Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts.
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.
Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr 15, 85748 Garching, Germany.
Boqiao
B
Sun
Christina
C
Kantzow
Sven
S
Bresch
Kathrin
K
Castiglione
Dirk
D
Weuster-Botz
D039903:Actinobacteria / Q000201:enzymology; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D019149:Bioreactors / Q000382:microbiology*; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D020585:Comamonas / Q000201:enzymology; D003685:Dehydrocholic Acid / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D009161:Mycobacterium / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014580:Ursodeoxycholic Acid / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014580:Ursodeoxycholic Acid; D006913:Hydroxysteroid Dehydrogenases; D003685:Dehydrocholic Acid