13TSB_SynBio- Novel Bacterial Hosts for Biobutanol Production

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

Abstract

Energy insecurity, global warming and fluctuations in oil prices and has resulted a rapid increase in the demand for the biofuel, bioethanol. However, the alcohol biobutanol is widely acknowledged as a 'superior biofuel'. It is traditionally produced via a sugar-based fermentation process using a bacterium Clostridium acetobutylicum. The process, however, remains uneconomic due to feedstock prices, which represents 60-80% of production costs.

Another member of the same family of bacteria, Clostridium pasteurianum is a robust, fast growing bacterium which is also able to grow on glycerol as well as sugars and produce valuable 3-carbon (1,3 propanediol) and 4-carbon chemicals (butyrate & butanol). The microbe has tremendous potential as a fermentation host but has not been exploited due to its limited substrate range and mixed array of chemical products that result in low butanol yield compared to current commercial. In this project we aim to use synthetic biology to introduce new metabolic pathways for starch utilisation and butanol production into C. pasteurianum with the aim to produce butanol in high yield from starch. Genetic manipulation based on gene knockout will also be used to inactivate competing pathways.

The partners have extensive experience with solventogenic Clostridia. Nottingham's CRG has developed an impressive range of gene tools and recently completed the determination of the complete genetic blueprint (genome sequence) of C. pasteuranium. GBL has identified novel pathways for both starch degradation and butanol production from its unique collection of commercial strains and leads recommercialisation efforts for the butanol fermentation. This collaboration between GBL and GRG enables, for the first time, a targeted and rational approach for strain improvement in a novel host using synthetic biology and metabolic engineering. This project clearly demonstrates the potential application of synthetic biology for industrial biotechnology. Fermentation performance from the engineered C. pasteurianum strains will be benchmarked against GBL's best performing commercial strains and if promising taken forward for further development. The project output will also support additional projects with this microbe focused on extending the substrate range to include cellulosic feedstocks and developing glycerol metabolism for the production of 3-carbon chemicals.

Technical Summary

This project is a collaboration between the Clostridia Research Group (CRG) at Nottingham and Green Biologics Ltd (GBL).

GENOME SEQUENCING & ANNOTATION: CRG has just completed sequencing of the C. pasteurianum ATCC 6013 genome. Data will be used to finalise the assembly and complete annotation of the genome.

DEVELOPMENT OF A METABOLIC MODEL: GBL will use the annotated genome sequence to construct a metabolic model. Engineered strains will be used to validate the model. The model will be used as a predictive tool for future manipulations.

PLASMID TRANSFORMATION: Existing CRG modular shuttle vectors will be used for gene transfer experiments. Optimisation of transformation will be by both the empirical alteration of electroporation parameters, and by instigating strategies designed to circumvent any barrier posed by restriction-modification systems identified in the genome.

GENE KNOCK-OUT/KNOCK-IN: CRG will use the genome sequence data and transformation system to identify any knockout genes encoding enzymes involved in by-product formation. The genes will initially be knocked out using ClosTron technology. Thereafter, using novel in-frame deletions methods.

BUTANOL PRODUCTION: In parallel, CRG will modify expression of enzymes involved in butanol formation, replacing C. pasteurianum 'butanol genes' with synthetic equivalents (identified from hyper-butanol producing clostridia). In addition, endogenous promoters will be replaced with stronger promoters.

STARCH UTLISATION: GBL have identified a novel operon containing four hydrolytic enzymes involved in starch metabolism. Synthetic genes and appropriate orthogonal promoters and terminators will be designed and assembled in BioBrick format using GBlock assembly. These will be integrated into the 6013 genome using ACE.

FERMENTATION PERFORMANCE TESTING: GBL will test fermentation performance with both wildtype & engineered strains at lab-scale with glucose, glycerol and starch.

Planned Impact

WHO WILL BENEFIT?

The overall aim of this project is to enhance and extend the capabilities of solventogenic bacteria in terms of fuel and chemical production from cost effective feedstocks. As this is an Industrial Partnership, the primary beneficiary is GBL. They will directly commercialise all useful strains that emerge from the project and will have first refusal on any foreground intellectual property that arises.

Both parties have extensive global networks of existing commercial contacts and strategic partners. For example, GBL have partnerships with Guangxi Jinyuan Biochemical and Lianyungang Union of Chemicals in China. Nottingham have partnerships/ collaborations with EBI, Lanxess and Genencor (N America), Evonik, Universities of Munich, Ulm and Berlin (Germany), TMO Renewables Ltd, Invista and Unilever (UK), Metabolic Explorer Ltd, INRA and CNRS (France), Chinese Academy of Sciences (Shanghai and Tsinghau, China), the Mumbai Institute of Chemical Technology (India) and LanzaTech (New Zealand). Working together, the partnership will seek to maximise these links for the benefit of both parties.

The successful commercialization of anticipated outputs will have a rapid and global impact for both humanity and the environment. It will reduce greenhouse gas emissions and environmental pollution, provide an alternative to the use of food or farm resources for the production of low cost low carbon fuels and chemicals. It is therefore of benefit to society, ultimately impacting on health and well-being.


HOW WILL THEY BENEFIT?

Project outcomes will allow improved fermentation process economics and product diversity, thus encouraging more rapid and wide spread adoption of clostridia-based butanol fermentation as a process to produce high volumes of low cost butanol as a chemical commodity and potentially as a biofuel. The partnership are anticipated to directly benefit from the outputs of the project through their commercial adoption via the pipeline established by GBL to scale-up and commercially produce fuel and chemical products by clostridia fermentation. Additionally the partnership intends to explore strategic licensing deals with third party organisations. These will take the form of up front and milestone payments as well as ongoing royalty streams.

The successful scale-up and commercialization of processes will assist the UK and Europe in meeting challenging 'greenhouse' gas reduction targets, and contribute to indirectly to food security. The generation of butanol from cellulosic feedstocks will additionally impact on reducing reliance on fossil reserves, and therefore increase national fuel security.

The use of low carbon fuels to displace petrol reduces localized pollution from transport, and has a positive influence on public health and thus national productivity, ie, EtOH petrol blends reduce smog formation (the American Lung Association credits ethanol-blended petrol with reducing smog-forming emissions by 25% since 1990), and toxic exhaust emissions (CO emissions by as much as 30%, toxic content by 13% (mass) and 21% (potency), and tailpipe fine particulate matter emissions by 50%).

Our programme is tailored to allow definitive benefits to be realized within the project's timeframe. Thus, our target is to improve the productivity of the existing GBL process, allowing its immediate transfer to commercial operations in China. The project will provide the opportunity for staff working directly on the project, together with indirectly affiliated postgraduate students, to become trained in the strategically important areas of 'Synthetic Biology' and 'Industrial Biotechnology and Bioenergy'. These skills will be translatable to many different areas outside of butanol-producing clostridia, enhancing future job prospects.
 
Description Energy insecurity, global warming and fluctuations in oil prices and has resulted a rapid increase in the demand for the biofuel, bioethanol. However, the alcohol biobutanol is widely acknowledged as a 'superior biofuel'. It is traditionally produced via a sugar-based fermentation process using a bacterium Clostridium acetobutylicum. The process, however, remains uneconomic due to feedstock prices, which represents 60-80% of production costs.

Another member of the same family of bacteria, Clostridium pasteurianum is a robust, fast growing bacterium which is also able to grow on glycerol as well as sugars and produce valuable 3-carbon (1,3 propanediol) and 4-carbon chemicals (butyrate & butanol). The microbe has tremendous potential as a fermentation host but has not been exploited due to its limited substrate range and mixed array of chemical products that result in low butanol yield compared to current commercial. In this project we aim to use synthetic biology to introduce new metabolic pathways for starch utilisation and butanol production into C. pasteurianum with the aim to produce butanol in high yield from starch. Genetic manipulation based on gene knockout will also be used to inactivate competing pathways.

The Nottingham component has been to implement a Roadmap for gene system development, formulated as part of the BBSRC Sustainable Bioenergy Centre (BSBEC) in Clostridium pasteurianum, and thereafter brin g about the necessary modifications to the organism to maximise butanol production. To date we have:-

[1] Completed the entire genome sequence of two Clostridium pasteurianum strains and completely annotated the genomes

[2] Isolated a super transforming derivative of the host strain and optimised transformation to be several orders of magnitude higher than the parent strain

[3] Identified the most appropriate plasmid replicons to be used

[4] Exemplified ClosTron technology by making a pyrE mutant

[5] Implemented Allele-Coupled Exchange (ACE) to make a pyrE mutant in the genome.

[6] Used the strain created to implement in-frame deletion technology using a negative selection marker based on a heterologous pyrE gene.

[7] Used the deletion technology to make in frame deletions in genes encoding
enzymes involved in by-product formation.

[8] Demonstrated functionality of a high level, orthogonal expression system

[9] Cloned heterologous genes involved in butanol formation ready for insertion into the genome using ACE

The end date of the project has been extended until 31st January 2015.
Exploitation Route Our findings represent an exemplar example of the suitability of Nottingham's Roadmap for gene system development in clostridial species, through its flawless adaptation to Clostridium pasteurianum. Currently, the number of researchers in the UK investigating butanol-producing clostridia is very restricted, limited to just a couple of groups. The fundamental findings of this project will help others in the UK to embrace clostridial research.

Although the commercial partner are only interested in starch as a feedstock, the organism is particularly adept at growing on glycerol. Glycerol is a versatile carbon and energy source and presently it is produced in large scale as the principle by-product (10% w/w) of the biodiesel industry where the rapidly expanding market for biodiesel has dramatically altered the cost (for crude glycerol, prices have decreased from $0.25 per pound to $0.05 per pound) and availability of glycerol. It is now essentially a waste product of biodiesel industry and it has been estimated that the production of crude glycerol from biodiesel industry will reach 37 billion gallons by 2016. Nottingham will therefore independently pursue this feedstock through the award of a Marie Curie International Incoming Fellow who will start at Nottingham in 2015.
Sectors Chemicals,Energy,Manufacturing, including Industrial Biotechology

 
Description Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1,3-propanediol, when grown on glycerol. Further increases in the yields of n-butanol produced by C. pasteurianum could be accomplished through rational metabolic engineering of the strain. Accordingly, in this project we developed and exemplified a robust tool kit for the metabolic engineering of C. pasteurianum and used the system to make the first reported in-frame deletion mutants of pivotal genes involved in solvent production, namely hydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase). We were, for the first time in C. pasteurianum, able to eliminate 1,3-propanediol synthesis and demonstrate its production was essential for growth on glycerol as a carbon source. Inactivation of both rex and hydA resulted in increased n-butanol titres, representing the first steps towards improving the utilisation of C. pasteurianum as a chassis for the industrial production of this important chemical.
First Year Of Impact 2016
Sector Chemicals,Energy,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description German Fellow
Amount £43,000 (GBP)
Funding ID SCHU 3122/1-1 
Organisation German Research Foundation 
Sector Public
Country Germany
Start 01/2016 
End 06/2017