Engineering Pseudomonas putida KT2440 for open-loop upcycling of mixed plastics

Current recycling technologies address less than 10% of global plastic waste, necessitating alternative valorization routes. Biological upcycling via enzymatic depolymerization combined with microbial conversion of the resulting monomers offers a promising pathway to transform mixed-plastic waste into valuable alternatives. In this research article, we employed a single engineered Pseudomonas putida KT2440 for simultaneous co-utilization of five plastic monomers including ethylene glycol, terephthalate, adipate, 1,4-butanediol, and l-lactic acid, which can be derived from enzymatic hydrolysis of polyethylene terephthalate (PET), poly(butylene adipate-co-terephthalate) (PBAT), polyester polyurethanes (PUs), and polylactic acid. Continuous fermentation over 21 days with alternating mixed-monomer feeds achieved steady-state growth and complete substrate depletion, yielding adaptive mutations that informed iterative strain improvement. Further engineering enabled the biosynthesis of (R)-3-hydroxybutyrate (R-3HB), and 0.70 g l^-1 R-3HB was produced directly from enzymatic hydrolysates of blended PET, PBAT, and thermoplastic PU. These results establish a viable bio-based approach for upcycling realistic mixed plastics into value-added bioproducts.