Cost-effective whole-cell biosynthesis of ursodeoxycholic acid using engineered Escherichia coli with a multienzyme cascade

Ursodeoxycholic acid (UDCA) can be used as a drug to treat various liver and bile diseases. Currently, the biological synthesis of UDCA is predominantly conducted via a two-step enzymatic process in which synthesis is catalyzed by 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) in succession, utilizing chenodeoxycholic acid (CDCA) as the substrate. In this study, an engineered <i>Escherichia coli</i> (<i>E. coli</i>) strain, designated UCA23, was constructed. This strain coexpressed four enzymes under the control of three independent T7 promoters: lactate dehydrogenase (LDH) derived from <i>Lactobacillus delbrueckii</i>, glucose dehydrogenase (GDH) derived from <i>Priestia megaterium</i>, 7α-HSDH derived from <i>E. coli</i>, and 7β-HSDH derived from <i>Ruminococcus torques</i>, enabling the whole-cell catalytic synthesis of UDCA from CDCA. This study systematically optimized the reaction parameters, including temperature, pH, and the addition of organic solvents and surfactants, for the whole-cell catalytic synthesis of UDCA by UCA23, and at the 2 L level, a UDCA conversion rate of 99% was achieved with 100 mM CDCA in 2 h, which is the highest level of conversion of a high-concentration CDCA substrate reported to date.