Effect of the SP20 module on the secretion efficiency of MHETase and PETase. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals (BBa_K3002212, BBa_K3002213, BBa_K3002214) introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs shown in (a) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformants C12 (BBa_K3002202) and A27 (BBa_K3002200) introduced in Figures 4 and 5, respectively, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.

The SP20 module increases the efficiency of protein secretion. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6. The constructs contain the coding sequence for a conventional 3xHA tag (C, K, L)(BBa_K3002202, BBa_K3002210, BBa_K3002211), or the 3xHA tag preceded by a SP20 tag to enhance glycosylation (M, N, O). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs C, K, L and M, N, O (BBa_K3002202, BBa_K3002210, BBa_K3002211, BBa_K3002212, BBa_K3002213, BBa_K3002214) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformant A27 introduced in Figures 4, served as positive control. The black arrow points to MHETase, the white arrow to MUT-PETase and the grey arrow to RPL1 (chloroplast ribosomal 50S protein L1). The RPL1 antibody was used to detect contamination from intracellular proteins.

Identification of MHETase and MUT-PETase by LC-MS/MS. (a) Transformants generated with construct L2N (BBa_K3002213) (d) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Protein bands corresponding to those detected with the anti-HA antibody in a gel run in parallel and stained with Coomassie brilliant blue were excised, in-gel digested with trypsin and analyzed by LC-MS/MS. Peptides identified by LC-MS/MS for MHETase (green) and MUT-PETase (purple) are indicated. (b, c) Sequences of MHETase and MUT-PETase with the peptides detected by LC-MS/MS are highlighted in green and purple, respectively.

Verification of secretion of MHETase and MUT-PETase into the medium. Transformants generated with constructs M, N, and O (BBa_K3002212, BBa_K3002213, BBa_K3002214)(Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant (s) lyophilized and resuspended in 2xSDS buffer. Cell pellets (p) were also resuspended in SDS-buffer. Both fractions were analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. The black arrow points to MHETase, the white arrow to MUT-PETase.

Quantification of secreted MHETase and MUT-PETase. (a) Transformants generated with constructs C, J, M, N, and O (BBa_K3002202, BBa_K3002208, BBa_K3002212, BBa_K3002213, BBa_K3002214)(Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Whole-cell extracts of strain B1-TIG-HA for which concentrations of the HA-tagged TIG protein are known are loaded next to the lyophilized supernatants. The black arrow points to MHETase, the white arrows to MUT-PETase. The supernatant of a culture with the UVM4 strain were loaded as negative control. (b) Maximum cell densities, doubling times, daily growth rates, yields of MHETase and PETase and daily productivity of both combined were calculated for the transformant lines indicated.

Analysis of secreted enzymes of transformant N6 transformed with construct AI. (b) Clones generated with transformant N6 (Figure 8) and construct L2AI (BBa_K3002234) (a) were grown in TAP medium for four days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformant C12 introduced in Figure 5, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.

Analysis of secreted MUT-PETase and MHETase with secretion signals cCA, ARS and GLE in the CC-4533 strain background. Transformants generated in the CC-4533 strain background with constructs M (BBa_K3002212) and N (BBa_K3002213) (Figure 8) were grown in TAP medium for four days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. The supernatant of a culture with the CC-4533 strain were loaded as negative control. The black arrow points to MHETase, the white arrow to MUT-PETase.

Growth and secretion of MUT-PETase and MHETase in UVM4 transformant N6 under different conditions. (a) Growth curves of the UVM4 recipient strain and UVM4 transformant N6 (BBa_K3002213) (Figure 8) at 25°C, 80 µE and 33°C, 170 µE. UVM4 and transformant N6 were inoculated in 50 mL with 2*10⁵ cells/mL. Growth was measured by counting cells for 8 days. Error bars represent the standard error of three biological replicates. (b) Time course of MHETase and MUT-PETase secretion into TAP medium. 2 mL of each sample was lyophilized, desalted and resuspended in 2xSDS loading buffer. 10 µl of each sample were separated via SDS-PAGE and analyzed by immunoblotting with an anti-HA antibody. An antibody against chloroplast ribosomal 50S protein L1 (RPL1) was used to detect contaminations from cellular proteins. The black arrow points to MHETase, the white arrow to MUT-PETase and the grey arrow to RPL1. (c-f) Bright-field images of strains UVM4 and N6 grown grown for 3 days at 25°C and 89 µE or at 33°C and 170 µE.