Development of oxidases for synthesis of bioplastics intermediates
Lead Research Organisation:
University of Liverpool
Department Name: Chemistry
Abstract
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.
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.
Technical Summary
The aim of this proposal is to use directed evolution to deliver robust and highly active new enzymes for a bioprocess for the commercial production of FDCA from cellulose-derived HMF. FDCA is a key bio-derived building block for biodegradable plastics such as poly(ethylene furanoate) (PEF) and poly(butylene adipate-co-butylene furandicarboxylate) (PBAF). The new enzymes will be characterised and demonstrated as suitable drop-in replacements for existing metal-dependent enzymes that we have used to develop the bioprocess to date. The existing enzymes (GOaseM3,5 and PaoABC) were difficult to manufacture on scale with consistent activity and GOaseM3,5 requires an ancillary enzyme (HRP). We devised a panel screen for non-metal, flavin-dependent alcohol oxidases and have several promising hits. The most promising hit catalyses all 3 oxidation steps from HMF to FDCA but all with low activity; another hit catalyses the first 2 steps and has higher activity. We will use advanced molecular biology methods for directed evolution of the enzymes and apply a ultra high throughput screening approach for the evolution of these flavin-dependent alcohol oxidases. The screening will involve using Fluoresence Activated Cell Sorting (FACS) to identify improved mutants. Improved genetic variants will be sequenced and sequences used to iteratively tune enzyme activities. Advanced evolved hits will be kinetically characterised and benchmarked against existing enzymes, for which Biome already have techno-economic assessment. New enzymes will be assessed for TRL 3-5 readiness by use as immobilised enzymes and as whole cell systems.
Planned Impact
With the rapid growth of the world's population and increasing consumption of fossil fuels, there is an urgent need to produce chemicals from renewable sources. Biomass is cheap and abundant and can be a potential substitute for chemicals' production. 75% of biomass is composed of carbohydrates so developing efficient approaches to transform biomass derived carbohydrates into value-added chemicals is a high priority.
Stora Enso recently announced a EUR 9 million investment to build a pilot plant for a chemical process producing FDCA from plant-based sugars for the production of PEF. This investment exemplifies the rapidly growing interest in bio-based materials for packaging. Our industrial partner Biome plans to launch a manufacturing process for the bio-based and biodegradable copolymer, PBAF, within 5 years. They are initially planning a 10 kilotonne pa. (pilot scale) continuous FDCA production facility based on ongoing research in order to meet market requirements. This project will help Biome develop a competitive process that is enzymatic rather than chemical and is commercially viable whilst being environmentally sustainable.
The BBSRC NIBB, LBNET commissioned the UKBioChem10 report which identified the top ten bio-based chemicals on which the UK should focus resources for maximum impact. The list was agreed by experts representing the chemicals industry, biotech startups, academia, government, biotechnology consultants and the BBSRC. FDCA, the focus of this research project, was the number 2 chemical on this list, highlighting its importance for the UK economy and the widespread interest in improving its production.
The knowledge exchange and impact team at the University of Liverpool is dedicated to developing relationships with businesses and other external organisations. We will continuously monitor for commercially exploitable information and knowledge arising from this project that can be used in discussions with Biome and other commercial partners. The enzymes we have chosen to develop have been carefully selected for their unique capabilities and freedom to operate within this space. We therefore hope to protect intellectual property surrounding the processes developed using these enzymes.
Stora Enso recently announced a EUR 9 million investment to build a pilot plant for a chemical process producing FDCA from plant-based sugars for the production of PEF. This investment exemplifies the rapidly growing interest in bio-based materials for packaging. Our industrial partner Biome plans to launch a manufacturing process for the bio-based and biodegradable copolymer, PBAF, within 5 years. They are initially planning a 10 kilotonne pa. (pilot scale) continuous FDCA production facility based on ongoing research in order to meet market requirements. This project will help Biome develop a competitive process that is enzymatic rather than chemical and is commercially viable whilst being environmentally sustainable.
The BBSRC NIBB, LBNET commissioned the UKBioChem10 report which identified the top ten bio-based chemicals on which the UK should focus resources for maximum impact. The list was agreed by experts representing the chemicals industry, biotech startups, academia, government, biotechnology consultants and the BBSRC. FDCA, the focus of this research project, was the number 2 chemical on this list, highlighting its importance for the UK economy and the widespread interest in improving its production.
The knowledge exchange and impact team at the University of Liverpool is dedicated to developing relationships with businesses and other external organisations. We will continuously monitor for commercially exploitable information and knowledge arising from this project that can be used in discussions with Biome and other commercial partners. The enzymes we have chosen to develop have been carefully selected for their unique capabilities and freedom to operate within this space. We therefore hope to protect intellectual property surrounding the processes developed using these enzymes.