ActinoGEN - Integrating genomics-based applications to exploit actinomycetes as a resource for new antibiotics

EC contribution: : € 9.384.133
Duration: : 60 months
Starting date: : 01/01/2005
Funding scheme: : Integrated Project
Keywords: : antibiotics, actinomycetes, Streptomyces, antibiotic resistance, genomics
Contract/Grant agreement number: : LSHM-CT-2004-005224
Project web-site: : http://www.swan.ac.uk/research/ActinoGEN/

Background:

ActinoGEN is an integrated project aimed at developing novel genomics-based approaches to exploit hitherto overlooked genetic resources for new antibiotics. Drug discovery will focus on (1) accessing new antibiotic biosynthetic pathways from diverse actinomycetes that have yet to be cultured; (2) activating cryptic pathways from well-characterised actinomycetes; and (3) engineering novel hybrid antibiotics by combinatorial biosynthesis. To greatly accelerate the drug discovery process, a parallel strategy will be to engineer generic hosts optimised to produce high antibiotic yields. With the complete genome sequence of the model actinomycete, Streptomyces coelicolor, and mobilization of a pan-European effort to apply newly developed multidisciplinary post-genomic technologies, a holistic understanding of the physiology and regulation of antibiotic biosynthesis is achievable for the first time. This will, in turn, permit rational intervention to engineer generic hosts for high-yield antibiotic production. This synergy of discovery linked to overproduction will place the European biotechnology sector at the forefront of developing much-needed new antibiotics to combat multi-drug resistant pathogens.

Problem:

Multiple drug resistant bacteria are a major threat to human health and a significant burden on already stretched medical budgets. This threat is predicted to increase in severity, and remedial actions of reducing antibiotic use in animal husbandry and limiting current prescribing activities for non-lethal human disease are both unlikely to reduce the danger in the short-term. Of major concern are antibiotic-resistant nosocomial infections. The economic and societal costs of these hospital-acquired infections are enormous: the UK National Health Service has estimated an annual cost of 1.5 billion € for extra patient care and that 5000 deaths result each year. In addition, the incidence of infection by multiple drug resistant strains of Mycobacterium tuberculosis, the causative agent of the tuberculosis, is rapidly increasing, particularly among the disadvantaged in society. Investment in R&D into antibiotic discovery by the major pharmaceutical companies has declined dramatically in the last 15 years as a perception has taken hold that easily obtained natural products may have been fully exploited. Hence conventional screening of natural products for new drugs is no longer considered economically worthwhile. Unfortunately, the downturn in drug discovery has coincided with a dramatic worldwide increase in the incidence of resistance to all the antibiotics currently used in medicine.

Aim:

The aim of this proposal is to combine new functional genomic technologies with chemical analysis in an integrated multidisciplinary approach both to exploit hitherto overlooked genetic resources for new antibiotics and, secondly, develop generic 'superhosts' to produce these new antibiotics in high yields. ActinoGEN proposes three parallel objectives to discover and develop new antibiotics based on exploiting the genetic resources of actinomycetes, hitherto the major source of existing antimicrobials. The first of these is to activate cryptic antibiotic biosynthetic pathways. Recent genome sequencing projects have revealed a genetic potential for actinomycetes to produce many more antibiotics than previously recognised. ActinoGEN will explore how different cryptic pathways can be activated and then determine the structures and activities of the resulting new antimicrobials. The second approach will rely on the discovery of new antibiotic biosynthetic pathways from diverse actinomycetes. The number of actinomycete species that have been isolated to date represents a small fraction of the total in the environment. ActinoGEN will exploit the untapped genetic resource of as yet uncultured species to obtain antibiotic biosynthetic gene clusters that can direct synthesis of new antimicrobials. A third route to new antimicrobials is by combinatorial biosynthesis. Biosynthetic genes from both new and existing pathways will be combined to direct synthesis of new antibiotics with predicted structures. The design of new hybrid molecules will be related to improving antimicrobial activity. A fourth major aim, underpinning the Drug Discovery objectives, is the engineering of generic Superhosts for antibiotic production. A rate-limiting step to developing a new antibiotic is yield improvement. Post-genomic analysis permits, for the first time, a concerted and holistic approach to engineering generic Superhosts for use in the production of high yields of a wide variety of antibiotics. As part of ActinoGEN, this complementary activity is vital to greatly accelerate the discovery and development of new drugs.

Expected and obtained results:

1. The establishment of generic procedures for the activation of cryptic antibiotic biosynthetic pathways:

Manipulating pleiotropic and pathway specific regulators has activated cryptic pathways in S. coelicolor and S. ambofaciens

2. Expression of a variety of heterologous cryptic pathways after their transfer to defined Superhost antibiotic production strains.

Products of cryptic pathways now being over-produced in Superhost strains

3. Optimised expression of new antimicrobials, and engineered variants thereof, derived from activation of cryptic pathways, together with structural analysis and antimicrobial spectra.

Structural elucidation of one 'cryptic' antibiotic now complete

4. The establishment of refined genomic-based procedures for analysis of metagenomes to identify new antibiotic biosynthetic pathways.

Generic methods developed and successfully applied in accessing metagenomic biosynthetic pathways

5. Expression of a variety of metagenomic pathways after their transfer to defined Superhost antibiotic production strains.

Two metagenomic pathways transferred to Superhost strains

6. Optimised expression of new antimicrobials, and engineered variants thereof, derived from metagenomic pathways, together with structural analysis and antimicrobial spectra.

Optimisation of expression underway prior to structural studies

7. Optimised expression of new combinatorial antibiotics, together with structural analysis and antimicrobial spectra:

Success achieved by combinatorial manipulation of biosynthetic pathways to produce several new antibiotic molecules

8. Generic antibiotic production Superhosts derived by rational genomics-driven manipulation of Streptomyces coelicolor.

Very successful exploitation of accumulated genomics data to generate antibiotic over-producing hosts

9. Refined Superhosts strains optimised for production of key new antimicrobials.

Derivative Superhost strains now being used to over-produce new antibiotics derived form several aspects of the ActinoGEN IP

Potential applications:

The development of new technologies for antibiotic discovery and production will benefit European SMEs in the biotechnology sector whose remit is to provide new antibiotics. Application of these new genomics-based procedures and technologies for discovery and exploitation of natural products can provide a platform for a renaissance in drug discovery after 15 years of stagnation in this area. The pharmaceutical world market is estimated to amount to 506 billion € in 2004. Antibiotics represent one of the principal and indispensable groups of pharmaceuticals. Hence the project can help to stimulate significant growth of European biotechnology SMEs. In addition, new antimicrobials discovered in the course of the project can potentially help alleviate the current crisis in treatment of multiple drug-resistant pathogens. New antibiotics can provide treatments of last resort for life-threatening diseases such as tuberculosis and nosocomial infections. The efficacy of new antimicrobials will depend on subsequent rigorous testing for toxicity and side effects. However, even in the case of a product with significant side effects, the compound can provide a lead for the subsequent development of safe but effective derivatives, either by chemical modification or by engineering biosynthetic modifications. Thus, there is the potential for these new antibiotics to make a major impact on healthcare in the EU, both at the level of the individual patient and also on healthcare budgets by reducing treatment times in hospitals.

Coordinator:

Prof. Paul Dyson
Swansea University
School of Medicine
Institute of Life Science
Singleton Park
Swansea, SA2 8PP, Wales, UK
Tel. +44-1792-295667
Fax +44-1792-602280
p.j.dyson@swansea.ac.uk

European Commission