Elucidating and Engineering Legonmycin Biosynthesis: a framework for heterobicyclic biotransformation

Many currently used drugs that we rely on for the treatment of cancer, bacterial infection, immune disorders and viral infections are either natural products or are derived from natural products. As such natural products still remain the major source of lead compounds for the development of new drugs and diagnostic molecules. However, natural products are often complex in terms of structure and composition making them difficult for synthetic chemists to access and thus develop further. In contrast bacteria can possess an amazing inventory of complex chemistry that organic chemists only dream of. In making new molecules, a key challenge is often to identify plausible new scaffolds or motifs (also known as chemical diversity) which is at the heart of drug discovery. We are going to study two bacterial enzymes that have the capability to convert linear peptide into heterobicyclic molecules under mild reactive conditions. These heterobicyclic ring systems are a common motif in biologically important compounds but there does not have any good way of making them by synthetic chemistry. The use of the bacterial enzymes to accomplish chemical tasks is well established in food industry and the benefits in terms of sustainable manufacturing are well document. However, the application of enzymatic process/ industrial biotechnology processes are still under developed the fine chemical, pharmaceutical and agricultural industries. By working out the catalytic capabilities of the two enzymes we will gain the ability to develop their industrial biotechnological potential. In doing this, we will be able to make novel building blocks (also known as scaffolds) and, access new biologically active compounds.

We propose to investigate the capabilities of two novel enzymes, the thioesterase LgnD-Te and FAD-dependent monooxygenase LgnC from the soil bacterium Streptomyces sp. MA37, responsible for the formation of heterobicycles. These heterobicyclic rings are critical components of many biologically important and industrially relevant molecules. We have made significant progress in understanding the chemical mechanism of LgnC, which is the first step towards harnessing the industrial potential this enzyme. We intend to continue this work and elucidate its mechanism in detail through site direct mutagenesis studies to locate the active site. We will also probe the capacity of this enzyme using modified substrates with structural diversity. From our preliminary result we have begun to understand the mechanism of the thioesterase LgnD-Te and our data indicates the formation of a heterobicyclic ring using a surrogate synthetic substrate. We will advance our initial mechanistic work by using substrates with different stereogenic centres and through site direct mutagenesis to locate its active site. Substrate tolerance is a key feature for a successful industrial biocatalyst and we will synthesize peptide thioesters equipped with different chemical handles (i.e. bromine/chlorine/hydroxyl/amine) and probe the substrate flexibility of LgnD-Te. Furthermore, we will identify new homologues of LgnC and LgnD-Te that possess enhanced kinetic profiles and broader substrate tolerance through genome mining and synthetic gene expression. This will not only give us exquisite control of the biochemical experiments but lead to more diverse chemical scaffolds being accessible thus broadening the molecule library generated for bioactivity screening.

Impact will be delivered by fulfilling the research program's key objective which is to provide stakeholders in various sectors with new bioactive compounds and knowledge in industrial biocatalysis.

The project will deliver impact across four main areas.

People: The program will deliver multidisciplinary training in the chemical and biological sciences to two PDRAs. The UK has identified synthetic biology and industrial biotechnology as key deficits in scientists' training for the future workforce and this program will address that need. The project is at the cutting edge of industrial biotechnology and it will provide excellent training for the PDRAs if they intend to continue to work in this research field (in industry or academia). The PDRAs will also have access to a variety of staff training courses run by both the University of Aberdeen and the University of Durham, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRAs have enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Jaspars are very active in outreach programmes that engage with school children. If funded we will offer summer placements (1-2 per year) to local children to experience research at the chemical-biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage. The Co-I (Cobb) regularly gives educational lectures to teachers as part of a program of University engagement with Secondary School Education. Interaction with local high schools will also help to enthuse the next generation of scientists. In this area the Co-I (Cobb) has prior experience having led various sixth form projects as part of the annual North East Schools Industry Partnership scheme (NESIP) held in the Durham University Chemistry Department. Students from secondary schools in the North-East of England, accompanied by their teachers, spend a full week undertaking research projects.

Society: The work will generate new biologically active molecules and moreover allow their generation in useful quantities using sustainable manufacturing methods (i.e. biocatalysts). This means they can be used in drug development programmes and in screening campaigns for the identification of new agents such as anti-biotics. The generation of new antimicrobial agents has the potential to reduce the burden of infection both in the National Health Service (NHS), and, more directly in everyday life. Delivering in these areas will in turn have a direct and considerable impact on the health of the UK population.

Economic: The generation of novel IP will help to develop new markets for commercial exploitation which help UK PLC to remain internationally competitive in the areas of chemical-biology and industrial biocatalysis. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would expect to transfer technology through service agreements (supply novel compounds) or by technology licensing. We will consider founding our own spin out company as the project develops.

Knowledge: On a fundamental level the work program will deliver exciting new insights into the, biosynthesis of HAs and specifically the mode of action of the novel enzymes, LgnD-Te and LgnC, This new knowledge will be of benefit to researchers both in academia and in industry who are engaged in enhancing biotransformation to access novel molecular scaffolds for biological screening.