Elucidating and engineering bottromycin biosynthesis

One of the greatest threats to public health in the 21st century is the rise of multi-drug resistant bacterial infections, which has been caused by a shortage in new types of antibiotics, as well as the improper use of antibiotics in medicine and agriculture. This has prompted the World Health Organisation to warn that "the need for action to avert a developing global crisis in health care is increasingly urgent" and the UK's Chief Medical Officer, Prof. Dame Sally Davies, to declare that "we are also not developing new drugs fast enough". One incredibly rich resource for antibiotics are microorganisms that live in soil, and the majority of clinically used antibiotics come from these bacteria. These bacteria have evolved the ability to produce natural products with excellent antibacterial activities, as the ability to kill surrounding bacteria is a big advantage when competing for nutrients.

One compound I am currently researching is an antibiotic called bottromycin. The molecule has some highly unusual structural features and is a very active antibiotic towards dangerous infections like the "superbug" MRSA (methicillin-resistant Staphylococcus aureus), which kills tens of thousands of people worldwide each year. Bottromycin has actually been known for many years but problems with its stability and availability have prevented it from being used clinically. However, its entirely novel structure and mode of action make it highly promising antibiotic of the future. An understanding of bottromycin biosynthesis would provide the information necessary for the pathway to be modified to alter the structure of this antibiotic and increase the amount that can be made.

Natural products are produced by the action of a series of enzymes (proteins), which are encoded by genes (DNA) in the bacterial genome. I have previously identified the genes required for bottromycin production and will now focus on the details of each biochemical step. I will also develop methods to make new bottromycin-like compounds. From a purely scientific perspective, the biochemical steps in this pathway are highly unusual, and an understanding of the enzyme mechanisms will increase our understanding of enzyme function. The experimental techniques involved in this work include the fermentation of bacterial cultures, the purification of enzymes and the analysis of biochemical reactions using mass spectrometry, which determines the mass of a compound and can provide important structural information.

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a class of natural products that have not yet been exploited clinically but do possess significant clinical promise. For example, bottromycin is a structurally unique peptide that possesses potent antibacterial activity towards life-threatening infections, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). It functions by blocking aminoacyl-tRNA binding to the A-site of the 50S ribosome, thus inhibiting bacterial protein synthesis. I have previously identified the biosynthetic gene cluster for bottromycin in Streptomyces scabies, a pathogen that is the causative agent of potato scab and demonstrated that bottromycin derives from a ribosomal precursor peptide. Uniquely, bottromycin originates from the N-terminus of a precursor peptide and it is proposed that a "follower" peptide has an analogous role to leader peptides in other RiPP pathways.

Bottromycin possesses some highly unusual structural features such as an unprecedented macrocyclic amidine and rare Beta-methylated amino acids. This research programme will biochemically characterise the enzyme(s) responsible for macrocycle formation and utilise this protein as an in vitro tool for the assembly of bottromycin like cyclic peptides. A mixture of techniques will be employed, including in vitro enzymology and in vivo co-expression with the precursor peptide. The pathway will also be engineered to generate novel bottromycins. The transcriptional regulation of the pathway will be investigated to inform the rational reprogramming of the pathway to enhance bottromycin yield.

The research programme described in this proposal will increase the understanding of how complex drug-like compounds are constructed from ribosomal precursors. Methods will be developed to rapidly modify an important biosynthetic pathway. The urgent requirement to combat multi-drug resistant bacteria, and the vital role natural products have in a variety of other therapeutic areas, means that the research will benefit a number of parties across academia, industry and government, as well as the wider public.

The multidisciplinary nature of this research means that post-doctoral researchers working on the project itself will develop a wide array of skills in the fields of microbiology, analytical chemistry, enzymology and molecular biology that are highly relevant for working in the biotechnology or pharmaceutical sectors. This will help support a strong knowledge-based UK economy, and assist in maintaining the UK's position as a world-leader in the biological sciences. In addition to the positive intellectual benefits this has, success in these fields generates significant wealth for the United Kingdom through the establishment of biotechnology companies and the creation of high-value pharmaceutical products.

It is feasible that a library of promising antimicrobial compounds will be generated, which could then be studied for their clinical and commercial potential. Routes include establishing a new spin-out company or a partnership/licensing agreement with a biotechnology company or a large pharmaceutical company. The Department of Molecular Microbiology at the John Innes Centre has strong recent track record in the establishment of companies based on commercially promising academic research, such as Novacta Biosystems and Procarta Biosystems.

The discovery of enzymes with novel functions will be of interest to members of the chemical industry that use enzymes for the production of fine chemicals and pharmaceutical ingredients. The generation of complex chemicals using biosynthesis or biocatalysis represents a much more environmentally benign approach to chemical manufacturing. Significant investment is being made in biocatalysis by large UK-based pharmaceutical companies such as GlaxoSmithKline at Stevenage and Dr. Reddy's Technology Centre in Cambridge. Biocatalysis assists in reducing the carbon footprint of chemical manufacture, so provides a widespread environmental benefit.

In the longer term, the identification of novel drug-like compounds and improved methods for ribosomal peptide pathway engineering will have a number of wider beneficiaries. This research will provide medical researchers and the pharmaceutical industry with new chemical scaffolds for therapeutic trials. Ultimately, this research could lead to improvements in the treatment of multi-drug resistant bacterial infections, and thus benefit the health of the general public. The development of novel antibiotics benefits the NHS, where a new antibiotic would increase the efficacy of infection treatment strategies and thus save money.

Finally, the research programme aligns with BBSRC strategic research priority 2: Bioenergy and Industrial Biotechnology, and will therefore be valuable to policy makers. More specifically, elucidating and engineering the pathway of a clinically-promising natural product fits with the BBSRC high priority of the "molecular and cellular basis of key biosynthetic processes and their regulation".