Award details

Development of Geobacillus thermoglucosidasius as a robust platform for production of chemicals from renewables through modelling and experimentation

ReferenceBB/J002410/1
Principal Investigator / Supervisor Professor Michael Danson
Co-Investigators /
Co-Supervisors
Institution University of Bath
DepartmentBiology and Biochemistry
Funding typeResearch
Value (£) 275,240
StatusCompleted
TypeResearch Grant
Start date 01/08/2012
End date 30/11/2015
Duration40 months

Abstract

The need to produce fuels and chemicals from renewable lignocellulose-derived feedstocks, thereby reducing the use of fossil fuels, is generally recognised. However, there are many challenges to achieving commercial viability, ranging from cost-effective release of fermentable carbohydrates to optimised re-direction of metabolic flux. Moving beyond ethanol production, the rationale for using yeast diminishes, due to its limited substrate range. Working with the applicants, TMO Renewables have engineered the thermophile, Geobacillus thermoglucosidasius, to create a metabolically versatile ethanologen and used this as the basis for a commercially-viable process using treated municipal solid waste as a substrate. This has served not only to demonstrate the potential of the organism, but also to highlight the problems of engineering a relatively poorly characterised organism. In this project, starting with the genome sequence of G. thermoglucosidasius NCIMB 11955 provided by TMO, we will: develop and experimentally validate a genome scale metabolic model to support future metabolic engineering of this organism; explore the mechanism(s) of catabolite repression operating in both simple laboratory and complex industrial media and; combine these findings to produce a strain optimally engineered for 2-butanol production from mixed sugars in the absence of catabolite repression. While the overall aim is to increase the potential of Geobacillus spp as industrial organisms, this will be done by improving our fundamental understanding of the biochemistry and physiology of this increasingly important genus. In particular, we will apply RNA-seq, supported by gene disruption and detailed enzyme characterisation, to improve the genome annotation and explore catabolite regulation. Combined with comparative bioinformatics against Bacillus spp, we expect to rapidly highlight the differences between the genera, enabling us to focus on features specific to Geobacillus spp.

Summary

In this project, researchers from Imperial College London and the University of Bath will work together with the company TMO Renewables Ltd to (a) understand fundamental aspects of the physiology and biochemistry of the thermophilic bacterium Geobacillus thermoglucosidasius, which the company uses in its current bio-ethanol process, and (b) develop computer based metabolic models, using a combination of genome sequence information and experimental measurements, which will be useful for predicting how to make changes to the organism so that it can produce a specific end-product from a variety of different substrates. While the company has been successful in creating a strain of Geobacillus thermoglucosidasius that can produce ethanol from renewable lignocellulose and fermentable components of waste, this was done with little understanding of how the organism behaves under complex fermentation conditions. During this process, many observations have been made that are not easy to explain from our limited current knowledge of the organism. As well as a financial contribution to the project, the company will provide the genome sequence for their parent strain. This is the first (available) complete genome sequence for this species of thermophile and provides the academic researchers with a significant platform from which to make new discoveries. Building on this platform, the research team will apply recently-developed methods for model building, model validation and physiological investigation. The latter will involve the newly-developed approach of 'transcriptomics' by 'RNA -sequencing' to understand how the organism regulates its metabolism and behaviour under different physiological conditions. Direct analysis of RNA (strictly speaking, it has to be converted to DNA before sequencing) using modern methods of high-throughput sequencing is an advance on the previous approach using microarrays, because it does not rely on initial deduction of which are bona-fide genesequences in a genome. Because the analysis is essentially blind to prior assumptions, it has revealed many unexpected features of regulation in different bacteria. Papers on the application of this method to bacteria only started appearing in 2009, and most of these either focus on methods development or pathogenic organisms. This project will give us the opportunity to look at an industrially relevant organism, addressing questions that are pertinent to industrial fuel and chemical production from biomass and ultimately testing hypotheses and strains in an industrial context. Therefore, there is a strong chance for discovering new and fundamental processes underlying the regulation of microbial growth and metabolism. One of the outputs from this project will be a set of metabolic models. In silico metabolic models can be useful for predicting how metabolic flux should be altered to achieve a specific outcome (eg enhanced growth or metabolite overproduction). So, as part of this exercise, we will use the models in a metabolic engineering programme to make a new metabolite, not normally produced by this strain. Using the model, we should be able to predict how flux through different pathways should be changed to accomplish the dual requirements of rapid growth and product formation. In addition to this, we hope to link the transcriptomic analysis to the models. Metabolic models are essentially static pictures that do not adequately incorporate the dynamic aspects of physiological regulation. By studying cells under different growth conditions, we can generate a set of 'condition-specific models' which can be linked through comparative analysis of the transcriptomic data. The team involves a systems biologist who is expert at integrating different types of data, who will explore the possibility of linking the two types of analysis in a meaningful manner.

Impact Summary

Who will benefit from this research? Wider Academic Community: This work will raise our understanding of the metabolic physiology of Geobacillus spp to a level approaching that of better characterised organisms such as Bacillus subtilis. It will produce a set of freely available genomic sequences, annotated with extensive experimental support, and a similarly supported set of genome-scale metabolic models. These will be released through online databases (eg NCBI and the RAST server), publication in relevant academic journals and presentations at national and international scientific meetings. Part of this work will focus on the issue of catabolite regulation during growth in the complex mixtures of monomeric and polymeric carbohydrates found in lignocellulosic hydrolysates, which should be of interest to both academic and industrial researchers working on Cleantech processes. Commercial Private Sector: This research will directly benefit the industrial partner, TMO Renewables Ltd (TMO), which has developed an integrated process technology for converting waste biomass into valuable products. TMO's business model is to license their technology to industrial partners who own and operate the facilities. While the initial focus has been on ethanol production, TMO wish to expand the range of chemicals that can be produced from their technology platform. The ability to switch products or add extra products without a major rebuild of the original plant is very attractive and would offer a more flexible (and hence lower risk) commercial proposition to their industrial partners. More broadly, the results of this programme should benefit all companies operating in the area of chemicals from renewables. Primarily, this will be through furthering our understanding of catabolite regulation (see above). However, a number of companies (including Biocaldol and Green Biologics in the UK) recognise that thermophiles such as Geobacillus spp, that express a repertoire of glycoside hydrolases, can offer process advantages in the conversion of lignocellulosic wastes. So, we expect that both the genomic information and the metabolic engineering approach will have wide industrial interest and potential for application. By collaborating with an established industrial partner, there is a realistic opportunity to rapidly exploit the modelling, regulatory information and metabolic engineering strategies arising for the production and commercialisation of new products. TMO has recently signed a commercial contract with a US Cleantech Company (Fiberight) to build multiple community-scale plants which will convert municipal waste into ethanol. There will therefore be multiple opportunities over the next 5 years to incorporate a demonstration scale application in these commercial plants. National and International Perspective: Climate change: A primary driver for the move from fossil fuels to fuels and chemicals from waste, or sustainably derived renewables, is the reduction in greenhouse gas (GHG) emissions. An efficiently operated biorefinery using cellulosic substrates should be able to deliver an 80% reduction in GHG emissions compared to its fossil fuel equivalent (based on ethanol production). This will help meet national and international targets for use of renewables and mitigation of climate change. Green jobs: The successful delivery of this project will have an impact on delivering green jobs within the UK and further afield - a more diversified, and hence valuable, technology platform will be more attractive to new customers and take up of the technology will be greater, promoting growth within the Cleantech sector. For the PDRAs, the possibility of working closely with an established and developing Cleantech company, including spending time working at TMO, will give them an excellent perspective of both academic and industrial research environments, which should be invaluable for their future employment prospects
Committee Research Committee B (Plants, microbes, food & sustainability)
Research TopicsBioenergy, Industrial Biotechnology, Microbiology, Systems Biology
Research PriorityBioenergy, Synthetic Biology, Systems Approach to Biological research
Research Initiative X - not in an Initiative
Funding SchemeIndustrial Partnership Award (IPA)
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