Engineering of 1,4‐Butanediol and Adipic Acid Metabolism in <i>Pseudomonas taiwanensis</i> for Upcycling to Aromatic Compounds

The overwhelming amount of plastic produced is an unprecedented challenge for humanity due to the lack of end-of-life solutions for heterogeneous plastic wastes. One possibility is feedstock recycling of mixed plastics and complex polymers with subsequent biological funnelling and upcycling. Major depolymerisation products of common plastics such as polyurethanes, polyesters and polyamides include aliphatic dicarboxylic acids or diols such as adipic acid (AA) and 1,4-butanediol (BDO), which can be metabolised by engineered Pseudomonas putida strains. However, the spectrum of upcycled compounds that can be produced from these monomers is still limited. Therefore, we extended the substrate spectrum of an aromatics-overproducing Pseudomonas taiwanensis strain to AA and BDO. Adaptive laboratory evolution (ALE) followed by genome sequencing was used to identify and reverse engineer key growth-enabling mutations. In this context, we observed a conflict between the dual objectives of fast growth on AA and efficient aromatics production, which materialised in the form of mutations in the ribosomal protein-encoding gene rpmE. These mutations promote faster growth on AA at the cost of aromatics production. In contrast to P. putida KT2440, knockout of the repressor gene psrA regulating expression of genes involved in β-oxidation had no positive effect on growth of P. taiwanensis on AA. Evolution for growth on BDO revealed several point mutations that affect expression of multiple oxidoreductases, with an identified key role for the dehydrogenase encoded by PVLB_10545. This dehydrogenase likely catalyses the initial oxidation of BDO, thus substituting for PedE, which is present in P. putida but absent in P. taiwanensis. Integration of RpcTAL into the Tn7 site enabled de novo production of 4-coumarate with a yield of 14.4% ± 0.1% (Cmol/Cmol) from BDO and 11.5% ± 0.3% (Cmol/Cmol) from AA. Thereby, the potential of these P. taiwanensis strains for upcycling plastic hydrolysates to value-added compounds was successfully demonstrated.