Cholic acid 1

Clostridium scindens VPI 12708 and Clostridium sporogenes ATCC 15579 were obtained from the Japan Collection of Microorganisms (JCM) and the American Type Culture Collection (ATCC), respectively. Engineered C. sporogenes strains used in this study are shown in Supplementary Table 3. They were cultured in TYG (3% w/v tryptone, 2% w/v yeast extract, 0.1% w/v sodium thioglycolate) broth at 37 ˚C in an anaerobic chamber from Coy Laboratories. Escherichia coli CA434 (HB101/pRK24) was cultured at 37 ˚C in LB broth supplemented with 12 μg/mL tetracycline and 100 μg/mL carbenicillin. In addition, 20 μg/mL chloramphenicol, 100 μg/mL spectinomycin or 250 μg/mL erythromycin was used for the selection of series of plasmids of pMTL83153, pMTL83353 or pMTL83253 respectively. Plasmids used in this study are shown in Supplementary Table 2. Cholic acid (1), chenodeoxycholic acid, deoxycholic acid (9) and lithocholic acid were purchased from Sigma-Aldrich. 3-oxo-cholic acid (3b) and 3-oxo-deoxycholic acid (8) were purchased from Steraloids. 3-oxo-4,5–6,7-didehydro-DCA (6) and 3-oxo-4,5-dehydro-DCA (7) were synthesized using previously reported procedures⁴⁴ . Structural assignments for the remaining pathway intermediates and derivatives shown in Fig. 2 and Fig. 3 are provisional, and were made on the basis of mass spectra, retention times, and comparison to chemically related standards.