Nature - USA (2020-06-25)

Nature | Vol 582 | 25 June 2020 | 569

which have important biological activities, including the induction of
regulatory T cells^35. 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 mono-

colonized 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

d

b

Retention time (min)

11 12 13 14 15 16 17

Selected ion abundance

MF012(C. sporogenes

+ baiG)

MF001(C. sporogenes

+ baiB-I)

Retention time (min)

11 12 13 14 15 16 17

Cholic DCA

acid

*

0 h24 h

48 h72 h

96 h120 h

m/z 407.28030Cholic acid m/zDCA 391.28538 c

*

a PspoIIE

baiG baiH baiI

Pfdx

baiB baiCD EA 2 baiF

PspoIIE

C. sporogenes +

bai genes (MF001)

Conjugate

plasmid 1

Conjugate

plasmid 2

Conjugate

C. sporogenes plasmid 3

e

Selected ion abundance

Retention time (min)

11 12 13 14 15 16 17

Retention time (min)

11 12 13 14 15 16 17

Scaled

10×

Scaled

10×

didehydro-DCA3-Oxo-4,5-6,7- 3-Oxo-4,5-dehydro-DCA 3-Oxo-4,5-dehydro-DCA 3-Oxo-DCA DCA

MF012

(C. sporogenes + baiG)

C. sporogenes + baiG/baiCD

C. sporogenes + baiG/baiH

MF012

(C. sporogenes + baiG)

C. sporogenes + baiG/baiCD

C. sporogenes + baiG/baiH

3-Oxo-CA

(3b)

3-Oxo-DCA

( 8 )

DCA

Cholyl-CoA( 2 ) ( 9 )

dehydro-CA3-Oxo-4,5-

(4b)

didehydro-deoxy-3-Oxo-4,5-6,7-

cholyl-CoA

( 5 )

didehydro-DCA3-Oxo-4,5-6,7-

( 6 )

dehydro-DCA3-Oxo-4,5-

Cholic acid( 1 ) ( 7 )

5

Ion abundance

×10^7

1.5

×10^4 ×10^6 ×10^3 ×10^3 ×10^5 ×10^4 ×10^5 ×10^6

5 2 4 4 1.5 5 2.5

No bai genes

baiIbaiHbaiFbaiEbaiA2baiCDbaiB

HO H OH

OH

O

HO H OH

SCoA

O

O H OH

SCoA

O

O

SCoA

O

O OH

SCoA

O

OH

O

OH

O

O

OH

O

O

OH

O

O H

OH

O

HO H

OH

O

1234

3b 4b

5

9876

Fig. 3 | Transferring the 7α-dehydroxylation

pathway into C. sporogenes. a, We divided the bai

operon among three plasmids: baiB–baiF in

pMTL83153 (plasmid 1, or pMF01), baiG in

pMTL83353 (plasmid 2, or pMF02) and baiH–baiI in

pMTL83253 (plasmid 3, or pMF03). These were under

the control of the spoIIE or fd x promoters (P). We

successively conjugated pMF01, pMF02 and pMF03

into C. sporogenes ATCC 15579 to create MF001.

b, Combined EICs showing the conversion of cholic

acid to DCA by MF001 versus a control strain of

C. sporogenes harbouring the transporter baiG

(MF012). The strains were grown with 1 μM cholic acid

for 72 h, extracted with acetone, and analysed by LC–

MS. The asterisk indicates isoDCA. The experiment

was repeated independently three times with similar

results. c, Combined EICs showing time-dependent

conversion of cholic acid to DCA by MF001. The strain

was grown with 1 μM cholic acid and aliquots from the

indicated time points were analysed as in b. The

experiment was repeated independently twice with

similar results. d, Top, simplified proposed pathway

for the 7α-dehydroxylation of cholic acid to DCA,

including the off-pathway hydrolysis products

observed here. Bottom, LC–MS ion abundances for

DCA, pathway intermediates and derivatives

produced by C. sporogenes strains with single gene

deletions within the bai operon (as indicated at the

left). Bars indicate means of three independent

biological replicates. e, Combined EICs showing the

conversion of 3-oxo-4, 5-6,7-didehydro-DCA to 3-oxo-

4, 5-dehydro-DCA by C. sporogenes plus baiG/baiH

(left), and the conversion of 3-oxo-4, 5-dehydro-DCA

to 3-oxo-DCA by C. sporogenes plus baiG/baiCD

(right). Each strain was cultivated with synthetic

3-oxo-4,5,6,7-didehydro-DCA (left) or 3-oxo-

4, 5-dehydro-DCA (right) for 72 h and culture extracts

were analysed as in b. The portions of the traces in the

grey boxes have been scaled up tenfold to make it

easier to visualize the peaks corresponding to

3-oxo-DCA and DCA. The experiment was repeated

independently twice with similar results.