Novel inducible gene expression systems based on furan inducers and the MmfR transcriptional repressor

Streptomyces bacteria are also known as the "antibiotic makers" as the majority of clinically-used antibiotics originate from natural products made by these micro-organisms. Interestingly, Streptomyces have developed fine-tuned regulatory mechanisms to adapt in their soil environment. In consequence they do not always produce antibiotics but we know, for instance, that the presence of a competitor in their environment can trigger antibiotic production. In order to fully exploit the potential of these bacteria and discover novel antibiotics, it is vital to understand the molecular processes involved in regulating these pathways.

A sub-family of proteins, known as ArpA-like, is responsible for controlling antibiotic production in Streptomyces bacteria. These proteins are characterised by two main components, a DNA-binding part and a ligand-binding part. When the ligand (or signalling molecule) is absent from the environment, these proteins are bound to the DNA and they silence the systems responsible for antibiotic production. However, as soon as minute amount of signalling molecules are present, these compounds interact with the ligand-binding part of ArpA-like proteins which are in turn released from the DNA. In other words the protein is either bound to the DNA or to the signalling molecule.

This project aims at understanding in details the interactions between ArpA-like protein and the DNA as well as the interactions between ArpA-like proteins and the signalling molecules.
In addition to understanding how antibiotic production is controlled and therefore contributing to the research for urgently needed novel antibiotics, this project aims at exploiting these proteins for controlling other processes in bacteria. Indeed these systems can in principle control any of the bacteria behaviour: for instance we have already shown that we can make them produce light upon addition of the signalling molecule.

This project will be focusing on a specific ArpA-like protein called MmfR as it responds to a particularly exciting class of signalling molecules which are easy to make using chemistry and which are very stable. We have also solved the three-dimensional structure of the MmfR protein in complex with its signalling molecule using X-ray crystallography.

This research is also very timely because sequencing the genome of streptomycetes bacteria has revealed the presence of many unsuspected machineries predicted to assemble novel antibiotic-like molecules. However many of these systems are not well expressed in the laboratory and are thought to be regulated by ArpA-like proteins. Furthermore novel biological research approaches (systems and synthetic biology) are in exponential development and will benefit immensely from this project. Indeed new biological parts are desperately needed to investigate biological networks or to control biological circuits.

In summary ArpA-like proteins, the specific DNA sequences they bind to and the specific signalling molecules that release these proteins from the DNA are really ideal switch mechanisms that will find a multitude of biotechnological applications.

Streptomyces bacteria have developed fine-tuned regulatory mechanisms to adapt in their soil environment. In consequence even if tens of biosynthetic pathways predicted to direct the assembly of antibiotic-like natural products can be identified in their genomes, these systems are often silent. In order to fully exploit their potential and discover novel antibiotics, it is therefore vital to understand the molecular processes involved in these regulatory mechanisms.

ArpA-like proteins are involved in controlling antibiotic production. These proteins are composed of a DNA-binding domain linked to a ligand-binding domain. In the absence of signalling molecule (ligand) in the environment, these proteins are bound to specific DNA sequences and silence the systems responsible for antibiotic production. However, when sub-micromolar concentrations of signalling molecule are present in the environment, these compounds interact with the ligand-binding domain of ArpA-like proteins which are in turn released from the DNA and antibiotic production is triggered.

This project aims at understanding in details the DNA/protein and ligand/protein molecular interactions. We will be focusing on the ArpA-like protein MmfR as it responds to a particularly exciting class of signalling molecules which are easy to synthesise and chemically stable. In preliminary work we have also solved the three-dimensional structure of the MmfR protein in complex with its signalling molecule using X-ray crystallography.

In addition to understanding how antibiotic production is controlled and therefore contributing to the research for urgently needed novel antibiotics, this project aims at exploiting these proteins for developing a new generation of inducible gene expression systems. These new biological parts will find diverse applications in particular in systems and synthetic biology where they will contribute to investigating biological networks or to controlling biological circuits.

Natural products made by streptomycete bacteria are the major source for antibiotic drugs, accounting for more than 70% of commercially available antibiotics. Over the last ten years, genome sequencing has revealed the presence of an untapped reservoir of "cryptic" natural products in these bacteria.

The outcomes of this project will directly and/or indirectly contribute to the activation of the production of novel antibiotics by streptomycete bacteria grown in laboratory culture conditions. This is particularly important given that, over the last 25 years, the discovery of novel antibiotics has declined dramatically and during the same period the incidence of drug-resistant bacterial strains has increased considerably. The development of novel antibiotic drugs would clearly impact on the nation's health but also on the nation's economy.

In addition to the research novel antibiotics, understanding new regulatory processes will result in the biotechnology and pharmaceutical industries gaining new approaches for strain improvement in order to enhance production of their compounds of interest. For instance several ArpA-like proteins are proposed to control the production of bioactive compounds in the industrially relevant Streptomyces avermitilis strain, which produces the widely used anti-parasitic agent avermectin. The impact of this research will be highly relevant towards the improvement of S. avermitilis related strains for academic and industrial uses.

The proposed project will contribute to a precise understanding of transcriptional regulation mechanisms mediated by signalling molecules. This will result in the development of a new generation of chemical-inducible expression systems that will benefit biotechnology companies as well as all users. Efficient systems are currently lacking for protein and for bioactive metabolite production in Streptomyces hosts.

This project will serve the scientific community (in particular academia, biotechnology and pharmaceutical industries) by providing new fundamental knowledge. The work will also be communicated to the general public via Outreach activities such as Café scientifiques and school visits.
All of this will significantly contribute to maintaining the UK's leading reputation in the field of natural products chemistry and biology.