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Aims: The role of a Acinetobacter johnsonii strain, isolated from a soil sample, in the biotransformation of bile acids (BAs) was already described but the enzymes responsible for these transformations were only partially purified and molecularly characterized. Methods and Results: This study describes the use of hybrid de novo assemblies, that combine long‐read Oxford Nanopore and short‐read Illumina sequencing strategies, to reconstruct the entire genome of A. johnsonii ICE_NC strain and to identify the coding region for a 12α‐hydroxysteroid dehydrogenase (12α‐HSDH), involved in BAs metabolism. The de novo assembly of the A. johnsonii ICE_NC genome was generated using Canu and Unicycler, both strategies yielded a circular chromosome of about 3.6 Mb and one 117 kb long plasmid. Gene annotation was performed on the final assemblies and the gene for 12α‐HSDH was detected on the plasmid. Conclusions: Our findings illustrate the added value of long read sequencing in addressing the challenges of whole genome characterization and plasmid reconstruction in bacteria. These approaches also allowed the identification of the A. johnsonii ICE_NC gene for the 12α‐HSDH enzyme, whose activity was confirmed at the biochemical level. Significance and impact or the study: At present, this is the first report on the characterization of a 12α‐HSDH gene in an A. johnsonii strain able to biotransform cholic acid into ursodeoxycholic acid, a promising therapeutic agent for several diseases. [ABSTRACT FROM AUTHOR]

12α‐Hydroxysteroid dehydrogenase (12α‐HSDH) has the potential to convert cheap and readily available cholic acid (CA) to 12‐oxochenodeoxycholic acid (12‐oxo‐CDCA), a key precursor for chemoenzymatic synthesis of the therapeutic bile acid ursodeoxycholic acid (UDCA). In this work, a native nicotinamide adenine dinucleotide (NAD+)‐dependent 12α‐hydroxysteroid dehydrogenase (Rr12α‐HSDH) from Rhodococcus ruber was identified using a structure‐guided genome mining (SSGM) approach, which is based on the structure of cofactor pocket and the conserved nicotinamide cofactor binding motif alignment. Rr12α‐HSDH was heterologously overexpressed in Escherichia coli BL21 (DE3), purified and characterized. The purified Rr12α‐HSDH showed a high oxidative activity of 290 U mg−1protein toward CA, with a catalytic efficiency (kcat/KM) of 5.10×103 mM−1 s−1. In a preparative biotransformation (100 mL), CA (200 mM, 80 g L−1) was efficiently converted to 12‐oxo‐CDCA in 1 h, with a 85% isolated yield and a space‐time yield (STY) of up to 1632 g L−1 d−1. Furthermore, Rr12α‐HSDH was shown to be able to catalyze the oxidation of other 12α‐hydroxysteroids at high substrate loads (up to 200 mM), giving the corresponding 12‐oxo‐hydroxysteroids in 71%–85% yields, indicating the great potential of Rr12α‐HSDH as a promising biocatalyst for the synthesis of various therapeutic bile acids. [ABSTRACT FROM AUTHOR]

Bile acids are important cholesterol-derived nutrient signaling hormones, synthesized in the liver, that act as detergents to solubilize dietary lipids. Bile acid 7α-dehydroxylating gut bacteria generate the toxic bile acids deoxycholic acid and lithocholic acid from host bile acids. The ability of these bacteria to remove the 7-hydroxyl group is partially dependent on 7α-hydroxysteroid dehydrogenase (HSDH) activity, which reduces 7-oxo-bile acids generated by other gut bacteria. 3α-HSDH has an important enzymatic activity in the bile acid 7α-dehydroxylation pathway. 12α-HSDH activity has been reported for the low-activity bile acid 7α-dehydroxylating bacterium Clostridium leptum; however, this activity has not been reported for high-activity bile acid 7α-dehydroxylating bacteria, such as Clostridium scindens, Clostridium hylemonae, and Clostridium hiranonis. Here, we demonstrate that these strains express bile acid 12α-HSDH. The recombinant enzymes were characterized from each species and shown to preferentially reduce 12-oxolithocholic acid to deoxycholic acid, with low activity against 12-oxochenodeoxycholic acid and reduced activity when bile acids were conjugated to taurine or glycine. Phylogenetic analysis suggests that 12α-HSDH is widespread among Firmicutes, Actinobacteria in the Coriobacteriaceae family, and human gut Archaea. IMPORTANCE 12α-HSDH activity has been established in the medically important bile acid 7α-dehydroxylating bacteria C. scindens, C. hiranonis, and C. hylemonae. Experiments with recombinant 12α-HSDHs from these strains are consistent with culture-based experiments that show a robust preference for 12-oxolithocholic acid over 12-oxochenodeoxycholic acid. Phylogenetic analysis identified novel members of the gut microbiome encoding 12α-HSDH. Future reengineering of 12α-HSDH enzymes to preferentially oxidize cholic acid may provide a means to industrially produce the therapeutic bile acid ursodeoxycholic acid. In addition, a cholic acid-specific 12α-HSDH expressed in the gut may be useful for the reduction in deoxycholic acid concentration, a bile acid implicated in cancers of the gastrointestinal (GI) tract. [ABSTRACT FROM AUTHOR]

Abstract: We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7α- and 12α-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12α-HSDH-mediated total oxidation of sodium cholate followed by the Wolf–Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7α-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield). [Copyright &y& Elsevier]

A simple colorimetric enzymatic assay for determination of serum 12α-hydroxy bile acids was developed using 12α-hydroxysteroid dehydrogenase (HSD). The enzymes were extracted from Bacillus sphaericus. The principle of the method is as follows: 12α-hydroxy bile acids are converted to 12-oxo bile acids using 12α-HSD with the conocomitant reduction of NAD to NADH, and then the hydrogen of the generated NADH is transferred by diaphorase to NTB to yield diformazan. Finally, the color of resultant diformazan was measured. The specificity and precision of this assay method were satisfactory. A linear relationship was noted between the amount of 12α-hydroxy bile acids and the degree of absorbance in the range of 6.7 to 215μM. The fasting values for serum 12α-hydroxy bile acid in 10 patients with liver diseases ranged widely from 7.6 to 91.1μM, and values obtained with this assay agreed closely with those obtained by gas-liquid chromatography (r=0.94, p< 0.001). The assay is convenient, rapid, and specific for the measurement of 12α-hydroxy bile acid concentrations in the serum of patients with liver diseases. [ABSTRACT FROM AUTHOR]