Identification and characterisation of novel oxidoreductase from Gluconobacter oxydans

Abstract

Plastic production and disposal have excessively contributed to climate change and other environmental problems. Therefore, the green route for the production of recyclable polymers, which is industrially and economically practical, is indispensable. 2,5-furan dicarboxylic acid (FDCA) synthesised from 5-Hydroxymethylfurfural (HMF) is a new alternative biobased resource for polymers. FDCA has been discovered as an excellent carbon-neutral resource for polyethylene furanoate for new-generation packaging, which has superior properties to polyethylene terephthalate (PET).
The synthesis of FDCA from a partially oxidized derivative of HMF at high specificity and yield, facilitated by a microorganism, Gluconobacter oxydans, is of scientific as well as industrial interest. Not only FDCA but the partially oxidized derivative 5-hydroxymethyl-2-furan carboxylic acid (HMFCA), produced by the organism from HMF, can also serve as a monomer for making novel polymers. HMF oxidation to FDCA has different pathways. Hence, acquiring a better understanding of HMF derivative oxidation is also essential for taking the next step toward green plastics.
The involvement of membrane-bound dehydrogenase was suggested in the oxidation of HMF and FFCA by the whole cells of G. oxydans DSM 50049. The whole genome sequence of G. oxydans DSM 50049 was recently revealed. In this study, the primary objectives were to identify the enzymes responsible for the oxidation of furan derivatives and characterise their catalytic activity.
Out of 14 genes screened, 3 enzymes were produced and tested for catalytic activity. The enzymes were annotated as membrane-bound aldehyde dehydrogenase (mALDH), coniferyl aldehyde dehydrogenase (CALDH), and xanthine dehydrogenase (XDH). mALDH and CALDH oxidised FFCA and HMF to FDCA and HMFCA, respectively. The enzymes catalysed HMF oxidation to HMFCA specifically, and there was no overoxidation to FDCA. Furthermore, the insoluble fraction of mALDH showed complete oxidation of 5 mM FFCA and the soluble fraction of CALDH had 2.4 times higher activity than its insoluble fraction. XDH reduced NAD+ to NADH in the presence of urea, suggesting oxidation/reduction between the enzyme and urea. Studying the unknown reaction of XDH with urea and purifying mALDH and CALDH would be the next challenge.

Popular Abstract

Towards the production of bioplastics: Discovery of new enzymes


Look around you. Plastics are everywhere. Do you know how much impact plastic products can make on climate change? For 1 kg of plastic you use, around 6 kg of carbon dioxide is emitted. 6 kg of CO2 is about the same amount of CO2 emissions as six people exhale in a day. Sustainability including green plastic has drawn significant attention as climate change has become a big global concern and people’s awareness of environmental issues increases. What can offer greener solutions? – microbes.

It is known that the microorganism, Gluconobacter oxydans, converts
5-Hydroxymethylfurfural (HMF, a chemical produced by sugar degradation) to another chemical 2,5-furan dicarboxylic acid (FDCA). Those have been studied in this field for a long time due to their various applications, such as being non-fossil-based building block monomers. This means that the enzymes from G. oxydans produce chemicals that can produce bioplastics.

Moreover, other important chemicals are produced throughout the process of FDCA production from HMF. Biopolymers made from those compounds are stronger and more temperature resistant than conventional fossil-based plastics. To further improve the bioprocess required for the production of FDCA, it is important to identify the enzymes from G. oxydans that will allow us to take the next step toward green plastic.

Which enzymes from G. oxydans do this reaction? – we don’t know.
So, our goal was to answer these questions.
• Which enzymes from G. oxydans are producing the chemicals for bioplastic?
• How do they look like and how do they work?

Here is how we did:
From previous research, we narrowed down and listed up the candidate enzymes (computationally). The genes identified were amplified from the G. oxydans genome. Finally, they were transferred, and the enzymes were produced in E. coli -to facilitate the handling and production of the target enzymes-. The activity of the enzymes was examined on the target compounds and analysed for the production of FDCA. Furthermore, the 3D structure of the enzymes was estimated using a programme (in a similar way to how AlphaFold works) to look deeper into why they work on certain compounds and not others, and for the future to help further improve the activity of those enzymes for industrial applications.

And…
Finally, we were able to identify two new enzymes that have excellent activities on our target compounds under study (like HMF). They are more open questions that remain to be answered. It is interesting to know how fast those enzymes are, and how to further improve the process to be acceptable on an industrial scale. More discoveries await!

Master’s Degree Project in Molecular Biology General 45 credits 2023
Department of Biology, Lund University

Advisor: Rajni Hatti-Kaul
Division of Biotechnology, Department of Chemistry, Lund University

• author: Kawano, Riko
• supervisor: Rajni Hatti-Kaul
• organization: Degree Projects in Molecular Biology
• course: MOBN02 20221
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9113571
• date added to LUP: 2023-04-18 11:33:07
• date last changed: 2023-04-18 11:33:07