Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440

Abstract

Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml^-1 OD^-1 compared with the signal peptide of V. cholerae CtxB (52 mU ml^-1 OD^-1 ). This positive effect was confirmed with a β-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher β-glucosidase activity (181 mU ml^-1 OD^-1 ) than with the CtxB signal peptide (42 mU ml^-1 OD^-1 ). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.

Figure 1

Effect of different translocator domains on the surface display of the esterase EstA.A. Domain structures of MATE‐EstA, MATE2‐EstA and MATE3‐EstA. The calculated molecular weight of each fusion protein is provided in kDa. The respective α‐helix and β‐barrel sequences are provided in Fig. S1.B. Modelled structure of EstP from P. putida, revealing its α‐helix (red) and β‐barrel (yellow). The arrow indicates the end of the protein sequence that was used in this study.C. Flow cytometric histograms of P. putida pMATE‐EstA, (D) pMATE2‐EstA and (E) pMATE3‐EstA. Cells were labelled with mouse anti‐6xHis and secondary DyLight 633‐conjugated antibodies. Histograms of control cells cultivated without induction of protein expression are shown in grey. Representative histograms from single experiments are shown. Additional control experiments are shown in figure S3.F. Time‐course of 4‐nitrophenolate formation in whole‐cell esterase activity assay and (G) resulting activities performed with P. putida pMATE‐EstA (red), pMATE2‐EstA (orange), pMATE3‐EstA (yellow) and control cells without induction of protein expression (grey).

Figure 2

Effect of different signal peptides on the surface display of the esterase EstA.A. Domain structures of MATE‐EstA, OprF^SP‐MATE‐EstA and EstA^SP‐MATE‐EstA. The calculated molecular weight of each fusion protein is provided in kDa.B. SDS‐PAGE of outer membrane proteins of P. putida expressing autotransporter fusion proteins with different signal peptides. An arrow indicates the protein band assigned to the outer membrane protein OprF used as loading control. P. putida with an empty vector was used as control. The asterisks depict the protein bands assigned to the recombinant fusion proteins.C. Flow cytometric histograms of P. putida pMATE‐EstA, (D) pOprF^SP‐MATE‐EstA and (E) pEstA^SP‐MATE‐EstA. Cells were labelled with mouse anti‐6xHis and secondary DyLight 633‐conjugated antibodies. Histograms of control cells cultivated without induction of protein expression are shown in grey. Representative histograms from single experiments are presented. Additional control experiments are shown in figure S3.F. Time‐course of 4‐nitrophenolate formation in whole‐cell esterase activity assay and (G) resulting activities of P. putida pMATE‐EstA (red), pOprF^SP‐MATE‐EstA (blue), pEstA^SP‐MATE‐EstA (green) and control cells without induction of protein expression (grey). Standard deviations of biological triplicates are indicated.

Figure 3

Effect of different signal peptides on the surface display of the β‐glucosidase BglA.A. SDS‐PAGE of outer membrane isolates. An arrow indicates the protein band assigned to the outer membrane protein OprF used as loading control. P. putida with an empty vector was used as control. The asterisks depict the protein bands assigned to the recombinant fusion proteins.B. Flow cytometric histograms of P. putida pMATE‐BglA and (C) pOprF^SP‐MATE‐BglA. Cells were labelled with mouse anti‐6xHis and secondary DyLight 633‐conjugated antibodies. Histograms of control cells cultivated without induction of protein expression are shown in grey. Representative histograms from single experiments are presented.D. Time‐course of 4‐nitrophenolate formation in whole‐cell β‐glucosidase activity assay and (E) resulting activities of P. putida pMATE‐BglA (red), pOprF^SP‐MATE‐BglA (blue) and control cells without plasmid (grey). Standard deviations of biological triplicates are indicated.