A universal tool for rapid functional characterisation of antibiotic production genes in the bacterial genus, Streptomyces

During this project we will develop a genetic tool that will accelerate and simplify the discovery of new antibiotics produced by the bacterial genus Streptomyces. Ever since the golden age of antibiotics in the mid-20th century, streptomycetes have provided the richest source of novel antimicrobial compounds. These natural products include clinically-important antibiotics (tetracyclines, streptomycins, & penicillins), immunosuppressants (FK506/520 & rapamycin) and anti-cancer drugs (doxorubicin). Perhaps the major medical challenge in the 21st century is to develop new antibiotics to combat bacterial antibiotic resistance. Genome sequencing of streptomycetes, coupled with mining for antibiotic biosynthetic genes has revealed great undiscovered biosynthetic potential in this genus and highlights their enormous possibilities for antibiotic development. However, there is a need to develop new genetic tools that will allow the rapid characterization of antibiotic biosynthetic gene function and facilitate exploitation of this biochemical potential. Currently, gene disruption in streptomycetes is a laborious process. We need to speed this process to more rapidly characterise antibiotic biosynthetic genes and so simplify the antibiotic development pipeline. Rapid mutagenesis of biosynthetic genes will allow manipulation of pathways to develop new compounds through genetic engineering. Alternatively gene manipulation of a producing-strain can be used to improve yield and thus commercial viability of desired antibiotics. Taking advantage of gene synthesis technology, we will develop a genetic tool that is universally applicable to all sequenced streptomycetes as well as some related bacteria. This tool will remove the need for laborious gene manipulations that is necessary to characterise antibiotic biosynthesis at the present time. In the first instance, we will synthesise mutagenesis cassettes targeted at the disruption of a number of characterised antibiotic biosynthetic gene clusters. We will demonstrate proof-of-principle of this system in both a model streptomycete, Streptomyces coelicolor, and an industrial strain, the oxytetracycline producer Streptomyces rimosus through the disruption and deletion of known antibiotic biosynthetic genes. Application of our tool to the latter organism will demonstrate the utility of our system in non-model, industrial organisms. We will assess the efficiency with which gene disruption takes place and identify the location and stability of the gene disruption using a range of molecular biological techniques. Finally, we will make the genetic tools developed during this project available to the academic and industrial scientific communities through a biological resource repository following dissemination of results in open access journals so as to achieve the greatest possible uptake of our system for the development of novel antibiotics.

During this project we will develop a genetic tool that will accelerate the discovery of new antibiotics from the bacterial genus Streptomyces, the most prolific natural source of antibiotics. Perhaps the major medical challenge in the 21st century is to develop new antibiotics to combat bacterial antibiotic resistance. Genome mining for antibiotic biosynthetic gene clusters has revealed great undiscovered biosynthetic potential in this genus. However, there is a need to develop new genetic tools that will facilitate exploitation of this biochemical potential. Currently, gene disruption in streptomycetes is a laborious process. We need to expedite this process to more rapidly characterise antibiotic biosynthetic genes and speed the antibiotic development pipeline. Taking advantage of gene synthesis technology, we will develop a genetic tool, based on group II introns, that is universally applicable to all sequenced streptomycetes. At the present time, group II introns are unsuitable for use in streptomycetes and we will synthesise a core-group II intron (targetron) specifically tailored for use in this genus. This tool will remove the need for laborious gene manipulations to generate targeted gene disruptions in non-model organisms. We will add intron insertion sites to the core-targetron targeted at the disruption of characterised antibiotic biosynthetic gene clusters. We will demonstrate proof-of-principle of this system in both the model streptomycete, Streptomyces coelicolor and an industrial strain, the oxytetracycline producer Streptomyces rimosus, through the disruption and deletion of known antibiotic biosynthetic genes and gene clusters. Application of our tool to the latter organism will demonstrate the utility of our system in non-model, industrial organisms. We will assess the efficiency with which gene disruption takes place and identify the location and stability of the gene disruption using a range of molecular biological techniques.

WHO WILL BENEFIT FROM THIS RESEARCH? We will generate outputs that benefit academic researchers working in the characterisation of antibiotic biosynthesis and applied scientists working in the antibiotic industry. Ultimately, we expect to speed the process of antibiotic discovery in conjunction with the advances in genome mining. This will result in reduced costs and development time for the introduction of antibiotics to market; this will benefit the UK pharmaceutical industry in addition to the health care benefits to the general public through combatting bacterial antibiotic resistance. Training of the PDRA in the bacterial genetics that underpin antibiotic biosynthesis will enhance the economic competitiveness of the UK by contributing to the knowledge economy either as an academic or industrial researcher.

HOW WILL THEY BENEFIT FROM THIS RESEARCH? This research will not only benefit streptomycete researchers but also the wider bacterial community through expediting gene functional analysis in this bacterial group. Streptomyces research has generated considerable economic benefits to the UK economy. A recent report (DTZ-BBSRC, April 2012) estimated the impact of Streptomyces research for potential antibiotic sales at £247m pa and better antibiotic productivity could save £44.7m. The antibiotic sector faces competition from overseas generic products and there is increased NHS drug price sensitivity, so even a small increase in antibiotic productivity will have a significant economic impact. The DTZ-BBSRC report also estimated that the value of a reduction in the number of deaths in the UK from MRSA could be worth £100m pa. As well as skills training of the PDRA, this project will benefit the knowledge economy through training of postgraduate, undergraduate and ERASMUS students.

WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT FROM THIS RESEARCH? We will share results by publication in high impact factor Open Access scientific journals. We will also present data at international scientific meetings and engage researchers from other disciplines. Finally we will make the genetic tools freely available to international researchers through sharing our gene disruption system through Addgene (https://www.addgene.org/), a non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. To increase understanding of how this research impacts on society, we will publicize findings through the Strathclyde Public Engagement Forum, the National Coordinating Centre for Public Engagement and the Glasgow Science Centre. Strathclyde is one of the UK's top 10 institutions for licence royalty income (cumulative income of £42m) and has formed over 50 spin-out companies. Strathclyde was 6th in the UK for spin-out formation (2000-2010) with sales of £20m per annum and employing around 500 people. One example is MGB Biopharma Ltd, established in 2009, to develop DNA Minor groove binders, a completely new class of antibiotics. Intellectual property is handled through Research and Knowledge Exchange services (RKES) and we will ensure that research findings are shared with RKES for potential commercialization. In addition to participation in Strathclyde's staff development program, the PDRA will be encouraged to become involved with the Strathclyde Vertically Integrated Project program which gives students from first year through to postgraduate level the opportunity to work with staff in multi-disciplinary R&D teams. Not only will this encourage undergraduates to learn from the PDRA, but will also give the PDRA educational skills necessary for their career development as an academic.