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Papra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Papra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Papra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Papra比lipAB的原始启动子PlipAB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 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Papra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Papra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Papra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 Papra is more efficient than the original promoter PlipAB in Pf5 strain. Furthermore, the present study provides an important prerequisite for scale production and industrial application of the lipase.