Upcycling of PBAT to 4-coumaric acid by combined enzymatic hydrolysis and monomer conversion with engineered Pseudomonas taiwanensis

Bioplastics present a very promising approach to help solving the plastic crisis. However, it is crucial to establish efficient recycling strategies for these materials to ensure circularity of the plastic economy. Microbial upcycling is an important option for the end of life of more complex polymers or blends as well as mixed plastic waste. We engineered the metabolism of the PBAT monomer 1,4-butanediol in an aromatic-producing Pseudomonas taiwanensis strain with the ultimate goal of upcycling PBAT to valuable aromatic compounds. Growth on 1,4-butanediol was achieved via adaptive laboratory evolution. Subsequent genome sequencing and reverse engineering revealed a mutation upstream of PVLB_10545 encoding a putative ethanol dehydrogenase as essential for growth on 1,4-butanediol. This dehydrogenase likely oxidizes 1,4-butanediol to 4-hydroxybutyrate thus substituting for PedE which is present in P. putida but not P. taiwanensis (1). Adding two further mutations enhanced growth on 1,4-butanediol to ALE-like growth. One of these mutations resulted in an amino acid substitution in a LysR family transcriptional regulator (PVLB_12690), the homologue of which was also mutated in P. putida evolved on 1,4-butanediol (1). After successful reverse engineering, the strain was utilized in combination with two other strains growing on the PBAT monomers adipate and terephthalate and the PBAT hydrolyzing enzyme HiC for a process intensification, in which PBAT was hydrolyzed and the monomers were upcycled to 4-coumarate within one reactor.