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。
To implement heterologous expression of Burkholderia glumae PG1 lipase operon lipAB in Pseudomonasprotegens Pf-5 via Red / ET homologous recombineering. The vector p15A-cm-lipAB was obtained using Red / ET directcloning technology. Then, two recombinant expression vectors pBBR1-km-lipAB and pBBR1-km-Papra -lipAB withdifferent promoters were constructed by subcloning technology, and electrotransformated the resultant expression vectorsinto P. protegens Pf-5. Transformants obtained by kanamycin or apramycin resistance screening. The tributyrin glyceryltrinitrate plate diffusion method and the p-nitrophenol method were used for the assay of the activities of lipase,and theeffect of promoter replacement on lipA expression was examined by qRT-PCR. We successfully cloned the lipase operonlipAB （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 extracellularlipase in Pf-5 was accomplished. Moreover, it was found that the expression level of lipA gene was 2. 1-fold the originallevel 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/ mLresulting in 3. 47-fold variation after promoter optimization. PG1 lipase gene lipA can be successfully heterologouslyexpressed in Pf-5 via genetic engineering. Results reveal that the constitutive promoter Papra is more efficient than theoriginal promoter PlipAB in Pf-5 strain. Furthermore, the present study provides an important prerequisite for scaleproduction and industrial application of the lipase.
作者
方倩
谢芝玲
陈汉娜
李建伟
潘登
丁学知
夏立秋
涂强
张友明
FANG Qian;XIE Zhiling;CHEN Hanna;LI Jianwei;PAN Deng;DING Xuezhi;XIA Liqiu;TU Qiang;ZHANG Youming(State Key Laboratory of Developmental Biology of Freshwater Fish,Hunan Provincial Key Laboratory ofMicrobial Molecular Biology,College of Life Science,Hunan Normal University,Changsha 410081,Hunan,China;Shandong University-Helmholtz Institute of Biotechnology,State Key Laboratory of Microbial Technology,School of Life Science,Shandong University,Qingdao 266237,Shandong,China,3.Shandong ProvinceSoil and Fertilizer Station,Jinan 250100,Shandong,China,4.Suzhou Institute of Shandong University,Suzhou 215123,Jiangsu,China)
出处
《激光生物学报》
CAS
2018年第5期442-450,共9页
Acta Laser Biology Sinica
基金
湖南省发展生物工程和新产品合作创新中心(20134486)
中国博士后科学基金(2018M632669)
山东省自然科学基金(ZR2017ZB0212)
高等学校学科创新引智计划B16030
苏州科技发展计划(SYG201507)
