Integration and coordination within complex antibiotic biosynthetic pathways

Lead Research Organisation: John Innes Centre
Department Name: Molecular Microbiology


The harmless soil bacteria called streptomycetes are vital to human welfare because they are the source of the vast majority of antibiotics used by doctors to cure infectious diseases, as well as providing us with numerous other medicines used, for example, to treat cancer, and to help organ transplant patients (immunosuppressants). Despite the importance of antibiotics, relatively little is understood about how these bacteria coordinate the activities of all the components of the machinery that make these compounds. Recent discoveries have revealed that the antibiotics themselves and intermediate compounds in the antibiotic pathways control the activities of key regulators (called 'transcription factors') that switch the genetic machinery of these useful bacteria to coordinate and integrate the production of antibiotics. The aim of this work is to find out exactly how these regulators work at the molecular level, how their activities are controlled, and how this serves to coordinate and integrate the synthesis of the antibiotics. We will perform these experiments in a model system on an antibiotic called simocyclinone, which is not yet used in human medicine. However, since there is strong evidence that the signalling mechanisms we are studying are widespread in antibiotic-producing streptomycetes, our results might allow pharmaceutical companies to make knowledge-based improvements in the yield of commercially important antibiotics that are used in human medicine, potentially making them less expensive and more widely available.

Technical Summary

Streptomyces are the most abundant source of antibiotics and other natural products used in human medicine. Research on signalling mechanisms involved in antibiotic biosynthesis has focussed on upstream events that trigger activation of the biosynthetic gene cluster and there has been little investigation of signalling mechanisms that coordinate and integrate events within the biosynthetic pathway. However, recent research has shown that biosynthetic intermediates and the mature antibiotic are likely to play key signalling roles that coordinate events within these long, complex pathways. This realisation hinges on the discovery that the activities of at least 2 classes of antibiotic pathway-specific transcription factors are controlled by the cognate antibiotic or its biosynthetic intermediates. Building on our recent data, we will establish a comprehensive understanding of the metabolite signalling mechanisms coordinating the complex biosynthetic pathway for simocyclinone (a gyrase inhibitor made by S. antibioticus). We will determine which genes (hence pathway steps) are regulated by the 3 pathway-specific regulators encoded within the biosynthetic cluster (SimR, SimR2 and SimR3), how their DNA-binding activities are controlled by simocyclinone and/or its intermediates, and which simocylinone-related compounds are made by simR, simR2 and simR3 null mutants. We have already shown that SimR represses the promoter of simX, encoding the drug efflux pump, and that simocyclinone abolishes DNA-binding by SimR, coupling biosynthesis of the drug to its export. As a secondary goal, exploiting our recent crystal structure of the SimR-drug complex, we will make SimR* variants that respond only to intermediates and not to simocyclinone, and use these intermediate-specific SimR* proteins as biosensors to test the feed-forward hypothesis of Nodwell, and to answer a key question in antibiotic research - are intermediates released into the cytoplasm during antibiotic production?

Planned Impact

WHO WILL BENEFIT FROM THIS RESEARCH, AND HOW? SimR, SimR2 and simR3 are likely to play crucial roles in the regulation and coordination of production of the antibiotic simocyclinone, a potent DNA gyrase inhibitor made by Streptomyces antibioticus. We have shown that SimR is regulated by simocyclinone and/or its intermediates, and SimR2 and SimR3 are strongly predicted to be. Streptomycetes and their actinomycete relatives produce 80% of the commercially important antibiotics, and are also a rich source of other types of bioactive molecules such as anticancer agents and immunosupressants, in total accounting for $40 billion of revenue in the pharmaceutical industry worldwide. Since there is good evidence that the signalling mechanisms we are studying are likely to be widespread in streptomycete antibiotic biosynthetic pathways, our results might allow pharmaceutical companies to make knowledge-based improvements in the yield of commercially important antibiotics. WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH? Academic research at the John Innes Centre (JIC) with potential commerical application is patented through Plant Biosciences Ltd (PBL), a technology transfer company based at JIC that is jointly and equally owned by the BBSRC, the Sainsbury Laboratory and the JIC. The purpose of Plant Bioscience Ltd is to bring the results of research in plant and microbial sciences at the Centre into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC. Streptomyces research is prominent in PBL's portfolio. As an illustration, two spin-out companies have been established based on JIC Streptomyces group patents: Novacta Biosystems Ltd, founded at JIC in 2003 and now based at Welwyn Garden City Biopark, where it employs about 30 people; and Procarta Biosystems, founded at JIC in 2008. Thus, there are well established routes for delivery of IP arising from Streptomyces research at the John Innes Centre.


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Le TB (2011) Crystallization and preliminary X-ray analysis of the TetR-like efflux pump regulator SimR. in Acta crystallographica. Section F, Structural biology and crystallization communications

Description Because most antibiotics are potentially lethal to the producing organism, there must be mechanisms to ensure that the machinery responsible for export of the mature antibiotic is in place at the time of biosynthesis. Simocyclinone is a potent inhibitor of DNA gyrase made by Streptomyces antibioticus. In this, we identified simR, encoding a TetR-like repressor, and the divergently transcribed simX, encoding a proton-dependent efflux pump, as the resistance genes associated with the antibiotic biosynthetic cluster in S. antibioticus. We showed that transcription of simX is controlled by SimR, which directly represses the simX and simR promoters by binding to two operator sites in the simX-simR intergenic region. Most importantly, we showed that simocyclinone abolishes DNA binding by SimR, providing an intimate mechanism that couples the biosynthesis of simocyclinone to its export. In addition, we showed that the biosynthetic intermediate, C4, which is inactive as a DNA gyrase inhibitor, also induces simX expression in vivo and relieves simX repression by SimR in vitro.

In order to understand the mechanism of simocyclinone-mediated derepression, in a second paper, we went on to solve 3 crystal structures: (i) SimR alone, (ii) SimR in complex with simocyclinone, and (iii) SimR in complex with the pathway intermediate C4. In addition to a conventional helix-turn-helix (HTH) DNA-binding motif, SimR carries a novel N-terminal extension that is absent from other studied members of the TetR-family. In solving the crystal structures of SimR alone and in complex with the antibiotic, we saw that this N-terminal extension was not ordered, yet when we deleted the disordered residues we found that DNA-binding affinity was reduced >100-fold. As a consequence, we decided to perform protease protection experiments, through which we showed that the N-terminal extension is hypersensitive to trypsin, but that it becomes protease-resistant upon DNA binding. From this, we concluded that the novel N-terminal extension must be involved in DNA binding. To see if this were true, we crystallised and solved the structure of the SimR-DNA complex, and discovered that the N-terminal extension of SimR binds in the minor groove adjacent to the major groove occupied by the classical HTH motif. This binding to the minor groove is mediated through arginine residues that sit at the tip of the N-terminal extension. We then carried out a comprehensive bioinformatic analysis of all known TetR family regulators (TFRs) and showed that an N-terminal extension rich in positively charged residues is a previously unrecognised feature of the majority of TFRs, suggesting they will all make use of the minor groove binding mechanism we discovered.

Having the structures of (i) SimR alone, (ii) the SimR-antibiotic complex and (iii) the SimR-DNA complex allowed us to understand the mechanism of simocyclinone-mediated derepression. Firstly, it became clear that the conformational changes associated with antibiotic-mediated derepression result from rigid-body rotation of the two subunits about the dimer interface. The binding of simocyclinone captures a conformational state in which the two recognition helices are 42 Å apart, a spacing that is not compatible with binding to two consecutive major grooves. The rigid body rotation that is required to bring two recognition helices 37 Å apart (the distance seen in the DNA-bound state), is prevented in the ligand-bound structure by the antibiotic threading through both subunits, acting as a locking pin, and, in a related mechanism, by the inter-digitation of the Arg122 side chains, which project across the dimer interface into pockets in the surfaces of the opposing monomers, effectively locking the subunits together.
Sectors Pharmaceuticals and Medical Biotechnology

Description Since there is now strong evidence that the metabolite signalling mechanisms we have investigated are likely to be widespread within streptomycete antibiotic biosynthetic pathways, our results are likely to prove relevant to knowledge-based improvements in the yield of commercially important antibiotics. Streptomycetes account for ~80% of commercially important antibiotics, and are also a rich source of other types of bioactive molecules such as anticancer agents and immunosuppressants, currently accounting for ~$40 billion of annual revenue annually in the pharmaceutical industry worldwide.
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic