WO2007028729A1 - Alcool deshydrogenase de nocardia globerula et son utilisation - Google Patents

Alcool deshydrogenase de nocardia globerula et son utilisation Download PDF

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WO2007028729A1
WO2007028729A1 PCT/EP2006/065712 EP2006065712W WO2007028729A1 WO 2007028729 A1 WO2007028729 A1 WO 2007028729A1 EP 2006065712 W EP2006065712 W EP 2006065712W WO 2007028729 A1 WO2007028729 A1 WO 2007028729A1
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nucleic acid
polypeptide
seq
polypeptides
acid sequence
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PCT/EP2006/065712
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Werner Hummel
Julia Schümers
Harald GRÖGER
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Evonik Degussa Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group

Definitions

  • the present invention relates to nucleic acids and the polypeptides encoded by them.
  • the polypeptides described have an alcohol dehydrogenase activity.
  • the polypeptides and, respectively, nucleic acids are from the organism Nocardia globerula or have been derived therefrom.
  • the present invention furthermore relates to expression systems, primers, a process for preparing improved nucleic acids or polypeptides encoded by them and to the use thereof for preparing alcohols.
  • ADHs are classified as class E. C. 1.1.1.1 and thus belong to the "oxidoreductases” . They can be found in a number of organisms (Enzyme Catalysis in Organic Synthesis, Ed. : K. Drauz and H. Waldmann, 1995, VCH, Vol. II, 595ff) . Of interest are "broadband" enzymes which stereoselectively convert a wide spectrum of substrates.
  • Alcohol dehydrogenases catalyze a multiplicity of biological reactions, with alcohol substrates being oxidized to the corresponding ketones or aldehydes or the reduction, in opposite direction, of the aldehyde or ketone to the alcohol being catalyzed.
  • Alcohol dehydrogenase- catalyzed biological processes include such important reactions as the last step of alcoholic fermentation, i.e. conversion of glucose to ethanol in yeasts, the reduction of all- trans-retinal to all- trans-retinol (vitamin Ai) in the retina or the degradation of blood alcohol in the liver.
  • the reactions described are usually reversible and take place in the presence of nicotinamide adenine dinucleotide (NAD + / NADH) or nicotinamide adenine dinucleotide phosphate (NADP + / NADPH) as co-enzyme (co- factor) .
  • NAD + / NADH nicotinamide adenine dinucleotide
  • NADP + / NADPH nicotinamide adenine dinucleotide phosphate
  • alcohol dehydrogenases are also regarded as interesting catalysts for the organochemical synthesis of preparing alcohols, ketones or aldehydes.
  • the enantioselective synthesis of optically active alcohols by catalytic reduction of the corresponding ketones in particular is of particular interest technically, in addition to the preparation of primary alcohols by way of reduction of aldehydes.
  • a multiplicity of alcohol dehydrogenases which are preferably suitable for reducing ketones with methyl as substituents in the vicinity of the carbonyl group (i.e. compounds containing an aceto substituent, e.g. acetophenone) have been reported previously.
  • alcohol dehydrogenases are of interest in connection with the use of a coupled enzymatic system (scheme 1), since they allow inter alia a more efficient preparation of enantiomerically enriched alcohols, starting from ketones or racemic alcohols, in such a system (DE10037101 ; for an up-to-date, comprehensive review on the prior art, see: W. Hummel, Adv. Biochem. Engineering / Biotechnology 1997, 58, 145-184.). It is moreover also possible to efficiently prepare primary alcohols using alcohol dehydrogenases and starting from aldehydes (WO2004/085662) . a) Preparation of enantiomerically enriched alcohols, starting from carbonyl compounds
  • Co-substrate red Co-substrate red.
  • alcohol dehydrogenases usually only a small proportion of alcohol dehydrogenases is in the recombinant form required for industrial purposes.
  • examples of known recombinantly available alcohol dehydrogenases are a Rhodococcus erythropolis (5) -selective alcohol dehydrogenase (EP1499716) and a Lactobacillus kefir (R) -selective alcohol dehydrogenase (EP456107). Both alcohol dehydrogenases are very well suitable for the reduction of aceto-containing ketone structures, i.e. of ketones containing a methyl group in the vicinity of the carbonyl group.
  • alcohol dehydrogenases which do not have the disadvantages of the enzymes of the prior art. More specifically such alcohol dehydrogenases should also accept ketones which have sterically demanding groups in addition to the carbonyl function.
  • the alcohol dehydrogenases should furthermore be superior to the known enzymes with regard to economic points of view, in particular regarding stability, activity and/or selectivity.
  • nucleic acid sequence coding for a polypeptide having alcohol dehydrogenase activity selected from the group consisting of:
  • nucleic acid sequence hybridizing with the nucleic acid sequence according to SEQ. ID. NO: 1 or the sequence complementary thereto under stringent conditions
  • nucleic acid sequence coding for a polypeptide which is at least 80% homologous at the amino acid level to the amino acid sequence depicted in SEQ. ID. NO: 2, without the activity and/or selectivity and/or stability of the polypeptide being substantially reduced compared to the polypeptide of SEQ. ID. NO: 2,
  • the nucleic acid sequences indicated enable recombinant alcohol dehydrogenases of the Nocardia globerula type to be obtained which can be expressed in extremely high yields in host organisms such as, for example, Escherichia coli and which can reduce, with an unusually high activity and with high selectivity, also sterically demanding ketones to give the corresponding enantiomerically enriched alcohols.
  • the stability of the alcohol dehydrogenases obtained in this way also makes a use on an industrial scale appear particularly advantageous from an economic point of view.
  • nucleic acids are also comprised which are alleles or functional variants of the nucleic acid sequences of the invention.
  • Functional variants preferably are more than 75%, more preferably more than 80%, 85%, and particularly preferably more than 90%, homologous to SEQ. ID. NO: 1.
  • Suitable mutagenesis methods are all methods available to the skilled worker for this purpose. They are in particular saturation mutagenesis, random mutagenesis, in vitro recombination methods and site-directed mutagenesis (Eigen, M. and Gardiner, W., Evolutionary molecular engineering based on RNA replication, Pure Appl . Chem. 1984, 56, 967-978; Chen, K. and Arnold, F., Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media.
  • SEQ. ID. NO: 1 may be utilized for generating primers in order to identify directly, for example in other Nocardia strains, and to clone allelic forms by means of PCR.
  • sequence information it is possible, owing to the sequence information, to utilize probes for finding further naturally occurring functional variants of the nucleic acids of the invention and thus the corresponding encoded enzyme variants.
  • a library of artificially generated functional enzyme variants may be obtained, for example via PCR by using a faulty DNA polymerase.
  • the invention also comprises nucleic acid sequences which hybridize with the single-stranded nucleic acid sequences of the invention or their complementary single-stranded nucleic acid sequences under stringent conditions.
  • Gene probes or primers inter alia can be regarded as such nucleic acid sequences.
  • the term "under stringent conditions" here has the meaning as described in Sambrook et al . (Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2 nd ed., Cold Spring Harbor Laboratory Press, New York) .
  • a hybridization according to the present invention is stringent when a positive hybridization signal is observed after washing with 1 x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.1% SDS (sodium dodecyl sulphate) at 50°C, preferably at
  • the application furthermore relates to the polypeptides (enzymes) selected from the group consisting of:
  • polypeptides having a homology of at least 80% to the polypeptide of SEQ. ID. NO: 2, without the activity and/or selectivity and/or stability of the polypeptide being substantially reduced compared to the polypeptide of SEQ. ID. NO: 2.
  • polypeptides of the invention can be employed very well in industrial processes owing to the already indicated stability and the expanded substrate spectrum.
  • the invention thus also comprises the allelic or functional variants of the polypeptides of the invention.
  • a functional variant means for the purposes of the present invention an alcohol dehydrogenase whose amino acid sequence is more than 80%, more preferably more than 85%, 87%, and very preferably more than 90%, homologous to SEQ. ID. NO: 1. It is possible to introduce by mutagenesis (see above) amino acid substitutions into the polypeptides of the invention, but the activity and/or selectivity and/or stability of the polypeptide must not be reduced substantially compared to the polypeptide of SEQ. ID. NO: 2.
  • amino acids which are not located at the active site and whose replacement by an amino acid "of the same kind" cannot be expected prima facie to result in a substantially altered three- dimensional structure may be replaced by an amino acid "of the same kind".
  • amino acids having non-polar side chains amino acids of the same kind
  • amino acids of the same kind can be expected to be replaced, for example alanine by valine, without this having a (substantial) influence on a deteriorated biological function of the enzyme in accordance with the invention.
  • the skilled worker may also draw corresponding conclusions on the substitution of other types of amino acids (for example the replacement of basic amino acids by other basic amino acids or of amino acids with uncharged polar side chains by other amino acids from this group) .
  • polypeptides having alcohol dehydrogenase activity may additionally have post-translational modifications such as, for example, glycosylations or phosphorylations.
  • the polypeptides according to the invention in addition comprise at least one heterologous amino acid section which characterizes these polypeptides as fusion proteins.
  • heterologous components of the fusion protein of the invention may be tags (e.g. His tag or Flag tag) which may be employed in the purification of the fusion proteins of the invention.
  • the heterologous components may have a separate enzymatic activity.
  • the two enzymatic components are preferably connected by a linker such as a flexible glycine or glycine-serine linker of 6-10 amino acids in length, in order to ensure functionality of the components.
  • a linker such as a flexible glycine or glycine-serine linker of 6-10 amino acids in length, in order to ensure functionality of the components.
  • the term "heterologous” may mean on the one hand that the components of the fusion protein do not naturally occur covalently linked together, and on the other hand that the components come from different species. Fusion proteins are usually prepared by recombinant DNA technology (see Sambrook et al . , loc . cit . ) .
  • the present invention relates to recombinant expression systems or recombinant plasmids/vectors having one or more of the nucleic acids of the invention.
  • An expressions system means a system for recombinant expression of the nucleic acids of the invention and thus for recombinant production of the polypeptides of the invention .
  • This production may preferably take place in microorganisms or other hosts transformed or transfected (the terms “transformation” and “transfection” are used in the same sense according to the present invention) with corresponding nucleic acid sequences or vectors (see below) .
  • the recombinant microorganism is preferably of prokaryotic origin.
  • Suitable host cells include cells of unicellular microorganisms such as bacterial cells.
  • Microorganisms which may be mentioned in this respect are prokaryotes such as E. coli, Bacillus subtilis .
  • Other bacteria which may be used for expression of the nucleic acid sequences of the invention are those of the genera/species Lactobacillus, Bacillus, Rhodococus, Campylobacter, Caulobacter, Mycobacterium, Streptomyces, Neisseria, Ralstonia, Pseudomonas, and Agrobacterium.
  • Appropriate strains are available in the prior art and may, at least partially, be obtained from the international deposition sites such as ATCC or DMSZ.
  • eukaryotes such as mammalian cells, insect cells or plant cells or organisms such as, for example, yeasts like Hansenula polymorphs, Pichia sp . , Saccharomyces cerevisiae, or fungi such as, for example, Aspergillus sp., for recombinant production of the polypeptides.
  • E. coll strains for this purpose. Very particular preference is given to: E. coli XLl Blue, NM 522, JMlOl, JM109, JM105, RRl, DH5CC, TOP 10- , HBlOl, BL21 codon plus, BL21 (DE3) codon plus, BL21, BL21 (DE3), MM294, W3110, DSM14459 (EP1444367) .
  • the cytoplasm of E. coli cells has the co-factors which are required for the enzymatic activity of the polypeptide of the invention. They are, in particular, NADH, NADPH, NAD + or NADP + .
  • recombinant production of the polypeptides of the invention may take place in a non-human host.
  • the non- human host may be a cell or a multi- to polycellular organism. Suitable polycellular organisms include model systems familiar in molecular biology, such as Drosophila melanogaster, zebra fish or C. elegans.
  • the host is a cell.
  • the host according to the invention is, in this preferred embodiment, a recombinant cell which has been transformed or transfected with one or more nucleic acid sequences of the invention or one or more vectors of the invention (see below) .
  • the cell is of eukaryotic origin. Suitable eukaryotic cells include CHO cells, HeLa cells and others.
  • the transformation or transfection described above may be carried out by known methods, for example by calcium phosphate co-precipitation, lipofection, electroporation, PEG/DMSO methods, particle bombardment or viral/bacteriophage infection.
  • the cell according to the invention may contain the recombinant nucleic acid in an extrachromosomal or a chromosomally integrated form.
  • the transfection/transformation may be a stable or transient transfection/transformation .
  • Transfection and transformation protocols are known to the skilled worker (Chan and Cohen. 1979. High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA. MoI Gen Genet.
  • the host is a transgenic non-human animal.
  • Transgenic non-human animals may be produced by methods known in the prior art.
  • the transgenic non-human animal according to the invention may have various genetic constitutions. It may (i) overexpress the gene of a nucleic acid sequence of the invention in a constitutive or inducible manner, (ii) contain the endogenous gene of a nucleic acid sequence according to the invention in an inactivated form, (iii) contain the endogenous gene of a nucleic acid sequence according to the invention, which has been completely or partially replaced by a mutated gene of a nucleic acid sequence according to the invention, (iv) have conditional and tissue-specific overexpression or underexpression of the gene of a nucleic acid sequence according to the invention or (v) have a conditional and tissue-specific knock-out of the gene of a nucleic acid sequence according to the invention.
  • the transgenic animal additionally contains an exogenous gene of a nucleic acid sequence according to the invention under the control of a promoter allowing overexpression .
  • the endogenous gene of a nucleic acid sequence according to the invention may be overexpressed by activating or/and replacing its own promoter.
  • the endogenous promoter of the gene of a nucleic acid sequence according to the invention preferably has a genetic modification resulting in increased expression of the gene.
  • the said genetic modification of the endogenous promoter comprises both a mutation of individual bases and deletion and insertion mutations.
  • the host according to the invention in a particularly preferred embodiment, is a transgenic rodent, preferably a transgenic mouse, a transgenic rabbit, a transgenic rat, or is a transgenic sheep, a transgenic cow, a transgenic goat or a transgenic pig.
  • mice have numerous advantages over other animals. They are easy to keep and their physiology is regarded as a model system for that of humans. The generation of such gene- manipulated animals is sufficiently known to the skilled worker and is carried out by customary methods (for this, see for example, Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994), Manipulating the Mouse-Embryo; A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; WO91/08216) .
  • cell culture systems in particular human cell culture systems, for the applications described for the non-human transgenic animal according to the invention.
  • the coding nucleic acid sequences may be cloned into conventional plasmids/vectors and expressed in cell culture after transfection of microorganisms or other host cells with such vectors.
  • Suitable plasmids or vectors are in principle any embodiments available to the skilled worker for this purpose. Plasmids and vectors of this kind may be found, for example, in Studier and co-workers (Studier, W. F.; Rosenberg A. H.; Dunn J. J.; Dubendroff J.
  • Plasmids which may be used for cloning the gene construct having the nucleic acid sequence according to the invention into the host organism in a very preferred manner are: pUC18 (Roche Biochemicals) , pKK-177-3H (Roche
  • Suitable vectors are also, for example, pET- 21a (+) for E. coll but other expression vectors for prokaryotic unicellular organisms and vectors for eukaryotes, such as, for example, yeasts and insect or mammalian cells may also be used. Examples of vectors which have proved suitable for yeasts are the pREP vector and the pINT vector.
  • Baculovirus vectors such as in EP127839 or EP549721 have been disclosed, and SV40 vectors which are generally obtainable, for example, are suitable for expression in mammalian cells. Particular preference is given to vectors for unicellular eukaryotic organisms, in particular from the group of pET vectors for transformation of E. coli cells.
  • the nucleic acid sequence of the invention which has been introduced into the vector, is additionally fused to a histidine tag provided by the vector.
  • a histidine tag provided by the vector.
  • the vectors may contain further functional nucleotide sequences for regulating, in particular repressing or inducing, expression of the ADH gene and/or of a reporter gene.
  • regulatable weak promoters such as, for example, the rha promoter or the nmtl promoter, or regulatable strong promoters such as, for example, the lac, ara, lambda, pL, T7 or T3 promoter.
  • regulatable strong promoters such as, for example, the lac, ara, lambda, pL, T7 or T3 promoter.
  • the coding DNA fragments must be transcribable from a promoter in the vectors.
  • Other examples of proven promoters are the
  • Baculovirus polyhedrin promoter for expression in insect cells see, for example, EP127839 or the early SV40 promoter or LTR promoters, for example, of MMTV (Mouse Mammary Tumour Virus; Lee et al . (1981) Nature, 294 (5838), 228-232) .
  • MMTV Mammary Tumour Virus
  • the expression vectors of the invention may contain further functional sequence regions such as, for example, an origin of replication, operators or termination signals.
  • the present invention relates to partial sequences of the nucleic acid sequences according to the invention, which partial sequences preferably consist of at least 5 or 10, preferably 50, more preferably 100, very preferably 150, contiguous nucleotides, particularly preferably of at least 300 contiguous nucleotides of the nucleic acids according to the invention.
  • the primers are derived from the 3' and 5' ends of the nucleic acid sequences according to the invention. Preferably, they additionally have common cleavage sites such as, for example, Ndel on the 5' end, and a BamRI cleavage site on the 3' end. Particular preference is given to the following primers:
  • the present invention relates to a process for preparing improved rec polypeptides (recombinant polypeptides) having alcohol dehydrogenase activity, starting from nucleic acid sequences according to the invention, wherein a) the nucleic acid sequences are subjected to a mutagenesis, b) the nucleic acid sequences obtainable from a) are cloned into a suitable vector which is transferred to a suitable expression system, and c) the produced polypeptides having improved activity and/or selectivity and/or stability are detected and i s o l ated .
  • the invention likewise relates to rec polypeptides or nucleic acid sequences encoding them, which can be obtained by a process as described before.
  • the present invention concerns the use of the (rec) polypeptides of the invention for preparing alcohols, in particular enantiomerically enriched alcohols.
  • the conversion of carbonyl compounds, in particular ketones, to alcohols, in particular enantiomerically enriched alcohols, with the aid of alcohol dehydrogenases is known in principle to the skilled worker (see references indicated above) .
  • Examples of alcohols, in particular enantiomerically enriched alcohols, that can be prepared from the corresponding ketones are likewise familiar to the skilled worker. They can be subsumed under the following general formula
  • R and R' are different from one another, in particular H, (Ci-C 20 ) -alkyl, (Ci-C 8 ) -alkoxy, HO-(Ci-C 8 ; alkyl, (C 2 -C 8 ) -alkoxyalkyl, (Ci-C 8 ) -alkoxycarbonyl, (C 6 -Ci 8 ) -aryl, (C 7 -Ci 9 ) -aralkyl, (C 3 -Ci 8 ) -heteroaryl, (C 4 -Ci 9 ) -heteroaralkyl, (Ci-C 8 ) -alkyl- (C 6 -Ci 8 ) -aryl, (Ci-C 8 ) -alkyl- (C 3 -Ci 8 ) -heteroaryl, (C 3 -C 8 ) -cycloalkyl, (Ci-C 8 ) -alkyl-al
  • Particularly preferred substrates for the alcohol dehydrogenases of the invention are acyclic (C3-C20) - alkanones, it being possible for the particular carbon radicals to be substituted within the scope of the invention.
  • Particularly preferred substrates are likewise keto ester compounds, in particular CC-, ⁇ -, ⁇ -keto ester compounds, it being possible for a hydrocarbon radical corresponding to the invention being incorporated between the keto group and the ester group.
  • the substrates ethyl 8-chloro-6-oxooctanoate and ethyl 4-phenyl-2-oxobutyrate are exemplary representatives of this latter class of compounds .
  • the present invention concerns the use of the (rec) polypeptides of the invention for preparing carbonyl compounds.
  • the conversion of alcohols to the corresponding carbonyl compounds, in particular ketones and aldehydes, with the aid of alcohol dehydrogenases is known in principle to the skilled worker (see references indicated above) .
  • a racemic alcohol is employed, the said embodiment then resulting, due to enantioselective conversion, in a mixture of a carbonyl compound and an enantiomerically enriched alcohol. Accordingly, this process, starting from racemic alcohols, thus also results in enantiomerically enriched alcohols.
  • Another development of the invention relates to the use of the nucleic acid sequences of the invention for preparing whole-cell catalysts and to the use of the nucleic acid sequences of the invention for mutagenesis.
  • the invention likewise relates to a whole-cell catalyst having a cloned gene coding for a polypeptide having alcohol dehydrogenase activity according to the invention, and a cloned gene for an enzyme suitable for regenerating NAD(P)H, in particular a formate dehydrogenase, a glucose dehydrogenase or an NAD (P) + -regenerating enzyme such as NAD(P)H oxidase.
  • the further preferred whole-cell catalyst is distinguished by the fact that the gene is one that codes for a Nocardia globerula polypeptide having alcohol dehydrogenase activity, in particular one that codes for the alcohol dehydrogenase of the invention according to SEQ. ID. NO: 2.
  • the whole-cell catalyst containing a formate dehydrogenase should preferably derive from Candida boidinii formate dehydrogenase and, if an NADH oxidase is present, from Lactobacillus brevis NADH oxidase. If a glucose dehydrogenase is present, preference is given to considering a corresponding Bacillus, in particular Bacillus subtilis, glucose dehydrogenase.
  • a preferred host organism used for the whole-cell catalyst is an organism as mentioned in DE10155928.
  • the advantage of an organism of this kind is simultaneous expression of both polypeptide systems, thus requiring growing only one rec organism for the reaction.
  • the correspondingly encoding nucleic acid sequences may be put on different plasmids with different copy numbers and/or promoters with different strengths may be used for expressing the nucleic acid sequences at different levels.
  • a possibly inhibitory intermediate is not accumulated in enzyme systems adjusted in this way, and the observed reaction may proceed with an optimal overall rate.
  • the nucleic acid sequences of the invention can preferably be employed for preparing rec polypeptides.
  • Recombinant techniques which are sufficiently known to the skilled worker produce organisms which are capable of making available the contemplated polypeptide in an amount adequate for an industrial process.
  • the rec polypeptides of the invention are produced by genetic engineering processes known to the skilled worker (Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2 nd ed., Cold Spring Harbor Laboratory Press, New York; Balbas, P. and Bolivar, F. (1990), Design and construction of expression plasmid vectors in E.coli, Methods Enzymol. 185, 14-37; Rodriguez, R. L.
  • the present invention relates to a coupled enzymatic reaction system having a co- factor-dependent enzymatic transformation of a carbonyl compound, in particular of a ketone, with a polypeptide according to the invention and an enzymatic regeneration of the co-factor (NAD(P)H).
  • a reaction system may mean any vessel in which the reaction according to the invention can be carried out, i.e. reactors of any kind (loop reactor, stirred tank, enzyme membrane reactor etc.), or diagnostic kits in any form.
  • the co-factor is regenerated using formic acid or salts thereof as reductants.
  • a formate dehydrogenase the co-factor is regenerated using formic acid or salts thereof as reductants.
  • other enzymatic or substrate-based co-factor- regenerating systems e.g. glucose dehydrogenase.
  • Another use of the (rec) polypeptides having alcohol dehydrogenase activity according to the invention or of the whole-cell catalyst according to the invention relates to their use in a process for reducing carbonyl compounds, preferably for asymmetrically reducing ketones or for reducing aldehydes.
  • Suitable for conversion of the carbonyl compounds, preferably ketones, are aqueous solvents which are buffered accordingly.
  • the reduction is preferably carried out at a temperature of between 10 and 85°C, particularly preferably between 20 and 50 0 C (see Fig. 4) . It is particularly surprising here that the alcohol dehydrogenase of the invention has high activities in a wide and high temperature range from 15°C to approx. 55°C (with maximum activity at approx. 35°C; see Fig. 4) .
  • the preferred pH for the enzymatically catalyzed reduction of carbonyl compounds with the alcohol dehydrogenase according to the invention is between pH 4 and pH 9, particularly preferably between pH 5.5 and pH 8 (see Fig. 3) .
  • the contemplated polypeptide may be used in its native form as homogeneously purified compounds or as recombinantly produced enzyme for application.
  • the (rec) polypeptide may furthermore also be used as component of an intact guest organism or in connection with the disrupted cell mass of the host organism, which has been purified to any degree.
  • It is likewise possible to use the enzymes in immobilized form (Sharma B. P.; Bailey L. F. and Messing R. A. (1982), Immobilinstrumente Biomaterialien -techniken und füren [Immobilized biomaterials - techniques and applications], Angew. Chem. 94, 836-852) .
  • the immobilization is advantageously carried out by way of lyophilization (Paradkar, V. M.; Dordick, J. S. (1994), Aqueous-Like
  • CLEC Cofactor-bound cross- linked enzyme crystals
  • the procedure for converting carbonyl compounds, preferably ketones, with the polypeptides of the invention is preferably as follows.
  • the polypeptides are added in the desired form (free, immobilized, in host organisms or as whole-cell catalyst) to the aqueous solution.
  • the carbonyl compound, where appropriate the co-factor and where appropriate the co-factor regenerating agents are added to this mixture, while maintaining the optimal temperature and pH ranges.
  • the alcohol obtained may be isolated from the reaction mixture by methods known to the skilled worker (crystallization, extraction, chromatography) .
  • the expressible proteins of the invention were shown to possess an enzymatic alcohol dehydrogenase activity.
  • the alcohol dehydrogenases of the invention reduce in particular acyclic aliphatic ketones to give the corresponding enantiomerically enriched acyclic aliphatic alcohols.
  • the alcohol dehydrogenases are suitable to a particular degree for reducing ethyl 8-chloro-6- oxooctanoate and ethyl 4-phenyl-2-oxobutyrate (see experimental section) .
  • the claimed alcohol dehydrogenases are furthermore distinguished by their excellent stereoselectivity. Thus, even sterically demanding substrates are converted with high enantioselectivities of >95% ee to the desired, corresponding optically active alcohols (in this context, see also example in the experimental section and scheme 3) .
  • the present invention in a next aspect, furthermore relates to a coupled enzymatic reaction system having a co- factor-dependent enzymatic transformation of an alcohol, in particular of a racemic alcohol, with a polypeptide according to the invention and an enzymatic regeneration of the co-factor (NAD(P) + ).
  • enzymatic regeneration of the co-factor should be carried out using the enzymes discussed above in connection with the whole-cell catalyst, but may also be carried out electrochemically or by chemical reduction without the use of enzymes.
  • a reaction system may mean any vessel in which the reaction according to the invention can be carried out, i.e. reactors of any kind (loop reactor, stirred tank, enzyme membrane reactor etc.), or diagnostic kits in any form.
  • the co-factor is regenerated using oxygen as oxidant.
  • oxygen as oxidant.
  • a final use of the (rec) polypeptides having alcohol dehydrogenase activity according to the invention or of the whole-cell catalyst according to the invention relates to their use in a process for oxidizing alcohols, preferably for oxidizing racemic alcohols, to yield a mixture of carbonyl compound and an enantiomerically enriched form of the alcohol.
  • Suitable for conversion of the alcohols, preferably racemic alcohols, are aqueous solvents which are buffered accordingly.
  • the oxidation is preferably carried out at a temperature of between 10 and 85°C, particularly preferably between 20 and 50°C.
  • the preferred pH for the enzymatically catalyzed reduction of carbonyl compounds with the alcohol dehydrogenase according to the invention is between pH 4 and pH 9, particularly preferably between pH 5.5 and pH 8.
  • the contemplated polypeptide may be employed in the forms already described above for the reduction of carbonyl compounds .
  • the procedure for converting alcohols, preferably racemic alcohols, with the polypeptides of the invention is preferably as follows.
  • the polypeptides are added in the desired form (free, immobilized, in host organisms or as whole-cell catalyst) to the aqueous solution.
  • the alcohol, where appropriate the co-factor and where appropriate the co-factor regenerating agents are added to this mixture, while maintaining the optimal temperature and pH ranges.
  • the carbonyl compound and/or the remaining, enantiomerically enriched alcohol obtained may be isolated from the reaction mixture by methods known to the skilled worker (crystallization, extraction, chromatography) .
  • Optically enriched (enantiomerically enriched) compounds mean within the scope of the invention the presence of one optical antipode in a mixture with the other one in >50 mol%.
  • nucleic acid sequence of the invention may be a DNA or an RNA molecule. Preference is given to the nucleic acid molecule being a cDNA molecule or an mRNA molecule. According to the invention, the DNA molecule may furthermore be a genomic DNA molecule. The invention further comprises embodiments in which the DNA molecule is a PNA molecule or another derivative of a DNA molecule.
  • complementarity means according to the invention that the complementarity extends over the entire region of the nucleic acid molecule of the invention, without gaps. In other words: preference is given according to the invention to complementarity extending 100% over the entire region of the sequence of the invention, i.e. from the 5' end depicted to the 3' end depicted.
  • Improved activity and/or selectivity and/or stability means according to the invention that the polypeptides are more active and/or more selective and/or are more stable under the reaction conditions used. While the activity and stability of the enzymes should naturally be as high as possible for industrial application, an improvement with respect to selectivity is said to be the case if either substrate selectivity is reduced but the enantioselectivity of the enzymes is increased. The same applies mutatis mutandis to the term not substantially reduced, which is used in this context.
  • the invention also comprises those sequences which are more than 91%, preferably more than 92%, 93% or 94%, more preferably more than 95% or 96% and particularly preferably more than 97%, 98% or 99%, homologous (excluding natural degeneracy) to any of these sequences, as long as the mode of action or purpose of such a sequence is retained.
  • nucleic acid sequences which code for polypeptides includes any sequences that appear possible in accordance with the degeneracy of the genetic code .
  • (Ci-Cs) -Alkyl radicals to be considered are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl, including all of their structural isomers.
  • a (C1-C20) -alkyl radical is, within the scope of the definition according to the invention, a corresponding radical having from 1 to no more than 20 carbon atoms.
  • a (C3-C20) -alkyl radical is, within the scope of the definition according to the invention, a corresponding radical having from 3 to no more than 20 carbon atoms.
  • the (Ci-Cs) -alkoxy radical corresponds to the (Ci-Cs) -alkyl radical, with the proviso that the latter is bound via an oxygen atom.
  • (C2-C8) -Alkoxyalkyl means radicals in which the alkyl chain is interrupted by at least one oxygen function, although two oxygen atoms may not be joined together.
  • the number of carbon atoms indicates the total number of carbon atoms present in the radical.
  • a (C3-C5) -alkylene bridge is a carbon chain having from three to five carbon atoms, which chain is bound via two different carbon atoms to the molecule concerned.
  • radicals described above may be mono- or polysubstituted with halogens and/or (Ci-Cs) -alkoxycarbonyl and/or N-, O-, P-, S-, Si-containing radicals.
  • halogens and/or (Ci-Cs) -alkoxycarbonyl and/or N-, O-, P-, S-, Si-containing radicals are, in particular, alkyl radicals of the type mentioned above, whose chain contains one or more of these heteroatoms or which are bound via one of these heteroatoms to the molecule .
  • (C3-C8) -Cycloalkyl means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl radicals etc. They may be substituted with one or more halogens and/or N-, O-, P-, S-, Si-containing radicals and/or their ring may have N, O, P, S atoms, such as, for example, 1-, 2-, 3-, 4- piperidyl, 1-, 2-, 3-pyrrolidinyl, 2-, 3-tetrahydrofuryl, 2-, 3-, 4-morpholinyl .
  • a (C3-C8) -cycloalkyl- (Ci-Cs) -alkyl radical refers to a cycloalkyl radical as illustrated above which is bound via an alkyl radical as indicated above to the molecule.
  • a (C6-Cis) -aryl radical means an aromatic radical having from 6 to 18 carbon atoms. This includes in particular compounds such as phenyl, naphthyl, anthryl, phenanthryl, biphenyl radicals or systems of the above-described type which are anellated to the molecule in question, such as, for example, indenyl systems which may or may not be substituted with ( (Ci-C 8 ) -alkyl, (Ci-C 8 ) -alkoxy, (C 2 -C 8 )- alkoxyalkyl, NH (Ci-C 8 ) -alkyl, N ( (Ci-C 8 ) -alkyl) 2 , OH, 0(Ci-C 8 ) -alkyl, NO 2 , NH (Ci-C 8 ) -acyl, N ( (Ci-C 8 ) -acyl) 2 , F, Cl, CF 3 , (Ci-C 8
  • a (C 7 -Ci 9 ) -aralkyl radical is a (Ce-Ci 8 ) -aryl radical bound via a (Ci-C 8 ) -alkyl radical to the molecule.
  • a (C 3 -Cis) -heteroaryl radical refers, within the scope of the invention, to a five-, six- or seven-membered aromatic ring system of from 3 to 18 carbon atoms, whose ring has heteroatoms such as, for example, nitrogen, oxygen or sulphur.
  • Radicals which are regarded as heteroaromatics of this kind are radicals such as 1-, 2-, 3-furyl, such as 1-, 2-, 3-pyrrolyl, 1-, 2-, 3-thienyl, 2-, 3-, 4-pyridyl, 2-, 3-, 4-, 5-, 6-, 7-indolyl, 3-, 4-, 5-pyrazolyl, 2-, 4-,
  • heteroaromatics may be substituted in the same way as the (C ⁇ -Cis) -aryl radicals mentioned above.
  • a (C4-C19) -heteroaralkyl means a heteroaromatic system corresponding to the (C7-C19) -aralkyl radical.
  • Suitable halogens are fluorine, chlorine, bromine and iodine .
  • aqueous solvent means water or a solvent mixture consisting mainly of water with water-soluble organic solvents such as, for example, alcohols, in particular methanol or ethanol, or ethers such as THF or dioxane.
  • the host used for transformation with DNA plasmids was the E. coli strain BL21 (DE3) (Wood, W. B. (1966) J. MoI. Biol. 16, 118-133; Leahy, D. J., Hendrickson, W. A., Aukhil, I., and Erickson, H. P. (1992) Science 258, 987-991; Phillips, T. A., Van Bogelen, R. A., and Neidhardt, F. C. (1984) J. Bacteriol. 159, 283-287).
  • the cells are cultured in liquid LB medium or on LB agar plates at 37 0 C.
  • the vector used for cloning of DNA fragments is the plasmid pET-21a(+) (US 4,952,496, US 5,693,489 and US 5,869,320) which contains the IPTG-regulatable T7 promoter (Studier, F. W., Rosenberg, A. H., Dunn, J.J. and Dubendorff, J. W. (1990) Meth. Enzymol . 185, 60-89; Dubendorff, J. W. and Studier, F. W. (1991) J. MoI. Biol. 219, 45-59).
  • the catalyzing enzyme Since a ketone is reduced to an alcohol in this reaction, the catalyzing enzyme must be an alcohol dehydrogenase (ADH) . It was named NGADH, derived from the name of the bacterium in which it was originally expressed. The said enzyme was purified in several chromatographic steps (hydrophobic interaction chromatography, anion exchange chromatography, hydroxylapatite chromatography and gel filtration) . After blotting to a PVDF membrane, the band corresponding to ADH was excized and the N-terminus of the enzyme was partially sequenced by means of Edman degradation. On the basis of the deduced DNA sequence, the complete sequence of the NGADH gene was determined by means of the BD Genome Walker Universal Kit (Barnes, W. M.
  • NGADH gene was amplified by means of polymerase chain reaction
  • E. coli BL21 (DE3) cells were transformed with the pNGADH vector and cultured in ampicillin- containing LB medium. The cells were incubated to an optical density (at 600 nm) of 0.5 at 37°C and NGADH expression was induced with 1 mM IPTG solution once the said optical density was reached. Subsequently, the cell suspension was incubated at 30 0 C for 18 hours in order to achieve the optimal NGADH yield.
  • crude extract containing the recombinant Nocardia globerula alcohol dehydrogenase is prepared by disrupting cells of the strain E. coli BL21 (DE3) pNGADH.
  • 3.6 ml of phosphate buffer pH 7.0, 100 mM
  • 0.72 g of the cell pellets which is resuspended therein to give a biomass concentration of approx. 200 g/1.
  • 0.8 ml of this solution are admixed with 1 g of glass beads in an Eppendorf vessel and disrupted by treatment in a vibratory ball mill (frequency 30 s '1 ; duration 5 min; 3 cycles) .
  • centrifugation 13 200 rpm; 5 min
  • a supernatant is obtained which is subsequently used as enzyme solution for determining the activity and for preparative experiments.
  • the activity is determined by firstly weighing in 680 mg (0.1 mol/1) of dipotassium hydrogen phosphate trihydrate, adding 5.6 ml of sodium hydroxide solution and filling up with VE water to a volume of 20 ml, resulting in a pH of 6.0. This is followed by preparing a substrate solution containing the particular ketone to be studied (1.5 mmol/1) in the above-described buffer solution (pH 6.0). Separately, 44.5 mg (12.5 mmol/1) of NAD + trihydrate are weighed and dissolved in 5 ml of VE H2O.
  • the enzyme activity was determined spectrophotometrically with ethyl 8-chloro-6-oxooctanoate .
  • the measurement was carried out at 37 0 C for one minute, with oxidation of NADH being monitored via the decrease in extinction at 340 nm.
  • the measurement was carried out at various pH values and, correspondingly, in various buffers listed in the table below.
  • Glycine-NaOH pH 8.5-11.0 The corresponding graph in Fig. 3 shows that NGADH is active in the pH range from 4.0 to 9.5 and that the activity is particularly high in the pH range from 6.0 to 7.5.
  • the enzyme activity was determined spectrophotometrically with ethyl 8-chloro-6- oxooctanoate .
  • the measurement was carried out at pH 6.5 for one minute, with oxidation of NADH being monitored via the decrease in extinction at 340 nm.
  • the activity was determined at various temperatures in the range from 5°C to 55°C.
  • NGADH was shown to be active at temperatures of from 5 to 55°C, the activity being particularly high between 25 and 50 0 C.
  • the temperature dependence of NGADH is depicted as a graph in Fig . 4.
  • a reaction mixture consisting of ethyl 8-chloro-6-oxo- octanoate (200 mM) and of NAD + trihydrate (0.02 equivalents, based on the ketone) , sodium formate (3 equivalents, based on the ketone) with enzyme amounts of 58 U/mmol of Nocardia globerula recombinant alcohol dehydrogenase (recombinant NGADH, expressed in E. coli; enzyme solution according to preparation above) and 60 U/mmol of a Candida boidinii formate dehydrogenase (wild type enzyme) is stirred in 20 ml of VE water (pH 7.0) at a reaction temperature of 30 0 C for a period of 24 hours.
  • VE water pH 7.0
  • a constant pH (pH 6-8) is maintained during the reaction.
  • the reaction mixture is subsequently extracted with 50 ml of methyl tert-butyl ether, the aqueous phase is discarded and the organic phase (after washing with wash water (3x) and acidic aqueous solution of pH 3-4) is dried over magnesium sulphate.
  • Volatile components are removed in vacuo from the filtrate obtained after filtration, and the resulting residue is analysed with respect to the rate of formation by 1 H-nuclear magnetic resonance spectroscopy. A rate of formation of >95% was determined (scheme 2) .
  • a reaction mixture consisting of ethyl 4-phenyl-2-oxo- butyrate (200 mM) and of NAD + trihydrate (0.02 equivalents, based on the ketone) , sodium formate (3 equivalents, based on the ketone) with enzyme amounts of 58 U/mmol of Nocardia globerula recombinant alcohol dehydrogenase (recombinant NGADH, expressed in E. coli; enzyme solution according to preparation above) and 60 U/mmol of a Candida boidinii formate dehydrogenase (wild type enzyme) is stirred in 20 ml of VE water (pH 6.7) at a reaction temperature of 30 0 C for a period of 2 hours.
  • VE water pH 6.7
  • Volatile components are removed in vacuo from the filtrate obtained after filtration, and the resulting residue is analysed with respect to the rate of formation by 1 H- nuclear magnetic resonance spectroscopy and with respect to enantioselectivity by chiral HPLC. A rate of formation of >95% and an enantioselectivity of 96% ee were determined (scheme 3) .
  • Fig. 1 depicts the plasmid map of the pNGADH vector composed of the pET-21a(+) vector and the NGADH gene according to SEQ. ID. NO: 1.
  • Fig. 2 depicts overexpression of Nocardia globerula recombinant alcohol dehydrogenase in E. coli BL21 (DE3) in an SDS gel.
  • Fig. 3 depicts the influence of pH on the enzymatic activity of NGADH on the basis of the substrate ethyl 8-chloro-6-oxooctanoate .
  • Fig. 4 depicts the influence of temperature on the enzymatic activity of NGADH on the basis of the substrate ethyl 8-chloro-6-oxo-octanoate .

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Abstract

La présente invention concerne un alcool déshydrogénase de Nocardia globulera. Cette invention concerne également de nouveaux polypeptides présentant une activité alcool déshydrogénase, des acides nucléiques codant ces polypeptides, des amorces ou des sondes servant à préparer les séquences d'acides nucléiques, des hôtes non humains ou des cellules hôtes et des systèmes de réaction qui peuvent être utilisés pour préparer les produits souhaités. Les alcools déshydrogénases dépendants de cofacteurs de ce type peuvent être utilisés avantageusement pour obtenir des alcools, en particulier des alcools énantiomériquement enrichis, qui peuvent être utiles lorsqu'ils sont employés en synthèse organique, par utilisation d'une enzyme de régénération du cofacteur dans un système enzymatique couplé. Dans une variante, il est possible d'utiliser les polypeptides de cette invention pour la réaction inverse, autrement dit l'oxydation d'alcools avec formation de composés carbonyles.
PCT/EP2006/065712 2005-09-10 2006-08-28 Alcool deshydrogenase de nocardia globerula et son utilisation WO2007028729A1 (fr)

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EP3222712A1 (fr) * 2016-03-22 2017-09-27 Universität zu Köln Alcohol déhydrogénase de pichia pastoris et son utilisation
US10294479B2 (en) 2015-03-04 2019-05-21 East China University Of Science And Technology Candida carbonyl reductase and method for preparing (R)-lipoic acid precursor

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DE102010039833A1 (de) * 2010-08-26 2012-03-01 Symrise Ag Ganzzell-Biotransformation von Fettsäuren zu den um ein Kohlenstoffatom verkürzten Fettaldehyden
DE102010035702A1 (de) * 2010-08-27 2012-03-01 Zyrus Beteiligungsgesellschaft Mbh & Co. Patente I Kg Ganzzell-Biokatalysator

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10294479B2 (en) 2015-03-04 2019-05-21 East China University Of Science And Technology Candida carbonyl reductase and method for preparing (R)-lipoic acid precursor
EP3222712A1 (fr) * 2016-03-22 2017-09-27 Universität zu Köln Alcohol déhydrogénase de pichia pastoris et son utilisation

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