Characterisation and exploitation of a promiscuous non-ribosomal peptide cyclase

The majority of clinically used antibiotics are derived from natural products produced by Streptomyces species and other closely related soil bacteria. These drugs were primarily discovered and introduced into the clinic during a 'golden era' of antibiotic discovery that spanned 1940-1960. The utility of these agents has been eroded over the last half-century due to misuse. As a consequence, there is now an urgent need to discover new antibiotics to treat drug resistant bacterial infections. Growing concerns about resistance to antibacterial agents combined with the failure to find new leads from the screening of large libraries of synthetic compounds has led to a renewed interest in natural products discovery. Unfortunately, the overwhelming majority of microbes have yet to be cultured, and for those that have, only a small fraction of their natural products are produced in the laboratory. Conventional approaches to overcome this problem, typically rely upon time consuming genetic modification of the producing organism or the use of poorly understood 'elicitor' compounds to switch on production. The bottleneck with this approach is the fact that a large amount time can be spent on one biosynthetic pathway whose product could never be produced or is not an antibiotic. Moreover, even if success in activating an antibiotic pathway is achieved, discovery of a lead compound is only the first stage of drug discovery. Many antibiotics are derived from a class of microbial natural products called non-ribosomal peptides. Once an exciting prospect is identified, future development is ultimately dictated by its accessibility. Microbial fermentation rarely provides sufficient compound to move forward and therefore chemical synthesis is typically used to produce the quantity, and importantly, the chemical diversity of analogues necessary for testing the clinical potential of a discovery lead. The problem here is that the vast majority of non-ribosomal peptides are cyclic and cyclisation reactions are typically very problematic and produce a low yield of the final compound. In nature, the cyclisation reaction is carried out by a part of the biosynthetic pathway called a thioesterase domain. We recently identified a novel cyclase enzyme, which is promiscuous with respect to the peptide substrates it cyclises. This is exciting and we want to understand how this enzyme works so we can harness its potential for biotechnology. For example, to improve chemical synthesis of antibiotics. We believe this could ultimately help more medicines reach the clinic, possibly making them less expensive and more widely available.

Surugamides are cyclic octa- and decapeptide antibiotics produced by an unusual non-ribosomal peptide synthetase biosynthetic pathway that lacks a C-terminal thioesterase domain. Instead, cyclisation of the terminal biosynthetic intermediates is carried out by a novel cyclase enzyme named SurE, which turns out to be remarkably widespread within Actinobacteria. The fact that SurE can cyclise up to six octapeptides and a decapeptide means that it is promiscuous and could be used for biotechnology.

We will interrogate substrate utilisation by the SurE cyclase and two related cyclases. To achieve this, we will perform in vitro cyclisation assays using purified cyclase enzyme and a series of peptide substrates that we will chemically synthesise. We will track the reactions by LC-HRMSMS and characterise cyclised products by NMR. This will allow us to deduce any rules that govern substrate utilisation.

The substrate utilisation rules for the cyclase will allow us to generate a platform for the production of cyclic synthetic non-ribosomal peptide antibiotics. The cyclase combined with peptide synthesis will be used to produce two known peptide antibiotics as well as a cryptic antibiotic encoded by a strain of Streptomyces, which we will test for bioactivity against a cross-section of drug-resistant bacterial pathogens. The cryptic antibiotic pathway will also be activated in the producing organism by promoter engineering using CRISPR/Cas9/homology-direct repair expression, which will allow us to compare our synthetically made compound to the natural product.

The cyclase technology we will develop during this project has the potential to provide rapid access to otherwise inaccessible non-ribosomal peptide antibiotics without the need for time consuming genetic manipulation of the producing organism.

WHO WILL BENEFIT FROM THIS RESEARCH?
Longer term, the outputs of this research should benefit both the pharmaceutical industry and society as a whole. Shorter term, this work will be of value to fundamental and applied scientists in academia and industry.

HOW WILL THEY BENEIFT FROM THIS RESEARCH?
The value of the global pharmaceutical industry exceeds £200 billion per year. More than half of all drugs critical for human health and wellbeing are derived from or inspired by natural products produced by bacteria and in particular by Streptomyces species. We have only uncovered ~20% of the biochemical diversity these microorganisms offer. Accessing the remainder of these chemical entities is paramount, for example, to fight the war against antimicrobial resistance. Non-ribosomal peptides comprise a large proportion of these unknown potential drugs and the most efficient way to access them is by peptide synthesis. The problem here is that the vast majority of non-ribosomal peptides are cyclic and cyclisation reactions are very challenging suffer from low yield of the final compound. In nature, the cyclisation reaction is carried out by a part of the biosynthetic pathway called a thioesterase domain. The novel peptide cyclase enzyme we identified can cyclise a wide range of substrates. The deliverables from this project will enable us to understand how the enzyme works so that we can harness its potential for biotechnology. For example, to improve chemical synthesis of antibiotics and other therapeutics. We believe this could ultimately circumvent the frequent stumbling block of not having enough material for clinical trials. Our research could help more medicines reach the clinic, possibly making them less expensive and more widely available.

WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH?
We will disseminate the results of this project to the scientific community through publications and presentations at conferences and workshops. We will publish our data in open access journals when possible in order to increase their availability. The commercial potential of our work will be identified during regular self-assessments of progress and appropriate discoveries will be discussed (with a view to patenting) with Commercialisation Services at the University of Leeds and partner company IP Group Plc. The purpose of IP Group Plc is to bring scientific results from Leeds-based scientists into public use for public benefit. This is an established route within the University, which currently boasts >30 spin-out companies. The potential for future financial links with the industrial sector will be explored through the Astbury Centre for Structural Molecular Biology's Research and Innovation Hub, which is dedicated to pharmaceutical and biotechnology development, and through the High Value Biorenewables Network. The research team will work closely with the University of Leeds Media Relations Communication Team and the Faculty Marketing Team to maximise publicity and press coverage for the high impact papers we expect to publish from this work to audiences outside academia, for example in print (e.g. London Evening Standard, Yorkshire Evening Post, New Scientist), online (e.g. BBC, Daily Telegraph). The PI will ensure the wider public benefit from this work by becoming involved in initiatives to inspire school children to study science including workshops and science fairs coordinated through National Science and Engineering Week, such as Discovery Zone and the Leeds Festival of Science.