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Engineering new capacities for solar energy utilisation in bacteria

Reference	BB/M000265/1
Principal Investigator / Supervisor	Krebs Professor of B Christopher Hunter
Co-Investigators / Co-Supervisors	Professor Per Bullough, Professor Ashley Cadby, Professor Mark Dickman, Professor Wei Huang, Professor Matthew Johnson, Dr Jim Reid
Institution	University of Sheffield
Department	School of Biosciences
Funding type	Research
Value (£)	3,380,116
Status	Completed
Type	Research Grant
Start date	02/02/2015
End date	31/03/2021
Duration	74 months

Abstract

This sLoLa programme is organised into four interdependent sections. In the first, we will combine chlorophyll (Chl), bacteriochlorophyll (BChl) and carotenoid biosynthesis genes from bacteria and plants to create new pigment pathways tailored that extend the spectral range for absorbing solar energy. The morphology, organisation and performance of altered photosynthetic complexes and membranes comprising these novel pigments will be assessed in the second section using functional, proteomic and state-of-the-art imaging approaches, including electron microscopy, affinity mapping AFM, and super-resolution optical imaging. By applying selection pressure to grow under defined, restricted light regimes we will select engineered strains with improved photosynthetic performance. In the third part of the programme we will compile and then test multigene constructs for biosynthesis of proteorhodopsins, pigments, lipids, assembly factors and photosystem apoproteins. Following co-expression of these gene assemblies in heterotrophic bacteria such as E. coli and Ralstonia eutropha the outcomes will be assessed using simple growth tests, mass spectrometry, atomic force and electron microscopy, and spectroscopy. The fourth part of the programme, to be conducted in parallel with the other sections, proceeds initially by optimising the light-driven production of alkanes in Rhodobacter sphaeroides and Synechocystis, and by establishing an electro-biosynthesis platform for metabolite production in Ralstonia eutropha and Rhodobacter sphaeroides. Then this section builds on the design and construction of new photosynthetic systems in the other sections; proteorhodopsins, engineered pigment biosynthesis and photosystem pathways, augmented by hitherto undiscovered genes, will be used to enhance solar-powered production of metabolites in photosynthetic bacteria and in heterotrophs converted for absorbing and using light.

Summary

Photosynthesis captures the power of sunlight to drive the growth of plants on land and single-celled bacteria and plankton in the oceans, underpinning all global food chains and providing the oxygen we breathe. Because our planet Earth is mostly covered in water, the quantity and activity of water based photosynthetic bacteria is stupendous; billions of tonnes of photosynthetic bacteria grow in the oceans every year. These bacteria have to compete with each other for sunlight, and have evolved to live at different depths and environments, even growing in extreme conditions 100 metres or more below the surface. Sunlight is made up of a spectrum of many different colours of light and different bacteria have evolved specialised chemicals called pigments that absorb a particular colour of the spectrum. Future biotechnological applications of photosynthesis are likely to require multicoloured bacteria containing multiple pigments that can harvest more of the solar spectrum than evolution has demanded of them. That way they could use more solar energy for making chemicals useful for man. Achieving this would mean putting together 'mix and match' combinations of pigments from different bacteria inside one cell. This is now possible because we have been finding out how photosynthetic bacteria make each type of pigment - chlorophylls, bacteriochlorophylls, bilins and carotenoids. They do it by using sets of biological machines called enzymes that work together in a production line called a biosynthetic pathway. We have found that we can create new pigment biosynthesis pathways by combining the genetic codes for enzymes from more than one type of photosynthetic bacterium. This teaches us more about how the natural enzymes and pathways work and being able to build or make something is the ultimate test of whether you understand it. The first part of this research programme will create new pathways and combinations of pigments in a photosynthetic bacterium. The second partwill find out how these new pigment combinations work together to absorb new colours of light from the solar spectrum both inside the cell, and on biomimetic silicon chips. The third part starts the process of converting a bacterial cell such as E. coli, which is colourless and lives by respiring oxygen the way humans do, into a photosynthetic cell. The simple way to do this is by importing a primitive light-powered protein called proteorhodopsin from oceanic bacteria, but we will also begin the more ambitious large-scale genetic engineering of E. coli and similar bacteria so they can make bacteriochlorophyll, bilin and carotenoid pigments. Such cells will have internal solar panels that allow them to use sunlight for the first time. These light-powered cell factories have great potential for future biotechnology and bioenergy applications such as the production of, for example, alcohols, alkanes and novel pharmaceuticals. In the last part of this research programme we will take something that is already useful, in this case photosynthetic cells that make biodiesel, and use our pigment biosynthesis engineering to make them more efficient at using light to drive biodiesel production. We will go prospecting for new pigment biosynthesis genes, since we have only scratched the surface in terms of the number of pigment pathway genes out there in the oceans. New genes can be found using a machine that sees the colour of cells and plucks valuable single bacteria out of seawater so their DNA can be sequenced to look for new pigment pathways. We hope to use the genes we discover, as well as the genes we already know about, to build new bacteria that can capture and use solar energy. This knowledge is important to us all, not just because capturing and using solar energy fuels life, but it also holds the secret of using cells that one day could give us clean, unlimited energy and valuable chemicals from sunlight.

Impact Summary

Economic impacts Nanotechnology: Development of nanotechnological tools for imaging and functional measurements of biological samples. AFM, including affinity-mapping, and super-resolution optical microscopy, as well as picosecond lifetime and spectral imaging, will be developed and applied to membranes from plants and bacteria, and to enzymes and surface-attached nanoarrays of membrane protein complexes. AJC will test new optical microscope technologies and develop new applications and data analysis approaches for industrial partner Andor Technology. Bruker Nano Surfaces Division test new developments in probe manufacture in the CNH laboratory. Separation technologies. We collaborate with Thermofisher on innovative methodologies for separating peptides prior to MS analysis, and with Bruker for mass spectrometry. Synthetic biology. Dr Pin Yang, CEO of InCelliGEN, a biotechnology company specialising in genome and pathway synthesis, has expressed interest in our programme to construct 'photosynthesis modules' for expression in a variety of bacteria. High-Throughput Techniques. Development of novel high-throughput screening methods of single colonies of mutant bacterial strains (CNH/WH) will be undertaken in collaboration with BMG LabTech. Utilising next generation fluorescent plate readers which can produce fluorescent spectra will decrease the time taken to screen for new fluorescent pigments or knockouts. Biofuels, CO2 remediation and Biosensors. WH and CNH will work with Tata Steel, who are looking to cut CO2 emissions by 50% by 2015. Waste CO2 could be used as a carbon source for electrobiosynthesis in R. eutropha or R. sphaeroides. Societal Impacts The sLoLa team encompasses molecular genetics and biosynthetic pathway engineering (Huang, Hunter), biochemistry, spectroscopy, structural studies using electron microscopy and atomic force microscopy (Bullough, Hunter, Johnson), mass spectrometry (Dickman), and super-resolution/spectral imaging/lifetime microscopy (Cadby). The multidisciplinary nature of this research project will train young researchers in a wide variety of practical skills, providing an outstanding training environment for PhD students and postdoctoral scientists, working to meet ambitious technical goals and targets. Communication and engagement with potential beneficiaries Synthetic biology, food and energy security and the nano-scale world are highly topical in the media, and the work in this application will be communicated by, for example, the Krebs Festival, to be held in November 2015. This three-month, large-scale public engagement and outreach activity will be supported by this sLoLa; Matt Johnson and named PDRA Dr Nathan Adams are part of the steering committee. Ongoing University outreach activities for younger audiences (Festival of the Mind, Mobile University, Sheffield Science and Engineering Festival, Researchers Night) will be supported by PIs (Reid/Cadby/Johnson), and PDRAs and PhD students will be encouraged to present their research at these events over the course of the sLola. Nathan Adams is a presenter at national science festivals; in 2013 he undertook 28 science presenter/public engagement activities at the British Science Festival, Bang!, Cheltenham Science Festival, CBBC Live, Edinburgh Festival, TV and Radio with the support of the University and professional bodies, reaching over 2 million people through media appearances and demonstrations of science. NA will continue his science presenting on TV and radio during the sLoLa. PIs and PDRAs will disseminate important findings via press releases and interviews with the broadcast and print media, in collaboration with the office for public engagement.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Bioenergy, Industrial Biotechnology, Microbiology, Plant Science, Structural Biology, Synthetic Biology
Research Priority	X – Research Priority information not available
Research Initiative	Longer and Larger Grants (LoLas) [2007-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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