Characterisation and Harnessing of 5-Hydroxymethylfurfural Metabolism in Pseudomonas umsongensis GO16 for the Production of 2,5-Furandicarboxylic Acid

In the search for biobased alternatives to traditional fossil plastics, 2,5-furandicarboxylic acid (FDCA) represents a potential substitute to terephthalic acid (TPA), a monomer of the ubiquitous polyester, polyethylene terephthalate (PET). Pseudomonas umsongensis GO16, which can metabolise TPA and ethylene glycol (EG), can also oxidise 5-hydroxymethylfurfural (HMF), a precursor to FDCA. The enzymes involved in the oxidation to FDCA, PsfA and PsfG, were identified and characterised. Deletion of FDCA decarboxylase HmfF involved in the conversion of FDCA to furoic acid, and subsequently to a central metabolic intermediate, 2-ketoglutarate, allowed for the accumulation of FDCA. GO16 ΔhmfF cells were grown on glycerol, TPA, EG or mock PET hydrolysate, and the catalyst was then used for the biotransformation of HMF to FDCA. When TPA was used as a growth substrate and to power the biotransformation, the transport of 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) into the cytoplasm represented a rate-limiting step in HMF oxidation. De-bottlenecking transport limitations through in trans overexpression of the HMFCA transporter (HmfT) along with the PsfA aldehyde dehydrogenase and PsfG alcohol dehydrogenase allowed 100% conversion of 50 mM HMF to FDCA within 24 h when TPA, EG or mock PET hydrolysate were used to grow the biocatalyst and subsequently to power the biotransformation. This expands the repertoire of valuable products obtained from engineered P. umsongensis GO16 in the strategy to bio-upcycle post-consumer PET.