Enabling the P450 complement of Beauveria bassiana and Rhodococcus jostii for biocatalysis Claudia Spandolf PhD University of York Chemistry October 2015 I II Abstract Enzyme discovery today is proceeding at an enormous pace due to ever-growing technology development. As a result of this, more than 21000 cytochrome P450s have been identified in all kingdoms of life to date, making a wide range of enzyme resources with outstanding potential for biocatalytical implementation available. P450s from filamentous fungi, such as Beauveria bassiana, represent particularly compelling targets for the discovery of novel enzymes as these organisms have a long history of application in industrial hydroxylation reactions, many of which are believed to be P450-dependent. In addition, the genome sequence of B. bassiana has recently been completed revealing 83 putative P450s. In order to uncover new cytochrome P450-based biocatalysts from the fungus Beauveria bassiana extensive bioinformatics analysis of the Beauveria CYPome were performed. As a result 7 genes encoding for heme domains with possible alkane hydroxylase function and one encoding a naturally fused P450 with homology to P450foxy from Fusarium oxysporum could be identified for subsequent cloning, heterologous expression and characterization. Different expression hosts as well as various expression conditions have been investigated. Despite our efforts, delivery of active biocatalysts could not be realized. However, empirical data acquired in this project will be of value for future studies of fungal P450s. In addition, 23 cytochrome P450 heme domains from Rhodococcus jostii fused to the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784 have been investigated in a further strand of this work and provided a screening platform that could be applied for industrial purposes. III IV Table of Contents Abstract ..........................................................................................................III Table of Contents ........................................................................................... V List of Figures ................................................................................................ IX List of Tables .............................................................................................. XIII Acknowledgements ...................................................................................... XV Author’s Declaration ................................................................................. XVI 1. Introduction ................................................................................................. 1 1.1 Cytochrome P450 ............................................................................................................ 1 1.1.1 Discovery and Nomenclature ................................................................................... 1 1.1.2 Structure and Classes ............................................................................................... 5 1.1.3 Catalytic Reaction Mechanism .............................................................................. 12 1.1.4 Evolution and Catalysed Reactions ....................................................................... 16 1.1.5 Applications ........................................................................................................... 18 1.1.5.1 Potential and Limitations of P450s in biocatalysis .................................................................... 18 1.1.5.2 Commercial applications ........................................................................................................... 21 1.1.5.3 Medicine .................................................................................................................................... 28 1.1.5.4 Bioremediation .......................................................................................................................... 29 1.1.6 The fungal Kingdom .............................................................................................. 31 1.1.6.1 Fungi – the double-edged sword ................................................................................................ 31 1.1.6.2 Fungal P450 ............................................................................................................................... 33 1.2 Beauveria bassiana ....................................................................................................... 35 1.3 Rhodococcus jostii ........................................................................................................ 37 1.4 Project Aim ................................................................................................................... 38 2. General Material and Methods ................................................................ 39 2.1 Chemicals ...................................................................................................................... 39 2.2 Strains and Plasmids ..................................................................................................... 39 2.2.1 Escherichia coli strains .......................................................................................... 39 2.2.2 Yeast strains ........................................................................................................... 40 2.2.3 Plasmids ................................................................................................................. 40 2.3 Media ............................................................................................................................ 42 2.3.1 Escherichia coli ..................................................................................................... 42 2.3.2 Yeast ...................................................................................................................... 43 2.3.3 Supplements ........................................................................................................... 44 V 2.4 Glycerol stocks ............................................................................................................. 44 2.5 Working with nucleic acids .......................................................................................... 45 2.5.1 Enzymatic restriction of DNA ............................................................................... 45 2.5.2 Plasmid DNA preparation ..................................................................................... 45 2.5.3 Purification of DNA fragments ............................................................................. 45 2.5.4 Agarose gel electrophoresis ................................................................................... 46 2.5.5 In vitro-Amplification of DNA by PCR ................................................................ 46 2.5.6 Ligation independent cloning method ................................................................... 47 2.5.7 In Fusion cloning ................................................................................................... 49 2.6 Preparation of recombinant microorganisms ................................................................ 51 2.6.1 Preparation of CaCl2-competent E. coli-cells ........................................................ 51 2.6.2 Plasmid transformation into BL21 (DE3) and Rosetta2 (DE3) competent E. coli cells 52 2.6.3 Plasmid transformation into NovaBlue competent E. coli cells ............................ 52 2.6.4 Plasmid transformation into Saccharomyces cerevisiae WAT11 ......................... 52 2.7 Working with Proteins .................................................................................................. 53 2.7.1 Cell growth and protein expression ....................................................................... 53 2.7.1.1 Small scale expression test ........................................................................................................ 54 2.7.1.2 Scaled up protein expression ..................................................................................................... 54 2.7.2 Protein purification ................................................................................................ 54 2.7.3 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ....... 55 2.7.4 Western blot ........................................................................................................... 57 2.7.4.1 Western blot set up and protein transfer .................................................................................... 57 2.7.4.2 Immunoprecipitation .................................................................................................................. 58 3. Selection of Beauveria bassiana P450 targets .......................................... 60 3.1 In situ analysis of CYPome .......................................................................................... 60 3.2 Phylogenetic tree .......................................................................................................... 64 3.3 Discussion ..................................................................................................................... 65 4. Construction of a library of B. bassiana P450 genes and their expression in E.coli ........................................................................................ 69 4.1 Materials and Methods ................................................................................................