Introducing Chlamydomonas reinhardtii as a secretion platform was one of our main goals. Even though Chlamydomonas is a widely used model organism the secretion of recombinant proteins requires more research. This field is even more important when utilizing the green algae for synthetic biology by implementing it in a bioreactor, by doing so the secreted proteins can be easily purified. As a result, a continuous culture can be established with ongoing protein production. Through proving our project idea with valuable data, we hope to introduce this interesting possibility to the iGEM community and the industry.
A fundamental part of our project was to verify the tolerance of our algae to the resulting substrates, ethylene glycol, and terephthalic acid. By performing an expanded toxicity test we were able to prove that the products of PET degradation are non-toxic for Chlamy. Using different substrates concentrations, three technical and three biological replicates the results are highly significant.
Suitability of Chlamydomonas as PET degrader
MoClo and Part Improvement
The modular cloning system (MoClo) is an efficient and reliable assembly method for creating gene fragments in a short time. Thereby MoClo is the optimal base for a successful project because of its modular structure we are able to switch through different secretion signals and tags.
To accomplish a successful expression of our proteins of interest it was necessary to optimize the codon usage according to Chlamydomonas reinhardtii and to insert introns to the coding sequences. Furthermore, we decided to take advantage of the PAR promoter which is a fusion of the endogen promoters HSP70A and RBSC2. By taking all these factors into consideration we observed a high yield of our enzymes.
Design of project collection
Screening for Secreted Proteins
The aim of our project was the secretion of proteins into the culture medium.
Therefore, we decided to use different endogen secretion signals, namely cCA (carbo anhydrase), GLE (gametic lytic enzyme) and ARS (arylsulfatase). When we were first screening for positive transformants, we observed a successful secretion of MHETase but no PETase could be detected. To analyze this further we decided to do fluorescence microscopy, thereby we could observe that the PETase seemed to be stuck in the ER.
Troubleshooting of secretion process
When searching for a solution, we found the possibility of an sp20 tag. This contains repeats of serine and hydroxyproline, whereby hydroxyproline gets glycosylated. Through the posttranslational modifications both of our enzymes could be detected in the supernatant and we even increased the yield of protein. Thus, we reached a milestone for our project realization. We further could confirm the appearance of PETase and MHETase in supernatant by mass spectroscopy.
Enhancing secretion and identification
Another problem occurred when we wanted to analyze the supernatant in search of our proteins, this is why we established several screening protocols. Initially, we used TCA-precipitation but considering the amount of time and loss of proteins we decided to optimize the process. Then we started to lyophilize, despite the possibility to detect positive transformants fast the enriched salts of the culture medium seemed to be problematic, resulting in blurred bands. By adding an additional acetone precipitation, we further improved our protocol to be efficient and fast. So, we were finally able to detect the enzymes and showed their successful secretion. While doing so we created 78 blots and screened several hundred transformants.
Even though getting our enzymes secreted is a great success and a further advancement in introducing Chlamydomonas as a secretion platform for industry, analysis was needed. Only functional enzymes would lead us to a PET degrading chassis. Therefore we did slightly differing experiments all ending in HPLC measurements, where we were checking for reaction products and thereby verifying enzymatic activity. Area peaks for TPA are indicating MHETase activity, while PETase activity can result in BHET, MHET and TPA appearance when degrading PET. Cleaving of BHET leads to MHET. Most of our transformants secretes both PETase and MHETase, so products of PETase are further converted to TPA. In this manner activity for MHETase and PETase against BHET could be shown.
Consequently, our project shows the magnificent possibilities of synbio, as we bridged the gap from Ideonella sakaiensis, a prokaryote living in Japanese landfills, to Chlamydomonas rheinhardtii, a eukaryote who feels at home in the worlds ponds and lakes. This way, we were able to degrade MHET and BHET and we even were able to detect degradation products of PET, which makes for a proof of principle of our biorecycling. Continuation of our research efforts could lead to a new era of plastic management. We are giving waste a value again so that everyone will profit.
Activity of secreted enzymes of Chlamydomonas
Additionally, we transformed E. coli T7 Shuffle with a plasmid containg PETase and MHETase. We expressed the proteins cytoplasmically and checked their acitivity as described above. Thereby we created a positive control for PET digestion and had enzymes available that share the same Amino acid sequence to our Chlamy enzymes but are not posttranslationally modified.
Activity measurements of E. coli