A synthetic biology approach to enhancing chemical production by anaerobic bacteria (SynBio-AnOx)

Electricity, fuels and chemicals are at present mostly made from oil, gas and coal, which will become increasingly expensive and eventually run out. Consuming these fossil carbon sources also releases carbon dioxide into the atmosphere and oceans, adversely affecting the environment and climate. New, sustainable technologies are needed to generate electricity, heat our homes, power our vehicles, and make the chemicals needed in all kinds of products and industries. Developing and adopting these technologies is important for our well-being, economic prosperity and security.

Microbial fermentation is an important technology, involving the conversion of sugars to alcohol or other chemicals by yeast or bacteria in the absence of oxygen. Fermentation has been used for centuries in the production of bread, alcoholic drinks, and dairy products. Using modern techniques including genetic modification, it is now possible to develop new fermentation processes in which microbes convert feedstocks (sugars, starch or crop waste) into useful fuels and chemicals that are conventionally made from oil. These processes can be operated in a sustainable way, and could be part of a carbon-neutral economy.

The overall aim of this research programme is to produce bacterial strains and knowledge useful in the development of new fermentation processes for the sustainable manufacture of chemicals. Previous research on improving fermentation strains has made the best progress with facultative anaerobes, which are organisms able to grow either in the presence or absence of oxygen. This programme aims to make similar progress in techniques for improving strict (obligate) anaerobes, which are organisms that only grow in the absence of oxygen, because some strict anaerobes have useful properties for the sustainable manufacture of chemicals.

By the end of this research programme we aim to produce one or more new strains of the strictly anaerobic bacterium Clostridium acetobutylicum that continuously, efficiently and reliably convert feedstocks to the useful chemical butanol, without making by-products.

To achieve these aims, we will use and develop techniques from a new area of science and engineering called synthetic biology. We will develop methods to fine-tune and measure gene expression in Clostridium acetobutylicum, and new approaches to increase, decrease and otherwise modify the expression of selected genes. These new techniques will allow us to make progress that would not be possible using previous approaches.

If successful, we will work with a UK company to test the suitability of the new bacterial strains for commercial use.

This research has several potential applications and benefits. The new bacterial strains could directly or indirectly lead to improved industrial processes for the microbial manufacture of butanol, which is a valuable chemical and an excellent fuel. One of the world's leading microbial butanol manufacturing companies is based in the UK, so this research could lead to UK jobs and other contributions to the UK economy and UK wealth. More broadly, the knowledge produced by this research should help researchers and companies develop new fermentation processes for the sustainable manufacture of other chemicals.

The overall aim of this programme is to develop, validate and apply novel and existing synthetic biology approaches to the metabolic engineering of obligate anaerobes, particularly for sustainable manufacturing of chemicals at high yields. Previous research on improving anaerobic fermentation strains has made the best progress with facultative anaerobes such as E. coli. This programme aims to make similar progress in improving obligate anaerobes, which have various advantages, but also challenges for genetic and metabolic engineering that have not been overcome by conventional approaches. To overcome these challenges, more sophisticated synthetic biology approaches will be developed and applied.

The model obligate anaerobe Clostridium acetobutylicum will be engineered to achieve a homo-butanol fermentation from carbohydrate substrate.

1. We will develop core synthetic biology tools for anaerobes, using oxygen-independent fluorescent reporters to develop promoter libraries for native and dedicated (orthogonal) expression systems and to validate computational RBS design in Clostridium acetobutylicum.

2. We will develop and implement a system for pyruvate node redox flux redistribution that is compatible with the biochemistry and metabolism of obligate anaerobes. This will allow reducing equivalents to be directed to butanol formation, resulting in a homo-butanol fermentation.

3. We will implement STABLE solutions to the key challenges of butanol pathway over-expression, megaplasmid stability, and resilience to acid crash; and combine these with known modifications and the novel redox flux partitioning system to maximise butanol yield.

Improved butanol-producing strain(s) generated in this programme will be evaluated for industrial use by our industrial partner.

The overall aim of this programme is to develop, validate and apply novel and existing synthetic biology approaches to the metabolic engineering of obligate anaerobes, particularly for sustainable manufacturing of chemicals at high yields. The model obligate anaerobe Clostridium acetobutylicum will be engineered to achieve a homo-butanol fermentation from carbohydrate substrate.

The anticipated outcomes of this programme will have impacts on a variety of beneficiaries.

The new approaches developed in the research will benefit academic and industrial researchers in the related fields of Clostridium and other anaerobes, synthetic biology, metabolic engineering and industrial biotechnology. In these fields, academic and industrial researchers are closely linked, and academic research is often directly relevant to industrial research. The new approaches will improve metabolic engineering of obligate anaerobes, allowing progress not possible using previous techniques, thereby improving technology for sustainable manufacturing of chemicals.

The high-yield biological manufacture of bulk chemicals using anaerobic fermentation is an industrial technology of great value and importance to numerous companies. There are several such companies worldwide, including some based in the UK, or otherwise active in the UK. The approaches developed in the research programme could directly benefit the existing manufacturing processes of such companies, and could be important in the development of new manufacturing processes, which might involve other organisms, feedstocks and products. These benefits could in turn lead to UK jobs and other contributions to the UK economy and UK wealth.

Sustainable manufacturing is not only of economic benefit, but is also an important part of the necessary transition to sustainability and away from finite fossil carbon. This is crucial for the long-term energy security, food security, well-being and economic prosperity of the UK and nations worldwide.

The research will benefit the expertise and development of those directly involved in performing the research, and those who are part of relevant research communities at Imperial College. These benefits contribute to the UK skill base in the important areas of synthetic biology and industrial biotechnology.

The research is at the cutting-edge of synthetic biology and metabolic engineering, and will influence the advanced education of students at Imperial College by the principal investigator John Heap (JH) who lectures both undergraduate and postgraduate students on these topics.