US20130040341A1 - NOVEL 7beta-HYDROXYSTEROID DEHYDROGENASES AND THEIR USE - Google Patents

NOVEL 7beta-HYDROXYSTEROID DEHYDROGENASES AND THEIR USE Download PDF

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US20130040341A1
US20130040341A1 US13/512,166 US201013512166A US2013040341A1 US 20130040341 A1 US20130040341 A1 US 20130040341A1 US 201013512166 A US201013512166 A US 201013512166A US 2013040341 A1 US2013040341 A1 US 2013040341A1
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hsdh
acid
keto
udca
sequence
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Luo Liu
Rolf Schmid
Arno Aigner
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Pharmazell GmbH
PhamaZell GmbH
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    • 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
    • C12P33/00—Preparation of steroids
    • 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
    • C12P33/00—Preparation of steroids
    • C12P33/02—Dehydrogenating; Dehydroxylating
    • 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
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01201—7-Beta-hydroxysteroid dehydrogenase (NADP+) (1.1.1.201)

Definitions

  • the invention relates to novel 7 ⁇ -hydroxysteroid dehydrogenases obtainable from bacteria of the genus Collinsella , in particular of the strain Collinsella aerofaciens , the sequences coding for these enzymes, methods for the production of the enzymes and the use thereof in enzymatic conversions of cholic acid compounds, and in particular in the production of ursodesoxycholic acid (UDCA); also a subject of the invention are novel methods for the synthesis of UDCA.
  • ursodesoxycholic acid and the corresponding diastereomer chenodesoxycholic acid (CDCA) have for many years been used for the medicinal treatment of gallstone problems.
  • the two compounds differ only in the configuration of the hydroxy group on C atom 7 (UDCA: ⁇ configuration, COCA: ⁇ configuration).
  • UDCA ursodesoxycholic acid
  • COCA chenodesoxycholic acid
  • the CA is first oxidized by 7 ⁇ -HSDH from Bacteroides fragilis ATCC 25285 (Zhu, D., et al., Enzymatic enantioselective reduction of - ketoesters by a thermostable 7- hydroxysteroid dehydrogenase from Bacteroides fragilis . Tetrahedron, 2006. 62(18): p. 4535-4539) and 12 ⁇ -HSDH to 7,12-diketo-LCA. These two enzymes are each NADH-dependent. After the reduction by 7 ⁇ -HSDH (NADPH-dependent) from Clostridium absonum ATCC 27555 (DSM 599) (MacDonald, I. A. and P. D.
  • Hirano et al also disclosed K M and V max values only for NADP + (0.4 and 0.2 respectively) but not for NADPH. Those skilled in the art thus recognize that the enzyme described by Hirano et al is not suitable for the catalysis of the reduction of DHCA in the 7 position to 3,12-diketo-7 ⁇ -CA.
  • the purpose of the invention is the provision of a novel method for the production of UDCA which avoids the aforesaid disadvantages.
  • a novel enzyme which catalyzes the stereospecific reduction of DHCA in the 7-position to 3,12-diketo-7 ⁇ -CA should be provided.
  • a further purpose consists in the provision of novel 7 ⁇ -HSDH enzymes which for example are usable in the preparation of UDCA, and in particular catalyze the stereo- and enantioselective oxidation/reduction of cholic acid derivatives in the 7-position.
  • a novel method for UDCA production is in particular provided, which can be represented schematically as follows:
  • UDCA can thus be synthesized from CA in just three steps.
  • the enzymatic reaction enables high conversion, displays high selectivity and does not form byproducts.
  • the recombinantly produced enzymes 7 ⁇ -HSDH and 3 ⁇ -HSDH surprisingly advantageously enable the large-scale production of UDCA.
  • FIG. 1 a shows the amino acid sequence of the 7 ⁇ -HSDH from Collinsella aerofaciens and FIG. 1 b the coding nucleic acid sequence for the amino acid sequence of FIG. 1 a ;
  • FIG. 1 c shows the amino acid sequence of the 3 ⁇ -HSDH from Comanomonas testosteroni and FIG. 1 d the coding nucleic acid sequence for the amino acid sequence of FIG. 1 c ;
  • FIG. 1 e shows the amino acid sequence of the 3 ⁇ -HSDH from Rattus norvegicus and FIG. 1 f the coding nucleic acid sequence for the amino acid sequence of FIG. 1 e.
  • FIG. 2 shows the SDS gel of a purified 7 ⁇ -HSDH prepared according to the invention, namely on track 1: crude cell extract, track 2: purified protein, and track M: Page RoulerTM, molecular weight marker (Fermentas, Germany).
  • FIG. 3 shows the sequence alignment of 7 ⁇ -HSDH from Collinsella aerofaciens DSM 3979 and selected HSDH proteins. conserveed residues in the sequences are highlighted in color.
  • sequence deposition numbers apply: 11 ⁇ -HSDH from Homo sapiens, GenBank NP — 005516; 11 ⁇ -HSDH from Mus — musculus , GenBank NP — 001038216; 11 ⁇ -HSDH from Cavia porcellus , GenBank AAS47491; 7 ⁇ -HSDH from Brucella melitensis, GenBank NP — 698608; 7 ⁇ -HSDH from Escherichia coli, GenBank NP — 288055; 7 ⁇ -HSDH from Clostridium sordellii , GenBank P50200; 3 ⁇ /20 ⁇ -HSDH from Streptomyces exfoliates , Swiss-Port P19992; 3 ⁇ /17 ⁇ -HSDH from Comamonas testosteroni GenBank AAA25742; 3 ⁇ -HSDH from
  • FIG. 4 shows the phylogenetic tree on the basis of an alignment of HSDH protein sequences and illustrates the relatedness between the selected HSDH proteins.
  • 7 ⁇ -hydroxysteroid dehydrogenase obtainable from an anaerobic bacterium, in particular of the genus Collinsella , such as the strain Collinsella aerofaciens DSM 3979 (ATCC 25986) and functional equivalents derived therefrom.
  • the 7 ⁇ -HSDH obtainable according to the invention from Collinsella aerofaciens DSM 3979 is characterized in particular by at least one more of the following properties, such as for example 2, 3, 4, 5, 6 or 7 or all such properties:
  • a 7 ⁇ -HSDH displays the following properties or combinations of properties; a); b); a) and b); a) and/or b) and c); a) and/or b) and c) and d); a) and/or b) and c) and d) and e); a) and/or b) and c) and d) and e) and f).
  • NAD(P)H is regenerated by coupling with an NAD(P)H-regenerating enzyme selected from an NAD(P)H dehydrogenase and in particular an alcohol dehydrogenase (ADH), in particular in situ.
  • an NAD(P)H-regenerating enzyme selected from an NAD(P)H dehydrogenase and in particular an alcohol dehydrogenase (ADH), in particular in situ.
  • NAD(P)H-regenerating enzyme is selected from natural or recombinant, isolated or enriched a) alcohol dehydrogenases (ADH; EC.1.1.1.2) and b) functional equivalents derived therefrom (in particular functional domains). 19.
  • R stands for alkyl, NR 1 R 2 , H. an alkali metal ion or N(R 3 ) 4 + , wherein the residues R 3 are the same or different and stand for H or alkyl,
  • DHCA in the presence of at least one 7 ⁇ -HSDH according to the definition in one of embodiments 1 to 3 is NADPH-dependently reduced to the 3,12-diketo-7 ⁇ -cholanic acid (3,12-diketo-7 ⁇ -CA) of the formula (4)
  • 3,12-diketo-7 ⁇ -CA in the presence of at least one 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSDH) is NADPH-dependently or NADH-dependently (depending on the type of 3 ⁇ -HSDH used) reduced to the corresponding 12-keto-ursodesoxycholic acid (12-keto-UDCA) of the formula (5)
  • step b) is coupled with a cofactor regeneration step, in particular in situ, in which NADPH is regenerated by alcohol dehydrogenase (ADH) with consumption of a sacrificial alcohol (in particular isopropanol and formation of acetone) and wherein optionally the removal of acetone from the reaction equilibrium is promoted (e.g. by raising the temperature).
  • ADH alcohol dehydrogenase
  • step c) is coupled with a cofactor regeneration step, in particular in situ, in which depending on the type of 3 ⁇ -HSDH used, NADH is regenerated by formate dehydrogenase (FDH) with consumption of formate (and formation of gaseous CO 2 ); or in which NADPH is regenerated by alcohol dehydrogenase (ADH) with consumption of a sacrificial alcohol (in particular isopropanol and formation of acetone) and wherein optionally the removal of acetone from the reaction equilibrium is promoted (e.g. by raising the temperature).
  • FDH formate dehydrogenase
  • ADH alcohol dehydrogenase
  • a sacrificial alcohol in particular isopropanol and formation of acetone
  • CA Cholic acid DHCA Dehydrocholic acid 3,12-diketo-7 ⁇ -CA 3,12-diketo-7 ⁇ -cholanic acid 12-keto-UDCA 12-keto-ursodeoxycholic acid UDCA
  • Ursodeoxycholic acid CA methyl ester Cholic acid methyl ester 3,7-diacetyl-CA methyl ester 3,7-diacetyl-cholic acid methyl ester* 12-keto-3,7- diacetyl-CA methyl ester 12-keto-3,7-diacetyl-cholanic acid methyl ester*
  • CDCA Chenodeoxycholic acid 7-keto-LCA 7-keto-lithocholic acid 7,12-diketo-LCA 7,12-diketo-lithocholic acid 12-keto-CDCA 12-keto-chenodeoxycholic acid
  • the term “711-HSDH” designates a dehydrogenase enzyme which catalyzes at least the stereospecific and/or nag reduction of DHCA to 3,12-diketo-7 ⁇ CA in particular with stoichiometric consumption of NADPH, and optionally the corresponding reverse reaction.
  • the enzyme can be a natural or recombinantly produced enzyme.
  • the enzyme in principle be present mixed with cellular, such as for example protein impurities, but preferably in pure form. Suitable detection methods are for example described in the experimental section below or known from the literature (e.g.
  • 3 ⁇ -HSDH designates a dehydrogenase enzyme which catalyzes at least the stereospecific and/or regiospecific reduction of 3,12-diketo-7 ⁇ CA to 12-keto-UDCA, in particular with stoichiometric consumption of NADH and/or NADPH, and optionally catalyzes the corresponding reverse reaction.
  • Suitable detection methods are for example described in the experimental section below or known from the literature.
  • Suitable enzymes are for example obtainable from Comanomonas testosteroni (e.g. ATCC11996).
  • An NADPH-dependent 3 ⁇ -HSDH is for example known from the rodents and is also usable.
  • a “pure form” or a “pure” or “essentially pure” enzyme is understood to mean an enzyme with a purity of more than 80, preferably more than 90, in particular more than 95, and above all more than 99 wt. %, based on the total protein content, determined by means of normal protein estimation methods, such as for example the biuret method or the protein estimation after Lowry et al. (see description in R. K. Scopes, Protein Purification, Springer Verlag, New York, Heidelberg, Berlin (1982)).
  • a “redox equivalent” is understood to mean a small molecule organic compound usable as an electron donor or electron acceptor, such as for example nicotinamide derivatives such as NAD + and NADH + or the reduced forms thereof NADH and NADPH respectively.
  • NAD(P) + here stands NAD + and/or NADP +
  • NAD(P)H here stands for NADH and/or NADPH.
  • a “cholic acid compound” is understood to mean compounds with the basic carbon skeleton, in particular the steroid structure of cholic acid and the presence of keto and/or hydroxy or acyloxy groups at ring position 7 and optionally the ring positions 3 and/or 12.
  • a compound of a specific type such as for example a “cholic acid compound” or an “ursodesoxycholic acid compound” is in particular also understood to mean derivatives of the underlying starting compound (such as for example cholic acid or ursodesoxycholic acid).
  • Such derivatives include “salts”, such as for example alkali metal salts such as lithium, sodium and potassium salts of the compounds; and ammonium salts, where an ammonium salt is understood to include the NH 4 + salt and those ammonium salts wherein at least one hydrogen atom can be replaced by a C 1 -C 6 alkyl residue.
  • Typical alkyl residues are in particular C 1 -C 4 alkyl residues, such as methyl, ethyl, n- or i-propyl-, n-, sec- or tert-butyl, and n-pentyl and n-hexyl and the singly or multiply branched analogs thereof.
  • Alkyl ester compounds according to the invention are in particular low alkyl esters, such as for example C 1 -C 6 alkyl esters.
  • low alkyl esters such as for example C 1 -C 6 alkyl esters.
  • methyl-, ethyl-, n- or i-propyl-, n-, sec- or tert-butyl esters, or longer chain esters, such as for example n-pentyl- and n-hexyl ester and the singly or multiply branched analogs thereof, can be named.
  • Amides are in particular conversion products of acids according to the invention with ammonia or primary or secondary monoamines.
  • Such amines are for example mono- or di-C 1 -C 6 alkyl monoamines, wherein the alkyl residues can mutually independently be optionally further substituted, for example by carboxy, hydroxy, halogen (such as F, Cl, Br or I), nitro and sulfonate groups.
  • “Acyl groups” are in particular non-aromatic groups with 2 to 4 carbon atoms, such as for example acetyl, propionyl and butyryl, and aromatic groups with an optionally substituted mononuclear aromatic ring, wherein suitable substituents are for example selected from hydroxy, halogen (such as F, Cl, Br or I), nitro- and C 1 -C 6 alkyl groups, such as for example benzoyl or toluoyl.
  • suitable substituents are for example selected from hydroxy, halogen (such as F, Cl, Br or I), nitro- and C 1 -C 6 alkyl groups, such as for example benzoyl or toluoyl.
  • hydroxysteroid compounds used or produced according to the invention such as for example cholic acid, ursodesoxycholic acid, 12-keto-chenodesoxycholic acid, chenodesoxy-cholic acid and 7-keto-lithocholic acid can be used in the method according to the invention in stereoisomerically pure pure form or mixed with other stereoisomers or obtained therefrom.
  • the compounds used or prepared are used or isolated in essentially stereoisomerically pure form.
  • an “immobilization” is understood to mean the covalent or non-covalent binding of a biocatalyst used according to the invention, such as for example an 7 ⁇ -HSDH to a solid, i.e. essentially insoluble in the surrounding liquid medium, support material.
  • the present invention is not limited to the specifically disclosed proteins or enzymes with 7 ⁇ -HSDH activity or 3 ⁇ -HSDH activity, but rather also extends to functional equivalents thereof.
  • “functional equivalents” or analogs of the specifically disclosed enzymes are polypeptides different therefrom, which moreover possess the desired biological activity, such as for example 7 ⁇ -HSDH activity.
  • “functional equivalents” is understood to mean enzymes which in the test for 7 ⁇ -HSDH activity used display an activity of an enzyme comprising an amino sequence defined herein higher or lower by at least 1%, such as for example at least 10% or 20%, such as for example at least 50% or 75% or 90%.
  • functional equivalents are preferably stable between pH 4 to 11 and advantageously have a pH optimum in a range from pH 4 to 6 or pH 6 to 10, such as for example 8.5 to 9.5, and a temperature optimum in the range from 15° C. to 80° C. or 15° C. to 40° C., 20° C. to 30° C. or 20° C. to 70° C., such as for example about 45 to 60° C. or about 50 to 55° C.
  • the 7 ⁇ -HSDH activity can be detected by means of various known tests. Without being limited thereto, a test with use of a reference substrate, such as for example CA or DHCA, under standardized conditions as defined in the experimental section, may be mentioned.
  • “functional equivalents” is understood also in particular to mean “mutants” which in at least one sequence position of the aforesaid amino acid sequences have an amino acid other than that specifically named but nonetheless have one of the aforesaid biological activities.
  • “functional equivalents” include the mutants obtainable by one or more, such as for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, amino acid additions, substitutions, deletions and/or inversions, where said changes can occur in any sequence position, so long as they lead to a mutant with the property profile according to the invention.
  • there is also functional equivalence when the reactivity pattern between mutants and unchanged polypeptide qualitatively coincide, i.e. for example the same substrates are converted at different rates. Examples of suitable amino acid substitutions are summarized in the following table:
  • precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
  • salts is understood to mean 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 produced in a manner known per se and comprise inorganic salts such as for example sodium, calcium, ammonium, iron and zinc salts and 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 also subjects of the invention.
  • “Functional derivatives” of polypeptides according to the invention can also be produced on functional amino acid side groups or on the N- or C-terminal ends thereof by known techniques.
  • Such derivatives for example comprise aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, produced by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups, produced by reaction with acyl groups.
  • “Functional equivalents” naturally also include polypeptides which are accessible from other organisms, and naturally occurring variants. For example, through sequence comparison zones of homologous sequence regions can be identified and equivalent enzymes determined on the basis of the specific stipulations of the invention.
  • “Functional equivalents” also include fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention, which for example display the desired biological function.
  • “functional equivalents” are fusion proteins which have one of the aforesaid 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 functional impairment of the fusion protein parts).
  • heterologous sequences are for example signal peptides, histidine anchors or enzymes.
  • homologs to the specifically disclosed proteins are also comprised with “functional equivalents”. These possess at least 60%, preferably at least 75% in particular at least 85%, such as for example 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the specifically disclosed amino acid sequences, calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448.
  • a percentage homology or identity of a homologous polypeptide according to the invention means in particular percentage identity of the amino acid residues based on the overall length of one of the amino acid sequences specifically described herein.
  • the percentage identity values can also be determined on the basis of BLAST alignments, algorithm blastp (protein-protein BLAST), or by application of the clustal adjustments stated below.
  • “functional equivalents” include proteins of the type indicated above in deglycosylated or glycosylated form and modified forms obtained by alteration of the glycosylation pattern.
  • Homologs of the proteins or polypeptides according to the invention can be created by mutagenesis, e.g. by point mutation, elongation or truncation of the protein.
  • Homologs of the proteins according to the invention can be identified by screening of combinatorial banks of mutants, such as for example truncation mutants.
  • a variegated bank of protein variants can be created by combinatorial mutagenesis at the nucleic acid level, such as for example by enzymatic ligation of a mixture of synthetic oligonucleotides.
  • degenerated gene set enables the provision of all sequences in a mixture which code for the desired set of potential proteins.
  • Methods for the synthesis of degenerated oligonucleotides are known to those skilled in the art (e.g. Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).
  • REM Recursive ensemble mutagenesis
  • nucleic acid sequences which code for an enzyme with 7 ⁇ -HSDH or 3 ⁇ -HSDH activity.
  • the present invention also relates to nucleic acids with a defined degree of identity to the specific sequences described herein.
  • Identity between two nucleic acids is understood to mean the identity of the nucleotides over the whole nucleic acid length in question, in particular the identity which is calculated by comparison by means of Vector NTI Suite 7.1 Software from Informax (USA) with use of the Clustal method (Higgins D G, Sharp P M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1) with adjustment of the following parameters:
  • nucleic acid sequences mentioned herein are producible in a manner known per se by chemical synthesis from the nucleotide building blocks, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can for example be effected in known manner by the phosphoamidite method (Voet. Voet, 2 nd Edition, Wiley Press New York, pages 896-897).
  • nucleic acid sequences single and double strand DNA- and RNA sequences, such as for example cDNA and mRNA
  • cDNA and mRNA single and double strand DNA- and RNA sequences, such as for example cDNA and mRNA
  • nucleic acid molecules according to the invention can also contain untranslated sequences from the 3′ and/or 5′ end of the coding gene region.
  • the invention further includes the nucleic acid molecules complementary to the specifically described nucleotide sequences or a section thereof.
  • nucleotide sequences according to the invention enable the creation of probes and primers which are usable for the identification and/or cloning of homologous sequences in other cell types and organisms.
  • probes or primers usually comprise a nucleotide sequence region which under “stringent” conditions (see below) hybridizes to 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 a corresponding antisense strand.
  • a nucleic acid molecule according to the invention can be isolated by means of standard molecular biology techniques and the sequence information provided according to the invention.
  • cDNA can be isolated from a suitable cDNA bank by using one of the specifically disclosed complete sequences or a section thereof as a hybridization probe and standard hybridization techniques (as for example described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2 nd Edn., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • nucleic acid molecule comprising one of the disclosed sequences or a section thereof can be isolated by a polymerase chain reaction wherein the oligonucleotide primers which were created on the basis of this sequence are used.
  • the nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • the oligonucleotides according to the invention can be produced by standard synthesis methods, e.g. with an automatic DNA synthesizer.
  • Nucleic acid sequences according to the invention or derivatives thereof, homologs or parts of these sequences can for example be isolated from other bacteria for example with usual hybridization methods or the PCR technique, e.g. via genomic or cDNA banks. These DNA sequences hybridize under standard conditions with the sequences according to the invention.
  • Hybridization is understood to mean the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under standard conditions, while under these conditions nonspecific bindings between non-complementary partners does not occur.
  • the sequences can be 90-100% complementary.
  • the property of complementary sequences of being able to bind specifically to one another is for example exploited in the Northern or Southern blot technique or in the primer binding in PCR or RT-PCR.
  • oligonucleotides of the conserved regions are advantageously used.
  • longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • These standard conditions vary depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or depending on what type of nucleic acid DNA or RNA are used for the hybridization.
  • the melting temperatures for DNA:DNA hybrids lie ca. 10° C. lower than those of DNA:RNA hybrids of the same length.
  • the hybridization conditions for DNA:DNA hybrids lie at 0.1 ⁇ SSC and temperatures between about 20° C. and 45° C., preferably between about 30° C. to 45° C.
  • the hybridization conditions advantageously lie at 0.1 ⁇ SSC and temperatures between about 30° C. and 55° C., preferably between about 45° C.
  • hybridization can in particular be effected under stringent conditions.
  • stringent conditions are for example described in Sambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • “Stringent” hybridization conditions are in particular understood to mean: incubation at 42° C. overnight in a solution consisting of 50% formamide, 5 ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt solution, 10% dextran sulfate and 20 g/ml denatured, sheared salmon sperm DNA, followed by a filter washing step with 0.1 ⁇ SSC at 65° C.
  • a subject of the invention are derivatives of the specifically disclosed or derivable nucleic acid sequences.
  • a subject of 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 natural variation. These natural variations usually cause a variance of 1 to 5% in the nucleotide sequence of a gene.
  • nucleic acid sequence according to the invention with the sequence SEQ ID No.: 1 should for example be understood to mean allelic variants which display at least 60% homology at the derived amino acid level, preferably at least 80% homology and quite especially preferably at least 90% homology over the whole sequence region (concerning homology at the amino acid level, reference may be made to the above explanations). Over part regions of the sequences, the homologies can advantageously be higher.
  • derivatives should also be understood to mean homologs of the nucleic acid sequences according to the invention, in particular of SEQ ID No.: 1, for example fungal or bacterial homologs, truncated sequences or single strand DNA or RNA of the coding and non-coding DNA sequence.
  • SEQ ID No.: 1 possess a homology of at least 40%, preferably of at least 60%, particularly preferably of at least 70%, quite especially preferably of at least 80% over the whole DNA region stated in SEQ ID No.: 1.
  • those skilled in the art can introduce random or also targeted mutations into genes or also non-coding nucleic acid regions (which for example are important for the regulation of expression) and then create gene banks.
  • the molecular biology methods necessary for this are known to those skilled in the art and for example described in Sambrook and Russell, Molecular Cloning. 3 rd Edition, Cold Spring Harbor Laboratory Press 2001.
  • directed evolution (described inter alia in Reetz M T and Jaeger K-E (1999), Topics Curr Chem 200:31; Zhao H. Moore J C, Volkov A A, Arnold F H (1999), Methods for optimizing industrial enzymes by directed evolution, In: Demain A L, Davies J E (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology) those skilled in the art can also create functional mutants in a directed manner and also on a large scale.
  • gene banks of the proteins in question are first created, for which for example the aforesaid methods can be utilized.
  • the gene banks are expressed in a suitable manner, for example by bacteria or by phage display systems.
  • the relevant genes of host organisms which express functional mutants with properties which largely correspond to the desired properties can be subjected to a further round of mutation.
  • the steps of mutation and selection or screening can be iteratively repeated until the present functional mutants display the desired properties to an adequate extent.
  • a limited number of mutations such as for example 1 to 5 mutations, can be performed stepwise and assessed and selected for their influence on the relevant enzyme property.
  • the selected mutant can then be subjected to a further mutation step in the same manner. The number of individual mutants to be tested can thereby be significantly reduced.
  • results according to the invention yield important information with regard to the structure and sequence of the enzymes concerned, which is necessary in order specifically to generate further enzymes with desired modified properties.
  • so-called “hot spots”, i.e. sequence sections which are potentially suitable for modifying an enzyme property via the introduction of targeted mutations, can be defined.
  • an “expression unit” is understood to mean a nucleic acid with expression activity which contains a promoter as defined herein and after functional linkage with a nucleic acid or a gene to be expressed regulates the expression, that is the transcription and the translation, of this nucleic acid or this gene. Therefore in this context reference is also made to a “regulative nucleic acid sequence”.
  • a promoter as defined herein and after functional linkage with a nucleic acid or a gene to be expressed regulates the expression, that is the transcription and the translation, of this nucleic acid or this gene. Therefore in this context reference is also made to a “regulative nucleic acid sequence”.
  • other regulative elements such as for example enhancers, can also be contained.
  • an “expression cassette” or “expression construct” is understood to mean an expression unit which is functionally linked with the nucleic acid to be expressed or the gene to be expressed.
  • an expression cassette thus includes not only nucleic acid sequences which regulate transcription and translation, but also the nucleic acid sequences which are to be expressed as protein as a result of the transcription and translation.
  • the terms “expression” or “overexpression” describe the production or increase of the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA.
  • a gene can be introduced into an organism, an existing gene be replaced by another gene, the copy number of the genes or the genes increased, a strong promoter used or a gene used which codes for a corresponding enzyme with a high activity and these measures can optionally be combined.
  • constructs according to the invention contain a promoter 5′ upstream from the respective coding sequence and a terminator sequence 3′ downstream, and optionally other normal regulative elements, also operatively linked to the coding sequence.
  • Nucleic acid constructs according to the invention comprise in particular sequence SEQ ID No.: 1 or derivatives and homologs thereof, and the nucleic acid sequences derivable therefrom which have advantageously been operatively or functionally linked with one or more regulation signals for controlling, e.g. increasing the gene expression.
  • regulating sequences are for example contained in the gram-positive promoters amy and SPO 2 , and in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28 and ADH. Artificial promoters can also be used for the regulation.
  • the nucleic acid construct is advantageously inserted into a vector, such as for example a plasmid or a phage which enables optimal expression of the genes in the host.
  • a vector such as for example a plasmid or a phage which enables optimal expression of the genes in the host.
  • vectors should also be understood to mean all other vectors known to those skilled in the art, for example viruses such as SV40. CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or chromosomally replicated. These vectors are a further embodiment of the invention.
  • Suitable plasmids are for example: in E. coli pET28a(+), pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-II 113 -B1, ⁇ gt11 or pBdCl, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, plL2 or pBB116, in yeasts 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23,
  • Said plasmids represent a small selection of the possible plasmids. Further plasmids are well known to those skilled in the art and can for example be taken from the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
  • an expression cassette according to the invention is effected by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or poly-adenylation signal.
  • standard recombination and cloning techniques as for example described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987) are used.
  • microorganism can be understood to mean the starting microorganism (wild type) or a genetically modified, recombinant microorganism or both.
  • recombinant microorganisms are producible, which for example are transformed with at least one vector according to the invention and can be used for the production of the polypeptides according to the invention.
  • the recombinant constructs according to the invention described above are introduced into a suitable host system and expressed.
  • common cloning and transfection methods such as for example coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like known to those skilled in the art are preferably used in order to bring said nucleic acids to expression in the expression system in question. Suitable systems are for example described in Current Protocols in Molecular Biology, F.
  • telomeres As recombinant host organisms for the nucleic acid or the nucleic acid construct according to the invention, in principle all prokaryotic or eukaryotic organisms are possible.
  • microorganisms such as bacteria, fungi or yeasts are used as host organisms.
  • gram-positive or gram-negative bacteria preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus are used.
  • the genus and species is Escherichia coli .
  • Further advantageous bacteria are moreover to be found in the group of the alpha protobacteria, beta protobacteria or gamma protobacteria.
  • the host organism or the host organisms according to the invention here preferably contain at least one of the nucleic acid sequences, nucleic acid constructs or vectors described in this invention which code for an enzyme with 7(3-HSDH activity according to the above definition.
  • the organisms used in the method according to the invention are grown or cultured in a manner known to those skilled in the art depending on the host organism.
  • Microorganisms are as a rule cultured in a liquid medium which contains 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 and magnesium salts and optionally vitamins, at temperatures between 0° C. and 100° C., preferably between 10° C. to 60° C. with oxygen aeration.
  • the pH of the nutrient liquid can be kept at a fixed value, that is be regulated during the culturing, or not.
  • the culturing can be effected “batch”-wise, “semi batch”-wise or continuously.
  • Nutrients can be provided at the start of the fermentation or fed in semicontinuously or continuously thereafter.
  • hydroxy groups of CA are oxidized to carbonyl group with chromic acid or chromates in acidic solution (e.g. H 2 SO 4 ) in a manner known per se by the classical chemical route. As a result, DHCA is formed.
  • acidic solution e.g. H 2 SO 4
  • DHCA is specifically reduced by 3 ⁇ -HSDH and 7 ⁇ -HSDH to 12-keto-UDCA in the presence of NADPH or NADH respectively.
  • the cofactor NADPH or NADH can be regenerated by an ADH or FDH from isopropanol or sodium formate respectively.
  • the 12-carbonyl group of 12-keto-UDCA is removed by Wolff-Kishner reduction in a manner known per se, and thereby UDCA is formed from 12-keto-UDCA.
  • the carbonyl group is firstly converted to the hydrazone with hydrazine.
  • the hydrazone is heated to 200° C. in the presence of a base (e.g. KOH), and thereby nitrogen is cleaved off and UDCA is formed.
  • a base e.g. KOH
  • a subject of the invention are methods for the recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, if necessary expression of the polypeptide is induced and this is isolated from the culture.
  • the polypeptides can thus also be produced on a large industrial scale, if this is desired.
  • the culture medium to be used must appropriately satisfy the needs of the strains in question. Descriptions of culture media of various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” from the American Society for Bacteriology (Washington D.C., USA, 1981).
  • These media usable according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Preferred carbon sources are sugars such as mono-, di- or polysaccharides. Very good carbon sources are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, saccharose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other byproducts of sugar refining. It can also be advantageous to add mixtures of different carbon sources.
  • oils and fats such as for example soya oil, sunflower oil, peanut oil and coconut fat, fatty acids such as for example palmitic acid, stearic acid or linolic acid, alcohols such as for example glycerin, methanol or ethanol and organic acids such as for example acetic acid or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials which contain these compounds.
  • nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soya meal, soya protein, yeast extract, meat extract, and others.
  • the nitrogen sources can be used singly or as a mixture.
  • Inorganic salt compounds which can be contained in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • inorganic sulfur-containing compounds such as for example sulfates, sulfites, dithionites, tetrathionates, thiosulfates and sulfides, but also organic sulfur compounds such as mercaptans and thiols, can be used.
  • phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used.
  • Chelating agents can be added to the medium in order to hold the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols such as catechol or proto-catechuate, or organic acids such as citric acid.
  • the fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters including for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
  • growth factors and salts often derive from complex media components such as yeast extract, molasses, corn steep liquor and the like. Apart from this, suitable precursors can be added to the culture medium.
  • suitable precursors can be added to the culture medium.
  • the exact composition of the media compounds depends strongly on the particular experiment and is decided individually for each specific case. Information about media optimization is obtainable from the textbook “Applied Microbiol. Physiology, A Practical Approach” (Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) S. 53-73, ISBN 0 19 963577 3).
  • Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
  • All media components are sterilized, either by heat (20 mins at 1.5 bar and 121° C.) or by sterile filtration.
  • the components can either be sterilized together or if necessary separately. All media components can be present at the start of the culturing or optionally be added continuously or in batches.
  • the culture temperature normally lies between 15° C. and 45° C., preferably at 25° C. to 40° C. and can be kept constant or varied during the experiment.
  • the pH of the medium should lie in the range from 5 to 8.5, preferably around 7.0.
  • the pH for the culturing can be controlled during the culturing by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid.
  • antifoamants such as for example fatty acid esters can be used.
  • suitable selectively acting substances such as for example antibiotics, can be added to the medium.
  • oxygen or oxygen-containing gas mixtures such as for example ambient air
  • the culture temperature normally lies at 20° C. to 45° C.
  • the culturing is continued until a maximum of the desired product has formed. This target is normally reached within 10 hours to 160 hours.
  • the fermentation broth is then further processed.
  • the biomass can be wholly or partly removed from the fermentation broth by separation methods, such as for example centrifugation, filtration, decantation or a combination of these methods, or be entirely left therein.
  • the cells can also, if the polypeptides are not secreted into the culture medium, be disintegrated and the product recovered from the lysate by known protein isolation methods.
  • the cells can optionally be disintegrated by high frequency ultrasound, by high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by combination of several of the stated methods.
  • a purification of the polypeptides can be achieved by known chromatographic methods such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other usual methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis.
  • chromatographic methods such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other usual methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis.
  • Suitable methods are for example described in Cooper, F. G., Biochemical Working Methods, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
  • vector systems or oligonucleotides which lengthen the cDNA by certain nucleotide sequences and hence code for modified polypeptides or fusion proteins which for example serve for simpler purification.
  • suitable modifications are for example so-called “tags” functioning as anchors, such as for example the modification known as the hexa-histidine anchor or epitopes which can be recognized as antigens by 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 the attachment of the proteins onto a solid support, such as for example a polymer matrix, which can for example be filled into a chromatography column, or can be used on a microtiter plate or on another support.
  • these anchors can also be used for recognition of the proteins.
  • normal markers such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors for derivatization of the proteins can be used.
  • the enzymes according to the invention can be used free or immobilized in the methods described herein.
  • An immobilized enzyme is understood to mean an enzyme which is fixed onto an inert support. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and from the literature references cited therein. In this regard, reference is made to the disclosure of these publications in its entirety.
  • suitable support materials are for example clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, and synthetic polymers such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene.
  • the support materials are normally used for the production of the supported enzymes in a finely divided, particulate form, with porous forms being preferable.
  • the particle size of the support material is usually not more than 5 mm, in particular not more than 2 mm (size distribution curve).
  • a free or immobilized form can be selected.
  • support materials are Ca alginate, and carrageenan.
  • Enzymes, like cells, can also be directly crosslinked with glutaraldehyde (crosslinking to CLEAs). Similar and other immobilization methods are for example described in J. Lalonde and A. Margolin “Immobilization of Enzymes” and in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim.
  • the cloning steps performed in the context of the present invention such as for example restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids onto nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of microorganisms, culturing of microorganisms, proliferation of phages and sequence analysis of recombinant DNA can be performed as described in Sambrook et al. (1989) loc. cit.
  • the genomic DNA from Collinsella aerofaciens DSM 3979 was obtained from the German Collection for Microorganisms and Cell Cultures (DSMZ).
  • DSMZ German Collection for Microorganisms and Cell Cultures
  • UDCA and 7-keto-LCA are starting compounds known per se and described in the literature. All other chemicals were obtained from Sigma-Aldrich and Fluka (Germany). All restriction endonucleases, the T4 DNA ligase, the Tag DNA polymerase and isopropyl*D-1-thiogalactopyranoside (IPTG) were obtained from Fermentas (Germany).
  • the Escherichia. coli strain DH5 ⁇ (Novagen, Madison, Wis., USA) was proliferated at 37° C. in LB medium containing suitable antibiotics.
  • the reaction mixture contains a total volume of 1 ml:
  • the increase in the extinction at 340 nm is measured and the activity is calculated as enzyme unit (U, i.e. ⁇ mol/min) using the molar extinction coefficient of 6.22 ml ⁇ 1 ⁇ cm ⁇ 1 .
  • the samples were mixed with BCA reagent (from Interchim) and incubated at 37° C. for 45 mins.
  • the protein content was determined at 562 nm against a calibration curve (BSA) in the concentration range of the assay used.
  • 7 ⁇ -HSDH coding sequences were PCR-amplified.
  • the PCR products were obtained using the genomic DNA of Collinsella aerofaciens ATCC 25986 (DSM 3979) as template and the primers 5′-gggaattc CATATG AACCTGAGGGAGAAGTA-3′ (SEQ ID No.:3) and 5′-ccc AAGCTT CTAGTCGCGGTAGAACGA-3′ (SEQ ID No.:4).
  • the NdeI and HindIII cleavage sites in the primer sequences are underlined.
  • the PCR product was purified with PCR Purification Kit (Qiagen) and then cleaved with the enzymes NdeI and HindIII.
  • the relevant vector was also cleaved with the NdeI and The products were applied on to an agarose gel, separated, cut out from this and purified.
  • the cleaved PCR product and the cleaved vector were ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5 ⁇ .
  • the resulting vector (contains the gene of 7 ⁇ -HSDH) was confirmed by sequencing and transformed into E. coli BL21(DE3) and induced with IPTG and expressed.
  • the expression was performed in 50 ml of LB medium.
  • For the preparation of preculture one colony on an LB agar plate was picked and incubated overnight at 37° C. and 160 Rpm in 5 ml of LB medium (contains relevant antibiotics) a.
  • the 50 ml of LB medium (contains relevant antibiotics) was inoculated with 500 ⁇ l of preculture.
  • the culture was incubated at 37° C. and 160 Rpm. Up to OD600 ca. 0.8, expression was induced by addition of 0.5 mM IPTG.
  • the 7 ⁇ -HSDH displayed the activity 60 U/ml against UDCA, activity 35 U/ml against 7-keto-LCA and activity 119 U/ml against DHCA in the presence of NADP + or NADPH.
  • the activity against CA is not detectable.
  • the gene which codes for the 7 ⁇ -HSDH was recloned in pET28a+ with His-Tag, in order to enable rapid purification.
  • This 7 ⁇ -HSDH with His-Tag was actively expressed in E. coli BL21(DE3) as was described above.
  • the purification was performed with a Talon column. The column was first equilibrated with potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl). After the cell lysate had been loaded, the column was washed with potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl). The 7 ⁇ -HSDH was eluted with potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl and 200 mM imidazole). The imidazole in the eluate was removed by dialysis. The purification yield was 76% with purity ca. 90%.
  • the 20 ml conversion mixture contains 50 mM 7-keto-LCA (ca. 0.4 g), 5 U/ml 7 ⁇ -HSDH and 0.05 mM NADP + .
  • 4 U/ml ADH and 1% isopropanol was used (see Scheme 1). The reaction was performed in the fume cupboard at pH 8 and 24° C. with stirring. Since acetone evaporates faster than isopropanol, the reaction is shifted towards formation of UDCA. 1% isopropanol was again added after 24 hrs, 48 hrs and 72 hrs.
  • the product was analyzed by TLC (Kieselgel 60, Merck, mobile phase petroleum ether and ethyl acetate 1:10, vol:vol). On TLC the product was compared with authentic references 7-keto-LCA. UDCA and CDCA. The TLC analysis shows that UDCA was formed from 7-keto-LCA by the 7 ⁇ -HSDH. The enantiomer COCA is not detectable on TLC.
  • the 50 ml conversion mixture contains 50 mM DHCA (1 g), 5 U/ml 7 ⁇ -1-ISDH and 0.05 mM NADP + .
  • 4 U/ml ADH and 1% isopropanol was used (see Scheme 2). The reaction was performed in the fume cupboard at pH 8 and 24° C. with stirring. Since acetone evaporates faster than isopropanol, the reaction is shifted towards formation of 3,12-diketo-7 ⁇ -CA.
  • the intermediate 3,12-diketo-7-CA (prepared according to conversion example 2) was converted further to 12-keto-UDCA by a 3 ⁇ -HSDH (SEQ ID Nos.:5 and 6) from Comamonas testosteroni (Mobus, E. and E. Maser, Molecular cloning, overexpression, and characterization of steroid - inducible 3 alpha - hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. A novel member of the short - chain dehydrogenase/reductase superfamily . J Biol Chem, 1998. 273(47): p. 30888-96).
  • This 3 ⁇ -HSDH requires cofactor NADH, which was regenerated by the FDH (see FIG. 3 ).
  • 4 U/ml 3 ⁇ -HSDH, 1 U/ml FDH(NADH-dependent, Codexis), 200 mM sodium formate and 0.05 mM NAD + were added to the reaction.
  • the product was acidified to pH 2 with 2M HCl and extracted with 6 times 10 ml ethyl acetate. After evaporation, 1.07 g of product were obtained.
  • the product 12-keto-UDCA was analyzed and confirmed by TLC and NMR.
  • the 3alpha-HSDH was produced analogously to the production of 7 ⁇ -HSDH, but with the plasmid pET22b+, and used without further purification.
  • the PCR product was once again purified as described above and digested with the restriction endonucleases NdeI and HindIII.
  • the digested PCR product was once again purified and cloned into the pET-28a(+) vector using the T4 ligase, in order to create an expression vector.
  • the resulting expression construct was than transformed into E. coli DH5 ⁇ cells.
  • the protein to be expected should have 20 amino acid residues comprising a signal peptide and an N-terminal 6 ⁇ His-Tag and a thrombin cleavage site. The sequence of the inserted DNA was checked by sequencing.
  • E. coli BL21(DE3) was transformed with the expression construct.
  • the E. coli BL21(DE3) strain containing the expression construct was proliferated in LB medium (2 ⁇ 400 ml in 2 liter shaker bottles) containing 30 ⁇ g/ml kanamycin.
  • the cells were harvested by centrifugation (10,000 ⁇ g, 15 mins, 4° C.).
  • the pellet was resuspended in 20 ml of phosphate buffer (50 mM, pH 8, containing 0.1 mM PMSF).
  • the cells were disintegrated with constant cooling by one-minute ultrasound treatment (40 W power, 40% interval and 1 min pause) with the use of a Sonifier 250 ultrasound device (Branson, Germany). The disintegration was repeated three times.
  • the cell extract was centrifuged (22,000 ⁇ g, 20 mins, 4° C.). The supernatant was loaded onto a Talon column (Clontech. USA) equilibrated with loading buffer (50 mM potassium phosphate, 300 mM NaCl, pH 8). The process was carried out at 24° C. Non-bound material was washed out by washing of the column with loading buffer (3 column volumes). Weakly binding protein was removed by washing with washing buffer (20 mM imidazole in the loading buffer; 3 column volumes). The His-Tag-7) 3 —HSDH protein was eluted with elution buffer (200 mM imidazole in the loading buffer). The eluate was dialyzed overnight at 4° C.
  • loading buffer 50 mM potassium phosphate, 300 mM NaCl, pH 8. The process was carried out at 24° C. Non-bound material was washed out by washing of the column with loading buffer (3 column volumes). Weakly binding protein was removed by washing with washing buffer (20 mM
  • the molecular weight of 7 ⁇ -HSDH was determined by comparison of its elution volume with that of protein standards (serum albumin (66 kDa), ⁇ -amylase from Aspergillus otyzae (52 kDa), trypsin from pig pancreas (24 kDa) and lysozyme from chicken egg (14.4 kDa)).
  • protein standards serum albumin (66 kDa), ⁇ -amylase from Aspergillus otyzae (52 kDa), trypsin from pig pancreas (24 kDa) and lysozyme from chicken egg (14.4 kDa)).
  • the conversion of 7-keto-LCA by 7 ⁇ -HSDH was performed in order to verify the biochemical function of 7 ⁇ -HSDH.
  • 0.4 g of 7-keto-LCA were suspended in 10 ml of potassium phosphate buffer (50 mM, pH 8) and the pH was adjusted to pH 8 by addition of 2M sodium hydroxide.
  • 0.2 ml of isopropanol, 100 U of 7 ⁇ -HSDH and 80 U of alcohol dehydrogenase (ADH-TE) from Thermoanaerobacter ethanolicus (kindly donated by Dr. K. Momoi, ITB University, Stuttgart) and 1 ⁇ mol NADP + were added.
  • the same buffer was added, so as to obtain a total reaction volume of 20 ml.
  • a fusion protein provided with a His-Tag at the N-terminus was obtained with a 78-HSDH yield of 332.5 mg (5828 U) per liter of culture.
  • the 7 ⁇ -HSDH provided with the His-Tag was purified in one step by means of one immobilized metal ions affinity chromatography (purity >90%, yield 76%, see FIG. 2 .).
  • the main bands of tracks 1 and 2 represent the expected expression product at 30 kDa, which corresponds to the predicted molecular weight derived from the amino acid sequence of the gene. However, by gel filtration a molecular weight of 56.1 kDa is determined for the 7 ⁇ -HSDH. This confirms the dimeric nature of the 7 ⁇ -HSDH from Collinsella aerofaciens DSM 3979.
  • the amino acid sequence of the 7 ⁇ -HSDH according to the invention was compared with known HSDH sequences (see FIG. 3 ).
  • the sequence similarity observed indicates that the enzyme according to the invention belongs to the family of the short-chain dehydrogenases (SDR).
  • SDRs display very low homology and sequence identity (Jornvall. H., B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J. Jeffery, and D. Ghosh. 1995. Short-chain dehydrogenases/reductases (SDR). Biochemistry 34:6003-13 and Persson, B., M. Krook, and H. Jornvall. 1991. Characteristics of short-chain alcohol dehydrogenases and related enzymes.
  • FIG. 4 The evolutionary tree based on the alignment of FIG. 3 is shown in FIG. 4 .
  • 7 ⁇ -HSDH from Clostridium sordellii, Brucella melitensis and Escherichia coli belong to the same subgroup. Both 3 ⁇ -HSDHs shows a more marked relationship than other HSDHs.
  • the prokaryotic 7 ⁇ -HSDH is related to the animal 11 ⁇ -HSDH subgroup, comprising Cavia porcellus, Homo sapiens and Mus musulus.
  • the 7 ⁇ -HSDH activity for various substrates as a function of pH was determined with purified enzyme (see FIG. 5 ).
  • optimal activity was observed in the range from pH 9 to 10 with a gradual decline on the acidic side.
  • optimal activity was found in the range from pH 4 to 6 with a sharp fall on the acidic side and a gradual decline on the alkaline side.
  • Different buffers have only a slight influence on the activity of the 7 ⁇ -HSDH at the same pH.
  • the NADP-dependent 7 ⁇ -HSDH according to the invention displays the following stability behavior: after 400 mins, the activity at 30° C. was about 30% lower than at 23° C. The enzyme was completely inactivated at 30° C. after 1500 mins, while at 23° C. and 1500 mins the remaining activity was 20%. No significant activity loss was observed during the storage at ⁇ 20° C. in potassium phosphate buffer (50 mM, pH 8) over a period of a few months after multiple freezing and thawing.

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US13/512,166 2009-11-30 2010-11-30 NOVEL 7beta-HYDROXYSTEROID DEHYDROGENASES AND THEIR USE Abandoned US20130040341A1 (en)

Applications Claiming Priority (5)

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EP09177544A EP2327790A1 (de) 2009-11-30 2009-11-30 Neuartige 7ß-Hydroxysteroid Dehydrogenasen und deren Verwendung
EP09177544.5 2009-11-30
EP10008837 2010-08-25
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