Development of oxidases for synthesis of bioplastics intermediates

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Due to environmental pressure, there is an urgent need to develop novel, high-performance, biodegradable and sustainable plastics from bio-renewable feedstocks. Plant lignocellulose-derived 2,5-furan dicarboxylic acid (FDCA) has great potential for replacing petroleum-derived chemicals in various plastic polymers, for instance in polyethylene terephthalate (PET). Bio-derived and biodegradable plastics incorporating FDCA, such as poly(ethylene furanoate) (PEF) and poly(butylene adipate-co-butylene furandicarboxylate) (PBAF) are being developed. However, currently there are no large-scale producers of the FDCA monomer which is needed to support large scale production of these potentially high-value polymers. This project addresses the need to a develop novel process for the production of FDCA from biomass which will feed into bioplastic manufacturing pipelines for which there is growing demand.Enzymatic conversions confer advantages over traditional industrial chemical routes as they require less energy and are carried out in milder conditions. Enzymes often permit highly selective production of desired target compounds and deliver a higher level of purity. This is particularly important when producing monomers such as FDCA since impurities can interfere with the polymerisation process. Recent research at Liverpool in collaboration with Biome Bioplastics and Leeds University showed proof-of-concept for a process that uses a combination of 4 enzymes to produce FDCA, some of which were difficult to produce on scale. At Liverpool we identified a single new enzyme (E56) which could replace 3 of these enzymes. While the enzyme is unique in this ability, its efficiency is currently low. We also discovered another new enzyme (E5), with higher activity, but that works for part of the pathway and could be used as a combination with the other new enzyme. This project seeks to employ advanced synthetic biology techniques and state-of-the art equipment to improve the enzymes' capabilities through directed evolution. This technique allows evolution of enzyme catalytic activity in the laboratory and the approach will involve generating and screening a very large number of genetic variants. Having identified new enzymes, we will test their activity and suitability as a drop-in replacement for the previously used enzymes.The project outcomes will include development of improved enzymes that can be used in the production of bioplastic precursors, and establishment of an efficient ultra-high throughput screening platform that can be applied to other enzymes in need of improvement.