Extracellular expression of Ideonella sakaiensis PETase mutant in photosynthetic microalga Chlorella vulgaris

The first PET degrading enzyme was first identified in 2017. Subsequently, various enzyme engineering studies were carried out on PETase enzyme. Three different mutations were determined from two different studies with high activity improvement, and combined within one PETase aa sequence. After being evaluated through in silico modeling and molecular docking analysis, their integration into the encoding nucleotide sequence of PETase enzyme was performed. Subsequently, the newly created PETase DNA fragment was commercially synthesized as a synthetic gene and called as PETase3M. Upon PCR amplification, PETase3M was cloned into the pJET1.2 cloning vector for long term maintenance. The corresponding DNa fragment of PETase3M enzyme was transferred into the expression vector pOPT_cCA_mRuby2_Paromy for expression in microalgae hosts. In this vector, PETase3M DNA fragment was positioned behind the HSP70/RBCS2 fusion promoter system. For this task, native DNA region encoding Red Fluorescent Protein (RFP) in pOPT_cCA_mRuby2_Paromy vector was removed using BglII and EcoRI restriction enzymes, and the DNA fragment of PETaz3M was ligated into this place. In this way, the DNA fragment encoding PETase3M was integrated between HSP70/RBCS2 promoter and the 3' untranslated region (3'UTR), making it ready for transformation into eukaryotic microalgae. Transformation of C. vulgaris microalgae cells with recombinant pOPT_cCA_mRuby2_Paromy_PETase3M vector was performed by a modified electroporation process. PETase3M positive transformants of C. vulgaris 211/11J cells were selected for three consecutive generations on TAP agar containing 50 µg/ml paromomycin. The presence of PETase3M insert within microalgae nuclear genome was confirmed through the amplification of PETase3M gene by PCR. Upon His tag based prufıication, the successful and extracellular expression of recombinant PETase3M in C. vulgaris transformants was confirmed by SDS-PAGE analysis of microalgae culture medium. PET films treated with purified PETase3M and degraded to a certain degree under light microscopy. In conclusion, extracellular and functional production of a PETase3M mutant was carried out in C. vulgaris with high tolerance to extreme conditions for the first time. Thus, the devloped microalgae strain could find extensive utilizations in large-scale bioremediation and recycling applications of environmental PET wastes photosynthetically.