Investigation of Fengycin Mechanism Using Biological, Chemical and Biophysical Tools

Control of plant pathogens is vital for effective crop production and whilst many products currently available for the elimination of fungal pathogens are synthetic chemicals, there are some biological control products ('biofungicides'). One of these is the product Serenade, which is manufactured by the company AgraQuest. This product is a bacterium, Bacillus subtilis QST713, which produces a mixture of bioactive lipopeptides (surfactins, iturins and fengycins). It has been established that the lipopeptides target the membranes of phytopathogens and considerable research effort has enabled a deep understanding of both the biosynthesis and mechanism of action of surfactins and iturins. However, much less is known about fengycin, which is the second most abundant lipopeptide produced by the bacterium. This project aims to deepen our knowledge of the anti-fungal mechanism of fengycin and to generate new fluorinated fengycin derivatives, via a combination of synthetic chemistry and biosynthesis, with improved bioactivity. The research will build on a highly successful collaboration between researchers in Durham (Steven Cobb) and Dublin (Cormac Murphy) who have expertise in synthetic fluorine chemistry and microbial biosynthesis, respectively. The project will employ a PDRA in Durham for the synthetic and biophysical aspects of the work and a PhD student in Dublin to conduct the microbiology/molecular biology experiments. In addition the PhD student (Dublin) will work closely with colleagues in Durham's Biophysical Science Institute, which has the specialist facilities necessary to probe the interaction between the lipopeptide and membrane. This will have the added benefit for the student to gain proficiency in a range of research tools that will boost chances of subsequent employment. The research will be of significant interest to academic researchers involved in natural product biosynthesis and those investigating the antifungal action of fengycins, and to companies that commercialise lipopeptides as biocontrol agents. Furthermore, the new fluorofengycins could open possibilities in the healthcare sector for the treatment of fungal infections.

The lipopeptide fengycin is produced by numerous Bacillus spp. and has considerable antifungal activity; indeed it is partly responsible for the bioactivity of the commercial biofungicide product Seranade. Despite its useful properties, its mechanism of action is not well understood and in this project we will use a combination of synthetic fluorine and peptide chemistry, microbial biosynthesis and biophysical analysis to both probe the interaction of fengycin with biological membranes and generate novel fluorofengycin analogues with improved antifungal activity. Fluorine is a key element in pharma- and agro-chemical compounds, where it can affect the compound's bioactivity, bioavailability and metabolic stability. Whilst the methods to improve the synthesis of small fluorinated molecules are effective, the incorporation of fluorine into larger biological molecules is more challenging. However, it is possible to incorporate smaller fluorinated building blocks, e.g. fluorinated amino acids, into large natural products, such as non-ribosomal peptides, via precursor-directed biosynthesis. Thus, by combining the expertise of the PIs in synthetic fluorine chemistry and biosynthesis, it is proposed to generate a series of new fengcin derivatives in which fluorine is incorporated into the peptide portion and the lipid chain of the molecule. These new molecules will be employed in investigations with model membranes using solid state NMR to shed light on the interactions between them and account for the antifungal activity of fengycin. The bioactivity of the fluorofengycins will be established, and since the composition of the fungal membrane influences fengycin sensitivity it might be possible to tailor the fluorofengycin to target a particular fungus.

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 enhanced knowledge in the fields of synthetic biology, natural products chemistry and bioorganic fluorine chemistry. The project will delivery impact across four main areas: People: The program will deliver multidisciplinary training in the chemical and biological sciences to one PDRA (Durham) and one PhD student (Dublin). The BBSRC 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 recruited researchers if they intend to continue to work in this research field (in industry or academia). The recruited researchers will also have access to a variety of staff/ graduate training courses run by both the University of Durham and University College Dublin, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited researchers have enhanced job prospects upon completion of the program and this will help to enhance both the UK and Ireland's scientific skill base. Both the Cobb and Murphy labs regularly host undergraduates from the UK or Europe through schemes such as ERASMUS. To further support the next generation of scientific researchers we will aim to offer summer placements (1 per year at both institutes) to enable undergraduates to gain real hands on experience of research at the chemical-biology interfaces. The placements will be support by either available local/ institutional schemes or national schemes (e.g. Nuffield). Cobb regularly engages in outreach activities including bringing science to students and teachers in partnership with local schools through; a) direct participation in school lessons ("What do Chemists do?" school kids aged 8-9) and b) educational lectures to teachers as part of a program of University wide engagement with Secondary School Education. Cobb has prior experience in leading various sixth form projects as part of the annual North East Schools Industry Partnership scheme. Students from secondary schools in the North-East of England, accompanied by their teachers, spend a full week undertaking research projects. The aforementioned interactions with local schools will help to enthuse the next generation of scientists (please note that specific outreach actions that the team will engage in are detailed in the Pathways to Impact document). Society: The project will look to develop a greater understanding of fengycin microbial action at a fundamental level. In the longer term this information could play an important role in the generation of new anti-fungal agents which would help to reduce the burden of infection both in the National Health Service (NHS), and, more directly in our everyday lives. Delivering in these areas (while a potential long term impact goal) would have a direct and considerable impact on the health of the UK population. In addition fengycin, is already used in a commercial agrochemical product SerenadeTM. Given this the generated novel fluoro-fengycins could have potential applications in the agro-tech industry, potentially providing new agents to ensure both long term national and global Agriculture and Food Security. Economic: The generation of novel IP will help to develop new markets for commercial exploitation in the UK. In particular the work will be of significant relevance to the numerous SMEs currently working on the developed of new (e.g. NovaBiotics Ltd, Aberdeen) as it will help them to remain internationally competitive. Knowledge: Please see Academic Beneficiaries section.