Difference between revisions of "Part:BBa K3589108"

Revision as of 17:39, 27 October 2020

Mutant laccase from Botrytis aclada for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the laccase from the ascomycete Botrytis aclada mutant L499F (B3 – B4). This part is codon-optimized for Chlamydomonas reinhardtii. Combined with a promoter and a terminator, this basic part should mediate the oxidation of a wide variety of substrates including phenolic compounds and aromatic amines. As this part contains the introns 1 – 3 of RBCS2, it perfectly matches the part BBa_K3002027 (pAR promoter A1-B2), resulting in a high expression (Eichler-Stahlberg et al., 2009). To detect the target protein a 3xHA-tag from the Kaiser Collection (BBa_K3002017) is recommended. We recommend using the pAR promoter (A1-A3) (BBa_K3002003).
This part was designed to allow synthesis due to a high GC-level.

Summary of the Results from Team Kaiserslautern 2020 for part BBa_K3589108

• Expression could be achieved in C. reinhardtii when combined with a pAR promotor, a 3xHA-tag and a RPL23 terminator (level 1), as well as a spectinomycin resistance (spec^R, level 2); intracellular expression
• No expression could be achieved in C. reinhardtii when combined with the cCA-secretion signal
• Intracellular protein showed no activity in ABTS-assay

Cytosolic Expression:

We first designed constructs without any secretion signal and transformed them into C. reinhardtii to make sure that the green alga is capable of producing both enzymes. We chose the strain UVM4 ¹ as a recipient.

Fig. 1: Proof for the expression of BaLac in C. reinhardtii.
a) Level 2 MoClo construct containing the coding sequence for BaLac and a 3xHA-tag for detection as well as the pAR-promotor and the tRPL23-terminator. Spec^R is a spectinomycin resistance cassette built from the level 0 parts PPSAD, aadA and PSAD_ter introduced by team TU Kaiserslautern 2019 as part of the Kaiser Collection.
b) Immunoblot of 12 randomly picked spectinomycin-resistant colonies transformed with construct a). They were inoculated in TAP medium under mixotrophic conditions. Total protein samples were analyzed via SDS page and immunoblotting using anti-HA antibody. 2 µg of chlorophyll were loaded onto the gel. Expression of BaLac (~ 70kDa) is visible in transformants 5, 9 and 11. The recipient strain served as a negative control, a 3xHA-tagged protein as a positive control.

To measure the activity of the expressed cytosolic laccases, we performed an ABTS-assay. The assay is based on the change in color which appears when ABTS is oxidized to its corresponding radical cation. We performed this assay in a 96-well plate. A commercially available laccase from the fungus Trametes versicolor served as a positive control, supernatant of the lysate from the recipient strain as a negative control. First, we tried to lyse the cells by vortexing them with glass beads. The supernatant was then used for the assay.

Fig. 2: Activity assay for the cytosolically expressed enzymes BaLac and marLac.
a) Cytosolic level 2 constructs of and BaLac. b) ABTS activity assay performed in a 96-well plate. Absorption was measured at 415 nm. The error bars represent the standard deviation of four independent replicates for each sample. The concentration in the parentheses represents the final concentration of the whole protein in the 96-well plate. For the positive control, 75 µg of commercially available Trametes versicolor laccase was added to the 375 µg/ml of whole cell protein in 200 µl of lysate from the recipient strain. Only one sample was measured for the negative control. Cells were lysed using glass beads. The assay was performed at pH 7. Normalization of the data was done by dividing each absorption value by the initially measured value for that replicate.

The assay allowed to monitor the activity of the commercially available laccase, as documented by the blue line in Figure 2 which indicates that the assay worked. Unfortunately, no activity could be detected for the recombinant protein BaLac when they are localized in the cytosol. We therefore tried different lysis methods, i.e. sonication and freeze and thaw to test if it impacted the amount of protein in the lysate or its activity. With the sonication method, we were able to load up to 500 µg of whole protein into the assay. At this concentration, chlorophyll hindered the measurement because of its own absorption at 415 nm, rendering the method useless. We therefore tried the freeze and thaw method to decrease the amount of chlorophyll in the lysate. The method was successful at reducing chlorophyll levels, giving us the ability to more whole protein into the assay. Unfortunately, we were not able to detect any activity with this method, either.

From these results, we concluded that either the concentration of the expressed laccases was too low or that the enzymes are not active when expressed in the cytosol. The immunoblot in Fig. 1 indicates that the expression levels of both enzymes, especially that of marLac, are very low when compared to the positive control. It is also possible that, when the laccase is expressed in the cytosol, it is inactive because of the lack of essential post-translational modifications, which usually occur during the secretion process, i.e. gylcosylation. Laccases are usually heavily glycosylated. They typically contain 10 – 45 % carbohydrates, depending on their origin. The carbohydrates are believed to play a key role in the stability of the laccases and protect them from proteolysis and inactivation by radicals.2 Since both laccases are not produced by C. reinhardtii natively, it is also possible that chaperones required for the insertion of the copper ions are missing. To check the validity of one of these explanations, the assays could be repeated with concentrated enzymes, e.g., via affinity chromatography. When the results are still negative with a much higher laccase concentration, one can assume that the enzymes are in fact not active when expressed in Chlamydomonas.

Protein Secretion:

For these experiments, we switched to the CC-4533 strain. TU Kaiserslautern’s 2019 iGEM team was able to produce good results with that strain for secreted proteins. It still has parts of its cell wall and is therefore more robust and better suited for its usage in a photobioreactor than the UVM4 strain.
First, we designed four different constructs: two for each laccase. Each construct contained a N-terminal secretion signal from the enzyme carbonic anhydrase (cCA) and an HA-8xHis tag for purification. The constructs pAR-BaLac -SP20-HA-RGS-8His additionally harbor a module consisting of a repeat of 20 serine and proline residues (SP20). This module was reported to strongly increase the secretion of proteins.3 TU Kaiserslautern’s 2019 iGEM team was able to increase the secretion of their proteins significantly when using this module. The other construct, pAR-BaLac -HA-RGS-8His, was designed to verify the results of TU Kaiserslautern’s 2019 iGEM team regarding the positive effect of the SP20 portion on secretion efficiency.

Fig. 3: The laccase cannot be detected in the supernatant when fused to an N-terminal cCA secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag.
a) and b) shows the constructs consisting of a spectinomycin-resistance cassette (Spec^R), the pAR-promotor, the coding sequence for the respective laccase, and the tRPL23 terminator. c) and d) shows secreted proteins precipitated from the medium of twelve independent transformants generated with each of the four constructs. The respective constructs are shown above each immunoblot. 6 mL of supernatant were harvested, lyophilized and resuspended in SDS sample buffer. 15 µL of sample were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. Secreted proteins precipitated from medium of the recipient CC-4533 strain served as a negative control, that of a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin as a positive control. Controls were treated like the samples.

Fig 3 shows that neither BaLac nor marLac could be detected in the supernatant. We therefore lysed the cell pellets of the transformants to see if the proteins are produced but not secreted or if the cells do not produce the proteins at all.

Fig 4: Neither laccase could be detected in the whole cell body when fused to an N-terminal cCA secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag.
a) shows the construct .c) the proteins of the lysed cell pellets were analyzed by immunoblotting. Cell cultures were lysed by boiling them with SDS sample buffer. For the samples and controls, proteins corresponding to 2 µg of chlorophyll were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. In panel c), lysate from a culture expressing BaLac served as a positive control. Note that no dedicated signal for the positive control could be detected, likely due to a weak signal. Since we observed a signal derived from a cross-reaction of the antibody with a protein of around 30 kDa in all samples including the negative control, we can conclude that there was no problem with the detection itself. The recipient CC-4533 strain served as a negative control in both cases.

In the course of the project we screened in total the supernatants of around 100 randomly picked spectinomycin-resistant colonies transformed with the two different constructs for BaLac secretion. The laccase could not be detected in any case.
Since we were able show that BaLac is expressed in the cytosol, we have three possible explanations for our inability to detect them in the supernatant: (i) the expression levels of the enzymes are so low that they cannot be detected via immunoblotting. (ii) the N-terminal cCA-tag does not efficiently drive secretion of the enzymes. (iii) Laccases are not compatible with the secretion pathway in C. reinhardtii, for example because chaperones required for the insertion of the copper ions are missing. The apoproteins lacking these copper ions might be rapidly degraded in the ER. To test the validity of the first explanation, we designed new constructs with the secretion signals ARS and GLE, introduced by TU Kaiserslautern‘s 2019 iGEM team. We also tried going back to a 3xHA-tag instead of the one containing an 8xHis portion. Because expression could not be observed, neither with nor without the SP20-portion of the C-terminal tag, we reasoned that it is not causing the problem in expression. We therefore chose the SP20-3xHA tag. As TU Kaiserslautern’s 2019 iGEM team has shown, the SP20 repeat greatly enhanced the secretion of their extracellular proteins. That’s why we did not want to waive it. Unfortunately, we were not able to screen transformants with these new constructs because of time restrictions.

Verifying Media Copper Concentration was not a Limiting Factor

Meanwhile, we contacted Dr. Dietmar Schlosser, an expert on fungal laccases. Because laccases are multi-copper enzymes, he suggested that we increase the copper concentration in the medium to identify whether copper is a limiting factor for the expression of our laccases. The medium we used before was the revised TAP medium by Kropat et. al.4 The copper concentration in this medium is 2 µM. Dr. Schlosser suggested that we should try a concentration of around 20 µM copper. Back in the laboratory, we made a modified version of the TAP medium containing 20 µM copper.

Fig. 5: Immunoblots of the supernatant from 12 randomly picked spectinomycin-resistant colonies transformed with the respective constructs and grown in TAP medium with 20 µM copper.
a) shows the construct, c) shows secreted proteins precipitated form medium for each transformant. The respective constructs are shown above each immunoblot. 6 mL of supernatant were harvested, lyophilized and resuspended in SDS sample buffer. 15 µL of sample were loaded onto the gel and analyzed via immunoblotting using an HA-antibody. Secreted proteins precipitated from medium of the recipient CC-4533 strain served as a negative control, that of a transformant expressing cCA and SP20-HA-8His-tagged brazil nut 2S albumin as a positive control. Controls were treated equally to the samples. For the positive control, only 5 µL were loaded onto the gel.

With an increased copper concentration in the medium, no signal could be detected for the laccase. From this finding we concluded that copper was not a limiting factor for the expression of BaLac.

Summary and Outlook

Over the course of iGEM, we were able to produce BaLac in the cytosol of the green alga Chlamydomonas reinhardtii. We were not able to detect any activity for the enzyme using an ABTS assay. The enzyme was not expressed when fused to a N-terminal cCA-secretion signal and a C-terminal (SP20)-HA-RGS-8His-tag. An increased copper concentration did not change these results.

To test whether C. reinhardtii can express and secrete laccases, more experiments are required. Transformants of the designed constructs with different secretion signals, i.e. GLE and ARS and/or different tags, i.e. an SP20-3xHA-tag should be screened. Another large obstacle could be the activity of the enzymes. Expression of the laccases does not automatically mean that they show any activity. Prof. Dr. Antonio Pierik, an expert on iron-sulfur proteins explained to us that the incorporation of metallic ions into a protein is not a trivial process. As laccases are multi-copper enzymes, it could be that secreted laccases expressed by C. reinhardtii would not be active at all. A screening for activity should therefore always follow a proof of expression.

(1) Neupert, J.; Karcher, D.; Bock, R. Generation of Chlamydomonas Strains That Efficiently Express Nuclear Transgenes. The Plant Journal 2009, 57 (6), 1140–1150. https://doi.org/10.1111/j.1365-313X.2008.03746.x. (2) Agrawal, K.; Chaturvedi, V.; Verma, P. Fungal Laccase Discovered but yet Undiscovered. Bioresour. Bioprocess. 2018, 5 (1), 4. https://doi.org/10.1186/s40643-018-0190-z. (3) Ramos-Martinez, E. M.; Fimognari, L.; Sakuragi, Y. High-Yield Secretion of Recombinant Proteins from the Microalga Chlamydomonas Reinhardtii. Plant Biotechnol J 2017, 15 (9), 1214–1224. https://doi.org/10.1111/pbi.12710. (4) Kropat, J.; Hong-Hermesdorf, A.; Casero, D.; Ent, P.; Castruita, M.; Pellegrini, M.; Merchant, S. S.; Malasarn, D. A Revised Mineral Nutrient Supplement Increases Biomass and Growth Rate in Chlamydomonas Reinhardtii: A Revised Mineral Nutrient Supplement for Chlamydomonas. The Plant Journal 2011, 66 (5), 770–780. https://doi.org/10.1111/j.1365-313X.2011.04537.x.

Sequence and Features