Engineering adipic acid metabolism in Pseudomonas putida.

Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant β-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h, reaching a final biomass yield of 0.27 ± 0.00 g g. In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts.