FROM P450 DISCOVERY TO SCALE-UP FOR DELIVERY OF CHIRALINTERMEDIATES

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

AbstractP450 monooxygenase (P450s or CYPs) enzyme applications in pharmaceutical and metabolite synthesis still capture the imagination of syntheticchemists due to their ability to perform regio- and enantioselective oxidation of unactivated carbons [1]. The present poster highlights P450senzyme panel building, including enzyme cloning and expression through to screening. Novel panels of diverse P450 enzymes from different speciesof plant, bacteria and fungus have been built using in-house bioinformatics to screen biocatalytic routes for chiral intermediates. The enzymeshave 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 isolationis highlighted to access chiral intermediates with moderate to high titres with high conversion and selectivities.

IntroductionCytochrome P450 monooxygenase (P450) enzymes are a super family ofheme-containing enzymes that introduce a single oxygen atom derivedfrom molecular oxygen into an organic molecule. They catalyse avariety of reactions including hydroxylation of aliphatic and aromaticcarbons, oxidation of organic nitrogen and sulphur, epoxidation andBaeyer-Villiger oxidation as summarized in Figure 1 [2,3].

P450 enzymes were identified from in-house bioinformatics, andmolecular modelling was used to build mutant libraries which have awide 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 expressionwas verified by SDS-PAGE and dot blot analysis before screening thepanel 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

ResultsFigure 4 shows the protein expression analysed by SDS-PAGE analysis.High level expression was observed for both Class II and Class III withbands corresponding to the correct size for each enzyme. Expression ofsoluble protein was optimised by varying parameters such astemperature, induction time, concentration of inducer and additives tothe culture broth (Figure 4c).The panel of enzymes were screened against compound 1 shown inScheme 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 IIP450 enzymes and b) soluble expression of Class III P450 enzymes by SDS-PAGEanalysis. C) optimisation of expression conditions as analysed by dot blot probed withanti-his antibody.

Reductase

P450

a)

b)

BAW-103

BAW-109

Cont

rols

[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 wasused for subsequent process development and scale up. Processdevelopment involved selection and optimisation of pH, temperature,medium and mode of aeration. Improved availability of the poorlywater-soluble substrate 1 was achieved by addition of PEG-400 andalcohol co-solvent.Agitator design proved critical for successful scale up. At final 1 m3

batch size and 12 g/L substrate loading, the reaction completed within21 hours with high selectivity (Figure 6).

A diverse panel of P450 enzymes has been built around Class II andClass III enzymes as shown in Figure 3.

SummaryA diverse panel of P450 enzymes has been developed with high levelsof expression and activity. Screens using the panel have returned highnumbers of hits. To date, one of these has been developed and scaledup to 1 m3.

heterocyclehydroxylation

epoxidation

Wildtypes

Microsomes

Recombinant enzymes (P450/Peroxidases)

Metalloporphyrins

O OH

NNH2

HN

R'NH

RO

R'NH

ROOH

OR

HOR

XO

O

R'

RXO

O

R'

R

R

XO

OH

O

R

R

XO

O

R'

R

R

HO

OH

HORR

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

XR

O

R

XR

O

R

HO

P450, O2