Omori Ken, Isoyama-Tanaka Junko, Ihara Fumio, Yamada Yasuhiro, Nihira Takuya
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biosci Bioeng. 2005 Sep;100(3):323-30. doi: 10.1263/jbb.100.323.
Pseudomonas sp. strain 109 secretes lactonizing lipase (LipL), which catalyzes efficient intramolecular transesterification of omega-hydroxyfatty acid esters to form macrocyclic lactones. Because Escherichia coli was found to be unsuitable as an expression host due to the predominant formation of inactive LipL-inclusion bodies and a lack of proper secretion machinery which is also required for the formation of active LipL, Pseudomonas strains were surveyed as expression hosts. Pseudomonas sp. strain 109, an original LipL producer, showed a 7.1-fold higher level of active LipL when the lipL gene under the control of tac-lacUV5 tandem promoter was introduced together with a limL gene encoding a LipL-specific chaperon. Pseudomonas aeruginosa ADD 1976 containing a T7 RNA polymerase gene in the chromosome and plasmid-borne lipL-limL genes under the control of T7 promoter showed a 13-fold higher level of active LipL. Several combinations in the number of lipL and/or limL genes on the plasmid were investigated, and (lipL)3-limL was found to be most efficient, yielding a 67-fold greater production of active LipL than that obtained by the wild-type Pseudomonas sp. strain 109.
假单胞菌属菌株109分泌内酯化脂肪酶（LipL），该酶催化ω-羟基脂肪酸酯的高效分子内酯交换反应以形成大环内酯。由于发现大肠杆菌作为表达宿主不合适，因为主要形成无活性的LipL包涵体且缺乏形成活性LipL所需的适当分泌机制，因此对假单胞菌菌株作为表达宿主进行了研究。当在tac-lacUV5串联启动子控制下的lipL基因与编码LipL特异性伴侣蛋白的limL基因一起导入时，原始LipL产生菌假单胞菌属菌株109显示出活性LipL水平提高了7.1倍。在染色体中含有T7 RNA聚合酶基因且在T7启动子控制下含有质粒携带的lipL-limL基因的铜绿假单胞菌ADD 1976显示出活性LipL水平提高了13倍。研究了质粒上lipL和/或limL基因数量的几种组合，并发现（lipL）3-limL最有效，其活性LipL的产量比野生型假单胞菌属菌株109高67倍。
