Tandem Chemical Deconstruction and Biological Upcycling of Poly(ethylene Terephthalate) to ..beta..-Ketoadipic Acid by Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2IIA3IIBIIA1II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, ..beta..-ketoadipic acid (..beta..KA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L ..beta..KA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to ..beta..KA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.