(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 27 January 2011 (27.01.2011) WO 2011/009849 A2
(51) International Patent Classification: (74) Common Representative: BASF SE; 67056 Lud C12P 7/62 (2006.01) wigshafen (DE). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/EP2010/060458 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) Date: International Filing CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 20 July 2010 (20.07.2010) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Language: English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 09166015.9 2 1 July 2009 (21 .07.2009) EP SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/227,797 23 July 2009 (23.07.2009) US TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants (for all designated States except US): BASF (84) Designated States (unless otherwise indicated, for every SE [DE/DE]; 67056 Ludwigshafen (DE). MAX- kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN E.V. [DE/DE]; Hofgarten- ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, str. 8, 80539 Mϋnchen (DE). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, (75) Inventors/Applicants (for US only): STURMER, Rain- SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, er [DE/DE]; Hauptstrasse 153, 67127 Rόdersheim- GW, ML, MR, NE, SN, TD, TG). Gronau (DE). SCHNEIDER, Nina [DE/DE]; Carl-Blos- Str. 3, 77654 Offenburg (DE). BOY, Matthias [DE/DE]; Published: Ernst-Ludwig-Str. 39 a, 64625 Bensheim (DE). — without international search report and to be republished ACHATZ, Brigitte [DE/DE]; Windeckstr. 26, 681 63 upon receipt of that report (Rule 48.2(g)) Mannheim (DE). RABUS, RaIf [DE/DE]; Hinrich- — with sequence listing part of description (Rule 5.2(a)) Schmidt-Str. 52, 26 160 Bad Zwischenahn (DE). HEI- DER, Johann [DE/DE]; Spiegelslustweg 24b, 35039 Marburg (DE).
(54) Title: METHOD FOR PREPARING OPTICALLY ACTIVE HYDROXY ACID ESTERS (57) Abstract: The invention relates to a method for preparing optically active 2-hydroxy acid ester derivatives of the formula Rl- C(OH)-C(O)-O-R2 (I),comprising the bringing into contact of 2-oxo acid ester derivatives of the formula R1-C(O)-C(O)-O-R2 (II) with an enzyme (E) selected from the class of dehydrogenases, in the presence of reduction equivalents, where the compound of the formula (II) is enzymatically reduced to the compound of the formula (I), and the reduction equivalents consumed in the course of the reaction are regenerated again by converting a reducing agent (RA) to the corresponding oxidation product (OP) with the help of the enzyme (E). The invention further comprises a dehydrogenase which reduces 2-oxo acid esters in the presence of reduction equivalents to the corresponding S- 2-hydroxy acid esters, and also a nucleic acid encoding the dehydrogenase. Method for preparing optically active hydroxy acid esters
The present invention relates to a method for obtaining optically active hydroxy compounds through enantioselective reduction of organic keto compounds and methods for obtaining these hydroxy compounds in the two-phase system using enzymes with dehydrogenase activity.
Background of the invention
Optically active hydroxy compounds, such as, for example, ethyl 2-hydroxy-4- phenylbutyrate, of very high enantiomer purity are important precursors in the synthesis of drugs, in particular inhibitors of the so-called angiotensin converting enzyme (ACE), which are used for treating patients with hypertension. Many of these inhibitors, such as enalapril, ramipril, cilazapril, quinapril and lysinapril, have a common general structural feature which is responsible for improved application properties. The feature common to such inhibitors is the S-enantiomer form of the 2-amino-4-phenylbutyrate with the structural formula (Ia). The S-2-hydroxy-4-phenylbutyric acid of the formula Ib (R enantiomer) is used for preparing isomeric compounds.
Obtaining chiral compounds through stereospecific microbiological reduction is known (for an overview cf. Simon et al., Angew. Chemie 97, 541 , 1985). The biocatalysts used are often intact microorganisms, for example fungi (e.g. Mucor, Geotrichum, Saccharomyces, Candida) or bacteria (e.g. Proteus, Pseudomonas). It is also possible to use microbial extracts. Electron donors are, for example, carbohydrates (e.g. glucose), formate, ethanol, hydrogen or the cathode of an electrochemical cell. Reduction of the substrate takes place through a reductase, e.g. through a substrate-specific dehydrogenase. In general, the reduction equivalents required by the reductase are supplied by a coenzyme, e.g. by pyridine nucleotides such as NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) or by flavin nucleotides such as FMNH (flavin mononucleotide) and FADH (flavin adenine dinucleotide). The reduced nucleotides for their part are usually formed in a series of enzyme-catalyzed steps with the formation of competing electron acceptors or by electron transfer via natural or synthetic mediators (e.g. ferredoxin, viologens). Also known are final reductases which can absorb electrons directly from the mediators.
For example, Lacerda et al., Tetrahedron: Asymmetry 17, 2006, pages 1186-1 188, describes a method for the microbial reduction of 2-oxo-4-phenylbutyrates using various bacterial strains.
The European patent specification EP 0 347 374 describes a method of preparing R-2- hydroxy-4-phenylbutyric acid in which the substrate is reduced with the enzyme D-lactate dehydrogenase from Staphylococcus epidermidis in the presence of an electron donor and an enzyme-substrate system for regenerating the electron donor.
Furthermore, WO2005/049816 discloses a NADPH-dependent dehydrogenase from Metschnikowia zobellii which, in the presence of water and NADPH, catalyzes the stereoselective reduction of carbonyl compounds to the corresponding chiral hydroxy compounds.
EP 0 645 453 discloses an enantioselective alcohol dehydrogenase which is suitable for the reduction of organic keto compounds to the corresponding hydroxy compounds, this reduction leading enantioselectively to the corresponding R compounds.
Short description of the Invention:
The object of the invention was to find a route for the enantioselective reduction of 2-oxo acid esters, in particular ethyl 2-oxo-4-phenylbutyrate, wherein the reaction method should lead as quantitatively as possible to the product by a cost-effective route.
This object was achieved by the surprising finding that enzymes with dehydrogenase activity, in particular those which can be prepared from microorganisms of the genus Azoarcus, are capable of the enantioselective catalysis of the above reaction with simultaneous cofactor regeneration.
The invention firstly provides a method for preparing optically active 2-hydroxy acid ester derivatives of the formula (I),
R1-C(OH)-C(O)-O-R2
or a salt thereof, wherein R 1 and R2, independently of one another, are a) -(C1-C20)-alkyl, in which alkyl is straight-chain or branched, b) -(C2-C20)-alkenyl, in which alkenyl is straight-chain or branched and comprises one, two, three or four double bonds depending on chain length, c) -(C2-C20)-alkynyl, in which alkynyl is straight-chain or branched and optionally comprises one, two, three or four triple bonds, d) -(C6-C14)-aryl, e) -(C1-C8)-alkyl-(C6-C14)-aryl, f) -(C5-C14)-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or g) -(C3-C7)-cycloalkyl, wherein the residues specified above under a . to g . are unsubstituted or are mono- to trisubstituted, independently of one another, by 1. -OH, 2 . halogen, such as fluorine, chlorine, bromine or iodine, 3 . -NO2, 4 . -C(O)-O-(CI -C20)-alkyl, in which alkyl is straight or branched and is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or 5 . -(C5-C14)-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro
the method comprising the bringing into contact of 2-oxo acid ester derivatives of the formula (II)
R1-C(O)-C(O)-O-R2 with an enzyme (E) selected from the class of the dehydrogenases, in the presence of reduction equivalents, wherein the compound of the formula (II) is enzymatically reduced to the compound of the formula (I), and the reduction equivalents consumed in the course of the reaction are regenerated again by converting a reducing agent (RA) to the corresponding oxidation product (OP) with the help of the enzyme (E).
Depending on the dehydrogenase used, the -OH group of the formula (I) is in the S configuration (Ia) or in the R configuration (Ib) relative to the carbon atom to which it is bonded.
Enzymes (E) suitable according to the invention are in particular the enzymes of the families of the aldo-keto reductases of the aldo-keto reductase superfamily (K.M.Bohren, B.Bullock, B.Wermuth and K.H.Gabbay J.Biol. Chem. 1989, 264, 9547-9551) and of the short-chain alcohol dehydrogenases/reductases (SDR). The latter enzyme group is described in detail, for example, in H.Jδrnvall, B.Persson, M.Krook, S.Atrian, R.Gonzalez-Duarte, J.Jeffery and D.Ghosh, Biochemistry, 1995, 34, pp. 6003-6013 or U.Oppermann, C.Filling, M .HuIt, N.Shafqat, X.Q.Wu, M.Lindh, J.Shafqat, E.Nordling, Y.Kallberg, B.Persson and H.Jornvall, Chemico-Biological Interactions, 2003, 143, pp. 247-253. Within these specified enzyme classes, the short-chain alcohol dehydrogenases are particularly highly suitable.
In these methods, preference is given to using an enzyme with dehydrogenase activity which can be prepared from microorganisms of the genera Azoarcus (also known to the person skilled in the art under the newer name Aromatoleum), Azonexus, Azospira, Azovibrio, Dechloromonas, Ferribacterium, Petrobacter, Propionivibrio, Quadricoccus, Rhodocyclus, Sterolibacterium, Thauera and Zoogloea. Particular preference is given to dehydrogenases from species of the genus Azoarcus. On account of their amino acid sequence, the phenylethanol dehydrogenase from Azoarcus sp EbN1 can be included in the short-chain alcohol dehydrogenases/reductases (SDR). The enzyme group is described in detail, for example, in H.Jδrnvall, B.Persson, M.Krook, S.Atrian, R.Gonzalez-Duarte, J.Jeffery and D.Ghosh, Biochemistry, 1995, 34, pp. 6003- 6013 or U.Oppermann, C.Filling, M .HuIt, N.Shafqat, X.Q.Wu, M.Lindh, J.Shafqat, E.Nordling, Y.Kallberg, B.Persson and H.Jornvall, Chemico-Biological Interactions, 2003, 143, pp. 247-253. Within the group of SDR, the amino acid sequences of the individual representatives can differ greatly from one another. Nevertheless, it is known that certain amino acids or amino acid regions within the group of SDR are strongly preserved. C.Filling, K.D.Berndt, J.Benach, S.Knapp, T.Prozorovski, E.Nordling, R.Ladenstein, H.Jornvall and U.Oppermann, Journal of Biological Chemistry, 2002, 277, pp. 25677-25684 describes important preserved regions of the SDR.
Examples of Azoarcus species are Azoarcus anaerobius, Azoarcus buckelii, Azoarcus communis, Azoarcus evansii, Azoarcus indigens, Azoarcus toluclasticus, Azoarcus tolulyticus, Azoarcus toluvorans, Azoarcus sp., Azoarcus sp. 22LJn, Azoarcus sp. BH72, Azoarcus sp. CC-1 1, Azoarcus sp. CIB, Azoarcus sp. CR23, Azoarcus sp. EB1 , Azoarcus sp. EbN1 , Azoarcus sp. FL05, Azoarcus sp. HA, Azoarcus sp. HxN1 , Azoarcus sp. mXyN1 , Azoarcus sp. PbN 1, Azoarcus sp. PH002, Azoarcus sp. T and Azoarcus sp. ToN 1.
Particular preference is given to using dehydrogenases from Azoarcus sp EbN1 .
A suitable embodiment of the invention is the use of enzymes (E) in the method specified above, wherein E has a polypeptide sequence (i) SEQ ID NO: 2 or 4 or (ii) has a polypeptide sequence in which up to 25% of the amino acid residues are altered compared with SEQ ID NO:2 or 4 through deletion, insertion, substitution or a combination thereof and which still has at least 50% of the enzymatic activity of SEQ ID NO:2 or 4 .
In a particularly preferred embodiment of the method, the enzyme with dehydrogenase activity is selected from enzymes which comprise an amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:4 or a sequence derived therefrom in which up to 25%, preferably up to 20%, particularly preferably up to 15%, in particular up to 10, 9 , 8 , 7 , 6 , 5 , 4 , 3 , 2 , 1% , of the amino acid residues have been altered through a deletion, a substitution, an insertion or a combination of deletion, substitution and insertion, wherein the polypeptide sequences altered compared with SEQ ID NO:2 or SEQ ID NO:4 still have at least 50%, preferably 65%, particularly preferably 80%, in particular more than 90%, of the enzymatic activity of SEQ ID NO:2 or SEQ ID NO:4.
In this connection, enzymatic activity of SEQ ID NO:2 or SEQ ID NO:4 should be understood as meaning the ability to reduce the ketones of the formula (II) enantioselectively to the (S)-alcohol (formula Ia) or the (R)-alcohol (formula Ib). The method according to the invention is carried out with the addition of reduction equivalents, in particular of NADH or NADPH. To regenerate the reduction equivalents consumed in the reaction, a sacrificial alcohol, preferably 1-isopropanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, n-heptane, is used, which is oxidized under the reaction conditions by the enzyme (E) to give the corresponding sacrificial ketone.
In a preferred embodiment of the invention, the sacrificial alcohol added is used not only to regenerate the consumed reduction equivalents, but also as cosolvent. Preference is given to working in a liquid two-phase system, wherein the one phase consists of water or water- miscible solvent, and the other phase consists of the sacrificial alcohol. Preference is given to using 2-pentanol, 2-butanol or n-heptane as sacrificial alcohol.
The reduction equivalents, in particular of NADH or NADPH, are preferably used in an amount of from 0.001 to 100 mmol, particularly preferably from 0.01 to 1 mmol, of reduction equivalents per mole of ethyl 2-oxo-4-phenylbutyrate (II) used.
It is a preferred embodiment to allow the method according to the invention to take place in the presence of a microorganism which is selected from bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Lactobacillaceae, Streptomycetaceae, Rhodococcaceae and Nocardiaceae. The microorganism may in particular be a recombinant microorganism which has been transformed with a nucleic acid construct which codes for an enzyme with dehydrogenase activity in accordance with the above definition.
Furthermore, the invention relates to expression cassettes comprising, in operative linkage with at least one regulative nucleic acid sequence, a coding nucleic acid sequence according to the above definition.
The invention further provides recombinant vectors comprising at least one such expression cassette.
The invention also relates to prokaryotic or eukaryotic hosts which have been transformed with at least one vector according to the invention.
The invention further provides the use of an enzyme with dehydrogenase activity according to the above definition or of a microorganism producing this enzyme for preparing compounds of the formulae Ia or Ib.
Description of the Drawing Figure 1: Screening of various alcohol dehydrogenases Figure 2 : Further screening of alcohol dehydrogenases Figure 3 : Comparison of single-phase and two-phase mixture Figure 4 : Comparison of 2-butanol with 2-butanol/10% 2-propanol Figure 5 : Reduction of OPB with the help of Ebn1_para in the 0.5 I mixture Figure 6 : OPB reactions at various temperatures Figure 7 : OPB reaction at 1O0C and 2 O0C Figure 8 : OPB reaction at pH 6.0 and pH 7.0 Figure 9 : Comparison of the acid fraction at various pHs Figure 10: Reaction in various solvents Figure 11: OPB reaction with 2-pentanol or 2-butanol as regenerating agent Figure 12: Enzymatic OPB reduction at various substrate concentrations Figure 13: OPB reduction at substrate concentrations up to 1M Figure 14: Comparison of reduction with crude extract and purified enzyme Figure 15: Course of the ee values following the addition of 1 g/l of amano lipase Figure 16: Course of the enzymatic reduction of OPB Figure 17: Course of the saponification by means of amano lipase Figure 18: Comparison of different lipases
Description of the Sequences
SEQ ID NO : 1 EbN1_para, encoding nucleic acid
SEQ ID NO : 2 EbN1_para, amino acid sequence
SEQ ID NO : 3 ChnA, encoding nucleic acid
SEQ ID NO: 4 ChnA, amino acid sequence
SEQ ID NO: 5 Vector comprising SEQ ID NO: 1
Detailed description of the Invention
A . General terms and definitions
Unless stated otherwise, the following general meanings apply:
"Halogen" is fluorine, chlorine, bromine or iodine, in particular fluorine or chlorine.
"Lower alkyl" is straight-chain or branched alkyl residues having 1 to 6 carbon atoms, such as methyl, ethyl, isopropyl or n-propyl, n-, iso, sec- or tert-butyl, n-pentyl or 2-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-ethylbutyl.
"(C1-C20)-Alkyl" is a hydrocarbon residue whose carbon chain is straight-chain or branched and comprises 1 to 20 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, tertiary-butyl, pentyl, hexyl, heptyl, octyl, nonenyl or decanyl.
"(C3-C7)-Cycloalkyl" is cyclic hydrocarbon residues such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
"Lower alkenyl" is the mono- or polyunsaturated, preferably mono- or diunsaturated, analogs of the aforementioned alkyl residues having 2 to 6 carbon atoms, wherein the double bond can be in any desired position on the carbon chain.
"Lower alkoxy" is the oxygen-terminated analogs of the above alkyl residues.
"Aryl" is aromatic carbon residues having 6 to 14 carbon atoms in the ring. -(C6-C14)-Aryl residues are, for example, phenyl, naphthyl, for example 1-naphthyl, 2-naphthyl, biphenylyl, for example 2-biphenylyl, 3-biphenylyl and 4-biphenylyl, anthryl or fluorenyl. Biphenylyl residues, naphthyl residues and in particular phenyl residues are preferred aryl residues.
"(C5-C14)-Heterocycle" is a monocyclic or bicyclic 5-membered to 14-membered heterocyclic ring which is partially saturated or completely saturated. Examples of heteroatoms are N, O and S. Examples of the terms -(C5-C14)-heterocycle are residues derived from pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole, 1,2,3,5-oxathiadiazole 2-oxides, triazolones, oxadiazolones, isoxazolones, oxadiazolidinediones, triazoles, which are substituted by F, -CN, -CF3 or -C(O)-O-(CI -C4)-alkyl, 3-hydroxypyrro-2,4-diones, 5-oxo-1 ,2,4-thiadiazoles, pyridine, pyrazine, pyrimidine, indole, isoindole, indazole, phthalazine, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, -carboline and benzo-fused, cyclopenta-, cyclohexa- or cyclohepta-fused derivatives of these heterocycles. In particular, preference is given to the residues 2- or 3-pyrrolyl, phenylpyrrolyl such as 4- or 5-phenyl-2-pyrrolyl, 2-furyl, 2-thienyl, 4-imidazolyl, methylimidazolyl, for example 1-methyl-2-, -4- or -5-imidazolyl, 1,3-thiazol-2-yl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-, 3- or 4-pyridyl N-oxide, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl, 2-, 3- or 5-indolyl, substituted 2-indolyl, for example 1-methyl-, 5-methyl-, 5-methoxy-, 5-benzyloxy-, 5-chloro- or 4,5-dimethyl-2-indolyl, 1-benzyl-2- or -3-indolyl, 4,5,6,7- tetrahydro-2-indolyl, cyclohepta[b]-5-pyrrolyl, 2-, 3- or 4-quinolyl, 1-, 3- or 4-isoquinolyl, 1-oxo-1 ,2-dihydro-3-isoquinolyl, 2-quinoxalinyl, 2-benzofuranyl, 2-benzothienyl, 2-benzoxazolyl or benzothiazolyl or dihydropyridinyl, pyrrolidinyl, for example 2- or 3-(N- methylpyrrolidinyl), piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl or benzodioxolanyl. Preferred 2-oxo acid esters of the formula (II) comprise, for example, ethyl 2-oxovalerate, ethyl 2-oxo-4-phenylbutyrate (formula (III)), ethyl pyruvate, ethylphenyl glyoxylate, ethyl-2- oxo-3-phenylpropionic acid, ethyl 8-chloro-6-oxooctanoate, ethyl 2-oxobutyrate, ethyl 2-oxohexanoate, methylphenyl glyoxylate, methyl 2-oxovalerate, methyl pyruvate, methyl 2-oxo-4-phenylbutyrate, methyl-2-oxo-3-phenylpropionic acid, methyl 8-chloro-6- oxooctanoate, methyl 2-oxobutyrate or methyl 2-oxohexanoate.
Formula III (ethyl 2-oxo-4-phenylbutyrate; OPB)
The S- and R-2-hydroxy acid esters formed correspondingly by reduction comprise, for example, ethyl S- or R-2-hydroxyvalerate, ethyl S-2 or R-hydroxy-4-phenylbutyrate, ethyl L-lactate or ethyl S-mandelate.
In a particularly preferred embodiment, the invention provides a method of preparing optically active ethyl S-2 or R-hydroxy-4-phenylbutyrate (formulae 1a and 1b), the salt of ethyl S-2 or R-hydroxy-4-phenylbutyric acid. This is also known to the person skilled in the art under the name S- or R-2-hydroxy-4-phenylbutyric acid ethyl ester.
Formula Ia (HPB; S enantiomer)
Formula 1b (HPB; R enantiomer)
Within the context of the present invention, "enantioselectivity" means that the enantiomer excess ee (in %) of one of the two possible enantiomers is at least 50%, preferably at least 80%, in particular at least 90% and specifically at least 95%. The ee value is calculated according to:
ee (%) = Enantiomer A - Enantiomer B / (Enantiomer A + Enantiomer B) x 100
B. Suitable enzymes with dehydrogenase activity
Particularly suitable dehydrogenases (EC 1. 1 .X .X) are primarily NAD- or NADP-dependent dehydrogenases (E.C. 1. 1 . 1 .x), in particular alcohol dehydrogenases (E.C.1. 1 .1. 1 or E.C.1. 1 .1.2), which effect the selective reduction of OPB to HPB. The dehydrogenase is preferably obtained from a microorganism, particularly preferably from a bacterium, a fungus, in particular a yeast, in each case listed in strain collections or obtainable from isolates of a natural source, such as soil samples, biomass samples and the like or by de novo gene synthesis.
Preferred enzymes with dehydrogenase activity comprise an amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:4, or a functional equivalent thereof.
The dehydrogenase can be used in purified or partially purified form or in the form of the original microorganism or of a recombinant host organism which expresses the dehydrogenase. Methods for obtaining and purifying dehydrogenases from microorganisms are sufficiently known to a person skilled in the art, e.g. from K . Nakamura & T. Matsuda, "Reduction of Ketones" in K . Drauz and H . Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol.Ill, 991-1032, Wiley-VCH, Weinheim. Recombinant methods for producing dehydrogenases are likewise known, for example from W. Hummel, K . Abokitse, K . Drauz, C. Rollmann and H . Grδger, Adv. Synth. Catal. 2003, 345, No. 1 + 2 , pp. 153-159.
Suitable bacteria are, for example, those of the orders of the Burkholderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales or Rhodocyclales.
Particular preference is given to dehydrogenases from the family of the family of Rhodocyclaceae.
Particular preference is given to dehydrogenases from the genera Azoarcus Azonexus, Azospira, Azovibrio, Dechloromonas, Ferribacterium, Petrobacter, Propionivibrio, Quadricoccus, Rhodocyclus, Sterolibacterium, Thauera and Zoogloea.
Particular preference is given to dehydrogenases from species of the genera Azoarcus.
The reduction with the dehydrogenase usually takes place in the presence of a suitable cofactor (also referred to as cosubstrate). Usually, NADH and/or NADPH serves as cofactor for the reduction of the ketone. In addition, dehydrogenases can be used as cellular systems which inherently comprise cofactors, or alternative redox mediators can be added (A. Schmidt, F. Hollmann and B. Buhler Oxidation of Alcohols" in K . Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol.Ill, 991-1032, Wiley-VCH, Weinheim).
Moreover, the reduction with the dehydrogenase usually takes place in the presence of a suitable reducing agent which regenerates the cofactor oxidized in the course of the reduction. Examples of suitable reducing agents are sugars, in particular the hexoses, such as glucose, mannose, fructose, and/or oxidizable alcohols, in particular ethanol, propanol, butanol, pentanol or isopropanol, and also formate, phosphite or molecular hydrogen. For the oxidation of the reducing agent and, associated therewith, for the regeneration of the coenzyme, a second dehydrogenase can be added, such as e.g. glucose dehydrogenase when using glucose as reducing agent, phosphite dehydrogenase when using phosphite as reducing agent or formate dehydrogenase when using formate as reducing agent. This can be used as free or immobilized enzyme or in the form of free or immobilized cells. Their preparation can take place either separately or through coexpression in a (recombinant) dehydrogenase strain.
The dehydrogenases used according to the invention can be used in free or immobilized form. An immobilized enzyme is understood as meaning an enzyme which is fixed to an inert support. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1 149849, EP-A-1 069 183 and DE-A 100193773 and also from the literature references cited therein. Reference is made to the disclosure of these specifications in its entirety in this regard. Suitable support materials include, for example, clays, clay minerals, such as kaolinite, diatomaceous earth, perlite, silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anionic exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene. For preparing the supported enzymes, the support materials are usually used in a finely divided, particulate form, with porous forms being preferred. The particle size of the support material is usually not more than 5 mm, in particular not more than 2 mm (sieve grade). Analogously, when using dehydrogenase as whole-cell catalyst, a free or immobilized form may be chosen. Support materials are, for example, Ca alginate, and carrageenan. Enzymes and also cells can also be crosslinked directly with glutaraldehyde (crosslinking to CLEAs). Corresponding and further immobilization methods are described, for example, in J. Lalonde and A . Margolin "Immobilization of Enzymes" in K . Drauz and H . Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol.Ill, 991-1032, Wiley-VCH, Weinheim.
Also included according to the invention are likewise "functional equivalents" of the specifically disclosed enzymes with dehydrogenase activity and the use of these in the methods according to the invention. Within the context of the present invention, "functional equivalents" or analogs of the specifically disclosed enzymes are polypeptides different therefrom which furthermore have the desired biological activity, such as, for example, substrate specificity. Thus, for example "functional equivalents" are understood as meaning enzymes which reduce from 3-chloro-1- (thien-2-yl)propan-1-one to the corresponding S-alcohol and which has at least 50%, preferably 60%, particularly preferably 75%, very particularly preferably 90%, of the activity of an enzyme with the amino acid sequence listed in SEQ ID NO:2 or SEQ ID NO:4. Moreover, functional equivalents are preferably stable between pH 4 to 10 and advantageously have a pH optimum between pH 5 and 8 and a temperature optimum in the range from 2 O0C to 8 O0C.
According to the invention, "functional equivalents" are in particular also understood as meaning mutants which have a different amino acid to those specifically mentioned in at least one sequence position of the aforementioned amino acid sequences but nevertheless have one of the aforementioned biological activities. "Functional equivalents" thus comprise the mutants obtainable by one or more amino acid additions, substitutions, deletions and/or inversions, it being possible for the said alterations to occur in any sequence position provided they lead to a mutant with the profile of properties according to the invention. Functional equivalence is in particular also present if the reactivity patterns between mutant and unaltered polypeptide are in qualitative agreement, i.e. for example identical substrates are reacted at a different rate.
Examples of suitable amino acid substitutions can be found in the table below: "Functional equivalents" in the above sense are also "precursors" of the described polypeptides and also "functional derivatives" and "salts" of the polypeptides.
Here, "precursors" are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
The expression "salts" is understood as meaning both salts of carboxyl groups and also acid addition salts of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be prepared in a manner known per se and comprise inorganic salts, such as, for example, sodium, calcium, ammonium, iron and zinc salts, and also salts with organic bases, such as, for example, amines, such as triethanolamine, arginine, lysine, piperidine and the like. Acid addition salts, such as, for example, salts with mineral acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are likewise provided by the invention. "Functional derivatives" of polypeptides according to the invention can likewise be prepared on functional amino acid side groups or on their N- or C-terminal end with the help of known techniques. Derivatives of this type comprise, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable through reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups, prepared by reaction with acyl groups.
"Functional derivatives" of polypeptides according to the invention can likewise be prepared on functional amino acid side groups or on their N- or C-terminal end with the help of known techniques. Derivatives of this type comprise, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable through reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups, prepared by reaction with acyl groups.
"Functional equivalents" naturally also comprise polypeptides which are accessible from other organisms, and also naturally occurring variants. For example, through sequence comparison, it is possible to establish areas of homologous sequence regions and, in accordance with the specific details of the invention, determine equivalent enzymes.
"Functional equivalents" likewise comprise fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which, for example, have the desired biological function.
Moreover, "functional equivalents" are fusion proteins which have one of the aforementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different therefrom, heterologous sequence in functional N- or C-terminal linkage (i.e. without mutual essential functional impairment of the fusion protein parts). Nonlimiting examples of such heterologous sequences are, for example, signal peptides or enzymes.
"Functional equivalents" also included according to the invention are homologs to the specifically disclosed proteins. These have at least 60%, preferably at least 75%, in particular at least 85%, such as, for example, 90%, 95% or 99%, homology to one of the specifically disclosed amino acid sequences, calculated according to the algorithm by Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. A percentage homology of a homologous polypeptide according to the invention means in particular percentage identity of the amino acid residues, based on the total length of one of the amino acid sequences specifically described herein.
In the case of a possible protein glycosylation, "functional equivalents" according to the invention comprise proteins of the type referred to above in deglycosylated or glycosylated form and also modified forms obtainable by altering the glycosylation pattern.
Homologs of the proteins or polypeptides according to the invention can be produced by mutagenesis, e.g. by point mutation or shortening of the protein.
Homologs of the proteins according to the invention can be identified by screening combinatorial libraries of mutants, such as, for example, truncation mutants. For example, a variegated library of protein variants can be produced by combinatorial mutagenesis at the nucleic acid level, such as, for example, by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a large number of methods which can be used for producing libraries of potential homologs from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerated gene sequence can be carried out in an automated DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector. Using a degenerate gene set makes it possible to provide all sequences in a mixture which code for the desired set of potential protein sequences. Methods for synthesizing degenerate oligonucleotides are known to the person skilled in the art (e.g. Narang, S.A . (1983) Tetrahedron 39:3; ltakura et al. (1984) Annu. Rev. Biochem. 53:323; ltakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).
Several techniques for screening gene products of combinatorial libraries which have been produced by point mutations or truncation, and for screening cDNA libraries for gene products with a selected property are known in the prior art. These techniques can be adapted to the rapid screening of gene libraries which have been generated through combinatorial mutagenesis of homologs according to the invention. The most often used techniques for screening large gene libraries, which are subject to analysis with high throughput, comprise the cloning of the gene library in replicatable expression vectors, transformation of the suitable cells with the resulting vector library and expression of the combinatorial genes under conditions under which detection of the desired activity facilitates the isolation of the vector which encodes the gene whose product has been detected. Recursive ensemble mutagenesis (REM), a technique which increases the frequency of functional mutants in the libraries, can be used in combination with the screening tests in order to identify homologs (Arkin and Yourvan (1992) PNAS 89:781 1-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
C. Nucleic acid sequences coding for the dehydrogenases
The invention provides in particular nucleic acid sequences (single- and double-stranded DNA and RNA sequences, such as, for example, cDNA and mRNA) which code for an enzyme with dehydrogenase activity according to the invention. Preference is given to nucleic acid sequences which code, for example, for amino acid sequences according to SEQ ID NO:2 or SEQ ID NO:4 or characteristic part sequences thereof, or comprise nucleic acid sequences according to SEQ ID NO:1 or SEQ ID NO:3 or characteristic part sequences thereof, or the complementary strand thereof (a)). Further preferred are nucleic acids, comprising a sequence which hybridizes with the DNA sequence according to SEQ ID NO:1 or its complementary strand, wherein the hybridization takes place under stringent conditions (b)), or a DNA sequence which, due to the degeneracy of the genetic code, encodes a protein which is also encoded by a DNA sequence according to a) or b).
All of the nucleic acid sequences mentioned herein can be prepared in a manner known per se through chemical synthesis from the nucleotide building blocks, such as, for example, through fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. The chemical synthesis of oligonucleotides can take place, for example, in a known manner in accordance with the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The addition of synthetic oligonucleotides and filling of gaps with the help of the Klenow fragment of the DNA polymerase and ligation reactions and also general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
The invention also provides nucleic acid sequences (single- and double-stranded DNA and RNA sequences, such as, for example, cDNA and mRNA), coding for one of the above polypeptides and their functional equivalents, which are accessible, for example, using artificial nucleotide analogs.
The invention relates both to isolated nucleic acid molecules which code for polypeptides and proteins according to the invention or biologically active sections thereof, and also nucleic acid fragments which can be used, for example, for use as hybridization probes or primers for the identification or amplification of coding nucleic acids according to the invention.
Moreover, the nucleic acid molecules according to the invention can comprise untranslated sequences of the 3' and/or 5' end of the coding gene region.
Furthermore, the invention comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences, or a section thereof.
The nucleotide sequences according to the invention allow the production of probes and primers which can be used for the identification and/or cloning of homologous sequences in other cell types and organisms. Such probes and primers usually comprise a nucleotide sequence region which under "stringent" conditions (see below) hybridizes onto at least about 12, preferably at least about 25, such as, for example, about 40, 50 or 75, consecutive nucleotides of a sense strand of a nucleic acid sequence according to the invention or of a corresponding antisense strand.
An "isolated" nucleic acid molecule is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid and can, moreover, be essentially free from other cellular material or culture medium if it is prepared by recombinant techniques, or be free from chemical precursors or other chemicals if it is chemically synthesized.
A nucleic acid molecule according to the invention can be isolated by means of molecular biological standard techniques and the sequence information provided according to the invention. For example, cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences or a section thereof as hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule comprising one of the disclosed sequences or a section thereof can be isolated by polymerase chain reaction, in which case the oligonucleotide primers which have been created on the basis of this sequence are used. The nucleic acid amplified in this way can be cloned into a suitable vector and be characterized by DNA sequence analysis. The oligonucleotides according to the invention can also be prepared by standard synthesis methods, e.g. using an automated DNA synthesis instrument.
The nucleic acid sequences according to the invention can in principle be identified and isolated from all organisms. The nucleic acid sequences according to the invention or the homologs thereof can be advantageously isolated from fungi, yeasts, archaea or bacteria. Bacteria which may be mentioned are Gram-negative and Gram-positive bacteria. Preference is given to the nucleic acids according to the invention from Gram-negative bacteria advantageously from [alpha]-proteobacteria, [beta]-proteobacteria or [gamma]- proteobacteria, particularly preferably from bacteria of the orders of the Burkholderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales or Rhodocyclales. Very particularly preferably from bacteria of the family of Rhodocyclaceae. Particularly preferably from the genus Azoarcus (Aromatoleum). Especially preferably from species Azoarcus anaerobius, Azoarcus buckelii, Azoarcus communis, Azoarcus evansii, Azoarcus indigens, Azoarcus toluclasticus, Azoarcus tolulyticus, Azoarcus toluvorans, Azoarcus sp., Azoarcus sp. 22LJn, Azoarcus sp. BH72, Azoarcus sp. CC-1 1, Azoarcus sp. CIB, Azoarcus sp. CR23, Azoarcus sp. EB1 , Azoarcus sp. EbN1 , Azoarcus sp. FL05, Azoarcus sp. HA, Azoarcus sp. HxN1 , Azoarcus sp. mXylMI , Azoarcus sp. PbN1 , Azoarcus sp. PH002, Azoarcus sp. T and Azoarcus sp. ToN1 .
Particular preference is given to using dehydrogenases from Azoarcus sp EbNL
Nucleic acid sequences according to the invention can be isolated, for example, using customary hybridization methods or the PCR technique from other organisms, e.g. via genomic or cDNA libraries. These DNA sequences hybridize under standard conditions with the sequences according to the invention. For the hybridization, short oligonucleotides of the preserved regions, for example from the active center, which can be ascertained by means of comparisons with a dehydrogenase according to the invention in the manner known to the person skilled in the art, are advantageously used. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization. Depending on the nucleic acid used (oligonucleotide, relatively long fragment or complete sequence) or depending on which nucleic acid type DNA or RNA are used for the hybridization, these standard conditions vary. Thus, for example, the melting temperatures for DNA:DNA hybrids are ca. 1O0C lower than those of DNA:RNA hybrids of identical length.
Standard conditions are to be understood as meaning, for example, depending on the nucleic acid, temperatures between 42 and 580C in an aqueous buffer solution with a concentration between 0.1 to 5 * SSC ( 1 * SSC = 0.15 M NaCI, 15 mM sodium citrate, p 420C in 5 * SSC, 50% formamide. The hybridization conditions for DNA:DNA hybrids are advantageously 0.1 * SSC and temperatures between about 2 O0C to 450C, preferably between about 3 O0C to 450C. For DNA:RNA hybrids, the hybridization conditions are advantageously 0.1 * SSC and temperatures between about 3 O0C to 550C, preferably between about 450C to 550C. These stated temperatures for the hybridization are, by way of example, calculated melting temperature values for a nucleic acid with a length of ca. 100 nucleotides and a C + G content of 50% in the absence of formamide. The experimental conditions for the DNA hybridization are described in the relevant genetics textbooks, such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated in accordance with formulae known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrids or the G + C content. Further information relating to hybridization can be found by the person skilled in the art in the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991 , Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.
The invention also provides derivatives of the specifically disclosed or derivable nucleic acid sequences.
Thus, further nucleic acid sequences according to the invention can be derived from SEQ ID NO:1 or NO:3 and can differ therefrom through addition, substitution, insertion or deletion of one or more nucleotides, but still code for polypeptides with the desired profile of properties.
Also included according to the invention are those nucleic acid sequences which comprise so-called silent mutations or have been altered corresponding to the codon usage of a specific source organism or host organism, compared to a specifically specified sequence, as well as naturally occurring variants, such as, for example, splice variants or allele variants.
Also provided are sequences obtainable by conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of identical charge, size, polarity and/or solubility).
Also provided by the invention are the molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population on account of the natural variation. These natural variations usually bring about a variance of from 1 to 5% in the nucleotide sequence of a gene.
Derivatives of a nucleic acid sequence according to the invention are to be understood as meaning, for example, allele variants which have at least 40% homology at the derived amino acid level, preferably at least 60% homology, very particularly preferably at least 80, 85, 90, 93, 95 or 98% homology over the entire sequence range (with regard to homology at the amino acid level, reference may be made to the above statements relating to the polypeptides). The homologies may advantageously be higher over part ranges of the sequences.
Furthermore, derivatives are also to be understood as meaning homologs of the nucleic acid sequences according to the invention, for example fungal or bacterial homologs, shortened sequences, single-strand DNA or RNA of the coding and noncoding DNA sequence. They had e.g. at the DNA level a homology of at least 40%, preferably of at least 60%, particularly preferably of at least 70%, very particularly preferably of at least 80%, over the entire stated DNA region. Moreover, derivatives are to be understood as meaning, for example, fusions with promoters. The promoters which are located upstream of the stated nucleotide sequences may have been altered by one or more nucleotide exchanges, insertions, inversions and/or deletions without, however, impairing the functionality and/or effectiveness of the promoters. Furthermore, the promoters can be increased in their effectiveness by altering their sequence or exchanged completely for more effective promoters, including those of organisms of other species.
Derivatives are also to be understood as meaning variants whose nucleotide sequence has been altered in the range from - 1 to - 1000 bases upstream of the start codon or 0 to 1000 bases downstream after the stop codon such that the gene expression and/or the protein expression is altered, preferably increased.
Furthermore, the invention also comprises nucleic acid sequences which hybridize with coding sequences specified above under "stringent conditions". These polynucleotides can be found upon screening genomic or cDNA libraries and, if appropriate, can be replicated therefrom using suitable primers by means of PCR and then be isolated, for example using suitable probes. Moreover, polynucleotides according to the invention can also be synthesized by a chemical route. This property is understood as meaning the ability of a poly- or oligonucleotide to bind under stringent conditions to a virtually complementary sequence whereas nonspecific bonds between noncomplementary partners do not take place under these conditions. For this, the sequences should be 70-100%, preferably 90- 100%, complementary. The property of complementary sequences to be able to bond specifically to one another is utilized, for example, in the Northern- or Southern-Blot technique or during primer bonding in PCR or RT-PCR. Usually, for this, oligonucleotides above a length of 30 base pairs are used. Under stringent conditions is understood, for example in the Northern-Blot technique, as meaning the use of a 50-70 0C, preferably 60- 650C warm washing solution, for example 0 .I x SSC buffer with 0.1% SDS (2Ox SSC: 3M NaCI, 0.3M Na citrate, pH 7.0) for the elution of nonspecifically hybridized cDNA probes or oligonucleotides. Here, as mentioned above, only highly complementary nucleic acids remain bonded to one another. The establishment of stringent conditions is known to the person skilled in the art and is described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
D. Embodiments of constructs according to the invention
Moreover, the invention provides expression constructs comprising, under the genetic control of regulative nucleic acid sequences, a nucleic acid sequence coding for a polypeptide according to the invention; and also vectors comprising at least one of these expression constructs.
Preferably, such constructs according to the invention comprise 5'-upstream of the respective coding sequence, a promoter and 3'-downstream a terminator sequence, and, if appropriate, further customary regulative elements, in each case operatively linked to the coding sequence.
An "operative linkage" is understood as meaning the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulative elements such that each of the regulative elements can properly perform its function in expressing the coding sequence. Examples of operatively linkable sequences are targeting sequences and also enhancers, polyadenylation signals and the like. Further regulative elements comprise selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
A nucleic acid construct according to the invention is in particular to be understood as meaning one in which the gene for a dehydrogenase according to the invention have been operatively or functionally linked to one or more regulation signals for controlling, e.g. increasing, the gene expression.
In addition to these regulatory sequences, the natural regulation of these sequences may still be present upstream of the actual structural genes and, if appropriate, may have been genetically altered, such that the natural regulation has been switched off and the expression of the genes has been increased. However, the nucleic acid construct can also be simpler in design, i.e. no additional regulation signals have been inserted before the coding sequence and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence is mutated such that regulation no longer takes place and gene expression is increased.
A preferred nucleic acid construct advantageously also comprises one or more of the already mentioned "enhancer" sequences, functionally linked to the promoter, which permit increased expression of the nucleic acid sequence. It is also possible to insert additional advantageous sequences at the 3' end of the DNA sequences, such as further regulatory elements or terminators. The nucleic acids according to the invention may be present in one or more copies in the construct. In the construct it is also possible for further markers to be present, such as antibiotic resistances or auxotrophy complementing genes, if appropriate for selection on the construct.
Advantageous regulatory sequences for the method according to the invention are present, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacl
For expression in a host organism, the nucleic acid construct is advantageously inserted into a vector, such as, for example, a plasmid or a phage, which permits optimal expression of the genes in the host. Apart from plasmids and phages, vectors are also to be understood as meaning all other vectors known to the person skilled in the art, thus e.g. viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or cicular DNA. These vectors can be replicated autonomously in the host organism or be chromosomally replicated. These vectors represent a further embodiment of the invention. Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-IIK1 13>-B1 , Igt1 1 or pBdCI, in Streptomyces plJ101 , plJ364, plJ702 or plJ361 , in Bacillus pUB1 10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1 , plL2 or pBB1 16, in yeasts 2alphaM, pAG-1 , YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHIac<+>, pBIN19, pAK2004 or pDH51 . The specified plasmids are a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H . et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
For the expression of the further genes present, the nucleic acid construct advantageously additionally also comprises 3'- and/or 5'-terminal regulatory sequences for increasing expression, which are selected for optimal expression depending on the selected host organism and gene or genes.
These regulatory sequences are intended to permit the targeted expression of the genes and of the protein expression. Depending on the host organism, this may mean, for example, that the gene is expressed or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.
The regulatory sequences and/or factors can here preferably have a positive influence, and thereby increase, the gene expression of the introduced genes. Thus, an enhancement of the regulatory elements can advantageously take place at the transcription level by using strong transcription signals such as promoters and/or "enhancers". In addition, however, an enhancement of the translation is also possible by, for example, improving the stability mRNA.
In a further embodiment of the vector, the vector comprising the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be introduced into the microorganisms in the form of a linear DNA and be integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid according to the invention.
For optimal expression of heterologous genes in organisms, it is advantageous to alter the nucleic acid sequences corresponding to the specific "codon usage" used in the organism. The "codon usage" can be readily determined by reference to computer analyses of other known genes from the organism in question.
The preparation of an expression cassette according to the invention takes place through fusion of a suitable promoter with a suitable coding nucleotide sequence and also a terminator or polyadenylation signal. For this, customary recombination and cloning techniques are used, as are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M .L. Berman and L .W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M . et al., Current Protocols in Molecular Biology, Greene Publishing Assoc and Wiley lnterscience (1987). For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which allows optimal expression of the genes in the host. Vectors are well known to the person skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P. H . et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
E. Host organisms which can be used according to the invention
With the help of the vectors or constructs according to the invention, it is possible to prepare recombinant microorganisms which are transformed, for example, with at least one vector according to the invention and can be used for the production of the polypeptides according to the invention. The above-described recombinant constructs according to the invention are advantageously introduced into a suitable host system and expressed. Here, customary cloning and transfection methods, such as, for example, coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like, known to the person skilled in the art are preferably used in order to express said nucleic acids in the particular expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley lnterscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
According to the invention, it is also possible to prepare homologously recombined microorganisms. For this, a vector is prepared which comprises at least one section of a gene according to the invention or of a coding sequence in which, if appropriate, at least one amino acid deletion, addition or substitution has been inserted in order to alter the sequence according to the invention, e.g. to functionally disrupt it ("knockout" vector). The introduced sequence can, for example, also be a homolog from a related microorganism or derived from a mammal, yeast or insect source. The vector used for the homologous recombination can alternatively be configured such that the endogenous gene is mutated or altered in some other way during homologous recombination, but still codes for the functional protein (e.g. the regulatory region positioned upstream can be altered in such a way that the expression of the endogenous protein is thereby altered). The altered section of the gene according to the invention is in the homologous recombination vector. The construction of suitable vectors for the homologous recombination is described, for example, in Thomas, K.R. and Capecchi, M .R. (1987) Cell 5 1:503.
Suitable recombinant host organisms for the nucleic acid according to the invention or the nucleic acid construct are in principle all prokaryotic or eukaryotic organisms. The host organisms used are advantageously microorganisms such as bacteria, fungi or yeasts. Gram-positive or Gram-negative bacteria are advantageously used, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus.
Very particularly preferably, the genus and species is Escherichia coli. Further advantageous bacteria, moreover, can be found in the group of alpha-proteobacteria, beta- proteobacteria or gamma-proteobacteria.
The host organism or the host organisms according to the invention comprise here preferably at least one of the nucleic acid sequences described in this invention, nucleic acid constructs or vectors which code for an enzyme with dehydrogenase activity according to the invention.
The organisms used in the method according to the invention can be grown or cultivated depending on the host organism in the manner known to the person skilled in the art. Microorganisms are usually grown in a liquid medium which comprises a carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and, if appropriate, vitamins, at temperatures between O0C and 100 0C, preferably between 1O0C to 6 O0C with oxygen gassing. Here, the pH of the nutrient liquid can be kept at a fixed value, i.e. may or may not be regulated during cultivation. The cultivation can take place batchwise, semibatchwise or continuously. Nutrients can be initially introduced at the start of the fermentation or be fed in afterwards semicontinuously or continuously. The ketone can be added directly for the cultivation or advantageously after cultivation. The enzymes can be isolated from the organisms by the method described in the examples or be used as crude extract for the reaction.
F. Recombinant preparation of the polypeptides according to the invention The invention further provides methods for the recombinant preparation of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultivated, if appropriate the expression of the polypeptides is induced and these are isolated from the culture. The polypeptides can thus also be produced on an industrial scale, if desired.
The recombinant microorganism can be cultivated and fermented by known methods. Bacteria can be replicated, for example, in TB or LB medium and at a temperature of from 20 to 4 O0C and a pH of from 6 to 9 . Suitable cultivation conditions are described in detail, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
Then, if the polypeptides are not secreted into the culture medium, the cells are disrupted and the product obtained from the lysate by known protein isolation methods. If desired, the cells can be disrupted by high-frequency ultrasound, by high pressure, such as, for example, in a French pressure cell, by osmolysis, through the effect of detergents, lytic enzymes or organic solvents, by homogenizers or by combining two or more of the listed methods.
Purification of the polypeptides can be achieved using known chromatographic methods, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and also using other customary methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, F. G., Biochemische Arbeitsmethoden [Biochemical working methods], Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
To isolate the recombinant protein, it may be advantageous to use vector systems or oligonucleotides which extend the cDNA by certain nucleotide sequences and thus code for altered polypeptides or fusion proteins which serve, for example, for simpler purification. Suitable modifications of this type are, for example, so-called "tags" functioning as anchors, such as, for example, the modification known as hexa-histidine anchor, or epitopes which can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can serve for attaching the proteins to a solid support, such as, for example, a polymer matrix, which can be poured, for example, into a chromatography column, or can be used on a microtiter plate or some other support.
At the same time, these anchors can also be used for recognizing the proteins. Moreover, for recognizing the proteins it is also possible to use customary markers, such as fluorescent dyes, enzyme markers which form a detectable reaction product following reaction with a substrate, or radioactive markers, alone or in combination with the anchors for derivatization of the proteins.
G . Carrying out the method according to the invention for preparing ethyl S or R-2-hydroxy- 4-phenylbutyrate (formulae Ia and Ib)
The reaction can take place in aqueous or nonaqueous reaction media or in two-phase systems or (micro)emulsions. The aqueous reaction media are preferably buffered solutions which generally have a pH between pH 4 and 12, preferably between 4.5 to 9 , particularly preferably between 5 to 8 . Besides water, the aqueous solvent can moreover comprise at least one alcohol, e.g. ethanol or isopropanol or dimethyl sulfoxide.
Nonaqueous reaction media are understood as meaning reaction media which comprise less than 1% by weight, preferably less than 0.5% by weight, of water, based on the total weight of the reaction medium. The reaction is preferably carried out in an organic solvent. Suitable solvents are, for example, aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, halogenated aliphatic hydrocarbons, preferably having one or two carbon atoms, such as dichloromethane, chloroform, tetrachloromethane, dichloroethane or tetrachloroethane, aromatic hydrocarbons, such as benzene, toluene, the xylenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran or esters, such as ethyl acetate or n-butyl acetate, or ketones, such as methyl isobutyl ketone, or dioxane or mixtures thereof.
For example, the reduction with the dehydrogenase is carried out in an aqueous-organic, in particular aqueous, reaction medium.
The ketone to be reduced is preferably used in the enzymatic reduction in a concentration of from 0.1 g/l to 500 g/l, particularly preferably from 1 g/l to 100 g/l and can be conveyed continuously or discontinuously.
The enzymatic reduction usually takes place at a reaction temperature below the deactivation temperature of the dehydrogenase used and is preferably at least -1O0C. It is particularly preferably in the range from 0 to 100 0C, in particular from 5 to 6 O0C and specifically from 10 to 4 O0C, and very particularly preferably between 30° and 4 O0C.
For the procedure, the ketone can, for example, be initially introduced with the dehydrogenase, the solvent and, if appropriate, the coenzymes, if appropriate a second dehydrogenase for regenerating the coenzyme and/or further reducing agents and the mixture can be thoroughly mixed, e.g. by stirring or shaking. However, it is also possible to immobilize the dehydrogenase(s) in a reactor, for example in a column, and to feed through the reactor a mixture comprising the ketone and, if appropriate, coenzymes and/or cosubstrates. For this, the mixture can be circulated through the reactor until the desired conversion is reached. During this, the keto group of the ketone is reduced to an OH group with essentially one of the two enantiomers of the alcohol being formed. As a rule, the reduction will be carried out up to a conversion of at least 70%, particularly preferably of at least 85% and in particular of at least 95%, based on the ketone present in the mixture. The progress of the reaction, i.e. the sequential reduction of the ketone, can be monitored here by customary methods such as gas chromatography or high-pressure liquid chromatography.
In the method according to the invention, enantiomerically pure or chiral products or optically active alcohols are to be understood as meaning enantiomers which exhibit an enantiomer enrichment. Preferably, in the method, enantiomer purities of at least 70% ee, preferably of at least 80% ee, particularly preferably of at least 90% ee, very particularly preferably at least 98% ee, are achieved.
The inventors have, moreover, recognized that the enantiomer unit in particular of the R enantiomer (formula Ib) can be considerably increased if, at the end of the dehydrogenase reaction, a lipase is added to the mixture which saponifies the undesired S enantiomer to the free acid, which can be separated off in the subsequent work-up in accordance with methods known to the person skilled in the art [J. Org. Chem. 1990, 55, 812-815].
Saponification is generally understood as meaning the basic hydrolysis of esters into their constituents alcohol and acid. The reaction is irreversible.
For the method according to the invention, preference is given to enzymes of the class of the hydrolases (EC 3), specifically of the esterases (EC 3.1), particularly preferably of the class (EC 3.1 . 1 .3: triacylglycerol acyl hydrolases), which specifically cleave fats (triglycerides) into glycerol and fatty acids. The person skilled in the art is familiar with a series of lipase from fungi (Aspergillis, Candida, Geotrichum, Humicola, Mucor, Rhizopus) and from bacteria (Chromobacterium, Pseudomonas), which are suitable for the method according to the invention.
For the method according to the invention, it is possible to use growing cells which comprise the nucleic acids, nucleic acid constructs or vectors according to the invention. Resting or disrupted cells can also be used. Disrupted cells are to be understood as meaning, for example, cells which have been rendered permeable via a treatment with, for example, solvents, or cells which have been broken open via an enzyme treatment, via a mechanical treatment (e.g. French press or ultrasound) or via some other method. The crude extracts obtained in this way are advantageously suitable for the method according to the invention. Purified or partly purified enzymes can also be used for the method. Likewise suitable are immobilized microorganisms or enzymes, which can be used advantageously in the reaction.
If free organisms or enzymes are used for the method according to the invention, then these are expediently separated off prior to extraction, for example via a filtration or centrifugation.
The product ethyl S-2-hydroxy-4-phenylbutyrate (abbreviated to HPB) prepared in the method according to the invention can advantageously be obtained from the aqueous reaction solution by means of extraction or distillation. The extraction can be repeated several times to increase the yield. Examples of suitable extractants are solvents, such as toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or acetic ester, without being limited thereto. Particular preference is given to MTBE as extractant.
Alternatively, the product (HPB) prepared in the method according to the invention can advantageously be obtained from the organic phase of the reaction solution by means of extraction or distillation and/or crystallization. The extraction can be repeated several times to increase the yield. Examples of suitable extractants are solvents, such as toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or acetic ester, without being limited thereto.
After concentrating the organic phase, the products can generally be obtained in good chemical purities, i.e. greater than 80% chemical purity. Following extraction, the organic phase containing the product can, however, also only be partly evaporated and the product crystallized out. For this, the solution is advantageously cooled to a temperature of from O0C to 1O0C. The crystallization can also take place directly from the organic solution or from an aqueous solution. The crystallized-out product can be taken up again in the same solvent or in a different solvent for recrystallization and be crystallized again. As a result of the subsequent advantageous crystallization, which is carried out at least once, the enantiomer purity of the product can be further increased if required.
In the case of these specified types of work-up, the product of the method according to the invention can be isolated in yields of from 60 to 100%, preferably from 80 to 100%, particularly preferably from 90 to 100%, based on the substrate OPB used for the reaction. The isolated product is characterized by a high chemical purity of > 90%, preferably > 95%, particularly preferably of > 98%. Furthermore, the products have a high enantiomer purity, which can advantageously, if required, be further increased through the crystallization.
The method according to the invention can be operated batchwise, semibatchwise or continuously.
The method can advantageously be carried out in bioreactors, as described, for example, in Biotechnology, Volume 3 , 2nd Edition, Rehm et al., Ed., (1993), in particular Chapter II. The description above and the examples below serve only to illustrate the invention. The numerous possible modifications obvious to the person skilled in the art are likewise encompassed according to the invention.
The advantage of the method according to the invention is the particularly high yield of the optically active HPB of the formula (Ia, Ib) or of the virtually quantitative conversion of OPB (II).
The invention will be illustrated in more detail by reference to the examples below, without limiting the invention thereto.
Examples
Example 1: Screening of alcohol dehydrogenases
In each case 20 µl of cell-free crude extract and 5 µmol ( 1 mg) of ethyl 2-oxo-4- phenylbutyrate (1) were dissolved in a 0.5 ml mixture (50 mM KH PO4 pH 6.0, 10 mM NADH/NADPH). The solution was incubated at 3 O0C for 20 min and the course of the reaction was monitored by means of HPLC. To determine the enantioselectivities, samples were measured by GC. Later on, all ee values were determined by means of chiral HPLC. The results of the screenings are shown in figure 1.
The EbN1_para (LU13150) here was identified as a suitable biocatalyst for preparing the S enantiomer (1a). This dehydrogenase completely converts the ketone with ee > 95%. The enzyme is the paralogous protein to EbN1 , which originates from an Azoarcus library. The ChnA (LU13283) has been identified as the only R-selective enzyme and likewise originates from the aforementioned Azoarcus library.
To guarantee the findings, a second screening was carried out with a further series of dehydrogenases.
Fig. 2 shows both the ee values from the GC and also from the chiral HPLC. It can be seen that ChnA produces the highest ee values for the R enantiomer. The SDR enzymes (short chain dehydrogenases) which, like the ChnA, also originate from the Azoarcus library exhibit very poor enantioselectivities apart from SDR2. However, in this screening, the Re-ADH from Rhodococcus (IEP) surprisingly exhibits a high activity and enantioselectivity for the S enantiomer.
The symbol ">" means that the actual activities are greater since the substrate was already completely converted. Example 2 : Preparation of ethyl S-2-hydroxy-4-phenylbutyrate (1a)
(1) Experiments in organic phase The solubility of OPB and HPB in water is very limited at about 10 mM (2 g/l). In order to achieve higher substrate concentrations in solution, various solvents (2-propanol and 2- butanol) were tested. In each case 6 g/l of bio dry mass cells (see table 1) and 0.5 mmol (105 mg) of ethyl 2-oxo-4-phenylbutyrate (1) were dissolved in 0.5 ml of buffer (50 mM
NaH2PO4 pH 6.0, 0.2 mM NAD). 0.5 ml of solvent (2-butanol or 2-butanol with 10% isopropanol) or 0.5 ml of buffer with 10% 2-propanol were added to this solution as regenerating agent and incubated at 3 O0C.
Figure 3 shows that by using a solvent the conversion proceeds significantly more rapidly than in the single-phase mixture. One reason for this may be the higher solubility of the substrate in 2-butanol.
As can be seen in figure 4 , an ee value of 94% is achieved here. The reaction with 2-butanol achieves somewhat higher yields than that with 10% 2-propanol. Consequently, for the preparation of a sample amount, 2-butanol was used as solvent and simultaneously regenerating agent.
(2) Preparation of a sample amount of S-2-hydroxy-4-phenylbutyric acid (0.5 I mixture) 2 g/l (bio dry mass) of cells and 250 mM (125 mmol, 26 g) of ethyl 2-oxo-4-phenylbutyrate
(1) were initially introduced in 250 ml of buffer (20 mM in NaH2PO4 pH 6.5, 0.2 mM NAD) in a 0.5 I reactor with pH control. 250 ml of 2-butanol (for regenerating the cofactor and as second phase) were added thereto. The mixture was stirred at room temperature for 24 h . Samples were taken, stopped with cone. HCI and analyzed by means of HPLC. Figure 5 shows the course of the reduction.
For the subsequent saponification of the ester, the reduction discharge was firstly centrifuged for 20 min at 10 000 rpm and then the organic phase was separated off. The solvent was removed on a rotary evaporator. This gave a brown oil (20.57 g).
The residue obtained in this way was dissolved in 200 ml of ethanol (abs.) and admixed with 10 ml of sodium hydroxide solution (50% strength) (ca. 1.2 eq). The cloudy solution was stirred at 4 O0C for 6 h , then continued stirring overnight at room temperature. Ca. 150 ml of ethanol were then removed on the rotary evaporator. The residue obtained therefrom was dissolved in 200 ml of water and extracted once with MTBE at pH 14. Following phase separation, the organic phase was discarded.
In the ice bath, the aqueous phase was slowly brought to pH 1.0 with continuous stirring using cone sulfuric acid. The free acid was then extracted at room temperature twice using in each case 200 ml of MTBE. The combined organic phases were dried using sodium sulfate and then the solvent was removed on a rotary evaporator. This gave pale yellow crystals (16.4 g = 9 1 mmol, 73% of theory).
Example 3 : Preparation of ethyl R-2-hydroxy-4-phenylbutyrate (1b)
(1) Biocatalyst The identified enzyme ChnA (LU13283) originates from the Azoarcus library from which EbN1 , biocatalyst in the preparation of duloxetine alcohol, also originates. [4] The biocatalyst is expressed in an Escherichia coli production strain LU12037. The sequence of the nucleic acid coding for the ChnA has the SEQ ID NO: 1.
(2) Temperature dependency Since the ee value (~ 8 1%) from the screening was relatively low, a series of experiments in the 1 ml mixture was firstly carried out with the aim of improving the ee value. In each case 10 µl of crude extract ( 1 .2 g/l of BTM) and 10 mM (5 µmol, 1 mg) of ethyl
2-oxo-4-phenylbutyrate (1) were dissolved in a 0.5 ml mixture (50 mM NaH PO4 pH 6.0, 10 mM NADH). The solution was incubated for 25 min at 20°C/30°C or 4 O0C and measured by means of HPLC. To determine the enantioselectivity, samples were measured in GC, or by means of chiral HPLC.
Fig. 6 shows that somewhat better ee values are achieved at lower temperatures. Consequently, two mixtures were tested at even lower temperatures.
At a temperature of 1O0C, although a better ee value is achieved, the reaction rate is so slow that even within 24 h complete conversion (300 mM in the organic phase) is not achieved (fig. 7). In the case of the two 1 ml mixtures, in each case 150 mM of OPB were used in a two-phase system with 50% 2-pentanol. 2-Pentanol serves here simultaneously as solvent and regenerating agent. The amount of biocatalyst (crude extract) was 2.4 g/l of BTM.
(3) pH In each case 10 µl of crude extract ( 1 .2 g/l of BTM) and 10 mM (5 µmol, 1 mg) of ethyl
2-oxo-4-phenylbutyrate (1) were dissolved in a 0.5 ml mixture (50 mM NaH PO4 pH 6.0 or pH 7.0, 1O mM NADH). The solution was incubated at 3 O0C for 25 min and measured by means of HPLC. To determine the enantioselectivity, samples were measured in GC.
At pH 7.0, the reaction of OPB proceeds significantly more rapidly than at pH 6.0 and a somewhat higher ee value is attained. At higher pH values (pH > 8.0), however, the product is saponified to the hydroxy acid. Consequently, a pH in the neutral to slightly acidic range is preferred. At higher pH values, the ee values, however, are somewhat higher. A second pH series was carried out to establish further parameters in a two-phase system. Figure 9 µ shows the formation of the free acid at higher pH values. For this, 500 l of 20 mM NaH PO4 (pH 5-7) with 0.2 mM NAD 500 mM (500 µmol, 105 mg) of OPB were dissolved in a 1 ml mixture and 400 µl of n-heptane as solvent with 100 µl of 2-butanol as regenerating agent were added. The reaction was started by adding the biocatalyst in the form of an untreated fermenter discharge (7.5 g/l of BTM) and shaken at 4 O0C for 24 h .
(4) Comparison of various solvents (two-phase) In each case 1.4 g/l of bio dry mass and 150 mM (150 µmol, 30 mg) of ethyl 2-oxo-4-
phenylbutyrate (1) were dissolved in a 1 ml mixture (50 mM NaH PO4 pH 6.0, 0.2 mM NAD). 400 µl of solvent (2-butanol, 2-pentanol or n-heptane) and 100 µl of 2-pentanol for the regeneration were added thereto. The solution was incubated at 4 O0C for 20 h . Samples were taken, stopped with cone. HCI and measured by means of HPLC. To determine the enantioselectivities, samples were measured in the GC.
As can be seen in fig. 10, the reaction in n-heptane with 10% 2-pentanol proceeds the fastest. However, the best ee value is achieved when using 2-pentanol both as solvent and as regenerating agent. However, 2-pentanol is more expensive than 2-butanol or n-heptane by a factor of approximately 8-1 0 , meaning that the use of 2-pentanol would only pay off for very large production quantities since here the solvent can be recycled. Initially, further processing was with n-heptane as second phase.
(5) Comparison of 2-pentanol and 2-butanol as regenerating agent Furthermore, instead of 2-pentanol as solvent, 2-butanol was tested since 2-butanol is considerably less expensive. 5 g/l (bio dry mass) of cells and 150 mM (75 mmol, 15.5 g) of ethyl 2-oxo-4-phenylbutyrate (1) were initially introduced in 250 ml of buffer (50 mM
NaH2PO4 pH 7.0, 0.2 mM NAD) in a heatable 0.5 I reactor with pH control. 50 ml (10%) of 2- butanol or 2-pentanol (for regenerating the cofactor) and 400 ml of n-heptane as second phase were added thereto. The mixture was stirred for 30 h at 4 O0C. Samples were taken, stopped with HCI and analyzed by means of HPLC. The reaction with 2-pentanol as regenerating agent proceeds more quickly at the start, with 2-butanol a complete conversion is achieved (fig. 11).
(6) Amount of substrate In order to see whether the substrate has an inhibitory effect, mixtures with different substrate concentrations were tested. In each case 7.5 g/l of bio dry mass and 100 µmol to 500 µmol (100 mM to 500 mM, 20 mg to 100 mg) of ethyl 2-oxo-4-phenylbutyrate (1) were µ dissolved in a 1 ml mixture (20 mM NaH2PO4 pH 7.0, 0.2 mM NAD). 400 l of n-heptane and 100 µl of 2-butanol for the regeneration were added thereto. The solution was incubated at 4 O0C for 24 h . Samples were taken, stopped with cone. HCI and measured by means of HPLC.
Fig. 12 shows the concentrations in the organic phase, i.e. 1 M corresponds approximately to a total concentration of 500 mM. As can be seen in figure 12, 500 mM of OPB can be converted without problem. For this reason, a further experimental series with substrate concentrations up to 1 M was prepared. Substrate concentrations above 0.5 M cannot be reacted overnight with the same amount of biomass (7.5 g/l of BTM) (fig. 13).
(7) OPB reduction with purified enzyme In order to exclude an unselective background reaction by further dehydrogenases which are possibly likewise expressed in the production strain, a reduction with purified enzyme as biocatalyst was carried out compared to a crude extract. In each case 2.4 g/l of bio dry mass or 0.4 g/l of purified enzyme and 200 mM (200 µmol, 40 mg) of ethyl 2-oxo-4-phenylbutyrate (1) were dissolved in a 1 ml mixture (50 mM Tris pH 8.0, 0.2 mM NAD). 500 µl of 2-pentanol as solvent/regenerating agent were added thereto. The solution was incubated for 24 h at 1O0C. Samples were taken, stopped with cone. HCI and measured by means of HPLC. As can be seen in figure 14, the reaction with purified enzyme proceeds significantly more quickly than with crude extract since here "pure" biocatalyst was used. The ee value is somewhat worse (ee ~ 92%) than in the case of the reaction with crude extract ee ~ 94%). The reaction rate possibly correlates to the ee value.
(8) Saponification of the R enantiomer with amano lipase Since the ee value (ee ~ 96%) at the end of the dehydrogenase reaction does not correspond to the specification (ee > 97.5%), 1 g/l of amano lipase are added to the mixture and stirred at 4 O0C. The saponification of OPB with amano lipase from Pseudomonas is described in the literature. Prior inactivation of the dehydrogenase, as unpublished experiments have shown, is not necessary. The course of the ee values is shown in figure 15 . The lipase saponifies the undesired enantiomer to give the free acid, which can then be separated off in a subsequent work-up. After about 4 days, in the case of the mixture with 2- butanol as solvent, an ee > 99% is achieved (blue line). In the mixture with 2-pentanol (red line), the lipase reaction proceeds considerably more slowly. Figure 18 (a)-(c) shows a comparison of different lipases and lipase batches (a) squares: Pseudomonas Lipase, as described in WO 95/08636, used at a cone of 0,125 g/L at 5 O0C ; triangles: amano lipase as used above). Amano Lipase was used at a cone of 2,5 g/L. Lipases were added after 24h of substrate reduction reaction, reaction was performed at 4 O0C or 5 O0C (reaction conditions:
10Vol% DF cells chnA enzyme / 18mM NaH2PO4 pH6 / 0,2mM NAD / 50OmM OPB / 40 Vol% n-Heptan / 10 Vol% 2-Butanol / 4 O0C).
(9) Preparation of a sample amount in the 4 I reactor 1.6 1of n-heptane as solvent and 400 ml of 2-butanol (as regenerating agent for the
cofactor) in 20 mM NaH PO4 buffer (pH = 7.0) were initially introduced in a heatable 4 I reactor with stirrer. 0.2 mM NAD and 500 mM (2 mol, 400 g) OPB were added. By adding the biocatalyst (7.5 g/l of BTM) in the form of whole cells (untreated fermenter discharge), the reaction was started. The two-phase reaction mixture was stirred at 4 O0C. The pH was checked using a pH titrator and kept constant between pH~6.5-7.0. Each hour, a sample was taken, stopped with cone. HCI and analyzed by means of HPLC (LJ31366, method mOPB). Figure 16 shows the course of the enzymatic reduction. About 10% of the acid or of an acetal from the oxo compound are formed as secondary component.
After the enzymatic reduction, the amano lipase was added in portions (see fig. 17) without inactivating the dehydrogenase beforehand. The dehydrogenase does not disturb the saponification.
The course of the ee value is shown in figure 17. After 4 days, an ee value > 99% with a lipase concentration of 2.5 g/l is achieved.
Following the phase separation, ca. 2 I of the organic phase from the run 070219 were freed from the solvent in vacuo and then distilled at 1-3 mbar over a 10 cm column. The main run was collected at a bath temperature of 16O 0C and a transition temperature of 116-12O 0C. Yield: 295.3 g = 75.3% of theory of a pale yellow oil with a content of >98% (GC; NMR). The angle of rotation is - 19.9° (c = 1 in CHCb); this value is in good agreement with data in the literature: -20.1° in Yanagisawa, H.; Ishihara, S.; Ando, A.; Kanazaki, T.; Miyamoto, S.; et al.; J. Med. Chem.; 30; 11; 1987; 1984-1991 ; -19.8 ° in Blaser, H.-U.; Burkhardt, S.; Kimer, Hans J.; Moessner, T.; Studer, M.; Synthesis 11; 2003; 1679-1682.
Two biocatalysts have been identified for preparing both ethyl S-L and R-2-hydroxy-4- phenylbutyrate (Ebn1_para and ChnA). Both stem from the Azoarcus library, from which the catalyst for preparing duloxetine alcohol also originates. An overview of the processing parameters is given in table 2 . The substrate concentrations for both enantiomers are ~ 100 g/l. The yields refer to the reduction, in the work-up about 10% is lost.
Since the ee value after the reduction is only about 95-96%, a lipase step has to be added in order to achieve an ee value of more than 99%. Amano lipase from Pseudomonas selectively saponifies the S enantiomer to give the acid, moreover it is very cost-effective and available in large amounts.
Tab. 1 Summary of the results *ln the preparation of the sample amount S-HPB, only a substrate concentration of 50 g/l was used (tab. 2) since at this point a larger amount of OPB was not available. However, experiments on a smaller scale have shown that in principle 100 g/l can be reacted here too.
The saponification is relatively slow in the two-phase system, meaning that an ee > 99% with 1 g/l of amano lipase is only achieved after 4 days. This step can be optimized by means of methods known to the person skilled in the art, e.g. amount of lipase or solvent used. Further lipases can be tested and compared. The experiments show that the initial ee value (81%) can be increased to more than 95% by optimizing the conditions. Claims
1. A method for preparing optically active 2-hydroxy acid ester derivatives of the formula (I),
R1-C(OH)-C(O)-O-R2 (I)
wherein R 1 and R2, independently of one another, are a) -(C1-C20)-alkyl, in which alkyl is straight-chain or branched, b) -(C2-C20)-alkenyl, in which alkenyl is straight-chain or branched and comprises one, two, three or four double bonds depending on chain length, c) -(C2-C20)-alkynyl, in which alkynyl is straight-chain or branched and optionally comprises one, two, three or four triple bonds, d) -(C6-C14)-aryl, e) -(C1-C8)-alkyl-(C6-C14)-aryl, f) -(C5-C14)-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or g) -(C3-C7)-cycloalkyl, and wherein the residues specified under a) to g) may be unsubstituted or mono- to trisubstituted, and wherein the -OH group is in the S or R configuration relative to the carbon atom to which it is bonded,
the method comprising the bringing into contact of 2-oxo acid ester derivatives of the formula (II) R1-C(O)-C(O)-O-R2 (II) wherein R 1 and R2 have the meaning as in formula I,
with an enzyme (E) selected from the class of the dehydrogenases, in the presence of reduction equivalents, wherein the compound of the formula (II) is enzymatically reduced to the compound of the formula (I), and the reduction equivalents consumed in the course of the reaction are regenerated again by converting a reducing agent (RA) to the corresponding oxidation product (OP) with the help of the enzyme (E), wherein the enzyme (E) comprises a polypeptide sequence of the SEQ ID NO: 2 or 4 or a polypeptide sequence in which up to 25% of the amino acid residues have been altered compared with SEQ ID NO:2 or NO:4 through deletion, insertion, substitution or a combination thereof and which also has at least 50% of the enzymatic activity of SEQ ID NO:2 or 4 .
2 . The method according to claim 1, wherein the enzyme (E) is encoded by a nucleic acid comprising a sequence according to SEQ ID NO:1 or NO:3, or a functional equivalent of NO:1 or NO:3. 3 . The method according to claim 1 or 2 , wherein the reduction of the compound of the formula (II) takes place in the presence of a microorganism which is selected from bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Lactobacillaceae, Streptomycetaceae, Rhodococcaceae and Nocardiaceae.
4 . The method according to any one of claims 1 to 3 , wherein the microorganism is a recombinant microorganism which has been transformed with a nucleic acid which codes for an enzyme as defined in any one of claims 1 to 3 .
5 . The method according to any one of claims 1 to 4 , wherein the method comprises an additional step, this comprising the bringing into contact with a further enzyme (E2) which catalyzes the saponification of a 2-hydroxy acid ester of the formula (I).
6 . The method according to claim 5 , wherein the enzyme (E2) is a lipase of the enzyme class E.C.3.1 .
7 . A dehydrogenase which reduces 2-oxo acid esters in the presence of reduction equivalents to the corresponding S-2-hydroxy acid esters, wherein the oxidoreductase has an amino acid sequence which comprises a polypeptide sequence of the SEQ ID NO: 2 or a polypeptide sequence in which up to 25% of the amino acid residues are altered compared with SEQ ID NO:2 through deletion, insertion, substitution or a combination thereof and which also has at least 50% of the enzymatic activity of SEQ ID NO:2.
8 . An isolated nucleic acid which codes for a dehydrogenase as defined in claim 7 .
9 . An isolated nucleic acid, wherein the nucleic acid comprises a sequence which is selected from the group a) DNA sequence which has the nucleotide sequence according to SEQ ID NO:1 or the complementary strand to SEQ ID NO:1 , b) DNA sequence which hybridizes with the DNA sequence according to SEQ ID NO:1 or its complementary strand, wherein the hybridization takes place under stringent conditions, and c) DNA sequence which, due to the degeneracy of the genetic code, encodes a protein which is also encoded by a DNA sequence according to a) or b).
10 .A vector which comprises a nucleic acid as defined in claim 8 or 9 .
11.The vector according to claim 10, wherein the vector has the sequence according to SEQ ID NO:5.