Towards polyethylene terephthalate valorisation into PHB using an engineered Comamonas testosteroni strain

The abundant production of plastic materials, coupled with their recalcitrant nature, makes plastic waste a major challenge as a pollutant. Polyethylene terephthalate (PET) is a polyester formed by polycondensation of terephthalic acid (TPA) and ethylene glycol (EG). This plastic polymer can be completely depolymerized to its monomers using microbial enzymes. In this study, we verified in silico and in vivo that the bacterium Comamonas testosteroni RW31 is able to assimilate TPA and to produce the bioplastic polyhydroxybutyrate (PHB). This bacterium was engineered to heterologously express a fusion of the PET-degrading enzymes FAST-PETase and IsMHETase. We verified that our strain successfully secretes the enzymes and depolymerize PET both in vitro and in vivo, achieving a weight loss of 37.1 % and 0.83 %, respectively. We also studied its capacity to form biofilm. Furthermore, our strain can employ bis(2-hydroxyethyl) terephthalate (BHET), an intermediate of PET degradation, as feedstock to accumulate PHB up to 12.03 % of its dry weight in 14 h. Our findings highlight C. testosteroni RW31 as a promising chassis for synthetic biology strategies aimed at upcycling PET waste.