CN113278571A - 一种棒状杆菌工程菌构建方法及应用 - Google Patents
一种棒状杆菌工程菌构建方法及应用 Download PDFInfo
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Abstract
本发明涉及一种棒状杆菌工程菌构建方法及应用。本发明的棒状杆菌工程菌，是在棒状杆菌宿主菌中，替换PC基因5′端序列中起始密码子GTG前‑73位～‑61位核苷酸序列，降低PC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，其中K指代T或G。该棒状杆菌工程菌与原始菌株相比，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
本发明涉及一种棒状杆菌工程菌构建方法及应用，属于生物工程技术领域。
背景技术
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，此法特点是工艺简单，但原料来源有限，仅适合小规模生产。后又出现了化学合成法、酶法，使用的化学合成法主要有荷兰的DMS法和日本的东丽法，这种方法最大的缺点是使用剧毒原料光气，可能残留催化剂，产品安全性差，存在严重的环保问题。1960年，日本首先采用微生物发酵法生产L-赖氨酸。微生物发酵生产氨基酸是人为地解除氨基酸生物合成的代谢控制机制，使其积累大量所需氨基酸。氨基酸的L型立体专一性决定了发酵法生产氨基酸较化学合成的工艺更简单、快捷。我国于20世纪60年代中期开始进行赖氨酸菌株选育和发酵的研究，但因产量较低难以工业化，直到70年代末80年代初世界赖氨酸实现工业化后我国研究才取得突破。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 regarded assafe”(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)。
虽然目前已有很多关于提高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，本发明提供了一种棒状杆菌工程菌构建方法及应用。本发明的棒状杆菌工程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丙酮酸羧化酶(Pyruvate Carboxylase，PC)基因5′端序列的部分核苷酸序列，获得高产L-赖氨酸的重组棒状杆菌。
术语说明：
丙酮酸羧化酶(Pyruvate Carboxylase，PC)：是糖异生的限速酶，广泛存在于细菌、酵母和动物植物中，可在L-赖氨酸和L-谷氨酸的工业发酵生产中补充消耗的草酰乙酸。
本发明技术方案如下：
一种改造PC基因5′端序列的棒状杆菌工程菌，是在棒状杆菌宿主菌中，将PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列-73GKGGTTACGATAC-61替换为- 73TGTGGTATAATGG-61，降低PC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，其中K指代T或G。
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PC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PC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99.2％的氨基酸序列。
根据本发明优选的，所述PC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PC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97.6％的核苷酸序列。
根据本发明优选的，所述PC基因5′端序列为SEQ ID NO.3或SEQ ID NO.10。
根据本发明优选的，所述PC基因5′端序列为与SEQ ID NO.3或SEQ ID NO.10的序列一致性大于等于96.7％的核苷酸序列。
上述棒状杆菌工程菌的构建方法，包括如下步骤：
(1)合成上游同源臂--73TGTGGTATAATGG-61-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PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列的前后各一段长度500～600bp的核苷酸序列；
(2)将上游同源臂--73TGTGGTATAATGG-61-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棒状杆菌工程菌。
根据本发明优选的，步骤(2)中上游同源臂--73TGTGGTATAATGG-61-下游同源臂的核苷酸序列连接至pK19mobsacB载体的Hind III、EcoRI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)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。
本发明中，步骤(4)采用条件培养基筛选验证后，还可采用PCR扩增技术进一步验证。
上述棒状杆菌工程菌在生产L-赖氨酸中的应用。
根据本发明优选的，所述应用是将棒状杆菌工程菌接种至液体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)2SO425g/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PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列-73GKGGTTACGATAC-61替换为-73TGTGGTATAATGG-61，能够降低PC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PC基因5′端序列的棒状杆菌工程菌，是将PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列-73GKGGTTACGATAC-61替换为- 73TGTGGTATAATGG-61，该棒状杆菌工程菌与原始菌株相比，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，其中丙酮酸羧化酶(PC)的氨基酸序列为SEQ ID NO.1，PC基因的核苷酸序列为SEQ ID NO.2，PC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，其中丙酮酸羧化酶(PC)的氨基酸序列为SEQ ID NO.8，PC基因的核苷酸序列为SEQ ID NO.9，PC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99.2％，SEQ ID NO.2与SEQ IDNO.9核苷酸序列一致性为97.6％，SEQ ID NO.3与SEQ ID NO.10核苷酸序列一致性为96.7％。
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：含有拟替换序列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--73TGTGGTATAATGG-61-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谷氨酸棒状杆菌CICC23604 PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列-73GTGGTTACGATAC-61的前后各一段长度500～600bp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--73GTGGTTACGATAC-61-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--73TGTGGTATAATGG-61-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Hind III、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-PC1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--73TGTGGTATAATGG-61-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针对谷氨酸棒状杆菌CGMCC1.15647PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列-73GGGGTTACGATAC-61的前后各一段长度500～600bp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--73GGGGTTACGATAC-61-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--73TGTGGTATAATGG-61-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Hind III、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-PC2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-PC1或pK19mobsacB-PC2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，针对谷氨酸棒状杆菌CICC23604转化子，所述PCR扩增的引物序列如下：
F2：5′-GGGGAATCGTGTGGTATAATGG-3′，
R2：5′-TTAGGAAATGACGACGATCAAGTC-3′，
F2引物3′末端序列含有替换后的位点-73TGTGGTATAATGG-61；
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4min，30个循环；72℃延伸10min，4℃保存；
挑选在约3505bp处的特异性条带，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棒状杆菌工程菌PC1。
针对谷氨酸棒状杆菌CGMCC1.15647转化子，所述PCR扩增的引物序列如下：
F3：5′-GGGGAATCGTGTGGTATAATGG-3′，
R3：5′-CTAGGAAATGACGACGATCAAGTC-3′，
F3引物3′末端序列含有替换后的位点-73TGTGGTATAATGG-61；
所述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4min，30个循环；72℃延伸10min，4℃保存；
挑选在约3505bp处的特异性条带，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棒状杆菌工程菌PC2。
实施例5：重组菌的稳定性验证
将上述筛选出并验证正确的棒状杆菌工程菌PC1和PC2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，分别用F2和R2、F3和R3为引物进行菌落PCR验证。结果显示，使用引物F2和R2扩增棒状杆菌工程菌PC1、使用F3和R3扩增棒状杆菌工程菌PC2能够分别扩增出一条特异性基因条带，大小约为3505bp，与理论值相符，证明替换的-73TGTGGTATAATGG-61已成功整合到棒状杆菌工程菌PC1和PC2基因组上，并稳定存在。
其中，所述PCR扩增体系如下：
表4 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4min，30个循环；72℃延伸10min，4℃保存。
实施例6：L-赖氨酸发酵测试
将上述制备的棒状杆菌工程菌PC1和PC2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％接种量分别接种至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5、表6所示。
表5不同时间棒状杆菌工程菌PC1与原始菌L-赖氨酸的平均产量
表6不同时间棒状杆菌工程菌PC2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，棒状杆菌工程菌PC1发酵液中L-赖氨酸的含量达到47.3g/L，较原始菌提高了10.5％，棒状杆菌工程菌PC2发酵液中L-赖氨酸的含量达到0.95g/L，是原始菌的2.16倍。这表明将谷氨酸棒状杆菌PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列替换为-73TGTGGTATAATGG-61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PC基因5′端序列(SEQ ID NO.3与SEQ ID NO.10)起始密码子GTG前-73位～-61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中PC基因5′端序列起始密码子GTG前-73位～-61位核苷酸序列替换为-73TGTGGTATAATGG-61后，5′端序列的最小自由能(minimum free energy)由-35.40kcal/mol提高至-34.60kcal/mol；谷氨酸棒状杆菌CGMCC1.15647中PC基因5′端序列起始密码子GTG前-73位～-61位核苷酸序列替换为- 73TGTGGTATAATGG-61后，5′端序列的最小自由能(minimum free energy)由-32.90kcal/mol提高至-28.90kcal/mol。这表明替换序列能够降低5′端序列的稳定性，进而使得PC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> 一种棒状杆菌工程菌构建方法及应用
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 1140
<212> PRT
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 1
Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu
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Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu
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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
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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 Glu 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 Ala 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 Ala 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 His 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
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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
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Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro
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Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val
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Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro
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Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser
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Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu
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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
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Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser
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Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln
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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
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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
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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
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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 Leu
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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
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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
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Ser Ser Asn Lys Gly His Val Ala Ala Pro Phe Ala Gly Val Val
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Thr Val Thr Val Ala Glu Gly Asp Glu Val Lys Ala Gly Asp Ala
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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 Ile Ser
1130 1135 1140
<210> 2
<211> 3423
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 2
gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180
attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240
gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 300
cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360
cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 420
gttttggcgg aatccacccc gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc 480
cagacttacc ccatctttgt gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt 540
gttgcttcac ctgatgagct tcgcaaatta gcaacagaag catctcgtga agctgaagcg 600
gctttcggcg atggcgcggt atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa 660
gtgcagatcc ttggcgatca cactggagaa gttgtacacc tttatgaacg tgactgctca 720
ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780
ctgcgtgatc gcatttgtgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840
gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat 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 aacattggtt tcttgcgtgc gttgctgcgt 1320
gaagaggact tcacttccaa gcgcatcgcc accggattca ttgccgatca cccgcacctc 1380
cttcaggctc cacctgctga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440
accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagctcctat cgataagctg 1500
cctaacatca 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 tgccagctcc 1920
ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980
atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040
gatctctctg atccaaatga aaagctctac accctggatt actacctaaa gatggcagag 2100
gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160
ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacactgc gggtggccag ctggcaacct 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 cggacagttg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgcgg atcgtttcga actcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700
gctgccgatc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760
cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880
gatgattcca aggaacgtcg caatagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000
ttctacggcc tggtcgaagg cctcgagact 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
atcatcgaag ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcatttcc 3420
taa 3423
<210> 3
<211> 150
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 3
aagtcgtgca ggtcagggga gtgttgcccg aaaacattga gaggaaaaca aaaaccgatg 60
tttgattggg ggaatcggtg gttacgatac taggacgcag tgactgctat cacccttggc 120
ggtctcttgt tgaaaggaat aattactcta 150
<210> 4
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 4
atcggtggtg tcgtggtggc accgaccgcg tctgagctga tcctaccgat cgctgtggca 60
gtgaccaacc gtctgacagt tgctgatctg gctgatacct tcgcggtgta cccatcattg 120
tcaggttcga ttactgaagc agcacgtcag ctggttcaac atgatgatct aggctaattt 180
ttctgagtct tagattttga gaaaacccag gattgctttg tgcactcctg ggttttcact 240
ttgttaagca gttttgggga aaagtgcaaa gtttgcaaag tttagaaata ttttaagagg 300
taagatgtct gcaggtggaa gcgtttaaat gcgtttaact tggccaaatg tggcaacctt 360
tgcaaggtga aaaactgggg cggggttaga tcctgggggg tttatttcat tcactttggc 420
ttgaagtcgt gcaggtcagg ggagtgttgc ccgaaaacat tgagaggaaa acaaaaaccg 480
atgtttgatt gggggaatcg 500
<210> 5
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 5
taggacgcag tgactgctat cacccttggc ggtctcttgt tgaaaggaat aattactcta 60
gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 120
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 180
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 240
attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 300
gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 360
cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 420
cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 480
gttttggcgg aatccacccc 500
<210> 6
<211> 1013
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CICC23604
<400> 6
atcggtggtg tcgtggtggc accgaccgcg tctgagctga tcctaccgat cgctgtggca 60
gtgaccaacc gtctgacagt tgctgatctg gctgatacct tcgcggtgta cccatcattg 120
tcaggttcga ttactgaagc agcacgtcag ctggttcaac atgatgatct aggctaattt 180
ttctgagtct tagattttga gaaaacccag gattgctttg tgcactcctg ggttttcact 240
ttgttaagca gttttgggga aaagtgcaaa gtttgcaaag tttagaaata ttttaagagg 300
taagatgtct gcaggtggaa gcgtttaaat gcgtttaact tggccaaatg tggcaacctt 360
tgcaaggtga aaaactgggg cggggttaga tcctgggggg tttatttcat tcactttggc 420
ttgaagtcgt gcaggtcagg ggagtgttgc ccgaaaacat tgagaggaaa acaaaaaccg 480
atgtttgatt gggggaatcg gtggttacga tactaggacg cagtgactgc tatcaccctt 540
ggcggtctct tgttgaaagg aataattact ctagtgtcga ctcacacatc ttcaacgctt 600
ccagcattca aaaagatctt ggtagcaaac cgcggcgaaa tcgcggtccg tgctttccgt 660
gcagcactcg aaaccggtgc agccacggta gctatttacc cccgtgaaga tcggggatca 720
ttccaccgct cttttgcttc tgaagctgtc cgcattggta ccgaaggctc accagtcaag 780
gcgtacctgg acatcgatga aattatcggt gcagctaaaa aagttaaagc agatgccatt 840
tacccgggat acggcttcct gtctgaaaat gcccagcttg cccgcgagtg tgcggaaaac 900
ggcattactt ttattggccc aaccccagag gttcttgatc tcaccggtga taagtctcgc 960
gcggtaaccg ccgcgaagaa ggctggtctg ccagttttgg cggaatccac ccc 1013
<210> 7
<211> 1013
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (501)..(513)
<223> 将原始核苷酸序列GTGGTTACGATAC替换为TGTGGTATAATGG
<400> 7
atcggtggtg tcgtggtggc accgaccgcg tctgagctga tcctaccgat cgctgtggca 60
gtgaccaacc gtctgacagt tgctgatctg gctgatacct tcgcggtgta cccatcattg 120
tcaggttcga ttactgaagc agcacgtcag ctggttcaac atgatgatct aggctaattt 180
ttctgagtct tagattttga gaaaacccag gattgctttg tgcactcctg ggttttcact 240
ttgttaagca gttttgggga aaagtgcaaa gtttgcaaag tttagaaata ttttaagagg 300
taagatgtct gcaggtggaa gcgtttaaat gcgtttaact tggccaaatg tggcaacctt 360
tgcaaggtga aaaactgggg cggggttaga tcctgggggg tttatttcat tcactttggc 420
ttgaagtcgt gcaggtcagg ggagtgttgc ccgaaaacat tgagaggaaa acaaaaaccg 480
atgtttgatt gggggaatcg tgtggtataa tggtaggacg cagtgactgc tatcaccctt 540
ggcggtctct tgttgaaagg aataattact ctagtgtcga ctcacacatc ttcaacgctt 600
ccagcattca aaaagatctt ggtagcaaac cgcggcgaaa tcgcggtccg tgctttccgt 660
gcagcactcg aaaccggtgc agccacggta gctatttacc cccgtgaaga tcggggatca 720
ttccaccgct cttttgcttc tgaagctgtc cgcattggta ccgaaggctc accagtcaag 780
gcgtacctgg acatcgatga aattatcggt gcagctaaaa aagttaaagc agatgccatt 840
tacccgggat acggcttcct gtctgaaaat gcccagcttg cccgcgagtg tgcggaaaac 900
ggcattactt ttattggccc aaccccagag gttcttgatc tcaccggtga taagtctcgc 960
gcggtaaccg ccgcgaagaa ggctggtctg ccagttttgg cggaatccac ccc 1013
<210> 8
<211> 1140
<212> PRT
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 8
Met Ser Thr Asn 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 Ala 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 Ile Ser
1130 1135 1140
<210> 9
<211> 3423
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 9
gtgtcgacta acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg ctgtccgtgc 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 gcatttgtgc ggatgcagta aagttttgcc gctccattgg ttaccagggc 840
gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat tgaaatgaac 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 cgattttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260
gctgaattca ccgtgtctgg tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgt 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 gcagcttgga 1560
ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620
ttccgcgatg cgcaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680
gcggcagagg ccgtcgcaaa gctaactcct gagttgttgt ccgtggaggc ctggggcggt 1740
gcgacctatg 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
atcgatgcgg tcctggagac caacactgca gtcgccgagg tggcgatggc ttattctggt 2040
gatctttccg atccgaatga aaagctctac accctggatt actacctgaa gatggcagag 2100
gagatcgtca agtctggcgc tcacattctg gctattaagg atatggctgg tctgcttcgc 2160
ccagctgcag ccaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacactgc gggtggccag ttggctacct actttgctgc agctcaagct 2280
ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340
ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg atacaggttt gagcctcgag 2400
gcggtttctg acctcgagcc atactgggaa gcagtgcgcg gactgtactt gccatttgag 2460
tctggaaccc caggtccaac tggtcgcgtc taccgccatg aaatcccagg cggacagctg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgctg atcgcttcga gctcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700
gctgccgatc cacaaaagta cgacatccca gattctgtta tcgcgttcct gcggggcgag 2760
cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca ccttgacgct 2880
gatgattcca aggaacgtcg caacagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgcgaattc 3000
ttctacggcc tggtcgaagg ccgcgagact ttgatccgac tgccagatgt gcgcacccca 3060
ctgcttgttc gcctggatgc gatctctgag ccagacgaca agggtatgcg caatgttgtg 3120
gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180
accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240
ggtgttgtca ctgtgactgt tgctgaaggt gatgaggtca aggctggaga cgcagtcgca 3300
atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcatttcc 3420
tag 3423
<210> 10
<211> 150
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 10
aagtcgtgca ggtcagggga gtgttgcccg aaaagattga gaggaaaaca aaaaccgatg 60
tttgattggg ggaatcgggg gttacgatac taggacgaag tgactgctat cacccttggt 120
ggtctcttgt tgaaaggaac aattactcta 150
<210> 11
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 11
atcggtggtg tcgtggtggc accgaccgcg tctgagctga tcctaccgat cgctgtggca 60
gtgaccaacc gtctgacagt tgctgatctg gctgatacct tcgcggtgta cccatcattg 120
tcaggttcga ttactgaagc agcacgtcag ctggttcaac atgatgatct aggctaattt 180
ttctgagtct cagattttaa gaaaccccag gattgctttg tgcactcctg ggttttcact 240
ttgttaagca gttttgggga aaagtgcaaa gtttgcaaag tttagaaata ttttaagagg 300
taagatgtct gcaggtggaa gcgtttaaat gcgttaaact tggccaaatg tggcaacctt 360
tgcaaggtga aaaactgcgg cggggttaga tcctgggggg tttatttcat tcactttggc 420
ttgaagtcgt gcaggtcagg ggagtgttgc ccgaaaagat tgagaggaaa acaaaaaccg 480
atgtttgatt gggggaatcg 500
<210> 12
<211> 500
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 12
taggacgaag tgactgctat cacccttggt ggtctcttgt tgaaaggaac aattactcta 60
gtgtcgacta acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 120
ggcgaaatcg ctgtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 180
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 240
attggtactg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 300
gctaaaaaag ttaaagcaga tgctatttac ccgggatatg gcttcctgtc tgaaaatgcc 360
cagcttgccc gcgagtgcgc ggaaaacggc attactttta ttggcccaac cccagaggtt 420
cttgatctca ccggtgataa gtctcgtgcg gtaaccgccg cgaagaaggc tggtctgcca 480
gttttggcgg aatccacccc 500
<210> 13
<211> 1013
<212> DNA
<213> 谷氨酸棒状杆菌(Corynebacterium glutamicum) CGMCC1.15647
<400> 13
atcggtggtg tcgtggtggc accgaccgcg tctgagctga tcctaccgat cgctgtggca 60
gtgaccaacc gtctgacagt tgctgatctg gctgatacct tcgcggtgta cccatcattg 120
tcaggttcga ttactgaagc agcacgtcag ctggttcaac atgatgatct aggctaattt 180
ttctgagtct cagattttaa gaaaccccag gattgctttg tgcactcctg ggttttcact 240
ttgttaagca gttttgggga aaagtgcaaa gtttgcaaag tttagaaata ttttaagagg 300
taagatgtct gcaggtggaa gcgtttaaat gcgttaaact tggccaaatg tggcaacctt 360
tgcaaggtga aaaactgcgg cggggttaga tcctgggggg tttatttcat tcactttggc 420
ttgaagtcgt gcaggtcagg ggagtgttgc ccgaaaagat tgagaggaaa acaaaaaccg 480
atgtttgatt gggggaatcg ggggttacga tactaggacg aagtgactgc tatcaccctt 540
ggtggtctct tgttgaaagg aacaattact ctagtgtcga ctaacacatc ttcaacgctt 600
ccagcattca aaaagatctt ggtagcaaac cgcggcgaaa tcgctgtccg tgctttccgt 660
gcagcactcg aaaccggtgc agccacggta gctatttacc cccgtgaaga tcggggatca 720
ttccaccgct cttttgcttc tgaagctgtc cgcattggta ctgaaggctc accagtcaag 780
gcgtacctgg acatcgatga aattatcggt gcagctaaaa aagttaaagc agatgctatt 840
tacccgggat atggcttcct gtctgaaaat gcccagcttg cccgcgagtg cgcggaaaac 900
ggcattactt ttattggccc aaccccagag gttcttgatc tcaccggtga taagtctcgt 960
gcggtaaccg ccgcgaagaa ggctggtctg ccagttttgg cggaatccac ccc 1013
<210> 14
<211> 1013
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (501)..(513)
<223> 将原始核苷酸序列GGGGTTACGATAC替换为TGTGGTATAATGG
<400> 14
atcggtggtg tcgtggtggc accgaccgcg tctgagctga tcctaccgat cgctgtggca 60
gtgaccaacc gtctgacagt tgctgatctg gctgatacct tcgcggtgta cccatcattg 120
tcaggttcga ttactgaagc agcacgtcag ctggttcaac atgatgatct aggctaattt 180
ttctgagtct cagattttaa gaaaccccag gattgctttg tgcactcctg ggttttcact 240
ttgttaagca gttttgggga aaagtgcaaa gtttgcaaag tttagaaata ttttaagagg 300
taagatgtct gcaggtggaa gcgtttaaat gcgttaaact tggccaaatg tggcaacctt 360
tgcaaggtga aaaactgcgg cggggttaga tcctgggggg tttatttcat tcactttggc 420
ttgaagtcgt gcaggtcagg ggagtgttgc ccgaaaagat tgagaggaaa acaaaaaccg 480
atgtttgatt gggggaatcg tgtggtataa tggtaggacg aagtgactgc tatcaccctt 540
ggtggtctct tgttgaaagg aacaattact ctagtgtcga ctaacacatc ttcaacgctt 600
ccagcattca aaaagatctt ggtagcaaac cgcggcgaaa tcgctgtccg tgctttccgt 660
gcagcactcg aaaccggtgc agccacggta gctatttacc cccgtgaaga tcggggatca 720
ttccaccgct cttttgcttc tgaagctgtc cgcattggta ctgaaggctc accagtcaag 780
gcgtacctgg acatcgatga aattatcggt gcagctaaaa aagttaaagc agatgctatt 840
tacccgggat atggcttcct gtctgaaaat gcccagcttg cccgcgagtg cgcggaaaac 900
ggcattactt ttattggccc aaccccagag gttcttgatc tcaccggtga taagtctcgt 960
gcggtaaccg ccgcgaagaa ggctggtctg ccagttttgg cggaatccac ccc 1013
Claims (10)
1.一种改造PC基因5′端序列的棒状杆菌工程菌，其特征在于，是在棒状杆菌宿主菌中，将PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列-73GKGGTTACGATAC-61替换为-73TGTGGTATAATGG-61，降低PC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，其中K指代T或G。
2.如权利要求1所述的棒状杆菌工程菌，其特征在于，所述棒状杆菌宿主菌为谷氨酸棒状杆菌；
优选的，所述谷氨酸棒状杆菌为谷氨酸棒状杆菌CICC23604或谷氨酸棒状杆菌CGMCC1.15647。
3.如权利要求1所述的棒状杆菌工程菌，其特征在于，所述PC的氨基酸序列为SEQ IDNO.1或SEQ ID NO.8；
优选的，所述PC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99.2％的氨基酸序列。
4.如权利要求1所述的棒状杆菌工程菌，其特征在于，所述PC基因的核苷酸序列为SEQID NO.2或SEQ ID NO.9；
优选的，所述PC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97.6％的核苷酸序列。
5.如权利要求1所述的棒状杆菌工程菌，其特征在于，所述PC基因5′端序列为SEQ IDNO.3或SEQ ID NO.10；
优选的，所述PC基因5′端序列为与SEQ ID NO.3或SEQ ID NO.10的序列一致性大于等于96.7％的核苷酸序列。
6.权利要求1所述的棒状杆菌工程菌的构建方法，其特征在于，包括如下步骤：
(1)合成上游同源臂--73TGTGGTATAATGG-61-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PC基因5′端序列中起始密码子GTG前-73位～-61位核苷酸序列的前后各一段长度500～600bp的核苷酸序列；
(2)将上游同源臂--73TGTGGTATAATGG-61-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棒状杆菌工程菌。
7.如权利要求6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--73TGTGGTATAATGG-61-下游同源臂的核苷酸序列连接至pK19mobsacB载体的Hind III、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)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，NaCl10g/L。
8.权利要求1所述的棒状杆菌工程菌在生产L-赖氨酸中的应用。
9.如权利要求8所述的应用，其特征在于，所述应用是将棒状杆菌工程菌接种至液体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)2SO425g/L，L-亮氨酸0.34g/L，KH2PO4 2g/L，MgSO4·7H2O 1.5g/L，生物素0.001g/L。
10.如权利要求9所述的应用，其特征在于，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℃。
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| CN115261247A (zh) * | 2021-04-30 | 2022-11-01 | 大象株式会社 | L-赖氨酸生产能力得到提高的谷氨酸棒状杆菌突变株及利用其的l-赖氨酸的生产方法 |
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| CN115261247B (zh) * | 2021-04-30 | 2024-04-02 | 大象株式会社 | L-赖氨酸生产能力得到提高的谷氨酸棒状杆菌突变株及利用其的l-赖氨酸的生产方法 |
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Application publication date: 20210820 |