Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts

Authors

  • Boqiao Sun,

    1. Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany; telephone: 49-0-89-289-15712; fax: 49-0-89-289-15714
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  • Christina Kantzow,

    1. Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany; telephone: 49-0-89-289-15712; fax: 49-0-89-289-15714
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  • Sven Bresch,

    1. Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany; telephone: 49-0-89-289-15712; fax: 49-0-89-289-15714
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  • Kathrin Castiglione,

    1. Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany; telephone: 49-0-89-289-15712; fax: 49-0-89-289-15714
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  • Dirk Weuster-Botz

    Corresponding author
    1. Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany; telephone: 49-0-89-289-15712; fax: 49-0-89-289-15714
    • Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany; telephone: 49-0-89-289-15712; fax: 49-0-89-289-15714
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Abstract

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non-surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven-step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo-enzymatic preparation of UDCA—the two-step reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid using a mutant of 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens and 3α-hydroxysteroid dehydrogenase (3α-HSDH) from Comamonas testosteroni. Three different one-pot reaction approaches were investigated using whole-cell biocatalysts in simple batch processes. We applied one-biocatalyst systems, where 3α-HSDH, 7β-HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two-biocatalyst systems, where 3α-HSDH and 7β-HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one-pot reaction. The best result was achieved by the one-biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12-keto-UDCA within 4.5 h in a simple batch process on a liter scale. Biotechnol. Bioeng. 2013; 110: 68–77. © 2012 Wiley Periodicals, Inc.

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