Team:TU Kaiserslautern/Engineering

Our goal was to have Chlamydomonas reinhardtii secrete the laccase BaLac or marLac. These should then oxidize micropollutants such as diclofenac in the wastewater and render them harmless. For this purpose, constructs for C. reinhardtii, as well as for Escherichia coli were created. E. coli was used as a rapidly growing organism to produce a large amount of the laccases. Those purified proteins are used to implement an activity assay for the proteins produced by C. reinhardtii.

With our results, we hope to open a new door for a cleaner wastewater treatment and an overhaul of old, outdated methods.

E. coli BaLac in pGEX-6P-1

For E. coli BaLac, we used the construct BBa_K3589105. We transformed the gene of interest containing vector pGEX-6P-1_baLac into the E. coli BL21 (DE3) strain. After producing BaLac, we harvested the cells and purified the protein. We saw the protein on the SDS-Gel and on the immunoblot (Fig. 1). The protein was on the SDS-PAGE at the level of a relative molecular mass of the marker between 75 and 100 kDa (BaLac, size 89.3 kDa). An immunological detection confirms this observation.

Fig. 1: Purification of BaLac.

The SDS-PAGE shows the samples taken after every step. The fusion protein (BaLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). (A) shows the first step of the purification before dialysis with PreScission Protease. The cell lysate was applied to the column. The fusion protein with GST-tag was able to bind to the column, the remaining lysate passed the column (flow through). This was followed by a washing step with washing buffer. Finally, the fusion protein was eluted with elution buffer containing glutathione (eluates 1-6). This was followed by dialysis with the PreScission protease to separate the laccase from the GST-tag. (B) shows the second step after dialysis. The GST binds to the glutathione agarose due to its affinity. The laccase flows through the column (D1-3). After a washing step with PBS (D4), the GST is eluted using an elution buffer containing glutathione (elution). The produced BaLac is shown in the red boxes. Marker: New England BioLabs ^® Blue Protein Standard Broad Range.

After purification of BaLac, the Assay Team worked with the protein and performed an ABTS assay. A visible color change occured and was noted (Fig. 2).

Fig. 2: BL21 ABTS assay performed in pH 4 Phosphate-Citrate buffer

1: Positive (+) control is 40 μM T. versicolor 2: BaLac BL21 sample containing 30 μM. 3: Negative (-) control contains no enzyme. Each well in row contains 250 μM ABTS, while all B rows have ABTS substituted with buffer.

We visualised our raw data and normalized it for a better comparison. The graphs showed what we already expected: the produced laccase BaLac has almost an activity as good as the positive control from T. versicolor.(Fig. 3).

a)

b)

Fig. 3: BL21 ABTS assay analysis.

a) Raw data: Positive (+) control is 40 μM T. versicolor. Negative (-) control contains no enzyme. BaLac (30 μM determined by spectra) shows similar activity to positive control. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates a change from initial to visualize the reaction more clearly, showing the BaLac and T. versicolor sample reacting at similar rates over the course of 4 hours

After those promising results, we continued to implement the HPLC. This demonstrated that the produced laccase BaLac degraded diclofenac.

Fig. 4: Diclofenac elution peak during reaction with BaLac.

Each sample held 3.5 μM BarLac BL21 laccase and 250 μM diclofenac and reactions were halted at assigned time with heat treatment and filtration. Diclofenac retention time was 13.1 minutes and aligned with the literature value of around 14.1 minutes. ¹ No obvious product peaks are visible and the area underneath the initial noise between 2 and 4 minutes does not change. Area underneath the diclofenac peaks appear to decrease at a linear rate until T10 at which time they are consistently below 100 mAU*s. BaLac t30 does not have a peak at 13.1, but instead at 13.4 with an area that corresponds with t60.

So for the laccase BaLac, we can conclude that we not only produced the protein with E.coli BL21, which we see very clear on the SDS gel and the immunoblot, but we also could purify the laccase and showed a very promising activity in both assays. Future assays are of course needed to corroborate these results.

E.coli marLac in pGEX-6P-1

For E. coli marLac, we used the construct BBa_K3589106. We transformed the gene of interest containing vector pGEX-6P-1_marLac in the E. coli BL21 (DE3) strain and we purified it after the production. The protein was visible on the SDS-PAGE relative to the molecular mass marker 75 kDa (BaLac, size 75.8 kDa). An immunological detection confirms this observation (Fig. 5).

Figure 5: Purification of marLac.

The SDS-PAGE shows the samples taken after every step. The fusion protein (marLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). (A) shows the first step of the purification before dialysis with PreScission Protease. The cell lysate was applied to the column. The fusion protein with GST-tag was able to bind to the column, the remaining lysate passed the column (flow through). This was followed by a washing step with washing buffer. Finally, the fusion protein was eluted with elution buffer containing glutathione (eluates 1-6). This was followed by dialysis with the PreScission protease to separate the laccase from the GST-tag. (B) shows the second step after dialysis. The GST binds to the glutathione agarose due to its affinity. The laccase flows through the column (D1-3). After a washing step with PBS (D4), the GST is eluted using an elution buffer containing glutathione (elution). The produced marLac is shown in the red boxes. Marker: New England BioLabs^® Blue Protein Standard Broad Range.

As with BaLac, we did an ABTS assay, but this time we had no colour change in the well that contains marLac (Fig. 6).

Fig. 6: ABTS assay for both marLac and BaLac (strain AD494).

The assay was performed in pH 7 and 4 Phosphate-Citrate buffer respectively to provide the manufactured enzymes optimal conditions. Positive (+) control is both 3570 μM in pH 7 buffer (C1) for marLac and 40 μM T. versicolor in pH 4 buffer (A1) for BaLac. All wells except D1, B3, and B4 contained 250 μM ABTS to act as an ABTS negative control. There was not enough produced enzyme of marLac to run an ABTS negative control. Negative (-) controls contained no enzyme, and tested ABTS against both pH 4 (A4) and pH 7 (B4). A light blue tint in the BaLac well (A3) indicates a reaction over the 4 hour assay.

After that we had a look at the graphs. Both the raw data and the normalized data didn´t show any activity for marLac (Fig. 7).

a)

b)

Fig. 7: marLac ABTS assay analysis.

a) Raw data: Positive (+) control is 3570 μM T. versicolor. Negative (-) control contains no enzyme. The marLac sample had its concentration (23.85 μM) determined by Bradford assay and shows similar activity to negative control, beginning at a slightly higher absorbance likely due to eluting proteins. b) Normalized data (Raw absorbance/Initial absorbance) demonstrates the change from initial to visualize the reaction more clearly, showing the marLac sample reacting at similar rate as the negative sample over the course of 4 hours.

For the laccase marLac we can say the protein was very clear on the SDS Gel and the Western Blot, but we did not measure any activity at all. Future teams should spend time optimizing production and produce a confirmation activity assay.

Mutant BaLac for C. reinhardtii

This part is the composite of the basic parts BBa_K3589107 (mutant BaLac for C. Reinhardtii part 1) and BBa_K3589110 (mutant BaLac for C. reinhardtii part 2). It contains the coding sequence of the mutant form L499F of the laccase from the ascomycete Botrytis aclada (hereafter referred to as BaLac). This part has been codon-optimized for Chlamydomonas reinhardtii. This basic part facilitates the oxidation of a wide variety of substrates, such as phenolic compounds and aromatic amines. The mutated laccase L499F has a high redox potential and shows activity near neutral pH levels. ² This part can be used with the Modular Cloning System for C. reinhardtii .

Design of the constructs

For the recombinant expression of the mutated laccase from Botrytis aclada (BaLac), the Modular Cloning System (MoClo) was used. As such, a Level 2 construct was created that included the coding sequence for BaLac and a 3xHA-tag for detection. The 3xHA tag allows for detection via Immunoblotting, using a primary antibody (anti-HA, mouse) and a secondary antibody (anti-mouse, rabbit) which has a conjugated horseradish peroxidase, which allows detection via Chemiluminescence.
The expression is regulated by the pAR-promotor and the RPL23-terminator. The pAR-promotor is a fusion of the promoters from the proteins HSP70, a heat shock protein, and RBCS2, which is expressed constitutively. ³ For the selection of cells containing the Lv. 2 construct a spectinomycin resistance cassette was used.

Fig. 8: Level 2 construct of cytosolic BaLac (BBa_K3589207).

It includes the constitutive pAR-promotor, the enzyme fused with a 3xHA tag for detection and a spectinomycin-resistance for selection of positive cells.

Cytosolic Expression

First, we needed to proof that C. Reinhardtii is able to produce the enzyme BaLac. As such we transformed the Level 2 construct (BBa-K3589207) into the green algae, specifically the C. Reinhardtii strain UVM4. The colonies to be tested were inoculated in TAP-Medium under mixotrophic conditions.
To see whether the protein was produced, an SDS-Page and Immunoblotting with an anti-HA antibody was conducted as can be seen in the Fig. 9 below.

Fig. 9: Western Blot intracellular BaLac.

It shows the Immunoblot of 12 spectinomycin-resistant colonies, which were transformed with construct (BBa-K3589207) the Level 2 construct BBa-K3589207 (as seen in Fig. 8). 2 µg Chlorophyll were always loaded onto the gel. At ca. 70 kDA (indicated with an arrow) is the expression of BaLac in the transformants 5,9 and 11. A 3xHA-tagged protein was used as positive control, while the recipient strain (UVM4) was used as a negative control.

Sources of Error

Since no activity could be detected for the intracellular BaLac, even though it is expressed, this suggests perhaps the laccase is folded incorrectly. This could be explained by several things. The concentration of BaLac could have simply been to low in comparison to the bought Trametes versicolor laccase. Furthermore, laccases need posttranslational modification, which do not occur in the cytosol.

Engineering Success

Using Chlamydomonas reinhardtii as a chassis was a conscious decision made in hopes to create a multipurpose organism that would not only oxidize certain micropollutants but also digest microplastics (integrating our previous 2019 TU Kaiserslautern team project).

Because of this, we used the promising laccase from Botrytis aclada and implemented it into Chlamydomonas reinhardtii. Though we could not prove activity of the cytosolic BaLac of Chlamydomonas reinhardtii, this is an important first step for research in the area of battling micropollutants. After all, through working with E. coli, we were able to prove that BaLac is a viable combatant to micropollutants.

References

(1) Hahn, V.; Meister, M.; Hussy, S.; Cordes, A.; Enderle, G.; Saningong, A.; Schauer, F. Enhanced Laccase-Mediated Transformation of Diclofenac and Flufenamic Acid in the Presence of Bisphenol A and Testing of an Enzymatic Membrane Reactor. AMB Express 2018, 8 (1). https://doi.org/10.1186/s13568-018-0546-y.

(2) Scheiblbrandner, S.; Breslmayr, E.; Csarman, F.; Paukner, R.; Führer, J.; Herzog, P. L.; Shleev, S. V.; Osipov, E. M.; Tikhonova, T. V.; Popov, V. O.; Haltrich, D.; Ludwig, R.; Kittl, R. Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells. Scientific reports 2017, 7 (1), 13688. DOI: 10.1038/s41598-017-13734-0.

(3) Schroda, M.; Blöcker, D.; Beck, C. F. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. The Plant journal : for cell and molecular biology 2000, 21 (2), 121–131. DOI: 10.1046/j.1365-313x.2000.00652.x.