MODIFIED MONOOXYGENASES FOR THE MANUFACTURE OF HYDROXYLATED HYDROCARBONS

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 having an amino acid sequence as defined by SEQ ID NO: 1 or an amino acid sequence with at least 90% sequence identity with SEQ ID NO. 1, wherein the asparagine at position 199 or at the homologous position is substituted 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, wherein said functional mutation leads to an improved reactivity on hydroxylation of aromatic hydrocarbons.

2. The modified P450 monooxygenase of claim 1, wherein the asparagine at position 199 is substituted by an amino acid selected from the group consisting of glutamine, isoleucine, leucine, phenylalanine, histidine, methionine, arginine, serine, tryptophan, alanine, and valine.

3. The modified P450 monooxygenase of claim 2, wherein the asparagine at position 199 is substituted by glutamine or isoleucine.

4. The modified P450 monooxygenase according to claim 1, wherein additionally a) glutamic acid at position 88 is substituted by an amino acid selected from the group consisting of alanine, serine, histidine, threonine, cysteine, methionine, and asparagine, and/or b) glutamine at position 209 is substituted by alanine.

5. The modified P450 monooxygenase according to claim 1, wherein the sequence having at least 90% sequence identity with the sequence defined by SEQ ID NO. 1 is derived from SEQ ID NO. 1 by conservative substitutions of amino acids.

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 having an increased activity on at least one hydrocarbon selected from the group consisting of naphthalene, 7-ethoxy-hydroxycoumarin, acenaphthene, fluorene, 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 or the expression construct of claim 10.

12. A microorganism comprising the nucleic acid of claim 9 or the expression construct of claim 10 or the vector according to claim 11.

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 the step of incubating the recombinant microorganism according to claim 12 under conditions suitable for the expression of the monooxygenase.

15. A method for the hydroxylation of an aromatic hydrocarbon, comprising a1) having at least one of the modified P450 monooxygenases according to claim 1 mixed and reacted with said aromatic hydrocarbon and having said aromatic hydrocarbon thus hydroxylated; or a2) having at least one of the recombinant microorganisms according to claim 12 mixed and reacted with 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, fluorene, indene, methylbenzene, ethylbenzene, and mixtures thereof.

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

18. Use of the modified P450 monooxygenase according to claim 1 for the hydroxylation of an aromatic carbon.

Description

EXAMPLES

[0072] Construction of Nucleic Acids Encoding Modified P450 Monooxygenases

[0073] 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.

[0074] 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.

[0075] Recombinant Expression of Modified P450 Monooxygenases

[0076] 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 (˜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.

[0077] Assessment of the Activity of Recombinant P450 Monooxygenases

[0078] 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.sup.−1. Products were detected by monitoring the absorbance at 272 nm.

TABLE-US-00002 TABLE 1 Effects of substitution of different amino acids by alanine on enzyme activity 1-Naphthol production 7-Hydroxycoumarin Mutants (mg .Math. L.sup.−4 .Math. h.sup.−1) production (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 190A 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 H 196A 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 E208A 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

TABLE-US-00003 TABLE 2 Effect of substitutions at amino acid position 88 on enzyme activity 1-Naphthol production 7-Hydroxycoumarin Mutants (mg .Math. L.sup.−4 .Math. h.sup.−1) production (mg .Math. L.sup.−1 .Math. h.sup.−1) Wild- 0.51 ± 0.05 46.98 ± 3.4 type E88A 0.99 ± 0.04 153.98 ± 1.14 E88D 0.49 ± 0.03  62.52 ± 2.13 E88S 0.86 ± 0.02  77.44 ± 2.29 E88H 0.81 ± 0.05 121.15 ± 4.87 E88G 0.31 ± 0.08  38.12 ± 1.34 E88R 0.37 ± 0.03  61.69 ± 9.31 E88T 0.79 ± 0.05  102.78 ± 12.02 E88P 0    3.8 ± 0.63 E88C 1.05 ± 0.05 126.38 ± 1.48 E88Y 0.43 ± 0.11  71.18 ± 3.07 E88V 0.51 ± 0.05  44.33 ± 2.33 E88M 0.95 ± 0.03  120.87 ± 22.18 E88K 0.71 ± 0.08  77.55 ± 5.89 E881 0.48 ± 0.09   37.52 ± 13.85 E88L 0.75 ± 0.05  86.15 ± 1.23 E88F 0.06 ± 0.02  11.14 ± 0.61 E88N 0.61 ± 0.07 112.95 ± 0.84 E88Q 0.46 ± 0.02  51.49 ± 1.88 E88W 0.09 ± 0.02  26.27 ± 1.29

TABLE-US-00004 TABLE 3 Effect of substitutions at amino acid position 199 on enzyme activity 1-Naphthol production 7-Hydroxycoumarin Mutants (mg .Math. L.sup.−4 .Math. h.sup.−1) production (mg .Math. L.sup.−1 .Math. h.sup.−1) Wild- 0.51 ± 0.05 46.98 ± 3.4 type N199A 1.73 ± 0.01 134.04 ± 2.54 N199D 0  28.95 ± 1.68 N1995 2.66 ± 0.03  193.47 ± 15.83 N199H 3.46 ± 0.79 191.55 ± 1.71 N199G 0.34 ± 0.07 60.29 ± 6   N199R 3.22 ± 0.44 178.73 ± 3.93 N199T 2.48 ± 0.06  86.71 ± 1.99 N199P 2.71 ± 0.58 65.09 ± 3.2 N199C 0.42 ± 0.11  61.59 ± 6.89 N199Y 2.49 ± 0.08 263.26 ± 9.77 1N99V 1.21 ± 0.43 135.94 ± 0.31 N199M 3.5 ± 0.3 150.44 ± 5.63 N199K 1.11 ± 0.18  66.55 ± 5.05 N199I 4.51 ± 0.37 212.94 ± 15.9 N199L 3.95 ± 0.26 267.08 ± 27   N199F 4.02 ± 0.18 217.51 ± 9.95 N199Q 6.64 ± 0.59  303.72 ± 39.38 N199E 3.21 ± 0.98  119.34 ± 14.43 N199W 2.69 ± 0.18  168.17 ± 12.37

TABLE-US-00005 TABLE 4 Effect of substitutions of multiple amino acids on enzyme activity 1-Naphthol production 7-Hydroxycoumarin Mutants (mg .Math. L.sup.−4 .Math. h.sup.−1) production (mg .Math. L.sup.−1 .Math. h.sup.−1) Wild- 0.51 ± 0.05 46.98 ± 3.4 type CMA 6.59 ± 0.45 134.67 ± 4.39 CMB    7 ± 0.51 149.91 ± 2.39 CMC 1.99 ± 0.15  73.48 ± 1.11 CMABC 7.19 ± 0.11 160.53 ± 5.05 CMA: E88C/N199Q CMB: N1990/Q209A CMC: E88C/Q209A CMABC: E88C/N199Q/Q209A