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Structural and Biochemical Characterization of Members of the Cannabinoid Biosynthetic Pathway to Inform Their Application in Synthetic Biology

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Structural and Biochemical Characterization of Members of the Cannabinoid Biosynthetic Pathway to Inform Their Application in Synthetic Biology Structural and Biochemical Characterization of Members of the Cannabinoid Biosynthetic Pathway to Inform their Application in Synthetic Biology A thesis submitted to the University of Manchester for the degree of Master of Philosophy in the faculty of science and engineering 2017 Lewis J. Kearsey School of Chemistry 1 Contents List of tables .................................................................................................................................................................. 4 List of figures ................................................................................................................................................................ 4 List of abbreviations .................................................................................................................................................. 7 Abstract ........................................................................................................................................................................... 9 Declaration ................................................................................................................................................................. 10 Copyright statement ............................................................................................................................................... 10 Acknowledgements ................................................................................................................................................ 11 1. Introduction ........................................................................................................................................................... 12 1.1 Fundamentals of synthetic biology .................................................................................................... 12 1.2 Uses of synthetic biology........................................................................................................................ 13 1.3 Microbial chassis used in synthetic biology .................................................................................. 13 1.4 Synthetic biology approaches .............................................................................................................. 14 2. Structural biology ............................................................................................................................................... 16 2.1 Principles of structural biology ............................................................................................................. 16 2.2 Crystallization ............................................................................................................................................... 16 2.3 X ray crystallography ................................................................................................................................ 20 2.4 Macromolecule structure solving ........................................................................................................ 22 3. Cannabinoids ....................................................................................................................................................... 25 3.1 The role of cannabinoids in plants ..................................................................................................... 25 3.2 Endocannabinoid system ....................................................................................................................... 27 3.3 The therapeutic potential of cannabinoids ..................................................................................... 30 4. Cannabinoid biosynthetic pathway ........................................................................................................... 36 4.1 Hexanoyl-CoA and malonyl-CoA ....................................................................................................... 37 4.2 Tetraketide synthase ................................................................................................................................ 38 4.3 Olivetolic acid cyclase .............................................................................................................................. 41 4.4 Aromatic prenyltransferase ................................................................................................................... 43 4.5 Tetrahydrocannabinolic acid synthase ............................................................................................ 43 4.6 Cannabidiolic acid synthase ................................................................................................................. 45 4.7 THCA and CBDA decarboxylation..................................................................................................... 47 5. Further work on the pathway ....................................................................................................................... 48 6. Objectives of this study ................................................................................................................................... 49 6.1 Expression of functional proteins ....................................................................................................... 49 6.2 Determining the structure of TKS ....................................................................................................... 49 6.3 Mutant design to improve efficiency of TKS ................................................................................. 50 2 7. Materials and methods .................................................................................................................................... 51 7.1 Genes, vectors and expression strains ........................................................................................... 51 7.2 Transformation and expression protocol ........................................................................................ 51 7.3 Harvesting cells and protein purification ......................................................................................... 52 7.4 SDS-PAGE .................................................................................................................................................... 53 7.5 Removal of protein tags .......................................................................................................................... 53 7.6 Size exclusion chromatography .......................................................................................................... 53 7.7 TKS crystallization and structure determination ......................................................................... 54 7.8 Adding soluble tags to OAC .................................................................................................................. 55 7.9 OAC size exclusion chromatography ............................................................................................... 57 7.10 Biotransformations .................................................................................................................................. 57 7.11 Mass spectrometry of organic products ....................................................................................... 58 7.12 TKS mutant design and activity testing ........................................................................................ 58 8. Results and discussion ................................................................................................................................... 61 8.1 TKS expression and purification ......................................................................................................... 61 8.2 OAC expression and purification ........................................................................................................ 66 8.3 Introduction of soluble tags to OAC .................................................................................................. 68 8.4 Point mutations of TKS ............................................................................................................................ 76 8.5 Liquid chromatography mass spectrometry of biotransformations ................................... 82 8.6 TKS structure ............................................................................................................................................... 89 9. Conclusions ........................................................................................................................................................ 105 10. Future work ...................................................................................................................................................... 107 Bibliography ............................................................................................................................................................. 108 Word count: 30,796 3 List of tables Table 1 - X-ray data collection and refinement statistics…………………………………………..53 Table 2 - Primers for OAC amplification…………………………………………………………..…..54 Table 3 - HPLC conditions……………………………………………………………………..………...57 Table 4 - Primers to introduce TKS mutations……………………………………………………….58 List of figures Figure 1.1 Siting drop vapour diffusion well diagram……………………………………….……..20 Figure 1.2 Chemical structures of THCA and CBDA………………………………………………..25 Figure 1.3 Chemical structures of anandamide and 2-AG…………………………………………28 Figure 1.4 Endocannabinoid retrograde
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