一种高产L-赖氨酸枯草芽孢杆菌的构建研究

A Strain of Bacillus subtilis Producing High-Yield L-lysine: Construction

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。

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 pK18 mobsacB 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/pK18 mobsacB-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 nonresistance, high yield, good genetic stability and low purification cost. Therefore, the strain exhibits outstanding potential for future applications.