Synthetic Biology Applications of Engineered Cytochrome P450 Enzymes

The project involves the engineering of a high-activity cytochrome P450-cytochrome P450 reductase fusion enzyme (P450 BM3) in order to redirect its substrate specificity. P450 BM3 is the fastest catalyst in the cytochrome P450 superfamily of monooxygenase enzymes, and naturally binds and oxidizes long chain fatty acid substrates. However, studies by our group (and many others) have demonstrated that BM3 is a malleable enzyme and that its substrate selectivity profile can be altered substantially by mutagenesis - including through rational mutagenesis of the active site and by introduction of mutations that lead to conformational readjustments to the enzyme. The major aim of this collaborative project with Syngenta is to generate mutants of the P450 BM3 enzyme in which substrate selectivity is switched towards the recognition of selected herbicide molecules. This will be achieved through the testing of a group of available BM3 mutants (in which substrate selectivity profiles are considerably altered from those of the wild-type enzyme) and by both rational mutagenesis and directed evolution approaches in order to produce variants that can bind/oxidize herbicide compounds from a group of such compounds that are of particular interest to Syngenta (the industrial partner). Activity towards target compounds will be established using substrate binding assays (in which the heme prosthetic group produces spectral shifts consistent with binding of the molecules close to the heme in the active site, and indicative of their substrate-like nature) and by using analytical methods (GC- and LC-MS) to confirm that the relevant P450 BM3 mutants can oxidatively transform target herbicides to inactive derivatives. The overarching aim of the project is to produce a series of BM3 mutants with specificity towards the binding and oxidation of a set of important herbicides. In the longer term, the plan is to test the effectiveness of these mutants in herbicide degradation through integration of relevant BM3 mutants into important crop plants and to establish that these plants develop herbicide tolerance.

In this collaborative project with Syngenta, the student will be involved in work to design and express mutant P450 BM3 enzymes and to identify variants with activity towards key herbicide molecules at the University of Manchester. These studies will extend to areas including the determination of herbicide binding affinity, spectroscopic analysis of BM3 variant-herbicide complexes, steady-state and stopped-flow kinetic studies, and structural studies by crystallization of P450 BM3 heme domain mutants bound to herbicide substrates. Work done by the PhD student at Syngenta will include detailed analytical studies to identify metabolites formed from targeted herbicide substrates, and plant cell engineering studies to investigate whether BM3 mutant constructs can effectively metabolize herbicides in vivo.