FROM P450 DISCOVERY TO SCALE-UP FOR DELIVERY OF CHIRAL INTERMEDIATES Jill M. Caswell, Stefan Mix, Thomas S. Moody, Jane Mueller, Derek J. Quinn Department of Biocatalysis and Isotope Chemistry, Almac Sciences, 20 Seagoe Industrial Estate, Craigavon, BT63 5QD, Northern Ireland, UK Abstract P450 monooxygenase (P450s or CYPs) enzyme applications in pharmaceutical and metabolite synthesis still capture the imagination of synthetic chemists due to their ability to perform regio- and enantioselective oxidation of unactivated carbons [1]. The present poster highlights P450s enzyme panel building, including enzyme cloning and expression through to screening. Novel panels of diverse P450 enzymes from different species of plant, bacteria and fungus have been built using in-house bioinformatics to screen biocatalytic routes for chiral intermediates. The enzymes have been successfully cloned and expressed in the host of choice some as self-sufficient and others coupled with different reductase domains. Screening of the enzyme panels has led to successful hits for biooxidation. Work on process development, scale-up and oxidative product isolation is highlighted to access chiral intermediates with moderate to high titres with high conversion and selectivities. Introduction Cytochrome P450 monooxygenase (P450) enzymes are a super family of heme-containing enzymes that introduce a single oxygen atom derived from molecular oxygen into an organic molecule. They catalyse a variety of reactions including hydroxylation of aliphatic and aromatic carbons, oxidation of organic nitrogen and sulphur, epoxidation and Baeyer-Villiger oxidation as summarized in Figure 1 [2,3]. P450 enzymes were identified from in-house bioinformatics, and molecular modelling was used to build mutant libraries which have a wide substrate specificity for hydroxylation (Figure 2). [1] Caswell, J.M., et al, Curr. Opin. Chem. Biol, 17,271-275, 2013 [2] Bernhardt, R. J. Biotechnol. 124, 128-145, 2006. [3] Chefson, A.; Auclair, K. Mol. Biosys. 2, 462-469, 2006. The panel of P450 enzymes were expressed in E. coli and expression was verified by SDS-PAGE and dot blot analysis before screening the panel of enzymes against compound of interest(X) for hydroxylation. Class II Class III Figure 3: Phylogenetic tree analysis of novel panel of P450 enzymes Figure 2: Bioinformatic analysis of P450 panel Results Figure 4 shows the protein expression analysed by SDS-PAGE analysis. High level expression was observed for both Class II and Class III with bands corresponding to the correct size for each enzyme. Expression of soluble protein was optimised by varying parameters such as temperature, induction time, concentration of inducer and additives to the culture broth (Figure 4c). The panel of enzymes were screened against compound 1 shown in Scheme 1 and the observed reactivity profile is shown in Figure 5 (green = >10% product, yellow = 1-10% product, red = <1% product). Figure 4: Expression of P450 enzyme panel a) shows soluble expression of Class II P450 enzymes and b) soluble expression of Class III P450 enzymes by SDS-PAGE analysis. C) optimisation of expression conditions as analysed by dot blot probed with anti-his antibody. Reductase P450 a) b) BAW-103 BAW-109 Controls [IPTG] Temp 0.6 0.8 1.0 OD600nm c) 1 2 3 4 5 6 7 8 9 10 11 12 A BAW-01 BAW-09 BAW-17 BAW-25 BAW-33 BAW-41 BAW-49 BAW-57 BAW-65 BAW-73 BAW-81 BAW-89 B BAW-02 BAW-10 BAW-18 BAW-26 BAW-34 BAW-42 BAW-50 BAW-58 BAW-66 BAW-74 BAW-82 BAW-90 C BAW-03 BAW-11 BAW-19 BAW-27 BAW-35 BAW-43 BAW-51 BAW-59 BAW-67 BAW-75 BAW-83 BAW-91 D BAW-04 BAW-12 BAW-20 BAW-28 BAW-36 BAW-44 BAW-52 BAW-60 BAW-68 BAW-76 BAW-84 BAW-92 E BAW-05 BAW-13 BAW-21 BAW-29 BAW-37 BAW-45 BAW-53 BAW-61 BAW-69 BAW-77 BAW-85 BAW-93 F BAW-06 BAW-14 BAW-22 BAW-30 BAW-38 BAW-46 BAW-54 BAW-62 BAW-70 BAW-78 BAW-86 BAW-94 G BAW-07 BAW-15 BAW-23 BAW-31 BAW-39 BAW-47 BAW-55 BAW-63 BAW-71 BAW-79 BAW-87 BAW-95 H BAW-08 BAW-16 BAW-24 BAW-32 BAW-40 BAW-48 BAW-56 BAW-64 BAW-72 BAW-80 BAW-88 BAW-96 Figure 5: Illustration of screening results against compound 1 Scheme 1: Hydroxylation of compound 1 BAW-85 enzyme showed highest conversion and selectivity, and was used for subsequent process development and scale up. Process development involved selection and optimisation of pH, temperature, medium and mode of aeration. Improved availability of the poorly water-soluble substrate 1 was achieved by addition of PEG-400 and alcohol co-solvent. Agitator design proved critical for successful scale up. At final 1 m 3 batch size and 12 g/L substrate loading, the reaction completed within 21 hours with high selectivity (Figure 6). A diverse panel of P450 enzymes has been built around Class II and Class III enzymes as shown in Figure 3. Summary A diverse panel of P450 enzymes has been developed with high levels of expression and activity. Screens using the panel have returned high numbers of hits. To date, one of these has been developed and scaled up to 1 m 3 . heterocycle hydroxylation epoxidation Wildtypes Microsomes Recombinant enzymes (P450/Peroxidases) Metalloporphyrins O OH N NH 2 H N R ' N H R O R ' N H R O OH O R HO R X O O R ' R X O O R ' R R X O OH O R R X O O R ' R R HO OH HO R R O O-dealkylation aromatic hydroxylation Aliphatic hydroxylation N-dealkylation O-dealkylation Figure 1: Summary of biooxidation reactions mediated by P450 enzymes Figure 6: Scale-up of biooxidation with P450 enzyme BAW-85 X R O R X R O R HO P450 , O 2