcatt 1020
cggcgacggt tcggtatatg tcgaacgtgc tgtgattaac ccccagcaca ttgaagtgca 1080
gatccttggc gatcgcactg gagaagttgt acacctttat gaacgtgact gctcactgca 1140
gcgtcgtcac caaaaagttg tcgaaattgc gccagcacag catttggatc cagaactgcg 1200
tgatcgcatt tgcgcggatg cagtaaagtt ctgccgctcc attggttacc agggcgcggg 1260
aaccgtggaa ttcttggtcg atgaaaaggg caaccacgtt ttcatcgaaa tgaacccacg 1320
tatccaggtt gagcacaccg tgactgaaga agtcaccgag gtggacctgg tgaaggcgca 1380
gatgcgcttg gctgctggtg caaccttgaa ggaattgggt ctgacccaag ataagatcaa 1440
gacccacggt gcagcactgc agtgccgcat caccacggaa gatccaaaca acggcttccg 1500
cccagatacc ggaactatca ccgcgtaccg ctcaccaggc ggagctggcg ttcgtcttga 1560
cggtgcagct cagctcggtg gcgaaatcac cgcacacttt gactccatgc tggtgaaaat 1620
gacctgccgt ggttccgact ttgaaactgc tgttgctcgt gcacagcgcg cgttggctga 1680
gttcaccgtg tctggtgttg caaccaacat tggcttcttg cgcgcgctgc tgcgggaaga 1740
ggacttcact tccaagcgca tcgccaccgg atttatcggc gatcacccac acctccttca 1800
ggctccacct gcggatgatg agcagggacg catcctggat tacttggcag atgtcaccgt 1860
gaacaagcct catggtgtgc gtccaaagga tgttgcagca ccaatcgata agctgcccaa 1920
catcaaggat ctgccactgc cacgcggttc ccgtgaccgc ctgaagcagc ttggcccagc 1980
cgcgtttgct cgtgatctcc gtgagcagga cgcactggca gttactgata ccaccttccg 2040
cgatgcacac cagtctttgc ttgcgacccg agtccgctca ttcgcactga agcctgcggc 2100
agaggccgtc gcaaagctga ctcctgagct tttgtccgtg gaggcctggg gcggcgcgac 2160
ctacgatgtg gcgatgcgtt tcctctttga ggatccgtgg gacaggctcg acgagctgcg 2220
cgaggcgatg ccgaatgtaa acattcagat gctgcttcgc ggccgcaaca ccgtgggata 2280
caccccgtac ccagactccg tctgccgcgc gtttgttaag gaagctgcca cctccggcgt 2340
ggacatcttc cgcatcttcg acgcgcttaa cgacgtctcc cagatgcgtc cagcaatcga 2400
cgcagtcctg gagaccaaca ccgcggtagc cgaggtggct atggcttatt ctggtgatct 2460
ctctgatcca aatgaaaagc tctacaccct ggattactac ctgaagatgg cagaggagat 2520
cgtcaagtct ggcgctcaca tcttggccat taaggatatg gctggtctgc ttcgcccagc 2580
tgcggtaacc aagctggtca ccgcactgcg ccgtgaattc gatctgccag tgcacgtgca 2640
cacccacgac accgcgggtg gccagctggc tacctacttt gctgcagctc aagctggtgc 2700
agatgctgtt gacggtgctt ccgcaccact gtctggcacc acctcccagc catccctgtc 2760
tgccattgtt gctgcattcg cgcacacccg tcgcgatacc ggtttgagcc tcgaggctgt 2820
ttctgacctc gagccgtact gggaagcagt gcgcggactg tacctgccat ttgagtctgg 2880
aaccccaggc ccaaccggtc gcgtctaccg ccacgaaatc ccaggcggac agctgtccaa 2940
cctgcgtgca caggccaccg cactgggcct tgctgatcgc ttcgagctca tcgaagacaa 3000
ctacgcagcc gttaatgaga tgctgggacg cccaaccaag gtcaccccat cctccaaggt 3060
tgttggcgac ctcgcactcc acctggttgg tgcgggtgta gatccagcag actttgctgc 3120
agacccacaa aagtacgaca tcccagactc tgtcatcgcg ttcctgcgcg gcgagcttgg 3180
taaccctcca ggtggctggc cagaaccact gcgcacccgc gcactggaag gccgctccga 3240
aggcaaggca cctctgacgg aagttcctga ggaagagcag gcgcacctcg acgctgatga 3300
ttccaaggaa cgtcgcaaca gcctcaaccg cctgctgttc ccgaagccaa ccgaagagtt 3360
cctcgagcac cgtcgccgct tcggcaacac ctctgcgctg gatgatcgtg aattcttcta 3420
cggcctggtc gaaggccgcg agactttgat ccgcctgcca gatgtgcgca ccccactgct 3480
tgttcgcctg gatgcgatct ctgagccaga cgataagggt atgcgcaatg ttgtggccaa 3540
cgtcaacggc cagatccgcc caatgcgtgt gcgtgaccgc tccgttgagt ctgtcaccgc 3600
aaccgcagaa aaggcagatt cctccaacaa gggccatgtt gctgcaccat tcgctggtgt 3660
tgtcactgtg actgttgctg aaggtgatga ggtcaaggct ggagatgcag tcgcaatcat 3720
cgaggctatg aagatggaag caacaatcac tgcttctgtt gacggcaaaa tcgatcgcgt 3780
tgtggttcct gctgcaacga aggtggaagg tggcgacttg atcgtcgtcg tttcctaata 3840
aatcgactac tcacatag 3858
<210> 8
<211> 3858
<212> DNA
<213> Artificial Sequence
<400> 8
cccatttaag gagtccgatt ttttccgagt cttagatttt gagaaaaccc aggattgctt 60
tgtgcactcc tgggttttca ctttgttaag cagttttggg gaaaagtgca aagtttgcaa 120
agtttagaaa tattttaaga ggtaagatgt ctgcaggtgg aagcgtttaa atgcgttaaa 180
cttggccaaa tgtggcaacc tttgcaaggt gaaaaactgg ggcggggtta gatcctgggg 240
ggtttatttc attcactttg gcttgaagtc gtgcaggtca gaggagtgtt gcccgaaaac 300
attgagagga aaacaaaaac cgatgtttga ttgggggaat cgggggttac gatactagga 360
cgaagtgact gctatcaccc ttggcggtct cttgttgaaa ggaacaatta ctctagtgtc 420
gactcacaca tcttcaacgc ttccagcatt caaaaagatc ttggtagcaa accgcggcga 480
aatcgcggtc cgtgctttcc gtgcagcact cgaaaccggt gcagccacgg tagctattta 540
cccccgtgaa gatcggggat cattccaccg ctcttttgct tctgaagctg tccgcattgg 600
tactgaaggc tcaccagtca aggcgtacct ggacatcgat gaaattatcg gtgcagctaa 660
aaaagttaaa gcagatgcta tttacccggg atatggcttc ctgtctgaaa atgcccagct 720
tgcccgcgag tgcgcggaaa acggcattac ttttattggc ccaaccccag aggttcttga 780
tctcaccggt gataagtctc gtgcggtaac cgccgcgaag aaggctggtc tgccagtttt 840
ggcggaatcc accccgagca aaaacatcga tgacatcgtt aaaagcgctg aaggccagac 900
ttaccccatc tttgtaaagg cagttgccgg tggtggcgga cgcggtatgc gctttgtttc 960
ttcacctgat gagctccgca aattggcaac agaagcatct cgtgaagctg aagcggcatt 1020
cggcgacggt tcggtatatg tcgaacgtgc tgtgattaac ccccagcaca ttgaagtgca 1080
gatccttggc gatcgcactg gagaagttgt acacctttat gaacgtgact gctcactgca 1140
gcgtcgtcac caaaaagttg tcgaaattgc gccagcacag catttggatc cagaactgcg 1200
tgatcgcatt tgcgcggatg cagtaaagtt ctgccgctcc attggttacc agggcgcgag 1260
aaccgtggaa ttcttggtcg atgaaaaggg caaccacgtt ttcatcgaaa tgaacccacg 1320
tatccaggtt gagcacaccg tgactgaaga agtcaccgag gtggacctgg tgaaggcgca 1380
gatgcgcttg gctgctggtg caaccttgaa ggaattgggt ctgacccaag ataagatcaa 1440
gacccacggt gcagcactgc agtgccgcat caccacggaa gatccaaaca acggcttccg 1500
cccagatacc ggaactatca ccgcgtaccg ctcaccaggc ggagctggcg ttcgtcttga 1560
cggtgcagct cagctcggtg gcgaaatcac cgcacacttt gactccatgc tggtgaaaat 1620
gacctgccgt ggttccgact ttgaaactgc tgttgctcgt gcacagcgcg cgttggctga 1680
gttcaccgtg tctggtgttg caaccaacat tggcttcttg cgcgcgctgc tgcgggaaga 1740
ggacttcact tccaagcgca tcgccaccgg atttatcggc gatcacccac acctccttca 1800
ggctccacct gcggatgatg agcagggacg catcctggat tacttggcag atgtcaccgt 1860
gaacaagcct catggtgtgc gtccaaagga tgttgcagca ccaatcgata agctgcccaa 1920
catcaaggat ctgccactgc cacgcggttc ccgtgaccgc ctgaagcagc ttggcccagc 1980
cgcgtttgct cgtgatctcc gtgagcagga cgcactggca gttactgata ccaccttccg 2040
cgatgcacac cagtctttgc ttgcgacccg agtccgctca ttcgcactga agcctgcggc 2100
agaggccgtc gcaaagctga ctcctgagct tttgtccgtg gaggcctggg gcggcgcgac 2160
ctacgatgtg gcgatgcgtt tcctctttga ggatccgtgg gacaggctcg acgagctgcg 2220
cgaggcgatg ccgaatgtaa acattcagat gctgcttcgc ggccgcaaca ccgtgggata 2280
caccccgtac ccagactccg tctgccgcgc gtttgttaag gaagctgcca cctccggcgt 2340
ggacatcttc cgcatcttcg acgcgcttaa cgacgtctcc cagatgcgtc cagcaatcga 2400
cgcagtcctg gagaccaaca ccgcggtagc cgaggtggct atggcttatt ctggtgatct 2460
ctctgatcca aatgaaaagc tctacaccct ggattactac ctgaagatgg cagaggagat 2520
cgtcaagtct ggcgctcaca tcttggccat taaggatatg gctggtctgc ttcgcccagc 2580
tgcggtaacc aagctggtca ccgcactgcg ccgtgaattc gatctgccag tgcacgtgca 2640
cacccacgac accgcgggtg gccagctggc tacctacttt gctgcagctc aagctggtgc 2700
agatgctgtt gacggtgctt ccgcaccact gtctggcacc acctcccagc catccctgtc 2760
tgccattgtt gctgcattcg cgcacacccg tcgcgatacc ggtttgagcc tcgaggctgt 2820
ttctgacctc gagccgtact gggaagcagt gcgcggactg tacctgccat ttgagtctgg 2880
aaccccaggc ccaaccggtc gcgtctaccg ccacgaaatc ccaggcggac agctgtccaa 2940
cctgcgtgca caggccaccg cactgggcct tgctgatcgc ttcgagctca tcgaagacaa 3000
ctacgcagcc gttaatgaga tgctgggacg cccaaccaag gtcaccccat cctccaaggt 3060
tgttggcgac ctcgcactcc acctggttgg tgcgggtgta gatccagcag actttgctgc 3120
agacccacaa aagtacgaca tcccagactc tgtcatcgcg ttcctgcgcg gcgagcttgg 3180
taaccctcca ggtggctggc cagaaccact gcgcacccgc gcactggaag gccgctccga 3240
aggcaaggca cctctgacgg aagttcctga ggaagagcag gcgcacctcg acgctgatga 3300
ttccaaggaa cgtcgcaaca gcctcaaccg cctgctgttc ccgaagccaa ccgaagagtt 3360
cctcgagcac cgtcgccgct tcggcaacac ctctgcgctg gatgatcgtg aattcttcta 3420
cggcctggtc gaaggccgcg agactttgat ccgcctgcca gatgtgcgca ccccactgct 3480
tgttcgcctg gatgcgatct ctgagccaga cgataagggt atgcgcaatg ttgtggccaa 3540
cgtcaacggc cagatccgcc caatgcgtgt gcgtgaccgc tccgttgagt ctgtcaccgc 3600
aaccgcagaa aaggcagatt cctccaacaa gggccatgtt gctgcaccat tcgctggtgt 3660
tgtcactgtg actgttgctg aaggtgatga ggtcaaggct ggagatgcag tcgcaatcat 3720
cgaggctatg aagatggaag caacaatcac tgcttctgtt gacggcaaaa tcgatcgcgt 3780
tgtggttcct gctgcaacga aggtggaagg tggcgacttg atcgtcgtcg tttcctaata 3840
aatcgactac tcacatag 3858
<210> 9
<211> 783
<212> DNA
<213> Artificial Sequence
<400> 9
tgatcgtcgt cgtttcctaa taaatcgact actcacatag ggtcgggcta gtcattctga 60
tcagcgaatt ccacgttcac atcgccaatt ccagagttca caaccagatt cagcattgga 120
ccttctagat cagcattgtg ggcggtgaga tctccaacat cacagcgcgc tgtgcccaca 180
ccggcggtac aacttaggct cacgggcaca tcatcgggca gggtgaccat gacttcgccg 240
atccctgagg tgatttggat gttttgttcc tgatccaatt gggtgaggtg gctgaaatcg 300
aggttcattt cacccacgcc agaggtgtag ctgctgagga gttcatcgtt ggtggggatg 360
agattgacat cgccgattcc agggtcgtct tcaaagtaga tgggatcgat atttgaaata 420
aacaggcctg cgagggcgct catgacaact ccggtaccaa ctacaccgcc gacaatccat 480
ggccacacat ggcgcttttt ctgaggcttt tgtggaggga cttgtacatc ccaggtgttg 540
tattggtttt gggcaagtgg atcccaatga ggcgcttcgg gggtttgttg cgcgaagggt 600
gcatagtagc cctcaacggg ggtgatagtg cttagatctg gttggggttg tgggtagaga 660
tcttcgtttt tcatggtggc atcctcagaa acagtgaatt cagtggtgag tagtccgcgg 720
ggtggaagtg gttgtttctt atgcagggta ccgagctcga attcgtaatc atggtcatag 780
ctg 783
<210> 10
<211> 1431
<212> DNA
<213> Artificial Sequence
<400> 10
cagtgccaag cttgcatgcc tgcaggtcga ctctagtcct ttggctacta acccacgcgc 60
caagatgcgt tccctgcgcc acggttttgt gaagctgttc tgccgccgta actctggcct 120
gatcatcggt ggtgtcgtgg tggcaccgac cgcgtctgag ctgatcctac cgatcgctgt 180
ggcagtgacc aaccgtctga cagttgctga tctggctgat accttcgcgg tgtacccatc 240
attgtcaggt tcgattactg aagcagcacg tcagctggtt caacatgatg atctaggcta 300
attttccgag tcttagattt tgagaaaacc caggattgct ttgtgcactc ctgggttttc 360
actttgttaa gcagttttgg ggaaaagtgc aaagtttgca aagtttagaa atattttaag 420
aggtaagatg tctgcaggtg gaagcgttta aatgcgttaa acttggccaa atgtggcaac 480
ctttgcaagg tgaaaaactg gggcggggtt agatcctggg gggtttattt cattcacttt 540
ggcttgaagt cgtgcaggtc agaggagtgt tgcccgaaaa cattgagagg aaaacaaaaa 600
ccgatgtttg attgggggaa tcgggggtta cgatactagg acgaagtgac tgctatcacc 660
cttggcggtc tcttgttgaa aggaacaatt actctaacct ttctgtaaaa agccccgctt 720
cttcctcatg gaggaggcgg ggctttttgg gtcaagatgg gagatgggtg agttggattt 780
ggtctgattc gacactttta aggactgaga tttgaagatc gagaccaagg ctcaaaggga 840
atccatgccg tcttgattta atactgcacc ccgctaatga aaatcattac tattaggtgt 900
catgatggac tatgcacacg attcctgctc accaactctg cgccgtgact tggaggtcac 960
cggacaagtc caacctgaga aagctgtgga tttagcagcg tcgtacgagg ggaaggttgc 1020
cgcaataacg aaggtgacct cctcaaatat ggagcatgcc atcacgcagg cctcaaaagc 1080
taaggaggtg gtggtgctca ttggtcactc cctgctgccc acatttcagg atttggaaaa 1140
agacattctg cactttcagg caggtaataa agggcgattt tctgtagcga ttgttgatcc 1200
tgatcgcagt gcagatgtgg ttgccagatt taggccaaaa cagattccgg tggcatacgt 1260
ggtgaaagat ggcgccagca ttgcggagtt caactcgctc aacaaggagc cggttgcaca 1320
atggcttgat cattttgtgt cgcgggaaac gatctccaat gaaaaagagg gggacgtcga 1380
taagcaaata gacgggtacc gagctcgaat tcgtaatcat ggtcatagct g 1431
Claims (10)
1.一种YH66_03760突变体，是将YH66_03760蛋白质第282位氨基酸残基由甘氨酸突变为其他氨基酸残基得到的蛋白质；
所述YH66_03760蛋白质为如下A1)-A3)中的任一种：
A1)SEQ ID No.2所示的氨基酸序列组成的蛋白质；
A2)将A1)所示的氨基酸序列经过除第282位氨基酸残基以外的一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与细菌产精氨酸相关的蛋白质；
A3)来源于细菌且与A1)或A2)具有95％以上同一性且与细菌产精氨酸相关的蛋白质。
2.根据权利要求1所述的YH66_03760突变体，其特征在于：所述YH66_03760突变体是将YH66_03760蛋白质第282位氨基酸残基由甘氨酸突变为精氨酸得到的蛋白质。
3.与权利要求1所述YH66_03760突变体相关的生物材料，所述生物材料为如下B1)至B4)中的任一种：
B1)编码权利要求1或2所述YH66_03760突变体的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物。
4.根据权利要求3所述的生物材料，其特征在于：所述核酸分子为如下C1)或C2)中的任一种：
C1)核苷酸序列为SEQ ID No.3的DNA分子；
C2)将SEQ ID No.3所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与C1)所示的DNA分子具有90％以上的同一性，且具有相同功能的DNA分子。
5.YH66_03760蛋白质或与YH66_03760蛋白质相关的生物材料或权利要求1所述的YH66_03760突变体或权利要求2所述的生物材料在如下X1)至X4)中任一种中的应用：
X1)调控细菌精氨酸产量；
X2)构建产精氨酸工程菌；
X3)制备精氨酸；
所述YH66_03760蛋白质为权利要求1中所述YH66_03760蛋白质；
与YH66_03760蛋白质相关的生物材料为如下D1)至D4)中的任一种：
D1)编码所述YH66_03760蛋白质的核酸分子；
D2)含有D1)所述核酸分子的表达盒；
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体；
D4)含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物。
6.提高YH66_03760蛋白质或YH66_03760突变体含量和/或活性的物质或提高YH66_03760基因或YH66_03760突变体基因表达量的物质在如下Y1)至Y4)中任一种中的应用：
Y1)提高细菌精氨酸产量；
Y2)构建产精氨酸工程菌；
Y3)制备精氨酸；
所述YH66_03760蛋白质为权利要求1中所述YH66_03760蛋白质；
所述YH66_03760突变体为权利要求1所述YH66_03760突变体；
所述YH66_03760基因为编码权利要求1中所述YH66_03760蛋白质的基因；
所述YH66_03760突变体基因为编码权利要求1所述YH66_03760突变体的基因。
7.一种提高细菌精氨酸产量的方法，为如下M1)或M2)：
所述M1)包括如下步骤：将细菌基因组中的YH66_03760基因替换为YH66_03760突变体基因，实现细菌精氨酸产量的提高；
所述M2)包括如下步骤：提高细菌中YH66_03760蛋白质或YH66_03760突变体含量和/或活性，或提高细菌中YH66_03760基因或YH66_03760突变体基因表达量，实现细菌精氨酸产量的提高；
所述YH66_03760蛋白质为权利要求1中所述YH66_03760蛋白质；
所述YH66_03760突变体为权利要求1所述YH66_03760突变体；
所述YH66_03760基因为编码权利要求1中所述YH66_03760蛋白质的基因；
所述YH66_03760突变体基因为编码权利要求1所述YH66_03760突变体的基因。
8.一种产精氨酸工程菌的构建方法，为如下N1)或N2)：
所述N1)包括如下步骤：将细菌基因组中的YH66_03760基因替换为YH66_03760突变体基因，得到所述产精氨酸工程菌；
所述N2)包括如下步骤：提高细菌中YH66_03760蛋白质或YH66_03760突变体含量和/或活性，或提高细菌中YH66_03760基因或YH66_03760突变体基因表达量，得到所述产精氨酸工程菌；
所述YH66_03760蛋白质为权利要求1中所述YH66_03760蛋白质；
所述YH66_03760突变体为权利要求1所述YH66_03760突变体；
所述YH66_03760基因为编码权利要求1中所述YH66_03760蛋白质的基因；
所述YH66_03760突变体基因为编码权利要求1所述YH66_03760突变体的基因。
9.按照权利要求8所述方法构建得到的产精氨酸工程菌在制备精氨酸中的应用。
10.一种制备精氨酸的方法，包括如下步骤：发酵培养按照权利要求8所述方法构建得到的产精氨酸工程菌，得到所述精氨酸。
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| CN1370236A (zh) * | 1999-06-25 | 2002-09-18 | Basf公司 | 编码参与膜合成和膜转运的蛋白质的谷氨酸棒杆菌基因 |
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