@misc{9239675,
  abstract     = {{The transition towards a sustainable bio-based economy requires efficient conversion of renewable biomass into high-value platform chemicals. One such compound is 2,5-furandicarboxylic acid (FDCA), a key precursor for the production of polyethylene furanoate (PEF), a promising alternative to petroleum-derived plastics such as Polyethylene terephthalate (PET). The oxidation of 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FDCA can be catalysed by HMF oxidoreductase (HmfH), a flavin-dependent enzyme originating from Cupriavidus basilensis. This thesis aimed to improve the catalytic performance of HmfH towards FDCA production through random mutagenesis and to evaluate complementary enzyme systems involved in the pathway. A mutant library of the hmfH gene was generated using error-prone PCR and cloned into expression vectors using both Gibson Assembly and Golden Gate Assembly. While Gibson Assembly resulted in low cloning efficiency, Golden Gate Assembly succeeded in successfully creating and transforming mutant library with over 470 variations. A 2,4- dinitrophenylhydrazine (DNPH) based colorimetric assay designed to measure the production of the intermediate 5-formyl-2-furancarboxylic acid (FFCA) was used to screen for mutant activity. The mutants showed a huge degree of variance in the apparent activity of the enzyme. Further HPLC verification and analysis could not confirm activity, with the reason most likely being difference in lysis methods causing WT and mutants to have different results and difference in growth conditions. Expressing hmfH along with hmfT was found to increase the production of FDCA compared to hmfH alone or psfG along with hmfT, with the presence of hmfT increasing transport of furanic compounds and hence availability to the enzyme.}},
  author       = {{Kartha, Shantanu}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Engineering of HMF Oxidoreductase (hmfH) for Enhanced Oxidation of 5-Hydroxymethyl-2-furancarboxylic Acid (HMFCA) to 2,5-Furandicarboxylic Acid (FDCA)}},
  year         = {{2026}},
}