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Mining the Arabidopsis Genome for Cytochrome P450 Biocatalysts

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Mining the Arabidopsis Genome for Cytochrome P450 Biocatalysts Mining the Arabidopsis genome for cytochrome P450 biocatalysts Maria Magdalena Razalan PhD University of York Biology September 2016 Abstract Cytochromes P450 (CYPs) constitute a wide group of NAD(P)H-dependent monooxygenases, found throughout all kingdoms of life. Among the most important functions of CYPs are the synthesis of bioactive compounds and the conversion of xenobiotics. These functions can be translated into biotechnological applications, such as the production of highly regio- and stereo-specific drug metabolites for the pharmaceutical industry, or to confer activity towards toxic compounds for agronomics and bioremediation purposes. Plants possess a large number of CYP sequences, but most still remain uncharacterised, due to the difficulty in the isolation of the membrane-bound enzyme and in the reconstitution of an active and efficient redox system. In this project, a fusion construct for the co-expression of the CYP with a suitable reductase was created. The construct consisted of a C-terminal Arabidopsis ATR2 reductase (codon-optimised for expression in E. coli and truncated of the N-terminal membrane anchor) connected through a poly-GlySer linker to the heme domain. An N-terminal Im9 peptide replaced the natural membrane-binding domain of the CYP. When CYP73A5 from Arabidopsis was cloned into the construct, it was able to convert almost 60 % of the substrate cinnamic acid to the hydroxylated derivative, in whole cell assays. This result demonstrated that this expression platform enables the expression of active redox self-sufficient P450 catalysts and it can be further utilised for the characterisation of orphan CYPs. Following from gene expression studies and reports on the existence of oxidative derivatives of TNT, the potential involvement of CYP81D11 in the detoxification of TNT was explored with different in planta assays, employing transgenic Arabidopsis lines and tobacco leaf discs. The results obtained were contrasting and did not provide a clear picture on the role of CYP81D11. Further studies have to be carried out in the future, using CYP81D11-knockout lines, as well as the purified enzyme. 2 List of Contents Abstract ......................................................................................................................... 2 List of contents ............................................................................................................... 3 List of tables ................................................................................................................... 8 List of figures ................................................................................................................. 9 Acknowledgements ...................................................................................................... 12 Declaration .................................................................................................................. 13 Chapter 1: Introduction ................................................................................................ 14 1.1 History of cytochrome P450 enzymes: the discovery ...................................... 14 1.2 Structure, nomenclature and organisation of CYP enzymes ........................... 18 Structure ............................................................................................................. 18 Nomenclature .................................................................................................... 22 Organisation ....................................................................................................... 23 Cytochrome P450 redox partners ...................................................................... 25 1.3 Catalytic cycle of cytochromes P450 ................................................................ 31 1.4 Human Cytochromes P450............................................................................... 33 1.5 Bacterial Cytochromes P450 ............................................................................ 37 1.6 Plant Cytochromes P450 .................................................................................. 42 1.7 Fungal Cytochromes P450 ................................................................................ 48 1.8 Challenges of using CYPs for biotechnological applications ............................ 49 Aim of the project .................................................................................................. 50 Chapter 2: General Materials and Methods ................................................................... 51 2.1 Chemical reagents ............................................................................................ 51 2.2 Organisms ......................................................................................................... 51 2.3 Media ............................................................................................................... 52 3 Media for bacterial growth ................................................................................ 52 Media for plant growth and assays .................................................................... 53 2.4 Molecular Biology techniques .......................................................................... 54 2.4.1 Polymerase Chain Reaction (PCR) & colony PCR ....................................... 54 2.4.2 DNA restriction digestion .......................................................................... 54 2.4.3 Dephosphorylation of linearised vector ................................................... 54 2.4.4 Agarose gel electrophoresis ...................................................................... 54 2.4.5 DNA isolation ............................................................................................. 55 2.4.6 Cloning: ligation, InFusion ......................................................................... 56 2.4.7 Preparation of competent cells ................................................................. 56 2.4.8 DNA transformation .................................................................................. 57 2.4.9. DNA sequencing ....................................................................................... 57 2.5 Biochemistry techniques .................................................................................. 58 2.5.1 Protein heterologous expression .............................................................. 58 2.5.2 Cell harvest & lysis..................................................................................... 58 2.5.3 Spectrophotometric evaluation of protein expression ............................. 59 2.5.4 SDS-PAGE verification ................................................................................ 59 2.5.5 Western blot verification........................................................................... 60 2.5.6 Protein purification ................................................................................... 60 2.5.7 Protein quantification ............................................................................... 62 2.5.8 Activity assay ............................................................................................. 62 Chapter 3: Soluble expression of Cytochromes P450 ...................................................... 63 3.1 Introduction: Bottlenecks in the heterologous expression of plant CYPs ....... 63 3.2 Materials and methods used for the soluble expression of plant CYPs .......... 68 3.2.1 Sequence analysis, PCR and cloning ......................................................... 68 3.2.2 Expression trials in E. coli: Rosetta 2, BL21(DE3), Arctic express .............. 72 4 3.2.3 Expression trials in yeast (Saccharomyces cerevisiae WAT11 modified strain) ................................................................................................................. 72 Media and buffers for yeast growth and harvest ............................................... 73 3.2.4 Purification ................................................................................................ 74 3.2.5 Protein characterisation: UV-Vis spectrophotometry and activity ........... 74 3.3 Results .............................................................................................................. 75 3.3.1 Sequence analysis and cloning .................................................................. 75 3.3.2 Expression trials ........................................................................................ 82 3.3.3 Expression in yeast .................................................................................... 85 3.3.4 Purification of the S-CYP73A5tr construct ................................................ 86 3.3.5 Activity assay ............................................................................................. 90 3.4 Discussion ......................................................................................................... 90 Gene expression in bacteria ............................................................................... 91 Gene expression in yeast.................................................................................... 92 Chapter 4: Expression screening and characterisation of P450- reductase fusion proteins 94 4.1 Introduction: plant P450-reductase fusions .................................................... 94 4.2 Materials and Methods used for the expression screening of the plant fusions ...............................................................................................................................
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