Development of a new platform technology for plant Cytochrome P450 fusions

摘要: 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 wealth of biocatalysts covering diverse range chemistries. Characterisation, however, has greatly hindered by poor solubility many P450s, result membrane anchoring region common all P450s. Fusions heme domains an appropriate reductase without hydrophobic anchor could provide basis for developing robust, soluble enzyme systems substrate screens discover novel activities that are also benefit industry. In this project, two predominantly expressed Arabidopsis reductases ATR1 ATR2 cloned, anchor, in Escherichia coli. These truncated enzymes purified assessed activity found be active ATR1. was chosen engineering into vector platform high throughput applications, whereby P450s can easily quickly swapped using ligation independent cloning techniques. Four different (CYP93C1, CYP73A5, CYP82E4 CYP81D8) were selected validate technology, fusions CYP93C1 (Isoflavone synthase I from Glycine max) CYP73A5 (cinnamate-4-hydroxylase Arabidopsis) shown. The presence fused verified through purification further studies showed it associated activity. Additionally, bacterial RhF Rhodococcus sp. E. coli compared – fusion protein. plant-bacterial first example bacterium. This technology will possibility characterisation eukaryotic unknown function discovery new activities.