Metabolic Engineering of Pseudomonas umsongensis GO16 for the Production of the Bioplastic Monomer, 2,5-Furandicarboxylic Acid

2,5-Furandicarboxylic acid (FDCA) is a biobased analog to terephthalic acid (TPA), a monomer of the ubiquitous plastic, polyethylene terephthalate (PET). FDCA can substitute TPA in PET, producing polyethylene 2,5-furanoate (PEF). However, the use of FDCA for plastic applications is limited by high production costs and poor conversion efficiencies. Biotechnology offers an alternative production route by using more selective biocatalysts, milder conditions, and cheap, waste feedstocks for upcycling potential. Pseudomonas umsongensis GO16, which can natively metabolise the PET monomers ethylene glycol (EG) and TPA as growth and energy substrates, has been used to upcycle these molecules to valuable compounds previously. Building on this ability, this thesis focuses on the engineering of P. umsongensis GO16 as a microbial chassis for the synthesis of FDCA. A precursor of FDCA is 5-hydroxymethylfurfural (HMF). P. umsongensis GO16 possesses an hmf operon, enabling it to utilise HMF as a sole growth substrate, with FDCA as an intermediate. Characterisation of this pathway uncovered key proteins and rate limiting steps. Metabolic engineering by gene deletions and overexpression generated a strain capable of efficient production of FDCA from HMF, even when using cells grown on PET monomers. To generate a strain better capable of tolerating the toxic starting substrate HMF, an adaptive laboratory evolution (ALE) campaign was conducted to uncover novel tolerance mechanisms. Whole genome sequencing of the strains revealed mutations contributing to a phenotype better able to deal with increased starting HMF concentrations. Attempts to improve the performance of P. umsongensis GO16 using a rational strain engineering approach were also performed. Deletion of non-essential traits in P. umsongensis GO16 in a biotechnology context was conducted. Comparing the growth performance of the mutants against the wild type gave mixed performances. Finally, proof of concept gram scale production of FDCA using the best performing P. umsongensis GO16 strain was achieved litre scale in a bioreactor. Purification of the product was achieved through an acid precipitation protocol. Holistically, the results presented herein demonstrate the first case of FDCA production using a biocatalyst derived from PET monomers. This thus represents a novel avenue to produce biobased products from a waste stream, thus contributing to the concept of the circular bioeconomy as society attempts to transition towards a greener future.