Identification of the tliDEF ABC Transporter Specific for Lipase in Pseudomonas fluorescens SIK W1

Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25°C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37°C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.

荧光假单胞菌（Pseudomonas fluorescens）是一种革兰氏阴性嗜冷菌，可向胞外培养基（extracellular medium）分泌耐热脂肪酶（thermostable lipase）。本团队此前的研究中，我们将荧光假单胞菌SIK W1的脂肪酶基因克隆并在大肠杆菌（Escherichia coli）中表达，但该脂肪酶以无活性包涵体（inclusion bodies）的形式积累。对该脂肪酶的氨基酸序列分析显示，其含有一段可被ATP结合盒（ATP-binding cassette, ABC）转运蛋白识别的潜在C端靶向序列。我们对脂肪酶基因周边的基因座进行了检索，成功鉴定出一个分泌相关基因。对一段8.5kb的DNA片段进行核苷酸测序后发现，在脂肪酶基因tliA的上游，存在ABC转运蛋白的三个组分：tliD、tliE与tliF。此外，在tliDEF的上游还存在编码蛋白酶（protease）与蛋白酶抑制剂（protease inhibitor）的基因。tliDEF与铜绿假单胞菌（Pseudomonas aeruginosa）碱性蛋白酶、菊欧文氏菌（Erwinia chrysanthemi）蛋白酶、粘质沙雷氏菌（Serratia marcescens）脂肪酶以及荧光假单胞菌CY091蛋白酶对应的ABC转运蛋白具有较高的序列同源性。仅将tliDEF与脂肪酶结构基因构建在同一操纵子（operon）中，即可使大肠杆菌细胞实现脂肪酶的分泌。此外，仅携带脂肪酶基因的大肠杆菌，可通过互补表达另一质粒上的tliDEF来完成脂肪酶的分泌。荧光假单胞菌的ABC转运蛋白最适功能温度为20℃与25℃，而铜绿假单胞菌的ABC转运蛋白AprD、AprE与AprF则可在20℃至37℃的宽泛温度范围内介导脂肪酶的分泌，不受温度影响。上述实验结果表明，荧光假单胞菌SIK W1的ABC转运蛋白与脂肪酶结构基因tliA共同组成操纵子，负责介导脂肪酶的分泌，且其分泌功能具有温度依赖性。