Bioengineering of next generation lipoglycopeptide antibiotics

One of the major challenges in healthcare is the provision of new antimicrobial agents that can combat antibiotic-resistant pathogens (superbugs), which are widely recognised as a major global threat. Despite this, the majority of antimicrobial agents used today belong to old classes of antibiotics discovered before 1970. Consequently, there is an urgent need for new approaches that can deliver the next generation of antibiotics. To address this, we propose to investigate the biosynthesis and bioengineering of lipoglycopeptide antibiotics of the ramoplanin and enduracidin family.

The lipoglycopeptides are highly potent antibiotics which have considerable clinical potential, with ramoplanin having entered phase III clinical trials. However, these highly complex natural products are very difficult to modify using traditional synthetic chemistry, and are inaccessible through total synthesis on the scale required for drug development. Consequently, efforts to generate modified and improved second-generation lipoglycopeptide antibiotics are severely limited. In this project we will develop alternative biosynthetic engineering approaches to enable the rapid structural diversification of this class of antibiotics, providing access to large numbers of lipoglycopeptide variants with potentially improved antimicrobial activities, for subsequent development with industrial partners. Initially, we will sequence the genome of the most productive enduracidin producer, S. fungicidicus, and use this sequence to optimise production of lipoglycopeptides, by deleting competing pathways and introducing other mutations to further increase product yields. We will also explore key steps in the biosynthesis of lipoglycopeptides, including glycosylation, halogenation and assembly of the fatty acid moiety. The biosynthetic insights will then be used to guide the development of novel biosynthetic engineering methods in the optimised S. fungicidicus host. We will develop bioengineering methodology to alter the glycosylation, halogenation and lipidation patterns, as well as the peptide sequence of hybrid lipoglycopeptides. We will also explore approaches aimed at replacing the lactone peptide core, which is prone to inactivation through hydrolysis, with more stable lactam structures, to increase the longevity in vivo and improve the activity of these important antibiotics.

The large repertoire of new biosynthetic engineering methodologies that we develop will be generic, and so can also be used to generate a wide range of derivatives for other promising classes of antibiotics (e.g. vancomycins and mannopeptimycins), as well as other natural product variants with therapeutic applications, for example new immunosuppressive, antiviral and anticancer agents. Moreover, the methodology could be adopted to produce new herbicides, fungicides and insecticides for agricultural use.

This project also addresses key environmental issues, by providing methods that can lead to the more environmentally benign and sustainable biomanufacture of pharmaceuticals and other bioactive products from renewable resources, via fermentation. This is particularly important given our over reliance on fossil fuels, not just for energy, but also for the petrochemicals used in the synthesis of pharmaceuticals, agrochemicals, and other products that are essential in our lives today. The irreversible depletion of metals and other elements used in synthesis, along with the toxic pollution generated in traditional chemical manufacturing processes, are also major environmental concerns that necessitate the development of alternative approaches, such as those described in this proposal.

It is essential that new approaches are developed that can deliver the next generation of antibiotics, which are urgently required to combat emerging drug-resistant pathogens. This project aims to develop novel biosynthetic engineering methodologies to generate structurally diverse variants of the enduracidin and ramoplanin family of lipoglycopeptide antibiotics, which have entered phase III clinical trials. Despite their high potency and significant clinical potential, the lipoglycopeptide antibiotics are highly structurally complex natural products. Consequently traditional semisynthesis and total synthesis approaches are unlikely to deliver more effective variants. To this end, we will explore the biosynthesis of the lipoglycopeptides, characterising the key enzymes involved in lipoglycopeptide assembly. The new biosynthetic insights will be used to guide the development of bioengineering strategies aimed at altering the glycosylation, halogenation and lipidation patterns, as well as the amino acid sequence of the lipoglycopeptides. The long-term objective is to produce more effective next generation lipoglycopeptide antibiotics, for subsequent development with industrial partners.

The bioengineering methodologies that we develop will be generic and can also be used to engineer a wide range of derivatives for other promising classes of antibiotics (e.g. vancomycins and mannopeptimycins), as well as other natural product variants for alternative therapeutic and agrochemical applications. This project also addresses key environmental issues, by providing methods that can lead to the more environmentally benign and sustainable biomanufacture of pharmaceuticals, agrochemicals, and other products from renewable resources, via fermentation.

The main industrial beneficiaries will be project partners GSK. The collaboration with GSK will involve interactions with the Synthetic Biochemistry team (Stevenage), who aim to develop environmentally sustainable and economically viable processes for producing therapeutic lead compounds and intermediates. We will also collaborate with the Biotechnology & Environmental Shared Services (BESS) group, within GSK manufacturing (Worthing). BESS are involved in the discovery and manufacture of pharmaceutically relevant fermentation products, using a variety of microorganisms including Streptomyces. GSK have begun to use genome sequencing to guide metabolic engineering in Streptomyces, and they will benefit from collaboration through our development of a generic set of methodologies, including genome mining, strain optimisation and pathway engineering, that could be applied within their own research and manufacturing programmes.

There are also other companies in the pharma, biotech and agrochemical sectors that have specific interests in the methodology we propose to develop, these include: Novacta, AZ, Pfizer, Ingenza, Lonza, DSM, BASF, Novartis, Syngenta, Dow, Dr Reddys. These companies will be introduced to this research through natural product synthetic biology workshops, which we will establish as part of this project. The objectives of these workshops will be to foster new collaborations between the broader academic and industrial communities, as well as raising the profile of natural product biosynthetic engineering and synthetic biology research. The workshops can thus benefit a much wider network of researchers, and potentially also policy makers. We will work with University KT staff to secure intellectual property rights for all new inventions we discover. Having secured IP, future development work can take place, and several routes to commercialisation will be explored. This could include partnerships and/or licensing agreements with GSK. In addition to this, we will seek partnerships with other companies through the workshops and other activities.

We will deliver public lectures and write articles for popular magazines aimed at non-specialists, alongside presenting a series of School lectures targeting a younger audience. Our public engagement work will highlight the importance of research in this project, drawing on key topical themes (antibiotics, drug discovery, synthetic biology, sustainability & biomanufacturing). Ultimately, this project can benefit the UK economy through the provision of trained scientists and inventions, which can lead to the creation of wealth and new job opportunities. In the long term, the methods that we develop can be used in the environmentally sustainable biomanufacturing of antibiotics, other medicines, and agrochemicals, which can benefit society by improving both health and the food supply. The UK is already world leading in natural product research and biosynthetic engineering. This project, through its cutting edge research, workshops, industrial collaborations and other activities, will further strengthen the UK's position in this competitive field.