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Phd Thesis Martin G. Klompmaker Insights Into Carotenoid

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Phd Thesis Martin G. Klompmaker Insights Into Carotenoid PhD thesis Martin G. Klompmaker Insights into carotenoid sequestration mechanisms in Tomato fruit (Solanum lycopersicum) and the effect of ectopic expression approaches on carotenoid biosynthesis This thesis was submitted for the degree of Doctor of Philosophy at Royal Holloway University of London, June 2017 1 Declaration of authorship I hereby declare that the work presented in this thesis is the original work of the author unless otherwise stated. Original material used in the creation of this thesis has not been previously submitted either in part or whole for a degree of any description from any institution. _________________ Martin G. Klompmaker 2 Abstract Carotenoids are involved in a wide range of plant processes varying from development to defence, pollination and seed dispersal. The consumption benefits of carotenoids for human health and nutrition have led to development of an interest in the industrial application of carotenoids in food, feed and cosmetics. Tomato (Solanum lycopersicum) is the model plant for carotenoids related studies with its fruits which contain high levels of carotenoids. Carotenoid sequestration is an important process linked to carotenoid biosynthesis which requires further elucidation. A selection of tomato lines perturbed in carotenoid biosynthesis was studied for their differences in sequestration mechanisms. Premature accumulation of carotenes in a tomato line constitutively expressing PSY1 led to the differentiation of chloroplasts to chromoplast in immature fruit to create a higher capacity for carotenoid accumulation. Tomato lines rr, ogC and tan, knock out lines for PSY1, LCY-B and CRT-ISO respectively, demonstrated different distributions between plastoglobules and crystalline membrane structures associated with cis-carotenes and trans-carotenes. The role of carotenoid accumulation and changes in carotenoid profiles suggests the plastid can adapt to changes in carotenoid content through plastid differentiation and preferential sequestration. Ectopic expression of the MEP and bacterial carotenoid (crtE, crtB, crtI) pathway in the cytosol has hereby been attempted to enhance carotenoid content as an alternative to metabolic engineering of endogenous key enzymes. The isoprenoid biosynthesis clusters are spread over various cellular compartments in plants. A modular cloning system (GB Cloning) was used for the assembly of vectors with the genes of the MEP and bacterial carotenoid pathway in single or multi gene combinations. The ectopic expression of crtB led to the accumulation of phytoene (14-fold), phytofluene and lycopene, and increased levels of lutein and β-carotene. The impact of the enzyme on pathway intermediates beyond its function suggest a signalling cascade based on cytosolic apocarotenoids derived from the CRTB activity. 3 Acknowledgements After finishing my master’s degree in the Netherlands I was looking for a PhD project within the field of Plant Biotechnology focussing on metabolic engineering. I am very happy with the opportunity provided by Professor Paul Fraser to pursue a PhD-project in his laboratory at the Royal Holloway University and the support of the EU consortia DISCO and MultiBioPro. A PhD-project is a personal achievement, but I would have never managed without help throughout the years I have worked on my project. I thank Professor Paul Fraser and Professor Peter Bramley, my supervisor and advisor respectively, for their support and questions to keep me focussed during the course of my PhD. Furthermore I would like to thank Dr Genny Enfissi and Dr Marilise Nogueira for the helpful discussions during our project meetings for the EU DISCO project and their help in the laboratory. Also Chris Gerrish for his very helpful technical support and my project student Veronica Czernik for her work on the cloning of carotenoid biosynthesis vectors. I am grateful for the fantastic group of colleagues who have all contributed to my project and in general to three pleasant years within this team. I thank my family and friends, who have been supportive, helped me stay optimistic and have been great company to calm down after stressful moments. Doing a PhD is a big challenge, but I am sure I can implement all the valuable lessons in my future carrier and life. Thank you! Martin Klompmaker 4 Table of Contents Declaration of authorship ............................................................................................................. 2 Abstract ......................................................................................................................................... 3 Acknowledgements ....................................................................................................................... 4 Table of Contents .......................................................................................................................... 5 List of Figures ................................................................................................................................ 7 List of tables ................................................................................................................................ 10 List of abbreviations .................................................................................................................... 11 1. Introduction ........................................................................................................................ 14 1.1. The Isoprenoid network .............................................................................................. 15 1.2. Carotenoids: biosynthesis and sequestration ............................................................. 24 1.3. Isoprenoid regulation and carotenoid functions in plants ......................................... 32 1.4. Applications of carotenoids in health and industry .................................................... 39 1.5. Genetic engineering and model plants ....................................................................... 44 1.6. Aims and objectives .................................................................................................... 48 1.7. Scientific and economic rationale ............................................................................... 49 2. Materials and Methods ....................................................................................................... 53 2.1. Starting materials and sampling ................................................................................. 54 2.2. General molecular biology techniques ....................................................................... 55 2.3. Competent cells and transformations of bacteria ...................................................... 58 2.4. Golden Braid Cloning .................................................................................................. 60 2.5. Subplastid fractionation .............................................................................................. 62 2.6. Transient transformation in N.benthamiana .............................................................. 63 2.7. Metabolite extractions ................................................................................................ 64 2.8. Chromatography techniques ...................................................................................... 66 2.9. Data analysis and statistics ......................................................................................... 68 3. Analysis of plastidial carotenoid sequestration in mutants and transgenic varieties with different carotenoid contents. .................................................................................................... 70 3.1. Introduction ................................................................................................................ 71 3.2. Resources: tomato mutant lines perturbed in carotenoid biosynthesis .................... 72 3.3. Subplastid fractionation: description of expected outcomes ..................................... 75 5 3.4. Results ......................................................................................................................... 77 3.5. Discussion .................................................................................................................. 102 4. Ectopic expression of isoprenoid pathways: the development of a synthetic biology vector library using the Golden Braid modular cloning system. .......................................................... 108 4.1. Introduction .............................................................................................................. 109 4.2. Golden Braid cloning ................................................................................................. 110 4.3. Results: modular cloning library for isoprenoid biosynthesis ................................... 113 4.4. Discussion .................................................................................................................. 143 5. Ectopic expression of isoprenoid pathway genes: screening and validation of an isoprenoid Synthetic Biology vector library .............................................................................. 148 5.1. Introduction .............................................................................................................. 149 5.2. Transient leaf infiltrations in N. benthamiana .......................................................... 150 5.3. Results ......................................................................................................................
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