CN113308426B - 一种改造tk基因5′端序列的重组棒状杆菌及其应用 - Google Patents
一种改造tk基因5′端序列的重组棒状杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及一种改造TK基因5′端序列的重组棒状杆菌及其应用。本发明的重组棒状杆菌，是在棒状杆菌宿主菌中，替换TK基因5′端序列中起始密码子TTG前‑92位～‑85位核苷酸序列，降低TK基因5′端序列二级结构的稳定性，使其更易于被转录或表达。该重组棒状杆菌与原始菌株相比，在L‑赖氨酸合成效率上有一定提高，进而提高L‑赖氨酸产量，降低生产成本，而且与现有的高产L‑赖氨酸的改造方法没有冲突，但是与现有的改造方法是否有相互促进的作用，仍需要进一步验证。
Description
技术领域
本发明涉及一种改造TK基因5′端序列的重组棒状杆菌及其应用，属于生物工程技术领域。
背景技术
L-赖氨酸是人体必需氨基酸之一，是世界上仅次于味精的第二大氨基酸品种。目前世界L-赖氨酸产品约90％用作饲料添加剂，10％用于食品和药品中间体，我国饲用赖氨酸市场起步于20世纪90年代中期，其原因主要是当时的鱼粉价格昂贵，动物营养学家和饲料企业为降低饲料成本，致力于通过在饲料中添加赖氨酸和蛋氨酸开发无鱼粉日粮，并取得了巨大成功，而且在饲料中的应用越来越广泛。L-赖氨酸最初是从蛋白质水解产物中分离得到的，蛋白质水解法一般以动物血粉为原料，但由于使用剧毒原料光气，可能残留催化剂，产品安全性差，存在严重的环保问题。目前，世界生产赖氨酸的企业大多采用发酵法，生产的为L型赖氨酸，生产工艺已基本成熟。
用于发酵法生产L-赖氨酸的微生物包括多个种属，例如棒状杆菌、芽孢杆菌、埃希氏菌等，但是，野生型菌株产L-赖氨酸的能力差，代谢副产物多，难以实现高纯度和高产量L-赖氨酸的制备。因此，通常需要获得高产L-赖氨酸的菌株。目前，获得高产L-赖氨酸的菌株的方法主要包括诱变筛选育种或者基因工程育种。诱变筛选育种是指通过紫外照射或者其他外界条件刺激，诱导菌株发生不特定的基因位点突变，然后通过筛选获得高产菌株，这种方法缺乏方向性，基因突变位点难以控制，对菌株性能的预期性差。基因工程育种是通过明确的基因改造方式来优化选育菌株，例如通过增加拷贝或者定点突变导入酶活性高的有益酶基因，或者通过敲除不利基因使酶活性/表达消失。目前，谷氨酸棒状杆菌CICC 23604已经被广泛应用于工业发酵生产各类氨基酸，其产品被FDA认证为“generally regardedas safe”(GRAS)安全级别。因此，运用代谢工程手段构建重组谷氨酸棒状杆菌是生产食品安全级L-赖氨酸的有效途径。
基因5′端序列一般是指位于结构基因5′端上游，通常包含有RNA聚合酶识别、结合和起始转录位点的一段DNA序列，它含有RNA聚合酶特异性结合和转录起始所需的保守序列，其本身不被转录。它的特性最初是通过能增加或降低基因转录速率的突变而鉴定的。启动子一般位于基因5′端序列内部，长度因生物种类而异，一般不超过200bp，是典型的顺式作用元件，与转录因子(反式作用因子)相结合调控基因表达的水平、部位及方式(中国专利文献CN109385424A)。基因5′端序列替换可被用于以特定的方式调节基因的表达，如它们的条件表达或过表达。以PCR为基础的基因靶向，通过同源重组实现开放阅读框(ORF)上游调控序列的染色体整合，可以使基因组发生稳定的改变(中国专利文献CN111655860A)。
虽然目前已有跟多关于提高L-赖氨酸产量的报道，但是开发新的提高L-赖氨酸产量的方法仍然有其必要性。
发明内容
针对现有技术的不足，本发明提供了一种改造TK基因5′端序列的重组棒状杆菌及其应用。本发明的重组棒状杆菌是通过基因工程改造棒状杆菌宿主菌获得，具体策略为运用基因5′端序列替换的方法替换转酮醇酶(Transketolase，TK)基因5′端部分核苷酸序列，获得高产L-赖氨酸的重组棒状杆菌。
术语说明：
转酮醇酶(Transketolase，TK)：是一种在戊糖磷酸循环以及光合成的还原型戊糖磷酸循环中起着重要作用的酶，广泛存在于细菌、酵母和动物植物中，催化二碳单元在酮糖(供体)和醛糖(受体)间的转移，是平衡细胞中酮糖和醛糖含量的关键酶，有研究结果表明，转酮醇酶表达量提高能增强L-赖氨酸产量。
本发明技术方案如下：
一种改造TK基因5′端序列的重组棒状杆菌，是在棒状杆菌宿主菌中，将TK基因5′端序列中起始密码子TTG前-92位～-85位核苷酸序列-92CCAATTAA-85替换为-92TGTGGTAT-85，降低TK基因5′端序列二级结构的稳定性，使其更易于被转录或表达。
根据本发明优选的，所述棒状杆菌宿主菌为谷氨酸棒状杆菌；进一步优选为谷氨酸棒状杆菌CICC23604或谷氨酸棒状杆菌CGMCC1.15647。
根据本发明优选的，所述TK的氨基酸序列为SEQ ID NO.1或SEQ ID NO.8。
根据本发明优选的，所述TK的氨基酸序列为与SEQ ID NO.1或SEQ ID NO.8的序列一致性大于等于99.7％的氨基酸序列。
根据本发明优选的，所述TK基因的核苷酸序列为SEQ ID NO.2或SEQ ID NO.9。
根据本发明优选的，所述TK基因的核苷酸序列为与SEQ ID NO.2或SEQ ID NO.9的序列一致性大于等于99.6％的核苷酸序列。
根据本发明优选的，所述TK基因5′端序列为SEQ ID NO.3或SEQ ID NO.10。
上述重组棒状杆菌的构建方法，包括如下步骤：
(1)合成上游同源臂--92TGTGGTAT-85-下游同源臂的核苷酸序列，其中上游同源臂和下游同源臂为TK基因5′端序列中起始密码子TTG前-92位～-85位核苷酸序列- 92CCAATTAA-85的前后各一段长度500～600bp的核苷酸序列；
(2)将上游同源臂--92TGTGGTAT-85-下游同源臂的核苷酸序列连接至pK19mobsacB载体中，构建替换载体；
(3)将替换载体转化棒状杆菌宿主菌感受态细胞，筛选具有卡那霉素抗性的阳性转化子，获得发生了第一次同源单交换的重组菌；
(4)将发生了第一次同源单交换的重组菌自然传代后，筛选能在10％蔗糖培养基生长但不能在具有卡那霉素抗性的培养基生长的菌落，验证后得到完成两次同源单交换的重组棒状杆菌。
根据本发明优选的，步骤(2)中上游同源臂--92TGTGGTAT-85-下游同源臂的核苷酸序列连接至pK19mobsacB载体的Sal I、EcoR I酶切位点之间。
根据本发明优选的，步骤(3)中以卡那霉素抗性基因引物采用PCR扩增技术筛选具有卡那霉素抗性的阳性转化子，所述引物序列如下：
F1：5′-ATGATTGAACAAGATGGATTGC-3′，
R1：5′-TCAGAAGAACTCGTCAAGAAGGCG-3′。
进一步优选的，所述PCR扩增的体系为：2×HiFi-PCRmaster10μL，10μmol/L F1 1μL，10μmol/L R1 1μL，模板1μL，ddH2O 7μL；
所述PCR扩增的程序为：95℃预变性5min；94℃变性30sec，56℃退火30sec，72℃延伸1min，30个循环；72℃延伸10min，4℃保存。
根据本发明优选的，步骤(4)所述验证是采用PCR扩增技术验证，PCR扩增的引物序列如下：
F2：5′-AAATTTGAATGTGGTAT-3′，
R2：5′-TTAACTGTTAATGGAGTCCT-3′。
进一步优选的，所述PCR扩增的体系为：2×HiFi-PCRmaster10μL，10μmol/L F2 1μL，10μmol/L R2 1μL，模板1μL，ddH2O 7μL；
所述PCR扩增的程序为：95℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸3min，30个循环；72℃延伸10min，4℃保存。
根据本发明优选的，步骤(4)中所用培养基为LBG培养基：葡萄糖5g/L，蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L。
上述重组棒状杆菌在生产L-赖氨酸中的应用。
根据本发明优选的，所述应用是将重组棒状杆菌接种至液体LBG培养基中进行种子培养，其后按体积百分比2～5％接种量接种至发酵培养基发酵培养；
所述LBG培养基：葡萄糖5g/L，蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L；
所述发酵培养基：葡萄糖100g/L，蛋白胨20g/L，玉米浆30mL/L，尿素5g/L，(NH4)2SO4 25g/L，L-亮氨酸0.34g/L，KH2PO4 2g/L，MgSO4·7H2O 1.5g/L，生物素0.001g/L。
根据本发明优选的，所述种子培养的条件为200～220rpm、28～30℃下培养18-25h；所述发酵培养的条件为200～220rpm、28～30℃。
本发明的技术原理是：
基因5′端序列包含基因的启动子区序列，启动子区序列的二级结构能够影响启动子启动效率，进而影响启动子后基因的表达活性。本发明通过将TK基因5′端序列中起始密码子TTG前-92位～-85位核苷酸序列-92CCAATTAA-85替换为-92TGTGGTAT-85，能够降低TK基因5′端序列二级结构的稳定性，进而使其更易于被转录或表达。
有益效果：
本发明提供了一种改造TK基因5′端序列的重组棒状杆菌，是将TK基因5′端序列中起始密码子TTG前-92位～-85位核苷酸序列-92CCAATTAA-85替换为-92TGTGGTAT-85，该重组棒状杆菌与原始菌株相比，在L-赖氨酸合成效率上有一定提高，进而提高L-赖氨酸产量，降低生产成本，而且与现有的高产L-赖氨酸的改造方法没有冲突，但是与现有的改造方法是否有相互促进的作用，仍需要进一步验证。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述，但本发明所保护范围不限于此。实施例中的涉及的试剂及药品，若无特殊说明，均为普通市售产品；实施例中涉及的实验方法，若无特殊说明，均为本领域常规技术手段。
微生物来源：
谷氨酸棒状杆菌CICC23604，购自中国工业微生物菌种保藏管理中心，菌种编号CICC23604，其中转酮醇酶(TK)的氨基酸序列为SEQ ID NO.1，TK基因的核苷酸序列为SEQID NO.2，TK基因5′端序列为SEQ ID NO.3。
谷氨酸棒状杆菌CGMCC1.15647，购自中国普通微生物菌种保藏管理中心，菌种编号CGMCC1.15647，其中转酮醇酶(TK)的氨基酸序列为SEQ ID NO.8，TK基因的核苷酸序列为SEQ ID NO.9，TK基因5′端序列为SEQ ID NO.10。
SEQ ID NO.1与SEQ ID NO.8氨基酸序列一致性为99.7％，SEQ ID NO.2与SEQ IDNO.9核苷酸序列一致性为99.6％，SEQ ID NO.3与SEQ ID NO.10核苷酸序列一致性为100％。
实施例中涉及的玉米浆为发酵专用玉米浆，可购自诸城东晓生物科技有限公司。
实施例1：含有替换核苷酸序列的同源臂基因合成及替换载体构建
1.1谷氨酸棒状杆菌CICC23604
针对谷氨酸棒状杆菌CICC23604，合成上游同源臂--92TGTGGTAT-85-下游同源臂的核苷酸序列，上游同源臂和下游同源臂为TK基因5′端序列中起始密码子TTG前-92位～-85位核苷酸序列-92CCAATTAA-85的前后各一段长度500bp的核苷酸序列，其中，上游同源臂的核苷酸序列为SEQ ID NO.4，下游同源臂的核苷酸序列为SEQ ID NO.5，原始的上游同源臂-- 92CCAATTAA-85-下游同源臂的核苷酸序列为SEQ ID NO.6，设计合成的上游同源臂-- 92TGTGGTAT-85-下游同源臂的核苷酸序列为SEQ ID NO.7。SEQ ID NO.7序列由上海生工生物工程有限公司合成，并连接至pK19mobsacB载体(GenBank:LC257601.1)的Sal I、EcoRI酶切位点之间。将连接产物转化至大肠杆菌DH5α感受态细胞中，取200μL转化液使用已灭菌涂布器涂布于含卡纳霉素抗生素(终浓度为50μg/mL)的固体LB平板上，置于37℃的培养箱中过夜培养，筛选获得阳性转化子，用于替换载体pK19mobsacB-TK1的提取和保存。
1.2谷氨酸棒状杆菌CGMCC1.15647
针对谷氨酸棒状杆菌CGMCC1.15647，合成上游同源臂--92TGTGGTAT-85-下游同源臂的核苷酸序列，上游同源臂和下游同源臂为TK基因5′端序列中起始密码子TTG前-92位～-85位核苷酸序列-92CCAATTAA-85的前后各一段长度500bp的核苷酸序列，其中，上游同源臂的核苷酸序列为SEQ ID NO.11，下游同源臂的核苷酸序列为SEQ ID NO.12，原始的上游同源臂--92CCAATTAA-85-下游同源臂的核苷酸序列为SEQ ID NO.13，设计合成的上游同源臂-- 92TGTGGTAT-85-下游同源臂的核苷酸序列为SEQ ID NO.14。SEQ ID NO.14序列由上海生工生物工程有限公司合成，并连接至pK19mobsacB载体(GenBank:LC257601.1)的Sal I、EcoR I酶切位点之间。将连接产物转化至大肠杆菌DH5α感受态细胞中，取200μL转化液使用已灭菌涂布器涂布于含卡纳霉素抗生素(终浓度为50μg/mL)的固体LB平板上，置于37℃的培养箱中过夜培养，筛选获得阳性转化子，用于替换载体pK19mobsacB-TK2的提取和保存。
实施例2：制备谷氨酸棒状杆菌CICC23604/CGMCC1.15647感受态细胞
(1)挑取谷氨酸棒状杆菌单菌落，接种于10mL种子培养基中，37℃、220r/min，过夜培养；
种子培养基(1000mL)，组分如下：蛋白胨10g、酵母粉5g、氯化钠10g、山梨醇91g；
(2)取1mL上述菌液转接到100mL种子培养基中，37℃、220r/min培养至OD600＝0.9；
(3)将菌液转移至100mL离心管，冰浴15-20min，使菌体停止生长；
(4)冰浴后4℃、5000r/min、5min离心，收集菌体；
(5)离心后的菌体用预冷的电转缓冲液(ETM)洗涤3次；
电转缓冲液(1000mL)，每升组分如下：山梨醇91g、甘露醇91g、甘油100mL；
(6)洗涤结束后，使用1000μL电转缓冲液重悬菌体，得感受态细胞；
(7)将制备好的感受态细胞分装100μL/管，-80℃保存，备用。
实施例3：替换载体电转谷氨酸棒状杆菌感受态细胞
首先利用核酸超微量分光光度计测定替换载体pK19mobsacB-TK1或pK19mobsacB-TK2片段浓度，达到300μg/mL浓度后1800V电击5ms，进行电转化，分别转化至谷氨酸棒状杆菌CICC23604感受态细胞和谷氨酸棒状杆菌CGMCC1.15647感受态细胞中，得到的细胞使用液体复苏培养基30℃复苏培养1h后，取100μL涂布在含25μg/mL卡那霉素的LB固体培养基上，在37℃培养2天，分别筛选具有卡那霉素抗性的谷氨酸棒状杆菌CICC23604转化子和谷氨酸棒状杆菌CGMCC1.15647转化子。
其中液体复苏培养基，每1000mL组分如下：蛋白胨10g、酵母粉5g、氯化钠10g、山梨醇91g、甘露醇69.4g。
实施例4：阳性重组菌的培养及鉴定
(1)卡那霉素抗性初筛
挑取实施例3中卡那霉素抗性平板上的单菌落分别接种于含25μg/mL卡那霉素的液体LBG培养基中，提取基因组作为模板DNA，并采用卡那霉素抗性基因引物进行PCR扩增来验证，在795bp处有目的条带即为阳性转化子；
其中，所述LBG培养基：葡萄糖5g/L，蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L；
所述卡那霉素抗性基因引物序列如下：
F1：5′-ATGATTGAACAAGATGGATTGC-3′，
R1：5′-TCAGAAGAACTCGTCAAGAAGGCG-3′；
所述的PCR扩增体系如下：
表1 PCR扩增体系
所述的PCR扩增程序如下：
95℃预变性5min；94℃变性30sec，56℃退火30sec，72℃延伸1min，30个循环；72℃延伸10min，4℃保存。
挑选在795bp处有特异性条带出现的菌株作为发生同源单交换的菌株进行进一步验证。
(2)蔗糖平板复筛
将上述验证正确的发生同源单交换的菌株分别接种到不含抗生素的液体LBG培养基中，自然传代3次，每代培养24h，最后取200μL菌液涂布到含10％蔗糖的固体LBG培养基(不含抗生素)上，18-24h后，挑选菌落点种至具有卡那霉素抗性(25μg/mL)的固体LBG培养基进行培养，筛选能在10％蔗糖LBG培养基生长但不能在具有卡那霉素抗性的LBG培养基生长的菌落，进行基因组的提取，采用PCR扩增进行验证；
其中，所述PCR扩增的引物序列如下：
F2：5′-AAATTTGAATGTGGTAT-3′，
R2：5′-TTAACTGTTAATGGAGTCCT-3′，
F2引物3′末端序列含有替换后的位点-92TGTGGTAT-85；
所述PCR扩增体系如下：
表2 PCR扩增体系
所述的PCR扩增程序如下：
95℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸3min，30个循环；72℃延伸10min，4℃保存；
挑选在2195bp处的特异性条带，分别连接至pMD18-T载体，并采用载体引物测通测序，验证后获得完成两次同源单交换的重组谷氨酸棒状杆菌TK1和TK2。
实施例5：重组谷氨酸棒状杆菌的稳定性验证
将上述筛选出并验证正确的重组谷氨酸棒状杆菌TK1和TK2分别进行传代培养，首先挑取平板上的单菌落接种于不含抗生素的液体LBG培养基中培养12h，之后按照体积百分比1％的接种量连续传代培养30代，取最后一代菌液进行基因组提取，用F2和R2为引物进行菌落PCR验证。结果显示，使用引物F2和R2能够扩增出一条特异性基因条带，大小约为2195bp，与理论值相符，证明替换的-92TGTGGTAT-85已成功整合到重组谷氨酸棒状杆菌TK1和TK2基因组上，并稳定存在；
其中，所述的PCR扩增体系如下：
表3 PCR扩增体系
所述的PCR扩增程序如下：
95℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸3min，30个循环；72℃延伸10min，4℃保存。
实施例6：L-赖氨酸发酵测试
将上述制备的重组谷氨酸棒状杆菌TK1和TK2分别接种至100mLLBG培养基(葡萄糖5g/L，蛋白胨10g/L，酵母膏5g/L，NaCl10g/L)中220rpm、30℃下进行种子培养20h，其后按体积百分比2％接种量接种至100mL发酵培养基(葡萄糖100g/L，蛋白胨20g/L，玉米浆30mL/L，尿素5g/L，(NH4)2SO425g/L，L-亮氨酸0.34g/L，KH2PO42g/L，MgSO4·7H2O1.5g/L，生物素0.001g/L)发酵培养48h，每隔12h取样，并通过生物传感分析仪SBA-40C(山东省科学院生物研究所制)测定发酵液中L-赖氨酸的含量，结果如表4、表5所示。
表4不同时间重组谷氨酸棒状杆菌TK1与原始菌L-赖氨酸的平均产量
表5不同时间重组谷氨酸棒状杆菌TK2与原始菌L-赖氨酸的平均产量
结果显示与原始菌相比，在发酵48h后，重组谷氨酸棒状杆菌TK1发酵液中L-赖氨酸的含量达到47.1g/L，较原始菌提高了10.05％，重组谷氨酸棒状杆菌TK2发酵液中L-赖氨酸的含量达到0.84g/L，是原始菌的1.91倍，这表明将谷氨酸棒状杆菌TK基因5′端序列起始密码子TTG前-92位～-85位核苷酸序列替换为-92TGTGGTAT-85后可以提高L-赖氨酸发酵的产酸水平，也是一种新的提高L-赖氨酸产量的方法。
使用RNAfold web server(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)对谷氨酸棒状杆菌CICC23604和CGMCC1.15647中TK基因5′端序列(SEQ ID NO.3与SEQ ID NO.10)起始密码子TTG前-92位～-85位核苷酸序列替换前后的序列进行稳定性分析，结果显示谷氨酸棒状杆菌CICC23604和CGMCC1.15647中TK基因5′端序列起始密码子TTG前-92位～-85位核苷酸序列替换为-92TGTGGTAT-85后，5′端序列的最小自由能(minimum free energy)由-20.10kcal/mol提高至-18.00kcal/mol。这表明替换序列能够降低5′端序列的稳定性，进而使得TK基因更容易被转录和翻译，最终提高了氨基酸发酵的产酸水平。
SEQUENCE LISTING
<110> 齐鲁工业大学
诸城东晓生物科技有限公司
<120> 一种改造TK基因5′端序列的重组棒状杆菌及其应用
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 697
<212> PRT
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 1
Met Thr Leu Ser Pro Glu Leu Gln Ala Leu Thr Val Arg Asn Tyr Pro
1 5 10 15
Ser Asp Trp Ser Asp Val Asp Thr Lys Ala Val Asp Thr Val Arg Val
20 25 30
Leu Ala Ala Asp Ala Val Glu Asn Cys Gly Ser Gly His Pro Gly Thr
35 40 45
Ala Met Ser Leu Ala Pro Leu Ala Tyr Thr Leu Tyr Gln Arg Val Met
50 55 60
Asn Val Asp Pro Gln Asp Thr Asn Trp Ala Gly Arg Asp Arg Phe Val
65 70 75 80
Leu Ser Cys Gly His Ser Ser Leu Thr Gln Tyr Ile Gln Leu Tyr Leu
85 90 95
Gly Gly Phe Gly Leu Glu Met Asp Asp Leu Lys Ala Leu Arg Thr Trp
100 105 110
Asp Ser Leu Thr Pro Gly His Pro Glu Tyr Arg His Thr Lys Gly Val
115 120 125
Glu Ile Thr Thr Gly Pro Leu Gly Gln Gly Leu Ala Ser Ala Val Gly
130 135 140
Met Ala Met Ala Ala Arg Arg Glu Arg Gly Leu Phe Asp Pro Thr Ala
145 150 155 160
Ala Glu Gly Glu Ser Pro Phe Asp His His Ile Tyr Val Ile Ala Ser
165 170 175
Asp Gly Asp Leu Gln Glu Gly Val Thr Ser Glu Ala Ser Ser Ile Ala
180 185 190
Gly Thr Gln Gln Leu Gly Asn Leu Ile Val Phe Trp Asp Asp Asn Arg
195 200 205
Ile Ser Ile Glu Asp Asn Thr Glu Ile Ala Phe Asn Glu Asp Val Val
210 215 220
Ala Arg Tyr Lys Ala Tyr Gly Trp Gln Thr Ile Glu Val Glu Ala Gly
225 230 235 240
Glu Asp Val Ala Ala Ile Glu Ala Ala Val Ala Glu Ala Lys Lys Asp
245 250 255
Thr Lys Arg Pro Thr Phe Ile Arg Val Arg Thr Ile Ile Gly Phe Pro
260 265 270
Ala Pro Thr Met Met Asn Thr Gly Ala Val His Gly Ala Ala Leu Gly
275 280 285
Ala Ala Glu Val Ala Ala Thr Lys Thr Glu Leu Gly Phe Asp Pro Glu
290 295 300
Ala His Phe Ala Ile Asp Asp Glu Val Ile Ala His Thr Arg Ser Leu
305 310 315 320
Ala Glu Arg Ala Ala Glu Lys Lys Ala Thr Trp Gln Val Lys Phe Asp
325 330 335
Glu Trp Ala Ala Ala Asn Pro Glu Asn Lys Ala Leu Phe Asp Arg Leu
340 345 350
Asn Ser Arg Glu Leu Pro Ala Gly Tyr Ala Asp Glu Leu Pro Thr Trp
355 360 365
Asp Ala Asp Glu Lys Gly Val Ala Thr Arg Lys Ala Ser Glu Ala Ala
370 375 380
Leu Gln Ala Leu Gly Lys Thr Leu Pro Glu Leu Trp Gly Gly Ser Ala
385 390 395 400
Asp Leu Ala Gly Ser Asn Asn Thr Val Ile Lys Gly Ser Pro Ser Phe
405 410 415
Gly Pro Glu Ser Ile Ser Thr Glu Thr Trp Ser Ala Glu Pro Tyr Gly
420 425 430
Arg Asn Leu His Phe Gly Ile Arg Glu His Ala Met Gly Ser Ile Leu
435 440 445
Asn Gly Ile Ser Leu His Gly Gly Thr Arg Pro Tyr Gly Gly Thr Phe
450 455 460
Leu Ile Phe Ser Asp Tyr Met Arg Pro Ala Val Arg Leu Ala Ala Leu
465 470 475 480
Met Glu Thr Asp Ala Tyr Tyr Val Trp Thr His Asp Ser Ile Gly Leu
485 490 495
Gly Glu Asp Gly Pro Thr His Gln Pro Val Glu Thr Leu Ala Ala Leu
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Arg Ala Ile Pro Gly Leu Ser Val Leu Arg Pro Ala Asp Ala Asn Glu
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Thr Ala Gln Ala Trp Ala Ala Ala Leu Glu Tyr Lys Glu Gly Pro Lys
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Gly Leu Ala Leu Thr Arg Gln Asn Ile Pro Val Leu Glu Gly Thr Lys
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Glu Lys Ala Ala Glu Gly Val Arg Arg Gly Gly Tyr Val Leu Val Glu
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Gly Ser Lys Glu Thr Pro Asp Val Ile Leu Met Gly Ser Gly Ser Glu
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Val Gln Leu Ala Val Asn Ala Ala Lys Ala Leu Glu Ala Glu Gly Val
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Ala Ala Arg Val Val Ser Val Pro Cys Met Asp Trp Phe Gln Glu Gln
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Asp Ala Glu Tyr Ile Glu Ser Val Leu Pro Ala Ala Val Thr Ala Arg
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Val Ser Val Glu Ala Gly Ile Ala Met Pro Trp Tyr Arg Phe Leu Gly
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Thr Gln Gly Arg Ala Val Ser Leu Glu His Phe Gly Ala Ser Ala Asp
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Tyr Gln Thr Leu Phe Glu Lys Phe Gly Ile Thr Thr Asp Ala Val Val
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Ala Ala Ala Lys Asp Ser Ile Asn Ser
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<210> 2
<211> 2094
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 2
ttgacgctgt cacctgaact tcaggcgctc actgtacgca attacccctc tgattggtcc 60
gatgtggaca ccaaggctgt agacactgtt cgtgtcctcg ctgcagacgc tgtagaaaac 120
tgtggctccg gccacccagg caccgcaatg agcctggctc cccttgcata caccttgtac 180
cagcgggtta tgaacgtaga tccacaggac accaactggg caggccgtga ccgcttcgtt 240
ctttcttgtg gccactcctc tttgacccag tacatccagc tttacttggg tggattcggc 300
cttgagatgg atgacctgaa ggctctgcgc acctgggatt ccttgacccc aggacaccct 360
gagtaccgcc acaccaaggg cgttgagatc accactggcc ctcttggcca gggtcttgca 420
tctgcagttg gtatggccat ggctgctcgt cgtgagcgtg gcctattcga cccaaccgct 480
gctgagggcg aatccccatt cgaccaccac atctacgtca ttgcttctga tggtgacctg 540
caggaaggtg tcacctctga ggcatcctcc atcgctggca cccagcagct gggcaacctc 600
atcgtgttct gggatgacaa ccgcatctcc atcgaagaca acactgagat cgctttcaac 660
gaggacgttg ttgctcgtta caaggcttac ggctggcaga ccattgaggt tgaggctggc 720
gaggacgttg cagcaatcga agctgcagtg gctgaggcta agaaggacac caagcgacct 780
accttcatcc gcgttcgcac catcatcggc ttcccagctc caactatgat gaacaccggt 840
gctgtgcacg gtgctgctct tggcgcagct gaggttgcag caaccaagac tgagcttgga 900
ttcgatcctg aggctcactt cgcgatcgac gatgaggtca tcgctcacac ccgctccctc 960
gcagagcgcg ctgcagagaa gaaggctaca tggcaggtca agttcgatga gtgggcagct 1020
gccaaccctg agaacaaggc tctgttcgat cgcctgaact cccgtgagct tccagcgggc 1080
tacgctgacg agctcccaac atgggatgca gatgagaagg gcgtcgcaac tcgtaaggct 1140
tccgaggctg cacttcaggc actgggcaag acccttcctg agctgtgggg cggttccgct 1200
gacctcgcgg gttccaacaa caccgtgatc aagggctccc cttccttcgg ccctgagtcc 1260
atctccaccg agacctggtc tgctgagcct tacggccgta acctgcactt cggtatccgt 1320
gagcacgcta tgggatccat cctcaacggc atttccctcc acggtggcac ccgcccatac 1380
ggcggaacct tcctcatctt ctccgactac atgcgtcctg cagttcgtct tgcagctctc 1440
atggagaccg acgcttacta cgtctggacc cacgactcca tcggtctggg cgaagatggc 1500
ccaacccacc agcctgttga aaccttggct gcactgcgcg ccatcccagg tctgtccgtc 1560
ctgcgtcctg cagatgcgaa cgagaccgcc caggcttggg ctgcagcact tgagtacaag 1620
gaaggcccta agggtcttgc actgacccgc cagaacattc ctgttctgga aggcaccaag 1680
gagaaggctg ctgaaggcgt tcgccgcggt ggctacgtcc tggttgaggg ttccaaggaa 1740
accccagatg tgatcctcat gggctccggc tccgaggttc agcttgcagt taacgctgcg 1800
aaggctctgg aagctgaggg cgttgcagct cgcgttgttt ccgttccttg catggattgg 1860
ttccaggagc aggacgcaga gtacatcgag tccgttctgc ctgcagctgt gaccgctcgt 1920
gtgtctgttg aagctggcat cgcaatgcct tggtaccgct tcttgggcac ccagggccgt 1980
gctgtctccc ttgagcactt cggtgcttct gcggattacc agaccctgtt tgagaagttc 2040
ggcatcacca ccgatgcagt cgtggcagcg gccaaggact ccattaacag ttaa 2094
<210> 3
<211> 150
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 3
gacgggtcag attaagcaaa gactactttc ggggtagatc acctttgcca aatttgaacc 60
aattaaccta agtcgtagat ctgatcatcg gatctaacga aaacgaacca aaactttggt 120
cccggtttaa cccaggaagg attgaccacc 150
<210> 4
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 4
tttaaaaagc ttcagacttt tatttccact tcataaaaac tgcctgtgac gattccgtta 60
aagattgtgc caaatcactg cgcaaaactc gcgcggaacc agaccttgcc atgctatcgc 120
ctattcacac tatttgagta atcggaaata gatgggtgta gacgcttgat tggcggacgg 180
ttcacagcgg acgatttcag gccctcgtag ctcgagagtt tgaaggggtc cgattcgttc 240
cgttcgtgac gctttgtgag gttttttgac gttgcaccgt attgcttgcc gaacattttt 300
cttttccttt cggtttttcg agaattttca cctacaaaag cccacgtcac agctcccaga 360
cttaagattg gtcacacctt tgacacattt gaaccacagt tggttataaa atgggttcaa 420
catcactatg gttagaggtg ttgacgggtc agattaagca aagactactt tcggggtaga 480
tcacctttgc caaatttgaa 500
<210> 5
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 5
cctaagtcgt agatctgatc atcggatcta acgaaaacga accaaaactt tggtcccggt 60
ttaacccagg aaggattgac caccttgacg ctgtcacctg aacttcaggc gctcactgta 120
cgcaattacc cctctgattg gtccgatgtg gacaccaagg ctgtagacac tgttcgtgtc 180
ctcgctgcag acgctgtaga aaactgtggc tccggccacc caggcaccgc aatgagcctg 240
gctccccttg catacacctt gtaccagcgg gttatgaacg tagatccaca ggacaccaac 300
tgggcaggcc gtgaccgctt cgttctttct tgtggccact cctctttgac ccagtacatc 360
cagctttact tgggtggatt cggccttgag atggatgacc tgaaggctct gcgcacctgg 420
gattccttga ccccaggaca ccctgagtac cgccacacca agggcgttga gatcaccact 480
ggccctcttg gccagggtct 500
<210> 6
<211> 1008
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 6
tttaaaaagc ttcagacttt tatttccact tcataaaaac tgcctgtgac gattccgtta 60
aagattgtgc caaatcactg cgcaaaactc gcgcggaacc agaccttgcc atgctatcgc 120
ctattcacac tatttgagta atcggaaata gatgggtgta gacgcttgat tggcggacgg 180
ttcacagcgg acgatttcag gccctcgtag ctcgagagtt tgaaggggtc cgattcgttc 240
cgttcgtgac gctttgtgag gttttttgac gttgcaccgt attgcttgcc gaacattttt 300
cttttccttt cggtttttcg agaattttca cctacaaaag cccacgtcac agctcccaga 360
cttaagattg gtcacacctt tgacacattt gaaccacagt tggttataaa atgggttcaa 420
catcactatg gttagaggtg ttgacgggtc agattaagca aagactactt tcggggtaga 480
tcacctttgc caaatttgaa ccaattaacc taagtcgtag atctgatcat cggatctaac 540
gaaaacgaac caaaactttg gtcccggttt aacccaggaa ggattgacca ccttgacgct 600
gtcacctgaa cttcaggcgc tcactgtacg caattacccc tctgattggt ccgatgtgga 660
caccaaggct gtagacactg ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc 720
cggccaccca ggcaccgcaa tgagcctggc tccccttgca tacaccttgt accagcgggt 780
tatgaacgta gatccacagg acaccaactg ggcaggccgt gaccgcttcg ttctttcttg 840
tggccactcc tctttgaccc agtacatcca gctttacttg ggtggattcg gccttgagat 900
ggatgacctg aaggctctgc gcacctggga ttccttgacc ccaggacacc ctgagtaccg 960
ccacaccaag ggcgttgaga tcaccactgg ccctcttggc cagggtct 1008
<210> 7
<211> 1008
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (501)..(508)
<223> 将原始核苷酸序列CCAATTAA替换为TGTGGTAT
<400> 7
tttaaaaagc ttcagacttt tatttccact tcataaaaac tgcctgtgac gattccgtta 60
aagattgtgc caaatcactg cgcaaaactc gcgcggaacc agaccttgcc atgctatcgc 120
ctattcacac tatttgagta atcggaaata gatgggtgta gacgcttgat tggcggacgg 180
ttcacagcgg acgatttcag gccctcgtag ctcgagagtt tgaaggggtc cgattcgttc 240
cgttcgtgac gctttgtgag gttttttgac gttgcaccgt attgcttgcc gaacattttt 300
cttttccttt cggtttttcg agaattttca cctacaaaag cccacgtcac agctcccaga 360
cttaagattg gtcacacctt tgacacattt gaaccacagt tggttataaa atgggttcaa 420
catcactatg gttagaggtg ttgacgggtc agattaagca aagactactt tcggggtaga 480
tcacctttgc caaatttgaa tgtggtatcc taagtcgtag atctgatcat cggatctaac 540
gaaaacgaac caaaactttg gtcccggttt aacccaggaa ggattgacca ccttgacgct 600
gtcacctgaa cttcaggcgc tcactgtacg caattacccc tctgattggt ccgatgtgga 660
caccaaggct gtagacactg ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc 720
cggccaccca ggcaccgcaa tgagcctggc tccccttgca tacaccttgt accagcgggt 780
tatgaacgta gatccacagg acaccaactg ggcaggccgt gaccgcttcg ttctttcttg 840
tggccactcc tctttgaccc agtacatcca gctttacttg ggtggattcg gccttgagat 900
ggatgacctg aaggctctgc gcacctggga ttccttgacc ccaggacacc ctgagtaccg 960
ccacaccaag ggcgttgaga tcaccactgg ccctcttggc cagggtct 1008
<210> 8
<211> 697
<212> PRT
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 8
Met Thr Leu Ser Pro Glu Leu Gln Ala Leu Thr Val Arg Asn Tyr Pro
1 5 10 15
Ser Asp Trp Ser Asp Val Asp Thr Lys Ala Val Asp Thr Val Arg Val
20 25 30
Leu Ala Ala Asp Ala Val Glu Asn Cys Gly Ser Gly His Pro Gly Thr
35 40 45
Ala Met Ser Leu Ala Pro Leu Ala Tyr Thr Leu Tyr Gln Arg Val Met
50 55 60
Asn Val Asp Pro Gln Asp Thr Asn Trp Ala Gly Arg Asp Arg Phe Val
65 70 75 80
Leu Ser Cys Gly His Ser Ser Leu Thr Gln Tyr Ile Gln Leu Tyr Leu
85 90 95
Gly Gly Phe Gly Leu Glu Met Asp Asp Leu Lys Ala Leu Arg Thr Trp
100 105 110
Asp Ser Leu Thr Pro Gly His Pro Glu Tyr Arg His Thr Lys Gly Val
115 120 125
Glu Ile Thr Thr Gly Pro Leu Gly Gln Gly Leu Ala Ser Ala Val Gly
130 135 140
Met Ala Met Ala Ala Arg Arg Glu Arg Gly Leu Phe Asp Pro Thr Ala
145 150 155 160
Ala Glu Gly Glu Ser Pro Phe Asp His His Ile Tyr Val Ile Ala Ser
165 170 175
Asp Gly Asp Leu Gln Glu Gly Val Thr Ser Glu Ala Ser Ser Ile Ala
180 185 190
Gly Thr Gln Gln Leu Gly Asn Leu Ile Val Phe Trp Asp Asp Asn Arg
195 200 205
Ile Ser Ile Glu Asp Asn Thr Glu Ile Ala Phe Asn Glu Asp Val Val
210 215 220
Ala Arg Tyr Lys Ala Tyr Gly Trp Gln Thr Ile Glu Val Glu Ala Gly
225 230 235 240
Glu Asp Val Ala Ala Ile Glu Ala Ala Val Ala Glu Ala Lys Lys Asp
245 250 255
Thr Lys Arg Pro Thr Phe Ile Arg Val Arg Thr Ile Ile Gly Phe Pro
260 265 270
Ala Pro Thr Met Met Asn Thr Gly Ala Val His Gly Ala Ala Leu Gly
275 280 285
Ala Ala Glu Val Ala Ala Thr Lys Thr Glu Leu Gly Phe Asp Pro Glu
290 295 300
Ala His Phe Ala Ile Asp Asp Glu Val Ile Ala His Thr Arg Ser Leu
305 310 315 320
Ala Glu Arg Ala Ala Glu Lys Lys Ala Ala Trp Gln Val Lys Phe Asp
325 330 335
Glu Trp Ala Ala Ala Asn Pro Glu Asn Lys Ala Leu Phe Asp Arg Leu
340 345 350
Asn Ser Arg Glu Leu Pro Ala Gly Tyr Ala Asp Glu Leu Pro Thr Trp
355 360 365
Asp Ala Asp Glu Lys Gly Val Ala Thr Arg Lys Ala Ser Glu Ala Ala
370 375 380
Leu Gln Ala Leu Gly Lys Thr Leu Pro Glu Leu Trp Gly Gly Ser Ala
385 390 395 400
Asp Leu Ala Gly Ser Asn Asn Thr Val Ile Lys Gly Ser Pro Ser Phe
405 410 415
Gly Pro Glu Ser Ile Ser Thr Glu Thr Trp Ser Ala Glu Pro Tyr Gly
420 425 430
Arg Asn Leu His Phe Gly Ile Arg Glu His Ala Met Gly Ser Ile Leu
435 440 445
Asn Gly Ile Ser Leu His Gly Gly Thr Arg Pro Tyr Gly Gly Thr Phe
450 455 460
Leu Ile Phe Ser Asp Tyr Met Arg Pro Ala Val Arg Leu Ala Ala Leu
465 470 475 480
Met Glu Thr Asp Ala Tyr Tyr Val Trp Thr His Asp Ser Ile Gly Leu
485 490 495
Gly Glu Asp Gly Pro Thr His Gln Pro Val Glu Thr Leu Ala Ala Leu
500 505 510
Arg Ala Ile Pro Gly Leu Ser Val Leu Arg Pro Ala Asp Ala Asn Glu
515 520 525
Thr Ala Gln Ala Trp Ala Ala Ala Leu Glu Tyr Lys Glu Gly Pro Lys
530 535 540
Gly Leu Ala Leu Thr Arg Gln Asn Val Pro Val Leu Glu Gly Thr Lys
545 550 555 560
Glu Lys Ala Ala Glu Gly Val Arg Arg Gly Gly Tyr Val Leu Val Glu
565 570 575
Gly Ser Lys Glu Thr Pro Asp Val Ile Leu Met Gly Ser Gly Ser Glu
580 585 590
Val Gln Leu Ala Val Asn Ala Ala Lys Ala Leu Glu Ala Glu Gly Val
595 600 605
Ala Ala Arg Val Val Ser Val Pro Cys Met Asp Trp Phe Gln Glu Gln
610 615 620
Asp Ala Glu Tyr Ile Glu Ser Val Leu Pro Ala Ala Val Thr Ala Arg
625 630 635 640
Val Ser Val Glu Ala Gly Ile Ala Met Pro Trp Tyr Arg Phe Leu Gly
645 650 655
Thr Gln Gly Arg Ala Val Ser Leu Glu His Phe Gly Ala Ser Ala Asp
660 665 670
Tyr Gln Thr Leu Phe Glu Lys Phe Gly Ile Thr Thr Asp Ala Val Val
675 680 685
Ala Ala Ala Lys Asp Ser Ile Asn Ser
690 695
<210> 9
<211> 2094
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 9
ttgacgctgt cacctgaact tcaggcgctc actgtacgca attacccctc tgattggtcc 60
gatgtggaca ccaaggctgt agacactgtt cgtgtcctcg ctgcagacgc tgtagaaaac 120
tgtggctccg gccacccagg caccgcaatg agcctggctc cccttgcata caccttgtac 180
cagcgggtta tgaacgtaga tccacaggac accaactggg caggccgtga ccgcttcgtt 240
ctttcttgtg gccactcctc tttgacccag tacatccagc tttacttggg tggattcggg 300
cttgagatgg atgacctgaa ggctctgcgc acctgggatt ccttgacccc aggacaccct 360
gagtaccgcc acaccaaggg cgttgagatc accactggcc ctcttggcca gggtcttgca 420
tctgcagttg gtatggccat ggctgctcgt cgtgagcgtg gcctattcga cccaaccgct 480
gctgagggcg aatccccatt cgaccaccac atctacgtca ttgcttctga tggtgacctg 540
caggaaggtg tcacctctga ggcatcctcc atcgctggca cccagcagct gggcaacctc 600
atcgtgtttt gggatgacaa ccgcatctcc atcgaagaca acactgagat cgctttcaac 660
gaggacgttg ttgctcgtta caaggcttac ggctggcaga ccattgaggt tgaggctggc 720
gaggacgttg cagcaatcga agctgcagtg gctgaggcta agaaggacac caagcgacct 780
accttcatcc gcgttcgcac catcatcggc ttcccagctc caaccatgat gaacaccggt 840
gctgtgcacg gtgctgctct tggcgcagct gaggttgcag caaccaagac tgagcttgga 900
ttcgatcctg aggctcactt cgcgatcgac gatgaggtca tcgctcacac ccgctccctc 960
gcagagcgcg ctgcagagaa gaaggctgca tggcaggtca agttcgatga gtgggcagcc 1020
gccaaccctg agaacaaggc tctgttcgat cgcctgaact cccgtgagct tccagcgggc 1080
tacgctgacg agctcccaac atgggatgca gatgagaagg gcgtcgcaac tcgtaaggct 1140
tccgaggctg cacttcaggc actgggcaag acccttcctg agctgtgggg cggttccgct 1200
gacctcgcag gttccaacaa caccgtgatc aagggctccc cttccttcgg ccctgagtcc 1260
atctccaccg agacctggtc tgctgagcct tacggccgta acctgcactt cggtatccgt 1320
gagcacgcta tgggatccat cctcaacggc atttccctcc acggtggcac ccgcccatac 1380
ggcggaacct tcctcatctt ctccgactac atgcgtcctg cagttcgtct tgcagctctc 1440
atggagaccg acgcttacta cgtctggacc cacgactcca tcggtctggg cgaagatggc 1500
ccaacccacc agcctgttga aaccttggct gcactgcgcg ccatcccagg tctgtccgtc 1560
ctgcgtcctg cagatgcgaa cgagaccgcc caggcttggg ctgcagcact tgagtacaag 1620
gaaggcccta agggtcttgc actgacccgc cagaacgttc ctgttctgga aggcaccaag 1680
gagaaggctg ctgaaggcgt tcgtcgcggt ggctacgtcc tggttgaggg ttccaaggaa 1740
accccagatg tgatcctcat gggctccggc tccgaggttc agcttgcagt taacgctgcg 1800
aaggctctgg aagctgaggg cgttgcagct cgcgttgttt ccgttccttg catggattgg 1860
ttccaggagc aggacgcaga gtacatcgag tccgttctgc ctgcagctgt gaccgctcgt 1920
gtgtctgttg aagctggcat cgcaatgcct tggtaccgct tcttgggcac ccagggccgt 1980
gctgtctccc ttgagcactt cggtgcttct gcggattacc agaccctgtt tgagaagttc 2040
ggcatcacca ccgatgcagt cgtggcagcg gccaaggact ccattaacag ttaa 2094
<210> 10
<211> 150
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 10
gacgggtcag attaagcaaa gactactttc ggggtagatc acctttgcca aatttgaacc 60
aattaaccta agtcgtagat ctgatcatcg gatctaacga aaacgaacca aaactttggt 120
cccggtttaa cccaggaagg attgaccacc 150
<210> 11
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 11
tttaaaaagc ttcagacttt tatttccact tcataaaaac tgcctgtgac gattccgtta 60
aagattgtgc caaatcactg cgcaaaactc gcgcggaacc agaccttgcc atgctatcgc 120
ctattcacac tatttgagta atcggaaata gatgggtgta gacgcttgat tggcggacgg 180
ttcacagcgg acgatttcag gccctcgtag ctcgagagtt tgaaggggtc cgattcgttc 240
cgttcgtgac gctttgtgag gttttttgac gttgcaccgt attgcttgcc gaacattttt 300
ctttttcttt cgggttttcg agaattttca cctacaaaag cccacgtcac agctcccaga 360
cttaaaattg gtcacacctt tgacacattt gaaccacagt tggttataaa atgggttcaa 420
catcactatg gttagaggtg ttgacgggtc agattaagca aagactactt tcggggtaga 480
tcacctttgc caaatttgaa 500
<210> 12
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 12
cctaagtcgt agatctgatc atcggatcta acgaaaacga accaaaactt tggtcccggt 60
ttaacccagg aaggattgac caccttgacg ctgtcacctg aacttcaggc gctcactgta 120
cgcaattacc cctctgattg gtccgatgtg gacaccaagg ctgtagacac tgttcgtgtc 180
ctcgctgcag acgctgtaga aaactgtggc tccggccacc caggcaccgc aatgagcctg 240
gctccccttg catacacctt gtaccagcgg gttatgaacg tagatccaca ggacaccaac 300
tgggcaggcc gtgaccgctt cgttctttct tgtggccact cctctttgac ccagtacatc 360
cagctttact tgggtggatt cgggcttgag atggatgacc tgaaggctct gcgcacctgg 420
gattccttga ccccaggaca ccctgagtac cgccacacca agggcgttga gatcaccact 480
ggccctcttg gccagggtct 500
<210> 13
<211> 1008
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 13
tttaaaaagc ttcagacttt tatttccact tcataaaaac tgcctgtgac gattccgtta 60
aagattgtgc caaatcactg cgcaaaactc gcgcggaacc agaccttgcc atgctatcgc 120
ctattcacac tatttgagta atcggaaata gatgggtgta gacgcttgat tggcggacgg 180
ttcacagcgg acgatttcag gccctcgtag ctcgagagtt tgaaggggtc cgattcgttc 240
cgttcgtgac gctttgtgag gttttttgac gttgcaccgt attgcttgcc gaacattttt 300
ctttttcttt cgggttttcg agaattttca cctacaaaag cccacgtcac agctcccaga 360
cttaaaattg gtcacacctt tgacacattt gaaccacagt tggttataaa atgggttcaa 420
catcactatg gttagaggtg ttgacgggtc agattaagca aagactactt tcggggtaga 480
tcacctttgc caaatttgaa ccaattaacc taagtcgtag atctgatcat cggatctaac 540
gaaaacgaac caaaactttg gtcccggttt aacccaggaa ggattgacca ccttgacgct 600
gtcacctgaa cttcaggcgc tcactgtacg caattacccc tctgattggt ccgatgtgga 660
caccaaggct gtagacactg ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc 720
cggccaccca ggcaccgcaa tgagcctggc tccccttgca tacaccttgt accagcgggt 780
tatgaacgta gatccacagg acaccaactg ggcaggccgt gaccgcttcg ttctttcttg 840
tggccactcc tctttgaccc agtacatcca gctttacttg ggtggattcg ggcttgagat 900
ggatgacctg aaggctctgc gcacctggga ttccttgacc ccaggacacc ctgagtaccg 960
ccacaccaag ggcgttgaga tcaccactgg ccctcttggc cagggtct 1008
<210> 14
<211> 1008
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (501)..(508)
<223> 将原始核苷酸序列CCAATTAA替换为TGTGGTAT
<400> 14
tttaaaaagc ttcagacttt tatttccact tcataaaaac tgcctgtgac gattccgtta 60
aagattgtgc caaatcactg cgcaaaactc gcgcggaacc agaccttgcc atgctatcgc 120
ctattcacac tatttgagta atcggaaata gatgggtgta gacgcttgat tggcggacgg 180
ttcacagcgg acgatttcag gccctcgtag ctcgagagtt tgaaggggtc cgattcgttc 240
cgttcgtgac gctttgtgag gttttttgac gttgcaccgt attgcttgcc gaacattttt 300
ctttttcttt cgggttttcg agaattttca cctacaaaag cccacgtcac agctcccaga 360
cttaaaattg gtcacacctt tgacacattt gaaccacagt tggttataaa atgggttcaa 420
catcactatg gttagaggtg ttgacgggtc agattaagca aagactactt tcggggtaga 480
tcacctttgc caaatttgaa tgtggtatcc taagtcgtag atctgatcat cggatctaac 540
gaaaacgaac caaaactttg gtcccggttt aacccaggaa ggattgacca ccttgacgct 600
gtcacctgaa cttcaggcgc tcactgtacg caattacccc tctgattggt ccgatgtgga 660
caccaaggct gtagacactg ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc 720
cggccaccca ggcaccgcaa tgagcctggc tccccttgca tacaccttgt accagcgggt 780
tatgaacgta gatccacagg acaccaactg ggcaggccgt gaccgcttcg ttctttcttg 840
tggccactcc tctttgaccc agtacatcca gctttacttg ggtggattcg ggcttgagat 900
ggatgacctg aaggctctgc gcacctggga ttccttgacc ccaggacacc ctgagtaccg 960
ccacaccaag ggcgttgaga tcaccactgg ccctcttggc cagggtct 1008
Claims (7)
1.一种改造TK基因5′端序列的重组棒状杆菌，其特征在于，是在棒状杆菌宿主菌中，将TK基因5′端序列中起始密码子TTG前-92位~-85位核苷酸序列-92CCAATTAA-85替换为- 92TGTGGTAT -85，降低TK基因5′端序列二级结构的稳定性，使其更易于被转录或表达；所述TK基因为转酮醇酶的编码基因；所述棒状杆菌宿主菌为谷氨酸棒状杆菌CICC23604，所述TK的氨基酸序列为SEQ ID NO.1，所述TK基因的核苷酸序列为SEQ ID NO.2，所述TK基因5′端序列为SEQ ID NO.3。
2.一种改造TK基因5′端序列的重组棒状杆菌，其特征在于，是在棒状杆菌宿主菌中，将TK基因5′端序列中起始密码子TTG前-92位~-85位核苷酸序列-92CCAATTAA-85替换为- 92TGTGGTAT -85，降低TK基因5′端序列二级结构的稳定性，使其更易于被转录或表达；所述TK基因为转酮醇酶的编码基因；所述棒状杆菌宿主菌为谷氨酸棒状杆菌CGMCC1.15647，所述TK的氨基酸序列为SEQ ID NO.8，所述TK基因的核苷酸序列为SEQ ID NO.9，所述TK基因5′端序列为SEQ ID NO.10。
3.权利要求1或权利要求2所述的重组棒状杆菌的构建方法，其特征在于，包括如下步骤：
（1）合成上游同源臂--92TGTGGTAT -85-下游同源臂，其中上游同源臂和下游同源臂为TK基因5′端序列中起始密码子TTG前-92位~-85位核苷酸序列-92CCAATTAA-85的前后各一段长度500~600bp的多核苷酸；
（2）将上游同源臂--92TGTGGTAT -85-下游同源臂连接至pK19mobsacB载体中，构建替换载体；
（3）将替换载体转化棒状杆菌宿主菌感受态细胞，筛选具有卡那霉素抗性的阳性转化子，获得发生了第一次同源单交换的重组菌；
（4）将发生了第一次同源单交换的重组菌自然传代后，筛选能在10%蔗糖培养基生长但不能在具有卡那霉素抗性的培养基生长的菌落，验证后得到完成两次同源单交换的重组棒状杆菌。
4.如权利要求3所述的构建方法，其特征在于，满足以下条件之一项或多项：
i. 步骤（2）中上游同源臂--92TGTGGTAT -85-下游同源臂连接至pK19mobsacB载体的SalI、EcoRI酶切位点之间；
ii. 步骤（3）中以卡那霉素抗性基因引物采用PCR扩增技术筛选具有卡那霉素抗性的阳性转化子，所述引物序列如下：
F1：5′- ATGATTGAACAAGATGGATTGC -3′，
R1：5′- TCAGAAGAACTCGTCAAGAAGGCG -3′；
所述PCR扩增的体系为：2×HiFi-PCRmaster10μL，10μmol/L F1 1μL，10μmol/L R1 1μL，模板 1μL，ddH2O 7μL；
所述PCR扩增的程序为：95℃预变性5min；94℃变性30sec，56℃退火30sec，72℃延伸1min，30个循环；72℃延伸10min，4℃保存；
iii. 步骤（4）所述验证是采用PCR扩增技术验证，PCR扩增的引物序列如下：
F2：5′- AAATTTGAATGTGGTAT -3′，
R2：5′- TTAACTGTTAATGGAGTCCT -3′；
所述PCR扩增的体系为：2×HiFi-PCRmaster10μL，10μmol/L F2 1μL，10μmol/L R2 1μL，模板 1μL，ddH2O 7μL；
所述PCR扩增的程序为：95℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸3min，30个循环；72℃延伸10min，4℃保存；
iv. 步骤（4）中所用培养基为LBG培养基：葡萄糖5g/L，蛋白胨10 g/L，酵母膏5 g/L，NaCl 10g/L。
5.权利要求1或权利要求2所述的重组棒状杆菌在生产L-赖氨酸中的应用。
6.如权利要求5所述的应用，其特征在于，所述应用是将重组棒状杆菌接种至液体LBG培养基中进行种子培养，其后按体积百分比2~5%接种量接种至发酵培养基发酵培养；
所述LBG培养基：葡萄糖5g/L，蛋白胨10 g/L，酵母膏5 g/L，NaCl 10g/L；
所述发酵培养基：葡萄糖100 g/L，蛋白胨20 g/L，玉米浆30 mL/L，尿素5g/L，(NH4)2SO4 25g/L，L-亮氨酸0.34g/L，KH2PO4 2g/L，MgSO4·7H2O 1.5g/L，生物素0.001 g/L。
7.如权利要求6所述的应用，其特征在于，所述种子培养的条件为200~220 rpm、28~30℃下培养18-25 h；所述发酵培养的条件为200~220 rpm、28~30℃。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110585325.0A CN113308426B (zh) | 2021-05-27 | 2021-05-27 | 一种改造tk基因5′端序列的重组棒状杆菌及其应用 |
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