CN110079566A - 用改变ppc启动子的细菌发酵生产L-赖氨酸的方法 - Google Patents
用改变ppc启动子的细菌发酵生产L-赖氨酸的方法 Download PDFInfo
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- CN110079566A CN110079566A CN201910407560.1A CN201910407560A CN110079566A CN 110079566 A CN110079566 A CN 110079566A CN 201910407560 A CN201910407560 A CN 201910407560A CN 110079566 A CN110079566 A CN 110079566A
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Abstract
本发明提供了发酵生产L‑赖氨酸的方法，其包括改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域；和，用改造而得到的谷氨酸棒杆菌发酵生产L‑赖氨酸。另外，本发明还提供了由该方法衍生的方法和应用，以及可以用在这些方法和应用中的谷氨酸棒杆菌、多核苷酸等。
Description
技术领域
本发明属于氨基酸发酵领域，具体而言，本发明涉及发酵生产L-赖氨酸的方法及其衍生的方法和应用，和可以用在这些方法和应用中的细菌等。
背景技术
通过产L-赖氨酸的细菌（如，埃希氏菌属的大肠杆菌和棒杆菌属的杆状细菌）发酵来生产L-赖氨酸已经得到了产业化应用。这些细菌，可以是从自然界分离的细菌，也可以是通过诱变或基因工程改造获得的细菌，或者两者兼而有之。
现有文献中有报道ppc（磷酸烯醇式丙酮酸羧化酶，phosphoenolpyruvatecarboxylase）基因、其调控元件及其编码蛋白的增加、减少或突变等用于氨基酸（包括L-赖氨酸）生产中，例如，WO0100844A2、WO2005058945A2、EP0358940A1和JP1998165180A等，然而，突变后的调控元件的性质是难以预料的，相应地，其对L-赖氨酸生产的影响也难以预料，尤其是，近十多年来，对ppc基因及其调控元件的突变对L-赖氨酸生产的影响的新的研究已经越来越少，表明研究人员对是否有新的ppc基因及其调控元件的突变来改善L-赖氨酸生产的兴趣已经越来越小了。
本发明人没有受到近十多年来的趋势影响，经过长期研究和实践，尤其凭借了一些运气，偶然发现对ppc基因调控元件的新的改造能够有助于提高谷氨酸棒杆菌发酵生产L-赖氨酸的产量。而且，该方法与现有改造的大量高产L-赖氨酸的谷氨酸棒杆菌的染色体改造位点没有冲突，可以叠加提高的效果，从而在实践上可用于谷氨酸棒杆菌发酵生产L-赖氨酸。
发明内容
本发明要解决的技术问题在于提供新的发酵生产L-赖氨酸的方法及其相关的方法，包括相对于未改造谷氨酸棒杆菌提高L-赖氨酸的发酵生产量的方法，改造的谷氨酸棒杆菌在发酵生产L-赖氨酸中的应用，改造的谷氨酸棒杆菌在相对于未改造谷氨酸棒杆菌提高L-赖氨酸的发酵生产量的应用，和/或，改造谷氨酸棒杆菌的方法等。另外，本发明还提供了可以用于上述方法中的突变蛋白、多核苷酸、载体和/或谷氨酸棒杆菌等。
具体而言，在第一方面，本发明提供了发酵生产L-赖氨酸的方法，其包括：
（1）改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸；和，
（2）用步骤（1）改造而得到的谷氨酸棒杆菌发酵生产L-赖氨酸。
在本文中，术语“改造”指的是是相应被改造的对象发生变化，从而达到一定的效果。改造位于染色体上的基因或调控元件的手段包括但是不限于，诱变、定点突变、和/或同源重组，优选是后两者。改造位于染色体上的基因或调控元件使得该基因或调控元件的核苷酸序列被添加、缺失或替换一个或多个核苷酸。这些技术手段广泛记载于分子生物学和微生物学文献中，有许多甚至已经商品化了。在本发明的具体实施方式中，根据同源重组的原理，可以采用Addgene公司商品化的pKOV质粒系统来进行改造，也可以采用pK18mobsacB质粒系统来进行改造，改造谷氨酸棒杆菌染色体上的野生型的ppc基因和/或其调控元件。因此，在本文中，优选改造是通过同源重组进行的改造。
本发明人经过长期研究发现，在谷氨酸棒杆菌中，使野生型的ppc基因启动子区域发生缺失，尤其是在对ppc基因改造的情况下，可以提高L-赖氨酸的。在本发明的具体实施方式中，改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，在ppc基因的第-1位缺失一个核苷酸。基因的-1位是基因编码区第一个密码子之前紧邻的一个核苷酸，这是本领域技术人员所熟知的。
野生型的ppc基因的启动子区域的核苷酸序列如SEQ ID NO：5的第2761-3360位的互补序列所示，野生型的ppc基因的启动子区域缺失一个核苷酸后的核苷酸序列如SEQ IDNO：6的第2761-3359位的互补序列所示，在本发明的具体实施方式中，用后者的核苷酸序列取代前者的核苷酸序列，实施改造。
相应地，本发明还提供了其他的应用或方法。例如，在第二方面，本发明提供了提高L-赖氨酸的发酵量的方法，其包括：
（1）改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸，优选在ppc基因的第-1位缺失一个核苷酸；和，
（2）用步骤（1）改造而得到的谷氨酸棒杆菌发酵生产L-赖氨酸。
L-赖氨酸作为细菌的重要代谢产物，大多数细菌或多或少都能够发酵产生一定量的L-赖氨酸，尤其是谷氨酸棒杆菌。尽管低产L-赖氨酸的细菌不适合有经济效益地生产L-赖氨酸，但是通过本发明的方法，仍旧能提高L-赖氨酸的发酵量，仍旧可以供对经济效益不敏感的地方使用。当然，在本文中，优选细菌是高产L-赖氨酸的细菌。通过本发明的方法，可以进一步提高其产量。另外，在本发明的方法或应用中，除了改造细菌染色体上ppc基因和/或其调控元件以外，可以不再进行其他改造，当然也可以进行其他改造。
又如，在第三方面，本发明提供了改造获得的谷氨酸棒杆菌在发酵生产L-赖氨酸中的应用，其中，所述改造获得是改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸，优选在ppc基因的第-1位缺失一个核苷酸，而获得。
改造获得的谷氨酸棒杆菌可以单独应用于发酵生产L-赖氨酸中，也可以和其他产L-赖氨酸的细菌混合发酵生产L-赖氨酸，或者以其他方式应用于发酵生产L-赖氨酸中。在本文中，如无特别限定（如未以“改造获得”来限定），术语“谷氨酸棒杆菌”是未改造或改造前的谷氨酸棒杆菌，其染色体的ppc基因及该基因座位前后的调控元件是是野生型的。
还如，在第四方面，本发明提供了改造获得的谷氨酸棒杆菌在提高L-赖氨酸的发酵量中的应用，其中，所述改造获得是改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸，优选在ppc基因的第-1位缺失一个核苷酸，而获得。
在本文中，谷氨酸棒杆菌可以是产L-赖氨酸的谷氨酸棒杆菌。诸如北京棒杆菌，其特性，尤其是野生型的ppc基因及其前后的调控元件，与谷氨酸棒杆菌基本相同，因此也可以纳入本发明的谷氨酸棒杆菌的范围内。
更本质地，在第五方面，本发明提供了改造谷氨酸棒杆菌的方法，其包括改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸。
本发明第五方面的方法改造而获得的谷氨酸棒杆菌能够用于发酵生产或产生L-赖氨酸。因此，在第六方面，本发明提供了本发明第五方面的方法改造而获得的谷氨酸棒杆菌。
优选在本发明中，步骤（1）进一步包括：改造谷氨酸棒杆菌染色体上野生型的ppc基因，使其编码的蛋白质的第482位发生突变。在本发明的具体实施方式中，野生型的ppc基因编码的蛋白质的第482位发生A482V突变，即丙氨酸残基（Ala）被取代为缬氨酸残基（Val）。
野生型的ppc基因所编码的氨基酸序列如SEQ ID NO：3所示，在本发明的具体实施方式中，野生型的ppc基因的核苷酸序列如SEQ ID NO：1的互补序列所示。另外，发生突变的ppc基因所编码的氨基酸序列如SEQ ID NO：4所示，在本发明的具体实施方式中，发生突变的ppc基因的核苷酸序列如SEQ ID NO：2的互补序列所示。
在本发明的具体实施方式中，用如SEQ ID NO：2的互补序列所示的核苷酸序列取代谷氨酸棒杆菌染色体上的如SEQ ID NO：1的互补序列所示的核苷酸序列，实施进一步的改造。
改造谷氨酸棒杆菌染色体上野生型的ppc基因在改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域的同时、之前或之后。在本发明的具体实施方式中，改造谷氨酸棒杆菌染色体上野生型的ppc基因实施在改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域之后。
另外，本发明还提供了可以用于上述方法中的中间产物，如突变蛋白、多核苷酸和/或载体等物质，以及它们的应用等。例如，在第七方面，本发明提供了突变蛋白，其氨基酸序列如SEQ ID NO：4所示。
在第八方面，本发明提供了多核苷酸，其编码本发明第七方面的蛋白。在本发明的具体实施方式中，本发明第八方面的多核苷酸的核苷酸序列如SEQ ID NO：2的互补序列所示。
在第九方面，本发明提供了载体，其包含本发明第八方面的多核苷酸。
另外，在第十方面本发明提供了多核苷酸，其核苷酸序列如SEQ ID NO：6的第2761-3359位的互补序列所示。该多核苷酸是野生型ppc基因的第-1位缺失一个核苷酸而得到的启动子区域，其不同于野生型的ppc基因的启动子区域。
相应地，在第十一方面，本发明提供了载体，其包含本发明第十方面的多核苷酸。
在第十一方面，本发明提供了本发明第七方面的突变蛋白和/或本发明第八和/或第十方面的多核苷酸和/或本发明第九和/或第十一方面的载体在本发明第一、二、三和/或四方面的方法或应用中的应用。即在本发明第一、二、三和/或四方面的方法或应用中，使用了本发明提供了本发明第七方面的突变蛋白和/或本发明第八和/或第十方面的多核苷酸和/或本发明第九和/或第十一方面的载体。
在第十二方面，本发明提供了本发明第七方面的突变蛋白和/或本发明第八和/或第十方面的多核苷酸和/或本发明第九和/或第十一方面的载体在制备本发明第五方面的谷氨酸棒杆菌中的应用。即在制备本发明第五方面的谷氨酸棒杆菌的过程中，使用了本发明第七方面的突变蛋白和/或本发明第八和/或第十方面的多核苷酸和/或本发明第九和/或第十一方面的载体。
本发明的有益效果在于，开辟并且实践证明了新的提高L-赖氨酸的发酵量的方式，而且与现有改造的大量高产L-赖氨酸的谷氨酸棒杆菌的染色体改造位点没有冲突，应用在现有高产L-赖氨酸的谷氨酸棒杆菌上观察到了可以进一步提高产量的效果，从而在实践上可用于谷氨酸棒杆菌发酵生产L-赖氨酸，便于推广应用。
为了便于理解，以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见的。
另外，本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
具体实施方式
以下通过实施例进一步说明本发明的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂，可参见《分子克隆实验指南（第3版）》（科学出版社）、《微生物学实验（第4版）》（高等教育出版社）以及相应仪器和试剂的厂商说明书等参考。
实施例1 包含点缺失的NCgl1523基因启动子的转化载体pK18-NCgl1523的构建
根据NCBI公布的谷氨酸棒杆菌ATCC13032的基因组序列，合成两对扩增NCgl1523基因启动子区片段（包含启动子和编码区的核苷酸序列如SEQ ID NO：5的互补序列所示，或可登陆https://www.ncbi.nlm.nih.gov/gene/*term=NCgl1523获取）的引物，以通过等位基因置换在菌株YPL-4染色体上的野生型的NCgl1523基因启动子区-1位敲除一个碱基（包含启动子和编码区的核苷酸序列如SEQ ID NO：6的互补序列所示）。引物设计如下（上海invitrogen公司合成）：
P1’: 5' CCGGAATTC GTGATGCGACGGCGGATGT 3' （EcoR I）
P2’: 5' CTCAATGTGAAAGAGTGTTTAAAGTAGTTAATGACTGA 3'
P3’: 5' TCAGTCATTAACTACTTTAAACACTCTTTCACATTGAG 3'
P4’: 5' CCCAAGCTT CGTTGGTGAGCCACTGGAAAT 3' (Hind III)
以谷氨酸棒杆菌ATCC13032为模板，分别以引物P1’/P2’及P3’/P4’，进行PCR扩增，获得两条含有NCgl1523基因启动子区分离的DNA片段，大小分别为586bp及554bp。再用引物P1’/P4’进行OVERLAP PCR得到整个等位替换的片段1100bp，片段包含NCgl1523基因的完整启动子区，且片段两端分别含有HindⅢ和EcoRⅠ酶切位点，此DNA片段导致野生型NCgl1523基因的启动子-1区点缺失。PCR反应结束后，对扩增的产物进行琼脂糖凝胶电泳，采用柱式DNA凝胶回收试剂盒进行纯化所需要的DNA片段，将片段双酶切（EcoRⅠ/ HindⅢ）后回收，与同样双酶切（EcoRⅠ/ HindⅢ）后的穿梭质粒pk18mobsacB质粒相连接，获得等位替换质粒pK18-NCgl1523，该质粒上含有卡那霉素抗性标记。
实施例2 包含点缺失的NCgl1523基因启动子的菌株的构建
将实施例1获得的质粒pK18- NCgl1523电转化入赖氨酸生产菌（谷氨酸棒杆菌）专利菌株YPL-4（即YP97136）中（其构建方法可参见WO2014121669A1；经测序确认该菌株染色体上保留有野生型的NCgl1523基因及其启动子），对培养产生的单菌落分别通过引物P1’/M13F进行鉴定，能扩增出1100bp大小条带的菌株为阳性菌株。将阳性菌株在含12%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，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' CCAGGTTAGCCAGCAGAGC 3'
P6:’ 5' TTGATGAGCCCATGAAAGC 3'
上述PCR扩增产物通过高温变性、冰浴后进行sscp电泳（以质粒pK18- NCgl1523扩增片段为阳性对照，野生型扩增片段为阴性对照，水作为空白对照），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PCR扩增阳性菌株目的片段，并连接到PMD19-T载体，并测序，通过序列比对，碱基序列发生突变的序列验证菌株的等位替换成功，并被命名为YPL-4-001。
实施例3 包含点突变的NCgl1523基因的转化载体pK18-NCgl1523A482V的构建
根据NCBI公布的谷氨酸棒杆菌ATCC13032的基因组序列，合成两对扩增NCgl1523基因编码区（其核苷酸序列如SEQ ID NO：1的互补序列所示，所编码的氨基酸序列如SEQ ID NO：3所示）片段的引物，以通过等位基因置换在菌株YPL-4染色体上的野生型NCgl1523基因编码区中引入A482V点突变（其核苷酸序列如SEQ ID NO：2的互补序列所示，所编码的氨基酸序列如SEQ ID NO：4所示）。引物设计如下（上海invitrogen公司合成）：
P1: 5' CGCGGATCCCCTGGGCTGGGCAAGAATC 3' （BamH I）
P2: 5' CCGAATTTCTTAACAACCTCCGACGCGGTG 3'
P3: 5' CACCGCGTCGGAGGTTGTTA AGAAATTCGG 3'
P4: 5' ACGCGTCGACCTGTCGGACCGCATGAATA 3' ( Sal I )
以谷氨酸棒杆菌ATCC13032为模板，分别以引物P1/P2及P3/P4，进行PCR扩增，获得两条含有NCgl1523基因部分编码区（ORF）分离的DNA片段，大小分别为472bp及572bp。再用引物P1/P4进行OVERLAP PCR得到整个等位替换的片段1044bp，片段包含NCgl1523基因的部分编码区，且片段两端分别含有BamH I和Sal I酶切位点，此DNA片段导致野生型NCgl1523基因的第1445位的核苷酸改变，最终导致编码蛋白的第482位氨基酸由丙氨酸（A）被缬氨酸（V）所取代。PCR反应结束后，对扩增的产物进行琼脂糖凝胶电泳，采用柱式DNA凝胶回收试剂盒进行纯化所需要的DNA片段，将片段双酶切（BamH I / Sal I）后回收，与同样双酶切（BamHI / Sal I）后的穿梭质粒pk18mobsacB质粒相连接，获得等位替换质粒pK18-NCgl1523A482V，该质粒上含有卡那霉素抗性标记。
实施例4 包含点突变的NCgl1523A482V基因编码区的菌株的构建
将实施例1获得的质粒pK18- NCgl1523A482V电转化入实施例2获得的YPL-4-001，对培养产生的单菌落分别通过引物P1/M13F进行鉴定，能扩增出1100bp大小条带的菌株为阳性菌株。将阳性菌株在含12%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，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' GGAG GTAGTTGCGGTAGAG 3'
P6: 5' TTCAGATGAATACAGCGAGGTC 3'
上述PCR扩增产物通过高温变性、冰浴后进行sscp电泳（以质粒pK18- NCgl1523扩增片段为阳性对照，野生型扩增片段为阴性对照，水作为空白对照），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PCR扩增阳性菌株目的片段，并连接到PMD19-T载体，并测序，通过序列比对，碱基序列发生突变的序列验证菌株的等位替换成功，并被命名为YPL-4-002。
实施例5 赖氨酸发酵实验
将实施例4构建的菌株和原始菌株在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所示的培养基和表2所示的过程进行发酵实验。每个菌株重复三次，结果如表3所示。
表1 发酵培养基配方
| 品名 | 配比 |
| 淀粉水解糖 | 30g/l |
| 硫酸铵 | 12g/L |
| 硫酸镁 | 0.87g/l |
| 糖蜜 | 20g/l |
| 酸化玉米浆 | 3ml/l |
| 磷酸 | 0.4ml/l |
| 氯化钾 | 0.53g/l |
| 消泡剂（2%泡敌） | 4ml/L |
| 硫酸亚铁 | 120mg/l |
| 硫酸锰 | 120mg/l |
| 烟酰胺 | 42mg/l |
| 泛酸钙 | 6.3mg/l |
| VB1 | 6.3mg/l |
| 铜、锌盐溶液 | 0.6g/L |
| 生物素 | 0.88mg/L |
表2 发酵过程
表3 赖氨酸发酵实验结果
结果如表3所示，对谷氨酸棒杆菌中NCgl1523A482V进行点突变和对谷氨酸棒杆菌中NCgl1523基因（启动子）-1处碱基进行敲除，有助于L-赖氨酸产量的提高。
序列表
<110> 黑龙江伊品生物科技有限公司
<120> 用改变ppc启动子的细菌发酵生产L-赖氨酸的方法
<130> CN
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 2760
<212> DNA
<213> 谷氨酸棒杆菌
<400> 1
ctagccggag ttgcgcagcg cagtggaaag accgttcatg gtcagctgaa tgttgcggga 60
cacttgctcg ctttggtcgc cttttcggta gcgtcgcatc atctctacct ggatcacgtt 120
gagtggaagc aggtaggggt atcggcgctg gacagagcgt gcgagaagtg ggttgtcatc 180
aagcagatca tcagagccgg tgattacgca gaacatcttc ttggtcagga agtactcctc 240
gcggatgacg gaatagactc gctcggctac ttccgtatct gggatcaggt ctgcgtagag 300
ctttgccaaa cgcagctctg ccttggacat cacctgagcc atgttatcca acactgaggt 360
gaaaaatggc caggactcat tgagtgtttg cagctcggca atgcgttggg tggcctgctc 420
cccttcgcca atccactgct ctaatgcggt tccgacacca aaccagcctg gcagcatgac 480
acgagactgt gaccagctga gcacccatgg gatggctcgc aaatcttcca ccgaggaggt 540
ctgcttgcgt gaggaaggcc tggatccgat gttgagggat ccaatctcct gcagcggcgt 600
ggactgggtg aagtaatcga tgaagccttg atcctcgtgc accaaggagg cgtacttctt 660
caagctgagc tcagagatct cactcatgat gtcgtacgcg cgttggtgat cggtgagttc 720
ggagacgtcg agaagcgatg cctcaagcgt ggctgagacc agggcttcga ggtttcggcg 780
cgcggtttcg gggttgccgt acttagcgga gatgatctcg ccctgctcgg tgatgcgcac 840
ggaaccttgg acagcccccc tgggctgggc aagaatcgcg tcgtaggaag gtccgccacc 900
gcggccgacg gtgccaccac ggccgtggaa caggcgaagc ttgaccccgg ctgatcggca 960
tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag tttgcggaga aatatccgcc 1020
atccttgttg gaatcggagt aaccgagcat gacttcctgg acgttgtcgc gctgcaggag 1080
gtagttgcgg tagagatcaa ttttccacag ttcgtcgagg attccggcgc cggcctggag 1140
atcttcgatg gtttcgaaca gtgggatgac atcgacggtg ccgcgtgggt tgtcgccgtt 1200
ggctgcgatg agtccgaatt ccttgagcaa caccatcggc tcgagcacat cggtgaccga 1260
tgatgccatg gagatgatgc agtgaggcac catccgtggc ccgaatttct taacagcctc 1320
cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc tcgctgtatt catctgaacc 1380
gtgcgggatc agcggacgag ggctgcgcag ttccttcagc agcacctcaa gcttctctgc 1440
ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt tcgaaaagct cggtgaggac 1500
gtcctcgtag ctttcggagt tttggcgcag atccagtgcg taaaggttga atccaaagct 1560
ctcgatggca gaaatcagca cagacaaacg atcatcggca atgagaacgt ccttggattc 1620
acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat tcttccggag atgcgtatgg 1680
agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg ccgatcagct cggccgtcgt 1740
cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga taaggctcat ccacgcggct 1800
tggcacgtcg ttgtgccctg catctgccag cgcaagcagc tgcggggtga ccttattcat 1860
gcggtccgac aggctgagct catgctcgag ggaatgcagc tggcgtgcat agtacttgag 1920
cacggtttcc gcagcgcggt gagtggaata ctcaactgtt tccgcggtga cataagggtt 1980
accgtcgtgg tctccaccaa tccaggaacc tggcttgacc acgggcttca aaggaacacc 2040
ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg ttgatacgtg gaatctcttc 2100
caaaaggctc agcttgtagt agcgcagccc tacttcgatc tcgtcctcga tacgtgggcg 2160
ggccacacga atcaacgcgg tctgccacaa aatggtgatg cgacggcgga tgttcttctc 2220
gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc gcagactgca aagcgtggcg 2280
ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa acagtgcggc ggcgagtctc 2340
agttgggtgc gcagtcagaa ccggcgccac ctcagcattg cgcagcacat cggccacagc 2400
ttctgcgcca acattgccct cattgagttt cagccaggtg gcatcaagag tgctgtccgg 2460
aggggtgtcg cctgcatcga gagcctgttc acgaagctct tcatcgtaga ggtcttccgc 2520
caggttagcc agcagagcga agtgggaaaa tgcgcgagca atcggtgttg ccttggctgg 2580
agtaatgccg tcgaaaacct gaaccaggct atccatttcg gcgttgccct tggcgatatc 2640
aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc tcctggcctt cttgttccgc 2700
aattacctca ccgaggattt gaccgaggaa cctgatgtca tcgcgtaaaa aatcagtcat 2760
<210> 2
<211> 2760
<212> DNA
<213> 谷氨酸棒杆菌
<400> 2
ctagccggag ttgcgcagcg cagtggaaag accgttcatg gtcagctgaa tgttgcggga 60
cacttgctcg ctttggtcgc cttttcggta gcgtcgcatc atctctacct ggatcacgtt 120
gagtggaagc aggtaggggt atcggcgctg gacagagcgt gcgagaagtg ggttgtcatc 180
aagcagatca tcagagccgg tgattacgca gaacatcttc ttggtcagga agtactcctc 240
gcggatgacg gaatagactc gctcggctac ttccgtatct gggatcaggt ctgcgtagag 300
ctttgccaaa cgcagctctg ccttggacat cacctgagcc atgttatcca acactgaggt 360
gaaaaatggc caggactcat tgagtgtttg cagctcggca atgcgttggg tggcctgctc 420
cccttcgcca atccactgct ctaatgcggt tccgacacca aaccagcctg gcagcatgac 480
acgagactgt gaccagctga gcacccatgg gatggctcgc aaatcttcca ccgaggaggt 540
ctgcttgcgt gaggaaggcc tggatccgat gttgagggat ccaatctcct gcagcggcgt 600
ggactgggtg aagtaatcga tgaagccttg atcctcgtgc accaaggagg cgtacttctt 660
caagctgagc tcagagatct cactcatgat gtcgtacgcg cgttggtgat cggtgagttc 720
ggagacgtcg agaagcgatg cctcaagcgt ggctgagacc agggcttcga ggtttcggcg 780
cgcggtttcg gggttgccgt acttagcgga gatgatctcg ccctgctcgg tgatgcgcac 840
ggaaccttgg acagcccccc tgggctgggc aagaatcgcg tcgtaggaag gtccgccacc 900
gcggccgacg gtgccaccac ggccgtggaa caggcgaagc ttgaccccgg ctgatcggca 960
tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag tttgcggaga aatatccgcc 1020
atccttgttg gaatcggagt aaccgagcat gacttcctgg acgttgtcgc gctgcaggag 1080
gtagttgcgg tagagatcaa ttttccacag ttcgtcgagg attccggcgc cggcctggag 1140
atcttcgatg gtttcgaaca gtgggatgac atcgacggtg ccgcgtgggt tgtcgccgtt 1200
ggctgcgatg agtccgaatt ccttgagcaa caccatcggc tcgagcacat cggtgaccga 1260
tgatgccatg gagatgatgc agtgaggcac catccgtggc ccgaatttct taacaacctc 1320
cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc tcgctgtatt catctgaacc 1380
gtgcgggatc agcggacgag ggctgcgcag ttccttcagc agcacctcaa gcttctctgc 1440
ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt tcgaaaagct cggtgaggac 1500
gtcctcgtag ctttcggagt tttggcgcag atccagtgcg taaaggttga atccaaagct 1560
ctcgatggca gaaatcagca cagacaaacg atcatcggca atgagaacgt ccttggattc 1620
acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat tcttccggag atgcgtatgg 1680
agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg ccgatcagct cggccgtcgt 1740
cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga taaggctcat ccacgcggct 1800
tggcacgtcg ttgtgccctg catctgccag cgcaagcagc tgcggggtga ccttattcat 1860
gcggtccgac aggctgagct catgctcgag ggaatgcagc tggcgtgcat agtacttgag 1920
cacggtttcc gcagcgcggt gagtggaata ctcaactgtt tccgcggtga cataagggtt 1980
accgtcgtgg tctccaccaa tccaggaacc tggcttgacc acgggcttca aaggaacacc 2040
ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg ttgatacgtg gaatctcttc 2100
caaaaggctc agcttgtagt agcgcagccc tacttcgatc tcgtcctcga tacgtgggcg 2160
ggccacacga atcaacgcgg tctgccacaa aatggtgatg cgacggcgga tgttcttctc 2220
gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc gcagactgca aagcgtggcg 2280
ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa acagtgcggc ggcgagtctc 2340
agttgggtgc gcagtcagaa ccggcgccac ctcagcattg cgcagcacat cggccacagc 2400
ttctgcgcca acattgccct cattgagttt cagccaggtg gcatcaagag tgctgtccgg 2460
aggggtgtcg cctgcatcga gagcctgttc acgaagctct tcatcgtaga ggtcttccgc 2520
caggttagcc agcagagcga agtgggaaaa tgcgcgagca atcggtgttg ccttggctgg 2580
agtaatgccg tcgaaaacct gaaccaggct atccatttcg gcgttgccct tggcgatatc 2640
aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc tcctggcctt cttgttccgc 2700
aattacctca ccgaggattt gaccgaggaa cctgatgtca tcgcgtaaaa aatcagtcat 2760
<210> 3
<211> 919
<212> PRT
<213> 谷氨酸棒杆菌
<400> 3
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Arg Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Gly Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Lys Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Thr Glu Val Ala
820 825 830
Glu Arg Val Tyr Ser Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 4
<211> 919
<212> PRT
<213> 谷氨酸棒杆菌
<400> 4
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Arg Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Gly Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Lys Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Val Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Thr Glu Val Ala
820 825 830
Glu Arg Val Tyr Ser Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 5
<211> 3360
<212> DNA
<213> 谷氨酸棒杆菌
<400> 5
ctagccggag ttgcgcagcg cagtggaaag accgttcatg gtcagctgaa tgttgcggga 60
cacttgctcg ctttggtcgc cttttcggta gcgtcgcatc atctctacct ggatcacgtt 120
gagtggaagc aggtaggggt atcggcgctg gacagagcgt gcgagaagtg ggttgtcatc 180
aagcagatca tcagagccgg tgattacgca gaacatcttc ttggtcagga agtactcctc 240
gcggatgacg gaatagactc gctcggctac ttccgtatct gggatcaggt ctgcgtagag 300
ctttgccaaa cgcagctctg ccttggacat cacctgagcc atgttatcca acactgaggt 360
gaaaaatggc caggactcat tgagtgtttg cagctcggca atgcgttggg tggcctgctc 420
cccttcgcca atccactgct ctaatgcggt tccgacacca aaccagcctg gcagcatgac 480
acgagactgt gaccagctga gcacccatgg gatggctcgc aaatcttcca ccgaggaggt 540
ctgcttgcgt gaggaaggcc tggatccgat gttgagggat ccaatctcct gcagcggcgt 600
ggactgggtg aagtaatcga tgaagccttg atcctcgtgc accaaggagg cgtacttctt 660
caagctgagc tcagagatct cactcatgat gtcgtacgcg cgttggtgat cggtgagttc 720
ggagacgtcg agaagcgatg cctcaagcgt ggctgagacc agggcttcga ggtttcggcg 780
cgcggtttcg gggttgccgt acttagcgga gatgatctcg ccctgctcgg tgatgcgcac 840
ggaaccttgg acagcccccc tgggctgggc aagaatcgcg tcgtaggaag gtccgccacc 900
gcggccgacg gtgccaccac ggccgtggaa caggcgaagc ttgaccccgg ctgatcggca 960
tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag tttgcggaga aatatccgcc 1020
atccttgttg gaatcggagt aaccgagcat gacttcctgg acgttgtcgc gctgcaggag 1080
gtagttgcgg tagagatcaa ttttccacag ttcgtcgagg attccggcgc cggcctggag 1140
atcttcgatg gtttcgaaca gtgggatgac atcgacggtg ccgcgtgggt tgtcgccgtt 1200
ggctgcgatg agtccgaatt ccttgagcaa caccatcggc tcgagcacat cggtgaccga 1260
tgatgccatg gagatgatgc agtgaggcac catccgtggc ccgaatttct taacagcctc 1320
cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc tcgctgtatt catctgaacc 1380
gtgcgggatc agcggacgag ggctgcgcag ttccttcagc agcacctcaa gcttctctgc 1440
ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt tcgaaaagct cggtgaggac 1500
gtcctcgtag ctttcggagt tttggcgcag atccagtgcg taaaggttga atccaaagct 1560
ctcgatggca gaaatcagca cagacaaacg atcatcggca atgagaacgt ccttggattc 1620
acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat tcttccggag atgcgtatgg 1680
agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg ccgatcagct cggccgtcgt 1740
cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga taaggctcat ccacgcggct 1800
tggcacgtcg ttgtgccctg catctgccag cgcaagcagc tgcggggtga ccttattcat 1860
gcggtccgac aggctgagct catgctcgag ggaatgcagc tggcgtgcat agtacttgag 1920
cacggtttcc gcagcgcggt gagtggaata ctcaactgtt tccgcggtga cataagggtt 1980
accgtcgtgg tctccaccaa tccaggaacc tggcttgacc acgggcttca aaggaacacc 2040
ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg ttgatacgtg gaatctcttc 2100
caaaaggctc agcttgtagt agcgcagccc tacttcgatc tcgtcctcga tacgtgggcg 2160
ggccacacga atcaacgcgg tctgccacaa aatggtgatg cgacggcgga tgttcttctc 2220
gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc gcagactgca aagcgtggcg 2280
ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa acagtgcggc ggcgagtctc 2340
agttgggtgc gcagtcagaa ccggcgccac ctcagcattg cgcagcacat cggccacagc 2400
ttctgcgcca acattgccct cattgagttt cagccaggtg gcatcaagag tgctgtccgg 2460
aggggtgtcg cctgcatcga gagcctgttc acgaagctct tcatcgtaga ggtcttccgc 2520
caggttagcc agcagagcga agtgggaaaa tgcgcgagca atcggtgttg ccttggctgg 2580
agtaatgccg tcgaaaacct gaaccaggct atccatttcg gcgttgccct tggcgatatc 2640
aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc tcctggcctt cttgttccgc 2700
aattacctca ccgaggattt gaccgaggaa cctgatgtca tcgcgtaaaa aatcagtcat 2760
taactacttt aaacactctt tcacattgag ggtgttggcg tgcatgagat aaaaaatcac 2820
ccagcaaaac aggtgtttag ctgggcgatt gatttcgatg gctttcatgg gctcatcaaa 2880
accgcacttc tacagtgctt gtgctttaag gctgtgcttt aaggctgtgc tttaaagctc 2940
tgtactttaa gcaacgctcg cagcgttggc agccagcttg gcgaatgctt caccgtcgag 3000
ggaagcgcca ccgacaagtc cgccgtcgac gtcaggctga ccgacgatct cagcgacggt 3060
ttctgcctta acagaaccac cgtaaagaat acgcaggccc tcagcgacct cgtcgcctgc 3120
aagctccacg atcagaccgc ggatagcctt gcacacttcc tgagcgtcag ctgcggaagc 3180
aaccttaccg gtgccgatag cccacactgg ctcatacgcg ataacggtgt tggccagctc 3240
agcagcatcc aggccagcaa gggacttacg ggtctgctcg acgacgtact caacgtgggt 3300
gccagcttca cggatttcca gtggctcacc aacgcagacg atcgggctga tgccgttgga 3360
<210> 6
<211> 3359
<212> DNA
<213> 谷氨酸棒杆菌
<400> 6
ctagccggag ttgcgcagcg cagtggaaag accgttcatg gtcagctgaa tgttgcggga 60
cacttgctcg ctttggtcgc cttttcggta gcgtcgcatc atctctacct ggatcacgtt 120
gagtggaagc aggtaggggt atcggcgctg gacagagcgt gcgagaagtg ggttgtcatc 180
aagcagatca tcagagccgg tgattacgca gaacatcttc ttggtcagga agtactcctc 240
gcggatgacg gaatagactc gctcggctac ttccgtatct gggatcaggt ctgcgtagag 300
ctttgccaaa cgcagctctg ccttggacat cacctgagcc atgttatcca acactgaggt 360
gaaaaatggc caggactcat tgagtgtttg cagctcggca atgcgttggg tggcctgctc 420
cccttcgcca atccactgct ctaatgcggt tccgacacca aaccagcctg gcagcatgac 480
acgagactgt gaccagctga gcacccatgg gatggctcgc aaatcttcca ccgaggaggt 540
ctgcttgcgt gaggaaggcc tggatccgat gttgagggat ccaatctcct gcagcggcgt 600
ggactgggtg aagtaatcga tgaagccttg atcctcgtgc accaaggagg cgtacttctt 660
caagctgagc tcagagatct cactcatgat gtcgtacgcg cgttggtgat cggtgagttc 720
ggagacgtcg agaagcgatg cctcaagcgt ggctgagacc agggcttcga ggtttcggcg 780
cgcggtttcg gggttgccgt acttagcgga gatgatctcg ccctgctcgg tgatgcgcac 840
ggaaccttgg acagcccccc tgggctgggc aagaatcgcg tcgtaggaag gtccgccacc 900
gcggccgacg gtgccaccac ggccgtggaa caggcgaagc ttgaccccgg ctgatcggca 960
tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag tttgcggaga aatatccgcc 1020
atccttgttg gaatcggagt aaccgagcat gacttcctgg acgttgtcgc gctgcaggag 1080
gtagttgcgg tagagatcaa ttttccacag ttcgtcgagg attccggcgc cggcctggag 1140
atcttcgatg gtttcgaaca gtgggatgac atcgacggtg ccgcgtgggt tgtcgccgtt 1200
ggctgcgatg agtccgaatt ccttgagcaa caccatcggc tcgagcacat cggtgaccga 1260
tgatgccatg gagatgatgc agtgaggcac catccgtggc ccgaatttct taacagcctc 1320
cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc tcgctgtatt catctgaacc 1380
gtgcgggatc agcggacgag ggctgcgcag ttccttcagc agcacctcaa gcttctctgc 1440
ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt tcgaaaagct cggtgaggac 1500
gtcctcgtag ctttcggagt tttggcgcag atccagtgcg taaaggttga atccaaagct 1560
ctcgatggca gaaatcagca cagacaaacg atcatcggca atgagaacgt ccttggattc 1620
acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat tcttccggag atgcgtatgg 1680
agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg ccgatcagct cggccgtcgt 1740
cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga taaggctcat ccacgcggct 1800
tggcacgtcg ttgtgccctg catctgccag cgcaagcagc tgcggggtga ccttattcat 1860
gcggtccgac aggctgagct catgctcgag ggaatgcagc tggcgtgcat agtacttgag 1920
cacggtttcc gcagcgcggt gagtggaata ctcaactgtt tccgcggtga cataagggtt 1980
accgtcgtgg tctccaccaa tccaggaacc tggcttgacc acgggcttca aaggaacacc 2040
ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg ttgatacgtg gaatctcttc 2100
caaaaggctc agcttgtagt agcgcagccc tacttcgatc tcgtcctcga tacgtgggcg 2160
ggccacacga atcaacgcgg tctgccacaa aatggtgatg cgacggcgga tgttcttctc 2220
gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc gcagactgca aagcgtggcg 2280
ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa acagtgcggc ggcgagtctc 2340
agttgggtgc gcagtcagaa ccggcgccac ctcagcattg cgcagcacat cggccacagc 2400
ttctgcgcca acattgccct cattgagttt cagccaggtg gcatcaagag tgctgtccgg 2460
aggggtgtcg cctgcatcga gagcctgttc acgaagctct tcatcgtaga ggtcttccgc 2520
caggttagcc agcagagcga agtgggaaaa tgcgcgagca atcggtgttg ccttggctgg 2580
agtaatgccg tcgaaaacct gaaccaggct atccatttcg gcgttgccct tggcgatatc 2640
aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc tcctggcctt cttgttccgc 2700
aattacctca ccgaggattt gaccgaggaa cctgatgtca tcgcgtaaaa aatcagtcat 2760
aactacttta aacactcttt cacattgagg gtgttggcgt gcatgagata aaaaatcacc 2820
cagcaaaaca ggtgtttagc tgggcgattg atttcgatgg ctttcatggg ctcatcaaaa 2880
ccgcacttct acagtgcttg tgctttaagg ctgtgcttta aggctgtgct ttaaagctct 2940
gtactttaag caacgctcgc agcgttggca gccagcttgg cgaatgcttc accgtcgagg 3000
gaagcgccac cgacaagtcc gccgtcgacg tcaggctgac cgacgatctc agcgacggtt 3060
tctgccttaa cagaaccacc gtaaagaata cgcaggccct cagcgacctc gtcgcctgca 3120
agctccacga tcagaccgcg gatagccttg cacacttcct gagcgtcagc tgcggaagca 3180
accttaccgg tgccgatagc ccacactggc tcatacgcga taacggtgtt ggccagctca 3240
gcagcatcca ggccagcaag ggacttacgg gtctgctcga cgacgtactc aacgtgggtg 3300
ccagcttcac ggatttccag tggctcacca acgcagacga tcgggctgat gccgttgga 3359
Claims (10)
1.发酵生产L-赖氨酸的方法或者提高L-赖氨酸的发酵量的方法，其包括：
（1）改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸，优选在ppc基因的第-1位缺失一个核苷酸；和，
（2）用步骤（1）改造而得到的谷氨酸棒杆菌发酵生产L-赖氨酸。
2.改造获得的谷氨酸棒杆菌在发酵生产L-赖氨酸或者提高L-赖氨酸的发酵量中的应用，其中，所述改造获得是改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸，优选在ppc基因的第-1位缺失一个核苷酸，而获得。
3.改造谷氨酸棒杆菌的方法，其包括改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域，使该启动子区域缺失一个或几个核苷酸，优选在ppc基因的第-1位缺失一个核苷酸。
4.权利要求1-3之任一所述的方法或应用，其中，野生型的ppc基因的启动子区域的核苷酸序列如SEQ ID NO：5的第2761-3360位的互补序列所示。
5.权利要求1-3之任一所述的方法或应用，其中，野生型的ppc基因的启动子区域缺失一个核苷酸后的核苷酸序列如SEQ ID NO：6的第2761-3359位的互补序列所示。
6.权利要求1-3之任一所述的方法或应用，其中步骤（1）进一步包括：改造谷氨酸棒杆菌染色体上野生型的ppc基因，使其编码的蛋白质的第482位发生突变，优选发生A482V突变。
7.权利要求6所述的方法或应用，其中，野生型的ppc基因的核苷酸序列如SEQ ID NO：1的互补序列所示；和/或，野生型的ppc基因所编码的氨基酸序列如SEQ ID NO：3所示。
8.权利要求6所述的方法或应用，其中，发生突变的ppc基因的核苷酸序列如SEQ IDNO：2的互补序列所示；和/或，发生突变的ppc基因所编码的氨基酸序列如SEQ ID NO：4所示。
9.权利要求6所述的方法或应用，其中，改造谷氨酸棒杆菌染色体上野生型的ppc基因在改造谷氨酸棒杆菌染色体上野生型的ppc基因的启动子区域之前或之后，优选之后。
10.权利要求3-9之任一所述的方法改造而得到的谷氨酸棒杆菌。
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