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.
The human gut microbiota harbour hundreds of metabolic pathways, most of which are encoded by genes that have not yet been identified5,6,7,8. Their small-molecule products are of interest for three reasons. First, most derive predominantly or exclusively from the microbiota (that is, there is no host source), and many enter the circulation, where they can have effects on peripheral tissues and organ systems. Second, their concentrations are similar to or exceed those of a typical drug; for example, indoxyl sulfate can accumulate in the human host at 130 mg per day9. Moreover, their concentration ranges are large, typically more than tenfold10, which could help to explain microbiome-mediated biological differences among people. Finally, of the few high-abundance molecules whose biological functions are well understood, most are ligands for a key host receptor; for example, short-chain fatty acids modulate host immune function via GPR41/GPR43 (refs. 11,12,13). Thus, high-abundance, microbiota-derived molecules are responsible for a remarkably broad range of phenotypes conferred on the host by bacteria.
Among these pathways, 7α-dehydroxylation of the primary bile acids cholic acid and chenodeoxycholic acid (CDCA) is particularly notable because the organisms that carry it out are present at very low abundance—an estimated ratio of 1:106 in a typical gut community14—yet they fully process a pool of primary bile acids that reaches concentrations of about 1 mM (ref. 15). Therefore, the flux through this pathway must be very high in the small subset of cells in which it operates, and the low-abundance organisms in the microbiome that perform this transformation have an unusually large impact on the pool of metabolites that enters the host. This pathway’s products—DCA and LCA—are the most abundant secondary bile acids in humans (up to 450–700 μM in caecal contents)16, and are known to be important in three biological contexts: prevention of C. difficile outgrowth1, induction of hepatocellular carcinogenesis2, and modulation of host metabolic and immune responses17,18,19. More broadly, DCA, LCA and their derivatives are a major component of the recirculating bile acid pool, representing more than 90% of the pool in the intestine and more than 25% in the gallbladder15. These microbiome-derived bile acids are therefore central to understanding the efficacy of therapeutics that target the bile acid pool and are approved or in clinical trials for treatment of primary biliary cholangitis and nonalcoholic steatohepatitis4.
In 1980 it was shown that the gut bacterium Clostridium scindens VPI 12708 carries out the 7α-dehydroxylation of cholic acid to produce DCA20. The knowledge that cholic acid serves as an inducer of 7α-dehydroxylation led to the discovery of a bile-acid-induced operon (termed bai) containing eight genes (Fig. 1 and Extended Data Fig. 1)21. Through heterologous expression and characterization of individual bai gene products, enzymes have been attributed to each step of the oxidative arm of the pathway22,23,24,25,26,27, but the reductive arm remained poorly characterized28. A complete understanding of the pathway would enable efforts to control the composition of the bile acid pool by engineering the microbiome.
Here, by purifying and assaying pathway enzymes under anaerobic conditions, we reconstituted 7α-dehydroxylation in vitro. We then transferred the pathway from its genetically intractable producer C. scindens into C. sporogenes, conferring production of DCA and LCA on a nonproducing commensal bacterial species. These data establish a complete pathway for two central components of the bile acid pool, and they provide a genetic basis for controlling the bile acid output of the microbiome.
Reconstitution of 7α-dehydroxylation
We first set out to de-orphan the remaining steps in the 7α-dehydroxylation pathway. Because previous studies of the bai enzymes involved expressing them individually in Escherichia coli, we reasoned that an alternative approach—in which enzymes are purified, mixed and assayed in vitro—could help to delineate the set of enzymes necessary and sufficient for 7α-dehydroxylation. Given that the eight-gene bai operon is shared among all known 7α-dehydroxylating strains, we focused our efforts on the enzymes encoded by the operon. We cloned three orthologues of each enzyme, expressed them individually in E. coli under microaerobic conditions, and purified them anaerobically as amino-terminal His6 fusions. Using this strategy, we obtained at least one soluble, purified orthologue of each Bai enzyme (Extended Data Fig. 2). When we incubated a mixture of the purified Bai enzymes with cholic acid, nicotinamide adenine dinucleotide (NAD)+, coenzyme A and ATP under anaerobic conditions and monitored the reaction by liquid chromatography with mass spectrometry (LC–MS), we observed the time-dependent conversion of cholic acid to DCA, indicating that the combination of BaiB, BaiCD, BaiA2, BaiE, BaiF, and BaiH is sufficient for 7α-dehydroxylation; no additional enzymes are required (Fig. 2a, b).
To test our hypotheses regarding the order of steps in the pathway, we performed stepwise reconstitutions in which enzymes were added one at a time and intermediates were allowed to build up at each step in the pathway (Fig. 2c). From these data, we draw two conclusions. First, the six enzymes used in the reconstitution are not just sufficient but also necessary, and the pathway proceeds according to the scheme shown in Fig. 2c. We directly observed mass ions consistent with each of the proposed intermediates, providing direct evidence for the previously proposed portion of the biosynthetic route. (See Supplementary Table 1 and Extended Data Fig. 3 for data supporting our provisional structural assignments; two important limitations are that we do not have authentic standards for all intermediates, and that the ability to distinguish bile acid isomers by LC–MS can be limited.) In spite of its conservation in all known dehydroxylating species, BaiI is dispensable for cholic acid dehydroxylation in vitro. As BaiI is a predicted Δ5-ketosteroid isomerase, it may process a substrate other than cholic acid, probably one with a 4,5- or 5,6-olefin.
Second, to our surprise, the absence of BaiH caused the pathway to stall at the highly oxidized intermediate 3-oxo-4,5-6,7-didehydro-DCA, and its addition resulted in two successive 2e− reductions to form 3-oxo-DCA. BaiH had previously been proposed to oxidize an alternative substrate, 3-oxo-4,5-dehydro-ursodeoxycholic acid25, so a potential role in the reductive arm of the pathway was unexpected. To explore this finding further, we incubated purified BaiH with synthetic 3-oxo-4,5-6,7-didehydro-DCA; we observed that the enzyme catalyses a 2e− reduction to 3-oxo-4,5-dehydro-DCA, but does not reduce this intermediate further (Extended Data Fig. 4). Notably, 3-oxo-4,5-dehydro-DCA does not build up in the reconstitution reaction containing BaiH, suggesting that another enzyme present in the mixture catalyses the second reductive step. Hypothesizing that the BaiH homologue BaiCD catalyses the second reductive step, we incubated it with synthetic 3-oxo-4,5-dehydro-DCA, revealing that it reduces this substrate to 3-oxo-DCA (Extended Data Fig. 4). Together, these data show that the pathway uses an unusual redox strategy in which the A and B rings of the steroid core are converted into a highly oxidized intermediate, 3-oxo-4,5-6,7-didehydro-DCA; and that the two key reductive steps are catalysed by two homologous enzymes in the Fe–S flavoenzyme superfamily, BaiH and BaiCD.
Finally, the last step in the pathway—reduction of 3-oxo-DCA to DCA—is carried out by BaiA2, as confirmed by assaying purified BaiA2 alone (Extended Data Fig. 5). Thus, BaiA2 and BaiCD both act twice in the pathway, catalysing its first two and last two redox steps.
Engineering the pathway into C. sporogenes
Having determined the set of enzymes that are necessary and sufficient for the pathway, we sought to gain genetic control over the pathway as a first step towards engineering the bile acid output of the gut community. We began by attempting to construct a mutation in the baiCD gene of the native producer, C. scindens, using the ClosTron group II intron system; however, we were unsuccessful owing to an inability to introduce DNA constructs into C. scindens by conjugation. As an alternative approach, we considered expressing the bai pathway in a gut commensal that is unable to carry out 7α-dehydroxylation; however, with notable exceptions29,30,31,32, methods for transferring pathways in Clostridium are underdeveloped. To our knowledge, no pathway from the human microbiome has been mobilized from one Clostridium species to another.
We selected C. sporogenes American Type Culture Collection (ATCC) strain 15579 as the recipient for two reasons: it is related to C. scindens, making it likely that ancillary metabolic requirements for the pathway (for example, cofactor biogenesis) would be met; and genetic tools have been developed that enable plasmids to be transformed into C. sporogenes33. Our initial attempts to clone the entire eight-gene bai operon (baiB–baiI) into an E. coli–C. sporogenes shuttle vector failed to yield clones harbouring the complete operon. Reasoning that there might be a gene in the cluster that is toxic to E. coli, we cloned various fragments of the cluster under the control of different promoters (detailed in Supplementary Table 2), eventually managing to split the cluster into three pieces, each in its own E. coli–C. sporogenes shuttle vector: baiB–baiF in pMTL83153 (pMF01), baiG in pMTL83353 (pMF02), and baiH–baiI in pMTL83253 (pMF03) (Fig. 3a and Extended Data Fig. 7). Genes in pMF01 and pMF03 were placed under the control of the spoIIE promoter from C. sporogenes ATCC 15579, which is expressed during the late stages of Clostridium growth34, while baiG in pMF02 was driven by the strong fdx promoter. We conjugated these plasmids sequentially into C. sporogenes to yield strain MF001.
When incubated with cholic acid, MF001 produces DCA in a time-dependent manner, in contrast with a control strain that contains only the transporter (baiG) (Fig. 3b, c), which does not. Additionally, MF001 converts CDCA to LCA (Extended Data Fig. 6). These data show that the eight genes in the core bai cluster (Fig. 1) are sufficient to confer bile acid 7α-dehydroxylation on C. sporogenes, although they do not rule out the participation of one or more genes endogenous to C. sporogenes.
Identifying branch points in the pathway
To uncover potential branch points for engineering the biosynthesis of non-native pathway products, we constructed a set of strains in which each of the eight genes was individually deleted (Extended Data Fig. 7). We grew these strains with cholic acid and assayed their culture supernatant for the build-up of intermediates (Fig. 3d). Deletion of genes in the oxidative arm of the pathway resulted in the build-up of early pathway intermediates, as expected. Two exceptions were the baiE mutant, which produced only cholyl-CoA; and the baiF-deficient strain, which generated a small quantity of the final product DCA, suggesting that there might be a compensatory CoA hydrolase or that nonenzymatic hydrolysis of the CoA thioester happens to some extent in vivo.
Intriguingly, the baiH mutant accumulates a key intermediate in the reductive arm of the pathway, 3-oxo-4,5-6,7-didehydro-DCA (Fig. 3d), supporting our finding that BaiH catalyses the first reductive step in the pathway. Moreover, strains of C. sporogenes expressing BaiG/BaiH and BaiG/BaiCD convert, respectively, 3-oxo-4,5-6,7-didehydro-DCA to 3-oxo-4,5-dehydro-DCA and 3-oxo-4,5-dehydro-DCA to 3-oxo-DCA (Fig. 3e), providing access to intermediates that do not accumulate in a culture of C. scindens. Notably, the fully oxidized and partially reduced intermediates are branch points for the production of allo (5α) bile acids, which have important biological activities, including the induction of regulatory T cells35. Thus, gaining genetic control over the pathway by expressing it in an alternative gut microbe provides opportunities for the rational and deliberate control of bile acid metabolism and the production of alternative molecules with distinct biological properties.
Colonizing mice with engineered C. sporogenes
Finally, we colonized germ-free mice with MF001 to see whether it would confer the production of pathway products on the host. We included two other experimental groups: as a negative control, germ-free mice monocolonized by C. sporogenes plus baiG (the bile acid transporter); and as a positive control, germ-free mice monocolonized by wild-type C. scindens, a native bai-operon-containing (7α-dehydroxylating) strain. As shown in Fig. 4, the engineered C. sporogenes plus baiB–I strain (MF001) conferred production of DCA on the host. The level of production was substantially lower than that observed from C. scindens; we suspect that this is because of the need to include additional genes, not yet known, that couple the pathway to a pool of reduced cofactor, increasing flux. Nonetheless, our data provide an important starting point for efforts to study and engineer the bile acid pool.
Engineering pathways from the microbiome
Our results reveal the complete bile acid 7α-dehydroxylation pathway, bringing it closer to the level of knowledge we have about endogenous human metabolic pathways. Key features of the pathway might serve as a model for other pathways that produce high-abundance metabolites in the gut (see Supplementary Discussion and Extended Data Fig. 8).
The gut microbiome harbours hundreds of pathways, many of which may modulate host biology, but so far only a few have been the target of engineering36,37. This stands in contrast to natural product pathways from terrestrial and marine microorganisms and plants, which are commonly expressed in heterologous hosts38,39 and engineered to generate non-native products40. Two technology gaps need to be overcome in order to make microbiome-derived pathways amenable to engineering: first, we need efficient strategies to identify pathways for known metabolites and small-molecule products of orphan gene clusters, and second, we need tools for transferring pathways into bacterial hosts native to the gut and manipulating them to produce novel molecules. The work described here is a starting point for these efforts. If it can be generalized to other Clostridia species, it could lead to a set of tools for de-orphaning, heterologously expressing, and engineering pathways from the microbiome.
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
Bacterial strains, culture conditions and bile acids
C. scindens VPI 12708 and C. 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 here 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. E. coli CA434 (HB101/pRK24) was cultured at 37 °C in LB broth supplemented with 12 μg ml−1 tetracycline and 100 μg ml−1 carbenicillin. In addition, 20 μg ml−1 chloramphenicol, 100 μg ml−1 spectinomycin or 250 μg ml−1 erythromycin was used for the selection of series of plasmids of pMTL83153, pMTL83353 or pMTL83253 respectively. Plasmids used here 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 reported procedures41. Structural assignments for the remaining pathway intermediates and derivatives shown in Figs. 2, 3 are provisional, and were made on the basis of mass spectra, retention times and comparison to chemically related standards.
Cloning of the bai operon
All amplification by polymerase chain reaction (PCR) was conducted using PrimeSTAR Max DNA polymerase (Takara Bio) according to the manufacturer’s instructions. Sequences of primers for target genes and cloning vectors are in Supplementary Table 4. For the heterologous expression of bai genes under the fdx promoter, pMTL vectors were amplified with primers 1 and 2. For the expression of bai genes under the spoIIE promoter, pMTL vectors harbouring the spoIIE promoter were constructed first. pMTL vectors were amplified with primers 1 and 3 to remove the fdx promoter, and the spoIIE promoter region, which is the 277-base-pair sequence upstream of CLOSPO_01065, was amplified with primers 4 and 5. Then these two PCR fragments were assembled by overlap PCR. The target gene sequences were amplified with primer pairs shown in Supplementary Table 4. PCR fragments were assembled with the amplified fragments of vectors using a Gibson assembly kit (New England Bio Labs). E. coli Stbl4 competent cells (Invitrogen) were transformed with the assembled plasmids by electroporation and transformants were confirmed by PCR. Positive clones containing the assembled plasmids were cultivated, with plasmids obtained by miniprep and verified by sequencing.
Heterologous expression in C. sporogenes
Bacterial cultures were incubated in a Coy anaerobic chamber under an atmosphere consisting of 10% CO2, 5% H2 and 85% N2. Growth media were prereduced by overnight preincubation in the anaerobic chamber. For the heterologous expression experiments, plasmids were transferred into C. sporogenes by conjugation using E. coli CA434, which was electroporated with the individual plasmids and recovered overnight in selective media. We collected 1 ml of overnight culture from the resultant transformants. The cell pellet was washed with phosphate-buffered saline (PBS) to remove residual antibiotics and resuspended with 200 μl of an overnight culture of C. sporogenes in anaerobic chamber. Eight drops of 25 μl of the suspension were pipetted on a TYG agar plate without antibiotics and the plate was incubated anaerobically at 37 °C for 2 days. The bacterial biomass was scraped up and resuspended in 300 μl of PBS. The whole cell suspension was then plated on TYG agar plates supplemented with 250 μg ml−1 d-cycloserine and appropriate antibiotics (15 μg ml−1 thiamphenicol for pMTL83153, 500 μg ml−1 spectinomycin for pMTL83353 or 5 μg ml−1 erythromycin for pMTL83253). After a few days, antibiotic-resistant colonies were picked and restreaked on agar containing the same antibiotic. The resulting clones were confirmed by PCR amplification using appropriate primers (Supplementary Table 4). Multiple plasmids were introduced sequentially, using the same procedure.
Extraction of metabolites
Engineered strains were cultured anaerobically in TYG medium supplemented with appropriate antibiotics from frozen glycerol stocks. We inoculated 10 μl of the overnight culture in 1 ml of TYG medium supplemented with appropriate antibiotics and 1 μM substrate. After 72 h, unless otherwise noted, the culture was extracted with 20% acetone and centrifuged. The supernatant was analysed by LC–MS.
LC–MS analysis of metabolite extracts
Metabolite extracts were analysed using an Agilent 1290 LC system coupled to an Agilent 6530 quadrupole time-of-flight (QTOF) mass spectrometer with a 1.7 μm, 2.1 mm × 100 mm Kinetex C18 column (Phenomenex). Water with 0.05% formic acid (A) and acetone with 0.05% formic acid (B) were used as the mobile phase at a flow rate of 0.35 ml min−1 over a 32-min gradient: 0–1 min, 25% B; 1–25 min, 25–75% B; 25–26 min, 75–100% B; 26–30 min, 100% B; 30–32 min 75–25% B. All data were collected in negative-ion mode.
For detection of CoA conjugates and flavin cofactors, a 1.8 μm, 2.1 mm × 50 mm ZORBAX SB-C18 column (Agilent Technologies) and water with 10 mM ammonium acetate pH 9.0 (A) and acetonitrile (B) was used. A flow rate of 0.3 ml min−1 was used over the 17-min gradient: 0–2 min, 15% B; 2–14 min, 15–50% B; 14–14.1 min 50–95% B, 14.1–17 min, 85% B. All data were collected in positive-ion mode.
Cloning of bai operon genes
To increase the probability of assembling a complete bai operon, we cloned the genes encoding baiB, baiA2, baiCD, baiE, baiF, and baiH from C. scindens VPI12708, Clostridium hylemonae and Clostridium hiranonis using the primers in Supplementary Table 5 and the KOD Xtreme Hot Start PCR kit (Millipore) according to the manufacturer’s protocol. Each PCR-amplified gene contains ligation-independent cloning (LIC) sites that are complementary to the pSGC vector. PCR products were purified with the Agencourt Ampure XP PCR clean-up kit (Beckman Coulter) according to the manufacturer’s protocol. The pSGC vector was prepared for LIC by linearization with the restriction enzyme BsaI. LIC sites were installed by adding T4 DNA polymerase (NEB) to 10 μg of linearized plasmid in a 50 μl reaction containing 2.5 mM GTP, 1× NEB buffer 2, and 1× bovine serum albumin (BSA) for 1 h at 22 °C. T4 DNA polymerase was heat-inactivated by incubation at 75 °C for 20 min. PCR products (in a volume of 2 μl) were treated with T4 DNA polymerase in a 10 μl reaction containing 2.5 mM CTP, 1× NEB buffer 2 and 1× BSA for 1 h at 22 °C. T4 DNA polymerase was heat-inactivated by incubation at 75 °C for 20 min. The LIC reaction was assembled by mixing 15 ng of digested vector DNA with roughly 40 ng of digested PCR product; the reaction mixture was then incubated at 22 °C for 10 min. A 30 μl aliquot of DH10B cells (NEB) was transformed with 2 μl of the LIC reaction mixture using standard bacterial transformation protocols. This cloning procedure adds a His6 tag to the amino terminus of each protein with the following sequence: MHHHHHHSSGVDLGTENLYFQS. All final constructs were sequence verified (Genescript).
Expression and purification of BaiH and BaiCD
BL-21(DE3) cells containing the pPH151 plasmid were transformed with the pSGC plasmid containing either BaiCD or BaiH. The transformants were selected on an LB/agar plate containing 50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol. A single colony was used to inoculate 20 ml of LB overnight culture containing the above antibiotics. The overnight culture was used to inoculate 2 l of Studier’s autoinduction media (ZYP-5052 supplemented with 1 mM flavin mononucleotide and 200 μM FeCl3) housed in a 2 l Pyrex media bottle. Cultures were grown with constant aeration using a sparging stone attached to a pressurized, 0.22-μm filtered air source, all in a water bath maintained at 37 °C. After 5 h, aeration was stopped and the culture was placed in an ice bath for 1 h. The culture was returned to a 22 °C water bath and light aeration was resumed. After 5 min, cysteine was added to a final concentration of 600 μM. The culture was grown at 22 °C for roughly 20 h before being harvested by centrifugation at 10,000g. Cell pellets were flash frozen and stored in liquid N2 until purification. All subsequent steps were carried out in an MBraun anaerobic chamber maintained at less than 0.1 ppm oxygen (MBraun, Stratham, NH). Plastics were brought into the chamber and allowed to sit for two weeks before use. All solvents and buffer stocks were degassed by sparging with argon gas for 4 h before being taken into the chamber.
In a typical purification, roughly 30 g of BaiCD or BaiH cell paste was resuspended in 30 ml of lysis buffer containing 50 mM HEPES, pH 7.5, 300 mM KCl, 4 mM imidazole, 10 mM 2-mercaptoethanol (BME), 10% glycerol, 1 mM flavin mononucleotide (FMN), 1 mM flavin adenine dinucleotide (FAD) and 1% Triton-X305. The resuspension was subjected to 50 rounds of sonic disruption (80% output, 3-s pulse on, 12-s pulse off) at 4 °C. The lysate was cleared by centrifugation at 4 °C for 1 h at 15,000g. The supernatant was loaded with an ÄKTA express fast protein liquid chromatography (FPLC) system onto a 5 ml fast-flow HisTrap column (GE Healthcare Life Sciences) equilibrated in lysis buffer lacking FMN, FAD and Triton-X305. The column was washed with 10 column volumes of lysis buffer before elution with 5 ml of buffer containing 50 mM HEPES, pH 7.5, 300 mM KCl, 300 mM imidazole, 10 mM BME and 10% glycerol. The fractions containing protein, based on absorbance at 280 nm, were pooled and reconstituted with iron and sulfur as described42. The reconstituted proteins were then passed over a HiPrep 16/60 Sephacryl S-200 HR column equilibrated in 20 mM HEPES, pH 7.5, 300 mM KCl, 5 mM dithiothreitol (DTT) and 10% glycerol. The proteins were concentrated to roughly 1 ml with a Vivaspin 20 concentrator (Sartorius Stedium Biotech). The protein concentration was estimated by absorbance at 280 nm (A280) using the extinction coefficient calculated on the basis of its corresponding amino-acid sequence. The presence of FAD and FMN was confirmed by precipitating purified enzyme with sulfuric acid and analysing the resulting supernatant by LC–MS, comparing analytes to authentic standards of FAD and FMN.
Purification of BaiB, BaiA2, BaiE and BaiF
BL-21(DE3) cells containing the pRIL plasmid were transformed with plasmids containing BaiB, BaiA2, BaiE or BaiF. Each transformant was selected on an LB/agar plate containing 50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol. A single colony was used to inoculate 20 ml of LB overnight culture containing the above antibiotics. The overnight culture was used to inoculate 2 l of Studier’s autoinduction media (ZYP-5052) housed in a 2l Pyrex media bottle. Cultures were grown with constant aeration using a sparging stone attached to a pressurized, 0.22-μm filtered air source in a water bath at 37 °C. After 5 h, aeration was stopped and the culture was placed in an ice bath for 1 h. The culture was returned to a 22 °C water bath and light aeration was resumed. The culture was grown at 22 °C for roughly 20 h before being harvested by centrifugation at 10,000g. Cell pellets were flash frozen and stored in liquid N2 until purification. All subsequent steps were carried out in an MBraun anaerobic chamber maintained at less than 0.1 ppm oxygen as above with minor modifications. Briefly, in a typical purification, roughly 30–40 g of cell paste was resuspended in 30–40 ml of lysis buffer containing 50 mM HEPES, pH 7.5, 300 mM KCl, 4 mM imidazole, 10 mM BME, 10% glycerol and 1% Triton-X305. The resuspension was subjected to 50 rounds of sonic disruption (80% output, 3-s pulse on, 12-s pulse off) at 4 °C. The lysate was cleared by centrifugation at 4 °C for 1 h at 15,000g. The supernatant was loaded with an ÄKTA express FPLC system onto a 5 mL fast-flow HisTrap column (GE Healthcare Life Sciences) equilibrated in lysis buffer lacking Triton-X305. The column was washed with 10 column volumes of lysis buffer before elution with 5 ml of buffer containing 50 mM HEPES, pH 7.5, 300 mM KCl, 300 mM imidazole, 10 mM BME and 10% glycerol. The fractions containing protein, based on absorbance at 280 nm, were pooled and immediately passed over a HiPrep 16/60 Sephacryl S-200 HR column equilibrated in 20 mM HEPES, pH 7.5, 300 mM KCl, 5 mM DTT and 10% glycerol. The proteins were concentrated to roughly 1 ml with a Vivaspin 20 concentrator (Sartorius Stedium Biotech). The protein concentration was estimated by A280 using the extinction coefficient calculated on the basis of its corresponding amino-acid sequence.
In vitro reconstitution of bile acid pathway
Six assays each contained 50 mM HEPES pH 7.5, 50 mM KCl, 200 μM NAD, 100 μM CoA and 200 μM ATP. In addition, each assay contained 0.1 mM of between one and six of the following enzymes: BaiB from C. scindens, BaiA2 from C. scindens, BaiCD from C. hiranonis, BaiE from C. hiranonis, BaiF from C. hylemonae and BaiH from C. scindens. All reactions were initiated with the addition of cholic acid and incubated at 22 °C for 30 min before being quenched by addition of an equal volume of 100% acetone. Each assay was performed in triplicate. Product formation was monitored by LC–MS as described above.
Bile acid pathway reconstitution kinetics
To determine the rate of DCA production by the in vitro pathway, we carried out assays with 50 mM HEPES pH 7.5, 50 mM KCl, 200 μM NAD, 100 μM CoA and 200 μM ATP, plus 0.1 mM of each of BaiB from C. scindens, BaiA2 from C. scindens, BaiCD from C. hiranonis, BaiE from C. hiranonis, BaiF from C. hylemonae and BaiH from C. scindens. Reactions were initiated by adding cholic acid and incubated at 22 °C. Samples of the reaction were removed and mixed with an equal volume of 100 mM H2SO4 at the designated times. Each assay was performed in triplicate. Product formation was monitored by LC–MS as above.
K M assay for BaiCD
Kinetic parameters for BaiCD from C. hiranonis were determined in assays that contained 0.45 μM enzyme, 50 mM HEPES pH 7.5, 50 mM KCl and 500 μM NADH. Reactions mixtures were incubated for 5 min at 22 °C before being initiated with 3-oxo-4,5-dehydro-deoxycholic acid. Concentrations of substrate were varied between 3.91 μM and 500 μM. We removed 20 μl of samples and mixed them with an equal volume of 100 mM H2SO4 to stop the reaction. Product formation was determined by LC–MS as above. Reactions were performed in triplicate and data were fit to the Michaelis–Menten equation by the least-squares method.
K M assay for BaiH
Kinetic parameters for BaiH from C. scindens were determined in assays that contained 0.45 μM enzyme, 50 mM HEPES pH 7.5, 50 mM KCl and 500 μM NADH. Reactions mixtures were incubated for 5 min at 22 °C before being initiated with 3-oxo-4,5,6,7-didehydro-deoxycholic acid. Substrate concentrations were varied between 0.78 μM and 100 μM. We removed 20 μl of samples and mixed them with an equal volume of 100 mM H2SO4 to stop the reaction. Product formation was determined by LC–MS as above. Reactions were performed in triplicate and data were fit to the Michaelis–Menten equation by the least-squares method.
Colonization of germ-free mice
Engineered C. sporogenes strains or C. scindens ATCC 15579 were cultured anaerobically in TYG medium (supplemented with appropriate antibiotics) or BHI medium from frozen glycerol stocks. Of the overnight cultures, 50 μl were used to inoculate 5 ml of fresh growth medium containing 50 μM cholic acid. After 48 h, bacterial cells were pelleted by centrifugation, washed twice with PBS, resuspended in 25% glycerol solution, and stored at −80 °C. Germ-free C57BL/6 mice (male, roughly 8 weeks of age, n = 6 or 7 per group) were obtained from Taconic Biosciences (Hudson, NY), and colonies were maintained in gnotobiotic isolators in accordance with the Administrative Panel on Laboratory Care (APLAC) of the Stanford Institutional Animal Care and Use Committee (IACUC). The mice were maintained on a Teklad Custom Diet (TD.180755, with 0.5% cholic acid). Mice were gavaged once daily for three successive days using the following procedure: glycerol stocks of engineered strains of C. sporogenes or C. scindens ATCC 15579 were thawed to room temperature, and mice were inoculated by oral gavage with roughly 200 μl of the thawed glycerol stock (about 1 × 107 colony-forming units). Six days after colonization, faecal pellets were collected and mice were euthanized humanely by CO2 asphyxiation. Caecal contents were also collected, snap-frozen in liquid nitrogen, and stored at −80 °C.
Murine faecal samples (roughly 30 mg) or intestinal contents (roughly 100 mg) were preweighed into a 2 ml screwtop tube containing six 6-mm ceramic beads (Precellys CK28 Lysing Kit). Then, 300 μl or 1 ml of a mixture of ice-cold acetonitrile, methanol and water (4/4/2, v/v/v) was added to each tube, and samples were homogenized by vigorous shaking using a Qiagen Tissue Lyser II at 25/s for 10 min. The resulting homogenates were centrifuged for 15 min at 14,000g at 4 °C, and 100 μl of the supernatant was combined with 100 μl of an internal standard solution (2 μM d4-cholic acid in H2O). The resulting mixtures were filtered through a Durapore PVDF 0.22-μm membrane using Ultrafree centrifugal filters (Millipore, UFC30GV00), and 5 μl was analysed by LC–MS as described above in the Methods section ‘LC–MS analysis of metabolite extracts’.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
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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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.).
M.A.F. is a co-founder and director of Federation Bio.
Peer review information Nature thanks Pieter Dorrestein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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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.
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.
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.
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).
Combined EICs showing the conversion of 3-oxo-DCA to DCA by recombinant BaiA2. This experiment was performed once.
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.
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.
This file contains the Supplementary Discussion and Supplementary References.
Data supporting our provisional chemical structure assignments.
Plasmids used in this study.
Bacterial strains used in this study.
Primers used for heterologous expression in C. sporogenes.
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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