Metabolic Engineering of Pseudomonas putida KT2440 for Valorization of Terephthalic acid to Produce Catechol and Muconic acid

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics, yet its environmental persistence necessitates sustainable end-of-life management strategies. Microbial upcycling of PET-derived monomers, particularly terephthalic acid (TPA), into high-value chemicals represents a promising alternative to conventional recycling. In this study, Pseudomonas putida KT2440 was engineered as a modular bioconversion platform for efficient TPA valorization into aromatic products, including catechol and muconic acid (MA). Introduction of the Comamonas sp. E6 tphBAabc operon combined with deletion of pcaHG in the chromosome of P. putida enabled conversion of TPA into protocatechuate (PCA). However, limited native TPA uptake was found. To overcome this bottleneck, nine TPA transporters were screened, identifying Acinetobacter baylyi ADP1 MucK mut and Rhodococcus opacus TpaK as superior transporters that enhanced TPA assimilation. Using these transporters, catechol biosynthesis was enabled by plasmid-based expression of Enterobacter cloacae aroY and chromosomal deletion of catA1 and catA2 . Notably, the tpaK -expressing strain achieved catechol production of 8.51 mM with an 86.0% molar yield. Extension of the pathway toward MA through plasmid-based expression of aroY, catA1 and catA2 , and chromosomal deletion of catB and catC enabled efficient MA production, reaching 8.45 mM MA with a 93.1% molar yield. In addition, TPA derived from chemically depolymerized post-consumer PET was effectively utilized for catechol production. Furthermore, fed-batch fermentation validated process scalability, achieving 15.04 mM catechol from 16.76 mM TPA. Collectively, this work demonstrates that recombinant P. putida KT2440 can be constructed as a robust chassis for sustainable PET upcycling through implementation of an efficient TPA transporter. • Pseudomonas putida KT2440 was engineered for TPA valorization • Acinetobacter baylyi ADP1 MucK mut and Rhodococcus opacus TpaK were identified as effective TPA transporters • PET-derived TPA was efficiently converted into catechol and muconic acid by engineered P. putida • Fed-batch fermentation demonstrated scalability of TPA valorization