激光生物学报, 2018, 27 (5): 442, 网络出版: 2018-11-25  

脂肪酶lipAB操纵子在防御假单胞菌中的克隆、表达及活性研究

Cloning, Expression and Activity of Lipase Operon lipAB in Pseudomonas protegens
作者单位
1 淡水鱼类发育生物学国家重点实验室, 湖南师范大学生命科学学院, 微生物分子生物学湖南省重点实验室, 湖南 长沙 410081
2 山东省土壤肥料总站, 山东 济南250100
3 山东大学生命科学学院, 山东大学亥姆霍兹生物技术研究所, 微生物技术国家重点实验室, 山东 青岛 266237
4 山东大学苏州研究院, 江苏 苏州 215123
摘要
对荚壳伯克氏菌PG1(Burkholderia glumae PG1)基因组中的脂肪酶操纵子lipAB片段进行直接克隆, 构建含有脂肪酶基因的分泌表达载体, 实现其在防御假单胞菌Pf-5(Pseudomonas protegens Pf-5)中的异源表达, 并研究重组工程菌的胞外脂肪酶活性。利用 Red/ET直接克隆技术获得克隆载体p15A-cm-lipAB; 再通过亚克隆技术构建重组表达载体pBBR1-km-lipAB和pBBR1-km-Papra-lipAB, 将这两个表达载体分别电转至Pf-5中, 通过卡那霉素或者阿伯拉霉素抗性筛选得到转化子, 以三丁酸甘油酯平板扩散法和对硝基苯酚法检测脂肪酶酶活, 并通过实时荧光定量PCR检测启动子的替换对lipA表达的影响。本研究从PG1中成功克隆了脂肪酶操纵子lipAB(GenBank accession number:AJK49931.1 and AJK49932.1); 成功构建了重组工程菌Pf-5/pBBR1-km-lipAB和Pf-5/pBBR1-km-Papra-lipAB, 并成功检测到两株工程菌的胞外脂肪酶活性; 以LB培养基培养至24 h时, 启动子优化后lipA基因表达量是原始水平的2.1倍; 在LB培养基摇瓶发酵至66 h时, Pf-5/pBBR1-km-lipAB的脂肪酶酶活最高且为13.51 U/mL, 而Pf-5/pBBR1-km-Papra-lipAB的酶活为46.85 U/mL, 是Pf-5/pBBR1-km-lipAB的3.47倍。初步实现基因lipA在Pf-5中的表达, 发现组成型启动子Papra比lipAB的原始启动子PlipAB效率更高, 为将来实现规模化生产奠定了技术基础。
Abstract
To implement heterologous expression of Burkholderia glumae PG1 lipase operon lipAB in Pseudomonas protegens Pf-5 via Red/ET homologous recombineering. The vector p15A-cm-lipAB was obtained using Red/ET direct cloning technology. Then, two recombinant expression vectors pBBR1-km-lipAB and pBBR1-km-Papra-lipAB with different promoters were constructed by subcloning technology, and electrotransformated the resultant expression vectors into P.protegens Pf-5. Transformants obtained by kanamycin or apramycin resistance screening. The tributyrin glyceryl trinitrate plate diffusion method and the p-nitrophenol method were used for the assay of the activities of lipase, and the effect of promoter replacement on lipA expression was examined by qRT-PCR. We successfully cloned the lipase operon lipAB (GenBank accession number:AJK49931.1 and AJK49932.1). After the achievement of engineering bacteria Pf-5/pBBR1-km-lipAB and Pf-5/pBBR1-km-Papra-lipAB, fermentation results indicated that the activity of extracellular lipase in Pf-5 was accomplished. Moreover, it was found that the expression level of lipA gene was 2.1-fold the original level after promoter optimization. When the flask in LB medium was fermented to 66 h, the lipase activity of Pf-5/pBBR1-km-lipAB supernatant was 13.51 U/mL, with that of Pf-5/pBBR1-km-Papra-lipAB supernatant was 46.85 U/mL resulting in 3.47-fold variation after promoter optimization. PG1 lipase gene lipA can be successfully heterologously expressed in Pf-5 via genetic engineering. Results reveal that the constitutive promoter Papra is more efficient than the original promoter PlipAB in Pf-5 strain. Furthermore, the present study provides an important prerequisite for scale production and industrial application of the lipase.

方倩, 谢芝玲, 陈汉娜, 李建伟, 潘登, 丁学知, 夏立秋, 涂强, 张友明. 脂肪酶lipAB操纵子在防御假单胞菌中的克隆、表达及活性研究[J]. 激光生物学报, 2018, 27(5): 442. FANG Qian, XIE Zhiling, CHEN Hanna, LI Jianwei, PAN Deng, DING Xuezhi, XIA Liqiu, TU Qiang, ZHANG Youming. Cloning, Expression and Activity of Lipase Operon lipAB in Pseudomonas protegens[J]. Acta Laser Biology Sinica, 2018, 27(5): 442.

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