To construct a high-efficiency secretion system in E. coli, we developed a method that combines signal peptide PelB (pectate lyase B signal peptide) and colicin release protein Kil. In this project, extracellular PETase was achieved by E. coli BL21(DE3) using a sec-dependent translocation signal peptide, pelB, for secretion, and the signal peptide is removed by a signal peptidase. The colicin release protein Kil releases periplasmic protein by inducing membrane solubilization; as a result, the recombinant proteins can be efficiently secreted into the culture medium when co-expressed with Kil protein.
The DNA sequence of SbPETase was inserted into the BamHI and XhoI sites of the pET22b vector (Figure 1A). The gene Kil was amplified by PCR from the plasmid containing the Kil gene and cloned into the NcoI and XhoI sites of the pRSFduet1 vector (Figure 1B).

We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase (Figure 3).We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase (Figure 3).

In order to test if Kil signal peptide can improve the yield of SbPETase proteins in the culture medium, we also co-transformed the recombinant plasmids pET22b-PelB-SbPETase and pRSFDeut1-Kil into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we purified the protein SbPETase we wanted and collected the data (Figure 4). In Figure4, when co_transformed pET22b-PelB-SbPETase and pRSFDeut1-Kil, the yield of protein SbPETase in the extracellular medium is higher than without it.

We set up an in vitro system and confirmed the ability to use the SbPETase to degrade PET materials. As shown in Figure 4, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small quantity of TPA through HPLC (Figure 5A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate) or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.

Due to the differences in substrate binding sites between SbPETase, and since reported mutants showed improved catalytic efficiency, we performed the following site-directed mutagenesis: Y60A, L61T, W132H, W132A, V181I, T212F, T212S, and R259A. All the SbPETase mutant proteins were obtained from the cultured medium directly and an in vitro activity assay was performed, which generated two mutants (SbPETaseW132H, SbPETaseR259A) with improved catalytic efficiency of degrading PET (Figure 6A). The activity of another mutant SbPETaseL61T was only improved towards degrading BHET (Figure 6A). We then combined these three mutants (SbPETaseW132H, SbPETaseR259A, SbPETaseL61T), to generate three double mutants and one triple mutant. An in vitro activity assay showed that the triple mutant had the highest catalytic efficiency towards PET and BHET degradation (Figure 6B)