Exploring Natures Silent Pharmacy

Fungi have proven to be an important source of bioactive compounds in the past, with penicillins, cephalosporins and statins amongst the best examples. Recent developments in the ease with which we can sequence the genomes of fungi have revealed that fungi house a hitherto unexpectedly large number of gene clusters which appear to encode pathways for secondary metabolites, yet their chemical products are unknown and have not been evaluated in drug-discovery programmes. This suggests that there are many beneficial products yet to be discovered and exploited and these may include new classes of antibiotic which could be deployed to help combat the ongoing problems with antibiotic resistance.
Based on genome sequence data already available for selected target fungi, plus with generation of such data for other selected species of interest, we will develop a pipeline to quickly catalogue such gene clusters and to then design plasmid vectors to allow their expression in the fungus Aspergillus oryzae, a species which is very amenable to lab and industrial-scale cultivation. The use of a lab-friendly host fungus is necessary because our experience is that these gene clusters are usually cryptic; not usually expressed under laboratory conditions by the native fungus, and with products that cannot be predicted with any degree of confidence from genome data alone. The target fungi are each predicted to contain 40-60 such gene clusters based on what is typical for other fungi.
The plasmid vectors will be constructed in a series of expression cassettes we have already developed and tested, and will be made using a combination of yeast-based homologous recombination cloning, augmented by Gibson Assembly where necessary. This will be achieved using PCR products derived directly from genomic DNA, or where this is not readily achievable, by use of synthetic DNA designed from genome data. Such approaches should be readily scalable for high throughput use if they prove to be successful in our studies.
Our vector sets will allow coordinated expression of up to four genes per plasmid, with four different selectable markers available, meaning we can expect to readily express pathways comprising 16 genes, and could upgrade this system for additional genes should this prove necessary, which is ample capacity for the majority of pathways encountered in fungi.
Transformants of A. oryzae will then be analysed to determine if a new product is produced, and if so, this will be purified by reverse-phase HPLC with analysis by MS and by nmr to elucidate the structure. Milligram quantities will be purified to allow antibacterial assays against a range of clinically-relevant pathogens to determine antibacterial efficacy for each compound. For compound displaying antibacterial properties, each compound will be evaluated against a range of bacteria displaying characterised resistance to antibiotics to quickly eliminate any compounds showing known modes of action or those where resistance is already prevalent.
For products passing this evaluation, we aim to fully characterise the biosynthetic pathway, including isolation of the intermediate stages in their biosynthesis and to identify products suited to further chemical modification to support studies into structure-activity relationships in this group of compounds.
Our aim is to design a production pipeline that will allow us to investigate every candidate gene cluster from an initial group of ten selected fungi. The isolates selected for this study have been chosen to span a range of differing lifestyles, including insect, fungal and plant pathogens, marine fungi and soil fungi. This would help to inform future choice of strains for a wider scale analysis in a second round of screening should there be time available.

Genome sequencing of fungi has revealed an unexpectedly large number of uncharacterised secondary metabolite pathways, particularly amongst ascomycete fungi. These pathways are usually not active during routine culture so are unlikely to have been assessed as part of conventional drug discovery programs. From genome data as a starting point, we aim to develop an expression system to engineer the deliberate over-expression of each cluster either in situ or in heterologous hosts to more readily access their resulting metabolites. This will be achieved by exploiting yeast-based recombination systems to directly clone large segments of DNA from genomic material, coupled with the use of synthetic DNA or PCR-derived fragments to allow refactoring of promoters where necessary to achieve the required expression. The resulting metabolite will then be purified by preparative HPLC and characterised using the full range of NMR methods to give structural data, whilst also profiling against a panel of medically relevant bacterial species including those with known modes of resistance, to assess both antibiotic activity and potentially novel modes of action. This will demonstrate whether there are indeed novel antibiotics yet to be discovered in fungi. Fungal species selected as candidates for analysis span a range of environmental niches, to maximise the range of likely selection pressures and hence metabolite profiles.
Where compounds display the desired antibiotic properties, we will characterise their biosynthetic pathways giving the possibility of genetic intervention at various stages to purify intermediates for subsequent chemical modification or for assisting with structure-activity relationships.
The expression systems developed will be both scalable and readily transferable to other labs to allow higher throughput in the future should this prove to be a successful approach.

This research by its very nature is highly applied, addressing a priority theme of combating the problem of antimicrobial resistance which is of paramount importance for security of health. Should this study generate useful lead compounds to support antibiotic discovery, the greatest impacts of such a study are likely to be between the academic research community and those involved in commercial R&D, specifically those where natural products are commonly deployed, be this in the pharmaceutical industry or those allied with animal health/veterinary medicine. The compounds themselves would be of immediate impact, providing starting materials for full evaluation and exploitation, but also the methods generated could be used to increase the supply of compounds for drug discovery programmes.

Given the status of the pharmaceutical sector in the UK, we are in an excellent position to be able to exploit such development, both supporting employment within this key sector, and in benefiting from the outputs - effective drugs to support the health and well-being of the population. Whilst the focus is on antibiotics, this approach could be equally applied to any area where biological molecules are exploited. This includes all other aspects of medicine, but also the AgChem sector, or alternatively other biotech areas such as detergents, pigments and flavourings: this type of discovery platform could deliver any such molecule.

Because we are also developing the methodologies underpinning the discovery, these approaches are also likely to impact on the wider biotechnology industry where advances in expression of individual genes or whole metabolic pathways in amenable hosts is of benefit in cost-efficient production of high value biological products. Should there be compounds directly from this research that show promise as antibiotics, then these will be made available to companies for further exploitations, but would need to go through the normal series of preclinical and clinical trials, so any direct impact would be at least five or more years down the line, and more realistically, it would be the efficacy of this screening method, which may become adopted by companies and lead to more products for evaluation in years to come. The impacts could be important, a new class of antibiotic would be of both financial and societal importance, generating a strong income stream and hence taxable revenue and sustainable employment within the sector, but equally a means to continue the fight against bacterial infections for human and animal health.

If successful in demonstrating that there is a wealth of yet to be exploited compounds within fungi, this work may also highlight the need for enhanced conservation of such biological resources both in the UK and world-wide. Fungi and other microorganisms are often overlooked when considering biodiversity and environmental protection. If we can demonstrate their potential value, this is likely to lead to a wider appreciation of their importance and hence to enhanced awareness not just in special interest groups such as the "amateur enthusiasts" e.g. Association of British Fungus Groups, but to awareness of these organisms amongst the wider public, and this will naturally lead to more credence from policy makers for this group of organisms.

Guaranteed impacts are on those involved in the research. This provides an opportunity to develop the research skills of postdoctoral and technical staff, giving them training in multi-disciplinary skills that can be deployed in synthetic biology and biotechnology and hence increasing their future employment opportunities. Our labs also host a large number of postgraduate and undergraduate researchers who would also learn from this research. Additionally all the applicants are all involved in research-led undergraduate teaching so impacting on the next generation of science graduates whatever their career destinations.