WO2019209105A2

WO2019209105A2 - NAD+ DEPENDENT 7β-HYDROXYSTEROID DEHYDROGENASE

Info

Publication number: WO2019209105A2
Application number: PCT/NL2019/050228
Authority: WO
Inventors: Fabio TONIN; Isabella Wilhelmina Christina Everdina Arends
Original assignee: Technische Universiteit Delft
Current assignee: Technische Universiteit Delft
Priority date: 2018-04-25
Filing date: 2019-04-18
Publication date: 2019-10-31
Anticipated expiration: 2020-10-25
Also published as: WO2019209105A3; NL2020823B1

Abstract

The present invention relates to a NAD+ dependent 7β- hydroxysteroid dehydrogenases and to methods of providing a 7β- hydroxysteroid dehydrogenase with a co-substrate specificity of NAD+ instead of NADP+. The invention further relates to methods for converting cholic acid (CA) and/or chenodeoxycholic acid (CDCA) into ursocholic acid (UCA) and/or ursodeoxycholic acid (UDCA) 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 7β- hydroxysteroid dehydrogenase.

Description

NAD+ dependent 7 -hydroxysteroid dehydrogenase

Technical field

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 (UDCA) 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.

Background of the disclosure

Ursodeoxycholic acid (UDCA) is a bile acid which solubilizes cholesterol gallstones and can improve liver function in cholestatic diseases. Currently, 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. In order to achieve chemical dehydroxylation, firstly CA has to be oxidized in position C-12 to the corresponding ketone, after which Wolff-Kishner reduction can be applied. 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 Cr0₃ (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.

Chemically, 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%. A further purification step is necessary for preparation of free UDCA: it can be easily obtained with sequential esterification, extraction and hydrolysis (yield 91%). The theoretical yield of the whole process fluctuates around 30 to 40%. However, as is clear from the above, the chemical synthesis of UDCA requires toxic and dangerous reagents.

In view thereof, several enzymatic systems have been proposed, but these enzymatic procedures typically require a set of a NAD+ and a NADP+ dependent enzyme, and therefore require a cofactor regeneration system. As general rule, the oxidative and reductive steps are coupled with a corresponding co-factor regeneration system. In this way, the equilibrium of the reaction can be pushed to the production of UDCA.

For example, the packed-bed flow-system reactor set up by Zheng et al.

(ChemBioChem, 19(4), 347-353} achieves full conversion of CDCA into UDCA for at least 12 hours. This represents an improvement compared to the use of the same biocatalysts under batch conditions. 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

coimmobilized enzymes, lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH), respectively.

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).

It is an objective of the present disclosure to overcome one or more of the problems in the prior art, and in particular to provide for a new and more efficient NAD+ dependent 7b- hydroxysteroid dehydrogenase in order to obtain a redox-neutral biosynthesis for UDCA with higher yield than obtained in the prior art.

Summary of the disclosure

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

- an Aspartic acid at a position corresponding to position 39 as shown in SEQ ID NO:1 or at a position corresponding to position 40 as shown in SEQ ID NO:5.

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.

General definitions

In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term“7a- hydroxysteroid dehydrogenase” refers to an enzyme catalyzing a conversion of any 7a-hydroxysteroid into any 7-oxosteroid. In the context of the present disclosure, 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. Preferably, the enzyme does not require NADP+ for the recited conversion, and/or is preferably dependent on NAD+ to perform the recited conversion.

The term“7b- hydroxysteroid dehydrogenase” refers to an enzyme catalyzing a conversion of any 7-oxosteroid into any 7b-hydroxysteroid, and/or vice versa. In the context of the present disclosure, 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. Preferably, the enzyme does not require NADP+ for the recited conversion, and/or is preferably dependent on NAD+ to perform the recited conversion.

The term“alcohol dehydrogenase” refers to an enzyme catalyzing a conversion of an aldehyde and/or a ketone into an alcohol, and/or vice versa. In the context of the present disclosure, 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. Preferably, the enzyme does not require NADP+ for the recited conversion, and/or is preferably dependent on NAD+ to perform the recited conversion.

The term“nucleic acid” (or nucleic acid sequence) 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. Or for example, 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. Next to exons, 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.

The terms “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. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides 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”). Alternatively percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence, it is intended that the 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. In other words, to obtain a polynucleotide 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. These 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. Similarly, by 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. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, 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. These 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 chemical structure of the compounds disclosed herein are further clarified below:

compound name abbrev. R R² R³ R⁴

cholic acid CA a-OH a-OH a-OH H

chenodeoxycholic acid CDCA a-OH a-OH H H

deoxycho!tc acid DCA a-OH H a-OH H

dehydrocho!ic acid DHCA keto keto keto H

lithocholic acid LCA a-OH H H H

ursocholic acid UCA a-OH b-OH a-OH H

ursodeoxycholic acid UDCA a-OH b-OH H H

g!ycocholate - a-OH a-OH a-OH glycine

glycochenodeoxycholate - a-OH a-OH H glycine

iaurochoiate - a-OH a-OH a-OH taurine

taurochenodeoxycholate - a-OH a-OH H taurine

7-oxo iithochoiic acid 7-oxo LCA a-OH

H H

7-oxo deoxycholic acid7-oxo DCA a-OH

(Based on a table disclosed in Beilstein J Org. Chem 2018, 14, 470-483).

For amino acids the following common abbreviations may be used throughout the text:

Ala A Alanine

Arg R Arginine

Asn N Asparagine

Asp D Aspartic acid (Aspartate)

Cys C Cysteine

Gin Q Glutamine

Glu E Glutamic acid (Glutamate)

Gly G Glycine

His H Histidine

lie I Isoleucine

Leu L Leucine

Lys K Lysine

Met M Methionine

Phe F Phenylalanine

Pro P Proline

Ser S Serine

Thr T Threonine

Trp W Tryptophan

Tyr Y Tyrosine

Val V Valine Asx B Aspartic acid or Asparagine

Glx Z Glutamine or Glutamic acid

Xaa X Any amino acid (sometime - is used to refer to any amino acid).

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Detailed description of the disclosure

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:

- an Alanine at a position corresponding to position 17 as shown in SEQ ID NO:1 and/or SEQ ID NO:3, and/or an Alanine at a position corresponding to position 18 as shown in SEQ ID NO:5; and/or

- an Aspartic acid at a position corresponding to position 39 as shown in SEQ ID NO:1 and/or SEQ ID NO:3, and/or an Aspartic acid at a position corresponding to position 40 as shown in SEQ ID NO:5.

It was found that 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.

In particular, 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. Specifically, the side chain of the added aspartic acid (D39) may form a hydrogen bond with the 2ΌH 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.

In view thereof, it will be clear to the skilled person that the present disclosure can be translated to other 7b- hydroxysteroid dehydrogenases than those specifically disclosed herein, as well as to other alcohol dehydrogenases, as long as they have an Alanine and/or Aspartic acid at the recited positions according to the present disclosure, particularly alcohol dehydrogenases and/or 7b- hydroxysteroid dehydrogenases having at least 30, 40, 50, 60, 70, 80, 90, 95, 99% sequence identity with SEQ ID NO: 1 , SEQ ID NO:3, and/or SEQ ID NO:5.

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+.

Additionally or alternatively, the nucleic acid according to the present disclosure may encode an NAD+ dependent alcohol dehydrogenase, preferably an NAD+ dependent 7b- hydroxysteroid dehydrogenase comprising:

- 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:1 and/or SEQ ID NO:3, or at a position corresponding to position 41 as shown in SEQ ID NO:5; and/or

- an Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine at a position corresponding to position 41 as shown in SEQ ID NO:1 and/or SEQ ID NO:3, or at a position corresponding to position 42 as shown in SEQ ID NO:5

Said amino acid(s) at the recited position(s) can further increase specificity of the enzyme to NAD+.

At the same time, or as a further alternative, the nucleic acid according to the present disclosure may encode an NAD+ dependent alcohol dehydrogenase, preferably an NAD+ dependent 7b- hydroxysteroid dehydrogenase comprising:

- an Aspartic acid at a position corresponding to position 18 as shown in SEQ ID NO:1 or at a position corresponding to position 19 as shown in SEQ ID NO:5, to further improve affinity of the enzyme to NAD+, e.g. by forming another hydrogen bond with the 3’-OH group of ribose. A skilled person will have no problem with determining corresponding positions according to the present disclosure in nucleic acids encoding other alcohol dehydrogenases, preferably 7b- hydroxysteroid dehydrogenases or in amino acids sequences of other alcohol dehydrogenases, preferably 7b- hydroxysteroid dehydrogenases, for example by aligning sequences of various alcohol dehydrogenases, preferably 7b- hydroxysteroid

dehydrogenases genes, in accordance with the data of the present sequence listings.

In practice, different numbers may be used to refer to corresponding amino acid positions in the amino acid sequences of a particular enzyme, particularly if amino acid sequences of corresponding enzymes of different organisms are aligned. Where in the present disclosure (or in the claims) reference is made to amino acid position 17 as shown in SEQ ID NO:1 and/or SEQ ID NO:3; position 18 as shown in SEQ ID NO:5; position 39 as shown in SEQ ID NO:1 and/or SEQ ID NO:3; position 40 as shown in SEQ ID NO:5; position 40 as shown in SEQ ID NO:1 and/or SEQ ID NO:3; position 41 as shown in SEQ ID NO:5; position 41 as shown in SEQ ID NO:1 and/or SEQ ID NO:3; and/or position 42 as shown in SEQ ID NO:5, it is to be construed that also is meant an amino acid at a position analogous to said position in an amino acid sequence that is substantially homologous to SEQ ID NO:1 , SEQ ID NO:3, and/or SEQ ID NO:5, as will be understood by the skilled person, for example by having an amino acid identity of at least 30, 40, 50, 60, 70%, preferably at least 75%, more preferably at least 80%, even more preferable 84%, 88%, 92%, 95%, 98% or 99% identity over its entire length with SEQ ID NO: 1 , SEQ ID NO:3, and/or SEQ ID NO:5, and/or is functional, in other words has alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase enzymatic activity.

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:

- a mutation that corresponds to a change of an amino acid at position 17 as shown in SEQ ID NO:1 ; and/or

- a mutation that corresponds to a change of an amino acid at position 39 as shown in SEQ ID NO:1.

Alternatively or additionally, the nucleic acid encoding an alcohol dehydrogenase, preferably a 7b- hydroxysteroid dehydrogenase, may comprise:

- a mutation that corresponds to a change of an amino acid at position 40 as shown in SEQ ID NO:1 ; - a mutation that corresponds to a change of an amino acid at position 41 as shown in SEQ ID NO:1 ; and/or

- a mutation that corresponds to a change of an amino acid at position 18 as shown in SEQ ID NO:1.

For said 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).

As will be understood by a skilled person, 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. 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.

Preferably, 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). By applying ODM, specific nucleotides can be altered in a nucleotide sequence encoding an alcohol dehydrogenase and/or 7b- hydroxysteroid dehydrogenase, whereby a mutation according to the present disclosure may be introduced.

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.

Once introduced into the cell, e.g. by electroporation or PEG-mediated oligonucleotide uptake, such 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). By deliberately designing a mismatch in the

oligonucleotide, 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.

Alternatively, 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).

Preferably, 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. This means that 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. Similarly, 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

- at least 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NO:4 and/or wherein the nucleic acid is (derived) from Clostridium sardiniense ; and/or

- at least 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:6 and/or wherein the nucleic acid is (derived) from Lactobacillus spicheri.

Additionally or alternatively, the nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to the present disclosure preferably has

- at least 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:4 and/or wherein the nucleic acid is (derived) from Clostridium sardiniense·, and/or

- at least 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:6 and/or wherein the nucleic acid is, or is not, (derived) from Lactobacillus spicheri. It is thus also foreseen that the nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to the present disclosure 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.

Accordingly, 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.

As will be clear, 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

- an Aspartic acid at a position corresponding to position 39 as shown in SEQ ID NO:3 or at a position corresponding to position 40 as shown in SEQ ID NO:5.

Additionally and/or alternatively, the alcohol dehydrogenase, preferably the 7b- hydroxysteroid dehydrogenase 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;

- an Alanine, Aspartic acid, Glutamic acid, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine, Asparagine, Serine, Threonine, Valine, or Tyrosine at a position corresponding to position 41 as shown in SEQ ID NO:3 or at a position corresponding to position 42 as shown in SEQ ID NO:5; and/or

- an Aspartic acid at a position corresponding to position 18 as shown in SEQ ID NO:3 or at a position corresponding to position 19 as shown in SEQ ID NO:5.

Preferably, the 7b- hydroxysteroid dehydrogenase according to the present disclosure is capable of catalyzing a conversion of 7-oxosteroid into 7b-hydroxysteroid. This means that 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%). The 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.

Similarly, the alcohol dehydrogenase according to the present disclosure is capable of catalyzing a 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. In accordance with the above, the present disclosure also provides for a 7b- hydroxysteroid dehydrogenase which comprises:

- a change of an amino acid at position 17 as shown in SEQ ID NO:1 ; and/or

- a change of an amino acid at position 39 as shown in SEQ ID NO:1.

Alternatively or additionally, said 7b- hydroxysteroid dehydrogenase may further comprise:

- a change of an amino acid at position 40 as shown in SEQ ID NO:1 ;

- a change of an amino acid at position 41 as shown in SEQ ID NO:1 ; and/or

- a change of an amino acid at position 18 as shown in SEQ ID NO:1.

For said 7b- hydroxysteroid dehydrogenase , it is preferred that

- 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;

- 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

- 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

- at least 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:1 and/or SEQ ID NO:3; and/or is (derived) from Clostridium sardiniense; and/or

- at least 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:5 and/or is, or is not, (derived) from Lactobacillus spicheri.

It is thus also foreseen that 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.

From the present disclosure it will be clear that also is provided for a method for changing co-substrate specificity of (a nucleic acid encoding) an alcohol dehydrogenase or a 7b- hydroxysteroid dehydrogenase, e.g. from NADP+ to NAD+, the method comprising:

a) providing a nucleic acid encoding an NADP+ dependent alcohol dehydrogenase, preferably an NADP+ 7b- hydroxysteroid dehydrogenase;

b) providing the following mutations in the nucleic acid encoding said enzyme:

- a mutation corresponding to a change of an amino acid at position 17 as shown in SEQ ID NO:1 , preferably wherein the amino acid that is changed at position 17 is Threonine and/or wherein the amino acid at position 17 is changed into Alanine;

- a mutation corresponding to a change of an amino acid at position 39 as shown in SEQ ID NO:1 , preferably wherein 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;

- optionally, 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;

- optionally, 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

- optionally, a mutation corresponding to a change of an amino acid at position 18 as shown in SEQ ID NO:1 , preferably wherein 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 present disclosure also provides for a method for producing a compound according to Formula 1 wherein R5 is b-OH, the method comprising:

a) optionally, providing a compound according to Formula 1 wherein R5 is a-OH;

b) optionally, converting the compound according to Formula 1 wherein R5 is a-OH into a compound according to Formula 1 wherein R5 is ketone, preferably by using a 7a- hydroxysteroid dehydrogenase, more preferably a NAD+ dependent 7a- hydroxysteroid dehydrogenase, most preferably 7a- hydroxysteroid dehydrogenase from

Stenotrophomonas maltophilia·,

c) providing a compound according to Formula 1 wherein R5 is ketone;

d) converting the compound according to Formula 1 wherein R5 is ketone into a compound according to Formula 1 wherein R5 is b-OH by using a 7b- hydroxysteroid dehydrogenase according to the present disclosure,

wherein Formula 1 is

wherein

Ri is chosen from a-OH, b-OH, ketone, AcO (acetyl), or H;

R₂ is chosen from H, F, Cl, I, or Br;

R3 is chosen from a-OH, b-OH, ketone, AcO (acetyl), or H;

R₄ 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

(e.g. 88.5 vs. 8 g L·¹ d ¹) despite the fact that the half-life (e.g. 23 h) is generally lower and the biocatalyst can be reused e.g. for only five cycles of conversions. In order to reduce the mechanical stress that might inactivate the immobilized enzymes, the flow-system represents a preferred technology.

For an economically and environmentally sustainable process volumetric productivities may be considered. In other words 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.

In comparison to CA, 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. Notably, HSDHs are relatively stable and active in 10-20% methanol. Moreover, 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. However, working with a diluted solution, may produce a large amount of wastewater that has to be treated.

Of no lesser importance, the increased amount of substrates and products up to relevant concentrations for industrial application, may inhibit the enzymes used in the biocatalytic process. Several examples are reported in literature about substrate or product inhibition of HSDHs.

A solution is represented by using a biphasic system: in these cases, the organic phase works as reservoir of reagents and products. In light of this background, 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

Stenotrophomonas maltophilia.

Preferably, 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) is not very high (eg. the reported solubility values for CDCA and CA in chloroform are 7.6 and 14.4 mM, respectively).

The methods according to the present disclosure may be carried out in the biphasic system as described above, for example the following specific method:

Finally, the present disclosure provides for a method for producing ursocholic acid and/or ursodeoxycholic acid, the method comprising:

a) optionally, converting cholic acid (CA) into 7-oxo-deoxycholic acid (7-oxo DCA) and/or converting chenodeoxycholic acid (CDCA) into 7-oxo-lithocholic acid (7-oxo LCA), e.g. by using a 7a- hydroxysteroid dehydrogenase, preferably an NAD+ dependent 7a- hydroxysteroid dehydrogenase, most preferably 7a- hydroxysteroid dehydrogenase from Stenotrophomonas maltophilia ;

b) converting 7-oxo-deoxycholic acid (7-oxo-DCA) into ursocholic acid (UCA) and/or converting 7-oxo-lithocholic acid (7-oxo LCA) into ursodeoxycholic acid (UDCA), e.g. by using a 7b- hydroxysteroid dehydrogenase according to the present disclosure;

c) optionally, converting cholic acid (CA) into chenodeoxycholic acid (CDCA) and/or ursocholic acid (UCA) into ursodeoxycholic acid (UDCA) e.g. by (enzymatic) dehydroxylation of CA to CDCA and/or UCA to UDCA, and/or 12-OH oxidation through a 12alpha-HSDH followed by Wolff-Kishner reduction.

CLAUSES

1. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase, characterized in that 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

- an Aspartic acid at a position corresponding to position 39 as shown in SEQ ID NO:3 or at a position corresponding to position 40 as shown in SEQ ID NO:5. 2. Nucleic acid according to clause 1 , characterized in that the 7b- hydroxysteroid dehydrogenase further comprises

3. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of the previous clauses, wherein the 7b- hydroxysteroid dehydrogenase catalyzes a conversion of 7-oxosteroid into 7b-hydroxysteroid.

4. 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

- at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% sequence identity with SEQ ID NO:6 and/or wherein the nucleic acid is from Lactobacillus spicheri.

5. Vector comprising a nucleic acid according to any of the previous clauses.

6. 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.

7. 7b- hydroxysteroid dehydrogenase, characterized in that the 7b- hydroxysteroid dehydrogenase comprises

8. 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;

9. 7b- hydroxysteroid dehydrogenase according to any one of clauses 7-8, wherein the 7b- hydroxysteroid dehydrogenase catalyzes a conversion of 7-oxosteroid into 7b- hydroxysteroid.

10. 7b- hydroxysteroid dehydrogenase according to any one of clauses 7-9, wherein the 7b- hydroxysteroid dehydrogenase has

- at least 40, 50, 60, 70, 80, 90, 95 or 99% sequence identity with SEQ ID NO:3 and/or wherein the 7b- hydroxysteroid dehydrogenase is from Clostridium sardiniense; and/or

- at least 40, 50, 60, 70, 80, 90, 95 or 99% sequence identity with SEQ ID NO:5 and/or wherein the 7b- hydroxysteroid dehydrogenase is from Lactobacillus spicheri.

11. 7b- hydroxysteroid dehydrogenase according to any one of clauses 7-10, wherein the 7b- hydroxysteroid dehydrogenase is expressed in a host different from Clostridium sardiniense and/or Lactobacillus spicheri, preferably Escherichia coli.

12. Method for changing co-substrate specificity of a 7b- hydroxysteroid dehydrogenase from NADP+ to NAD+, the method comprising:

a) providing a nucleic acid encoding a NADP+ 7b- hydroxysteroid dehydrogenase;

b) providing the following mutations in the nucleic acid encoding a NADP+ dependent 7b- hydroxysteroid dehydrogenase:

- a mutation corresponding to a change of an amino acid at position 17 as shown in SEQ ID NO:1 , preferably wherein the amino acid that is changed at position 17 is Threonine and/or wherein the amino acid at position 17 is changed into Alanine; - a mutation corresponding to a change of an amino acid at position 39 as shown in SEQ ID NO: 1 , preferably wherein 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;

- optionally, 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

13. Method for producing a compound according to Formula 1 wherein R5 is b-OH, the method comprising:

a) optionally, providing a compound according to Formula 1 wherein R5 is a-OH;

Stenotrophomonas maltophilia ;

c) providing a compound according to Formula 1 wherein R5 is ketone;

d) converting the compound according to Formula 1 wherein R5 is ketone into a compound according to Formula 1 wherein R5 is b-OH by using a 7b- hydroxysteroid dehydrogenase according to any one of clauses 7-11 ,

wherein Formula 1 is

wherein

Ri is chosen from a-OH, b-OH, ketone, AcO (acetyl) or H;

R₂ is chosen from H, F, Cl, I, or Br;

R3 is chosen from a-OH, b-OH, ketone, AcO (acetyl) or H;

R₄ 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. Methods of carrying out the conventional techniques used in methods of the present invention will be evident to the skilled worker, and are disclosed for example in Molecular

Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.;Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

Brief description of the figures

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.

In panel A, the activity in absence of MeOH was taken as 100%. Due to the lower solubility of UDCA in water, in all the other panels the activity in presence of 10% of MeOH was taken as 100%. Values represent the means of three independent experiments (mean ± standard error). 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. (D) Bioconversion time-course of 10 mM CA with 1 mM NAD⁺, employing 1 U of S/7i7a-HSDH (0.23 pg) and 0.6 U of ADLN variant of Cs7p-HSDH (790 pg) in 50 mM KPi, pH 8.0 at 25 °C. In all the cases, the substrates, products and the corresponding 7-OXO derivatives are represented by green triangle, blue diamond and with red squares, respectively.

Figure 5: Biphasic system for the epimerization of hydroxysteroids was developed. In the Figure,

1 indicates hydroxysteroid (CDCA), 2 indicates Octil-CDCA, 3 indicates Octil-UDCA, 4 indicates UDCA.

Sequences referred to:

SEQ ID NO:1 (amino acid sequence of 7b- hydroxysteroid dehydrogenase of Clostridium sardiniense)·.

MNFREKYGQWGIVLGATEGIGKASAFELAKRGMDVILVGRRKEALEELAKAIHEETGKEIRV

LPQDLSEYDAAERLIEATKDLDMGVIEYVACLHAMGQYNKVDYAKYEQMYRVNIRTFSKLLH HYIGEFKERDRGAFITIGSLSGWTSLPFCAEYAAEKAYMMTVTEGVAYECANTNVDVMLLSA

GSTITPTWLKNKPSDPKAVAAAMYPEDVIKDGFEQLGKKFTYLAGELNREKMKENNAMDRN

DLIAKLGKMFDHMA

SEQ ID NO:2 (nucleotide sequence encoding 7b- hydroxysteroid dehydrogenase of Clostridium sardiniense)·.

ATGAACTTCCGTGAAAAATACGGCCAGTGGGGCATCGTTCTGGGTGCTACCGAAGGTAT

CGGCAAAGCTAGTGCATTTGAATTAGCAAAGCGTGGCATGGATGTGATTTTGGTAGGTA

GACGTAAAGAGGCACTT GAAGAGTT AGCT AAAGCGATTCACGAAGAAACCGGT AAAGAA

ATCCGTGTTCTGCCGCAGGATCTGAGCGAATACGATGCGGCGGAACGTCTGATCGAAG

CGACCAAAGATCTGGATATGGGTGTTATCGAATACGTTGCGTGCCTGCACGCGATGGG

TCAGT ACAACAAAGTT GATT ACGCGAAAT ACGAACAGAT GT ACCGT GTT AAC ATCCGT AC

CTT CTCT AAACTGCTGCACCACT AC ATCGGT GAATT CAAAG AACGT GAT CGTGGTGCGT

TCATCACCATCGGTTCTCTGTCTGGTTGGACCAGCCTGCCGTTCTGCGCGGAATACGCG

GCT GAAAAAGCGT ACAT GAT GACCGTT ACCGAAGGGGTTGCGT ACGAATGCGCCAACA

CCAACGTTGATGTTATGCTGCTGTCTGCGGGTAGCACCATCACCCCGACCTGGCTGAAA

AACAAACCGTCTGATCCGAAAGCGGTTGCGGCTGCGATGTATCCGGAAGATGTTATCAA

AGATGGTTTCGAACAGCTGGGTAAAAAATTCACCTACCTGGCGGGTGAACTGAACCGTG

AAAAAATGAAAGAAAACAACGCGATGGATCGTAACGATCTGATTGCGAAACTGGGTAAA

ATGTTCGATCACATGGCG

SEQ ID NO:3 (amino acid sequence of ADLN-variant 7b- hydroxysteroid dehydrogenase of Clostridium sardiniense) ·.

MNFREKYGQWGIVLGAAEGIGKASAFELAKRGMDVILVDLNKEALEELAKAIHEETGKEIRVL PQDLSEYDAAERLIEATKDLDMGVIEYVACLHAMGQYNKVDYAKYEQMYRVNIRTFSKLLHH YIGEFKERDRGAFITIGSLSGWTSLPFCAEYAAEKAYMMTVTEGVAYECANTNVDVMLLSAG STITPTWLKN KPSDPKAVAAAMYPEDVIKDGFEQLGKKFTYLAGELNREKMKENNAMDRND LIAKLGKMFDHMA

SEQ ID NO:4 (nucleotide sequence encoding ADLN-variant 7b- hydroxysteroid

dehydrogenase of Clostridium sardiniense ):

ATGAACTTCCGTGAAAAATACGGCCAGTGGGGCATCGTTCTGGGTGCTGCCGAAGGTA TCGGCAAAGCTAGTGCATTTGAATTAGCAAAGCGTGGCATGGATGTGATTTTGGTAGAT TT GAAT AAAGAGGCACTT GAAGAGTT AGCT AAAGCGATT CACGAAGAAACCGGT AAAGA AATCCGTGTTCTGCCGCAGGATCTGAGCGAATACGATGCGGCGGAACGTCTGATCGAA GCGACCAAAGATCTGGATATGGGTGTTATCGAATACGTTGCGTGCCTGCACGCGATGG GTCAGT ACAACAAAGTT GATT ACGCGAAAT ACGAACAGAT GT ACCGT GTT AACATCCGT A

CCTTCTCT AAACTGCTGCACCACT ACATCGGT GAATTCAAAGAACGT GAT CGTGGT GCG

TTCATCACCATCGGTTCTCTGTCTGGTTGGACCAGCCTGCCGTTCTGCGCGGAATACGC

GGCTGAAAAAGCGTACATGATGACCGTTACCGAAGGGGTTGCGTACGAATGCGCCAAC

ACCAACGTTGATGTTATGCTGCTGTCTGCGGGTAGCACCATCACCCCGACCTGGCTGAA

AAACAAACCGTCT GAT CCGAAAGCGGTT GCGGCTGCGAT GT AT CCGGAAGAT GTT AT CA

AAGATGGTTTCGAACAGCTGGGTAAAAAATTCACCTACCTGGCGGGTGAACTGAACCGT

GAAAAAATGAAAGAAAACAACGCGATGGATCGTAACGATCTGATTGCGAAACTGGGTAA

AATGTTCGATCACATGGCG

SEQ ID NO:5 (amino acid sequence of 7b- hydroxysteroid dehydrogenase of Lactobacillus spicheri)·.

MTNFQQKYGQYAVILGAADGLGKQICYKLASLGLDIVCVDYSAEKINQFAATFPNDFPDRQLI

TLQADLSMDNVTDQIFRVTDAESVGFMSYVACLHQFGRLQETAWKDHQKMLNVNVINFLKC

LHHYMGIFAKQSRGGVLNLSSLTGVTASPYNAQYGAGKAYIKSITQAAGYEAEKENIDVMVA

TLGATSTPTELENQPGGSVGAAIQKIAMTPEKTVNEIFEHFGQVHSYYVGEHPKAQVKKWK

TELSEDDVAAYMGRFYE

SEQ ID NO:6 (nucleotide sequence encoding 7b- hydroxysteroid dehydrogenase of Lactobacillus spicheri)·.

ATGACCAACTTTCAGCAGAAATACGGTCAGTACGCTGTTATTCTGGGTGCGGCGGATGG

TCTGGGTAAACAGATCTGCTATAAACTGGCGTCTCTGGGTCTGGATATCGTTTGCGTTG

ATTATAGCGCTGAAAAAATCAACCAGTTCGCGGCGACCTTCCCTAACGATTTCCCGGAT

CGT CAGCT GAT CACCCTGCAGGCGGATTT AT CT ATGGAT AACGTT ACCGACCAGATCTT

CCGTGTTACCGATGCTGAAAGCGTTGGTTTCATGTCTTACGTTGCGTGCCTGCACCAGT

TCGGTCGTCTGCAGGAAACCGCGTGGAAAGATCACCAGAAAATGCTGAACGTTAACGTT

ATCAACTTCCTGAAATGCCTGCACCACTACATGGGTATCTTCGCGAAACAGTCTCGTGG

TGGTGTTCTGAACCTGTCTAGCCTGACCGGCGTTACCGCGAGCCCGTACAACGCGCAG

TACGGCGCGGGTAAAGCGTACATCAAATCTATCACCCAGGCGGCGGGTTACGAAGCGG

AAAAAGAAAACATCGATGTTATGGTTGCGACCCTGGGTGCAACCTCTACCCCGACCGAA

CTGGAAAACCAGCCGGGTGGTAGCGTTGGTGCAGCGATCCAGAAAATCGCGATGACCC

CGGAGAAAACGGTT AAT GAAATTTTT GAACACTTCGGT CAGGT ACAT AGCTACT AT GTT G

GT GAACACCCGAAAGCACAGGTT AAAAAATGGAAAACT GAACT GT CT GAAGAT GAT GTT

GCGGCGTACATGGGTCGCTTCTATGAA SEQ ID NO:7 (amino acid sequence of 7a- hydroxysteroid dehydrogenase of Stenotrophomonas maltophilia)·.

MSPQSHFDLSGKVAIVTGGGNGIGRASALM LAAYGAAVTIADLKLADAEKVAAEITAHGGRA

LALECNVLKDEDLVRTVERTAAELGGIHILVNNAGGGGAGRESPFEITVQQFERPFQINVFSA

WRLAQLCAPHMKKGGYGSIINMSSMSSINKSPAISAYAASKAAINHMTANLAHDYGPDNIRIN

AVGPGAVRTAALATVLTPEIEQRMLSHTPIKRLGEADDIAGAVLYFAAPISSWVSGQILFVNG

GGVQTLY

SEQ ID NO:8 (nucleotide sequence encoding 7a- hydroxysteroid dehydrogenase of Stenotrophomonas maltophilia)·.

ATGAGCCCGCAGAGCCACTTCGATCTGTCTGGTAAAGTTGCGATCGTTACCGGCGGTG

GCAACGGTATCGGCCGTGCATCTGCACTGATGCTGGCGGCGTATGGTGCAGCGGTTAC

TATCGCTGATCTGAAACTGGCGGATGCTGAAAAAGTTGCTGCGGAAATTACCGCGCACG

GTGGTCGTGCTCTGGCTCTGGAATGTAACGTTCTGAAAGATGAAGACCTGGTTCGTACC

GTTGAACGTACCGCAGCAGAACTGGGTGGTATCCACATCCTGGTTAACAACGCGGGTG

GTGGTGGTGCAGGTCGTGAATCTCCGTTCGAAATCACCGTTCAGCAGTTCGAACGTCC

GTTCCAGATCAACGTTTTCAGCGCCTGGCGTCTGGCACAGCTGTGTGCGCCGCACATG

AAAAAAGGCGGTTATGGTTCTATTATTAACATGAGCAGCATGTCTAGCATCAACAAATCT

CCAGCAATTT CTGCTT ATGC AGCTTCT AAAGCT GCAATT AATCACAT GACT GCT AACTT A

GCACATGATTATGGTCCAGATAACATTCGTATCAACGCAGTTGGTCCGGGTGCAGTTCG

TACCGCTGCGCTGGCTACCGTTCTGACTCCGGAAATTGAACAGCGTATGCTGAGCCACA

CCCCGATCAAACGTCTGGGTGAAGCTGATGATATCGCGGGTGCAGTTCTGTATTTCGCA

GCGCCGATTAGCTCTTGGGTTTCTGGTCAGATCCTGTTCGTTAACGGTGGTGGTGTTCA

GACCCTGTAT

SEQ ID NO:9 (degenerate primer Rnd1_fwd for in site-saturation mutagenesis, the mismatching codons are underlined):

GGATGTGATTTTGGTAGATDHKDHKAAAGAGGCACTTGAAGAGTTAGC

SEQ ID NO: 10 (degenerate primer Rnd1_rev for in site-saturation mutagenesis, the mismatching codons are underlined):

GCT AACT CTT CAAGTGCCTCTTTM DH M DH AT CT ACCAAAAT C AC AT CC

SEQ ID NO: 1 1 (degenerate primer Rnd2_fwd for in site-saturation mutagenesis, the mismatching codons are underlined):

CGTTCTGGGTGCTRMCGAAGGTATCGGC SEQ ID NO: 12 (degenerate primer Rnd2_rev for in site-saturation mutagenesis, the mismatching codons are underlined):

GCCGATACCTTCGKYAGCACCCAGAACG

SEQ ID NO: 13 (degenerate primer Rnd3_fwd for in site-saturation mutagenesis, the mismatching codons are underlined):

GGTGCTGCAGACGGTATCGGCAAACG

SEQ ID NO: 14 (degenerate primer Rnd3_rev for in site-saturation mutagenesis, the mismatching codons are underlined):

CGTTTGCCGATACCGTCTGCAGCACC

The following Examples illustrate the different embodiments of the invention. Unless stated otherwise all recombinant DNA techniques can be carried out according to standard protocols as described in e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor

Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA.

EXPERIMENTAL PART

EXAMPLE 1

1 MATERIALS AND METHODS

1.1 7a-hydroxysteroid dehydrogenase from Stenotrophomonas maltophilia (Sm7a- HSDH)

The Sm7a-HSDH and ί$7b-H5ϋH gene sequences were identified by multiple sequence analysis using BLASTp (NCBI, https://blast.ncbi.nlm.nih.gov):

For Sm7a-HSDH, protein sequences with known activity from Clostridium sardiniense (GenBank: AET80685.1), Clostridium difficile (GenBank: CAJ66880.1 , putative), Clostridium sordelli (GenBank: L12058.1), Eubacterium sp. VPI 12708 (GenBank: M58473.1),

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.

For .$7b-H80H, protein sequences from Clostridium sardiniense (GenBank: AET80684.1), Ruminococcus gnavus (Genbank: WP004843516.1), Ruminococcus torques (GenBank: WP015528793.1) and Collinsella aerofaciens (GenBank: WP006236005.1) together with the variant of the 057b-Hd0H obtained (see below) were used as query.

The 3D model of 057b-Hd0H (GenBank: AET80684.1) and /^b-HdϋH were built using the same server employing the entire structure of 7b-HdOH from Collinsella aerofaciens (PDB code 5GT9) as template.

1.2 7-p-hydroxysteroid dehydrogenase from Clostridium sardiniense (Cs7p-HSDH) and Lactobacillus spicheri (Ls7p-HSDH)

The synthetic cDNAs encoding the Sm7a-HSDH, 0$7b-HdϋH and .57b-HdOH were designed by in silico back translation of the amino acid sequences (GenBank: KRG42928.1 , AET80684.1 and WP045806907, respectively). In order to subclone into the pET24d(+) plasmid (Merck Millipore), 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). S/777a-HSDH, ObTb-HdϋH and Z bTb-HdOH cDNAs were cloned in the pET24d(+) vector using the Ncol and Xhol sites, resulting in 6.0-, 6.0- and 6.1 -kb construct (pET24-S/777a-HSDH, rET24-0$7b-Hd0H and rET24-ίb7b- HSDH). Six codons (encoding for six additional histidines) were added to the 3’-end of both genes during the subcloning process.

The 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. 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 Ni²⁺ metal ions and equilibrated with 50 mM KPi buffer, 1 M NaCI and 5% glycerol (v/v) 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.

During the purification procedure, 7a-HSDH and 7P-HSDH activities were assayed on 1.0 mM chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA), respectively, as substrate (see below).

1.4 Activity and kinetic measurements

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 crrf¹. 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 Na₂HP0₄) 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 Na₂HP0₄) 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.

1.5 SDS-PAGE

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).

1.6 Site-saturation mutagenesis and screening NAD^* dependent enzyme variants

Site-saturation mutagenesis was carried out at different amino acid positions of Cs7b-HSDH by using the whole plasmid PCR; the Cs7b-HSDH cDNA, subcloned into the pET24d vector as template and a set of degenerated synthetic oligonucleotides employed. A list of employed primes is reported in Table 1.

Table 1

List of degenerate primers employed in site-saturation mutagenesis experiments. The mismatching codons are underlined.

Primer name Sequence

Rnd1_fwd GGAT GT GATTTTGGT AGATDH KDH KAAAGAGGCACTT GAAGAGTT AGC

Rnd1_rev GCT AACTCTT CAAGTGCCTCTTTM DH M DH ATCT ACCAAAAT C AC AT CC

Rnd2_fwd CGTTCTGGGTGCTRMCGAAGGTATCGGC

Rnd2_rev GCCGATACCTTCGKYAGCACCCAGAACG

Rnd3_fwd GGTGCTGCAGACGGTATCGGCAAACG

Rnd3 rev CGTTTGCCGATACCGTCTGCAGCACC

Whole plasmid PCRs were carried out in a final volume of 20 pL: in all the cases, 1 pL of template DNA, 0.5 pL of each primer (final concentration 0.25 pM), 10 pL of Phusion Q-5 DNA polymerase mastermix and 8 pL of MilliQ water were added in sterile PCR tube. After an initial denaturation step (98 °C for 1 min), reaction was carried out for 30 cycles

(denaturation at 98 °C for 30 sec., annealing at 58 °C for 30 sec. and elongation at 72 °C for 7 min). A final elongation step (72 °C for 7 min.) was added. The template DNA was eliminated by enzymatic digestion with 1 pl_ Dpnl restriction enzyme at 37 °C for 2 hours; the PCR products were used to transform E. coli TOP10 cells. Subsequently, the recombinant plasmids were transferred to BL21 (DE3) E. coli cells, and these clones were used for the screening procedure. The introduction of the mutations was confirmed by automated DNA sequencing. 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). To 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. Cell lysis was performed incubating the plate for 30 minutes at 37 °C, 200 rpm. The crude extracts were centrifuged at 5,000*g for 30 min and then 50 pL of the supernatant was transferred to a 96-well Elisa plate. The activity was assayed on the crude extract by adding 150 pL of 1.33X substrate solution (1.33 mM UDCA, 2.66 mM NAD⁺,

13.3% MeOH in 50 mM KPi buffer, pH 8.0).

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.

1.7 Preparation of 7-oxo-DCA and 7-oxo-LCA

In a 2 L round-bottom flask 2.1 g of CA, 133 mg of NAD⁺ and 1.98 g of oxalacetate were dissolved in 50 mM of KPi buffer, pH 8.0 with 10% MeOH (final volume 1 L). The reaction was initiated adding 15 mg of Sm7a-HSDH (5100 Utot) and 200 Utot of malate

dehydrogenase (Sigma-Aldrich).

The reaction was gently stirred at 25 °C. Conversion was checked with HPLC. At complete reaction (~1 h) the solution was acidified with HCI till pH 2.0, leading to the formation of white suspension. In order to increase the precipitation 50 g of (NH^SC were added and dissolved. The product was filtered with a porous glass sept funnel and washed with 20 ml_ of 0.01 M HCI solution. The powder was then dried and crystallized in MeOH, yielding 1.9g of 7-oxo-DCA (95% yield).

The same procedure was used for the preparation of 7-oxo-LCA: from 1.9 g of CDCA, 1.6 g of 7-oxo-LCA were obtained (yield 84%). 1.8 Bioconversion of CDCA to UDCA.

Bioconversion of CDCA to UDCA were carried out employing 1 U_tot of purified Sm7a-HSDS and 0.6 Utot of purified .dTb-Hbϋb on 10 mM of CDCA, NAD⁺ (0.2 or 1.0 mM). As general procedure, 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-H8ϋH on 10 mM of CA, NAD⁺ (0.2 or 1.0 mM). As general procedure, 1 ml_ of reaction mixture containing 10% MeOH and 50 mM of KPi buffer, pH 8.0 was incubated at 25 °C. At fixed times 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. HPLC analyses were performed on a Shimadzu apparatus equipped with a LC20AT pump and an ELSD-LTII detector and fitted with a XTerra RP C18 column (length/internal diameter 150/4.6 mm, pore size 5 pm) under the following conditions: eluent H₂O/CH₃CN/TFA (60/30/0.1), flux 1.0 mL/min. Retention times CDCA=6.16 min, 7-OXO- LCA=4.74 min, UDCA=4.42 min, CA=3.59 min, 7-oxo-DCA=3.02 min, UCA= 2.74 min.

2 Results

2.1 Protein expression and purification

The gene coding for the Sm7a-HSDH, 0£7b-H80H and /^b-HbϋH 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.

Table 2

A) Purification of recombinant His-tagged Sm7a-HSDH from E. coli BL21 (DE3) cells (7 g corresponding to 1.0 L of fermentation broth).

Protein Specific activity Purification Yield

(mg) (U/mg)^a (fold) (%)

Cell extract 1050 233.2 - 100.0

HiTrap chelating 232.3 430.4 1.8 40.8

aActivity was assayed on 1.25 mM CDCA and 2.5 mM NAD⁺ as substrate in 50 mM KPi buffer, pH 8.0. B) Purification of recombinant His-tagged ΰ£7b-H80H from E. coli BL21 (DE3) cells (3.5 g corresponding to 0.5 L of fermentation broth).

Protein Specific activity Purification Yield

(mg) (U/mg)^a (fold) (%)

Cell extract 177 0.05 - 100.0

HiTrap chelating 10 0.74 14.8 83.3

aActivity was assayed on 1.0 mM UDCA and 2.0 mM NADP⁺ as substrate in 50 mM KPi buffer, pH 8.0.

C) Purification of recombinant His-tagged ^b-HbϋH from E. coli BL21 (DE3) cells (2.2 g corresponding to 0.3 L of fermentation broth).

Protein Specific activity Purification Yield

(mg) (U/mg)^a (fold) (%)

Cell extract 122 1.28 - 100.0

HiTrap chelating 29 3.10 2.4 57.9

aActivity was assayed on 1.0 mM UDCA and 2.0 mM NAD⁺ as substrate in 50 mM KPi buffer, pH 8.0.

Table 3

A) Purification of recombinant His-tagged ADLN variant of Cs7p-HSDH from E. coli

BL21 (DE3) cells (3.5 g corresponding to 0.5 L of fermentation broth).

Protein Specific Purification Yield

activity

(mg) (U/mg)^a (fold) (%)

Cell extract 209 0.1 1 100.0

HiTrap

31 0.27 2.4 35.4

chelating

aActivity was assayed on 1.0 mM UDCA and 2.0 mM NAD+ as substrate in 50 mM KPi buffer, pH 8.0.

B) Purification of recombinant His-tagged ADDLN variant of Cs7p-HSDH from E. coli BL21 (DE3) cells (3.5) g corresponding to 0.5 L of fermentation broth).

Protein Specific Purification Yield

activity

(mg) (U/mg)^a (fold) (%)

Cell extract 203 0.01 100.0

HiTrap

15 0.07 12.4 91.7

chelating

C) Purification of recombinant His-tagged ADDAA variant of Cs7p-HSDH from E. coli BL21 (DE3) cells (2.2 g corresponding to 0.3 L of fermentation broth).

Protein Specific Purification Yield

activity

(mg) (U/mg)^a (fold) (%)

Cell extract 678 0.10 100.0

HiTrap

146 0.21 2.0 43.0

chelating

aActivity was assayed on 1.0 mM UDCA and 2.5 mM NAD+ as substrate in 50 mM KPi buffer, pH 8.0.

2.2 Mutagenesis of Cs7p-HSDH

Since the three-dimensional structure of 7P-HSDH from C. sardiniense is unknown, a model of the protein was built by comparative modeling using the Swiss-model facility [Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714; and Schwede, T.; Kopp, J.; Guex, N.;

Peitsch, M. C. Nucleic Acids Res. 2003, 31 , 3381] 7P-HSDH from Collinsella aerofaciens (PDB code 5GT9) was chosen as template because of the high sequence identity (42%) with Cs7p-HSDH [Ferrandi, E. E.; Bertolesi, G. M.; Polentini, F.; Negri, A.; Riva, S.; Monti, D. Appl. Microbiol. Biotechnol. 2012, 95, 1221] 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). On the basis of the in silico analysis, site-saturation mutagenesis was performed at positions 39, 40 and 41 using the QuikChange kit and the wild-type Cs7p-HSDH cDNA as template. In the first round of mutagenesis an aspartate was specifically introduced in position 39 and, thanks to a degenerated primer with DHK codon, position 40 and 41 were mutated to different amino acid (A, D, E, F, I, K, L, M, N, S, T, V and Y). The use of DHK codon allows the introduction of 13 different amino acids with low repetitions, that are representative of the different chemistry and steric encumbrance. This drastically reduces the number of clones that have to be screened. The activity of 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. 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.

After the first round of mutagenesis and screening most of the enzyme variants obtained are inactive or have an activity lower than the control activity of wt Cs7p-HSDH on NAD⁺ (~1mll/mL in these conditions): however, four Cs7p-HSDH variants were isolated since they show a clear activity on NAD⁺. All, these four variants (G39D, R40L, R41 N - DLN variant; G39D, R40F, R41 F - DFF variant; G39D, R40K, R41S - DKS variant; G39D, R40K, R41V - DKV variant) were used as template for the second round of mutagenesis: from the molecular modelling of the variants it could be observed that hydroxyl group of threonine in position 17, interfere with aspartate 39 in the hydrogen binding of the free OH-group of ribose. A second pair of degenerated primers were design in order to mutated that threonine. For the second round of mutagenesis, (T17X), 380 clones (96 clones for each variant) were screened.

Finally, one variant (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).

As shown in Table 4, among the Cs7p-HSDH variants obtained by site-saturation

mutagenesis, the ADLN was identified because of a thirty-fold increase in specific activity for NAD+ compared to wild-type enzyme. Table 4

Kinetic parameters of purified recombinant 7b-H30H£ on NADP⁺ and NAD⁺.

is calculated as ratio between the catalytic effiency on the two cofactors bd = below detection.

In order to further increase the activity on NAD⁺, 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. However, the specific activity on NAD⁺ of this variant decreases (10-fold lower in comparison to previous isolated one). In order to restore the specific activity on NAD⁺ a fourth round of SSM was carried out employing the same primers used in the first round and the ADDLN variant as template. T17A, E18D,

G39D, R40A, R41A variant (ADDAA) was isolated. The specific activity in standard condition of this variants is 0.3 U/mg (Table 4). Interestingly the affinity for the co-substrate is lower than the one observed in the previous variants. However, the expression level of this protein is fifteen-fold higher than the wt 0b7b-H30H (486 vs. 20 mg/Lcuiture) .

2.3 Protein sequence prediction

ί$7b-H3ϋH 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-H3ϋH in the short chain dehydrogenase/reductase superfamily.

This enzyme was identified as a putative NADH dependent 7b-H3ϋH. 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-H3ϋH as responsible of cofactor recognition.

2.4 Biochemical characterization

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.

All the kinetics data were presented in Table 5.

Table 5

Kinetic parameters of purified recombinant Sm7a-HSDH

Vrnax Km \/ / '

(U/mg) (mM) (mM) ^{Vn m}

CA^a 278.2 ± 9.6 0.960 ± 0.110 / 289.8 ± 43.2

CDCA^a 488.7 ± 19.1 0.218 ± 0.024 11 ± 1.6 2241.7 ± 334.4

NAD^+b 528.4 ± 5.1 0.560 ± 0.017 / 943.6 ± 37.8 ^aThe kinetic parameters were determined in the presence of 2.5 mM NAD⁺. ^bThe kinetic parameters were determined in the presence of 2.0 mM CDCA

The pH and temperature dependence of Sm7a-HSDH activity was investigated: both the maximal activity and stability occurred at slightly alkaline pH values (Figure 1A).

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%. On the other hand, wt 057b-HdϋH 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).

Table 6

Kinetic parameters of purified recombinant Tb-HdOHe on different substrates.

aThe kinetic parameters were determined in the presence of 2.0 mM NAD⁺. ^bThe 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 pH and temperature dependence of 0$7b-Hd0H activity was investigated: both the maximal activity and stability occurred at slightly alkaline pH values (Fig. 1 B). Interestingly, the pH optimum for the reduction reactions was detected at pH 6-7. In presence of NADPH, this enzyme is able to reduce both 7-oxo-DCA and 7-oxo-LCA (see Table 6).

In general, 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, ϋ57b-H3ϋH maintained ca. 85 and 62% of its initial activity, respectively.

057b-H3ϋH 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.

UDCA, 7-oxo-DCA and 7-oxo-LCA..

Interestingly, 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).

As predicted by in silico analyses, ί$7b-HbOH showed a strict NAD⁺ activity (3.10 U/mg in standard condition). A weak activity was detached when NADP⁺ was used as electron acceptor (Table 4). Ls7b-HSDH showed a 0.15, 0.04 and 0.13 mM K_m for UDCA, 7-oxo-LCA and 7-oxo-DCA, respectively. ίb7b-H30H is inhibited by UDCA showing K, of 0.8 mM for this substrate. The K_m value for NAD⁺ is 0.08 mM. This enzyme shows the highest NAD7NADH activities for both the reduction and the oxidation of 7b-OH hydroxysteroids and derivatives. All the kinetics data were presented in Table 4 and Table 6.

From the biochemical characterization it can be observed that this enzyme showed similar features of the other isolated enzymes, with the exception of the pH dependence: the maximal activity for both oxidation and reduction reactions were observed at lower pH (6-7) (Fig. 1 F).

ί57b-H3ϋH 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).

2.5 Bioconversion of CDCA and CA

In order to assay the applicability of the enzymes previously described as biocatalyst for the production of UDCA and UCA in a batch bioreactor, lab-scale bioconversions were carried out: 10 pmol of CDCA in presence of 1 pmol of NAD⁺, were converted in UDCA (92% yield) in 200 min by 1 U of Sm7a-HSDH (0.23 pg) and 0.6 U of _$7b-H3ϋH (190 pg) (Fig. 4A). Under the same conditions, 10 pmol of CA were converted in UCA (yield 90.7%) (Fig. 4B). Biocatalytic conversions were also carried out on CDCA as substrate using 0.2 pmol of NAD⁺: in this case the conversion is slower, yielding 24% of UDCA in 20 h (Fig. 4C).

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).

3 Biphasic system

3.1 Biphasic system for the epimerization of hydroxysteroids

A novel biphasic system for the epimerization of hydroxysteroids was developed, with a typical substrate loading of 100 mM (5- to 10-fold higher than the state of art).

This system can be divided in 3 steps, where the central one is the most important:

- Step 1 : dry powder of hydroxysteroid (CDCA (1 in Figure 5) in particular) was added in to 1-octanol at the final concentration of 100 mM. CalB lipase (or other lipases) was added. The reaction was stirred at 30 °C for 2 h giving Octyl-CDCA (2 in Figure 5) (100% conversion). The Octyl-CDCA and derivatives are soluble in octanol at high concentration (>250 mM).

- Step 2 (main part): in order to carry out the epimerization reaction of Octyl-CDCA, a biphasic system was set-up:

Organic phase (1 mL):

o 100 mM Oct-CDCA

o Octanol

Aqueous phase (0.1 mL):

o 1 U_tot of Sm7a-HSDH

o 1 U_tot of Z_57b-H50H

o 1 mM NAD⁺

o 50 mM KPi pH 8.0

The concentration of each component in comparison with a monophasic system (water : methanol) is reported in the following Table 7:

Biphasic system

Volume mL 1 +1 mL

CDCA mGho 100 mih

NAD miho 0.1 prn

RATIO (CDCA : NAD⁺) 10 1000

The reaction is then shook for 60 minutes at 25 °C.

After 60 minutes 62% of Octyl-UDCA (3 in Figure 5) was obtained. The reaction can be carried out longer increasing the yield.

Using this system 3 different problems were solved:

o The substrate loading increase (10-fold, in comparison to a monophasic system);

o Reduced enzyme inhibition: since the Oct-derivatives are poorly soluble in water there is no inhibition of the enzymes used for the epimerization reaction (whereas, in the monophasic system inhibition effects were observed at concentration higher than 10 mM);

o 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.

- 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.

3.2 Notes

For the esterification/hydrolysis other methods can be used eg. Fisher esterification (acid catalysis).

- The epimerization reaction of Oct-CDCA can be performed using other HSDHs with different cofactor specificity and sacrificial substrate for the cofactor regeneration: anyway that will complicate the system. The solubility of the sacrificial substrates together with the stability of the additional enzymes used for the cofactor regeneration had to be assessed

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.

Claims

- an Aspartic acid at a position corresponding to position 39 as shown in SEQ ID NO:3 or at a position corresponding to position 40 as shown in SEQ ID NO:5,

wherein the nucleic acid is not from Lactobacillus spicheri.

2. Use of a nucleic acid encoding a 7b- hydroxysteroid dehydrogenase, wherein the nucleic acid is expressed in a host cell, for catalyzing a conversion of a 7-oxosteroid into a 7b-I^k^5ίbG0^, characterized in that the 7b- hydroxysteroid dehydrogenase comprises:

3. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to claim 1 , or use of a nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to claim 2, characterized in that the 7b- hydroxysteroid dehydrogenase further comprises

4. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 1 and 3, wherein the 7b- hydroxysteroid dehydrogenase catalyzes a conversion of 7- oxosteroid into 7b-hydroxysteroid.

5. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 1 , 3-4 or use of a nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 2 and 3, 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

- at least 40, 50, 60, 70, 80, 90, 95, or 99% sequence identity with SEQ ID NO:6.

6. Use of a nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 2, 3 and 5, wherein the nucleic acid is from Lactobacillus spicheri.

7. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 1 , 3-5 or use of a nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 2, 3, 5-6, wherein the nucleic acid is comprised in a vector.

8. Nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 1 , 3-5 and 7 or use of a nucleic acid encoding a 7b- hydroxysteroid dehydrogenase according to any one of claims 2, 3, 5-7, wherein the host is Escherichia coli.

9. 7b- hydroxysteroid dehydrogenase, characterized in that the 7b- hydroxysteroid dehydrogenase comprises

wherein the 7b- hydroxysteroid dehydrogenase is not from Lactobacillus spicheri.

10. Use of a 7b- hydroxysteroid dehydrogenase for catalyzing a conversion of a

7-oxosteroid into a 7b-hydroxysteroid, characterized in that the 7b- hydroxysteroid dehydrogenase comprises

11. 7b- hydroxysteroid dehydrogenase according to claim 9, or use of a 7b- hydroxysteroid dehydrogenase according to claim 10, characterized in that the 7b- hydroxysteroid dehydrogenase further comprises

12. 7b- hydroxysteroid dehydrogenase according to any one of claims 9 and 11 , wherein the 7b- hydroxysteroid dehydrogenase catalyzes a conversion of 7-oxosteroid into 7b- hydroxysteroid.

13. 7b- hydroxysteroid dehydrogenase according to any of claims 9, 11-12, or use of a 7b- hydroxysteroid dehydrogenase according to any one of claims 10 and 11 , wherein the 7b- hydroxysteroid dehydrogenase has

- at least 40, 50, 60, 70, 80, 90, 95 or 99% sequence identity with SEQ ID NO:5.

14. Use of a 7b- hydroxysteroid dehydrogenase according to any one of claims 10, 11 and 13, wherein the 7b- hydroxysteroid dehydrogenase is from Lactobacillus spicheri.

15. 7b- hydroxysteroid dehydrogenase according to any of claims 9, 11-12 or use of a 7b- hydroxysteroid dehydrogenase according to any one of claims 10, 11 , 13-14, wherein the 7b- hydroxysteroid dehydrogenase is expressed in a host different from Clostridium sardiniense and/or a host different from Lactobacillus spicheri, preferably Escherichia coli.

16. Method for changing co-substrate specificity of a 7b- hydroxysteroid dehydrogenase from NADP+ to NAD+, the method comprising:

a) providing a nucleic acid encoding a NADP+ 7b- hydroxysteroid dehydrogenase; b) providing the following mutations in the nucleic acid encoding a NADP+ dependent 7b- hydroxysteroid dehydrogenase:

- a mutation corresponding to a change of an amino acid at position 17 as shown in SEQ ID NO: 1 , preferably wherein the amino acid that is changed at position 17 is Threonine and/or wherein the amino acid at position 17 is changed into Alanine;

17. Method for producing a compound according to Formula 1 wherein R5 is b-OH, the method comprising:

a) optionally, providing a compound according to Formula 1 wherein R5 is a-OH;

Stenotrophomonas maltophilia ;

c) providing a compound according to Formula 1 wherein R5 is ketone;

d) converting the compound according to Formula 1 wherein R5 is ketone into a compound according to Formula 1 wherein R5 is b-OH by using a 7b- hydroxysteroid dehydrogenase as defined in any one of claims 9-15,

wherein Formula 1 is

wherein

Ri is chosen from a-OH, b-OH, ketone, AcO (acetyl) or H;

R₂ is chosen from H, F, Cl, I, or Br;

R3 is chosen from a-OH, b-OH, ketone, AcO (acetyl) or H;

R₄ is chosen from H, Methyl, Trifluoro Methyl, Ethyl, /-Propyl, Butyl, Allyl, Pentyl, Hexyl, Heptyl, Octyl, or Nonyl.

18. Method according to claim 17, wherein the method is carried out in a system comprising at least the following two phases:

- an aqueous phase, preferably comprising the 7b- hydroxysteroid dehydrogenase as defined in any one of claims 9-15 and optionally the 7a- hydroxysteroid dehydrogenase, more preferably a NAD+ dependent 7a- hydroxysteroid dehydrogenase, most preferably 7a- hydroxysteroid dehydrogenase from Stenotrophomonas maltophilia.

19. Method according to claim 18, wherein the organic solvent phase is an octanol phase.