The aim of the proposed research is to find new enzymes that have potential uses in industry by searching for the genes for these enzymes in the DNA extracted directly from soil, compost or other environments. Enzymes are very useful in biocatalysis which is a sustainable method of making chemicals in industry. If enzymes are used the eventual industrial process can be cleaner and greener as it avoids the use of toxic reagents such as metals needed for many chemical catalysis steps, and often uses water-based systems. Biocatalysis can also replace several steps in a chemical process with one enzyme step due to their selectivity and this has a major effect of saving money and time in the overall process for making high value chemicals such as bioactive compounds in the fine chemical and pharmaceutical industry. We will use a technique called metagenomics to find new enzymes for biocatalysis. Many enzymes are derived from microbial sources and these would normally be found by growing bacteria on agar plates and analysing the enzymes they contain using special assays. However, several years ago scientists studying soil microorganisms found that there was a very large difference between the numbers of bacteria they could grow from a soil sample compared with the numbers they could identify by analysing the DNA from the same quantity of soil. These DNA techniques showed that there were over 1,000 times more bacteria in the soil than can be grown on agar plates. So by using plating and growth techniques to find bacteria for biocatalytic enzymes were are missing over 99.9% of the potential enzymes. A technique called metagenomics was developed by several researchers which started with the extraction of DNA directly from a soil sample and this DNA would potentially contain all the genes of the bacteria including the genes from bacteria that cannot be grown in the laboratory. We will use this metagenomic technique to isolate DNA from soils and other environmental samples. The metagenomic DNA will be sequenced and potential genes for biocatalysis will be searched for using computer based techniques to analyse the metagenome. When we find what could be useful genes we will amplify the gene from a sample of the metagenomic DNA and put the amplified gene into a laboratory bacterium that we can grow in large amounts and test the activity of the new biocatalytic enzyme. We call this overall method Functional Metagenomics. The new biocatalysts will be tested in collaboration with researchers at Almac who use enzymes and chemistry to make pharmaceutical compounds. We will test the range of reactions the new biocatalysts can perform and test the chemicals made. A new concept called enrichment metagenomics will also be investigated where we will enrich for bacteria able to use a specific compound before doing the metagenomics. This has the potential to increase the number of bacteria with the desired biocatalytic enzyme. Another new concept called cDNA metagenomics will be tested where we extract messenger RNA from the sample and convert this into what is known as cDNA. This technique will allow us to look for genes from the microorganisms such as soil fungi that have introns in their DNA. This could enable us to find a hitherto unaccessed pool of new enzymes for biocatalysis.

Chiral amines are prevalent in natural products, which often display potent biological activity. Such chiral amine motifs are also frequently found in pharmaceutical drug compounds and chemical building blocks meaning that the development of environmentally benign and sustainable routes to produce these important motifs is extremely desirable. Nature synthesizes these complex and valuable molecules through the action of highly specialized enzymes. These natural catalysts enable an extremely efficient biosynthesis from simple starting materials, installing functional groups with exceptional levels of selectivity. Chemical catalysts are frequently designed to mimic the action of enzymes and are often capable of achieving impressive selectivity. However, unlike enzymes, processes involving these catalysts usually involve high temperatures, sub-optimal pH, organic solvent and complex purification methods. Enzymes called omega-transaminases (TAs) catalyze the conversion of commercially available or easily accessible starting materials to high-value amines. These biocatalysts require an additional donor molecule to provide the amine functional group. This donor is ultimately converted to a by-product and the desired amine product is formed. However, the reaction is freely reversible and unless this by-product is removed from the reaction, low yields of the desired amine will be isolated, as the enzyme will more readily catalyse the reverse reaction to regenerate starting materials. A number of elegant approaches have been reported which remove this ketone by-product and allow access to appreciable quantities of the chiral amine. These strategies include the addition of expensive enzymes or the use of extremely large quantities of the amine donor in combination with the technically challenging removal of ketone by-products. One such approach, which relies on an extensively modified TA, is currently used for the industrial synthesis of the antidiabetic drug compound, sitagliptin. However, the approach is far from efficient and the development of this heavily modified TA biocatalyst was enormously challenging, highlighting an immediate need for more sustainable strategies for performing these biotransformations and for developing suitable enzyme catalysts. This research will build upon recent work reported in our laboratory that describes arguably the most efficient approach to date for performing biotransformations involving TAs. The success of the approach is due to spontaneous precipitation of the by-product, which cannot regenerate starting materials. This polymer is also highly colored and has allowed the development of an effective high-throughput screening strategy that enables the rapid identification of active enzymes. Our focus now is to optimize the process further and make it more suitable for industrial application. Specifically, low cost amine donor molecules will be used that are spontaneously removed from the reaction in a similar way to our previously reported method. We will also apply a simple high-throughput screening strategy to assist in the genetic engineering of natural enzymes in order to increase the scope of the reactions that they can catalyze and make them suitable for industrial scale synthesis. The enzymes developed in this study will enable cost-effective, sustainable and environmentally neutral methods for the small/medium and industrial scale production of one of the most important compound classes.

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.

Light has been used for centuries to image the world around us, and continues to provide profound insights across physics, chemistry, biology, materials science and medicine. However, what are the limits of light as a measurement tool? For example, we can use light to image single bacteria, but can we also use light to trap a single bacterium, identify the bacterial strain and assess its susceptibility to antibiotics? How can we image over multiple length scales, from single cells to multiple cellular tissue, in order to comprehensively map all the neuronal connections in the brain? Can we use a combination of resonance with the wave nature and momentum of light to measure the forces associated with the natural and stimulated motion of a single neuronal cell, or even the extremely small forces associated with phenomena at the classical-quantum interface? This proposal aims to answer these questions by exploring new and innovative ways in which we can use light to measure the natural world. This research builds on our recent advances in photonics - the science of generating, controlling and detecting light - and in particular will exploit resonant structures and shaped light. These provide us with tools for controlling the interaction of light and matter with exquisite sensitivity and accuracy. We will run three research strands in parallel and by combining their outputs, we aim to address major Global Challenges in antimicrobial resistance, neurodegenerative disease, multimodal functional imaging and next generation force, torque and microrheology. Our work is supported by a suite of UK and International project partners (both academic and industry) who are enthused to work with us and have committed over £0.5M in kind to the programme.