Novel 7alpha-hydroxysteroid dehydrogenase knockout mutants and use thereof

A technology of hydroxysteroids and dehydrogenases, applied in the direction of enzymes, oxidoreductases, and the use of vectors to introduce foreign genetic materials, etc., can solve problems such as pollution and accumulation of by-products

Inactive Publication Date: 2013-05-08
CELL PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There is the following serious disadvantage: since the chemical oxidation is not selective, the carboxyl group as well as the 3α and 7α-hydroxyl groups must be protected by esterification
[0008] In this chemical process there is the problem that lithocholic acid (LCA) contaminates the valuable product UDCA produced in this way, for reasons not yet known
Among these is the problem that the recombinant E. coli host also produces endogenous 7α-HSDH, which produces the 3,12-diketo-CDCA by-product from dehydrocholic acid
This 3,12-diketone-CDCA is then no longer a substrate for 7β-HSDH, so that in addition to the desired product, undesired by-products also accumulate

Method used

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  • Novel 7alpha-hydroxysteroid dehydrogenase knockout mutants and use thereof
  • Novel 7alpha-hydroxysteroid dehydrogenase knockout mutants and use thereof
  • Novel 7alpha-hydroxysteroid dehydrogenase knockout mutants and use thereof

Examples

Experimental program
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Effect test

Embodiment approach

[0031] 1. Recombinant microorganisms, wherein the enzymatic activity of 7α-hydroxysteroid dehydrogenase (7α-HSDH) is inhibited, and simultaneously expressively contain functionally different hydroxysteroid dehydrogenases (for example, 3α-, 3β-, 7β-, 11α -, 11β-, 12α-, 12β-, 17α-, 17β-, 20α- or 20β-HSDH, especially 3α-, 7β- or 12α-HSDH) enzymatic activity.

[0032] The 7α-HSDH inhibited here can show any desired cofactor dependence, for example, on NADH or NADPH (or NAD + or NADP + ) dependencies.

[0033] Optionally, this modified microorganism can also be additionally modified so that it not only expresses the desired HSDH activity, but also other proteins or enzymes that can optionally function in conjunction with HSDH, such as assisting cofactor regeneration Enzymes (as explained in more detail below). Examples of such enzymes are eg alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH).

[0034] In particular, the present invention relates to recombinant microorg...

Embodiment 1

[0415] Example 1: Production of a 7α-HSDH deletion mutant of E. coli strain BL21(DE3) (E. coli BL21(DE3)Δ7α-HSDH) (type 1) (knockout by homologous recombination)

[0416] 1.1 Sequence information of 7α-HSDH from Escherichia coli BL21(DE3)

[0417] Amino acid sequence: (SEQ ID NO: 10)

[0418] Length: 255 amino acids

[0419] Type: protein

[0420] Source: Escherichia coli BL21 (DE3)

[0421] VFNSDNLRLDGKCAIITGAGAGIGKEIAITFATAGASVVVSDINADAANHVVDEIQQLGGQAFACRCDITSEQELSALADFAISKLGKVDILVNNAGGGGPKPFDMPMADFRRAYELNVFSFFHLSQLVAPEMEKNGGGVILTITSMAAENKNINMTSYASSKAAASHLVRNMAFDLGEKNIRVNGIAPGAILTDALKSVITPEIEQKMLQHTPIRRLGQPQDIANAALFLCSPAASWVSGQILTVSGGGVQELN

[0422] Nucleotide sequence (SEQ ID NO: 9)

[0423] Length: 768 base pairs

[0424] Type: nucleic acid

[0425] Source: Escherichia coli BL21 (DE3)

[0426] Accession number: NC_012971 Area: 1642470..1643237

[0427] gtgtttaatt ctgacaacct gagactcgac ggaaaatgcg ccatcatcac aggtgcgggt 60

[0428] gcaggtattg gtaaagaaat cgccattaca ttcg...

Embodiment 2

[0458] Example 2: Heterologous expression of 12α-HSDH using Escherichia coli BL21(DE3)Δ7α-HSDH (type 1)

[0459] The pET28a(+) vector, Novagen, Darmstadt, which contains the MCS under the control of the T7 promoter and the transcription start and the T7 terminator for the expression of 12α-HSDH. Expression is induced by isopropyl-β-D-thiogalactopyranoside (IPTG).

[0460] To this end, the sequence encoding 12α-HSDH (short form) was PCR amplified (see also Applicant's PCT / EP2009 / 002190). PCR products were obtained using genomic DNA of Clostridium group P strain 48–50 as template and the following primer pairs:

[0461] (5'-GGTATTCCATATGATCTTTGACGGAAAGGTCGC-3' (SEQ ID NO:5) and

[0462] 5'-CGGGATCCCTAGGGGCGCTGACCC-3') (SEQ ID NO: 6).

[0463] Nucleotide residues in italics are redundant. Residues in bold encode the cleavage site. The other residues are homologous sequences of 12α-HSDH.

[0464] The PCR products were applied to an agarose gel, separated and excised from the...

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Abstract

The invention relates to novel microbial 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) knockout mutants and to the use thereof for producing other HSDHs having various functionalities, such as 3alpha-, 7beta- or 12alpha-HSDH, and to the use of thus-produced HSDH enzymes in enzymatic reactions of cholic acid compounds, and in particular for producing ursodeoxycholic acid (UDCS). The invention relates in particular to novel methods for synthesizing UDCS.

Description

[0001] The present invention relates to novel microbial 7α-hydroxysteroid dehydrogenase (7α-HSDH) knockout mutants and their use for the production of other HSDHs of different functionality (e.g. 3α-, 7β- or 12α-HSDH) , and the use of the HSDH enzyme thus produced in the enzymatic reaction of cholic acid compounds, and in particular in the production of ursodeoxycholic acid (UDCA); the invention relates in particular to a novel method for the synthesis of UDCA. Background of the invention: [0002] The active substance ursodeoxycholic acid (UDCA) and the related diastereoisomer chenodeoxycholic acid (CDCA) have been used for many years in the pharmacological treatment of gallstones. These two compounds differ only in the configuration of the hydroxyl group on the C atom at position 7 (UDCA: β-configuration, CDCA: α-configuration). In the prior art, various methods of producing UDCA are described, either entirely chemically or consisting of a combination of chemical and enzymat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/245C12P33/00C12P33/06
CPCC12P33/00C12Y101/01159C12N9/0006C12P33/06C07K14/245C12N15/52C12N15/70
Inventor R·施密德M·布劳恩L·刘A·艾格纳D·沃伊斯特-博茨
Owner CELL PHARM CO LTD
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