Mode of action and engineering of key regulators in butanol-producing clostridia

Solvent-producing clostridia are promising micro-organisms for the sustainable production of biofuels and chemicals from waste materials and renewable resources. However, at present bacteria of the strain Clostridium acetobutylicum generate butanol relatively slowly and in low concentrations. The underlying hypothesis of this project is that in order to create production strains with improved performance by genetic engineering we need to better understand the organisms' physiology, in particular the mechanisms that govern timing and extent of solvent formation. Recent work of the team has shown that bacterial communication mechanisms play a crucial role in butanol and acetone formation. Interestingly, C. acetobutylicum uses a chemical language based on secreted signal molecules to help decide when and how much of these solvents are produced. Novel transcriptional regulators have been identified as being an important part of this communication system. The overall objective of this project is to investigate the molecular basis of the transcriptional regulator's mode of action to decipher this chemical language. Inactivation of these regulators almost completely abolishes the formation of both acetone and butanol. The protein appears to be inactive when cells are actively growing but adopts an active state when the bacteria enter stationary phase and begin to produce solvents. A detailed understanding of the underlying molecular mechanism will greatly facilitate successful engineering of improved solvent producing strains. The aims of this project are to (i) purify, crystallise and structurally characterise the regulatory protein in both active and inactive form (ii) identify amino acid residues, ligands, and/or covalent modifications required or responsible for activation, (iii) exploit the generated knowledge to generate strains in which butanol formation is maximized, for instance by engineering the protein's structure so that it becomes permanently locked in the activated state.