Back to soil: awakening the production of cryptic antibiotics in Streptomyces strains

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The increasing incidence of antibiotic-resistant bacterial pathogens has resulted in an urgent need for new, clinically useful antibiotics. Most of the antibiotics used in medicine are produced by actinomycetes, and particularly a genus of harmless bacteria called Streptomyces, which is abundant in all soil environments. Based on their genome sequences, each Streptomyces species has the potential to produce on average around 10-15 antibacterial agents of natural product origin. However, when these species are grown under laboratory conditions, only one or two antimicrobial compounds are usually detected. This is because most of these antibiotic gene clusters appear dormant (i.e. are not expressed) under laboratory conditions. The fact Streptomyces species maintain these intact antibiotic gene clusters in their genomes suggest the products are useful in nature, probably to attack competitor microorganisms in the harsh soil environment. These dormant or cryptic antibiotic gene clusters represent an untapped resource in terms of novel chemistry which could lead to the discovery of new antimicrobial compounds that could be very useful in the clinic.Preliminary work leading to this research proposal has allowed us to grow two of these Streptomyces species, for the first time, in ecologically relevant environments including sterile and non-sterile soils. This project aims to understand when previously uncharacterised antibiotic gene clusters encoded within the genomes of these Streptomyces species are expressed and produced in more natural environments. The data generated will provide us with key information which will allow us to generate genetically modified strains with the ability to produce detectable amounts of these compounds under laboratory and industrial fermentation conditions. The aims of this research are:1. To identify how these cryptic antibiotics are made by Streptomyces species in soil.2. To detect which cryptic antibiotics are produced under ecologically relevant conditions.3. To use the information obtained in objectives 1 and 2 in order to generate genetically modified Streptomyces variants with the ability to produce detectable amounts of these new antibiotics under laboratory and fermentation conditions.To accomplish these aims we will use RNA sequencing, a technique used to identify which genes are being expressed under a particular growth condition. This will allow us to relate genes that are co-expressed and will provide the information required to identify global and specific regulators of cryptic antibiotic clusters, as well as key metabolic pathways leading to the production of these compounds. We will use chromatography and mass spectrometry to identify new antibiotics produced by the bacterial cells when grown in both sterile and non-sterile soils. We will combine the above information in order to select gene candidates involved in the production of particular cryptic antibiotics. We will then genetically manipulate these candidate genes from the bacterial genome one at a time. In some cases, we will insert extra copies of these candidate genes and in others we will delete these genes from the original genome. After each experiment, the production of that particular antibiotic will be analysed to see if these genetic changes increase production of the compound or if any new compounds are produced. This will allow us to generate variants of these Streptomyces species able to produce detectable levels of new antibiotics under laboratory conditions. The results from this work will have particular significance in the field of antimicrobial resistance (AMR) as it aims to increase the number of novel natural products available to test and therefore provide many more lead candidates to take forward in order to produce much needed new clinically relevant antimicrobial compounds.