Physiological activity of E. coli engineered to produce butyric acid

Abstract

Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant bacteria in the human intestine, with its anti-inflammatory effects establishing it as a major effector in human intestinal health. However, its extreme sensitivity to oxygen makes its cultivation and physiological study difficult. F. prausnitzii produces butyric acid, which is beneficial to human gut health. Butyric acid is a short-chain fatty acid (SCFA) produced by the fermentation of carbohydrates, such as dietary fibre in the large bowel. The genes encoding butyryl-CoA dehydrogenase (BCD) and butyryl-CoA:acetate CoA transferase (BUT) in F. prausnitzii were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health using DSS-induced colitis model mice. The results from the E. coli Nissle 1917 strain, expressing BCD, BUT, or both, showed that BCD was essential, while BUT was dispensable for producing butyric acid. The effects of different carbon sources, such as glucose, N-acetylglucosamine (NAG), N-acetylgalactosamine (NAGA), and inulin, were compared with results showing that the optimal carbon sources for butyric acid production were NAG, a major component of mucin in the human intestine, and glucose. Furthermore, the anti-inflammatory effects of butyric acid production were tested by administering these strains to DSS-induced colitis model mice. The oral administration of the E. coli Nissle 1917 strain, carrying the expression vector for BCD and BUT (EcN-BCD-BUT), was found to prevent DSS-induced damage. Introduction of the BCD expression vector into E. coli Nissle 1917 led to increased butyric acid production, which improved the strain's health-beneficial effects.

Fig. 1

Butyric acid production by recombinant E. coli strains and F.prausnitzii. Butyric acid was quantified in RCM medium after incubation of E. coli strains for 6 h and F. prausnitzii A2‐165 for 48 h.

Fig. 2

Test of BCD and BUT activity in EcN‐carrying plasmids to confirm involvement in butyric acid production. A. Schematic of the FadB‐coupled assay used to assess BUT and BCD activity in the EcN strain carrying an expression vector for both BCD and/or BUT. B. Strain dependence of BUT and/or BCD activity. C. Substrate dependence of BUT and BCD activity. The increase in absorption intensity at 340 nm was recorded to measure the increase in NADH due to BCD and BUT activity from the FadB‐coupled reactions.

Fig. 3

Growth parameter and butyric acid production of wild‐type and recombinant EcN strains carrying BUT and/or BCD (A) Cell growth, (B) glucose consumption, and (C) production of butyric and (D) acetic acids.

Fig. 4

The effect of different substrates on EcN‐BCD‐BUT. A. Cell growth, (B) production of butyric acid and (C) acetic acid. Substrates used are indicated.

Fig. 5

Effect of E. coli strains with BCD and BUT on host parameters after administration of 2% DSS drinking water of the test mice. Strains indicated were orally administered each day for 9 days. A. Amount of butyric acid in the ileum and cecum as indicated, (B) body weight curve, (C) colon length; (D) amount of IL‐6, (E) of TNFα, and (F) of myeloperoxidase (MPO). All data were obtained from ten mice. *P < 0.05.

Fig. 6

Effect of orally administered E. coli with BCD and BUT on histological damage in the DSS‐induced colitis mouse model. Ascending colonic tissue was stained using H & E and microphotographs of histological cuts were taken at x200 magnification. A. Control mice; all other mice were treated with 2% DSS (B) and at the same time with (C) E. coli MG1655, (D) E. coli MG1655‐BCD‐BUT, (E) EcN, and (F) ECN‐BCD‐BUT. G. Histological scoring of colonic epithelial damage and inflammation assessed after H & E staining. All data were obtained from ten mice. *P < 0.05.