Overview of different level 2 MoClo constructs. We designed 35 different level 2 constructs by using the modular cloning system (MoClo) and transformed these into Chlamydomonas reinhardtii. These constructs contain promoters (PPSAD, PAR, PTub2), terminators (PSADter, RPL23ter, Tub2ter), and the coding sequences for selection markers (aadA, Hygro), tags (HA, His, SP20-HA, SP20-His), secretion signals (cCA, ARS, GLE) and the enzymes MHETase, wild-type PETase (WT-PETase), mutated PETase (Mut-PETase) and the mutated PETase from the iGEM team TJUSLS China 2016 (Mutate M).

Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii. (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). (b) The UVM4 strain was transformed with the construct shown in (a). 11 spectinomycin-resistant transformants were inoculated in TAP and samples taken after 3 days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW – molecular weight. The black arrow represents the MHETase, the white arrow the MUT-PETase. The expression of both MHETase (~70 kDa) and MUT-PETase (~35 kDa) is visible in colonies 18, 22 and 27. The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.

MUT-PETase destined for secretion gets stuck inside the cell. (a) Level 2 MoClo construct harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase genes. MUT-PETase and MHETase are equipped with the secretion signal from carbonic anhydrase (cCA). See Figure 1 for the description of other parts. (b) Seven days old cultures of transformants generated with the construct shown in (a) were centrifuged and proteins in the culture medium were precipitated by TCA and analysed by immunoblotting using an anti-HA antibody. The black arrow represents MHETase. (c) Whole-cell proteins of UVM4 cells transformed with construct L2C (BBa_K3002202) shown in (a) were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (BBa_K3002200) (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. (d) Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.

Analysis of the secretion of MUT-PETase with secretion signals cCA, GLE, and ARS. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase equipped with secretion signals from carbonic anhydrase (cCA), gamete lytic enzyme (GLE) and arylsulfatase (ARS). 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 and A27 introduced in Figures 4 and 5, respectively, served as positive controls. The white arrow points to the MUT-PETase.

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.

Growth and secretion of MUT-PETase and MHETase in CC-4533 transformant M8 under different conditions. (a) Growth curves of the CC-4533 recipient strain and CC-4533 transformant M8 (Figure 12) at 25°C, 80 µE and 33°C, 170 µE. CC-4533 and transformant M8 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 CC-4533 and M8 grown grown for 3 days at 25°C and 89 µE or at 33°C and 170 µE.

Growth and secretion of MUT-PETase and MHETase in CC-4533 transformant M8 in two photobioreactors. (a, b) Time course analysis of secreted MUT-PETase and MHETase in bioreactors A (a) and B (b). The cell density in bioreactor A was held at a higher cell density than that in bioreactor B. Samples were taken once or twice a day starting at 48.2 h after inoculation. Lyophilized cell-free media was resuspended in 2xSDS loading buffer and analysed by immuno-blotting using an HA-antibody. (c) Cell growth in the Bioreactors A and B at 25°C.

Purification of HA-tagged MUT-PETase and MHETase from Chlamydomonas and activity measurement against BHET by reversed-phase HPLC. (a) Affinity purification of MUT-PETase and MHETase from Chlamydomonas by anti-HA magnetic beads. Transformants M5, C12 and untransformed UVM4 were inoculated in TAP for seven days. Cultures were centrifuged and supernatants incubated with anti-HA magnetic beads for 1 h. Enzymes were purified via biomagnetic separation. Samples of the unconcentrated supernatant (S), of the washing step (W), of the eluted proteins (E) and after 96 h incubation with BHET (A.I.) were analyzed by immunoblotting using an anti-HA antibody. (b) Proteins eluted from Chlamydomonas transformant M5 (producing MUT-PETase and MHETase) and the UVM4 strain (not producing MUT-PETase and MHETase) were incubated with 1 mM BHET in sodium phosphate (NaPi) buffer at 30°C for 96 h. The standard containing 1 mM TPA, MHET and BHET dissolved in DMSO is shown on top.

Measurement of activity of MHETase and MUT-PETase from Chlamydomonas against PET by reversed-phase HPLC. Transformant M8 and parent strain CC-4533 (CliP) were inoculated in HMP medium for seven days. M8 contains construct L2M (BBa_K3002212) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 13). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b) The 20-fold concentrated medium of transformant M8 was incubated with PET film at 25°C for 96 h and measured with HPLC. (c) The 20-fold concentrated medium of parent strain CC-4533 (CliP) was incubated with PET film at 25°C for 96 h and measured with HPLC. (d) The glycine buffer was measured with HPLC. The same measurements are displayed, but the scaling of the axis was set to 2000 mAU on the left and to 50 mAU on the right.

Activity measurement of MHETase and MUT-PETase from Chlamydomonas against BHET by reversed-phase HPLC. Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M (BBa_K3002212) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b) The 20-fold concentrated medium of M5 was incubated with BHET at 30°C for 48 h and measured by HPLC. (c) The 20-fold concentrated medium of parent strain UVM4 was incubated with BHET and measured by HPLC. (d) The glycine buffer was measured with HPLC. (e) Peak areas of the shown measurements in mAu*s.

Activity measurement of MHETase from Chlamydomonas against MHET by reversed-phase HPLC. Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M (BBa_K3002212)encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with 1 mM MHET at 30°C for 48 h. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b) Measurement of M5 supernatant with HPLC. (c) Measurement of UVM4 supernatant with HPLC. (d) The glycine buffer was measured with HPLC. (e) Peak areas of the shown measurements in mAu*s.

Measurement of activity of MUT-PETase and MHETase from Chlamydomonas at 25°C and 33°C by reversed-phase HPLC. Transformant M8 and parent strain CC-4533 (CliP) were inoculated in HMP medium for seven days. M8 contains construct L2M (BBa_K3002212) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 13). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with PET film or MHET at 25°C or 33°C for 96 h. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. Measurement of M8 medium incubated with MHET at 25°C (b) and 33°C (c). Measurement of M8 medium incubated with PET at 25°C (d, e) and 33°C (f, g). measured by HPLC. The measurements shown in (d) and (e), and in (f) and (g) are the same, but the axis scaling was set to 2000 mAU in (d) and (f) and to 50 mAU in (e) and (g). Note that the shifts in retention times are due to the clogging of the HPLC.

Measurement of activity of MUT-PETase and MHETase from Chlamydomonas at 30°C and 40°C by reversed-phase HPLC. Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M (BBa_K3002212) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with 1 mM BHET at 30°C (left) or 40°C (right) for 96 h. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b, e) Measurement of M5 medium incubated with BHET at 30°C (b) and 40°C (e). (c, f) Measurement of UVM4 medium incubated with BHET at 30°C (c) and 40°C (f). (d, g) Measurement of glycine buffer incubated with BHET at 30°C (d) and 40°C (g). (h) Peak areas of the shown measurements in mAu*s.