Development of ABC transporter system for efficient extracellular production of recombinant proteins

Abstract

The ABC transporter apparatus of the Gram-negative bacterial type I secretion pathway has been used to secrete many recombinant proteins. The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1 mediated the secretion of a thermostable lipase (TliA). To increase the secretion level of TliA, we firstly optimized the expression level and expression timing of tliA and tliDEF. The four types of co-expression systems for tliA and tliDEF were constructed. When the relative expression levels were changed by adding different concentrations of IPTG, the secretion (16.9 U/ml) of TliA in E. coli [pTliDEFA-223 + pACYC184] was significantly higher than E. coli [pKK223-3 + pTliDEFA-184] secreting the lowest level of TliA (5.2 U/ml). The secretion level of TliA of E. coli [pTliDEFA-223 + pACYC184] was achieved to 26.4 U/ml by inducing gene expression at culture initiation time. Secondly, The ABC transporter (TliDEF) was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency for TliA. TliD mutants with increased secretion efficiency were identified by co-expressing the mutated tliD library with the wild-type tliA in E. coli, and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24 and T35, showed 3.2- (36.4 U/ml), 2.6- (29.2 U/ml), 2.9- (32.8 U/ml) and 3.0-fold (34.0 U/ml) increases of the secretion level of TliA, respectively, but had almost the same expression level of TliD in membrane as that of the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA was mediated by these transporter mutations. Each mutant had a single amino acid change within the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of target protein. Finally, we tried to produce...