Abstract
The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile1, promote hepatocellular carcinoma2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A–B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe–S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
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Data availability
Mass spectrometry data that support our findings have been deposited in MassIVE (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) under accession code MSV000085048. Source data are provided with this paper.
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Acknowledgements
We thank C. T. Walsh, D. Dodd, C. O’Loughlin and members of the Fischbach and Almo laboratories for helpful comments on the manuscript. This work was supported by National Institutes of Health (NIH) grants DP1 DK113598 (to M.A.F.), R01 DK110174 (to M.A.F.), P01 HL147823 (to M.A.F.), P01 GM118303-01 (to S.C.A.), U54 GM093342 (to S.C.A.), U54 GM094662 (to S.C.A.) and DP2 HD101401-01 (to C.G.); the Chan–Zuckerberg Biohub (to M.A.F.); a Howard Hughes Medical Institute (HHMI)–Simons Faculty Scholars Award (to M.A.F.); an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Foundation (to M.A.F.); and the Price Family Foundation (to S.C.A.).
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Contributions
M.F., T.L.G., S.C.A. and M.A.F. conceived and designed the experiments. M.F. developed the system for gene-cluster expression in Clostridium, and M.F., C.G. and Y.V. performed the bacterial genetics experiments. T.L.G. expressed and purified enzymes and set up biochemical reconstitution experiments. M.F. analysed the data from biochemical and microbiological experiments by LC–MS. M.E.M. and L.C.B. synthesized bile acid intermediates. M.W. and S.H. performed and analysed mouse experiments. M.F., T.L.G., M.W., S.C.A. and M.A.F. analysed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.
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M.A.F. is a co-founder and director of Federation Bio.
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Extended data figures and tables
Extended Data Fig. 1 Previously proposed pathway for 7α-dehydroxylation of cholic acid in C. scindens VPI 12708.
See main text for details and a summary of the previous literature. CA, cholic acid.
Extended Data Fig. 2 Purification of recombinant Bai proteins.
a, SDS–PAGE analysis of purified Bai proteins after Ni-affinity and size-exclusion purification, visualized by Coomassie blue staining. The image was generated using a Bio-Rad Gel Doc Universal Hood II Molecular Imager. MWM, molecular weight marker; 1, BaiB from C. scindens; 2, BaiB from C. hylemonae; 3, BaiCD from C. hiranonis; 4, BaiE from C. scindens; 5, BaiE from C. hiranonis; 6, BaiA2 from C. scindens; 7, BaiF from C. hylemonae; 8, BaiH from C. scindens; 9, BaiI from C. scindens; 10, BaiI from C. hiranonis.b, Ultraviolet–visible spectra of BaiCD from C. hiranonis (24 μM, left) and BaiH from C. scindens (13 μM, right). Features at 370 nm and 450 nm are indicative of flavin bound to BaiCD and BaiH, and are partially obscured by the presence of a [4Fe–4S] cluster, which has broad absorbance between 300 nm and 700 nm. c, The presence of FMN and FAD is confirmed by mass spectrometry. Experiments in a–c were repeated independent twice, with similar results.
Extended Data Fig. 3 Bile acid standards.
a, For each compound in the study for which we have an authentic standard, we show an EIC of the authentic standard and the experimentally observed compound. Because the data shown here were collected from samples run at different times, a drift in retention time may be responsible for the peak pairs that do not have identical retention times. b, We observed a drift in retention time in the LC–MS data collected for the experiment shown in Fig. 2c. For two representative compounds from that data set, we show an EIC of the experimentally observed compound and an authentic standard run contemporaneously, showing that the retention times remain consistent with our peak assignments.
Extended Data Fig. 4 Kinetic parameters for BaiCD and BaiH.
a, Michaelis–Menten analysis of the conversion of 3-oxo-4,5-dehydro-DCA to 3-oxo-DCA by BaiCD. Reaction mixtures contained 0.45 μM BaiCD and 1 mM NADH, with the substrate concentration varying between 15 μM and 500 μM. b, Michaelis–Menten analysis of the conversion of 3-oxo-4,5,6,7-didehydro-DCA to 3-oxo-4,5-dehydro-DCA by BaiH. Reaction mixtures contained 0.45 μM BaiH and 1 mM NADH, with the substrate concentration varying between 3 μM and 100 μM. Data indicate the average product level ± 1 s.d. (three biological replicates).
Extended Data Fig. 5 Biochemical analysis of 3-oxo-DCA reduction by BaiA2.
Combined EICs showing the conversion of 3-oxo-DCA to DCA by recombinant BaiA2. This experiment was performed once.
Extended Data Fig. 6 7α-dehydroxylation of CDCA in vivo.
Combined EICs showing the conversion of CDCA to LCA by a C. sporogenes strain harbouring the complete bai operon on three plasmids (MF001) versus a control strain of C. sporogenes harbouring the transporter baiG (MF012). The strains were cultivated with 1 μM cholic acid for 72 h; an acetone extract of the culture supernatant was analysed by high-performance LC (HPLC)/MS. The single asterisk indicates isoLCA; the peak indicated by the double asterisk is provisionally assigned as isoCDCA. This experiment was performed once.
Extended Data Fig. 7 Constructs for expressing the bai operon and portions thereof in C. sporogenes.
Each of the plasmids has replication origins (origin and repH) for E. coli and Clostridium, the traJ gene to enable conjugal plasmid transfer, and an antibiotic-resistance gene (catP, aad9 or ermB). The bai genes were introduced into these plasmids under the control of the fdx or spoIIE promoter. For the genetic analysis of baiCD and baiH function, pMTL83153-based plasmids were used.
Extended Data Fig. 8 Metabolic logic of the 7α-dehydroxylation pathway.
Highly oxidized metabolic intermediates as anaerobic electron acceptors. In the first half of the 7α-dehydroxylation pathway, two successive two-electron oxidations set up a vinylogous dehydration of the 7-hydroxyl, yielding the highly oxidized intermediate 3-oxo-4,5-6,7-didehydro-DCA. In the second half of the pathway, three successive two-electron reductions reduce this molecule to DCA, resulting in a net two-electron reduction. The first two of these reductions are carried out by Fe–S flavoenzymes, which comprise a suite of four cofactors that enable them to convert two-electron inputs to a one-electron manifold. The previously proposed pathway is shown in Extended Data Fig. 1.
Supplementary information
Supplementary Information
This file contains the Supplementary Discussion and Supplementary References.
Supplementary Table 1
Data supporting our provisional chemical structure assignments.
Supplementary Table 2
Plasmids used in this study.
Supplementary Table 3
Bacterial strains used in this study.
Supplementary Table 4
Primers used for heterologous expression in C. sporogenes.
Supplementary Table 5
Primers used for expression of Bai proteins in E. coli.
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Funabashi, M., Grove, T.L., Wang, M. et al. A metabolic pathway for bile acid dehydroxylation by the gut microbiome. Nature 582, 566–570 (2020). https://doi.org/10.1038/s41586-020-2396-4
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DOI: https://doi.org/10.1038/s41586-020-2396-4
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