MODIFIED MONOOXYGENASES FOR THE MANUFACTURE OF HYDROXYLATED HYDROCARBONS BASED ON SUBSTITUTION OF AMINO ACIDS BY ALANINE

Abstract

The present invention relates to novel monooxygenases which are useful in the hydroxylation of aromatic hydrocarbons. They are particularly useful for the production of 1-naththol and 7-hydroxycoumarin from naphthol and 7-Ethoxycoumarin, respectively.

Claims

1. A modified P450 monooxygenase, comprising a functional mutation wherein at least one amino acid selected from the group consisting of leucine 87, glutamic acid 88, lysine 89, isoleucine 90, threonine 91, proline 92, valine 93, serine 94, glutamic acid 96, threonine 98, threonine 100, leucine 101, arginine 103, tyrosine 104, aspartic acid 105, histidine 196, threonine 197, valine 198, asparagine 199, threonine 200, tryptophan 201, glycine 202, arginine 203, proline 204, proline 206, glutamic acid 207, glutamic acid 208, glutamine 209, valine 210, and combinations thereof is substituted by alanine, wherein said functional mutation leads to an improved reactivity on hydroxylation of aromatic hydrocarbons.

2. The modified P450 monooxygenase according to claim 1, wherein glutamine at position 209 is substituted by alanine.

3. The modified P450 monooxygenase according to claim 2, further comprising at least one of the following substitutions: a) substitution of glutamic acid at position 88 by an amino acid selected from the group consisting of alanine, serine, histidine, threonine, cysteine, methionine, and asparagine; and/or b) substitution of asparagine at position 199 by an amino acid selected from the group consisting of glutamine, isoleucine, leucine, phenylalanine, histidine, methionine, arginine, serine, threonine, tyrosine, tryptophan, alanine, valine, and lysine.

4. The modified P450 monooxygenase according to claim 1, wherein glutamic acid at position 88 is substituted by alanine.

5. The modified P450 monooxygenase according to claim 4, further comprising at least one of the following substitutions: a) substitution of glutamic acid at position 199 by an amino acid selected from the group consisting of glutamine, isoleucine, leucine, phenylalanine, histidine, methionine, arginine, serine, threonine, tyrosine, tryptophan, alanine, valine, and lysine; and/or b) substitution of glutamine at position 209 by alanine.

6. The modified P450 monooxygenase according to claim 1, having an addition of up to 35 amino acids at the N-terminus and/or the C-terminus.

7. The modified P450 monooxygenase according to claim 1, having a deletion of up to 35 amino acids at the N-terminus and/or the C-terminus.

8. The modified P450 monooxygenase according to claim 1, wherein the functional mutation leads to an increased activity on at least one hydrocarbon selected from the group consisting of naphthalene, 7-ethoxy-hydroxycoumarin, acenaphthene, florene, indene, methylbenzene, and ethylbenzene.

9. A nucleic acid sequence encoding any of the modified P450 monooxygenase according to claim 1 and its complementary nucleic acid sequence.

10. An expression construct, comprising the nucleic acid sequence of claim 9 under the genetic control of a regulatory nucleic acid sequence.

11. A vector, comprising the nucleic acid sequence of claim 9.

12. A microorganism comprising the nucleic acid of claim 9.

13. The microorganism according to claim 12, wherein the microorganism is-belongs to the genus Rhodococcus or Escherichia.

14. A method for producing the modified p450 monooxygenase of claim 1 comprising: incubating a recombinant microorganism comprising a nucleic acid encoding the modified p450 monooxygenase under conditions suitable for the expression of the modified p450 monooxygenase.

15. A method for the hydroxylation of an aromatic hydrocarbon, comprising: mixing at least one of the modified P450 monooxygenases according to claim 1 with said aromatic hydrocarbon under conditions to hydroxylate said aromatic hydrocarbon.

16. The method according to claim 15, wherein the aromatic hydrocarbon is selected from the group consisting of naphthalene, 7-ethoxy-hydroxycoumarin, acenaphthene, florene, indene, methylbenzene, ethylbenzene, and mixtures thereof.

17. The method according to claim 15, wherein the aromatic hydrocarbon is hydroxylated to form a hydroxylated aromatic hydrocarbon selected from the group consisting of 1-naphthol, 7-hydroxycoumarin, 1-acenaphthylene, 9-benflumetol, indenol, benzyl alcohol, 3-methylbenzyl alcohol, and mixtures thereof.

18. (canceled)

19. The modified p450 monooxygenase according to claim 1, wherein the at least one amino acid is selected from the group consisting of glutamic acid 88, lysine 89, threonine 100, leucine 101, arginine 103, asparagine 199, arginine 203, proline 204, glutamine 209, and combinations thereof.

20. The microorganism of claim 12, comprising an expression construct and/or a vector comprising the nucleic acid.

21. The method according to claim 15, wherein mixing comprises combining a recombinant microorganism with the aromatic hydrocarbon, said recombinant microorganism comprising a nucleic acid encoding the modified p450 monooxygenase.

Description

EXAMPLES

[0059] Construction of Nucleic Acids Encoding Modified P450 Monooxygenases

[0060] A full-length gene encoding P450 protein was synthesized and amplified by PCR using the following primers: 5-ctgGAATTCATGAGTGCATCAGTTCCGGCGT-3 (SEQ ID NO: 3) and 5-catcAAGCTTTCAGAGTCGCAGGGCCA-3 (SEQ ID NO: 4). The EcoRI and HindIII restriction endonuclease sites in the primer sequences are underlined. The PCR product was isolated and digested with EcoRI and HindIII restriction endonucleases, cloned into the pET28a(+) vector, and expressed in E. coli BL21(DE3) cells. The sequence of the insert DNA was subsequently confirmed by sequencing.

[0061] Mutagenesis was performed as generally known in the art by designing suitable primers and conducting whole plasmid PCR. Thereafter, the original plasmid was digested by DpnI.

[0062] Recombinant Expression of Modified P450 Monooxygenases

[0063] E. coli BL21 (DE3) containing the expression construct was grown in 100 mL Luria-Bertani medium, supplemented with 50 μg ml.sup.−1 kanamycin, at 37° C. and 120 rpm. Expression was induced with 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and cells were incubated for 24 h at 18° C. Cells were harvested by centrifugation (.sup.˜10,000×g), washed with phosphate-buffered saline (PBS) and resuspended into PBS. The cell final concentration was adjusted to OD.sub.600 20 before the reaction.

[0064] Assessment of the Activity of Recombinant P450 Monooxygenases

[0065] The whole-cell reaction was initiated by adding 0.15 g/L PAH from a 3 g/L stock in DMSO to 2 mL working volume in a 10 mL vial. After 2 h, the products were extracted with 2 mL methyl tert-butyl ether (MTBE) after vigorous vortexing for 5 min. After centrifugation, the organic phase was transferred to a fresh glass tube and evaporated to dryness. The remaining residue was resolubilized with methanol. Samples were quantified by HPLC using an Alltech series 1500 instrument equipped with a prevail C18 reverse-phase column maintained at 25° C. For detection, 50% methanol was applied as the mobile phase at a flow rate of 1.0 mL min′. Products were detected by monitoring the absorbance at 272 nm.

TABLE-US-00001 TABLE 1 modified P450 monooxygenases and their activities 1-Naphthol production 7- Hydroxycoumarin production Mutants (mg .Math. L.sup.−1 .Math. h.sup.−1) (mg .Math. L.sup.−1 .Math. h.sup.−1) Wild-type 0.51 ± 0.05 46.98 ± 3.40 L87A 0.23 ± 0.05 48.44 ± 1.78 E88A 0.99 ± 0.04 153.98 ± 1.14  K89A 0.55 ± 0.02 96.31 ± 4.72 I90A 0.65 ± 0.08 58.31 ± 3.07 T91A 0.54 ± 0.12 44.98 ± 4.16 P92A 0.49 ± 0.09 49.02 ± 3.07 V93A 0.20 ± 0.10 28.71 ± 4.16 S94A 0.52 ± 0.05 48.14 ± 3.18 E95A 0.82 ± 0.11 62.42 ± 0.46 E96A 0.56 ± 0.06 71.52 ± 4.46 T98A 0.26 ± 0.05 39.84 ± 2.37 T100A 0.65 ± 0.09 86.75 ± 4.00 L101A 0.68 ± 0.03 120.58 ± 3.78  R103A 0.45 ± 0.10 76.82 ± 4.29 Y104A 0.19 ± 0.01 35.74 ± 2.96 D105A 0.41 ± 0.13 36.46 ± 4.14 H196A 0.54 ± 0.05 46.16 ± 3.96 T197A 0.71 ± 0.08 48.43 ± 2.91 V198A 0.08 ± 0.04 38.70 ± 0.57 N199A 1.73 ± 0.01 134.04 ± 2.54  T200A 0.44 ± 0.04 46.72 ± 3.15 W201A 0.35 ± 0.12 33.79 ± 2.38 G202A 0.37 ± 0.10 35.65 ± 1.12 R203A 1.28 ± 0.04 144.63 ± 5.60  P204A 0.66 ± 0.07 182.97 ± 9.32  P206A 0.29 ± 0.13 39.31 ± 0.85 E207A 0.58 ± 0.05 62.62 ± 5.67 E28A 0.37 ± 0.04 24.84 ± 1.56 Q209A 1.90 ± 0.07 225.27 ± 3.04  V210A 0.49 ± 0.02 43.92 ± 2.93