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 naphthalene and 7-ethoxycoumarin, respectively.

Hydroxylated hydrocarbons, particularly 1-naththol and 7-hydroxycoumarin, are important raw materials in the chemical industry. Presently, said compounds are produced by purely chemical processes. The currently used chemical methods of producing 1-naphthol are mainly divided into three types. The most widely used method in large-scale production is based hydrogenation, oxidation and dehydrogenation of naphthalene. This method is characterized by high quality and continuous production, but low yield. Moreover, current methods of manufacturing naphthols require acids, bases and metal catalysts. These compounds may be expensive or may cause environmental problems if not disposed of properly. Proper disposal may be an additional cost factor.

Biotechnological methods have become more popular for the synthesis of chemical compounds. Generally, such methods are characterized by mild reaction conditions, thus saving energy, and high specificity so that few undesired side products are formed. A P450 monooxygenase capable of introducing hydroxyl groups into a variety of aromatic hydrocarbons has been isolated from Rhodococcus ruber (Liu et al., 2006, Appl. Microbiol. Biotechnol. 72: 876-882). In principle, this enzyme opens the route to biotechnological methods for manufacturing hydroxylated aromatic hydrocarbons. However, the wild-type enzyme has a low catalytic activity which is not sufficient for an economically viable production process.

The above-described problems are solved by the embodiments defined in the claims and in the description below.

In a first embodiment, the present invention relates to a modified P450 monooxygenase, 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 95, 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 and valine 210 is substituted by alanine, wherein said functional mutation leads to an improved reactivity on hydroxylation of aromatic hydrocarbons. SEQ ID NO. 1 defines the amino acid sequence of a P450 monooxygenase originally derived from Rhodococcus ruber. A "modified P450 monooxygenase" has an amino acid sequence which differs by at least one of the substitutions defined above from the amino acid sequence defined in SEQ ID NO. 1.

In addition to the sequence modifications set forth above and below in this application, the amino acid sequence of the modified P450 monooxygenase of the present invention may have further differences to the amino acid sequence of the wild-type enzyme as defined by SEQ ID NO. 1 provided that these sequence differences do not affect its function, i.e. the improved reactivity on hydroxylation of aromatic carbons. It is well known to the person skilled in the art that not all parts of the amino acid sequence of an enzyme are equally important. Sequence regions which are not part of the aforementioned regions may in many cases be altered or even deleted without impairing the enzymatic activity of the protein.

Therefore, the present invention also relates to proteins having at least 90 %, more preferably at least 95 % and most preferably at least 98 % sequence identity to the amino acid sequence defined by SEQ ID NO. 1, provided that such proteins still have an improved reactivity on the hydroxylation of aromatic hydrocarbons.

The person skilled in the art is aware that additions or deletions of amino acids from SEQ ID NO. 1 may shift the particular amino acids positions recited in this application. Therefore, any amino acid position referred to in this application based on the wild-type sequence must be understood as referring to the homologous amino acid position in a protein derived from SEQ ID NO. 1 by deleting or adding amino acids.

Variants of SEQ ID NO. 1 having the degrees of sequence identity set forth above are preferably derived from SEQ ID NO. 1 only by conservative substitutions of amino acids. A "conservative substitution" is a substitution on one amino acid by a different amino acid with similar properties. Preferably, it is an exchange of an amino acid with a non-polar side chain for another amino acid with a non-polar side chain, an exchange of an amino acid with an acidic side chain for another amino acid with an acidic side chain, an amino acid with a basic side chain for another amino acid with a basic side chain or an exchange of an amino acid with a polar side chain for another amino acid with a polar side chain. Because the properties of the side chains in conservative substitutions do not change much, the overall structure of the resulting protein will not be severely affected.

Variants of SEQ ID NO. 1 derived from this sequence by addition of amino acids and having the degrees of sequence identity set forth above are, preferably, derived from SEQ ID NO. 1 by addition of up to 35, more preferably up to 20 and most preferably up to 10 amino acids at the C-terminus and/or the N-terminus. Typical additions to a protein are additions of amino acid sequences which make the purification of the expressed protein easier. One particularly preferred modification is the addition of several histidines, a so-called "his-tag". Also preferred is the addition of peptide linkers.

Variants of SEQ ID NO. 1 derived from this sequence by deletion of amino acids and having the degrees of sequence identity set forth above are, preferably, derived from SEQ ID NO. 1 by deletion of up to 35, more preferably up to 20 and most preferably up to 10 amino acids at the C-terminus and/or the N-terminus.

"Polar amino acids" or "amino acids with polar side chains" as understood by the present application are glycine, serine, threonine, cysteine, asparagine, glutamine, tryptophan and tyrosine.

"Non-polar amino acids" "amino acids with polar side chains" as understood by the present application are alanine, valine, leucine, iso-leucine, phenylalanine, proline, and methionine.

Amino acids with acidic side chains as understood by the present application are aspartate and glutamic acid.

Amino acids with basic side chains as understood by the present application are lysine, arginine and histidine.

In a preferred embodiment of the present invention, at least one of the polar amino acids belonging to the group consisting of glutamic acid 88, lysine 89, threonine 100, leucine 101, arginine 103, asparagine 199, arginine 203, proline 204 and glutamine 209 is substituted by alanine.

If the transformation of naphthalene to 1-naphthol is intended, in the modified P450 monooxygenase according to the present invention at least one of the polar amino acids belonging to the group consisting of glutamic acid 88, asparagine 199, arginine 203 and glutamine 209 is substituted by alanine.

If the transformation of 7-Ethoxycoumarin to hydroxycoumarin is intended, in the modified P450 monooxygenase according to the present invention at least one of the polar amino acids belonging to the group consisting of glutamic acid 88, lysine 89, leucine 101, arginine 103, asparagine 199, arginine 203, proline 204 and glutamine 209 is substituted by alanine.

Since the substitutions of glutamic acid 88, asparagine 199, arginine 203 and glutamine 209 work well for both transformations, in a particularly preferred modified P450 monooxygenase at least one of the amino acids selected from this group is exchanged for a non-polar amino acid. In a particularly preferred modified monooxygenase according to the present invention at least two of the above-mentioned polar amino acids are by alanine. Preferred combinations are the combination of glutamic acid 88 and asparagine 199, the combination of glutamic acid 88 and arginine 203, the combination of glutamic acid 88 and glutamine 209, the combination of asparagine 199 and arginine 203, the combination of asparagine 199 and glutamine 209 or the combination of arginine 203 and glutamine 209.

In another particularly preferred modified monooxygenase according to the present invention at least three of the above-mentioned polar amino acids are exchanged for non-polar ones. Preferred combinations are the combination of glutamic acid 88, asparagine 199 and arginine 203, the combination of asparagine 199, arginine 203 and glutamine 209, the combination of glutamic acid 88, arginine 203 and glutamine 209 or the combination of glutamic acid 88, asparagine 199 and glutamine 209.

In another particularly preferred modified monooxygenase according to the present invention glutamic acid 88, asparagine 199, arginine 203 and glutamine 209 are exchanged for non-polar amino acids.

Substitution at position 209

In one preferred embodiment of the present invention, the modified P450 monoxygenase has an alanine at position 209.

Preferably, additionally at least one of the other amino acids 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 95, 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, and valine 210 is substituted by alanine.

In one particularly preferred embodiment of the present invention, in addition to the substitution of glutamine at position 209 by alanine, 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. More preferably, glutamic acid at position 88 is substituted by an amino acid selected from the group consisting of alanine, serine, histidine, threonine, cysteine and methionine. With these substitutions, the catalytic activity of the enzyme with naphthalene as wells as 7- ethoxycoumarin is increased. Most preferably glutamic acid at position 88 is substituted by an amino acid selected from the group consisting of alanine, cysteine and methionine. In another particularly preferred embodiment of the present invention, in addition to the substitution of glutamine at position 209 by alanine, asparagine at position 199 of the P450 monooxygenase defined by SEQ ID NO: 1 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. Preferably, the modified P450 monooxygenase carries a glutamine at position 199.

In this embodiment, it is particularly preferred that in addition to the substitution of glutamine at position 209 by alanine, both glutamic acid at position 88 and asparagine at position 199 are substituted as defined above.

A particularly preferred modified P450 monooxygenase carries the following substitutions:

(i) an amino acid selected from the group consisting of alanine, cysteine and methionine at position 88;

(ii) an amino acid selected from the group consisting of glutamine, isoleucine, leucine phenylalanine, histidine, methionine, arginine, serine, threonine, tyrosine, tryptophan, alanine, valine and lysine at position 199, preferably glutamine; and

(iii) alanine at position 209.

Substitution at position 88

In one preferred embodiment of the present invention, the modified P450 monoxygenase has an alanine at position 88.

Preferably, additionally at least one of the other amino acids selected from the group consisting of leucine 87, lysine 89, isoleucine 90, threonine 91, proline 92, valine 93, serine 94, glutamic acid 95, 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 and valine 210 is substituted by alanine.

In one particularly preferred embodiment of the present invention, in addition to the substitution of glutamic acid at position 88 by alanine, asparagine at position 199 of the P450 monooxygenase defined by SEQ ID NO: 1 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. Preferably, the modified P450 monooxygenase carries a glutamine at position 199. The term "reactivity with hydrocarbons" refers to the enzyme's ability to introduce an hydroxyl group into a hydrocarbon compound selected from the group consisting of naphthalene, 7- ethoxycoumarin, acenaphthene, fluorine, indene, toluene, ethylbenzene and m-xylene. Preferably, the modified P450 monooxygenase of the present invention has an improved reactivity on hydroxylation of naphthalene and/or 7-ethoxycoumarin.

The reaction hydroxylation of the aforementioned substrates with the P450 monooxygenase of the present invention can be found below:

The reactivity on hydroxylation of aromatic carbons is, preferably, determined in phosphate-buffered saline solution (PBS) with 0.15 g/l of the hydrocarbon to be tested. The hydrocarbon is, preferably, taken from a 3 g/l stock solution in DMSO. The preferred incubation time at 30 °C is 2 hours. The products are then extracted with methyl-tert butyl ether and analyzed by HPLC, preferably using a C18 reverse-phase column. An enzyme shows "improved reactivity" towards the hydrocarbon in question of its specific activity in the above-described assay is higher than that of the wild-type enzyme defined by SEQ. ID NO. 1. Preferably, the specific activity is determined using equal concentrations of a purified enzyme. However, a whole-cell assay as described in the examples may also be used. In another embodiment the present invention relates to nucleic acid sequence encoding any of the modified P450 monooxygenase defined above. The invention also relates nucleic acid sequences having a complementary sequence to the aforementioned nucleic acid sequence.

In yet another embodiment, the present invention relates to an expression construct, comprising the nucleic acid sequence of claim 5as defined above under the genetic control of a regulatory nucleic acid sequence.

The term "expression construct" is well known to the person skilled in the art. An "expression construct" is a nucleic acid molecule comprising a protein coding region and a regulatory sequence which enables the transcription of the protein coding region. Suitable regulatory sequences depend on the host cell which is intended to be used for the recombinant expression of the protein. The person skilled in the art is able to select suitable regulatory regions based on his common knowledge about transcription processes in the selected host cell. A preferred expression construct for the recombinant expression of a modified P450 monooxygenase according to the present invention in E. coli has a nucleic acid sequence as defined by SEQ. ID NO. 2

In yet another embodiment, the present invention relates to a vector, comprising the nucleic acid as defined above or the expression construct as defined above.

The term "vector" is well known to the person skilled in the art. It is a nucleic acid sequence which can be replicated in a host cell. Hence, it must comprise all genetic elements which are required for successful replication in the selected host cell. The person skilled in the art knows which vectors to use for a specific host cell.

In yet another embodiment, the present invention relates to a microorganism comprising the nucleic acid as defined above or the expression construct as defined above or the vector as defined above.

In principle, any microorganism which allows the recombinant expression of transgenes may be used. Thus, the suitable microorganism is one, for which regulatory elements as defined above are known and for which vectors as defined above may be constructed. Preferably, the microorganism is a prokaryote, more preferably a bacterium. Preferred bacteria belong to the genera Rhodococcus or Escherichia. A preferred yeast is Pichia pastoris. The microorganism is, most preferably, E. coli, R. ruber or Pichia pastoris.

In yet another embodiment, the present invention relates to a method for producing the modified p450 monooxygenase as defined above in this application comprising the step of incubating the recombinant microorganism as defined above under conditions suitable for the expression of the monooxygenase. The person skilled in the art knows that different microorganisms have different requirements with regard to the composition of the medium, energy and carbon sources as well as temperature and oxygen supply. He is well able to select suitable conditions based on his knowledge of microbial physiology. If an inducible promotor is used as regulatory element in the expression construct, the person skilled in the art knows the conditions required for the induction of translation.

In yet another embodiment, the present invention relates to a method for the hydroxylation of an aromatic hydrocarbon, comprising the step of al) having at least one of the modified P450 monooxygenases according to the present invention mixed and reacted with said aromatic hydrocarbon and having said aromatic hydrocarbon thus hydroxylated; or

a2) having at least one of the recombinant microorganisms as defined above mixed and reacted with said aromatic hydrocarbon.

The person skilled in the art is able to find suitable reaction conditions by simple experiments. The preferred reaction temperature is 30 °C. The preferred pH is 7.4. Preferably, the reaction takes place in the presence of potassium ions (25 mM). The preferred substrate concentration is 0.12 g/L. If whole bacterial cells are used (embodiment a2), the OD ₆₀₀ should be 30. The person skilled in the art is well aware that the enzyme retains at least some activity in conditions which deviate in one or more parameters from the conditions given above. Hence, the method of the present invention is not limited to those parameters and the particular parameters disclosed above provide only one of several embodiments of the invention. Using the methods disclosed in the present application, the person skilled in the art can easily test the enzyme's activity under different reaction conditions.

If the method according to a2) is used it is preferred that the microorganism has been incubated under conditions suitable for the expression of the modified P450 monooxygenase before mixing it with said aromatic hydrocarbon. It is also preferred to wash this microorganism in a suitable buffer before mixing it with aromatic hydrocarbon in order to limit the presence of undesired side products.

All definitions pertaining to the modified P450 monooxygenases of the present invention, suitable hydrocarbons and host cells given further above in this application also apply to this embodiment.

In yet another embodiment, the present invention relates to the use of the modified P450 monooxygenase according to the present invention for the hydroxylation of an aromatic carbon.

All definitions given above also apply to this embodiment. The following examples are only intended to illustrate the invention. They shall not limit the scope of the claims in any way.

Examples

Construction of nucleic acids encoding modified P450 monooxygenases

A full-length gene encoding P450 protein was synthesized and amplified by PCR using the following primers: 5-ctgG AATT CATG AGT GCAT CAGTT CCGGCGT -3 ' (SEQ ID NO: 3) and 5- ca tcAAGCTTT CAGAGT CGCAG GGCCA-3 ' (SEQ ID NO: 4). The EcoRI and Hind III restriction endonuclease sites in the primer sequences are underlined. The PCR product was isolated and digested with EcoRI and Hind III 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.

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 Dpn I.

Recombinant expression of modified P450 monooxygenases

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

Assessment of the activity of recombinant P450 monooxygenases

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 ^-1 . Products were detected by monitoring the absorbance at 272 nm. Table 1: modified P450 monooxygenases and their activities