Efficient production of ursodeoxycholic acid via a deep eutectic solvent-enabled 7α- and 7(3-hydroxysteroid dehydrogenase cascade system with enhanced solubility of 7-keto-lithocholic acid

초록

7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7(3-hydroxysteroid dehydrogenase (7(3-HSDH) are biological macromolecules capable of catalyzing the biosynthesis of ursodeoxycholic acid (UDCA) from chenodeoxycholic acid (CDCA) through a stereoselective oxidation-reduction cascade. This study introduces a deep eutectic solvent (DES)-assisted, high-level UDCA production system based on a one-pot enzymatic cascade. The system employs 7 alpha-HSDH from Escherichia coli and 7(3-HSDH from Ruminococcus gnavus as the core biocatalysts, supplemented with lactate dehydrogenase (LDH) from Lactobacillus delbrueckii and glucose dehydrogenase (GDH) from Pseudomonas sp. RIT288 for NADH and NADPH regeneration, respectively. However, UDCA biosynthesis via this enzymatic cascade in conventional aqueous buffer is limited by the poor solubility of the reaction intermediate, 7-keto-lithocholic acid (7-keto-LCA), and by pH fluctuations that reduce enzymatic efficiency. To overcome these limitations and improve the biocatalytic performance of the HSDHs, a DES composed of betaine and propylene glycol (B1P3DES) was integrated into the enzymatic cascade system. With the addition of 20 % DES, the solubility of 7-keto-LCA improved significantly, and the buffer capacity was enhanced, mitigating pH fluctuations and leading to improved UDCA production. Under optimized conditions with pH control, a 94 % bioconversion rate was achieved, compared to 60 % in aqueous buffer. Additionally, high-level UDCA production was demonstrated, with a 92 % conversion rate and a final titer of 46 g/L from 50 g/L CDCA. These findings highlight the potential of DES to enhance enzymatic cascade bioprocesses involving insoluble substrates or intermediates, offering a robust and scalable platform for the efficient biosynthesis of UDCA.

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