The aim of this proposal is to investigate the use of Near and Mid Infrared Spectroscopy (NIRS and MIRS) in monitoring commercial biocatalysis processes. Both IR spectroscopies have been shown to offer improved monitoring capabilities in bioprocessing, especially in bioprocess(fermentation) monitoring. In fermentations, the practical utility of NIR/MIR for monitoring analyte levels in (near) real time is clear, but few studies focus on such spectroscopic techniques in biocatalysis. Those that do, concentrate on qualitative methods to characterise products rather than quantify them or monitor reactions of limited commercial interest. This is surprising, since fermentation fluids are often complex, the recent advances in applying spectroscopic techniques to these challenging processes clearly points to the potential of such techniques in biocatalysis where the matrix tends to be simpler. The use of quantitative IR in biocatalysis could allow development of real time analysis for such reactions, permitting in-process control. Scientific Case Ingenza has been developing amine and amino acid manufacture using biocatalysis from technology established at Edinburgh University. This technology represents a powerful approach to manufacture enantiopure unnatural chiral amines and amino acids, which are high value pharmaceutical intermediates. However, one limiting aspect of the technology development is the analysis of the biocatalytic process, as no current method allows real time monitoring, thus, process improvements are slow using current analytical methods. IR offers significant improvement in biocatalytic processes via enhanced monitoring and in-process control. Initial feasibility studies carried out by Ingenza and Strathclyde University have shown the considerable potential of NIR/MIR in monitoring a robust, economical manufacturing process for L-aminobutyric acid ( L-ABA). The process comprises of a kinetic resolution in which a racemic mixture of DL-ABA is converted to L-ABA and ketobutyric acid (KBA). The unwanted D-enantiomer of DL-ABA is oxidised to imino-butyric acid by a D-amino acid oxidase, subsequently imino-butyric acid rapidly hydrolyses to KBA and ammonia. The L-ABA is easily isolated from the reaction mixture in high yield and excellent entantiomeric excess (e.e). The disappearance and appearance of the two key components (ABA and KBA), is vital to understanding the reaction kinetics, chemical efficiency, and volumetric productivity in this bioprocess. Distinct spectral signatures for each analyte could readily be detected in both IRS. On this basis, the formulation of models capable of predicting the concentrations of ABA and KBA should be possible. Since enantioselective enzymatic oxidation is a route of manufacture for major classes of chemicals, namely amines, amino acids and alcohols IR monitoring is likely to be broad reaching in its application. In addition, all of these classes of compounds are likely to have a strong IR absorbance, due to strong dipole moments that are apparent from the structure. This means in-situ IR monitoring has far reaching potential for biocatalysis monitoring. Accordingly, we wish to investigate the use of such techniques further in industrially important biocatalytic processes, including the amino acid oxidase type reactions described above. The investigation has the potential to be wide ranging in process application since Ingenza operate the kinetic resolution and deracemisation processes as a platform technology across a broad range of amino acids. It will enhance process development by examining what effect critical reaction parameters (e.g. temp, pH, substrate/enzyme loading, etc) have on the overall efficiency and productivity.

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.

The world's oceans contain many different types of cells and viruses which have developed the means necessary to survive under conditions which are very different from those found on land. As a result these cells and viruses contain many proteins and chemicals which are not found in land-based plants, animals, and bacteria. Scientists at Plymouth Marine Laboratory have become experts at finding and growing both bacteria and viruses from the sea. Working with U.K.-based companies Ingenza and Aquapharm Biosciences we plan to use these skills to look for bacteria and viruses which produce particular enzymes which could be used to make new chemicals and medicines which are not normally found in nature. The enzymes could also be used to make existing chemicals in new ways which would be more efficient and less polluting to the environment.

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.

Metals have a finite supply, thus metal scarcity and supply security have become worldwide issues. We have to ensure that we do not drain important resources by prioritizing the desires of the present over the needs of the future. To solve such a global challenge we need to move to a circular, more sustainable economy where we use the resources we have more wisely. One of the founding principles of a circular economy is that waste is an unused feedstock; that organic and inorganic components can be engineered to fit within a materials cycle, by the design, engineering and re-purposing of waste streams. In this fellowship I propose to design and engineer bacteria to repurpose our waste streams for us. I plan to use the new tools and techniques provided by advances in biology to engineer a microbe with the ability to upcycle critical metal ions from waste streams into high value nanoparticles. Certain bacteria have the ability to reduce metal cations and form precipitates of zero-valence, pure metals, as part of their survival mechanism to defend against toxic levels of metal cations. I will adopt the modular approach used in Synthetic Biology alongside iterative design, build and test cycles in order to enhance, manipulate and standardise the biomanufacture of these nanosize precipitates as high value products. With training in life cycle assessment, I will determine the financial benefits for business of adopting biological waste treatment methods with high value resource recovery and I will provide biogenic material to other researchers (academic and industrial) free of charge to encourage user pull for the technology.