Recently, a new field of science has emerged called Synthetic Biology, which aims to apply engineering principles (for example, the use of modular components, and a "design-build-test-modify" approach to improvement) to the development of biological systems for useful purposes. One major target in Synthetic Biology is the creation of genetically modified microorganisms, to produce valuable chemical substances economically, in high yield and with low environmental impact, or to carry out beneficial chemical transformations such as neutralization of pollutants in waste water. To create these organisms, it is often necessary to introduce a set of new genes (encoded in DNA sequence) and assemble them in specified positions within the organism's long intrinsic DNA sequence ('genome'). The genetic techniques currently available for this 'assembly' task are still quite primitive and inadequate, and gene assembly is considered to be a serious bottleneck in the work leading to the development of useful microorganisms. The first main aim of our proposed research programme is to establish a sophisticated new methodology for this gene assembly process which will achieve a step-change in the speed and efficiency of creating new microorganism strains. For this purpose we will adapt a remarkable group of bacterial enzymes called the serine integrases, whose natural task is to carry out this kind of genetic rearrangement but which have hitherto been underused as tools for Synthetic Biology. We will design rapid, robust and efficient ways of making gene cassettes that can be slotted in (using serine integrases) to any one of a number of different specified positions ('landing pads') in genome DNA. By doing this we can assemble collections of genes to order within a particular microorganism. Furthermore we can choose where to place the genes in the genome and in what order, and replace any individual parts with different versions. This permits much easier optimization of complex genetic systems than is currently possible. Using our new methods we intend to engineer microbial cells to make next-generation biofuels, to make chemicals for the plastics industry by microbial fermentation instead of by using fossil fuel, and to synthesise new antibiotics. A second major target in Synthetic Biology is to make 'smart cells' that can respond in clever ways to external signals (for example, light, high temperature, or a chemical in their environment), or that can 'remember' if they have been exposed to a particular signal and how many times. These smart cells could thus be switched on to perform a useful function only when we need it, or could be programmed to carry out an ordered series of tasks, rather like the wash-rinse-spin-dry cycles of a washing machine. The serine integrase-based tools that we will create for gene assembly lend themselves to the construction of simple yet highly effective intracellular devices for detecting and counting signals. So a second part of our programme is to show the way to the design and construction of these memory devices, and prove that they can work in the way we envisage.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

The chemical and pharmaceutical industries are currently reliant on petrochemical derived intermediates for the synthesis of a wide range of valuable chemicals, materials and medicines. Decreasing petrochemical reserves, and concerns over increasing cost and greenhouse gas emissions, are now driving the search for renewable and environmentally friendly sources of these critically needed compounds. This project aims to establish a range of new manufacturing technologies for efficient conversion of biomass in agricultural waste streams into sustainable sources of these valuable chemical intermediates. The UK Committee on Climate Change (2018) has highlighted the importance of the efficient use of agricultural biomass in tackling climate change. The work undertaken in this project will contribute to this effort and help the UK government achieve its stated target of 'net-zero emissions' by 2050. The new approaches will be exemplified using UK-sourced Sugar Beet Pulp (SBP) a renewable resource in which the UK is self-sufficient. Over 8 million tonnes of sugar beet is grown annually in the UK on over 3500 farms concentrated in East Anglia and the East Midlands. After harvest, the beet is transported to a small number of advanced biorefineries to extract the main product; the sucrose we find in table sugar. SBP is the lignocellulosic material left after sucrose extraction. Currently it is dried (requiring energy input) and then sold as a low-value animal feed. SBP is primarily composed of two, naturally occurring, biological polymers; cellulose and pectin. Efficient utilisation of this biomass waste stream demands that applications are found for both of these. This work will establish the use of the cellulose nanofibres for making antimicrobial coatings and 3D-printed scaffolds (in which cells can be cultured for tissue engineering and regenerative medicine applications). The pectin will be broken down into its two main components: L-arabinose and D-galacturonic acid. The L-arabinose can be used directly as a low-calorie sweetener to combat the growing problem of obesity. The D-galacturonic acid will be modified in order to allow formation of biodegradable polymers which have a wide range of applications. This new ability to convert SBP into a range of useful food, chemical and healthcare products is expected to bring significant social, economic and environmental benefits. In conducting this research we will adopt a holistic approach to the design of integrated biorefineries in which these new technologies will be implemented. Computer-based modelling tools will be used to assess the efficiency of raw material, water and energy utilisation. Techno-Economic Analysis (TEA) and Life Cycle Analysis (LCA) approaches will be employed to identify the most cost-effective and environmentally benign product and process combinations for potential commercialisation. The results will be widely disseminated to facilitate public engagement with the research and ethical evaluation. In this way the work will support the UK in its transition to a low-carbon, bio-based circular economy.

The chemical and pharmaceutical industries are currently reliant on petrochemical derived intermediates for the synthesis of a wide range of valuable products. Decreasing petrochemical reserves and concerns over costs and greenhouse gas emissions are now driving the search for renewable sources of organic synthons. This project aims to establish a range of new technologies to enable the synthesis of a range of chemicals from sugar beet pulp (SBP) in a cost-effective and sustainable manner. The UK is self-sufficient in the production of SBP which is a by-product of sugar beet production (8 million tonnes grown per year) and processing. Currently SBP is dried in an energy intensive process and then used for animal feed. The ability to convert SBP into chemicals and pharmaceutical intermediates will therefore have significant economic and environmental benefits. SBP is a complex feedstock rich in carbohydrate (nearly 80% by weight). The carbohydrate is made up of roughly equal proportions of 3 biological polymers; cellulose, hemicellulose and pectin. If the processing of SBP is to be cost-effective it will be necessary to find uses for each of these substances. Here we propose a biorefinery approach for the selective breakdown of all 3 polymers, purification of the breakdown compounds and their use to synthesise a range of added value products such as speciality chemicals, pharmaceuticals and biodegradable polymers. It is already well known that cellulose can be broken down into hexose sugars and fermented to ethanol for use in biofuels. Here we will focus on the release of galacturonic acid (from pectin) and arabinose (from hemicellulose) and their conversion, by chemical or enzymatic means, into added value products. We will also exploit the new principles of Synthetic Biology to explore the feasibility of metabolically engineering microbial cells to simultaneously breakdown the polymeric feed material and synthesise a desired product, such as aromatic compounds, in a single integrated process. In conducting this research we will adopt a holistic, systems-led, approach to biorefinery design and operation. Computer-based modelling tools will be used to assess the efficiency of raw material, water and energy utilisation. Economic and Life Cycle Analysis (LCA) approaches will then be employed to identify the most cost-effective and environmentally benign product and process combinations. The project is supported by a range of industrial partners from raw material producer to intermediate technology providers and end-user chemical and pharmaceutical companies. This is crucial in providing business and socio-economic insights regarding the adoption of renewable resources into their current product portfolios. The company partners will also provide the material and equipment resources for the large-scale verification of project outcomes and their ultimate transition into commercial manufacture.

The bacterial surface display of proteins has been successfully employed in the affinity-based screening of cell surface display libraries by means of immobilised beads. Typical combined selection and screening strategies for large libraries use biotinylated target proteins for sequential magnetic separation (MACS) with streptavidin-functionalized magnetic particles followed by fluorescence-activated cell sorting (FACS) of the enriched population or fine affinity resolution (1,2,3). The project aims to develop novel screening systems for protein based therapeutics and diagnostics using bacterial display technology to select for binding affinity and specificity. The system we are proposing to develop features a number of improvements to those which are currently used in research. First, we propose dispensing with magnetic beads and expressing both the target protein and the potential binding candidate proteins on the outside of bacterial cells - the former being magnetized cells, the latter being 'normal' non-magnetized cells; when the two sets of cells are mixed and binding takes place, the resultant conjugates being cells attached to cells rather than cells attached to beads. Second, we propose using an expression system for the candidate binder proteins that can express 'unnatural' proteins - i.e. those comprising fluorinated or brominated amino acids having greater functionalities than their natural, wild-type counterparts. Third, we propose being able to express the fluorescent proteins used in the FACS part of system both extracellularly and intracellularly as required. 1. Samuelson P, Gunneriusson E, Nygren PA, & Ståhl S. Display of proteins on bacteria. J Biotechnol. 2002 Jun 26;96(2):129-54. Review. 2. Ståhl S & Uhlén, M. Bacterial surface display: trends and progress 15, 5, 1997, 185-192 3. Dane KY, Chan LA, Rice JJ, Daugherty PS. Isolation of cell specific peptide ligands using fluorescent bacterial display libraries. J Immunol Methods. 2006 Feb 20;309(1-2):120-9. Epub 2006 Jan 11.