Flavin Oxidoreductase‐Mediated Regeneration of Nicotinamide Adenine Dinucleotide with Dioxygen and Catalytic Amount of Flavin Mononucleotide for One‐Pot Multi‐Enzymatic Preparation of Ursodeoxycholic Acid

Abstract

Ursodeoxycholic acid (UDCA), a pharmaceutical ingredient widely used in clinics, can be prepared from chenodeoxycholic acid (CDCA) by the epimerization of the 7α-OH group. In this study, a nicotinamide adenine dinucleotide (NAD + ) regeneration system was developed by using flavin oxidoreductase (FR) and flavin mononucleotide (FMN). Only catalytic amount of FMN is required for the effective NAD + recycling. FR/FMN system was then applied in the oxidation of CDCA to 7-ketolithocholic acid (7-keto-LCA) by NAD + -dependent 7α-hydroxysteroid dehydrogenase (Bs-7α-HSDH) from Brevundimonas sp., which showed extremely high enzyme activity toward CDCA (k cat /K m = 8050 s À 1 · mM À 1 ). When Escherichia coli whole cells coexpressing Bs-7α-HSDH and FR genes were used as biocatalyst, CDCA (50 mM) was completely converted to 7-keto-LCA with the turnover number of FMN being 227 and 58.8 g · L À 1 · d À 1 space-time yield of 7-keto-LCA. For the reduction of 7-keto-LCA, nicotinamide adenine dinucleotide phosphate (NADPH)-dependent 7-β-hydroxysteroid dehydrogenase (Cm-7β-HSDH) from Clostridium sp. Marseille was employed with alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) and iso-propanol as co-factor regeneration system. When E. coli whole cells coexpressing Cm-7β-HSDH and TbADH genes were used as biocatalyst, 40 mM 7-keto-LCA was reduced to UDCA with 26.8 g · L À 1 · d À 1 space-time yield. The oxidation and reduction were then carried in a one-pot concurrent mode, 12.5 mM CDCA was completely converted to UDCA. The epimerization of CDCA to UDCA proceeded to completion at the substrate concentration of 30 mM in the one-pot sequential process. Therefore, the complete conversion of CDCA to UDCA in one-pot has been realized by employing 7α-HSDH and 7β-HSDH of different co-factor specificities with independent co-factor recycling systems. The cholic acids, especially UDCA, exert inhibitive effect on the activities of these enzymes, preventing the complete epimerization of 7α-OH at higher substrate loading. This inhibition issue should be solvable by engineering the involved enzymes, that is currently pursued in our laboratory.

Sustainable and Robust Closed-Loop Enzymatic Platform for Continuous/Semi-continuous Synthesis of Ursodeoxycholic Acid

“…These results showed that the PBR performed better with a lower substrate loading. Similar phenomena for UDCA biosynthesis were also observed in other studies where high substrate loads often resulted in a very long reaction time, which impeded the efficiency of the biosynthesis of UDCA …”

“…Similar phenomena for UDCA biosynthesis were also observed in other studies where high substrate loads often resulted in a very long reaction time, which impeded the efficiency of the biosynthesis of UDCA. 43 Long-Term Continuous Production of UDCA. The robustness and service life of the biocatalyst are essential for successful bioconversion, especially for large-scale production.…”

Molecular Mechanism Study on Stereo-Selectivity of α or β Hydroxysteroid Dehydrogenases

“…[33] are able to catalyze the hydrogenation/dehydrogenation of carbonyl and hydroxyl on C3 position of bile acids, participating in the conversation of deoxycholate and lithocholate to isodeoxycholate and isolithocholate (https://biocyc.org/ META/NEW-IMAGE?type=PATHWAY&object=PWY-7755 (accessed on 29 January 2021)). A pair of epimerases, 7α-HSDH and 7β-HSDH, could specifically catalyze the reduction of C7=O or the oxidation of C7-OH on steroid-skeleton [34,35], with a substrate or product of CDCA(7α-OH) and UDCA(7β-OH). These two epimerases work together for the conversion of CDCA to UDCA in industry, with an intermediate 7-keto-lithocholic acid (7-KLA).…”

Laccase Did It again: A Scalable and Clean Regeneration System for NAD+ and Its Application in the Synthesis of 12-oxo-Hydroxysteroids

“…Other solutions for the regeneration of NAD + , employing molecular O 2 as final electron acceptor, were also identified. Profiting from the spontaneous auto-oxidation of FMN, a flavin reductase was employed to regenerate NAD + in the biocatalytic oxidation of CDCA to 7-oxo-LCA [8]. Laccase/mediator systems have been similarly applied in the same reaction [9,10].…”