Cytochrome P450 polypeptide with increased enzyme activity

Abstract

The present invention pertains to an isolated P450 enzyme comprising or consisting of an amino acid sequence at least 80% identical to SEQ ID NO: 1, wherein said sequence comprises a threonine at position corresponding to position 225 and/or an aspartic acid mutation at position corresponding to position 289. The invention also concerns an isolated nucleic acid comprising a sequence encoding said enzyme, a vector comprising said nucleic acid, and a host cell containing said nucleic acid or said vector. Methods for preparing said enzyme and methods for producing steroid hormone precursors using the enzyme or the host cells featured in the invention are also provided.

Claims

1. An isolated P450 enzyme comprising an amino acid sequence at least 97% identical to SEQ ID NO: 1, wherein said sequence comprises a threonine at a position corresponding to position 225 and/or an aspartic acid at a position corresponding to position 289.

2. The enzyme according to claim 1 comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

3. The enzyme according to claim 1 consisting of the sequence SEQ ID NO: 1.

4. A method for producing a steroid hormone precursor, comprising the steps of: a) contacting the P450 enzyme of claim 1 with an isolated adrenodoxin (Adx) polypeptide, an isolated adrenodoxin reductase (AdR) polypeptide and a substrate selected from the group consisting of polycyclic and unsaturated mono alcohols having an aliphatic side chain such as cholesterol, a cholesterol analogue and a cholesterol derivative in conditions allowing the transformation of said substrate into a steroid hormone precursor, and b) recovering the steroid hormone precursor.

5. The method according to claim 4, wherein the steroid hormone precursor is pregnenolone.

6. The enzyme according to claim 1, wherein said amino acid sequence is at least 98% identical to SEQ ID NO: 1.

7. The enzyme according to claim 1, wherein said amino acid sequence is at least 99% identical to SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: HPLC-chromatogram showing the in vitro conversion of 20 μM cholesterol by the cytochrome P450 polypeptides SA1 (SEQ ID NO:1) and SA4 (control having sequence SEQ ID NO: 4). Legend: S: substrate, P: main product, DOC: 11-deoxycorticosterone.

(2) FIG. 2: Pregnelonone quantification from HPLC-chromatogram showing the in vivo conversion of different substrates into pregnenolone by the cytochrome P450 polypeptide SA1 (SEQ ID NO:1).

(3) FIG. 3: HPLC-chromatogram showing the in vivo conversion of 300 μM cholesterol by the cytochrome P450 polypeptides SA1 (SEQ ID NO:1) and SA6 (control having sequence SEQ ID NO: 6) after 24 h. Legend: Chol.: cholesterol, Prog.: pregnenolone.

(4) FIG. 4: Time course for the in vivo conversion of 300 μM cholesterol by the cytochrome P450 polypeptides SA1 (SEQ ID NO:1) and SA6 (control having sequence SEQ ID NO: 6).

(5) FIG. 5: Vector map of pSMF2.1_CYP11A1BYM encoding for SA1 (SEQ ID NO: 1).

EXAMPLES

Example 1

Materials and Methods

(6) Protein Synthesis

(7) CYP11A1 variants were obtained by gene synthesis and cloned into pTRC99A, fused to a poly-His tag. C43DE3-E. coli cells were co-transformed with each vector and the chaperone-encoding pGro12. The purification was performed as described in Janocha et al. (Biochim Biophys Acta. (2011) January;1814(1):126-31). The purity was assessed by SDS-PAGE. Concentrations of the purified proteins were determined by CO-difference spectra, after treatment with sodium dithionite and exposure to CO.

(8) Adx and AdR were purified according to Uhlmann et al. (Biochem. Biophys. Res. Commun., 188 (1992), pp. 1131-1138) and Sagara et al. (Biol. Pharm. Bull., 16 (1993), pp. 627-630), respectively.

(9) In Vitro Conversion Assay

(10) For in vitro enzyme assays, 150 mM of HEPES buffer, adjusted to pH 7.4, containing 0.05% Tween-20 and 1 mM MgCl2, were applied as reaction buffer. 1 unit of glucose-6-phosphate dehydrogenase, with 5 mM glucose-6-phosphate as substrate, served as a NADPH-regenerating system.

(11) For WT P450scc (noted “SA4”, SEQ ID NO: 4) and P450scc-I1A-K193E (noted “SA6”, SEQ ID NO: 6) the concentrations of CYP11A1, Adx and AdR were of 1 μM, 20 μM and 0.5 μM, and for P450scc-R225T-N289D (noted “SA1”, SEQ ID NO: 1) the concentrations of CYP11A1, Adx and AdR were of 0.25 μM, 5 μM and 0.125 μM, respectively.

(12) 20 μM of cholesterol, dissolved in 45% 2-hydroxypropyl-β-cyclodextrin, served as substrate for CYP11A1.

(13) The samples were pre-warmed to 37° C. and the reaction was started by addition of NADPH to a final concentration of 100 μM. The mixtures were incubated at 37° C. with agitation for 30 s. The reaction was stopped by boiling the samples in water for 30 s.

(14) To allow photometric detection at 240 nm, the steroids were converted into their 3-keto-Δ4 derivatives using a cholesterol oxidase from Nocardia spec.

(15) 20 μl of a cholesterol oxidase solution (5 mg cholesterol oxidase and 5 mg Na-cholate dissolved in 5 ml of 50 mM HEPES buffer pH 7, containing 0.05% Tween-20) were added to the samples. After incubation at 37° C. for 1 h, 11-deoxycorticosterone (DOC) was added to the reaction mixtures as an internal standard, followed by a 2-times extraction with equal volumes of ethylacetate. After evaporation, the extracts were dissolved in acetonitrile/water.

(16) In Vivo Conversion Assay

(17) The in vivo conversion of 300 μM cholesterol into pregnelonone was evaluated by HPLC after 24 h. Bacillus megaterium was cultivated in TB-medium containing 10 μg/ml tetracycline at 37° C. with 180 rpm shaking. Protein expression was induced by adding 0.25 g of xylose dissolved in 1 mL water, followed by the subsequent addition of the substrate, dissolved in 2-hydroxypropyl-β-cyclodextrin.

Example 2

In Vitro Conversion of Cholesterol by the Cytochrome P450 Polypeptides SA1 (SEQ ID NO:1) and SA4 (WT Control, SEQ ID NO:4)

(18) After acute evaluation of the concentration of each P450scc preparation, SA1, SA4 and SA6 were used in the following in vitro assays. Each of these isoforms was reconstituted at a concentration of 1 μM in the presence of cholesterol as a substrate (20 μM) in saturating amounts of Adx and AdR.

(19) While SA4 and SA6 showed identical activity, SA1 showed a 2-3-fold higher activity compared to SA4 and SA6 (FIG. 1). Since the substrate is already depleted after the incubation time, it is difficult to assess the differences between the SA1 and SA1/SA4 polypeptides at longer incubation time.

Example 3

In Vivo Conversion of Cholesterol by the Cytochrome P450 Polypeptides SA1 (SEQ ID NO:1) and SA6 (Control, SEQ ID NO:6)

(20) As no measurable differences were observed between SA4 and SA6, the inventors focused their interest on comparing the SA1 and SA6 polypeptides. A new system was recently developed in which cholesterol is metabolized into pregnenolone by recombinant Bacillus megaterium expressing P450scc, Fdx1 and FdxR in the presence of solubilized cholesterol. SA1 and SA6 corresponding cDNAs were optimized for Bacillus megaterium codon BIAS and transferred into the plasmid pSFM2.1 using the appropriate restriction sites. Both PSFM2.1 plasmids bearing respectively the codon optimized SA1 and SA6 were transferred into Bacillus megaterium MS941 using a classical protoplast transformation protocol.

(21) The in vivo cholesterol conversion activity was assessed after 24 h and 48 h for the SA1 and SA6 polypeptides expressed in Bacillus megaterium.

(22) In accordance with the in vitro experiments, SA1 exhibited now a 2-fold higher activity compared to SA6 (FIGS. 3 and 4).

Example 4

In Vivo Conversion of Different Substrates into Pregnenolone by the Cytochrome P450 Polypeptide SA1 (SEQ ID NO:1)

(23) Finally the improved system was used to test the SA1 polypeptide conversion capacity with various substrates of biotechnological interest: campesterol, desmosterol, ergosta-5,24(28)-dien-3β-ol and a mixture of various 20, 22-OH oxysterols.

(24) Each substrate was converted to one main product with the same retention time as progesterone, indicating that CYP11A1 was able to cleave the side-chain of each of these substrates, yielding pregnenolone.

(25) Pregnenolone formation was quantified for every substrate (FIG. 2). The polar oxysterols were converted at a rate comparable to cholesterol (˜35 mg/L after 48 hours). Conversion of the more hydrophobic sterols campesterol, desmosterol and ergostadienol only occurred at a rate of approximately 19% after 48 hours, compared to cholesterol.

(26) Taken together, the results show that all of the tested steroids were able to permeate through the cell membrane of Bacillus megaterium and were converted to pregnenolone by the CYP11A1 SA1 mutant.

(27) The inventors thus report here a new P450scc protein, named SA1, which shows an increased conversion activity towards various sterols including cholesterol.