Engineering the bacterium Rhodopseudomonas palustris as a platform for electrosynthetic bioproduction

In the context of global climate change and population growth, there is a need to replace fossil fuels with renewable sources of energy such as wind and solar power. However, if renewables are to be more widely taken up, new storage technologies are required to manage the fluctuations in the power they supply compared to demand. A second global challenge is meeting the growing demand for sustainably produced chemicals, both for fuels and for manufacturing.

This project offers a comprehensive solution to the problems of energy storage and sustainable chemicals production, in the form of an electro-active biological material formed of genetically engineered bacteria. Electro-active bacteria can interact with metals in their environment and exchange electrons with their internal metabolism. Electrons that are taken up are used supply energy and to drive chemical reactions. Biological materials have the advantage of being self-assembling and self-repairing, and they capture carbon as they grow which decreases their environmental impact. The engineered bacteria would be grown in specialised electro-chemical reactors, allowing for secure containment and efficient use of land.

This project will use the bacterium Rhodopseudomonas palustris, which is naturally electro-active and also has broad metabolic capabilities. This includes being able to harness light to produce energy, and the ability to capture both carbon dioxide (just as plants do when they photosynthesise) and nitrogen gases. The organism can also use electrical current to convert simple molecules into useful chemicals for fuels or manufacturing, or growth of the cell. We aim to engineer Rh. palustris so that it is more effective at taking up electrical current, and efficiently uses the electrical input to produce large amounts of desirable molecules. This would enable electrical energy to be converted into and stored as fuels such as hydrogen gas, or used for the production of useful molecules such as bio-plastics. Transfer of electrical current between cells and an electrode has already been demonstrated in Rh. palustris, showing the feasibility of this project.

Synthetic biology is an approach to building designer organisms that uses standardised genetic parts and modular design to more predictably engineer their behaviour. This project requires the development of new genetic components for Rh. palustris. We will characterise libraries of genetic parts that will give us control over when and how strongly certain genes are expressed. We will also optimise methods for making genetic alterations to the Rh. palustris chromosome and expressing foreign genes. These tools will also be useful to other researchers who are working with Rh. palustris, and may be transferable to related organisms.

We will also do fundamental research into how Rh. palustris takes up electrons, at the level of gene expression. This will inform us about how we might need to alter the gene expression to channel the organism's energy and metabolic pathways into useful activities. Again, this will be useful to other researchers looking to engineer Rh. palustris to produce desirable chemicals.

Together, these genetic tools and data will enable us to introduce genes into Rh. palustris that give it the capability to effectively import electrical current, and to produce useful chemicals that it cannot naturally synthesise. We will also add genetic elements to control the switching between these different activities. Ultimately we aim to have integrated all these new functions together, producing a prototype organism for the conversion of electrical power to chemicals.

The project aims to build and characterise genetic part libraries to facilitate rational engineering of the bacterium Rhodopseudomonas palustris. We will apply these tools to create electrosynthetic strains capable of producing useful chemicals by taking up electrical current as a source of reducing power for the nitrogenase enzyme and carbon dioxide fixation.

Genetic parts will be designed using the BASIC standard. We will validate the function of plasmid origins of replication and antibiotic resistance genes, and characterise constitutive and inducible promoters, RBSs, and transcription terminators using high-throughput plate-reader assays. We will adapt the CRISPR-dCas9 programmable repressor for use in Rh. palustris to enable parallel knockdown of genes for metabolic flux redirection. We will adapt and characterise the function of TetR-family repressors in Rh. palustris to enable synthetic regulatory structures to control gene expression.

In order to showcase the potential of Rh. palustris for bioproduction applications, we will build synthetic operons for biosynthesis of methane, hydrogen, and (R)-3-hydroxybutyrate (3HB). Methane and hydrogen are produced by the nitrogenase (methane production requires a nifD variant allele). 3HB synthesis will use fixed carbon; it is the precursor to the bioplastic polyhydroxybutyrate but is more easily quantified. Variant operons with differing relative expression rates of component genes will be tested. To improve biosynthesis we will use dCas9 to knock down genes for competing pathways.

We will perform RNAseq analysis of Rh. palustris cells growing photoautotrophically on an electrode to reveal electron uptake mechanisms in addition to the known pio genes. Electron uptake genes will be overexpressed to increase the supply of reducing equivalents to the photosystem, and ultimately will be integrated with the biosynthetic pathway and metabolic flux channelling systems.

Our proposed research has potential for major economic and societal impact through the development of a biological catalyst for the conversion of electrical power to chemicals. This technology will help to mitigate the barriers to integration of renewable energy sources into electrical supply networks caused by mismatches in supply and demand: excess electrical power can be used to produce chemical fuels for storage and later use. Additionally, the same mechanism can be applied to the bio-production of bulk chemicals, which is a major source of anthropogenic greenhouse gases (GHGs). GHG emissions will therefore be reduced by encouraging renewable electricity supplies and providing a more sustainable route for chemicals production. This benefits society at large by helping to combat the climate change processes that are having massive destabilising effects around the world. With respect to the economy in general, this is a potential foundational technology that will facilitate the UK's ambitious commitments to decarbonise its economy, and will provide greater diversity and resilience in our capacity to produce energy and materials. Ensuring that basic needs of industry (i.e. energy and bulk chemicals) are met sustainably will have pervasive and wide-ranging benefits.

The UK aims to have a world-leading bio-economy, using sustainable and efficient biological processes to meet challenges ranging from food production to healthcare. There is a fundamental need to develop new strains of microorganism for the bio-industry, as different microorganisms will be suited to different applications. To meet its higher aim of engineering a sophisticated synthetic microbe for bio-electrochemical catalysis, this project will first produce foundational genetic tools for purple non-sulphur bacteria, a group of microorganisms that possess a wide-range of industrially relevant properties. The genetic tools and techniques, and bacterial strains we develop through this project will be beneficial to both industrial (and academic) researchers, and will form the basis for further translational work to create useful microorganisms for the bioeconomy.

In addition to these tangible benefits, university-led foundational work in synthetic biology also enables the emerging bio-economy through the professional development of researchers, and through contributions to national and international policy-making. The expertise of researchers involved in this work, and the College's strategy groups for industrial biotechnology and synthetic biology, will be used to direct the exploitation of discoveries made in this and similar projects, and will inform future policy-making processes.

Mature results arising from the project will be communicated to the industrial sector through channels such as peer-reviewed publications, international and national conferences, and Industry Clubs. The College's Enterprise department offers valuable resources to support longer term future commercial developments arising from this research. More informal links to industry will be fostered to sound out follow-on interest and opportunities such as iCASE studentships linked to the project area.