Abstract
Polyethylene terephthalate (PET) is one of the most widely circulated single-use plastics, notably in the form of plastic containers for food and beverages. Poor recycling of this plastic contributes to global plastic pollution and there is a need for alternative solutions for dealing with PET waste. To biologically degrade and assimilate PET plastic, I isolated, identified and sequenced seven different bacteria able to grow on the PET monomer terephthalate (TA) as the sole carbon source. The isolates were ranked according to their growth on TA as follows: Rhodococcus pyridinivorans HM1, Pseudomonas vancouverensis HM2, Pseudomonas umsongensis GS, Pseudomonas reinekei SH, Ralstonia insidiosa TS, Ralstonia insidiosa GS1, and Acinetobacter schindleri LK1. Three isolates, R. insidiosa TS, R. insidiosa GS1, and A. schindleri LK1, showed impaired growth on TA consistent with the lack of tph genes in their genomes. Some of the strains were genetically modified to express the thermostable esterases TfCut2, TCur, and LCC active against PET. To this end, all enzymes were recombinantly expressed in their active conformations in E. coli and in P. umsongensis GS both from a replicative plasmid (pSEVA238) and from a transposon allowing for chromosomal integration. The activity of the secreted enzymes was confirmed on polycaprolactone (PCL) and further on amorphous PET films at different temperatures. When the tested enzymes were constitutively expressed in the P. umsongensis GS, TCur outperforms LCC and TfCut2. Moreover, constitutively expressed enzymes show better hydrolysis on PCL than the same enzymes expressed from the plasmid vector in this same isolate. PET degradation assays showed that TCur hydrolysed 10% of partially crystalline PET film when used in-vitro (Goodfellow) five times more than LCC and three-fold more than TfCut2. The weight-loss of the polymer in an in-vivo degradation correlated with an increase in the optical densities of the culture, likely due to the use of the released monomers as growth substrates by the engineered P. umsongensis GS expressing the enzymes constitutively. These results show that PET can be hydrolysed by environmental bacteria secreting esterases, although further research is needed to turn PET into a reliable feedstock for microbial transformations using whole-cell catalysts.