Novel whole-cell biocatalysts with recombinant hydroxysteroid dehydrogenases for the asymmetric reduction of dehydrocholic acid

Abstract

Ursodeoxycholic acid is an important pharmaceutical so far chemically synthesized from cholic acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of dehydrocholic acid to 3,12-diketo-ursodeoxycholic acid was observed with the selected recombinant E. coli strain. In the optimal, slightly acidic pH range dehydrocholic acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial dehydrocholic acid concentration, and initial formate concentration. Complete conversion of 72 mM dehydrocholic acid was thus made possible at pH 6.4 in a whole-cell batch process within a process time of 1 h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50 mM dehydrocholic acid directly to 12-keto-ursodeoxycholic acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic acid.

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review

“…In this way, the use of lyophilized whole-cell containing recombinant HSDHs can represent a solution in the reduction of catalyst costs, maintaining a reasonable safety. This approach was followed by Braun et al and Sun et al obtaining the 12-oxo-UDCA with a yield of 99.5% using engineered E. coli cells [ 86 – 87 ]. In comparison to the systems that employed purified enzymes (see below), this approach allows higher substrate loading (70–100 mM).…”

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

“…Moreover, for each one of these positions, HSDHs usually show a practically absolute stereoselectivity by oxidizing only either the hydroxyl group above (β configuration) or below (α configuration) the plane of the steroid molecule . The reactions catalyzed by HSDHs are reversible, thus these enzymes can be applied in the regioselective reduction of the corresponding keto derivatives as well …”

Optimization of culture conditions for enhanced asymmetric bioreduction of acetophenone and its derivatives by growing cells of Pseudomonas sp. AP1

“…For this, extensive screening of microbial diversity, especially from unexplored organic rich sites, is an effective way to obtain novel and efficient isolates showing specificity for substrate of interest and having ability to sustain supply of reduced cofactor for these enzymes. Further, optimization of nutrient supplement and growth conditions could help in achieving desired level of transformation which could make process economically viable and competitive with other processes for "gram" scale production of chiral synthons (Braun et al, 2012).…”