Integration and coordination within complex antibiotic biosynthetic pathways

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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?

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