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PhD Projects for Bioenergy and Industrial Biotechnology

Dr Tuomas Knowles, Dept. of Chemistry

Second Supervisor/Collaborator: Chris Howe

Microfluidic biological photovoltaic cells

PhD Project Description: This project focuses on biological photovoltaic cells (BPVs), which allow the production of electricity directly from oxygenic photosynthetic microorganisms (e.g., cyanobacteria), in this case cyanobacteria (oxygen-producing photosynthetic bacteria). This technology has advantages over established photovoltaic systems (PV) in (i) not requiring energy-intensive photo active components (e.g., semiconductors), (ii) generating a more stable current output and (iii) being able to generate electricity in the dark (from metabolites stored by the microorganisms). At present, the maximal output power density from BPVs (ca 100 mW m-2) is not high enough for them to be practicable for bulk supply of energy, but there is significant theoretical potential to improve on this limitation. The aim of the present project is to bring together the rapidly burgeoning field of microfluidics and synthetic biology. Microfluidics allows us to work in volumes that are around a million times smaller than conventional systems, which transforms how these devices work. For example, the distances which charge carriers have to migrate within the devices can be shortened dramatically relative to conventional macroscopic cells, reducing resistive losses in the electrolyte. In addition, the readily achievable conditions for laminar flow in microfluidic devices permit operation without the use of a proton-exchange membrane to separate anode and cathode compartments, required in macroscopic systems. Dispensing with this membrane should reduce the resistive losses even further. These advantages combine to allow us to study the energy output from small numbers of cyanobacteria, and possibly even single cells, and so screen a heterogeneous population of cells for those with enhanced output.

Referees:

1) McCormick AJ, Bombelli P, Scott AM, Philips AJ, Smith AG, Fisher AC, Howe CJ (2011) Photosynthetic biofilms in pure culture harness solar energy in a mediatorless bio-photovoltaic (BPV) cell system. Energ Environ Sci 4:4699-4709.

2) Bombelli P, Mueller T, Herling TW, Howe CJ, Knowles TPJ (2014) A high power-density mediator-free microfluidic biophotovoltaic device for cyanobacterial cells. Advanced Energy Materials DOI: 10.1002/aenm.201401299.

3) McCormick AJ, Bombelli P, Bradley RW, Thorne R, Wenzel T and Howe CJ (2015) Biophotovoltaics: oxygenic photosynthetic organisms in the world of bioelectrochemical systems. Energy Environ. Sci., 2015, DOI: 10.1039/C4EE03875D.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www-knowles.ch.cam.ac.uk/ http://www.bioc.cam.ac.uk/howe

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Lucy Colwell, Dept. of Chemistry

Second Supervisor/Collaborator: Steven Lee

Design of nanobody-fluorescent protein interactions for use in next generation single-molecule super-resolution microscopy

PhD Project Description: Nanobodies are single-chain antibodies derived from camelids that bind to protein targets with high affinity and specificity [1]. These properties have recently been exploited to produce nanobodies that efficiently bind green fluorescent protein (GFP), thus enabling any GFP-tagged construct to yield nanometer spatial resolution in single-molecule super-resolution microscopy [2]. However, imaging of more complex biological phenomena: such as macromolecular complexes and other bio-interactions requires targeted development of new nanobodies specific for fluorescent protein variants whose spectral emission differ to allow efficient multicolour imaging. This project brings together theoretical and experimental tools in a synergistic way to better understand how sequence and structure relate to binding affinity in order to improve bio-imaging. Statistical inference techniques, based on amino acid coevolution, will be used to construct a model for the molecular specificity code that dictates nanobody-protein interaction specificity. The student will 1) create a database of known nanobody-protein interactions to characterise the specificity determining nanobody sequence residues, 2) use the resulting model to design nanobodies that interact specifically with the desired fluorescent protein targets and 3) express the designed nanobodies in bacteria and test their efficacy using super-resolution imaging tools in the Lee lab. The project has the potential to further our understanding of rational design of protein interactions in a broad context.

Referees:

[1] Muyldermans, Serge. "Nanobodies: natural single-domain antibodies." Annual review of biochemistry 82 (2013): 775-797.

[2] Ries, Jonas, et al. "A simple, versatile method for GFP-based super-resolution microscopy via nanobodies." Nature methods 9.6 (2012): 582-584.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.ch.cam.ac.uk/person/ljc37 http://www.ch.cam.ac.uk/person/sl591

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Prof Alison Smith, Dept. of Plant Sciences

Algal biotechnology

PhD Project Description: Many microalgae grow fast and produce large amounts of lipid molecules that might be used for biofuels or other industrial chemical purposes. Because these organisms are photosynthetic, they offer the potential of a more sustainable source for compounds currently obtained from fossil fuels. However, for this to become a commercial reality many challenges must be overcome, in particular the need to establish ways to cultivate microalgae at industrial scale, whilst avoiding contamination by bacteria, viruses or grazers, and ensuring reproducible synthesis of the desired chemicals. In my lab, we are studying many aspects of algal biotechnology, including: algal molecular biology and genomics – identifying and characterizing genes involved in lipid metabolism [ref 1 below] development of synthetic biology approaches for algal metabolic engineering, including use of riboswitches [ref 2] algal-bacterial symbiosis as a means to protect against contamination by unwanted bacteria [ref 3]. All the projects use a combination of molecular biology, biochemistry and algal physiology, and include either synthetic biology or modelling approaches, depending on the interests and experience of the student. All projects are interdisciplinary and will have a second supervisor in another Dept.

Referees:

1. Chen JE and Smith AG (2012) A look at diacylglycerol acyltransferases (DGATs) in algae. J Biotechnol 162: 28-39

2. Helliwell KE, Scaife MA, Sasso S, Ulian de Araujo AP, Purton S and Smith AG (2014) Unravelling vitamin B12­-responsive gene regulation in algae. Plant Physiol 165: 388-397

3. Kazamia E, Riseley AS, Howe C J and Smith AG (2014) An engineered community approach for industrial cultivation of microalgae. Indust Biotechnol 10: 184-190; doi:10.1089/ind.2013.0041

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.plantsci.cam.ac.uk/research/alisonsmith

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Professor Paul Dupree, Dept. of Biochemistry

Patterning of glucuronic acid decorations on xylan

PhD Project Description: Xylan and cellulose are the most abundant polymers on earth, and provide an emormous resource for food, feed and renewable materials, including biofuels. Nevertheless, the way the functional material properties arise from the structure and interaction of these polysaccharides is not understood. This project will use plant molecular genetics and biochemistry to manipulate the synthesis of GlcA side chains and hence structure of xylan. The consequences on material properties will be examined, leading to new hypthotheses of the cell wall structure: function relationship.

Referees:

Bromley JR, Busse-Wicher M, Tryfona T, Mortimer JC, Zhang Z, Brown D, Dupree P. (2013) GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns. Plant J. 74, 423–34.

Mortimer JC, Miles GP, Brown DM, Zhang Z, Segura MP, Weimar T, Yu X, Seffen KA, Stephens E, Turner SR, Dupree P (2010) Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass. Proc Natl Acad Sci U S A. 107, 17409-14

Brown DM, Goubet F, Wong VW, Stephens E, Goodacre R, Dupree P, Turner SR (2007) Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis. Plant Journal 52, 1154-1168

Link: http://www.bioc.cam.ac.uk/people/uto/dupree

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Dr Erwin Reisner, Dept. of Chemistry

Second Supervisor/Collaborator: Prof Chris Howe / Department of Biochemistry

Artificial Photosynthesis with Semi-Biological Devices

PhD Project Description: Artificial photosynthesis is a process that uses sunlight and abundant resources such as water and carbon dioxide to make renewable fuels, so called solar fuels. This renewable process requires the finely tuned combination of light absorption, charge separation and chemical catalysis, which is well evolved, but shows an overall poor efficiency for fuel synthesis in biology. The Reisner (Chemistry) and Howe (Biochemistry) laboratories are developing novel approaches to overcome major obstacles in biological fuel production by interfacing photosynthetic enzymes such as the water oxidation enzyme Photosystem II and cyanobacterial cells with electrodes.[refs 1,2] Rational strategies for the assembly of such semi-biological hybrid materials will be developed in this PhD project with an emphasis of understanding the bio-materials interface and developing the rationale to integrate biological materials in nanostructured electrode materials to optimize light harvesting and electricity generation. The biological hybrid electrodes can then be wired via an artificial electric circuitry to a range of non-platinum catalysts to generate energy vectors such as hydrogen from Earth abundant resources. The range of hydrogen evolving catalysts includes the hydrogen evolving enzyme hydrogenase[ref 3] and other chemical, enzymatic and microbial catalysts. This project will therefore develop the under-explored area of solar energy conversion with enzymes and cyanobacteria on semiconductor electrodes and develop new ways to produce sustainable fuels from the natural resources such as sunlight and water.

Referees:

(1) Kato, M.; Zhang, J. Z.; Paul, N.; Reisner, E. Chem. Soc. Rev., 2014, 43, 6485–6497.

(2) McCormick, A.J.; Bombelli, P.; Lea-Smith, D.J.; Bradley, R.W.; Scott A.M.; Fisher, A.C.; Smith, A.G.; Howe, C.J. Energy Environ. Sci., 2013, 6, 2682–2690.

(3) Caputo, C. A.; Gross, M. A.; Lau, V. W.; Cavazza, C.; Lotsch, B. V.; Reisner, E. Angew. Chem. Int. Ed., 2014, 53, 11538–11542.

Link: http://www-reisner.ch.cam.ac.uk/ http://www.bioc.cam.ac.uk/howe

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Martin Welch, Dept. of Biochemistry

Engineering plant cell wall-degrading bacteria to more effectively degrade cellulostic biomass.

PhD Project Description: As its name suggests, Pectobacterium atrosepticum (Pca) degrades the pectin and cellulose components of plant cell walls. It does so by secreting a welter of exceptionally potent extracellular enzymes (generically, pectinases, cellulases and proteases) that digest cellulsotic biomass. The synthesis and secretion of these cell wall degrading enzymes is under the dual control of two global control mechanisms; quorum sensing system and ppGpp. These two regulatory systems act in concert to ensure that plant cell wall degrading enzymes (PCWDEs) are only made when the population is (i) "quorate" and (ii) starving. The aim of this project is to manipulate these global control mechanisms to up-regulate PCWDE production, with the ultimate goal of generating strains that facilitate the maceration and digestion of cellulostic biomass. Pca is genetically amenable and the genome sequence of the lab strain is available.

Referees:

1, Steven D. Bowden, Alison Eyres, Jade C.S. Chung, Rita E. Monson, Arthur Thompson, George P.C. Salmond, David R. Spring and Martin Welch. Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp. Molecular Microbiology 90 : 457-471 (2013).

2, Steven D. Bowden, Nicola Hale, Jade C.S. Chung, James T. Hodgkinson, David R. Spring and Martin Welch. Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing. Microbiology 159 : 2375-2385 (2013).

3, Pérez-Mendoza D, Coulthurst SJ, Humphris S, Campbell E, Welch M, Toth IK, Salmond GP. A multi-repeat adhesin of the phytopathogen, Pectobacterium atrosepticum, is secreted by a Type I pathway and is subject to complex regulation involving a non-canonical diguanylate cyclase. Mol Microbiol (2011) 82(3):719-733.

Other relevant themes: Food Security

Link: http://www.bioc.cam.ac.uk/people/uto/welch

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Dr Erwin Reisner, Dept. of Chemistry

Second Supervisor/Collaborator: Dr Judy Hirst, Medical Research Council (MRC) Mitochondrial Biology Unit (MBU)

Reduction of carbon dioxide to renewable fuels using formate dehydrogenase enzymes

PhD Project Description: The reduction of the greenhouse gas CO2 is one of the most pressing challenges of the 21st century. Photosynthesis is the only large-scale process capable of converting CO2 from the atmosphere into a fuel, and the stability and inertness of CO2 has prevented the development of any comparable industrial processes. An industrial process that could use solar (or any other green) energy to make a fuel out of CO2 would revolutionise modern society. This project aims to explore a new approach for the development of such a process by exploiting formate dehydrogenases (FDHs), enzymes that catalyse the reduction of CO2 to the energy vector formate. Chemically, formate is one of the simplest hydrocarbons - it is already used as a chemical building block (feedstock) in industry, and formate 'fuel cells' are being developed. This project will be executed in a BBSRC-supported (BB/I026367/1 & BB/J000124/1) and highly collaborative setting between the Hirst and Reisner groups in Cambridge (at the Medical Research Council and Department of Chemistry, respectively) where we have recently reported the first breakthroughs and demonstrated the formidable CO2 reduction ability of tungsten and molybdenum FDHs (refs 1 & 2). The project will involve sophisticated biochemical, electrochemical and physical techniques to elucidate the mechanistic details of FDH catalysis and develop prototype devices with FDH that show how efficient CO2 reduction catalysis can be both powered by solar radiation (in a process called ‘artificial photosynthesis’) and exploited in fuel cells (ref 3).

Referees:

1: Reda, T.; Plugge, C. M.; Abram, N. J.; Hirst, J. Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 10654−10658.

2: Bassegoda, A.; Madden, C.; Wakerley, D. W.; Reisner, E.; Hirst, J. J. Am. Chem. Soc., 2014, 136, 15473–15476.

3: Kato, M.; Zhang, J. Z.; Paul, N.; Reisner, E. Chem. Soc. Rev., 2014, 43, 6485–6497

Other relevant themes: Bioenergy and Industrial Biotechnology

Link: http://www-reisner.ch.cam.ac.uk/ http://www.mrc-mbu.cam.ac.uk/people/hirst

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Professor Peter Leadlay FRS, Dept. of Biochemistry

Genome mining, enzymology and synthetic biology of antibiotic biosynthesis clusters from actinomycete bacteria

PhD Project Description: Natural products continue to be a source of novel chemical diversity and a valuable starting point for drug discovery. Further, most novel enzymes are ones engaged in specific biosynthesis. We have optimised protocols for rapid and complete in-house sequencing of 8-12 Mbp genomes of Streptomyces and allied filamentous soil bacteria, already responsible for over 70% of all known natural product-based drugs. Especially for polyketides and nonribosomal pepetides, whose biosynthesis follows the remarkable modular assembly-line paradigm, it is possible quickly to match observed compounds (studied by high-resolution mass spectrometry) with the gene cluster responsible, allowing rapid entry into the enzymology and engineering of individual bioactive metabolites, including immunosuppressants, antifungals and anticancer compounds. Conversely, the genome sequence allows an immediate overview of the total biosynthetic capacity of each strain and opens the way to targeted approaches to activate silent clusters that appear to encode novel molecules; and to engineer production of new analogues of selected lead molecules using cutting-edge technologies.

Link: http://www.bioc.cam.ac.uk/leadlay

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Professor Christopher Howe, Dept. of Biochemistry

Second Supervisor/Collaborator: Alison Smith (Plant Sciences), John Dennis (Chemical Engineering & Biotechnology)

PhD Project Description: Photosynthetic Organisms in Biotechnology and Health We are interested in a number of aspects of the molecular biology of photosynthetic organisms and their exploitation, including the malaria parasite Plasmodium. Specific areas include: (i) Direct electricity production from photosynthetic micro-organisms. Surprisingly, cyanobacterial and eukaryotic algal cells produce a small amount of external current on illumination, which can be harvested by an anode and used to drive an external circuit. We are interested in understanding how to make these ‘biophotovoltaic’ devices more efficient, and to drive additional processes, such as hydrogen production. We are also developing similar devices using land plants. (ii) Manipulation of photosynthetic microorganisms, including cyanobacteria and purple photosynthetic bacteria, for production of useful products. This includes work on light harvesting, electron transfer (including a novel c-type cytochrome we discovered) and metabolic engineering (including expressing heterologous genes and modifying endogenous ones). We have a wide range of molecular genetic platforms available for this. We are also trying to develop dinoflagellate algae as a biotechnology platform. (iii) Novel antimalarial targets. Surprisingly, the malaria parasite Plasmodium has a photosynthetic ancestry and a remnant chloroplast, which is essential for the parasite to survive. This offers an attractive target for antimalarials. We are focusing on transcription of the remnant chloroplast genome and post-transcriptional RNA processing. We have identified a number of candidate nuclear genes for chloroplast proteins that are likely to be involved in these processes.

Referees:

Hydrogen production through oxygenic photosynthesis using the cyanobacterium Synechocystis sp. PCC 6803 in a bio-photoelectrolysis cell (BPE) system. McCormick AJ, Bombelli P, Lea-Smith DJ, Bradley RW, Scott AM, Fisher AC, Smith AG, Howe CJ. (2013) Energy & Environmental Science 6:2682-2690.

Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities Lea-Smith DJ, Ross N, Zori M, Bendall DS, Dennis JS, Scott SA, Smith AG, Howe CJ. (2013) Plant Physiology 162:484-495.

The purification of crude glycerol derived from biodiesel manufacture and its use as a substrate by Rhodopseudomonas palustris to produce hydrogen. Pott RWM, Howe CJ, Dennis JS (2014) Bioresource Technology 152:464-470.

Link: http://www.bioc.cam.ac.uk/people/uto/howe

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