Award details

System biology of Clostridium acetobutylicum - a possible answer to dwindling crude oil reserves

Reference	BB/F003390/1
Principal Investigator / Supervisor	Professor Nigel Minton
Co-Investigators / Co-Supervisors	Dr Klaus Winzer
Institution	University of Nottingham
Department	Sch of Molecular Medical Sciences
Funding type	Research
Value (£)	364,436
Status	Completed
Type	Research Grant
Start date	01/04/2007
End date	30/06/2010
Duration	39 months

Abstract

Our strategy is to inactivate the genes responsible for AI production and response, and then determine the effect on expression using DNA arrays. Effects will be verified by quantitative RT-PCR and proteome analysis. Qualitative and quantitative data obtained will be used for computational modelling of QS and the major regulatory networks and events occurring during the transition to stationary phase. Predictions derived at different stages of the developing model will be tested, eg., by adjustment of growth conditions and further rounds of mutation. Target gene identification will follow established procedures, ie., generation of AI-deficient mutants and the addition of synthetic AI to mutant cultures. Components of the AI-response system will also be mutated to verify observed changes. Specifically, agrD and luxS mutants will be made and analysed for differential gene expression. Addition of synthetic AIP and AI-2, respectively, will identify those genes dependent on signal production and also allow us to establish threshold concentrations and dose-response relationships. Genes under AIP or AI-2 control will be confirmed by mutation of genes involved in the signal response, ie., agrA and agrC will be inactivated. Furthermore, mutants will be constructed that encodes AgrA locked in either the active or inactive state. Mutants and parent strain will be grown in a chemostat under a set of different conditions. These include growth at varying pH values (shift from acid to solvent formation), different growth rates, and most importantly, different cell densities. This will allow us to identify those conditions where the QS mechanisms are most active or suppressed by other regulatory systems. Such cross-regulatory mechanisms are likely to be revealed through studies undertaken in the other WPs of this proposal: mutation of other major regulatory pathways will identify commonly regulated target genes or even cross-regulation between the major regulators themselves.

Summary

The genus Clostridium are an ancient grouping of bacteria which evolved before the earth had an oxygen atmosphere. To them oxygen in the air we breathe is a poison, and they are therefore called 'anaerobes'. They are also characterised by an ability to produce a spore resting stage that enables them to survive exposure to the air. These spores are also resistant to many other physical and chemical agents. Some species cause devastating diseases, such as the superbug Clostridium difficile. On the other hand, most clostridia are entirely benign, and their ability to produce a wide range of diverse chemicals from plant material is being pursued by industry as an alternative to generating these chemicals from crude oil. Principle amongst these is C. acetobutylicum, an organism with a longstanding history in the commercial production of solvents, most notably 'butanol'. Butanol is an alcohol, which, like its counterpart ethanol may be used as a replacement for petrol as a fuel. Currently, the use of ethanol as a petrol additive is widespread in the developed world. The development of alternatives to petroleum as fuels is essential if we are to reduce our reliance on finite crude oil resources. However, butanol has many properties that make it far superior to ethanol. It has a higher energy content than ethanol, and its low vapour pressure and its tolerance to water contamination in petrol blends facilitate its use in existing petrol supply and distribution channels. Moreover, butanol can be blended into petrol at higher concentrations than existing biofuels, without the need to make expensive modifications to car engines. It also gives better fuel economy than petrol-ethanol blends. Despite their importance, our understanding of the biology of the Clostridium cell has lagged behind the data available for more recently evolved bacteria which 'breathe' oxygen. With the dawn of a new century the situation has changed. The complete genetic blueprint (genome sequence) of seven different Clostridium species has now been determined. The first was that of Clostridium acetobutylicum, a reflection of its commercial importance. It is the intention of this project to undertake an extensive analysis of the biological processes that take place when this Clostridium grows. In particular, we wish to understand the key events that occur during the transition between normal cell growth and the onset of both butanol production and spore formation. Our intention is to build a mathematical model of these processes such that the process may be recreated as a computer programme that mirrors the living cell. These aims will be progressed through a combination of different scientific disciplines (genetics, biochemistry, chemical engineering and mathematicians) deployed by a consortium of eleven European scientists, from three member states (UK, D & NL). At Nottingham and Lancaster, we will focus on how individual bacterial cells communicate with one another, and how the communication signals deployed control butanol production and spore formation. Other members of the consortium will focus on other interlinked biological processes. The ability to more effectively predict the behavioural and metabolic response of clostridia will enable the more effective exploitation of C.acetobutylicum in the commercial production of butanol and as an anti-cancer deliver vehicle. It will also lead to a greater understanding of the biology of those clostridia that cause disease and, ultimately, to the development of more effective methods of controlling infection.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Bioenergy, Industrial Biotechnology, Microbiology, Synthetic Biology, Systems Biology
Research Priority	X – Research Priority information not available
Research Initiative	Systems Biology of Microorganisms (SysMo) [2007-2008]
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/F003382/1 SysMO: Systems Biology of Clostridium acetobutylicum - a possible answer to dwindling crude oil reserves.

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