Genome-scale functional genomics in Streptomyces species using CRISPR interference

The majority of clinically used antibiotics are derived from chemicals 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. Although we know a lot about Streptomyces biology, but unfortunately there are still major gaps in our knowledge that prevent us from accessing new drugs and producing them at the quantity required to be commercially viable. We believe that understanding the role of each gene in an organism's genome is essential to for overcoming this roadblock to progress. This goal of our research is to create technology to quickly and simultaneously analyse the importance of all genes encoded within the genome of a Streptomyces species. Our technology will allow us to understand these microorganisms in greater detail, for example, by learning the genes essential for life. Our technology could one day be used for other purposes, for instance, to learn how production of various antibiotics is controlled so we can access new compounds more easily, and it will also enable the pharmaceutical industry to engineer Streptomyces strains that produce more drug in a shorter timeframe. We believe our research could help make more medicines reach the clinic, possibly making them less expensive and more widely available.

Growing concerns about antibiotic resistance combined with failure to find new leads from screening of large libraries of synthetic compounds has led to a renewed interest in microbial natural products discovery. This renaissance has been fuelled to a large extent by the relatively inexpensive cost to sequence genomes of strains that produce promising bioactive small molecules. Access to genomic data has enhanced our understanding of evolution of the genus and has also led to exquisite genetic insight into important processes such as morphological development, the perception of environmental stress, and secondary metabolism among other areas. However, overall the field has not yet been able to truly harness the 'power' of the genome sequence, because there is simply no methodology available for genome-scale functional genomics studies. We will overcome this roadblock by generating a CRISPR/Cas9 interference platform that we will use in combination with Illumina amplicon sequencing to create a step-change in the way in which functional genomics experiments are conducted in Streptomyces species. Bioinformatics and statistical analyses will enable us to determine the relative fitness contribution of each genome for growth in the conditions used in the study. As a proof of principle, we will access gene essentiality in a Streptomyces species and validate a subset of these results using merodiploid depletion assays.

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 in sufficient quantity is paramount, for example, to fight the war against antimicrobial resistance. Synthetic biology has the potential to enhance our ability to engineer a Streptomyces species to produce more of the desired compound or drug. This goal is currently unattainable because of an inability to perform genome-wide studies quickly and robustly. The main deliverable of this project is to create such a platform and we will use it to analyse the importance of all genes encoded within the genome of a Streptomyces species when cultivated in a various growth media. This will not only enable us to determine which of these genes are essential for life, but will produce a method for enhanced strain breeding of industrially important Streptomyces species such that they produce more drug in a shorter timeframe. We believe our research could help make 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.