Development of a new platform technology for plant Cytochrome P450 fusions Julia Schückel PhD Thesis University of York Department of Biology January 2012 ABSTRACT Abstract To date more than 15000 Cytochromes P450 have been identified and named so far, with one third belonging to the plant kingdom. This is a key biochemical resource, providing a wealth of biocatalysts covering a diverse range of chemistries. Characterisation, however, has been greatly hindered by the poor solubility of many P450s, a result of the membrane anchoring region common to all plant P450s. Fusions of plant P450 heme domains to an appropriate reductase without the hydrophobic membrane anchor could provide the basis for developing robust, soluble plant enzyme systems for substrate screens to discover novel activities that are also of benefit to industry. In this project, the two predominantly expressed Arabidopsis reductases ATR1 and ATR2 have been cloned, without the membrane anchor, and expressed in Escherichia coli. These two truncated enzymes have been purified and assessed for activity with ATR2 found to be more active than ATR1. ATR2 was chosen for engineering into a novel plant P450 reductase vector platform for high throughput applications, whereby the P450s can be easily and quickly swapped using ligation independent cloning techniques. Four different plant P450s (CYP93C1, CYP73A5, CYP82E4 and CYP81D8) were selected to validate this technology, and activity for the fusions of CYP93C1 (Isoflavone synthase I from Glycine max) and CYP73A5 (cinnamate-4-hydroxylase from Arabidopsis) with ATR2 have been shown. The presence of CYP73A5 fused to ATR2 was verified through purification and further studies showed that it has to be membrane associated for activity. Additionally, CYP93C1 and CYP73A5 were also fused with the bacterial RhF reductase from Rhodococcus sp. and expressed in E. coli and compared to the plant P450 – plant reductase fusion protein. These novel plant-bacterial fusion P450 systems are the first example of active plant P450s fused to a reductase from a bacterium. This platform technology will provide the possibility for characterisation studies of eukaryotic P450s with unknown function and the discovery of new activities. 2 TABLE OF CONTANTS Abstract .................................................................................... 2 List of Tables ........................................................................... 8 List of Figures .......................................................................... 9 Acknowledgements ............................................................... 14 Author’s Declaration ............................................................ 15 1 Introduction ...................................................................... 16 1.1 Biotransformations in Industry ............................................... 16 1.2 Cytochromes P450 .................................................................... 17 1.2.1 History and Nomenclature of Cytochromes P450 ....................... 17 1.2.2 Structure and Function of Cytochromes P450 ............................ 22 1.2.3 Reaction Mechanism ...................................................................... 25 1.2.4 Cytochromes P450 reactions ......................................................... 27 1.2.5 Interactions between Cytochromes P450 and reductases ........... 28 1.2.6 Cytochrome P450 Fusion Systems ................................................ 29 1.3 Applications of Cytochromes P450 in biocatalysis ................ 33 1.3.1 Hydroxylation ................................................................................. 33 1.3.2 Oxidation ......................................................................................... 35 1.4 Plant Cytochromes P450 .......................................................... 37 1.4.1 Cytochromes P450s in Arabidopsis thaliana ............................... 37 1.4.2 Fusions of plant Cytochromes P450 .............................................. 43 1.5 Recombinant Expression Systems ........................................... 47 1.5.1 Expression of P450s in Escherichia coli........................................ 48 1.5.2 Expression in Yeast ........................................................................ 48 1.6 Aim of the project ..................................................................... 50 2 Materials and Methods .................................................... 51 2.1 Reagents and consumables ....................................................... 51 2.2 Media, strains and plasmids .................................................... 52 2.2.1 Bacterial media ............................................................................... 52 2.2.2 Bacterial strains .............................................................................. 53 2.2.3 Plasmids for gene cloning and enzyme expression ...................... 55 2.3 Preparation of chemically competent Escherichia coli cells . 58 2.4 Plant media, growth conditions and strains ........................... 59 2.5 Plasmid DNA preparation ........................................................ 60 2.6 Purification of DNA fragments ................................................ 60 2.7 Detection of the mRNA transcript .......................................... 60 2.7.1 RNA extraction from Escherichia coli .......................................... 60 3 TABLE OF CONTANTS 2.7.2 RNA extraction from plant tissue ................................................. 61 2.7.3 Reverse transcription from plant mRNA to cDNA ..................... 62 2.8 Traditional cloning method ...................................................... 62 2.8.1 Designing primers for truncated P450 and reductase genes ...... 62 2.8.2 Polymerase chain reaction (PCR) ................................................. 62 2.8.3 Preparation of PCR products for cloning .................................... 63 2.8.4 Transformation of plasmid DNA into chemically competent Escherichia coli cells ....................................................................... 63 2.8.5 Screening transformants for recombinant genes by PCR .......... 64 2.8.6 Restriction endonuclease digest of DNA ....................................... 64 2.8.7 Dephosphorylation of DNA 5’ end of the vector .......................... 64 2.8.8 DNA ligation reaction..................................................................... 65 2.8.9 DNA sequencing and analysis........................................................ 65 2.9 Ligation independent cloning method .................................... 66 2.10 Protein expression and purification .................................... 67 2.10.1 Expression in Escherichia coli ....................................................... 67 2.10.2 Solubilisation buffers...................................................................... 68 2.10.3 Cell lysis by sonication ................................................................... 70 2.10.4 Protein purification in a batch process ......................................... 71 2.10.5 Protein purification in a continuous process ............................... 71 2.11 Agarose gel electrophoesis.................................................... 72 2.12 Protein detection ................................................................... 72 2.13 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ......................................................... 72 2.13.1 SDS PAGE with the Bio-Rad system ............................................ 72 2.13.2 SDS PAGE with the RunBlue system from Expedeon ................ 73 2.14 Western blot analysis ............................................................ 73 2.14.1 Protein detection with anti-poly Histidine peroxidase conjugate73 2.14.2 Protein detection with alkaline phosphatase conjugate .............. 74 2.15 Protein characterisation ....................................................... 75 2.15.1 Characterisation of the P450 reductases ...................................... 75 2.15.1.1 Spectrophotometric characterisation ........................................... 75 2.15.1.2 Activity assay with cytochrome c ................................................ 75 2.15.1.3 Temperature and pH optima of ATR1tr and ATR2tr .................. 76 2.15.1.4 Stability test of ATR2tr in different buffers ................................ 77 2.15.1.5 Kinetic studies of the reductases .................................................. 77 2.15.2 Protein identification by MALDI-MS analysis ............................ 78 2.15.3 P450 enzyme activity assays .......................................................... 78 2.15.3.1 Activity assay for CYP71D15 with limonene ............................. 78 4 TABLE OF CONTANTS 2.15.3.2 Activity assay for CYP81D8 and CYP81D11 with methyl-tolyl- suphide ......................................................................................... 79 2.15.3.3 Isoflavone synthase activity assay ............................................... 80 2.15.3.4 Cinnamate-4-hydroxylase (CYP73A5) activity assay ................. 81 2.15.3.5 Activity assay for CYP82E4 ........................................................ 82 2.15.3.6 Activity assay for CYP81D8 with TNT and aminodinitrotoluenes83 2.15.3.7 Activity assay for CYP81D8 with 7-ethoxycoumarin ................. 84 3 Chapter: Expression of Cytochromes P450 ................... 86 3.1 Introduction ..............................................................................