Microbiological process for enantioselective (S)-hydroxylation

Abstract

The invention relates to a microbiological process for preparing chiral arylalkanols by enantioselectively hydroxylating arylalkanes in the presence of a microorganism's host cells which are expressing the genes for oxygenases.

Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to a microbiological process for preparing chiral arylalkanols by enantioselectively hydroxylating arylalkanes in the presence of a microorganism's host cells which are expressing the genes for oxygenases.

[0003] 2. Brief Description of the Prior Art

[0004] It is known that the biochemical oxidation of aromatic and aliphatic compounds can be effected by using oxygenases which are formed by microorganisms. In this connection, aliphatic compounds can be oxidised to primary and secondary alcohols (W. A. Duetz et al., Current Opinion in Biotechnology, 12 (2001) 419-425; H. L. Holland, H. K. Weber, Current Opinion in Biotechnology, 11 (2000) 547-553; H. L. Holland, Current Opinion in Biotechnology, 3 (1999) 22-27).

[0005] EP-A 277 674 discloses a microbiological process for terminally hydroxylating apolar aliphatic compounds having from 6 to 12 C atoms.

[0006] EP-A 502 524 and EP-A 477 828 describe microbiological processes for terminally hydroxylating methyl and ethyl groups at 5- or 6-ring heterocycles. Recombinant Escherichia coli or Pseudomonas strains which are expressing the Pseudomonas putida xyl genes or the Pseudomonas oleovorans alk genes, respectively, are used for a catalysis.

[0007] While a variety of studies deal with the side-chain hydroxylation of alkyl aromatic compounds using bacterial strains, high enantioselectivities have only been achieved in exceptional cases and have then been associated with poor regioselectivities (W. Adam, Z. Lukacs, C. Kahle, C. R. Saha-Möller, P. Schreier, Journal of Molecular Catalysis B: Enzymatic 11 (2001) 377-385). Uzura et al., Journal of Bioscience and Bioengineering, Vol. 91, No. 2 (2001) 217-221 describe the enantioselective synthesis of (R)-1-phenylethanol and (R)-1-phenylpropanol, in enantiomeric excesses of >99% (R), from the corresponding alkylbenzenes using fungi and yeasts. However, these authors were only able to synthesise the (R) enantiomers. The microorganisms were identified in an extensive screening.

[0008] In Appl. Environ. Microbiol., Vol. 62, No. 9 (1996) 3101-3106, K. Lee and D. T. Gibson report that the naphthalene dioxygenase (NDO) from the Pseudomonas sp. strain NCIB 9816-4 is able to convert ethylbenzene into a mixture consisting of 1-phenylethanol, acetophenone, 2-hydroxyacetophenone, styrene and 1-phenyl-1,2-ethanediol. The phenylethanol in this mixture was present as (S)-1-phenyl-ethanol in an enantiomeric excess (ee) of 77%. The reaction was carried out using isolated enzymes, with both the high number of by-products and the low degree of enantioselectivity having a disadvantageous effect on the yield of (S)-1-phenyl-ethanol.

[0009] There has thus far been no report of any enantioselective enzymic synthesis of (S)-1-arylalkanols in high enantiomeric excesses.

[0010] Chiral alcohols are important intermediates in the production of pharmaceutical active compounds, which means that the selective preparation of the individual enantiomers is of great importance.

[0011] The object of the present invention therefore was to provide a simple process for the enantioselective synthesis of (S)-1-arylalkanols in high enantiomeric excesses.

[0012] The process according to the invention which is described below, and which offers the possibility, for the first time, of preparing (S)-1-homoarylalkan-1-ols or (S)-1-heteroarylalkan-1-ols in high enantiomeric excesses, has now been surprisingly found.

SUMMARY OF THE INVENTION

[0013] The present invention therefore relates to a process for preparing (S)-1-homoarylalkan-1-ols or (S)-1-heteroarylalkan-1-ols by means of enantioselectively hydroxylating 1-homoarylalkanes or 1-heteroarylalkanes, which comprises carrying out the hydroxylation in the presence of a microorganism which contains nucleic acids which encode an oxygenase which catalyses the enantioselective (S)-hydroxylation.

[0014] The process according to the invention can be carried out either in the presence of a wild type or in the presence of a transformed microorganism.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The following is a more detailed description of the invention with particular reference to its preferred embodiments.

[0016] The process according to the invention is preferably a process in which the hydroxylation is carried out in the presence of a transformed microorganism which contains nucleic acids which encode an oxygenase.

[0017] This is furthermore preferably a process in which the hydroxylation is carried out in the presence of bacteria, yeasts or fungi which contain nucleic acids which encode an oxygenase.

[0018] This is particularly preferably a process in which the hydroxylation is carried out in the presence of Gram-negative bacteria, in particular those of the genus Pseudomonas, Rhodopseudomonas, Burkholderia, Ralstonia, Comamonas, Acinetobacter, Rhizobium or Escherichia coli.

[0019] This is very particularly preferably a process in which the hydroxylation is carried out in the presence of an Escherichia coli K 12 strain or of a Pseudomonas putida strain.

[0020] The process according to the invention is carried out in the presence of the above-listed microorganisms, which contain nucleic acids which encode an oxygenase. According to the present invention, oxygenases are to be understood as meaning any proteins which are suitable for catalyzing oxidation reactions in which oxygen atoms are incorporated directly into the substrate or starting-compound molecule.

[0021] The process according to the present invention is preferably carried out in the presence of a microorganism which contains nucleic acids which encode a naphthalene dioxygenase.

[0022] The process is particularly preferably carried out in the presence of a microorganism which contains nucleic acids which encode a naphthalene dioxygenase derived from Pseudomonas putida G7 (DSM 4476) or Pseudomonas sp. NCIB 9816 (DSM8368) or similar enzymes possessing at least 70% identity, preferably 80% identity, particularly preferably 90% identity, over the entire length of the amino acid sequence. The corresponding sequence for the naphthalene dioxygenase from Pseudomonas putida G7 (DSM 4476) or Psuedomonas sp. NCIB 9816 (DSM 8368) is described, from base pair 853 to base pair 4316, under Seq ID: NCBI Acc. No. M83949.

[0023] The degree of the identity of the amino acid sequence is preferably determined using the GAP program from the GCG program package, Version 9.1 under standard settings (Devereux et al. (1984), Nucleic Acids Research 12, 387).

[0024] A transformed microorganism which is preferably employed in the process according to the invention may harbor expression vectors for expressing the gene.

[0025] The expression vectors possess sequences, such as at least one promoter for constitutive or inducible expression, or else enhancers, which are functionally linked to the oxygenase genes to be expressed.

[0026] Preference is given to expression vectors, such as pUC18, pUC19, pVLT31, pVLT33, pVLT35, pProEXHTabc (Invitrogen, Karlsruhe), TOPO expression vectors, e.g. pCRT7/CT-TOPO (Invitrogen, Karlsruhe), vectors of the pET series, for example pET3a, pET23a, pET28a, and pET32a (Novagen, Bad Soden), vectors of the pASK series, e.g. pASK-IBA7 (IBA, Gottingen), vectors of the pQE series, e.g. pQE30 pQE31, pQE70 and pREP4 (Qiagen, Hilden), vectors of the pBluescript series (Stratagen, Heidelberg), and the like, which enable the oxygenase genes to be expressed in Gram-negative bacteria. The vectors may optionally possess N-terminal or C-terminal tags, such as a Strep tag or His tag, or the like.

[0027] Particular preference is given to the expression vectors pVLT33 and pCRT7/CT-TOPO.

[0028] The process according to the invention makes it possible to prepare (S)-1-homo-arylalkan-1-ols or (S)-1-heteroarylalkan-1-ols, which are collectively referred to below as (S)-1-arylalkan-1-ols, by means of enantioselectively hydroxylating 1-homoarylalkanes or 1-heteroarylalkanes.

[0029] Preferably, (C1-C6)-alkyl-(C5-C14)-aromatics, particularly preferably (C1-C6)-alkyl-(C5-C6)-aromatics, which are ring-substituted once or more than once, identically or differently, and in which from 0 to 3 carbon atoms, i.e. 0, 1, 2 or 3 carbon atoms, is/are replaced, in the aromatic ring system, with heteroatoms from the group N, 0 and/or S, are employed as starting compounds in the process according to the invention.

[0030] Particular preference is given to using the process according to the invention to prepare (S)-1-homoarylalkan-1-ols of the general formula (I), 1

[0031] where

[0032] R1 is C1-C3-alkyl, particularly preferably methyl,

[0033] R2, R3, R4, R5 and R6 are, independently of each other, H, halogen, C1-C4-haloalkyl, C1-C4-alkyl, hydroxyl, C1-C4-alkoxy, thiol, C1-C4-thioalkoxy, amino or primary or secondary C1-C4-aminoalkyl, and at least one radical R2 to R6 is different from H, particularly preferably, independently of each other, H, halogen or C1-C4-haloalkyl, and at least one radical R2 to R6 is different from H,

[0034] by means of enantioselectively hydroxylating compounds of the general formula (II), 2

[0035] where

[0036] R1 has the meaning mentioned for formula I, and

[0037] R2, R3, R4, R5 and R6 have, independently of each other, the meaning mentioned for formula I.

[0038] In accordance with the invention, C1-C3-alkyl can be methyl, ethyl, n-propyl and iso-propyl, C1-C4-alkyl can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, isobutyl and sec-butyl, C1-C4-haloalkyl can be the abovementioned C1-C4-alkyl which is substituted, once or more than once, by fluorine, chlorine, bromine and/or iodine, primary or secondary C1-C4-aminoalkyl can be the abovementioned C1-C4-alkyl which possesses corresponding amino substituents, C1-C4-alkoxy can be methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, iso-butoxy and sec-butoxy, and C1-C4-thioalkoxy can be the corresponding abovementioned alkoxy groups containing sulfur instead of oxygen.

[0039] The process according to the invention is a simple process for enantioselectively synthesizing (S)-1-arylalkan-1-ols in unexpectedly high enantiomeric excesses. In accordance with the invention, high enantiomeric excesses are ee values of greater than or equal to 80% (>80%) (S-enantiomer), preferably ee values of greater than or equal to 90% (>90%) (S-enantiomer), particularly preferably of greater than or equal to 95% (>95%) (S-enantiomer). This thereby achieves the highest enantiomeric excesses thus far for the enantioselective synthesis of (S)-1-arylalkan-1-ols. All the previously disclosed attempts to prepare these S-enantiomers yielded markedly lower ee values.

[0040] Prior to the conversion of the abovementioned substances, the oxygenase-containing microorganisms are grown on complex or mineral nutrient media, using culturing methods which are customary for growing the relevant microorganisms (culturing in shaking flasks, batch fermentations, fed-batch fermentations or continuous fermentations), to an optical density of from 1 to 500, preferably of from 5 to 300, measured at a wavelength of 600 nm (OD600), and, where appropriate, concentrated after they have been grown.

[0041] If the oxygenase-containing microorganisms are transformed microorganisms, the natural, synthetic or semisynthetic nucleic acid is first of all obtained or isolated, where appropriate purified, then cloned using a suitable method known to the skilled person and, where appropriate, purified once again. Suitable methods which are known to the skilled person can then be used to introduce the isolated genes into cells of the same or different species in relation to the wild type. The transformed host cells are then grown as described in the preceding passage.

[0042] After a suitable cell density has been reached, expression of the gene is induced by adding an inducer molecule. Alternatively, the induction can also be effected by using inducing culture conditions or using media which has an inducing effect.

[0043] For the reaction, the starting compound is added to the cells, which are either present in the growth medium or, where appropriate, resuspended in an isotonic solution after previously having been sedimented. The mixture consisting of starting compound and cells is supplied with oxygen either by being shaken in an Erlenmeyer flask or by being aerated in a unit (e.g. in a fermenter) which is suitable for aerating cell suspensions.

[0044] The reaction can be effected by adding starting compound once, several times or continuously. The concentration of starting compound in the cell suspension can be between 1 and 900 mM, preferably between 2 and 500 mM, particularly preferably between 3 and 250 mM.

[0045] Auxiliary substances, such as cyclodextrins or DMSO (dimethyl sulfoxide), can be added during the reaction in order to increase the solubility of the starting compound.

[0046] The reaction can be effected in a pH range of from 3 to 11, preferably of from 4 to 10, particularly preferably of from 5 to 9. It is customarily carried out at a temperature of from 10 to 60° C., preferably of from 18 to 45° C.

[0047] After the reaction, the corresponding (S)-1-homoarylalkan-1-ols or (S)-1-hetero-arylalkan-1-ols are isolated from the cell suspension using suitable extracting agents and extraction methods. Preference is given to using commercially available solvents, such as toluene, ethyl acetate, dichloromethane, isobutyl ketone, cyclo-hexane and methyl-cyclo-hexane, inter alia, for this purpose. In this connection, the extraction can take place by supplying the extracting agent either continuously or discontinuously. In the simplest case, the purification is achieved by shaking, while at the same time extracting, with the abovementioned extracting agents. Ethyl acetate is preferably used for the extraction by shaking.

[0048] In accordance with the invention, the above-described reaction is to be understood as meaning the enantioselective hydroxylation of 1-homoarylalkanes or 1-heteroarylalkanes to give (S)-1-homoarylalkan-1-ols or (S)-1-heteroarylalkan-1-ols in accordance with the process according to the invention.

[0049] In order to improve comprehension, the meaning of particular words and terms which are used in the description, the examples and the claims will be explained in more detail below.

[0050] The term “vector” describes a nucleic acid element which is used for introducing exogenous nucleic acids in the host cells. A vector contains a nucleic acid sequence which encodes one or more polypeptides. Vectors which are able to control the expression of the genes which they contain are also termed “expression vectors”.

[0051] The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) or, if appropriate, to ribonucleic acid (RNA). The term also encompasses, in an equivalent manner, analogs of RNA or DNA which are prepared from nucleotide analogs and also, in the relevant case, single-stranded (sense and antisense) polynucleotides and double-stranded polynucleotides.

[0052] Within the context of the present invention, the terms “cell” and “host cell” can be used in the same sense. It goes without saying that these terms refer not only to a single cell but also to the descendants of such cells. While, due to particular modifications during the course of following generations (e.g. mutations), such descendants may possibly not be identical to the progenitor cell, they are nevertheless also encompassed by the present invention.

[0053] The term “promoter” refers to nucleic acid sequences which regulate the expression of a particular nucleic acid which is functionally linked to the promoter, by the promoter sequences being recognized, for example, as being a specific binding sequence and start site for the transcription. The term also encompasses “tissue-specific” promoters, i.e. promoters which only control the expression of the specific nucleic acid in particular cells (for example cells belonging to a particular tissue). The term also encompasses “tissue-non specific” promoters and promoters which lead to constitutive expression or which are inducible.

[0054] The term “enhancers” refers to nucleic acid sequences which regulate the expression of a particular nucleic acid by increasing the activity of the promoters. Enhancers are able to stimulate the expression of both homologous and heterologous genes (inserted foreign genes).

[0055] Within the context of the present invention, the terms “cloning” and “to clone” can be used in the same sense. They describe the isolation and subsequent propagation of nucleic acid fragments or genes. This can be effected using the methods which are customary in molecular biology or recombinant DNA technology; for example, PCR (polymerase chain reaction) can be used to carry out cell-free cloning or a cloning system can be used, by way of a great Variety of methods, to effect a cell-bound cloning, proceeding from the introduction of the foreign DNA into vectors, followed by the introduction of a vector into a host cell and subsequent propagation of the host cells.

[0056] The term “amplification” refers to increasing a gene dose by producing additional copies of nucleic acid fragments or genes, something which can be achieved, for example, by means of the previously described cloning or by means of carrying out a PCR.

EXAMPLES

[0057] Both in the above and in that which follows, “mM” stands for “millimolar” solutions (statement of concentration).

[0058] Preparing the Oxygenase-Expressing Microorganisms

[0059] 1. Preparing the Genomic DNA from Pseudomonas putida G7

[0060] 100 ml of Luria-Bertani (LB) medium (10 g of yeast extract/l; 5 g of peptone/l; 10 g of NaCl/l; pH 7.1) were added to a 1,000 ml Erlenmeyer flask, inoculated with the bacterial strain Pseudomonas putida (P. putida) G7 (DSM 4476) and incubated overnight, at 30° C. and 175 rpm, in a shaking incubator.

[0061] The culture which had grown overnight was sedimented at 0.12,000×g for 10 min in a centrifuge. The supernatant was discarded and the cell pellet was resuspended in 40 ml of extraction buffer (100 mM tris HCl (trishydroxymethylaminomethane chloride); 100 mM EDTA (ethylenediamine tetraacetate); 100 mM Na2HPO4/NaH2PO4 pH 8.0; 1.5 M NaCl; 1% CTAB (hexadecyltrimethylammonium bromide)). 140 &mgr;l of proteinase K (10 mg/ml) were added to break down protein. The samples were incubated at 37° C. for 30 min in a water bath and shaken at regular intervals. This was then followed by the addition of 2 ml of SDS (sodium dodecyl sulfate) and a further incubation, at 65° C. for 2 h, in the waterbath. The samples were once again shaken at regular intervals.

[0062] Following on from this, the samples were shock-frozen three times at −82° C. in an ethanol/dry ice bath, thawed in the 65° C. water bath and centrifuged at 8,000×g for 16 min. The supernatant was decanted. The pellet was resuspended with 10 ml of extraction buffer, incubated at 60° C. for 30 min in the waterbath and shaken from time to time. After the cell debris had been sedimented once again for 15 min at 8,000×g, the supernatant was combined with the supernatant from the first extraction and the sedimented cell debris were discarded.

[0063] The combined supernatants were extracted by shaking with one volume of chloroform/isoamyl alcohol (24/1), after which the upper aqueous phase was carefully removed and treated with 0.6 times the volume of isopropanol in order to precipitate the DNA.

[0064] These samples were shaken well and centrifuged at 18,000×g for 20 min. The supernatant was decanted and discarded while the DNA pellet was washed twice with 20 ml of ice-cold 70% ethanol. After the ethanol had been added, the tubes were shaken vigorously. The samples were subsequently centrifuged once again at 8,000×g for 10 min. The supernatant was decanted and discarded. The DNA pellets were air-dried until It was no longer possible to discern any smell of ethanol and taken up in 8 ml of TE buffer (10 mM Tris HCl, 1 mM, EDTA, pH 8.0). 40 &mgr;l of ribonuclease A solution (100 mg/ml) were added to the dissolved DNA in order to break down the RNA and the mixture was incubated at 37° C. for 15 min in a waterbath. After the RNAse treatment, the DNA solution was cooled on ice and stored at −20° C.

[0065] 2. Using Agarose Gel Electrophoresis to Purify the Genomic DNA.

[0066] 10 &mgr;l of blue marker (BlueJuice™, GibcoBRL, Karlsruhe) were added to 400 &mgr;l of the DNA preparation and the whole was loaded onto a preparative 1.0% agarose gel and fractionated at 100 V for 1 h. After the fractionation had come to an end, a sterile scalpel was used to excise the DNA bands from the gel in the form of small cubes. Approximately 100 mg of DNA-containing agarose were added to a “gel nebulizer” (millipore, Eschbom).

[0067] The agarbse gel piece-loaded “gel nebulizers” were transferred to “Ultrafree-MC® Centrifugal Filters” (millipore, Eschborn) and centrifuged at 15,000×g for 10 min in an Eppendorf tube. The eluted DNA solution which had collected in the Eppendorf tube was stored at −20° C.

[0068] 3. Cloning the PCR-Amplified Oxygenase Genes

[0069] The naphthalene dioxygenase-en coding genes (nahAa, nahAb, nahAc and nahad, collectively termed nahaa-d in that which follows) were amplified using the polymerase chain reaction (PCR). The genomic DNA of the wild-type strain (Pseudomonas putida G7) was used as the template DNA. The following synthetically prepared single-stranded DNA molecules were used as primers: 1 “forward primer”: 5′-GAATTCATGGAACTTCTCATCATACAGCCAAAC-3′ (SEQ ID NO: 1) “reverse primer”: 5′-CTGCAGTCACAGANAGACCATCAGATTGTG-3′ (SEQ ID NO: 2)

[0070] The sequence of the forward primer corresponds to the sequence at the beginning of the nahAa gene and the sequence of the reverse primer corresponds to the sequence at the end of the nahAd gene. In order to be able to carry out subsequent secondary clonings more easily, an EcoRI restriction cleavage site was added to the forward primer and a PstI restriction cleavage site was added to the reverse primer (restriction sites in each case underlined).

[0071] For the PCR amplification, the following reaction mixture was pipetted into a 1.5 ml Eppendorf tube on ice: 39 &mgr;l of H2O, 1 &mgr;l of 50 mM MgSO4, 1 &mgr;l of genomic DNA solution 1 &mgr;l of dNTP solution (in each case 10 mM), 1 &mgr;l of forward primer solution (100 ng/&mgr;l) and 1 &mgr;l of reverse primer solution (100 ng/&mgr;l).

[0072] The PCR assays were mixed thoroughly and incubated in a thermocycler (Biometra T3 Thermocycler, Biometra, Göttingen) in accordance with the following temperature program: 1 cycle: 120 s at 94° C.; 30 cycles: 30 s at 94° C., 30 s at 60° C., 210 s at 68° C.; 1 cycle: 210 s at 68° C.; maintain temperature at 4° C.

[0073] 4. Purifying the PCR Product

[0074] The DNA fragments which had been prepared by PCR were fractionated in an agarose gel and extracted from the gel using the “Minlute m Gel Extraction Kit” (Qiagen, Hilden).

[0075] In each case 50 &mgr;l of the PCR reaction were treated with in each case 5 &mgr;l of “10× Blue Juice™” (GibcoBRL, Karlsruhe) and loaded onto a 1% agarose gel and fractionated at 100 V for 1 h. After the electrophoresis had come to an end, a sterile scalpel was used to excise the DNA fragments from the gel, after which the gel bands were transferred to an Eppendorf tube and treated with 300 &mgr;l of QG buffer (Mat. No. 1007226, Qiagen, Hilden) per 100 mg of gel. After 10 min of incubation at 50° C. and regular mixing, in each case 1 vol of isopropanol was added to the samples, which were then carefully mixed.

[0076] Following on from this, the DNA solution was transferred to a “MinElute™ Spin Column” (Qiagen, Hilden), which was centrifuged at 13,000×g for 1 min.

[0077] The eluate was discarded. In a second step, 500 &mgr;l of QG buffer were added to the “MinElute™ Spin Column”, which was centrifuged at 30,000×g for 1 min. The eluate was once again discarded. In the same way, the “MinElute™ Spin Column” was loaded once again with 750 &mgr;l of PE buffer (Mat. No. 1015211, Qiagen, Hilden) and centrifuged twice at 13,000×g for 1 min.

[0078] In order to collect the purified DNA solution, the “MinElute™ Spin Column” was transferred to a fresh Eppendorf tube and the bound DNA was eluted with 10 &mgr;l of EB buffer (10 mM tris HCl, pH 8.5).

[0079] 5. Preparing the Expression Strain E. coli BL 21 (DE3)::pCR T7/CT-TOPO::nahAa-d

[0080] The PCR fragments were cloned into the “pCR®T7/CT-TOPO® vector” using the “pCR®T7/CT-TOPO®-TA Expression Kit” (Invitrogen, Karlsruhe) and transformed into Escherichia coli (E. coli) cells. In order to ligate the PCR product to the “pCR®T7/CT-TOPO® vector”, the following reaction was prepared, mixed and incubated at room temperature (previously and in that which follows, 23° C.) for

[0081] 10 min: 4 &mgr;l of DNA solution containing purified PCR fragment (see 4.), 1 &mgr;l of vector DNA solution (“pCR®T7/CT-TOPO®-TA expression kit”, Invitrogen, Karlsruhe), 1 &mgr;l of salt solution (“pCR®T7/CT-TOPO®-TA expression kit, Invitrogen, Karlsruhe).

[0082] For the purpose of transforming the ligated DNA into the bacterial strain E. coli DH5&agr;, 5 &mgr;l of the previously described ligation preparation were mixed with 50 &mgr;l of Max Efficiency E. coli DH5(x competent cells (from Invitrogen, Karlsruhe) in 15 ml sterile Falcon tubes, which were incubated on ice for 30 min, heated at 42° C. for 30 s and then once again placed on ice for 2 min.

[0083] For the regeneration, 450 &mgr;l of SOC Medium (glucose 18 g, tryptone 20 g, yeast extract 5 g, NaCl 0.5 g, and deionised H2O 950 ml, GibcoBRL, Karlsruhe) were added to the cells and the latter were incubated at 37° C. for 1 h. For the selection, 250 &mgr;l of the cell suspension were plated on solid LuriaBertani Medium in the presence of 100 &mgr;g of Ampicillin/ml and cultured at 30° C. overnight.

[0084] The plasmid mini preparation kit (Qiagen, Hilden) was used to isolate the plasmid DNA from several clones, after which the sequence was checked and the recombinant vector DNA was transformed into competent E. coli BL21 (DE3) cells (Stratagen, Heidelberg) using the above-described method.

[0085] The resulting expression strain was designated E. coli BL21(DE3)::pCRT7/CT-TOPO::nahAa-d or E. coli T7-NDO for short.

[0086] 6. Preparing the Expression Strain E. coli BL21 (DE3)::pVLT33:nahAa-d

[0087] The vectors pCRT7/CT-TOPO::nahAa-d and pVLT33 were digested with the restriction enzymes EcoRI (New England Biolabs, Frankfurt a.M.) and HINDIII (New England Biolabs, Frankfurt a.M.) (in each case 30 &mgr;l of DNA; 3 &mgr;l of HindIII enzyme; 3 &mgr;l of enzyme EcoRI; 7 &mgr;l of buffer 3; 27 &mgr;l of H2O; incubation at 37° C. for from 2 to 3 h). In this digestion, the pVLT33 was linearized and the naphthalene dioxygenase-encoding nahAa-d DNA fragment was excised from pCRT7/CT-TOPO::nahAa-d.

[0088] The DNA fragments which were obtained in the restriction digestion were fractionated electrophoretically in an agarose gel. The DNA fragments encoding the pVLT33 vector and the naphthalene dioxygenase gene were fractionated in an agarose gel using the QLAX II agarose gel extraction kit (Qiagen, Hilden), isolated from the gel (see 4.) and ligated to each other (15 &mgr;l of pVLT33-DNA solution, 6 &mgr;l of nahAa-d DNA solution, 3 &mgr;l of ligase buffer and 1 &mgr;l of T4 ligase (New England Biolabs, Frankfurt a.M.), incubation at 16° C. overnight).

[0089] As described under 5., this was then followed by transformation into E. coli DH5&agr;, preparation of the plasmid DNA from different clones, checking of the sequence and transformation into competent E. coli BL21(DE3) cells. The resulting expression strain was designated E. coli BL21(DE3)::pVLT33::nahAa-d.

[0090] 7. Preparing the Expression Strain P. putida KT2440::pVLT33::nahAa-d

[0091] In order to prepare the expression strain Pseudomonas putida KT2440::pVLT33::nahAa-d, the pVLT33::nahAa-d vector was electrotransformed in competent P. putida KT2440 cells.

[0092] In order to prepare competent P. putida KT2440 cells, a 50 ml preliminary culture of the strain was grown overnight at 25° C. in LB medium. A 500 ml main culture of the same medium was inoculated with 5 ml of the preliminary culture and cultured, at 25° C., up to an optical density (600 nm) of 0.650. The cells were cooled on ice for 30 min and then sedimented at 8,000×g and at 0° C. for 30 min. The supernatant was discarded and the cell pellet was resuspended in 250 ml of sterile ice-cold H2O. In order to wash the cells, the latter were sedimented a second time and once again resuspended in 250 ml of ice-cold H2O. There then followed 4 further sedimentation and resuspension steps, after which the cells were resuspended in 125 ml of ice-cold H2O, 125 ml of ice-cold H2O+10% glycerol and twice in 500 &mgr;l of ice-cold H2O+10% glycerol. After the last resuspension, the cells were frozen for storage at −80° C. in 50 &mgr;l portions.

[0093] For the electrotransformation, 2 &mgr;l of the pVLT33::nahAa-d DNA were mixed, in an 0.1 cm electroporation cuvette (Bio-Rad, Munich) on ice, with a 50 &mgr;l portion of competent P. putida KT2440 cells which had previously been thawed on ice. The electrotransformation took place at 25 kV in a 2510 Electroporator (Eppendorf, Hamburg).

[0094] Subsequently, the cells were immediately taken up in 950 &mgr;l of SOC medium (GibcoBRL, Karlsruhe), regenerated at 30° C. for 1 h and, after that, plated out, for the selection, on solid Luria-Bertani medium containing 100 &mgr;g of kanamycin/ml and incubated at 30° C.

Example 1

Using the Strain E. coli BL21(DE3)::pCRT7/CT-TOPO::nahAa-d to Oxidize Various Arylalkanes

[0095] The strain E. coli BL21(DE3)::pCRT7/CT-TOPO::nahAa-d was grown overnight at 30° C. in 20 ml of Luria-Bertani medium containing 100 &mgr;g of ampicillin/ml. 200 ml of Luria-Bertani medium, containing 500 &mgr;g of ampicillin/ml, were inoculated with this preliminary culture in a 1-1-Erlenmeyer flask, and likewise incubated at 30° C. while being shaken. After an optical density of 2, which was measured at 600 nm (OD600), had been reached, expression of the nahAa-d genes was induced by adding 100 &mgr;l of a 1M solution of IPTG (IPTG=isopropylthiogalactoside). After approx. 3 h, the culture reached an OD600 of 4 and was harvested by being centrifuged for 10 min at 10,000×g, and the cells were then suspended in {fraction (1/20)} Volume of M9 medium (200 ml of 5×conc. M9 salts (Na2HPO4×7H2O 64 g, KH2PO4 15 g, NaCl 2.5 g, NH4CL 5 g); 2 ml of 1M MgSO4; 0.1 ml of 1M CaCl2; 0.1 ml of 0.15% glyceryl; 750 ml of H2O).

[0096] For the conversion reaction, in each case 1 ml of the concentrated cell suspension was transferred to a 13 ml screw cap tube and treated with 20 &mgr;l of a 200 mM stock ethanolic solution of the given starting compound (cf. Tab. 1). The samples were incubated at 30° C. on a shaker. The reactions were stopped after 0 h and 17 h by adding 1 ml of ethyl acetate. The samples were extracted by shaking intensively and, after a phase-separation centrifugation (4,000×g), the upper ethyl acetate phase was removed and transferred to a sample tube. 400 &mgr;l of SM NaCl were added to improve the phase separation.

[0097] The ethyl acetate phases were analyzed using coupled gas chromatography/mass spectroscopy (GC-MS) and chiral gas chromatography. The results are listed in Table 1. 2 TAB 1 Hydroxylation of arylalkanes by recombinant bacteria Reaction ee Starting Conc. time Conversion Value [%] compound [mM] [h] Product [%] (enantiomer) Ethylbenzene 4 17 1-phenylethan-1-ol 48.9 >98 (S) 1-Ethyl-4- 4 17 1-(4-trifluoro- 20.2 >98 (S) trifluoromethyl- phenyl)ethan-1-ol benzene 1-Ethyl-4-fluoro- 4 17 1-(4-fluorophenyl)- 9.6 >98 (S) benzene ethan-1-ol 4-Chloro-1- 4 17 1-(4-chlorophenyl)- 76.5 >98 (S) ethyl-benzene ethan-1-ol Propylbenzene 4 17 1-phenylpropan-1-ol 3.9 >98 (S) 2-Brom-1-ethyl- 4 17 1-(2-bromphenyl)- 24.2 >98 (S) benzene ethan-1-ol

[0098] The starting compounds can be obtained commercially.

Example 2

Oxidation of 4-chloro-1-ethylbenzene by the Strains E. coli::PVLT33::nahAa-d and P. putida::pVLT33::nahAa-d

[0099] Growth, expression of the naphthalene dioxygenase, the conversion of the 4-chloro-1-ethylbenzene, and the analysis, were carried out using the expression strains E. coli BL21 (DE3)::pVLT33::nahAa-d and P. putida KT2440::pVLT33::nahAa-d as described in Example 3. 4-Chloroethylbenzene was added to the samples, at concentrations of 4 and 8 mM, respectively, from a 400 mM stock solution in ethanol. In contrast to Example 1, the samples were prepared for the analysis by adding 1 vol. of acetonitrile. 3 TAB 2 Hydroxylation of 4-chloro-1-ethylbenzene by recombinant bacteria ee Value Conc. Reaction Starting Conversion Product Strain [mM] time [h] compound/product [%] [%] E. coli BL21(DE3) 4 17 4-chloro-1-ethylbenene/ 59.0 >98(S) : : pVLT3 3: :NDO 1-(4-chlorophenyl)- ethan-1-ol E. coli BL21(DE3) 8 17 4-chloro-1-ethyl- 11.7 >98(S) : : pVLT33: :NDO benzene/1-(4-chloro- phenyl)-ethan-1-ol P. putida KT2440: : 4 17 4-chloro-1-ethyl- 21.8 >98(S) pVLT3 3: :NDO benzene/1-(4-chloro- phenyl)-ethan-1-ol P. putida KT2440: : 8 17 4-chloro-1-ethyl- 23.9 >98(S) pVLT33 : :NDO benzene/1-(4-chloro- phenyl)-ethan-1-ol

[0100] Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.

Claims

1. A process for preparing (S)-1-homoarylalkan-1-ols or (S)-1-heteroarylalkan-1-ols by enantioselectively hydroxylating 1-homoarylalkanes or 1-heteroarylalkanes, which comprises carrying out the hydroxylation in the presence of a microorganism which contains nucleic acids which encode an oxygenase which catalyses the enantioselective (S)-hydroxylation.

2. The process as claimed in claim 1, wherein the hydroxylation is carried out in the presence of a transformed microorganism which contains nucleic acids which encode an oxygenase.

3. The process as claimed in claim 1, wherein the hydroxylation is carried out in the presence of bacteria, yeast or fungi which contain nucleic acids which encode an oxygenase.

4. The process as claimed in claim 1, wherein the hydroxylation is carried out in the presence of Gram-negative bacteria.

5. The process as claimed in claim 1, wherein the hydroxylation is carried out in the presence of Gram-negative bacteria of the genus Pseudomonas, Rhodopseudomonas, Burkholderia, Ralstonia, Comamonas, Acinetobacter, Rhizobium or Escherichia coli.

6. The process as claimed in claim 1, wherein the hydroxylation is carried out in the presence of an Escherichia coli K12 strain or of a Pseudomonas putida strain.

7. The process as claimed in claim 1, wherein the microorganisms contain nucleic acids which encode a naphthalene dioxygenase.

8. The process as claimed in claim 1, wherein the microorganisms contain nucleic acids which encode a naphthalene dioxygenase from Pseudomonas putida G7 (DSM 4476) or Pseudomonas sp. NCIB 9816 (DSM 8368) or similar enzymes possessing at least 70% identity over the total length of the amino acid sequence.

9. The process as claimed in claim 1, wherein the starting compounds employed are (C1-C6)-alkyl-(C5-C14)-aromatics which are ring-substituted once or more than once, identically or differently, and in which from 0 to 3 carbon atoms are replaced, in the aromatic ring system, with heteroatoms from the group N, O and/or S.

10. The process as claimed in claim 1, wherein the starting compounds employed are (C1-C6)-alkyl-(C5-C6)-aromatics which are ring-substituted once or more than once, identically or differently, and in which from 0 to 3 carbon atoms are replaced, in the aromatic ring system, with heteroatoms from the group N, O and/or S.

11. The process as claimed in claim 1, wherein (S)-1-homoarylalkan-1-ols of the general formula (I)

3

where

R1 is C1-C3-alkyl,

R2, R3, R4, R5 and R6 are, independently of each other, H, halogen, C1-C4-haloalkyl, C1-C4-alkyl, hydroxyl, C1-C4-alkoxy, thiol, C1-C4-thioalkoxy, amino or primary or secondary C1-C4-aminoalkyl, or at least one radical R2 to R6 is different from H

are prepared by enantioselectively hydroxylating compounds of the general formula I,

4

where

R1 has the meaning mentioned for formula I, and

R2, R3, R4, R5 and R6 have, independently of each other, the meaning mentioned for formula I.

12. The process as claimed in claim 11, wherein R1 is C1-C3-alkyl and R2, R R4, R5-and R are, independently of each other, H, halogen or C1-C4-haloalkyl, or at least one radical R2 to R6 is different from H.

13. The process as claimed in claim 11, wherein R1 is methyl.