7α- and 12α-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α‐ and 7β‐HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α‐OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7β‐OH). With a view to developing robust and active biocatalysts, novel NADH‐active 7β‐HSDH species are necessary to enable a solely NAD+‐dependent redox‐neutral cascade for UDCA production. A wild‐type NADH‐dependent 7β‐HSDH from Lactobacillus spicheri (Ls7β‐HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD+‐dependent 7β‐HSDH enzyme in combination with 7α‐HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD+, resulting in high yields (>90 %) of UCA and UDCA.

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“…Similar results were shown by the Dangate et al . and Giovannini et al . in chemical and chemoenzymatic routes, respectively.…”

Section: Discussionsupporting

confidence: 90%

“…1 H and 13 C NMR spectra, in agreement with previous reports, were acquired with Agilent ProPulse 400 MHz (Santa Clara, US) and are reported in the Supporting Information.…”

Section: Methodsmentioning

confidence: 99%

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

2018

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“…7α‐HSDHs have been found in Escherichia coli , Clostridium absonum , Pseudomonas sp. B‐083, Acinetobacter calcoaceticus lwoffi , Eubacterium sp. strain VPI 12708 and Bacteroides fragilis .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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“…The advantages of enzymatic oxidation include the reduction of waste and mild reaction conditions with high conversion and yield utilizing catalysts easily produced by microorganisms. [8] The 12α-HSDHs belong to the family of oxidoreductases with NAD + or NADP + as electron acceptor. The NADP + -dependent activity is distributed among the strains of the genus Clostridium, while the NAD + -dependent activity has been observed and reported in Eubacterium sp.…”

mentioning

confidence: 99%

Clean Enzymatic Oxidation of 12α‐Hydroxysteroids to 12‐Oxo‐Derivatives Catalyzed by Hydroxysteroid Dehydrogenase

Tonin

Alvarenga

et al. 2019

Adv Synth Catal

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The C12 specific oxidation of hydroxysteroids is an essential reaction required for the preparation of pharmaceutical ingredients like ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA), which can be synthesized by Wolff-Kishner reduction of the obtained 12-oxo-hydroxysteroids. 12α-hydroxysteroid dehydrogenases (12α-HSDHs) have been shown to perform this reaction with high yields, under mild conditions and without the need of protection and deprotection steps, required in chemical synthesis. Here, the recombinant expression and biochemical characterization of the nicotinamide adenine dinucleotide (NAD + )dependent HSDH from Eggerthella lenta (El12α-HSDH) are reported. This enzyme shows comparable properties with the well-known nicotinamide adenine dinucleotide phosphate (NADP + )-dependent enzyme from Clostridium sp. 48-50. In order to perform a viable and atom efficient enzymatic hydroxysteroid oxidation, NAD(P)H oxidase (NOX) was employed as cofactor regeneration system: NOX uses oxygen (O 2 ) as sacrificial substrate and produces only water as side product. 10 mM of cholic acid was fully and selectively converted to 12-oxo-CDCA in 24 h. The possibility to employ this system on UCA and 7-oxo-deoxycholic acid (7oxo-DCA) as substrates was additionally investigated. The performance of the El12α-HSDH was evaluated also in combination with a "classical" regeneration system (oxaloacetate/malate dehydrogenase) showing full conversion in 4 h. Finally, the feasibility of a catalytic aerobic-NAD + -dependent enzymatic oxidation was shown on a preparative scale (oxidation of CA to 12-oxo-CDCA) employing the El12α-HSDH-NOX system in a segmentedflow-reactor.

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7α- and 12α-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

Cited by 30 publications

References 24 publications

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

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

Clean Enzymatic Oxidation of 12α‐Hydroxysteroids to 12‐Oxo‐Derivatives Catalyzed by Hydroxysteroid Dehydrogenase

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7α- and 12α-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

Cited by 30 publications

References 24 publications

NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

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

Clean Enzymatic Oxidation of 12α‐Hydroxysteroids to 12‐Oxo‐Derivatives Catalyzed by Hydroxysteroid Dehydrogenase

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NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids