Biosynthesis and mode of action of a new antifungal antibiotic produced by bacterial plant pathogens and rhizosphere bacteria

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

The global human population is increasing and it is estimated to reach around 9 Billion by mid-century. Demand for human and animal food crops will increase in line with this growth rate and our food security will be compromised unless we can significantly increase crop productivity, decrease crop losses due to disease and spoilage - or, preferably, both. Recent research has shown that fungal (and oomycete) plant pathogens play increasingly important roles in causing plant diseases that reduce crop production or lead to spoilage of harvested food crops. Current crop protection methods involve various approaches, including the use of some pesticides made by polluting chemistry that can have undesirable environmental impacts.

This project will involve a study of a novel antifungal molecule(s) that is made naturally by some species of bacteria that live in close association with plants. This naturally-made antifungal antibiotic is lethal to a range of fungi that kill pants, including crop plants, and so it might be useful in preventing or limiting crop diseases without recourse to synthetic toxic pesticides. The genes responsible for the formation of this new natural antifungal molecule have been discovered in a range of bacteria isolated from the environment and a hypothetical pathway to biosynthesis of the active molecule has been suggested.

We will overproduce this new molecule using bacterial genetics and physiology methods and then purify the antifungal to try to determine the chemical structure of the antibiotic using a range of chemical and physical methods. We will study how the new antibiotic is assembled in the bacteria that produce it. We have shown that the antifungal antibiotic can also kill simple yeast (fission yeast) in addition to fungi that cause plant disease. The simple yeast is easy to study using genetics and molecular biology methods and so we intend to exploit this by investigating the cellular target of the new antifungal in yeast cells. We expect this information will be also directly relevant to identifying the nature of the molecular target in the plant pathogenic fungi and this will help us in future to develop different natural and semi-synthetic molecules that may protect crop plants from disease and spoilage - and thereby enhance our food security.

Technical Summary

Fungi and oomycetes are major pathogens of crop plants and agents of post harvest decay that directly challenge our food security while causing tremendous economic losses. Crop protection processes can depend on man-made agrochemicals, but some of these are under threat through legislative changes because of environmental and toxicity concerns. There is therefore a growing need for new antifungals that might be useful in controlling disease and decay in important crops.

We have been investigating production of natural antifungal molecules made by Gram-negative, plant-associated bacteria. We have investigated a new broad spectrum antifungal activity ("matillamide") in strains of Serratia and the bacterial phytopathogen, Dickeya solani. Through a transposon mutagenesis programme coupled with bioassays on various hosts we have defined the gene cluster responsible for this new bioactivity and assessed its distribution in taxonomically related bacteria. In this project we will clone the matillamide production gene cluster in E. coli and overproduce the molecule (either in the heterologous host or by forced overproduction/deregulation in natural producers). Overproduction of the antifungal will enable isolation and purification of the new antifungal antibiotic and assist definition of its chemical structure. We will investigate the biosynthetic cluster in detail and will dissect its organization and the functionality of each gene in the putative biosynthetic operon using engineered in-frame deletion mutants and corresponding genetic complementation assays, to try to define the product assembly pathway. The antibiotic has a broad host range but it does not affect bacteria or nematodes. Its lethality in fission yeast will be exploited to try to define the eukaryotic target for the new molecule. We will make use of the genetics and molecular biology of the experimentally tractable Schizosaccharomyces pombe to identify the mode of action of the new antifungal.

Planned Impact

Who will benefit from this research?
There could be diverse beneficiaries from a productive research project of this nature. These could include crop producers (agriculture), the manufacturers and processors of food and, ultimately, consumers. If successful, this research work could have implications for food security through enhanced crop productivity and reduction in spoilage. The work will also impact on ecosystem health and food sustainability, and, consequently our nutrition and health. The project aspires ultimately to have impacts on food crop productivity (for man and animals) but of course it could have beneficial impacts on non-food crops and environmental/leisure lifestyle aesthetics where plants and trees are also susceptible to seriously impactful fungal pathogens. The research may also have impacts in biofuel and fibre production.

The project involves the analysis of genes encoding production of a new antifungal antibiotic; how this is made in the producer and how this acts to kill fungal plant pathogens. The relevant genes, enzymes and molecules may be potentially exploitable in the manufacture of novel natural products, by semi-synthesis, with agricultural or other value. The research could ultimately create new molecules for more natural control of plant diseases using regimes that may be less ecologically damaging, more sustainable and more eco-friendly than the currently used industrial agrochemicals (increasing numbers of which are under threat now due to restrictive EU legislation). The biosynthetic enzymes investigated in this study could have utility in translational process technologies in chemical and synthetic biology applications e.g. in specific bioconversions or for use in bacterial biorefineries.

How will they benefit from this research?
Crop losses due to fungal pathogens and post harvest decay processes are massive, on a global scale, and they can account for losses of around 10-30% of production - ecologically and agriculturally devastating in some locations. These losses may be significantly exacerbated through climate change at the worst possible time, when we have an increasing global demand for crops to feed a rapidly expanding human population. Our research could underpin better disease control technologies that may decrease crop losses and thereby increase profitability in food production and distribution. Cutting our dependence on potentially toxic xenobiotic agrochemicals is now legislatively pertinent, ecologically sensible and perhaps even beneficial in human and animal health terms. Any possibility of improving UK food security and decreasing use of xenobiotic agrochemicals must be a good impact target. Furthermore, crop disease control in emerging nations would enhance food production and nutritional health and both of these could have considerable impacts on human health and longevity. To realise such translational benefits, this work would eventually need industrial sector partnership and commercial exploitation over a long time scale.

Another area of impact, of course, will be in the training of research personnel employed in the project. The PDRAs will benefit from the development of diverse skills in bacteriology, genetics, chemistry and chemical biology, bioinformatics, and yeast molecular biology - coupled with experience in teaching, science communication and other career enhancing skills through personal development courses.

What will be done to ensure that they have the opportunity to benefit from this research?
As taxpayer-funded research work, our research outcomes will be disseminated to scientists in the public domain by peer-reviewed publications, lectures and posters presented in UK and international meetings. We will publish in open access journals, in line with BBSRC directives to ensure global access to the knowledge outputs we generate. We are very positive about industrial, and other stakeholder, collaborations, wherever possible.

Publications

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Matilla MA (2018) Genome Sequence of the Oocydin A-Producing Rhizobacterium Serratia plymuthica 4Rx5. in Microbiology resource announcements

 
Description This project is still in progress. However, we have defined the genes involved in antifungal biosynthesis and examined their distribution in other bacterial pathogens and bacterial saprophytes. In-frame deletion mutants have been constructed for all biosynthetic genes and biochemical/chemical characterisation of mutants and wild type has been/is being conducted. We have now gone a long way towards solving the final structure for the bioactive molecule (although that is proving to be less straightforward than we had hoped because of the novel chemistry involved). Approaches towards defining the eukaryotic target are in progress and we are also defining various genes involved in antibiotic regulation. Mutants have been made that exhibit enhanced antibiotic production and these are being used to enable elevated production of the bioactive molecule. We have exploited the new bacteriophages that infect the antifungal producer strain for strain engineering.
Exploitation Route If we do finally define the structure then we will pursue IP issues and the possibility of industrial interactions.
Sectors Agriculture, Food and Drink,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Work on the bacterial pathogens, their phages and antibiotics led into a collaborative Agri-Tech Seeding catalyst award working with a UK Biotechnology Company ("Fixed-Phage Ltd", Glasgow, UK) to investigate the immobilisation of virulent phages that kill potato pathogens (Pectobacterium and Dickeya species) for their potential use in phytopathogen detection, ecology and identification (award BB/SCA/Cambridge/17; internal reference RG92070). Over 50 new environmental, virulent phages infecting the potato pathogens were isolated, purified and genetically sequenced to investigate evolutionary relationships. Bacterial host range was assessed and preliminary studies on viral immobilisation using Fixed Phage technology were initiated. The collaboration with the biotechnology company opened up new opportunities for future collaborative projects, for which we hope to acquire funding, so the seeding catalyst provided a useful and important platform to enable that possibility. In addition to the collaborative developments with the Fixed Phage Ltd company, our expertise developed with antibiotics and environmental phages also helped in the development of a new collaborative project on bacteriophages of the commercial mushroom (Agaricus) pathogen, Pseudomonas tolaasii (the causative agent of the commercially important spoilage disease brown blotch of mushrooms). This new IAA award is BB/S506710/1 (internal reference RG96069). The P. tolaasii pathogen can be responsible for diminution of commercial mushroom production efficiencies and has an important deleterious impact on shelf life (and associated consumer acceptability) in the product. The new collaboration (which has just recently started in 2019) developed with G's Mushroom Company (Littleport, Ely) and with FERA (York). The eventual purpose is to try to develop a phage-based biocontrol system that may help diminish brown blotch disease incidence and thereby enhance commercial productivity, and extend product shelf life and consequent consumer acceptability. This project is (March 2019) in progress and, thus far, we have isolated around 50 new environmental bacteriophages that infect the mushroom pathogen. Our aims are to define the phages at a detailed molecular level (genomics) and by electron microscopy. Bacterial pathogen host ranges of the phages will be studied (using mushroom pathogen isolates) and the potential utility of appropriate new phages in biocontrol of mushroom brown blotch will be assessed in the future. This collaborative project strategy with G's Mushrooms and FERA has also been enabled and significantly enriched by our "know-how" that was further developed through previous BBSRC awards: Agri-Tech Seeding Catalyst award (BB/SCA/Cambridge/17; internal reference RG92070; with Fixed-Phage Ltd) and by previous (RG68461), or contemporary (RG80298; RG80298), generous graduate studentship support (RG8461) on antibiotics and phages of plant and animal pathogens.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Education,Environment
Impact Types Societal,Economic

 
Description BB/SCA/Cambridge/17 (Agri-Tech Seeding Catalyst) - Cambridge University internal reference RG92070
Amount £19,850 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2017 
End 02/2018
 
Description Developing new bacteriophage banks for Pseudomonas tolaasii: route to biocontrol of mushroom brown blotch. Cambridge University IAA Internal Reference RG96069
Amount £14,250 (GBP)
Funding ID BB/S506710/1 (Internal Ref RG96069) 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2019 
End 04/2019