Engineering of HMF Oxidoreductase (hmfH) for Enhanced Oxidation of 5-Hydroxymethyl-2-furancarboxylic Acid (HMFCA) to 2,5-Furandicarboxylic Acid (FDCA)
(2026) KBTM01 20261Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
- 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... (More)
- 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. (Less)
- Popular Abstract
- Plastics nowadays are made primarily out of fossil fuels, which are depleteing at an alarming rate and hence researchers are looking at alternative ways to create plastic using materials available in nature. One such material is 2,5-furandicarboxylic acid (FDCA), which can be used to manufacture polyethylene furanoate (PEF), a bio-based alternative to conventional plastics such as Polyethylene terephthalate (PET), which are used in clothes and bottles.
Materials of biological origin can be converted in to a chemical called Hydroxymethyl furfural or HMF. It is this HMF which is the starting point for the production of FDCA. A key challenge is producing FDCA efficiently and sustainably, as the purely chemical means to do so are quite harsh... (More) - Plastics nowadays are made primarily out of fossil fuels, which are depleteing at an alarming rate and hence researchers are looking at alternative ways to create plastic using materials available in nature. One such material is 2,5-furandicarboxylic acid (FDCA), which can be used to manufacture polyethylene furanoate (PEF), a bio-based alternative to conventional plastics such as Polyethylene terephthalate (PET), which are used in clothes and bottles.
Materials of biological origin can be converted in to a chemical called Hydroxymethyl furfural or HMF. It is this HMF which is the starting point for the production of FDCA. A key challenge is producing FDCA efficiently and sustainably, as the purely chemical means to do so are quite harsh to the environment and very expensive as well. Nature already provides enzymes, chemicals capable of catalysing out the required chemical reactions under mild conditions. In this project, an enzyme called HMF oxidoreductase or HmfH was studied, which can convert HMF into FDCA. The goal was to improve the enzyme through directed evolution – a method that mimics natural evolution in the laboratory by creating thousands of slightly different enzyme variants and screening them for improved performance, i.e. their ability to convert the biomass into FDCA, using a technique called error prone PCR.
From here, 470 variants of the HmfH protein containing bacterial strains were screened to check their ability to catalyse the conversion of a chemical called 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), which is created when converting HMF, to FDCA. No variant HmfH-containing bacterial strain could be conclusively shown to have improvements over the original enzymes, due to differences in experimental conditions. The study also compared different enzyme combinations involved in FDCA production and found that combining HmfH with a transport protein called hmfT significantly improved FDCA formation.
The results show both the challenges of engineering enzymes for sustainable chemical production as well as its potential in contributing to a more sustainable future. While further optimization is needed, this study helps contribute to the research of greener alternative materials and biotechnological process involved in their creation. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/student-papers/record/9239675
- author
- Kartha, Shantanu LU
- supervisor
- organization
- course
- KBTM01 20261
- year
- 2026
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- enzyme, environmentally friendly alternatives, FDCA, HMF oxidoreductase (HmfH) gene, polyethylene furanoate (PEF), sustainable methods, HMF Transporter (hmfT), aldehyde dehydrogenase (psfG), furanic compounds, random mutagenesis, error prone PCR, DNPH, FFCA, directed evolution, Golden gate assembly, biotechnology
- language
- English
- id
- 9239675
- date added to LUP
- 2026-08-11 09:07:35
- date last changed
- 2026-08-11 09:07:35
@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}},
}