NOVEL 7Beta-HYDROXYSTEROID DEHYDROGENASE MUTANTS AND PROCESS FOR THE PREPARATION OF URSODEOXYCHOLIC ACID

a technology of ursodeoxycholic acid and hydroxysteroid dehydrogenase, which is applied in the field of new 7beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid, can solve the problems of incomplete reaction of educt and higher processing cos

Inactive Publication Date: 2014-03-27
PHARMAZELL GMBH
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  • Summary
  • Abstract
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  • Claims
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AI Technical Summary

Benefits of technology

[0015]The above problems were solved, surprisingly, by the production and characterization of mutants of a novel regio- and stereospecific 7β-HSDH from aerobic bacteria of the genus Collinsella, especially of the strain Collinsella aerofaciens and use thereof in the reaction of cholic acid compounds, especially in the preparation of UDCA.
[0016]Furthermore, the above problem was solved by providing a biocatalytic (microbial or enzymatic) process, comprising the enzymatic conversion of DHCA via two partial reductive steps catalyzed by 7β-HSDH or 3α-HSDH, which can take place simultaneously or with a time delay in any order, to 12-keto-UDCA and cofactor regeneration using dehydrogenases, such as in particular formate dehydrogenase (FDH) enzymes or glucose dehydrogenase (GDH) enzymes, which regenerate the spent cofactor from the two partial reductive steps.

Problems solved by technology

A serious disadvantage is, among other things, the following: as the chemical oxidation is not selective, the carboxyl group and the 3α and 7α-hydroxyl group must be protected by esterification.
This method has the drawback that owing to the position of the equilibrium of the catalyzed reaction, a complete reaction is not possible, and that for the first step of the reaction it is necessary to use two different enzymes, which makes the process more expensive.
A disadvantage with the cofactor regeneration used there is that the resultant co-product can only be removed from the reaction mixture with great difficulty, so that the reaction equilibrium cannot be influenced positively, which results in incomplete reaction of the educt.
Another problem is to provide novel enzymatic and microbial synthesis routes, which in particular are also characterized by simplified cofactor regeneration in the reductive preparation of UDCA via DHCA.

Method used

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  • NOVEL 7Beta-HYDROXYSTEROID DEHYDROGENASE MUTANTS AND PROCESS FOR THE PREPARATION OF URSODEOXYCHOLIC ACID
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  • NOVEL 7Beta-HYDROXYSTEROID DEHYDROGENASE MUTANTS AND PROCESS FOR THE PREPARATION OF URSODEOXYCHOLIC ACID

Examples

Experimental program
Comparison scheme
Effect test

production example 1

Identification of 7β-HSDH Activity

[0274]The genomic DNA sequence of Collinsella aerofaciens ATCC 25986 was published in the year 2007 by “Washington University Genome Sequencing Center” for the “human gut microbiome project” at GenBank. HSDHs belong to the “short-chain dehydrogenases”. As the biochemical function of the “short-chain dehydrogenases” from Collinsella aerofaciens ATCC 25986 had not been annotated in GenBank, 9 candidates were cloned into vector pET22b+, and then expressed in E. coli BL21(DE3).

[0275]For this, 7β-HSDH coding sequences were PCR-amplified. The PCR products were obtained using the genomic DNA of Collinsella aerofaciens ATCC 25986 (DSM 3979) as template and the primers 5′-gggaattcCATATGAACCTGAGGGAGAAGTA-3′ (SEQ ID NO:3) and 5′-cccAAGCTTCTAGTCGCGGTAGAACGA-3′ (SEQ ID NO:4). The NdeI and HindIII cleavage sites in the primer sequences are underlined. The PCR product was purified using the PCR-Purification-Kit (Qiagen) and then cut with the enzymes NdeI and HindI...

production example 2

Preparative-Scale Cloning, Expression and Purification Of 7β-HSDH from Collinsella aerofaciens ATCC 25986 and Further Characterization of the Enzyme

2.1 Cloning and Production of an Expression Construct

[0280]The gene coding for 7β-HSDH was once again amplified from the genomic DNA by PCR and using primers, as described above for production example 1:

[0281]The PCR product was once again purified as described above and digested with the restriction endonucleases NdeI and HindIII. The digested PCR product was purified again and cloned into the pET-28a(+) vector using the T4-ligase, to produce an expression vector. The resultant expression construct was then transformed into E. coli DH5α cells. The protein to be expected should have 20 amino acid residues comprising a signal peptide and an N-terminal 6×His-Tag and a thrombin cleavage site. The sequence of the inserted DNA was verified by sequencing.

2.2 Overexpression and Purification of 7β-HSDH

[0282]E. coli BL21(DE3) was transformed with...

production example 3

Production of 7β-HSDH Mutants and Characterization Thereof

[0295]Position 39 of the amino acid sequence (comprising start methionine) (cf. SEQ ID NO:2) was mutated.

3.1 Primers

[0296]The mutagenesis primers stated below were used for the site-directed mutagenesis of 7β-HSDH. The primers were selected based on the 7β-HSDH gene sequence, so that they bring about the desired amino acid exchange. It was borne in mind that the base to be mutated is localized centrally in the primer, and that the melting points of the primer pairs are in the same region.

[0297]The primer pair 7beta_mut_G39A_fwd and 7beta_mut_G39A rev was used for preparing the G39A mutant. The primer pair 7beta_mut_G39S_fwd and 7beta_mut_G39S_rev was used for preparing the G39S mutant.

Glycine → AlanineForward: 7beta_mut_G39A_fwd:CGTCGTCATGGTCGCCCGTCGCGAGG.SEQ ID NO: 9)Reverse: 7beta_mut_G39A_rev:CCTCGCGACGGGCGACCATGACGACG.SEQ ID NO: 10)Glycine → SerineForward: 7beta_mut_G39S_fwd:CGTCGTCATGGTCAGCCGTCGCGAGG.SEQ ID NO: 11)Revers...

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Abstract

The invention relates to novel 7β-hydroxysteroid dehydrogenase mutants, to the sequences which encode these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic reactions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCS). The invention also relates to novel processes for the synthesis of UDCS using the enzyme mutants; and to the preparation of UDCS using recombinant, multiply-modified microorganisms.

Description

[0001]The invention relates to novel 7β-hydroxysteroid dehydrogenase mutants, to the sequences that code for these enzyme mutants, to processes for the preparation of the enzyme mutants and use thereof in enzymatic reactions of cholic acid compounds, and especially in the preparation of ursodeoxycholic acid (UDCA); the invention also relates to novel processes for the synthesis of UDCA using the enzyme mutants; and to the preparation of UDCA using recombinant, multiply-modified microorganisms.BACKGROUND OF THE INVENTION[0002]The active substances ursodeoxycholic acid (UDCA) and the related diastereomer chenodeoxycholic acid (CDCA), among others, have been used for many years for the drug treatment of gallstone disease. The two compounds differ only in the configuration of the hydroxyl group on carbon atom 7 (UDCA: β-configuration, CDCA: α-configuration). Various processes are described in the prior art for the preparation of UDCA, which are carried out purely chemically or consist o...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/04C12P33/02
CPCC12P33/02C12N9/0006C12P33/06C12Y101/01201C12N15/62C12N9/0008C12P33/00C12N15/52
Inventor WEUSTER-BOTZ, DIRKBRAUN, MICHAELAIGNER, ARNOSUN, BOQIAOKANTZOW, CHRISTINABRESCH, SVENBAKONYI, DANIELHUMMEL, WERNER
Owner PHARMAZELL GMBH
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