WO2019209105A2 - 7β-HYDROXYSTÉROÏDE DÉSHYDROGÉNASE DÉPENDANTE DE NAD+ - Google Patents

7β-HYDROXYSTÉROÏDE DÉSHYDROGÉNASE DÉPENDANTE DE NAD+ Download PDF

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WO2019209105A2
WO2019209105A2 PCT/NL2019/050228 NL2019050228W WO2019209105A2 WO 2019209105 A2 WO2019209105 A2 WO 2019209105A2 NL 2019050228 W NL2019050228 W NL 2019050228W WO 2019209105 A2 WO2019209105 A2 WO 2019209105A2
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seq
hydroxysteroid dehydrogenase
amino acid
position corresponding
nucleic acid
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PCT/NL2019/050228
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WO2019209105A3 (fr
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Fabio TONIN
Isabella Wilhelmina Christina Everdina Arends
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Technische Universiteit Delft
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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
    • 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 present disclosure relates to the field of 7b- hydroxysteroid dehydrogenases and to methods of providing a 7b- hydroxysteroid dehydrogenase with a co-substrate specificity of NAD+ instead of NADP+.
  • the disclosure further relates to methods for converting cholic acid (CA) and/or chenodeoxycholic acid (CDCA) into ursocholic acid (UCA) and/or
  • ursodeoxycholic acid respectively, and more specifically methods for converting 7- oxo-deoxycholic acid (7-oxo-DCA) and/or 7-oxo-lithocholic acid (7-oxo LCA) into ursocholic acid (UCA) and/or ursodeoxycholic acid (UDCA) respectively, by using an NAD+ dependent 7b- hydroxysteroid dehydrogenase.
  • Ursodeoxycholic acid is a bile acid which solubilizes cholesterol gallstones and can improve liver function in cholestatic diseases.
  • ursodeoxycholic acid (UDCA) is obtained by a multistep chemical synthesis starting from cholic acid (CA). Two main steps are involved: the dehydroxylation at C-12 and the epimerization of the 7-OH group.
  • CA cholic acid
  • This whole sequence comprises 5 steps: after the protection of the carboxylic group by acid catalyzed esterification (quantitative yield), the 3- and 7-OH groups are protected selectively with acetic anhydride and pyridine (yield 92%).
  • the 12-OH group is oxidized with Cr0 3 (yield 98%) and, after a deprotection step in alkaline environment, the formed ketone group can be removed by a Wolff-Kishner reaction yielding CDCA (yield 82%).
  • the overall yield of the dehydroxylation step is around 65%.
  • the second step of UDCA synthesis from CA is the epimerization of the 7-OH group.
  • the 7a-OH group of CDCA obtained by dehydroxylation of CA (see above“C-12 dehydroxylation”), is selectively oxidized in the presence of sodium bromate (yield 88%), N- Bromosuccinimide (ungiven yield) or 1 -hydroxy-1 , 2-benziodoxol-3(1 H)-one 1-oxide (yield 90%) and subsequently reduced with metallic sodium in presence of imidazole and 1- propanol (yield 80%) yielding the 7b-OH epimer (UDCA) as imidazole salt.
  • the overall yield of the epimerization step is around 70%.
  • the packed-bed flow-system reactor set up by Zheng et al.
  • This particular flow-system consists of two modular column reactors: firstly, CDCA is oxidized to 7-oxo-LCA by an immobilized NAD + dependent 7a- hydroxysteroid dehydrogenase (first reactor column); afterwards, 7-oxo-LCA is reduced to UDCA by an immobilized NADP + dependent 7b- hydroxysteroid dehydrogenase (second reactor column).
  • the cofactors are individually regenerated in each column by
  • LDH lactate dehydrogenase
  • GDH glucose dehydrogenase
  • the decoupling of the 2 reactions is an elegant way to spin the equilibrium but, in every catalytic cycle, the co-substrates used to regenerate the cofactor have to be added in great surplus, leading to additional costs and additional problems in the downstream process.
  • the most used enzymes for the cofactor regeneration are glucose dehydrogenase (glucose to glucuronic acid), lactate dehydrogenase (pyruvate to lactate), glutamate dehydrogenase (a- ketoglutarate to glutamate) and formate dehydrogenase (formate to CO2).
  • the present disclosure provides for a NAD+ dependent 7b- hydroxysteroid dehydrogenase which can be used for catalyzing a conversion of any 7-oxosteroid into any 7b- hydroxysteroid, characterized in that the 7b- hydroxysteroid dehydrogenase comprises - an Alanine at a position corresponding to position 17 as shown in SEQ ID NO:1 or at a position corresponding to position 18 as shown in SEQ ID NO:5; and
  • the NAD+ dependent 7b- hydroxysteroid dehydrogenase can for example be employed in a redox-neutral biocascade for the synthesis of UDCA with higher yield than obtained in the prior art. It was found that specifically providing an Alanine and an Aspartic acid at the recited positions determines co-factor specificity, and provides a 7b- hydroxysteroid dehydrogenase which is not dependent from NADP+ as co-factor, but depends on NAD+ as co-factor.
  • 7a- hydroxysteroid dehydrogenase refers to an enzyme catalyzing a conversion of any 7a-hydroxysteroid into any 7-oxosteroid.
  • the term preferably refers to a 7a- hydroxysteroid dehydrogenase from Stenotrophomonas maltophilia and/or a 7a- hydroxysteroid dehydrogenase having at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO: 7 and/or is encoded by a nucleic acid having at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO: 8.
  • the enzyme does not require NADP+ for the recited conversion, and/or is preferably dependent on NAD+ to perform the recited conversion.
  • 7b- hydroxysteroid dehydrogenase refers to an enzyme catalyzing a conversion of any 7-oxosteroid into any 7b-hydroxysteroid, and/or vice versa.
  • the term preferably refers to a 7b- hydroxysteroid dehydrogenase from Lactobacillus spicheri or Clostridium sardiniense and/or a 7b- hydroxysteroid
  • dehydrogenase having at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5 and/or is encoded by a nucleic acid having at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO:6.
  • the enzyme does not require NADP+ for the recited conversion, and/or is preferably dependent on NAD+ to perform the recited conversion.
  • alcohol dehydrogenase refers to an enzyme catalyzing a conversion of an aldehyde and/or a ketone into an alcohol, and/or vice versa.
  • the alcohol dehydrogenase preferably has at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:5 and/or is encoded by a nucleic acid having at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO:6.
  • the enzyme does not require NADP+ for the recited conversion, and/or is preferably dependent on NAD+ to perform the recited conversion.
  • nucleic acid refers to a DNA or RNA molecule in single or double stranded form.
  • the nucleic acid may be an isolated nucleic acid, which refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the isolated nucleic acid no longer comprises the nucleic acid sequence naturally flanking the nucleic acid in the natural environment, such as less than 100, 50, 25 or 10 nucleic acids (nucleotides) of the nucleic acid sequence naturally flanking the nucleic acid is present in the isolated nucleic acid.
  • the isolated nucleic acid is now in a bacterial host cell or in the plant nuclear or plastid genome, or the isolated nucleic acid is chemically synthesized.
  • the term “gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into a RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3’non-translated sequence comprising e.g. transcription termination sites.
  • a gene may also include introns, which are, for example spliced out before translation into protein. It is further understood that, when referring to“sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. nucleotides or amino acids) are referred to.
  • A“nucleic acid construct” or“vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell.
  • the vector backbone may for example be a binary or superbinary vector (see e.g. US 5591616, US 2002138879 and WO95/06722), a co integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence.
  • Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like.
  • protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin.
  • the protein or polypeptide may be an isolated protein, i.e. a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • sequence identity and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using alignment algorithms (when optimally aligned by for example the programs GAP or BESTFIT using default parameters).
  • GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimises the number of gaps.
  • the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).
  • Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program “needle”).
  • percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc.
  • nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence
  • nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference polypeptide sequence.
  • nucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: 1 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 1.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. Sequence identity can be determined over the entire length of the sequence(s) to be considered.
  • the present disclosure provides for a nucleic acid encoding an NAD+ dependent alcohol dehydrogenase, preferably an NAD+ dependent 7b- hydroxysteroid dehydrogenase, characterized in that the 7b- hydroxysteroid dehydrogenase comprises:
  • the above-mentioned amino acid(s) at the recited position(s) determine cofactor recognition, and provide for an NAD+ dependent alcohol dehydrogenase, preferably an NAD+ dependent 7b- hydroxysteroid dehydrogenase, and can change cofactor specificity from NADP+ to NAD+.
  • the advantage is that the enzyme no longer requires NADP+ to perform its enzymatic activity, and can be used in a redox neutral enzymatic cascade, e.g. together with an NAD+ dependent 7a- hydroxysteroid
  • dehydrogenase for conversion of various compounds not limited to the specific examples mentioned herein.
  • the binding mode of the co-substrate NAD+ may work through interaction of NAD+ with the Alanine and/or Aspartic acid at the recited positions.
  • the side chain of the added aspartic acid (D39) may form a hydrogen bond with the 2 ⁇ H group of ribose, and, in addition, the Alanine at position 17 (A17) may avoid interaction between the side chains (e.g. the threonine hydroxyl group interferes with the D39 side chain).
  • Other amino acid(s) at the recited positions may interfere with binding of NAD+ and instead determine NADP+ specificity.
  • NAD+ co-substrate specificity of an enzyme can be easily confirmed by the skilled person if enzymatic activity in the presence of NAD+ is higher than enzymatic activity in the absence of NAD+, and/or the enzymatic activity does not depend on the presence of NADP+.
  • nucleic acid according to the present disclosure may encode an NAD+ dependent alcohol dehydrogenase, preferably an NAD+ dependent 7b- hydroxysteroid dehydrogenase comprising:
  • Said amino acid(s) at the recited position(s) can further increase specificity of the enzyme to NAD+.
  • nucleic acid according to the present disclosure may encode an NAD+ dependent alcohol dehydrogenase, preferably an NAD+ dependent 7b- hydroxysteroid dehydrogenase comprising:
  • dehydrogenases genes in accordance with the data of the present sequence listings.
  • the present disclosure also provides for a nucleic acid encoding an alcohol dehydrogenase, preferably a 7b- hydroxysteroid dehydrogenase, characterized in that the nucleic acid comprises:
  • nucleic acid encoding an alcohol dehydrogenase may comprise:
  • nucleic acid encoding an alcohol dehydrogenase preferably a 7b- hydroxysteroid dehydrogenase, it is preferred that:
  • the amino acid that is changed at position 17 is Threonine (T) and/or wherein the amino acid at position 17 is changed into Alanine (A);
  • the amino acid that is changed at position 39 is Glycine (G) and/or wherein the amino acid at position 39 is changed into Aspartic acid (D);
  • the amino acid that is changed at position 40 is Arginine (R) and/or wherein the amino acid at position 40 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine (A, D, E, F, I, K, L, M, N, S, T, V, or Y);
  • the amino acid that is changed at position 41 is Arginine (R) and/or wherein the amino acid at position 41 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine (A, D, E, F, I, K, L, M, N, S, T, V, or Y); and/or
  • the amino acid that is changed at position 18 is Glutamic acid (E) and/or wherein the amino acid at position 18 is changed into Aspartic acid (D).
  • a mutation may be introduced in a nucleotide sequence encoding alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase as defined herein by the application of mutagenic compounds, such as ethyl methanesulfonate (EMS) or other compounds capable of (randomly) introducing mutations in nucleotide sequences.
  • mutagenic compounds such as ethyl methanesulfonate (EMS) or other compounds capable of (randomly) introducing mutations in nucleotide sequences.
  • Said mutagenic compounds or said other compound may be used as a means for creating cells harboring a mutation in a nucleotide sequence encoding an alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase.
  • Cell(s) harboring a mutation according to the disclosure may then be selected by means of sequencing.
  • introducing a mutation in a nucleotide sequence encoding an alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase according to the present disclosure is introduced by genome engineering techniques, such as techniques based on homologous recombination, or oligo-directed mutagenesis (ODM), for instance as described in W02007073170; W02007073149; W02009002150; and W02007073166).
  • ODM oligo-directed mutagenesis
  • Providing a mutation that corresponds to a change of an amino acid according to the present disclosure may be performed by Targeted Nucleotide Exchange (TNE), i.e. by introduction of at least one oligonucleotide capable of hybridizing to the nucleotide sequence encoding an alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase and comprising a mismatch with respect to said nucleotide sequence, wherein the position of the mismatch corresponds to the position of a mutation that corresponds to a change of amino acid(s) at a position according to the present disclosure, to a nucleotide sequence that encodes an alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase.
  • TNE Targeted Nucleotide Exchange
  • oligonucleotide can hybridize (basepair) with the complementary sequence of the nucleotide sequence to be altered (i.e. the target locus in the nucleotide sequence that encodes the respective enzyme).
  • the mismatch may impart a nucleotide conversion at the corresponding position in the target nucleotide sequence. This may result in the provision of a mutation that corresponds to a change of amino acid(s) at position(s) according to the present disclosure.
  • the oligonucleotide may have a length of between 10-500 nucleotides, preferably 15-250 nucleotides.
  • the TNE method is described e.g. in patent publications W02007073166, W02007073170, W02009002150.
  • the alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase and nucleotide sequence encoding said can be synthetically produced, or commercially obtained, e.g. form BaseClear B.V. (Baseclear B.V., Leiden, The Netherlands).
  • the nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to the present disclosure is capable of catalyzing a conversion of 7-oxosteroid into 7b- hydroxysteroid.
  • the nucleic acid encoding an alcohol dehydrogenase according to the present disclosure is capable of catalyzing a conversion of conversion of an aldehyde and/or a ketone into an alcohol. This means that the conversion of an aldehyde and/or a ketone into an alcohol, under suitable conditions as understood by the skilled person, can be more efficient or effective in the presence of the alcohol dehydrogenase according to the present disclosure.
  • the nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to the present disclosure preferably has
  • nucleic acid is (derived) from Clostridium sardiniense ;
  • nucleic acid is (derived) from Lactobacillus spicheri.
  • nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to the present disclosure preferably has
  • nucleic acid is (derived) from Clostridium sardiniense ⁇ , and/or
  • nucleic acid encoding a 7b- hydroxysteroid dehydrogenase is not from Lactobacillus spicheri and/or does not comprise SEQ ID NO:6 in its entire length.
  • the present disclosure also provides for a vector comprising a nucleic acid according to the present disclosure.
  • Said vector may be used to transfer a nucleotide according to the disclosure into another cell such as a host cell.
  • Different types of vectors include plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes.
  • the present disclosure also provides a host comprising a nucleic acid according to the present disclosure, or the vector as mentioned above, preferably wherein the host is Escherichia coti.
  • the present disclosure particularly provides for an alcohol dehydrogenase, preferably a 7b- hydroxysteroid dehydrogenase, characterized in that the alcohol dehydrogenase, preferably the 7b- hydroxysteroid dehydrogenase comprises - an Alanine at a position corresponding to position 17 as shown in SEQ ID NO:3 or at a position corresponding to position 18 as shown in SEQ ID NO:5; and/or
  • the alcohol dehydrogenase preferably the 7b- hydroxysteroid dehydrogenase comprises
  • the 7b- hydroxysteroid dehydrogenase according to the present disclosure is capable of catalyzing a conversion of 7-oxosteroid into 7b-hydroxysteroid.
  • the conversion of 7-oxosteroid into 7b-hydroxysteroid under suitable conditions as understood by the skilled person, can be more efficient or effective in the presence of the 7b- hydroxysteroid dehydrogenase according to the present disclosure.
  • the reaction conditions may include presence of NAD + (e.g. 1-30 mM) KPi buffer and/or methanol (e.g. 10-30 vol.%), preferably in water-containing medium (e.g. 50-90 vol%).
  • concentration of NAD+ may be e.g. at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 mM.
  • Oxalacetate and/or malete dehydrogense may be added in order to further shift the equilibrium of the reaction and/or to regenerate the cofactor.
  • the alcohol dehydrogenase according to the present disclosure is capable of catalyzing a conversion of an aldehyde and/or a ketone into an alcohol.
  • the present disclosure also provides for a 7b- hydroxysteroid dehydrogenase which comprises:
  • said 7b- hydroxysteroid dehydrogenase may further comprise:
  • the amino acid that is changed at position 17 is Threonine and/or wherein the amino acid at position 17 is changed into Alanine;
  • the amino acid that is changed at position 39 is Glycine and/or wherein the amino acid at position 39 is changed into Aspartic acid;
  • the amino acid that is changed at position 40 is Arginine and/or wherein the amino acid at position 40 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine, preferably Leucine;
  • amino acid that is changed at position 41 is Arginine and/or wherein the amino acid at position 41 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine, preferably Asparagine; and/or
  • the amino acid that is changed at position 18 is Glutamic acid and/or wherein the amino acid at position 18 is changed into Aspartic acid.
  • the 7b- hydroxysteroid dehydrogenase according to the present disclosure preferably has
  • the 7b- hydroxysteroid dehydrogenase according to the present disclosure is not from Lactobacillus spicheri and/or does not comprise SEQ ID NO:5 in its entire length.
  • the 7b- hydroxysteroid dehydrogenase according to the present disclosure may be expressed in a host, e.g. a bacterial host cell, preferably different from Clostridium sardiniense and/or Lactobacillus spicheri, and preferably the host cell is Escherichia coli.
  • a mutation corresponding to a change of an amino acid at position 40 as shown in SEQ ID NO:1 preferably wherein the amino acid that is changed at position 40 is Arginine and/or wherein the amino acid at position 40 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine, preferably Leucine;
  • a mutation corresponding to a change of an amino acid at position 41 as shown in SEQ ID NO:1 preferably wherein the amino acid that is changed at position 41 is Arginine and/or wherein the amino acid at position 41 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine, preferably Asparagine; and/or
  • Glutamic acid and/or wherein the amino acid at position 18 is changed into Aspartic acid are examples of Glutamic acid and/or wherein the amino acid at position 18 is changed into Aspartic acid.
  • the present disclosure also provides for a method for producing a compound according to Formula 1 wherein R5 is b-OH, the method comprising:
  • Ri is chosen from a-OH, b-OH, ketone, AcO (acetyl), or H;
  • R 2 is chosen from H, F, Cl, I, or Br;
  • R3 is chosen from a-OH, b-OH, ketone, AcO (acetyl), or H;
  • R 4 is chosen from H, Methyl, Trifluoro Methyl, Ethyl, /-Propyl, Butyl, Allyl, Pentyl, Hexyl, Heptyl, Octyl, Nonyl. .
  • the above-disclosed method can be carried out in the system comprising at least two phases as described below (biphasic system). Further, the methods according to the present disclosure may be carried out in a membrane reactor or flow system and/or the enzymes according to the present disclosure may be immobilized, e.g. on a solid substrate (which allows reuse of the enzymes).
  • Membrane reactors show high stability (enzymes in the membrane reactor have an half-life of 1-2 weeks) and the biocatalysts can be reused for at least eight cycles of conversions. On the other hand, immobilized enzymes generally show a higher productivity
  • the flow-system represents a preferred technology.
  • substrate loadings preferably are not too low. While it does not represent a problem in chemical synthesis (UDCA, CDCA and CA are pretty soluble in alcohols like methanol and ethanol), the water-based environment preferred by enzymes is an obstacle in the development of an even more efficient biocatalytic process.
  • the solubility of CDCA and UDCA at pH 8.0 (typically used for HSDHs) is lower (around 25 mM), and it could be increased when adding methanol or ethanol as co solvent.
  • HSDHs are relatively stable and active in 10-20% methanol.
  • the immobilization of the enzyme can provide a higher stability to the protein and makes the system work also at higher concentrations of co-solvent.
  • working with a diluted solution may produce a large amount of wastewater that has to be treated.
  • a solution is represented by using a biphasic system: in these cases, the organic phase works as reservoir of reagents and products.
  • the present disclosure further provides for:
  • a system comprising at least the following two phases:
  • an organic solvent phase preferably comprising the compound according to Formula 1 wherein R5 is a-OH, the compound according to Formula 1 wherein R5 is ketone and/or the compound according to Formula 1 wherein R5 is b-OH;
  • an aqueous phase preferably comprising a 7b- hydroxysteroid dehydrogenase according to the present disclosure and optionally a 7a- hydroxysteroid dehydrogenase according to the present disclosure, more preferably a NAD+ dependent 7a- hydroxysteroid
  • dehydrogenase most preferably 7a- hydroxysteroid dehydrogenase from
  • the organic solvent phase is an octanol phase, which may improve solubility of hydroxysteroids.
  • the solubility of hydroxysteroids in non-alcoholic organic solvents eg. ethers, alkanes, dichloromethane, chloroform
  • the reported solubility values for CDCA and CA in chloroform are 7.6 and 14.4 mM, respectively.
  • the present disclosure provides for a method for producing ursocholic acid and/or ursodeoxycholic acid, the method comprising:
  • CA cholic acid
  • DCA chenodeoxycholic acid
  • UCA ursocholic acid
  • UDCA ursodeoxycholic acid
  • Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase characterized in that the 7b- hydroxysteroid dehydrogenase comprises:
  • nucleic acid according to any one of the previous clauses, wherein the nucleic acid has - at least 40, 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with SEQ ID NO:4 and/or wherein the nucleic acid is from Clostridium sardiniense ⁇ and/or
  • nucleic acid is from Lactobacillus spicheri.
  • Host comprising a nucleic acid according to any one of clauses 1-4 or a vector according to clause 5, preferably wherein the host is Escherichia coli.
  • 7b- hydroxysteroid dehydrogenase characterized in that the 7b- hydroxysteroid dehydrogenase comprises
  • 7b- hydroxysteroid dehydrogenase according to clause 7, characterized in that the 7b- hydroxysteroid dehydrogenase further comprises - an Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine at a position corresponding to position 40 as shown in SEQ ID NO:3 or at a position corresponding to position 41 as shown in SEQ ID NO:5;
  • Method for changing co-substrate specificity of a 7b- hydroxysteroid dehydrogenase from NADP+ to NAD+ comprising:
  • a mutation corresponding to a change of an amino acid at position 40 as shown in SEQ ID NO:1 preferably wherein the amino acid that is changed at position 40 is Arginine and/or wherein the amino acid at position 40 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine, preferably Leucine;
  • a mutation corresponding to a change of an amino acid at position 41 as shown in SEQ ID NO: 1 preferably wherein the amino acid that is changed at position 41 is Arginine and/or wherein the amino acid at position 41 is changed into any of Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine, preferably Asparagine; and/or
  • Glutamic acid and/or wherein the amino acid at position 18 is changed into Aspartic acid are examples of Glutamic acid and/or wherein the amino acid at position 18 is changed into Aspartic acid.
  • Ri is chosen from a-OH, b-OH, ketone, AcO (acetyl) or H;
  • R 2 is chosen from H, F, Cl, I, or Br;
  • R3 is chosen from a-OH, b-OH, ketone, AcO (acetyl) or H;
  • R 4 is chosen from H, Methyl, Trifluoro Methyl, Ethyl, /-Propyl, Butyl, Allyl, Pentyl, Hexyl, Heptyl, Octyl, or Nonyl. 14. Method according to clause 13, wherein the method is carried out in a system comprising at least the following two phases:
  • an organic solvent phase preferably comprising the compound according to Formula 1 wherein R5 is a-OH, the compound according to Formula 1 wherein R5 is ketone and/or the compound according to Formula 1 wherein R5 is b-OH;
  • an aqueous phase preferably comprising the 7b- hydroxysteroid dehydrogenase according to any one of clauses 7-11 and optionally the 7a- hydroxysteroid dehydrogenase, more preferably a NAD+ dependent 7a- hydroxysteroid dehydrogenase, most preferably 7a- hydroxysteroid dehydrogenase from Stenotrophomonas maltophilia.. 15. Method according to clause 14, wherein the organic solvent phase is an octanol phase.
  • Figure 1 Effect of pH on the oxidative (grey bars) and reductive (black bars) activities of purified (A) Sm7a-HSDH, (B) wt Cs7p-HSDH, (C) ADLN, (D) ADDLN and (E) ADDAA variants of Cs7p- HSDH and (F) wt Ls7p-HSDH.
  • the activity at pH 8.0 was taken as 100%. Enzymatic activities were determined by measuring NAD(P) + reduction and oxidation in the same conditions described in materials and methods. Values represent the means of three independent experiments (mean ⁇ standard error).
  • Figure 2 Effect of temperature on the initial enzymatic activity of (A) Sm7a-HSDH, (B) Cs7p- HSDH and (C) Ls7p-HSDH determined by measuring NAD + reduction, at pH 8.0. The value at 25°C is taken as 100%.
  • Figure 3 Effect of MeOH concentration on the activities of purified (A) Sm7a-HSDH, (B) wt Cs7p- HSDH, (C) ADLN, (D) ADDLN and (E) ADDAA variants of Cs7p-HSDH and (F) wt Ls7p-HSDH.
  • Figure 4 Bioconversion time-courses of: (A) 10 mM CDCA with 1.0 mM NAD + , (B) 10 mM CA with 1.0 mM NAD + , (C) 10 mM CDCA with 0.2 mM NAD + , employing 1 U of S/r?7a-HSDH (0.23 pg) and 0.6 U of Ls7p-HSDH (190 pg) in 50 mM KPi, pH 8.0 at 25 °C.
  • 1 indicates hydroxysteroid (CDCA)
  • 2 indicates Octil-CDCA
  • 3 indicates Octil-UDCA
  • 4 indicates UDCA.
  • SEQ ID NO:1 amino acid sequence of 7b- hydroxysteroid dehydrogenase of Clostridium sardiniense
  • SEQ ID NO:2 (nucleotide sequence encoding 7b- hydroxysteroid dehydrogenase of Clostridium sardiniense) ⁇ .
  • SEQ ID NO:3 amino acid sequence of ADLN-variant 7b- hydroxysteroid dehydrogenase of Clostridium sardiniense
  • SEQ ID NO:4 nucleotide sequence encoding ADLN-variant 7b- hydroxysteroid
  • SEQ ID NO:5 amino acid sequence of 7b- hydroxysteroid dehydrogenase of Lactobacillus spicheri
  • SEQ ID NO:6 nucleotide sequence encoding 7b- hydroxysteroid dehydrogenase of Lactobacillus spicheri
  • GCGGCGTACATGGGTCGCTTCTATGAA SEQ ID NO:7 amino acid sequence of 7a- hydroxysteroid dehydrogenase of Stenotrophomonas maltophilia
  • SEQ ID NO:8 (nucleotide sequence encoding 7a- hydroxysteroid dehydrogenase of Stenotrophomonas maltophilia) ⁇ .
  • SEQ ID NO:9 degenerate primer Rnd1_fwd for in site-saturation mutagenesis, the mismatching codons are underlined
  • SEQ ID NO: 1 1 degenerate primer Rnd2_fwd for in site-saturation mutagenesis, the mismatching codons are underlined
  • Bacteroides fragilis (GenBank: AF173833.2) and Escherichia coii (Gene ID: ACI83195.1) were used as query, restricting the organisms list to Stenotrophomonas maltophilia strains (taxi d: 40324).
  • a 3D structure model of this Sm7a-HSDH enzyme was obtained using SwissModel (https://swissmodel.expasy.org/interactive), employing the crystal structure of the 7a-HSDH from Escherichia coli (PDB ID: 1AHI.1) as template.
  • the synthetic cDNAs encoding the Sm7a-HSDH, 0$7b-Hd ⁇ H and .57b-HdOH were designed by in silico back translation of the amino acid sequences (GenBank: KRG42928.1 , AET80684.1 and WP045806907, respectively).
  • sequences corresponding to Ncol and Xhol restriction sites were added at the 5’- and 3’-ends of the cDNAs, respectively.
  • the codon usage of the synthetic genes was optimized for expression in Escherichia coli and produced by BaseClear B.V. (Baseclear B.V., Leiden, The Netherlands).
  • sequences of recombinant Sm7a-HSDH and Z eTb-HdOH with the multiple sequence analysis are reported herein and in the sequence listing.
  • the obtained expression plasmids were then used to transform BL21 (DE3) E. coli cells.
  • Starter cultures (100 mL) were prepared from a single recombinant BL21(DE3) E. coli colony grown in LB medium containing kanamycin (30 pg/mL), under vigorous shaking (200 rpm) at 37 °C. These cultures were diluted to a starting ODeoo nm of 0.1 in 1 L of LB medium (LB, 10 g/L bacto-tryptone, 10 g/L NaCI and 5 g/L yeast extract) and then incubated at 37 °C on a rotatory shaker at 200 rpm until an ODeoo nm of 1.0 was reached.
  • LB medium LB, 10 g/L bacto-tryptone, 10 g/L NaCI and 5 g/L yeast extract
  • Protein expression was induced by adding 0.25 mM IPTG: cultures were grown for another 12 h at 25 °C with shaking (200 rpm). Cells were harvested by centrifugation at 10,000 x g for 10 min at 4 °C, washed with 50 mM KPi buffer pH 8.0 and stored at -20 °C for at least 1 day before purification. 1.3 Protein purification
  • E. coli cell pellets were resuspended in lysis buffer (50 mM KPi buffer, 1 M NaCI, 5% glycerol (v/v) and 10 pg/mL DNAse, pH 8.0) and disrupted by French press (2 cycles, 180 psi). The insoluble fraction of the lysates were removed by centrifugation at 39,000 x g for 30 min at 4 °C. Crude extract were loaded onto a HiTrap chelating affinity columns (GE Healthcare, Little Chalfont, UK), previously loaded with Ni 2+ metal ions and equilibrated with 50 mM KPi buffer, 1 M NaCI and 5% glycerol (v/v) pH 8.0.
  • lysis buffer 50 mM KPi buffer, 1 M NaCI, 5% glycerol (v/v) and 10 pg/mL DNAse, pH 8.0
  • the columns were washed with this buffer until the absorbance value at 280 nm was that of the buffer and the bound proteins were eluted with 50 mM KPi buffer, 250 mM imidazole and 5% glycerol (v/v), pH 8.0.
  • the fractions containing the desired activity were dialyzed overnight against 50 mM KPi buffer and 5% glycerol (v/v), pH 8.0, using a 3-kDa dialysis tube.
  • 7a-HSDH and 7P-HSDH activities were assayed on 1.0 mM chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA), respectively, as substrate (see below).
  • 7a-HSDH’s enzymatic activity in the crude extract and of the purified enzyme was determined at room temperature (25 °C) using 1.0 mM CDCA, 2.0 mM NAD + , in 50 mM KPi buffer and 10% methanol (v/v), pH 8.0.
  • 7p-HSDH’s enzymatic activity was determined at room temperature (25 °C) using 1.0 mM UDCA, 2.0 mM NAD(P) + , in 50 mM KPi buffer and 10% methanol (v/v), pH 8.0.
  • the extinction coefficients of NADH, at 340 nm is 6,220 M ⁇ 1 crrf 1 .
  • One unit (U) was defined as the amount of enzyme producing 1 pmol of product per minute at 25 °C and at pH 8.0. Blank measurements were performed in absence of CDCA or UDCA, NAD + and enzyme.
  • the kinetic parameters of the purified samples were determined at room temperature in the presence of: different concentrations of substrates (5-10000 mM), 2.0 mM NAD(P) + in 50 mM KPi buffer and 10% methanol (v/v), pH 8.0, at 25 °C; different concentrations of NAD(P) + (1-5000 mM), 2.0 mM CDCA (for 7a-HSDH) or UDCA (for 7p-HSDH) in 50 mM KPi buffer and 10% methanol (v/v), pH 8.0, at 25 °C.
  • the specific activity was expressed as unit per mg of protein (determined by spectrophotometric analysis at 280 nm).
  • the kinetic data were fitted to the Michaelis-Menten equation or to the one modified to account for substrate inhibition.
  • the effect of pH on the enzymatic activities was determined using 1.0 mM CDCA (for 7a- HSDH) or UDCA (for Tb-HSDH), 2.0 mM NAD(P) + , in 100 mM citrate-phosphate buffer (66 mM citrate, 34 mM Na 2 HP0 4 ) and 10% methanol (v/v), in the 3.0-9.0 pH range.
  • the effect of methanol concentration on the enzymes activity toward CDCA and UDCA was determined using 1.0 mM CDCA (for 7a-HSDH) or UDCA (for 7P-HSDH), 2.0 mM NAD(P) + in 50 mM KPi buffer and different concentration of methanol (0-50% (v/v)), pH 8.0, at 25 °C. Temperature dependence of the enzymatic activities was determined using 1.0 mM CDCA (for 7a-HSDH) or UDCA (for 7P-HSDH), 2.0 mM NAD + in 50 KPi buffer and 10.0% methanol (v/v), pH 8.0 in the 18-95 temperature range.
  • Enzymatic stability was measured by incubating the enzyme solution in 100 mM citrate- phosphate buffer (66 mM citrate, 34 mM Na 2 HP0 4 ) in the 3.0-9.0 pH range at 25 X, in 50 mM KPi buffer with different concentration of methanol (0-50% (v/v)) at pH 8.0 at 25 X and in 50 mM KPi buffer, at pH 8.0 at different temperatures: samples were withdrawn at different times and residual activity was determined using the enzymatic activity assay.
  • Proteins from crude extract and the purified enzyme fractions were separated by SDS- PAGE on 12% polyacrylamide resolving gel: samples were resuspended in an appropriate volume of Laemmli sample buffer and boiled. Proteins were visualized by staining with SimplyBlue safe stain (Novex, Carlsbed, US).
  • the mutant libraries obtained from site-saturation mutagenesis were screened by means of a rapid colorimetric assay based on the reduction on NAD + (as described before) and by means of an automated liquid-handler system (BioRAD).
  • a saturated E. coli culture (1 ml_, growth in 2ml_ DeepWell plate) 0.250 mM IPTG were added and the culture was then incubated at 25 °C for 18 h.
  • the culture was centrifuged at 5,000* g for 2 min, and the cell pellet was resuspended with 200 pL of 50 mM KPi buffer, pH 8.0 added of 1 mg/mL lysozyme.
  • the increase of the absorbance at 340 nm was measured for 5 min at 25 °C by a microtiter plate reader and compared with cultures expressing the wild-type 0£7b-H80H and untransformed cells as controls.
  • the selected variants were sequenced and biochemically characterized.
  • Bioconversion of CDCA to UDCA were carried out employing 1 U to t of purified Sm7a-HSDS and 0.6 Utot of purified .dTb-Hb ⁇ b on 10 mM of CDCA, NAD + (0.2 or 1.0 mM).
  • 1 ml_ of reaction mixture containing 10% MeOH and 50 mM of KPi buffer, pH 8.0 was incubated at 25 °C.
  • Bioconversion of CA to UCA were carried out employing 1 Utot of purified Sm7a-HSDS and 0.6 Utot of purified Ls7p-HSDS or ADLN variant of 0$7b-H8 ⁇ H on 10 mM of CA, NAD + (0.2 or 1.0 mM).
  • 1 ml_ of reaction mixture containing 10% MeOH and 50 mM of KPi buffer, pH 8.0 was incubated at 25 °C.
  • 50 pl_ of reactions were withdrawn, diluted with 250 pl_ of MeOH and centrifuged at 14000 xg for 2 min. 10 pL of the obtained samples were analyzed by HPLC.
  • the gene coding for the Sm7a-HSDH, 0£7b-H80H and / ⁇ b-Hb ⁇ H were cloned into pET24d(+) plasmids, yielding enzymes containing a C-terminal 6x His-tag.
  • the recombinant enzymes forms were produced in E. coli BL21 (DE3) host cells grown at 37 °C in LB medium, adding IPTG at the late exponential phase of growth and collecting the cells after another 18 h of incubation at 25 °C under shaking. The expression level under these conditions of the different proteins is reported in Tables 2 and 3.
  • the His-tagged enzymes were purified by HiTrap chelating chromatography: all of the enzymes were isolated with a > 95% purity, as was judged by SDS-PAGE analysis.
  • aActivity was assayed on 1.25 mM CDCA and 2.5 mM NAD + as substrate in 50 mM KPi buffer, pH 8.0.
  • aActivity was assayed on 1.0 mM UDCA and 2.0 mM NADP + as substrate in 50 mM KPi buffer, pH 8.0.
  • aActivity was assayed on 1.0 mM UDCA and 2.0 mM NAD + as substrate in 50 mM KPi buffer, pH 8.0.
  • BL21 (DE3) cells (3.5 g corresponding to 0.5 L of fermentation broth).
  • aActivity was assayed on 1.0 mM UDCA and 2.0 mM NAD+ as substrate in 50 mM KPi buffer, pH 8.0.
  • aActivity was assayed on 1.0 mM UDCA and 2.0 mM NAD+ as substrate in 50 mM KPi buffer, pH 8.0.
  • aActivity was assayed on 1.0 mM UDCA and 2.5 mM NAD+ as substrate in 50 mM KPi buffer, pH 8.0.
  • the binding mode of the co-substrate NADP + in the model of the Cs7p-HSDH active site was analyzed: the ribose bounded phosphate group, interacts with two arginine residues (R40 and R41).
  • site-saturation mutagenesis was performed at positions 39, 40 and 41 using the QuikChange kit and the wild-type Cs7p-HSDH cDNA as template.
  • Cs7p-HSDH variants on NAD + as co substrate was screened on a microtiter plate using a spectrophotometric method (increasing of absorbance at 340 nm) and an automated liquid-handler system.
  • a spectrophotometric method increasing of absorbance at 340 nm
  • an automated liquid-handler system For the first round of mutagenesis, (G39D, R40X and R41X), 769 clones were screened, a number that gives a probability of 91 % that every combination of amino acids is introduced.
  • the clones most active on NAD + as identified through the screening procedure were isolated and the substitutions were identified by automatic DNA sequencing.
  • T17A, G39D, R40L, R41 N - ADLN variant shows high activity towards NAD + as cosubstrate.
  • ADLN variant was expressed in E. coli BL21 (DE3) cells and purified by HiTrap chelating chromatography (>90 % purity). This variant shows an expression yield similar to that of the wild-type Cs7p-HSDH (in terms of purified protein/liter of fermentation broth, see Table 3 above).
  • T17A, E18D, G39D, R40L, R41 N - ADDLN variant was designed and recombinantly expressed: the substitution of the glutamate with an aspartate in position 18 can form a second hydrogen bond between the cofactor and the protein.
  • the comparison of the kinetic parameters of ADLN and ADDLN variants of 0$7b- HSDH indicates that the addition of a second hydrogen bond increase the affinity of the ADDLN variant for the NAD + (8-fold), see Table 4.
  • the specific activity on NAD + of this variant decreases (10-fold lower in comparison to previous isolated one).
  • a fourth round of SSM was carried out employing the same primers used in the first round and the ADDLN variant as template.
  • G39D, R40A, R41A variant (ADDAA) was isolated.
  • the specific activity in standard condition of this variants is 0.3 U/mg (Table 4).
  • the affinity for the co-substrate is lower than the one observed in the previous variants.
  • the expression level of this protein is fifteen-fold higher than the wt 0b7b-H30H (486 vs. 20 mg/Lcuiture) .
  • ⁇ $7b-H3 ⁇ H sequence was identified using the Basic Local Alignment Search Tool (BLAST): the predicted sequence analysis showed a 792 bp ORF corresponding to a protein of 264 amino acids residues. The predicted MW of 29 kDa and the predicted homodimeric quaternary structure, put 057b-H3 ⁇ H in the short chain dehydrogenase/reductase superfamily.
  • BLAST Basic Local Alignment Search Tool
  • This enzyme was identified as a putative NADH dependent 7b-H3 ⁇ H.
  • the prediction was based first on the aminoacid sequence: although it shows a high structural conservation, the amino acids relative to the binding and recognition of NADH are present. Specifically, the Alanine and Aspartatate in position 18 and 19 and the stretch DYS in position 40-42, previously identified in the analogue 057b-H3 ⁇ H as responsible of cofactor recognition.
  • Sm7a-HSDH showed a strict NAD + activity on both CDCA and CA, although the activity on CA is considerably lower (halved). No activity was detached when NADP + was used as electron acceptor. Sm7a-HSDH displayed a 0.22 and 0.96 mM K m for CDCA and CA, respectively. To our knowledge, this is the highest affinities reported for that enzymatic class. On the other way, Sm7a-HSDH did not show any substrate inhibition on CA, and of 11 mM on CDCA. The K m value for NAD + is 0.55 mM.
  • the kinetic parameters were determined in the presence of 2.5 mM NAD + .
  • the kinetic parameters were determined in the presence of 2.0 mM CDCA
  • the enzyme is quite thermophilic, showing an optimum at around 70 °C ( Figure 2A), and is quite stable: after 24 h incubation at 25 and 37 °C, the enzyme maintained ca. 100 and 70% of its initial activity, respectively. Otherwise, incubations at higher temperatures resulted in a complete lost of enzymatic activity.
  • the enzymatic activity of Sm7a-HSDH was also investigated in presence of different concentration of methanol, those could be used as co-solvent for increase the solubility of hydroxysteroids in water enviroment: the enzyme shows no loss of activity in presence 10% methanol and it conserves 90% of activity in presence of 20% methanol (Fig. 3A). It is also quite stable in presence of concentrations of methanol lower than 20%.
  • wt 057b-Hd ⁇ H showed a strict NADP + activity on UDCA (0.74 U/mg in standard condition).
  • the activity on NAD + is roughly 100-fold lower showing a K m of 2.6 mM and a specific activity of 0.023 U/mg. 0$7b-HdOH displayed a 0.16 mM K m for UDCA (Table 6).
  • a The kinetic parameters were determined in the presence of 2.0 mM NAD + .
  • b The kinetic parameters were determined in the presence of 0.5 mM of NADH.
  • the wt 057b-Hd0H was characterized adding by NADP instead of the NAD.
  • the enzyme is less thermophilic than the Sm7a-HSDH, showing an optimum at around 60 °C (Fig. 2B), and less stable: after 24 h incubation at 25 and 37 °C, ⁇ 57b-H3 ⁇ H maintained ca. 85 and 62% of its initial activity, respectively.
  • 057b-H3 ⁇ H shows a good tolerance to concentration of methanol higher than 10% and it conserve the 75% of activity in presence of 20% methanol.
  • the ADLN, ADDLN and ADDAA variants showed and increase activity towards NAD + and NADH as cosubstrate, with a little change in specificity for the different substrates, i.e.
  • the isolated variants showed a higher stability then the wt Cs7b-HSDH. From the comparison, it can be observed that the ADLN variant maintains 95% of activity after incubation for 24h at 25 °C (in the same conditions the wt enzyme keeps only the 85% of its initial activity).
  • ⁇ 57b-H3 ⁇ H is quite thermophilic, showing an optimum at around 70 °C (Fig. 2C), and is it stable at 25 and 37 °C, maintaining, after 24 h of incubation, ca. 98 and 72% of its initial activity, respectively.
  • Z.57b-H30H conserve the 60% of activity in presence of 20% methanol: this is limiting the amount of substrate that can be loaded in a biotransformation (Fig. 3F).
  • Bioconversions were also tested at different pHs (6 and 7) but no improvement were observed (after 150 min in presence of 1 mM NAD + , 77.1 %, 80.5% and 86.6% conversion were observed at pH 6, 7 and 8, respectively).
  • ADLN variant of Cs7b-HSDH was also tested in the same conditions for the epimerization of CDCA and CA: although limited conversion was observed when CDCA was used as substrate, 10 pmol of CA were converted into UCA (91 % yield) by 790 pg of enzyme 60 min (Fig. 4D). This behavior can be partially explained by the kinetic parameters of this variant (K, for the intermediate 7-oxo-LCA is 4 times lower than the one for 7-oxo-DCA).
  • This system can be divided in 3 steps, where the central one is the most important:
  • Aqueous phase (0.1 mL):
  • reaction is then shook for 60 minutes at 25 °C.
  • the Organic phase (Octanol - with the product) and the aqueous phase (with the enzymes and cofactor) can be easily separated by centrifugation, sedimentation or phase separation: this led to the possibility to reuse enzymes and cofactor mixture in different catalytic cycles.
  • the final product can be obtained as described in step 3.
  • Octyl-UDCA can be hydrolyzed by CalB lipase in water : MeOH environment leading to the production of UDCA (4 in Figure 5 - final product).
  • the Organic phase can be reused in the system.
  • esterification/hydrolysis other methods can be used eg. Fisher esterification (acid catalysis).
  • EXAMPLE 2 The DLN variant (i.e. the mutant in position 39/40/41 without the mutation of the T in position 17) was measured to have a specific activity for NAD + ⁇ 0.03 U/mg and a Km for the same compound >3.0 mM. The catalytic efficiency was found to be ⁇ 0.01.
  • the ADLN variant was found to be >26 times more efficient than the DLN variant. This can be imputed to the mutation T17A.

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Abstract

La présente invention concerne des 7β-hydroxystéroïde déshydrogénases dépendantes de NAD+ et des procédés de fourniture d'une 7β-hydroxystéroïde déshydrogénase ayant une spécificité de co-substrat de NAD+ au lieu de NADP+. L'invention concerne en outre des procédés de conversion de l'acide cholique (CA) et/ou de l'acide chénodésoxycholique (CDCA) en acide ursocholique (UCA) et/ou de l'acide ursodésoxycholique (UDCA) respectivement, et plus spécifiquement, des procédés de conversion de l'acide 7-oxo-désoxycholique (7-oxo-DCA) et/ou de l'acide 7-oxo-lithocholique (7-oxo LCA) en acide ursocholique (UCA) et/ou en acide ursodésoxycholique (UDCA) respectivement, en utilisant une 7β-hydroxystéroïde déshydrogénase dépendante de NAD+.
PCT/NL2019/050228 2018-04-25 2019-04-18 7β-HYDROXYSTÉROÏDE DÉSHYDROGÉNASE DÉPENDANTE DE NAD+ WO2019209105A2 (fr)

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CN113462665A (zh) * 2021-06-30 2021-10-01 中山百灵生物技术股份有限公司 一种7α-HSDH酶突变体及其编码基因和应用
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CN115521964A (zh) * 2022-09-19 2022-12-27 湖北共同生物科技有限公司 一种甾体激素药物中间体的制备方法
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