Abstract
Tauroursodeoxycholic acid (TUDCA) has been used to treat many diseases effectively. 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) are two key enzymes that drive the efficient biosynthesis of TUDCA from taurochenodeoxycholic acid (TCDCA) in vitro. In this study, a metagenomic approach was used to isolate 7α- and 7β-HSDHs from fecal samples of black bears. Five new 7α-HSDHs and one new 7β-HSDH enzyme were discovered and identified from the gut microbiota of black bears, and four of them presented good enzymatic properties. Our data also suggest cooperation in the biotransformation of TUDCA by the gut microbiota in black bears. In conclusion, this work expands the natural enzyme bank of HSDHs, provides promising candidate enzymes for application in the biosynthesis TUDCA and the epimerization reaction of bile acids at the C-7 position, and provides a data set for the discovery of novel enzymes in the gut micriobiome of black bears.
Highlights
The Asiatic black bear (Ursus thibetanus), called the moon bear or white-chested bear, is the only mammal known to produce significant amounts of TUDCA
After searching the datasets consisting of the predicted ORFs, we successfully cloned and purified six full-length HSDH genes from the three fecal samples (Table 1): two 7α-HSDH encoding genes from S1, two 7α-HSDH encoding genes from H1, one 7α-HSDH and one 7β-HSDH encoding gene from sample Y1
The six HSDH genes discovered above and the 7α-and 7β-HSDH encoding genes from C. absonum ATCC27555 were overexpressed as GSTfused recombination proteins, and PreScission protease was used to excise the GST tag from the recombinant proteins
Summary
The Asiatic black bear (Ursus thibetanus), called the moon bear or white-chested bear, is the only mammal known to produce significant amounts of TUDCA. The 7α-and 7β-HSDH encoding genes from C. absonum ATCC27555 were cloned in order to compare their enzymatic characteristics in further steps of this study. We tested the eight proteins (five 7α-HSDHs and one 7β-HSDH discovered in the present study, and one known 7α-HSDH (name as Clo.sa-a) and 7β-HSDH (name as Clo.sa-b) purified from C. absonum ATCC27555) for enzymatic activity (Fig. 1B).
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