Design of Project Collection
In the course of our project we generated 27 level 0 constructs, 25 level 1 constructs and 35 level 2
constructs by employing the modular cloning system (MoClo), which is based on Golden Gate Assembly
(Weber et al., 2011; Crozet et al., 2018). This allows us to provide iGEM with a total of 87 MoClo
constructs. The level 2 constructs, shown in Figure 1, were transformed into Chlamydomonas
reinhardtii (Chlamydomonas). We worked with the Chlamydomonas strains UVM4 in the CC-4350 background
and its sister strain in the CC-4533 background, hereafter referred to as Clip. UVM4 is a suitable
expression chassis and easy to transform (Neupert et al., 2009). The Clip sister strain is also a
suitable expression chassis, but more robust that UVM4 and therefore also suitable for growth in
photobioreactors.
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).
To realize the biodegradation of polyethylene terephthalate (PET) by Chlamydomonas, we
designed different MoClo constructs encoding the enzymes MHETase and PETase. PETase cleaves the
polymer PET into its monomers MHET. MHETase then cleaves MHET into the basic modules ethylene glycol
(EG) and terephtalic acid (TPA). We introduced three point mutations W159H, S238F and R280A into
wild-type PETase (WT-PETase) to generate a mutant form (MUT-PETase). These changes have been
reported to improve the activity of the enzyme (Austin et al., 2018; Joo et al., 2018). To secret
these enzymes into the medium, we used three secretion signals, namely of carbonic anhydrase (cCa),
arylsulfatase (ARS) and the gametic lytic enzyme (GLE) and removed the original secretion signal of
Ideonella sakaiensis (Yoshida et al., 2016). To increase the amount of secreted enzyme, we also
equipped the enzymes with a C-terminal glycomodule composed of tandem serine and proline repeats of
20 units (SP20-tag) (Ramos-Martinez et al., 2017).
Suitability of Chlamydomonas as PET Degrader
Toxicity test with TPA, EG and EtOH. Growth of Chlamydomonas strain UVM4 was tested in TAP medium containing 0 mM (control), 5 mM and 10 mM terephtalic acid (TPA), ethylene glycol (EG) and ethanol (EtOH). The pH in the medium containing TPA was adjusted to pH 7 with potassium acetate. (a) UVM4 cells were inoculated in 20 mL with 2*105 cells/mL. Growth was measured by counting cells for 5 days. Error bars represent standard error of three biological replicates. (b) Spot test analysis with the same concentrations of EG, EtOH and TPA as used in (a). Note that the UVM4 strain lacks a cell wall and that cells die if they are too diluted.
We first tested, whether the breakdown products of PET are toxic for Chlamydomonas. For this,
we cultivated Chlamydomonas in the presence of 5 mM and 10 mM of breakdown products TPA and EG and
determined growth rates. As shown in Figure 2, the growth rates were not significantly different to
those of cultures lacking PET breakdown products and we can conclude that they are not toxic.
Chlamydomonas reinhardtii shows spontaneous attachment to PET. Wild-type strain CC-4533 was incubated for 7 days in TAP medium with 1 g PET/l. The cells spontaneously attached to the microplastic surface. This renders Chlamydomonas reinhardtii as a perfect model organism for the production of plastic-degrading enzymes.
Furthermore, we could show that Chlamydomonas cells spontaneously attach to PET particles
(Figure 3), which is of great advantage to increase the efficiency of secreted PETase and MHETase on
microplastic particles. With these results we could demonstrate that Chlamydomonas is well-suited
for degrading PET in a photobioreactor, since it shows spontaneous attachment to PET, is easy to
engineer thanks to the MoClo system, and the PET breakdown products show no toxicity.
Cytosolic Expression
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.
As a first step to equip Chlamydomonas with plastic-degrading enzymes MUT-PETase and MHETase,
we transformed the UVM4 strain with construct L2A which targets the enzymes with a C-terminal 3xHA
tag to the cytosol. As shown in Figure 4, both enzymes accumulated in transformants, but to
different levels. This is typically resulting from position effects. Here we could observe a mass
shift for both enzymes compared with the Ideonella sakaiensis forms of ~5 kDa for MUT-PETase and ~10
kDa for MHETase. These are most likely caused by the 3xHA tag and post-translational modifications.
Troubleshooting of the Secretion Process
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 shown in (a) were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (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.
Next, we wanted to have both enzymes secreted and to this end generated construct L2C that
differs from L2A by the presence of sequences equipping MUT-PETase and MHETase with the N-terminal
secretion signal from carbonic anhydrase (cCA). As shown in Figure 5a, we could detect MHETase in
the medium, but no MUT-PETase. We reasoned that MUT-PETase might not be secreted and looked for it
in cell proteins. Indeed, MUT-PETase accumulated to high levels in the cell body (Figure 5b). We
next employed immunofluorescence to localize MUT-PETase in the cell and detected it in the ER
(Figure 5c).
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.
To solve this problem, we generated constructs L2H, L2I, and L2J for the expression of
MUT-PETase alone equipped with the secretions signals from carbonic anhydrase (cCA), gamete lytic
enzyme (GLE), and arylsulfatase (ARS), respectively. While a module encoding the cCA signal is
present in the MoClo kit (Crozet et al., 2018), those for GLE and ARS were generated by us.
Arylsulfatase is essential for the mineralization of sulfate by hydrolyzing sulfate esters under
conditions of sulfate deprivation (deHostos et al., 1988). Gamete lytic enzyme is a metalloprotease
that mediates digestion of the cell wall during gametogenesis (Kinoshita et al., 1992). While we
detected no MUT-PETase in the medium when it was equipped with the cCA signal, weak signals were
detected when it contained secretion signals GLE and ARS (Figure 6).
Enhancing Secretion and Identification of Secreted Proteins
Effect of the SP20 module on the secretion efficiency of MHETase. (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 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 and A27 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), 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 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.
In the meanwhile, we had learned about a report where repeats of serine and proline attached
to the C-terminus of secreted proteins strongly increases the yield of secretion (Ramos-Martinez et
al, 2017). That’s why we designed constructs L2M, N and O to express MUT-PETase and HMETase with
repeats of 20 serine and proline residues (SP20-tag). As shown in Figures 7 and 8, this strongly
improved the secretion efficiency for both enzymes. Proline in these repeats is hydroxylated and
thereby converted to hydroxyproline, which subsequently gets O-glycosylated. This engineering
technology of hydroxyproline-rich glycoproteins exploits the glycosylation code of cell wall
proteins and leads to their efficient export into the medium (Kieliszewski and Shpak, 2001).
Accordingly, we observed a strong increase in the apparent molecular mass for both enzymes that is
likely due to heavy protein glycosylation (Figure 8).
Identification of MHETase and MUT-PETase by LC-MS/MS. (a) Transformants generated with construct L2N (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.
Our detection of MUT-PETase and MHETase was based on an antibody against the 3xHA tags at
their C-termini. Due to the glycosylation of the proteins, we detected multiple protein bands and
could not distinguish between the two enzymes. Therefore, we identified the proteins in these bands
by mass spectrometry. We could detect eight specific peptides for MHETase and three specific
peptides for MUT-PETase and therefore could verify that both enzymes are secreted into the medium
(Figure 9).
Verification of secretion of MHETase and MUT-PETase into the medium. Transformants generated with constructs M, N, and O (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.
To verify effective secretion of both enzymes equipped with the SP20 tag, we analyzed their
presence in the medium and in the cell bodies in three tranformants generated with constructs L2M,
N, and O (Figure 8) differing only by the secretion signals for MUT-PETase. As shown in Figure 10,
hardly any protein was detected in cell bodies while we detected the bulk of the proteins in the
medium. The highest amounts of secreted, glycosylated PETase were found in transformant N6
expressing MUT-PETase with the ARS secretion signal.
Quantification of secreted MHETase and MUT-PETase. (a) Transformants generated with constructs C, J, M, N, and O (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.
To estimate the quantity of secreted enzymes we performed quantitative immunoblot blot
analysis. Transformants N6 and M5 had the highest amounts of secreted MUT-PETase and MHETase (Figure
11).
Analysis of secreted enzymes of transformant N6 transformed with construct AI. (b) Clones generated with transformant N6 (Figure 8) and construct L2AI (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.
Since MHETase accumulates at a higher level than MUT-PETase, we wanted to secret both enzymes
equally. Therefore, we transformed clone N6 (Figure 8) with construct L2AI (12a). Thereby, we could
demonstrate an equally strong secretion of MHETase and MUT-PETase from the transformants 4, 9 and
10. Furthermore we observed secretion of MUT-PETase in all transformants, excluding transformant 1.
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 and N (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.
On a long run we need to grow Chlamydomonas engineered with plastic degrading enzymes in a
bioreactor. Since the UVM4 strain is too fragile, we had to use another strain background and for
this used the more robust Clip sister line of UVM4. We transformed Clip with the constructs L2M and
L2N and could also in this strain background demonstrate successful secretion of both enzymes, with
MHETase accumulating to much higher levels than MUT-PETase (Figure 12).
Expanding Experiments into a Bioreactors
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 (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.
To determine the optimal conditions for secreting our enzymes, we analyzed secretion and
growth at different light and temperature regimes. Thanks to the advice of Prof. Dr. Zimmermann
(University of Leipzig, Institute of Biochemistry) we learned that at PETase is more active and PET
is better accessible at higher temperatures. That’s why we tested secretion and growth not only at
25 °C but also at 33 °C. UVM4 grew faster at 33°C and 170 µE light intensity but reached a lower
final cell density that when grown at 25°C and 80 µE light intensity (Figure 14A). Also Clip grew
faster at 33°C and 170 µE light intensity but reached the same final cell density as when grown at
25°C and 80 µE light intensity (Figure 15A). Transformants with constructs L2N or L2M both
accumulated more MHETase and less MUT-PETase when grown at 33°C and 170 µE light intensity, while
they both accumulated less MHETase and more MUT-PETase when grown at 25°C and 80 µE light intensity
(Figures 14b and 15b). Hence, growth conditions obviously have a strong impact on the amounts of
secreted proteins.
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.
After the successful production and secretion of MUT-PETase and HMETase in Clip strains, we
wanted to test the suitability of the engineered strains for growth in a photobioreactor. We did
this in parallel in two 1.9-L photobioreactors run in turbidostat mode, in which the cell density
was kept at different thresholds. We could show that both enzymes accumulated in the culture medium
about 65 hours after inoculation. Surprisingly, between 120 h and 243 h of cultivation, levels of
secreted enzymes declined for yet unknown reasons. Further experiments are required to elucidate the
mechanisms involved here.
First Activity Measurements
After the successful production and secretion of MUT-PETase and MHETase via transgenic
Chlamydomonas cells, we wanted to demonstrate the functionality of these enzymes. For this purpose,
we incubated the secreted enzymes with PET, BHET and MHET under different conditions. Since PETase
degrades PET and BHET into MHET and MHETase degrades MHET into EG and TPA, the resulting products
can be detected by reversed phase high-performance liquid chromatography (HPLC). The phenol moiety
in BHET, MHET, and TPA allows detection of these compounds by UV light. Since such a moiety is
missing in EG, EG is not detectable by reversed phase HPLC.
Expression of His-tagged MUT-PETase and MHETase in E.coli SHuffle T7 and purification via Ni-NTA affinity chromatography. E.coli SHuffle T7 cells containing expression vectors for hexa-histidine tagged MUT-PETase or MHETase were incubated in 1 L medium at 33°C and induced with 1 mM IPTG at OD600=1. Cultures were shifted to 17°C for another 14 h after they had reached OD600=2.5. Cell lysates were loaded onto a Ni-NTA column. (a) Purification of MHETase (~ 65 kDa, black arrowhead). (b) Purification of MUT-PETase (~30 kDa, white arrowhead). Samples taken pre- and post-induction, pre- and post-column purification, as well as samples taken from four washing steps and five elution steps were analyzed by SDS-PAGE and staining of the gel with Coomassie brilliant blue. MW - molecular weight marker.
We first wanted to test, whether we could detect degradation of PET, BHET, and MHET by HPLC
and for this needed the purified PETase and MHETase enzymes. We received an appropriate expression
vector from Dr. rer. nat. Gottfried J. Palm, University of Greifswald, for the production of
hexa-histidine-tagged MHETase. We produced the recombinant proteins MHETase and PETase containing
three point mutations in E.coli and purified them via Ni-NTA affinity chromatography (Figure 17).
For their expression, we used the five E.coli strains Rosetta-gami, Origami, ArcticExpress, ER2566
and SHuffle T7 but only succeeded with E.coli SHuffle T7.
Measurement of the activity of MUT-PETase from E.coli against PET by reversed-phase HPLC. (a) A standard of 1 mM TPA, MHET and BHET dissolved in DMSO was measured by HPLC. (b) Hexa-histidine tagged MUT-PETase purified from E.coli SHuffle T7 was incubated with PET film for 96 h at 25°C. An aliquot of the reaction buffer was analyzed by HPLC. (c) Purified MUT-PETase was denatured for 10 min at 95°C and incubated with PET film for 96 h at 25°C. An aliquot of the reaction buffer was analyzed by HPLC. Detections were performed at 254 nm. 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.
After the successful purification of MUT-PETase and MHETase from E.coli, we tested PETase
activity by incubating the enzyme with PET film and measuring putative degradation products BHET,
MHET, and TPA via reversed-phase HPLC (Figure 18). Since we could recognize peaks for all three
degradation products after lowering the scale, we can conclude that PETase is active on PET
film when produced in E.coli. This is supported by the finding that no degradation products were
detected when the protein had been thermally denatured before incubation with the PET film.
Moreover, we can state that our analytical platform is well-suited for the detection of PETase activity.
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.
Activity of Secreted Enzymes from Chlamydomonas
The next step was to analyze the activity of the secreted MUT-PETase and MHETase produced in
Chlamydomonas. For this, we had to extract the enzymes from Chlamydomonas transformant M5, which
secretes both enzymes into the culture medium. We did this by affinity purification using anti-HA
magnetic beads and used the parent strain UVM4 as control (Figure 19). We then measured the activity
of the eluted proteins against BHET by reversed-phase HPLC. A peak for MHET was detected in the
assay with eluates of the M5 strain and from the parent strain. This indicates an activity for the
conversion of BHET to MHET coming from the buffer or Chlamydomonas. However, in the assay performed
with eluates from the M5 strain we detected a peak for TPA, which was absent in the assay with
eluates from the parent strain. This indicates that there is MHETase activity. When comparing the
TPA peaks in plots with the same x-axis scale between E.coli-derived and Chlamydomonas-derived
enzymes, they are even comparable.
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 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.
Since affinity-purification of the enzymes from culture medium is tedious and expensive, we
used ultracentrifuge filters for this purpose and achieved a ~20-fold concentration. We then tested
the activity of MUT-PETase and MHETase in concentrated culture medium against PET (Figure 20). To
this end, the concentrates from transformant M8 and the parental strain were incubated with PET
film. We observed tiny peaks for BHET and TPA that were absent in the assays with concentrate from
the CC-4533 parent strain or buffer alone. This indicated very weak activity of both enzymes
secreted by Chlamydomonas.
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 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.
After we have shown a low activity of the enzymes secreted from CC-4533 transformant M8
against PET, we repeated the experiment with BHET under the same conditions with enzymes secreted
from UVM4 transformant M5 (Figure 21). Unexpectedly, we observed a small peak for MHET in the assays
performed with the concentrate from the parental strain or buffer alone, which was absent in the
assay with concentrate from transformant M5. In contrast, a peak for TPA was observed in the assay
with concentrate from transformant M5, which was absent in the assays with concentrate from the
parental strain or buffer alone. These findings indicate that there is activity for the conversion
of BHET to MHET already in the buffer. However, the activity for the conversion of MHET to TPA, i.e.
for MHETase, is only present in the concentrate from transformant M5. We suggest that there is a
enzymatic activity against BHET, since the peak area of TPA of M5 is major than the sum of the peak
areas of MHET and TPA in controls.
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 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.
To verify the activity for MHETase from transformant M5, we incubated the concentrated medium
from transformant M5 and from the UVM4 parent strain directly with MHET as substrate (Figure 22).
Thereby, we could observe a complete conversion from MHET to TPA only in the assays with
concentrates from M5 and no peaks for TPA in assays with concentrates from the parent strain. This
corroborates our conclusion that the MHETase secreted from transformant M5 shows indeed a high
activity.
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 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.
Temperature Dependent Activity
Thanks to Prof. Dr. Zimmermann from the University of Leipzig we know that PETase shows a
higher activity at higher temperature (Link zu IHP (Human practice)). We therefore tested the
activity of the secreted enzymes of transformant M8 against MHET and PET at 25°C and 33°C (Figure
23). We observed activity of MUT-PETase and MHETase against PET and MHET at 25°C and 33°C. However,
we couldn’t observe great differences in activity between the temperatures.
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 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.
Since there was no difference in enzymatic activity between 25°C and 33°C, we compared the
activity of MUT-PETase and MHETase of transformant M5 at 30°C and 40°C and used BHET as substrate
(Figure 24). As already described in Figure 21, at 30°C we observed a peak for MHET in the assays
performed with the concentrate from the UVM4 parent strain and buffer alone, which was missing in
the assay with the concentrate from transformant M5. Instead, a peak for TPA was only observed in
the assay with M5 concentrate, but not in those with buffer of concentrate from the UVM4 parent
strain. At 40°C, stronger peaks for MHET were detected in all assays and the peak for TPA in the
assay with M5 concentrate was smaller. These data indicate that spontaneous conversion of BHET to
MHET is enhanced at 40°C versus 30°C, but MHETase activity is impaired.
Enhancing Safety
Safety - Growth test of the auxotrophic Chlamydomonas strain cw325. (a) Spot test analysis with 0 µg, 10 µg, 25 µg, 50 µg and 100 µg arginine per ml. Arginine auxotrophic strain cw325 on the left and UVM4 (served as control) on the right. Note that the UVM4 strain lacks a cell wall and that cells die if they are too diluted. (b) Growth test of the Chlamydomonas strains UVM4 and cw325 with 100 µg/ml arginine and without arginine was tested in 10 ml TAP medium.
After we successfully secreted MUT-PETase and MHETase and proofed the activity of both enzymes we wanted to ensure the safety of our bio-recycling method. Therefore, we used an auxotrophic strain and tested its dependence on arginine. We proofed that Chlamydomonas strain cw325 can’t grow without the presence of arginine in the medium. Consequently, it could be implemented in our system to enhance safety.
Outlook
The following experiments could be carried out to optimize PET biodegradation via genetically engineered Chlamydomonas:
To enhance the secretion of both enzymes, especially of MUT-PETase, the constructs harboring
the coding sequences for each enzyme should be transformed sequentially into Chlamydomonas. We would
first screen for transformants expressing PETase to very high levels and then transform this strain
with the MHETase construct and screen again. We would need to equip the enzymes with different tags
to better distinguish between them.
The HA tag at the C-terminus of PETase might interfer with its activity. We would construct a
vector containing the coding sequence for PETase without any tags and raise an antibody against
recombinant PETase from E.coli to detect it in Chlamydomonas transformants.
Another approach, which already was planned but only partially realized, is to create a fusion
protein of MUT-PETase and MHETase separated by a linker. We hope that this will result in an equally
strong secretion of both enzymes and an improvement of their activity since the subsequent reaction
can immediately take place.
To gain a deeper understanding of our enzymes we could design constructs for the production
and secretion of the cutinase from Prof. Dr Zimmermann and compare its activity with that of
MUT-PETase.
PET has crystalline and amorphous structures, with the amorphous structures being better
accessible for degradation than the crystalline structures. Since PET exhibits amorphous structures
at higher temperatures, another approach would be to engineer thermostability into both enzymes. The
thermostable enzymes could be purified from the culture medium for activity assays at higher
temperatures.
In order to prevent possible glycosylation near the active center, the glycosylation patterns
discovered in the model could be removed by inserting point mutations. Furthermore, the activity of
MUT-PETase could be compared with WT-PETase.
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