Bioconversion of cholic acid into deoxycholic acid by a multi-enzyme catalytic system

Deoxycholic acid (DCA) is a key secondary bile acid with multifaceted physiological functions and therapeutic applications, most notably as the active ingredient in ATX-101, the only approved pharmaceutical for moderate-to-severe submental fat. Conventional chemical synthesis of DCA, however, relies on hazardous reagents and intricate purification processes, and raises environmental concerns. In this study, we established an efficient, environmentally benign biosynthetic pathway to convert cholic acid into DCA using the bai gene cluster derived from Clostridium scindens. Six key enzymes, BaiA2, BaiB, BaiCD, BaiE, BaiF, and BaiH, were heterologously co-expressed in Escherichia coli. A three-enzyme co-expression system achieved 87.91% substrate conversion and a 68.1% yield of DCA determined by HPLC after 96 h in a scale-up reaction. Aerobic cultivation was employed to enhance biomass accumulation without inactivating the oxygen-sensitive enzymes, while optimization of the enzyme ratio (BaiA2-BaiB-BaiE: BaiCD-BaiH-BaiF = 3:1) minimized intermediate accumulation. Integration of glucose dehydrogenase (GDH) enabled efficient cofactor regeneration, thereby improving process economy. Gram-scale production demonstrated the scalability and industrial applicability of the process, with the resulting DCA confirmed by MS and NMR analyses. This biosynthetic strategy avoids the use of organic solvents and hazardous reagents, offering a sustainable and efficient alternative for the industrial production of DCA.