Exploring Natures Silent Pharmacy

Lead Research Organisation: University of Bristol
Department Name: Biological Sciences


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.

Technical Summary

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.

Planned Impact

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.


10 25 50
Title development of CRISPR for manipulation of Aspergillus oryzae 
Description Modifications have been made to the model fungus Aspergillus oryzae by gene knowckout and by CRISPR to make it a better platform for heterologous expression of secondary metabolites 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact not as yet, publication is in progress 
Title fungal genome sequence data 
Description fungal genome sequence data submitted to NCBI, a public repository and open access 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact none as yet 
Description Training in use of yeast-based cloning methods 
Organisation Cardiff University
Country United Kingdom 
Sector Academic/University 
PI Contribution Training visiting PhD student in the use of our yeast-based cloning methods to support expression and analysis of natural products with potential antibiotic activity.
Collaborator Contribution N/A
Impact The research group in Cardiff are still using these methods and exploring other possible antibiotics using these approaches.
Start Year 2019
Description Antibiotics Unearthed, A school visit and workshop exploring the potential for new soil microbes as producers of antibiotic substances. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact a workshop sponsored by the Microbiology Society, culturing soil bacteria and investigating them to see if any were making antibiotic substances. This event was aimed at Y13 students as St Marys School, Calne, Wilts, taking A-level Biology. It demonstrated good microbiological practice, techniques such as serial dilution, bioassays and the like. The activity is ongoing and will likely be repeated. A presentation was included about the challenges of how to combat AMR, and led top lively debate, particularly from some student s interested in pursuing careers in medicine.
Year(s) Of Engagement Activity 2017
Description Training workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Training workshop for researchers from Sub0Saharan Africa - training in methods for cloning complex DNA constructs and hig-throughput cloning methods . Part of a wider GCRF training course.
Year(s) Of Engagement Activity 2019
Description Workshop on Multidisciplinary working - challanges and opportunities 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This workshop was part of a wider GCRF training course. I ran a sseminar about the clallanges and opportunities around multi-disciplinary approaches to research, and in particular challanges around project management in this sort of field. The ausience were young research staff from institues and NGOs from Sub-Saharan Africa and are involved in various aspects of extension work in their home countries.
Year(s) Of Engagement Activity 2019
Description presentation to the MRF-funded DTP in AMR 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presenting the aims and approaches in this project to the annual confrence for the MRF-funded DTP in AntiMicrobial Restistance
Year(s) Of Engagement Activity 2019
Description presented display at National Fungus Day event at FeedBristol, Sept 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact National Fungus Day is facilitated by the Bristish Mycological Society. We had a locally organized day as part of this national event. It was based at Feed Bristol which is an independent farming/gardening cooperative and was open to the public. It was advertised via a number of supporting organisations including various local conservation groups such as Avon Wildlife Trust. I presented display material relating to approaches for the discovery of useful antibiotics and other pharmaceuticals from fungi.
The event was attended by more than 300 visitors of all ages, and I was kept busy all day with conversations about the beneficial uses of fungi and why their conservation might be important as it allows their exploitation in the future for useful products.
It also severed as a useful platform to talk about antibiotics and the ongoing concerns about their use and many useful conversations were had discussing various aspect of this, including dealing with some major misunderstandings about their use in farming..
Year(s) Of Engagement Activity 2018
URL https://www.avonwildlifetrust.org.uk/events/2018/10/06/uk-fungus-day?instance=0