基于T7表达系统的洋葱伯克霍尔德菌G63脂肪酶同源高效表达

Homologous expression of Burkholderia cepacia G63 lipase gene based on T7 RNA polymerase expression system

为实现对洋葱伯克霍尔脂肪酶的可控高效表达,将目前被广泛使用的T7重组蛋白高效表达系统移植到洋葱伯克霍尔德菌(Burkholderia cepacia)G63中进行脂肪酶同源表达。首先采用PCR从大肠杆菌BL21(DE3)中得到T7 RNA聚合酶基因(T7 RNAP)并将其克隆到致死质粒pJQ200SK上,然后在T7 RNAP前后各加入500 bp用于同源重组的片段,再通过三亲本杂交把T7 RNAP整合到B.cepacia基因组上,使T7 RNAP受到脂肪酶基因(lipA)启动子调控。接着把lipA和它的伴侣基因lipB单独或全部克隆到载体pUCPCM和pBBR22b上,构建出pBBR22blipAB、pBBR22blipA、pUCPCMlipAB、pUCPCMlipA、pUCPCM△lipAlipB、pUCPCM△lipA、pUCPCM△lipB七种表达质粒,通过电转化将上述表达质粒转化到含T7 RNAP的B.cepacia宿主菌中,最终得到一系列脂肪酶基因工程菌。通过摇瓶诱导发酵发现含表达质粒pUCPCMlipAB的工程菌脂肪酶酶活最高,达到607 U/mg,与野生菌相比酶活力提高2.8倍,并且除含pUCPCM△lipB的工程菌外,其它工程菌的脂肪酶酶活均有不同程度提高。野生菌与工程菌pUCPCMlipAB的发酵液经硫酸铵沉淀,Sephadex G-75凝胶过滤纯化后,比酶活分别为29 984 U/mg和30 875 U/mg。以上结果表明,构建的基于T7表达系统的B.cepacia脂肪酶基因工程能有效提高脂肪酶的表达量,同时说明分泌信号PelB和增强转录的核糖体接合位点对脂肪酶的表达有促进作用。

In order to realize over-expression of Burkholderia cepacia （B. cepacia） lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene （T7 RNAP） from the BL21 （DE3） and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene （lipA） promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMAlipAlipB, pUCPCMAlipA, pUCPCMAlipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/rag, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMAIipB were enhanced to some extent. The specific activities of wild type B. cepacia and B cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.