14TSB_SynBio P2P: Pentoses to products

Synthetic biology provides extraordinary scope to bring engineering and biological approaches together to manufacture new devices and products. The earliest industrial impacts are expected to be in high value chemicals manufacturing, provided that reliable tools and services can be developed for biocatalyst development. Early success in synthetic biology applied to chemicals will have significant impact on the UK economy. The UK chemical industry comprises a major sector in the UK, with > 8% of the world market. In 2009, the UK had over 3,000 companies generating an annual turnover of around £55bn, and the sector has been growing at about 5% a year. However, the chemicals manufacturing industry is dependent on increasingly expensive oil and gas as the starting materials, and sustainable alternatives are needed. Fortunately, synthetic biology provides new opportunities to use wastes from agriculture, forestry and food processing to manufacture bio-based chemicals and drop-in biofuels. These wastes are rich in sugar polymers (cellulose and hemicellulose) that can be hydrolyzed to produce C5 and C6 sugars. Although the C6 sugars are already used widely as fermentation feedstocks, there are few economically viable uses for C5 sugars. Utilization of C5 sugars would transform the economics of bio-based manufacturing, by forming high value products from almost zero cost wastes. This project aims to develop innovative biocatalytic modules for conversion of C5 sugars to chemicals. Until now, the problem of engineering microorganisms for C5 utilization has employed a whole systems approach, based on metabolic engineering of naturally occurring pathways. Since these pathways are inefficient and the product range is restricted, we will use the engineering concepts of modularization, characterization and standardization to develop new, artificial pathways for C5 sugar conversion to chemicals. We will apply modularization by breaking the metabolic process down into
component metabolic units, to design flexible, interchangeable metabolic modules. These will then be assembled into complete biocatalytic systems to obtain optimum performance in manufacturing. The modules will comprise genes coding for enzymes needed to (a) convert C5 sugars to key metabolic intermediates (isocitrate or 2-oxoglutarate) and (b) convert the intermediates to useful chemical products. We will use a bioinformatics approach to design the most efficient and compatible sets of enzymes to assemble the modules. We will also check the metabolic map of E. coli to identify enzymes that would compete with our metabolic modules, so that we can delete the corresponding genes. We will then clone, express and characterize the enzymes (where literature information is missing), so that we can define and understand their behaviour and function when combined into the modules, and fine-tune the designs. We shall then proceed to construct defined, standardized core modules, and characterize their performance. Next, the modules will be interfaced with accessory metabolic modules to form useful chemical products, providing bespoke microbial systems for chemicals manufacturing. We will demonstrate a bespoke system for production of mesaconic acid, needed by Lucite for methacrylic acid production. This will identify any issues relating to context dependency and predictability, enabling the designs to be fine-tuned for robust, industrial biocatalysis. The new metabolic modules will provide standardized components for the chemicals manufacturing industry, enabling rapid, predictable assembly of new biocatalysts to manufacture chemical products. Modularity will greatly reduce development time for new biocatalytic processes and accelerate the journey to market. Overall, the project will reduce the commercial and technical risk associated with biocatalytic manufacturing.

Ingenza and the University of Nottingham will engineer microorganisms for the utilisation of xylose and its conversion to products of interest by fermentation. We will exemplify the approach by converting xylose to a key intermediate required by Lucite International for the manufacture of monomers using sustainable bioprocessing. Use of xylose, derived from waste lignocellulosic biomass, allows the production of chemicals by fermentation using sustainable raw materials which in no way compete with sugars produced for food use.

The primary impact of this project will be within the chemicals and fuels industry, by providing new tools to construct engineered biocatalysts for utilization of C5 sugars from lignocellulosic waste. Utilizing the hemicellulose fraction of food, agricultural and forestry wastes is a key target for chemicals manufacturers, and our innovative, engineered pathway modules will offer significant advantages over current pentose phosphate pathway-based routes. However, the mere
existence of the modular systems and the strain construction tools is insufficient to ensure large-scale uptake unless offered as part of a complete service package. Therefore, Ingenza will offer modular construction of bespoke biocatalysts as a service to chemicals manufacturers. As such, the project will result in revenue generation and new employment opportunities for the UK both at Ingenza and from new opportunities for bio-based chemicals manufacturing. Mesaconate, the example chosen for demonstration, is a key target for Lucite, one of the largest chemicals manufacturers in the UK, and the global leader in acrylics manufacturing. Manufacturing new, sustainable, bio-based acrylics provides a significant market opportunity, with further benefit to the UK. The route to commercialization will be through Ingenza's tools and services, and through Lucite's exploitation of the mesaconate process, since the industrial partners are much better placed to exploit the results than the academic partner. Ingenza is already a leading service provider for synthetic biology, so this augmentation of their technical services and tools can be exploited immediately. Both Ingenza and Nottingham have fermentation facilities, to allow testing and demonstration of small-scale fermentations from C5 sugars to chemicals. For commercialisation of bio-based acrylics, Lucite has already built a small scale test rig to convert organic acids to methacrylic acid, and full demonstration of the new, upstream mesaconate process will be achieved through the National Industrial Biotechnology Facility operated by CPI, adjacent to the Lucite research facility at Wilton. On success, Lucite will build a pilot manufacturing facility within 2 years, and aim for full-scale production within a further 5 years. IP will be shared between the partners, and UoN will benefit from revenue sharing based on royalties. In addition to economic impact, the link between Nottingham (UoN), Ingenza and Lucite will result in the output of trained people, directly from the project. In addition, Lucite have a rolling programme of 4 CASE studentships at UoN, so additional PhD students will be trained in association with this project. The project will also provide benefit for wider undergraduate, masters and PhD training programmes in the form of case studies for lectures, topics for engineering design projects and subjects for research projects. There is also scope for Ingenza and Lucite to deliver lectures and to cosupervise undergraduate design and research projects. Therefore, the project will have a significant impact on training the next generation of development scientists and plant operators.
The project will result in environmental benefits, via significant reductions in both energy and GHG emissions (>85%) compared with manufacturing methacrylic acid and other chemicals from petrochemicals, and from the ability to use lignocellulosic feedstocks. The delivery of economic and sustainable lignocellulosic chemicals manufacturing is strategically important for the UK and Europe. Production of low cost and sustainable renewable chemicals will create jobs and help meet our renewable obligations and GHG reduction targets. In addition, alternative uses of wood and woody residues will make a significant contribution to the new clean tech economy.