| 一种高产L-赖氨酸枯草芽孢杆菌的构建研究 |
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| 引用本文： | 夏树立,韩静,王丽学,杨春蕾,刘长悦,王海亮,吴贶,史慧玲.一种高产L-赖氨酸枯草芽孢杆菌的构建研究[J].中国农学通报,2020,36(30):98-105. |
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| 作者姓名： | 夏树立 韩静 王丽学 杨春蕾 刘长悦 王海亮 吴贶 史慧玲 |
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| 作者单位： | 1.天津市畜牧兽医研究所,天津 300380;2.天津市农业科学院信息研究所,天津 300192;3.天津科润农业科技股份有限公司黄瓜研究所,天津 300192;4.天津市现代农业科技创新基地管理中心,天津 301700;5.天津市农业科学院,天津 300192 |
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| 基金项目： | 天津市科技重大专项与工程“农村科技帮扶工程：家禽特色蛋品标准化生产技术集成与应用”(18ZXBFNC00350);天津市农业科学院青年科技创新重点项目“非抗性稳定高产L-赖氨酸枯草芽孢杆菌工程菌株的构建及应用”(201801) |
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| 摘 要： | 为了选育一种非抗性、高产出、遗传稳定性好、低生产成本的L-赖氨酸生产菌株,以辣椒花药和枯草芽孢杆菌为试验材料,以穿梭表达载体pHT43和基因敲除载体pK18mobsacB为媒介,将辣椒花药中的高赖氨酸基因CFLR转化到枯草芽孢杆菌中。经过2次细胞内同源交换和培养基筛选,最终获得不带质粒抗性标记的高产L-赖氨酸枯草芽孢杆菌株CMCC(B)63501/pK18mobsacB-ΔALA::CFLR,该菌株连续传代20次后仍具有明显高于野生型的CFLR基因拷贝数,经HPLC验证,重组菌株L-赖氨酸含量比野生菌株提高了64.89%。结果表明,本研究所构建的非抗性高产L-赖氨酸枯草芽孢杆菌株产量高、遗传稳定性好、低纯化成本,具有良好的应用潜力。
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| 关 键 词： | 辣椒高赖氨酸基因 枯草芽孢杆菌 基因克隆 同源交换 HPLC |
| 收稿时间： | 2019-10-09 |
A Strain of Bacillus subtilis Producing High-Yield L-lysine: Construction |
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| Xia Shuli,Han Jing,Wang Lixue,Yang Chunlei,Liu Changyue,Wang Hailiang,Wu Kuang,Shi Huiling.A Strain of Bacillus subtilis Producing High-Yield L-lysine: Construction[J].Chinese Agricultural Science Bulletin,2020,36(30):98-105. |
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| Authors: | Xia Shuli Han Jing Wang Lixue Yang Chunlei Liu Changyue Wang Hailiang Wu Kuang Shi Huiling |
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| Institution: | 1.Tianjin Institute of Animal Husbandry and Veterinary Medicine, Tianjin 300380;2.Tianjin Institute of Agricultural Sciences Information Institute, Tianjin 300192;3.Tianjin Branch of Tianjin Kerun Agricultural Technology Co., Ltd., Tianjin 300192;4.Tianjin Modern Agricultural Science and Technology Innovation Base Management Center, Tianjin 301700;5.Tianjin Academy of Agricultural Sciences, Tianjin 300192 |
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| Abstract: | To breed a strain producing L-lysine with non-resistance, high yield, good genetic stability, low production cost, pepper anther and Bacillus subtilis were used as test materials, the shuttle expression vector pHT43 and gene knockout vector pK18mobsacB was performed as a vector to transform lysine-rich protein gene (CFLR) into Bacillus subtilis. After two rounds of self-recombination and screening of culture medium, the final strain CMCC (B) 63501/pK18mobsacB-ΔALA::CFLR, with high yield of L-lysine was obtained without antibiotic resistance. After 20 generations of serial passage, the strain still expressed a significant higher number of CFLR copies than the wild type. The subsequent data of High-Performance Liquid Chromatography (HPLC) revealed that the content of L-lysine in recombinant strain was 64.89% higher than that of the wild strain. The above results indicated that the recombinant strain we constructed were featured with non-resistance, high yield, good genetic stability and low purification cost. Therefore, the strain exhibits outstanding potential for future applications. |
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| Keywords: | CFLR gene Bacillus subtilis gene cloning homologous exchange HPLC |
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