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

Lead Research Organisation: University of Nottingham
Department Name: Sch of Molecular Medical Sciences

Abstract

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.

Technical Summary

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.

Publications

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Heap JT (2009) A modular system for Clostridium shuttle plasmids. in Journal of microbiological methods

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Heap J (2010) The ClosTron: Mutagenesis in Clostridium refined and streamlined in Journal of Microbiological Methods

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Heap JT (2010) ClosTron-targeted mutagenesis. in Methods in molecular biology (Clifton, N.J.)

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Cartman ST (2010) The emergence of 'hypervirulence' in Clostridium difficile. in International journal of medical microbiology : IJMM

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Bradshaw M (2010) Construction of a nontoxigenic Clostridium botulinum strain for food challenge studies. in Applied and environmental microbiology

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Kuehne SA (2011) ClosTron-mediated engineering of Clostridium. in Methods in molecular biology (Clifton, N.J.)

 
Description The overall goal of the Nottingham was to lead a SysMO consortium workpackage concerned with the identification of the regulatory networks subject to quorum sensing (QS) control in Clostridium acetobutylicum, by focussing on the putative Agr and LuxS systems responsible for production of the diffusible signal molecules AIP (Auto-Inducer Peptide) and AI-2 (Auto-Inducer 2), respectively. An additional goal was to provide consortium members with requisite gene tools for the planned experiments.

The most significant achievement was the development of a robust series of gene technologies that have revolutionised the options available for Systems Biology approaches in Clostridium acetobutylicum. These included the design and implementation of a:

(i) modular system to allow the combinatorial construction of Clostridium-E. coli shuttle plasmids from a choice of standardized components;

(ii) refined and streamled ClosTron mutagenesis system for mutant generation, and;

(iii) new, patent filed method (Allele Coupled Exchange - ACE) which allows the insertion of DNA of any size or complexity into the clostridial genome, useful for the construction of complex synthetic operons.

In parallel, the ClosTron was used to demonstrate a regulatory role for an Agr Quorum Sensing system in spore formation, independent of solvent formation, motility, amylase production, and biofilm formation. The likely the chemical structure of the autoinducing peptide responsible was derived using a library of 18 synthetic peptides.
Exploitation Route During the course of the project, the versatility and breadth of tools available for manipulating clostridia was expanded for the benefit of the wider scientific community. Thus, ClosTron technology was refined and streamlined to minimise labour-intensity and maximize the accessibility of the mutagenesis method. This has included the addition of the facility to make multiple mutations and to deliver small amounts of heterologous 'cargo' DNA to the chromosome. Most significantly, procedures have been put in place for automated intron retargeting and plasmid construction through custom synthesis and vector construction. Design and construction of retargeted plasmids are now easily performed online in just a few minutes (http://www.clostron.com) with no laboratory work for the researcher and dispensing with the need to purchase Sigma Aldrich Targetron kits. Synthetically re-targeted plasmids typically arrive within 2 weeks. Mutant isolation simply requires the selection of erythromycin or lincomycin resistant transformants using selective media.

To improve the availability of shuttle plasmids with properties suitable for clostridial hosts and applications of interest, we specified, designed and implemented a modular system to allow the combinatorial construction of Clostridium-E. coli shuttle plasmids from a choice of standardized components. Our design was based on BioBrick standard assembly is a method by which elements or 'Parts' in a standardized format may be combined (by ligation of compatible DNA ends which do not regenerate a restriction site) to yield a single larger Part in the same format. A total of 400 vectors may be made from the 18 specified components, but distribution is achieved through the supply of a core set of just 5 plasmids.

Our major innovation has been the design, formulation and patent filing of a new, ground breaking method, Allele Coupled Exchange (ACE) technology which allows the insertion of DNA of any size or complexity into the clostridial genome, useful for the construction of complex synthetic operons encoding, for instance cellulosomes and novel metabolic pathways. The developed and validated ACE vectors allow direct (positive) selection to be used at every step of the integration procedure. The approach is robust, requires little labour, and results in stable insertions of constructs without limits on size or complexity.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description As part of the SysMo consortium of European partners, Nottingham focused on how individual Clostridium cells communicate with one another by using a language based on chemical signals, and whether this allows them to control butanol production and spore formation. We found that the ability to form spores - to survive harsh environmental conditions in a dormant form - was much reduced, and under certain conditions completely abolished, when the cells were unable to produce or respond to the chemical signals. Our data were used to generate a model that predicts the sporulation behaviour of a C. acetobutylicum population. This is important for future attempts to exploit the organism as an anti-cancer deliver vehicle, a project that heavily relies on the bacterium's ability to form spores. Spores are also an important factor that contributes to the spread of food-poisoning and infectious, pathogenic clostridia. Little was known about the factors that trigger sporulation in these species, but with our discovery a promising starting point for future research has been identified that will ultimately lead to the development of more effective countermeasures for controlling infection. Nottingham also focused on the development of a robust series of gene technologies for the benefit of both the consortium partners and the wider scientific community. Their availability has revolutionised the options available for Systems Biology approaches in Clostridium acetobutylicum. The second generation ClosTron plasmid pMTL007C-E2 has been deposited in with Genbank accession number HQ263410, while the modular vector series were deposited with Genbank accession numbers: pMTL80110, FJ797644; pMTL82254, FJ797646; pMTL83353, FJ797648; pMTL84422, FJ797650; pMTL85141, FJ797651; pMTL82151, FJ797645; pMTL83151, FJ797647; pMTL84151, FJ797649; pMTL85151, FJ797652. These vector systems have been distributed to 100s of groups around the, and paper describing the construction of the ClosTron is the most cited (222) paper in its journal since the original publication in 2007, whereas the refined and streamline paper published during the course of this project is now (2014) the second most cited (116) since its publication in 2010.
First Year Of Impact 2010
Sector Agriculture, Food and Drink,Chemicals,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC China Partnersip
Amount £26,518 (GBP)
Funding ID BB/G530341/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2009 
End 08/2013
 
Description BBSRC Sustainable Bioenergy Centre
Amount £2,127,704 (GBP)
Funding ID BB/G016224/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2009 
End 09/2014
 
Description ERANET SysMO2
Amount £452,694 (GBP)
Funding ID BB/I004475/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2010 
End 12/2013
 
Description Evonik - A Scoping Study to Exemplify Advanced Gene Tools
Amount £155,000 (GBP)
Organisation Evonik Industries 
Sector Private
Country Germany
Start 12/2010 
End 02/2012
 
Description Evonik Clostridium research project
Amount £180,000 (GBP)
Funding ID 310422601 
Organisation Evonik Industries 
Sector Private
Country Germany
Start 12/2011 
End 07/2014
 
Description Fully Funded Commercial Studentship (LanzaTech)
Amount £200,000 (GBP)
Organisation LanzaTech 
Sector Private
Country United States
Start 09/2011 
End 10/2015
 
Description Invista Commerical Contract
Amount £625,000 (GBP)
Funding ID PO NO.57603334 
Organisation Invista (UK) 
Sector Private
Country United Kingdom
Start 05/2012 
End 05/2015
 
Description KTP8497 TSB/Green Biologics
Amount £97,000 (GBP)
Funding ID KTP008497 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 08/2011 
End 09/2013
 
Description Lanxess Use of a carboxydotrophic organism
Amount £190,000 (GBP)
Funding ID 310441364 
Organisation Lanxess 
Sector Private
Country Germany
Start 02/2012 
End 08/2014
 
Description LanxessUse of Clostridium ljungdahlii
Amount £190,000 (GBP)
Organisation Lanxess 
Sector Private
Country Germany
Start 02/2012 
End 08/2014
 
Description Marie Curie Initial Training Network (ITN)
Amount £4,111,621 (GBP)
Funding ID 215697-2 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 07/2009 
End 08/2013
 
Title SysMO-DB 
Description SysMO-DB was a database set up as part of the EARNET SysMO. We deposited details of clostridial mutants made and protocols for their generation. 
Type Of Material Database/Collection of data 
Year Produced 2008 
Provided To Others? Yes  
Impact Methods and protocols for mutant generation in Clostridia shared with other members of SysMO