Team:TU Kaiserslautern/Improve



Our goal was to fight plastic pollution by using the enzyme, PETase, which is able to hydrolyze polyethylene terephthalate (PET) at low temperatures. To make this process even more efficient, we searched for potential mutations, which could increase the catalytic activity even more.

In this manner, we found the TJUSLS China iGEM team of 2016, who designed various versions of PETase by adding different point mutations and analysed their activity against PET degradation. Their most active enzyme, Mutate M (BBa_K1921003 ), is a PETase with the point mutation S181F, which mediates significantly higher activity.

By testing this PETase (Mutate M) construct in Chlamydomonas reinhardtii, no cytosolic expression could be observed (Figure 1). Only the clone J6, transformed with construct L2J, containing the HA-tagged codon optimized MUT-PETase, shows successfully expression.

To increase the efficiency and rate of cloning of our constructs, we decided to use the Modular cloning system (MoClo). We therefore took advantage of Type IIS restriction enzymes in order to design defined fusion sites, which, in turn, allow one-step-one-pot reactions. Our Mutant PETase (BBa_K3002014) was thus designed to be Type IIS compatible by optimization of the codon usage for Chlamydomonas reinhardtii, the addition of introns RBCS2 1-3 and three point mutations (R280A, S238F and W159H). The mutations, S238F and W159H, narrow the PETase active site to mediate a more productive interaction with PET (Joo, S. et al., 2018). The first mutation, S238F, corresponds to the S181F mutation of the TJUSLS China Team. The amino acid shift between 181 and 238 is due to the addition of the secretion signal protein domain. We introduced an additional mutation, namely R280A, to further improve enzyme efficiency. As arginine seems to inhibit the binding of PET, the exchange to alanine mediates a stronger binding of the enzyme to PET (Austin, H. et al, 2018) . The addition of the three introns of RBCS2 and incorporation of the promoter pAR (BBa_K3002027) allowed for high expression to be achieved (Eichler-Stahlberg et al., 2009). With all these adjustments, we obtained high yield of expressed protein in Chlamydomonas reinhardtii, as well as managing to secrete the PETase through the use of different secretion signals.

Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control.

In conclusion, we improved expression and activitiy of the PETase, BBa_K1921003, by finely modifying its expression in Chlamydomonas and significantly improving its PET degradation efficiency.
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Enhancing secretion and identification and
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