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

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.

Although there is plenty of transcriptomic data for most industrially relevant Streptomyces strains grown under laboratory conditions, there is no data regarding their growth in more ecologically relevant environments.

Understanding the conditions for expression of cryptic clusters and the regulatory pathways that lead to the production of antimicrobial compounds in industrially useful strains will allow us to generate knowledge to improve strains for the production of natural products. By conducting RNA sequencing combined with an unbiased metabolomics approach on wild-type and antibiotic overproducing mutant variants of these Streptomyces strains, we will identify regulators and metabolic pathways which can then be manipulated to increase production of these compounds under fermentation conditions.

This research project will add value to natural product research and industry in two ways: by providing information about the regulation and expression of current cryptic clusters in ecologically relevant environments we will be able to generate strains which have the ability to express previously untapped cryptic pathways for further exploitation. Moreover, the data generated in this study will allow the community, particularly industrial partners, to understand the regulation of production of antimicrobials already known. This should lead to an increase of yield of these compounds, thus increasing the chances of promising natural products to undergo clinical trials.

Proposed Research Plan:

1. Identification of global transcriptional patterns leading to the activation of cryptic clusters when S. venezuelae and S. rimosus are grown in an ecologically relevant environment using RNA sequencing.
2. Metabolomic analyses of S. venezuelae and S. rimosus when the strains are grown in ecologically relevant conditions using UPLC/HRMS.
3. Generation of genetically modified industrial Streptomyces strains based on the RNA sequencing and metabolomic data.

We believe that our research has the potential for long term far-reaching health, societal, economic and academic impacts - not just through the results generated, but through associated AMR related outreach and engagement activities.

The discovery of new antimicrobial compounds with novel chemistry and therefore possible new modes of action would be of value to the pharmaceutical industry. Streptomyces species account for over two thirds of clinically used antimicrobial compounds and are also an abundant source of other types of bioactive molecules including anticancer agents and immunosuppressants, currently accounting for billions of annual revenues generated worldwide by the pharmaceutical industry.

It is estimated that 700,000 people die of AMR infections every year (O'Neill report on AMR, May 2016). The World Bank predicts that if action is not taken, by 2030 AMR could cost the world economy $1 trillion every year. The current financial burden of antimicrobial resistant pathogens alone in the UK is considerable. In 2018, multiple drug resistant E. coli infections have cost the NHS £2.3 billion. A more effective treatment could reduce this cost substantially.

In addition, introduction of new classes of antibiotics to the clinic would mean that antibiotics of last resort, which are increasingly being used more frequently to treat MRSA and other multiple drug resistant infections, could once again be retained for emergency cases thus prolonging their efficacy.

Apart from the evident long term health, societal and economic impacts associated with the development of a successful new antibiotic we believe that the AMR specific outreach and public engagement associated with our research will contribute to raise awareness and understanding of the problems associated with misuse and over use of antibiotics by the general public. This should lead to a shift in behaviour and attitudes towards more responsible and informed use of antibiotics.

Dr. Fernandez-Martinez has a strong track record in engagement with primary and secondary school children through science festivals and national and international outreach activities and she plans to continue this throughout the project. Dr. Fernandez-Martinez will train and encourage both the RA and PDRA to engage in such activities. We believe that raising awareness in the next generation of antibiotic consumers will not only have a positive impact in their attitudes towards the use of antibiotics but will also inspire the next generation of natural product scientists through our outreach and engagement activities.