Regulation, biosynthesis and mode of action of formicamycins, promising new antibiotics with a high barrier to resistance

Most of the antibiotics used in medicine are made from the natural products produced by soil bacteria called Streptomyces, of which there are thousands of different strains. They were discovered during a golden age of antibiotic discovery between 1940 and 1960. By the 1960s, however, scientists began to rediscover the same species, making the same, known compounds. They gave up searching for new compounds because they thought they had found all the natural antibiotics. In fact, they only discovered the easy to find strains and antibiotics, the so-called low hanging fruit.

Everything changed in 2000 with the sequencing of bacterial genomes. A genome contains all the instructions for the life of the cell, and by reading the instructions for thousands of Streptomyces strains we now know these bacteria have instructions for many more antibiotics than they make in the lab. This means we have only discovered a small proportion of the antibiotics made by these bacteria in their natural habitats. This is good news because we must discover and develop new antibiotics to tackle an alarming number of drug resistant infections. Bacteria can adapt quickly and rapidly become resistant to antibiotics which means most of the antibiotics we previously discovered are no longer working. This is known as AntiMicrobial Resistance (AMR) and the UK government predicts that if we fail to tackle AMR now we will face 'Antibiotic Armageddon' by the 2050s - that antibiotics will no longer be effective and infectious diseases will become a bigger killer than cancer, causing around 10 million deaths worldwide every year. Ten priority areas of action have been suggested, including increasing public awareness and stimulating early stage antibiotic discover, both of which are covered by our project proposal.

To discover new antibiotics, we use genome mining, which means sequencing Streptomyces genomes and then looking for sets of instructions (biosynthetic gene clusters) to make new antibiotics that we haven't seen before. We find and sequence new Streptomyces strains living on insects or in plant roots and we manipulate these bacteria to switch on production of their antibiotics, e.g. by genetically engineering the strains to over-express the biosynthetic genes. We are searching in these environments because they have been previously overlooked, and because there is evidence that Streptomyces strains from these environments have an increased potential for producing new antibiotics.

Our research also allows us to make lots of any new antibiotic, so we can purify it, determine its activity and figure out how it kills bacteria (targets and mechanism of action). This information is essential if the antibiotics are to be developed as drugs. It is also essential to understand how the producing strain is resistant to the antibiotics (which it must be, to avoid suicide) and whether disease causing bacteria can acquire resistance easily. Antibiotics which are easily resisted are not desirable as drug candidates but sometimes we can modify them and make semi-synthetic antibiotics which are more effective.

We used genome mining to identify a new group of antibiotics called formicamycins. They have powerful activity against Gram-positive superbugs like MRSA and VRE and they do not become resistant to formicamycins in the lab. We are working with a synthetic chemist to make new versions that target harder to kill Gram-negative bacteria like E. coli. We also identified the biosynthetic gene cluster and now we need to understand how the genes are regulated and to identify the natural resistance genes. We will use this knowledge to engineer strains to over-produce formicamycins and intermediates in its biosynthesis. Then we will purify large amounts of the molecules for semi-synthesis and to determine targets and modes of action and resistance in disease-causing bacteria. These are essential first steps in determining whether they are good drug candidates.

The formicamycins, and their biosynthetic precursors, the fasmsycins, are produced by the new species Streptomyces formicae, and a single biosynthetic gene cluster (BGC) is responsible for their production. Both groups of compounds are active against MRSA and VRE. Fasamycins bind the active site of FabF and block essential fatty acid biosynthesis to kill Gram-positive bacteria. They also kill Gram-negative bacteria treated with outer membrane permeabilising compounds suggesting they could be modified to enable outer membrane penetration to kill drug resistant Gram-negative pathogens. Formicamycins are even more potent and, importantly, MRSA does not become resistant under laboratory conditions. Despite their linked biosynthesis, fasamycins and formicamycins have very different 3D structures and we propose they have a different cellular target and antibacterial mode of action.

We will determine the regulation of fasamycin/formicamycin production, as well as elucidating the entire biosynthetic pathway. To achieve this, we will utilise CRISPR/Cas9 for genome editing, and ascribe functions to all genes comprising the BGC. This will enable us to produce target compounds at elevated titres for purification and structure elucidation, and for biological activity studies. These will include pathway intermediates and new analogues generated through biosynthetic engineering. Compounds will also be transferred to our collaborator Prof Mike Krische (U of Texas, Austin), a highly experienced synthetic organic chemist specialising in natural products total- and semi-synthesis, who will generate additional derivatives that target Gram-negative bacteria.

Additionally, will identify auto-immunity genes and determine whether the two groups of compounds have different molecular targets and modes of action, and confirm the high barrier to resistance observed for the formicamycins. Such information is critical for the development of any natural product as a potential antibiotic.

The proposed work will generate a range of impacts. We describe who will benefit and how below:

1. INDUSTRY. The techniques we use will be of interest to industry, particularly antibiotic development companies such as Warp Drive BIO and Demuris who use genome mining for antibiotic discovery, and Isomerase Therapeutics who develop new molecules through biosynthetic engineering. We are at the forefront of efforts to use CRISPR/Cas9 genome editing for antibiotic discovery and development in Streptomyces bacteria, in particular with the work we have and will do with S. formicae. To our knowledge no one has used CRISPR/Cas9 editing to the same extent and with the same success in any other Streptomyces strain. Thus, our work will provide a proof of concept for this approach and will also yield new molecules and strains that will be of value to industry. We will disseminate our results through publications and at conferences, including industry focussed meetings, e.g. via the Industrial Biotechnology Alliance (IBA) on the Norwich Research (Hutchings and Wilkinson lead the IBA Anti-infectives theme), the Biotech and Life Science Sector group set up by the New Anglia Local Enterprise Partnership (Hutchings is a member) and through our own industry and knowledge exchange contacts. Hutchings has been Associate Dean for Innovation in Science at UEA since 2013 and Wilkinson is co-Director of the BBSRC Natural Products discovery and Bioengineering Network (NPRONET), and a co-founder of Isomerase Therapeutics, so they are well connected. We will share more widely through social media and press releases.

2. SOCIETY. Impact will be achieved long term by the development of new antibiotics either directly via our work on the fasamycins and formicamycins and indirectly when the techniques we are developing are adopted by others to stimulate early stage discovery. Identifying the molecular target for the formicamycins will allow others to develop alternative chemical classes with activity against this target. Impact will also be achieved through public and policy engagement with our research and education about antibiotics and AMR. Two of our co-supervised PhD students did 3-month internships at Westminster in 2017: Rebecca Devine worked with CMO Dame Sally Davies and Sarah Worsley worked in the Science Policy office. These are useful links and Dame Sally Davies has been highly supportive of our research and public engagement efforts, including a book we published on antibiotics with the Science, Art and Writing Trust (http://www.sawtrust.org/buy-the-books/saw-antibiotics/). We will use these contacts to highlight our work and the work of our colleagues in the UK around AMR. Hutchings and Wilkinson have strong track records in public engagement in schools, through public lectures and at major public science events including exhibits at the Royal Society Summer Science Exhibition 2014, the BBSRC Great British Bioscience Festival 2014, Big Bang Science Fair 2015, Norwich Science Festival 2016 and Latitude Festival 2017. Hutchings won a UEA award in 2015 for his outstanding contribution to public and community engagement and the applicants will both continue to engage widely with the public through all available avenues to talk about their work on antibiotics.

3. UEA and JIC. There are potential economic benefits to UEA and JIC through licensing of materials (strains, constructs, molecules) developed during this project. Intellectual property will be protected and licensed by the Research and Innovation Office at UEA and / or Plant Biotech Ltd at the JIC. Another benefit to our host institutes and the Norwich Research Park will be the publicity gained by our work through communication of results at public engagement events and through the media and social media.