Metabolic engineering of Pseudomonas putida KT2440 for upcycling of terephthalic acid into levulinic acid

초록

Terephthalic acid (TPA) is an aromatic dicarboxylic acid that is widely used as a monomer in the production of polyesters such as polyethylene terephthalate (PET) and poly (butylene adipate- co -terephthalate) (PBAT). However, its chemical stability causes TPA to persistently accumulate in the natural environment after plastic degradation and cause secondary pollution. Therefore, we engineered a Pseudomonas putida KT2440 strain capable of utilizing TPA as a substrate to produce levulinic acid (LA), a key intermediate in the synthesis of polymers, pharmaceuticals, solvents, plasticizers, and biofuels. Although P. putida KT2440 possesses robust central metabolic pathways, it lacks the enzymatic steps required for LA biosynthesis. To establish an LA producing pathway from TPA, phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were integrated into the pcaIJ locus and introduced tpaK , tphA , and tphB into a pcaIJ and lvaR knocked out P. putida KT2440 (GP102). The engineered P. putida KT2440 GP102 strain used TPA as the sole carbon source and produced 4.9 mM of LA from 6 mM of TPA after optimization. Under continuous TPA feeding, a maximum of 8.75 mM LA was produced, however, substrate accumulation inhibited cell growth and reduced LA productivity, resulting in 7.44 mM LA at 96 h. To address this limitation, glycerol was supplemented with TPA as a co-substrate, which enhanced bacterial growth and TPA utilization and yielded 25.40 mM of LA at 96 h. This study is the first to report biological conversion of TPA to LA in P. putida KT2440 and demonstrates the potential of TPA as a valuable platform chemical for sustainable bioprocessing. © 2026 Elsevier B.V.

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